U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES

	FOOD AND DRUG ADMINISTRATION

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	CENTER FOR BIOLOGICS EVALUATION AND RESEARCH (CBER)

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	ALLERGENIC PRODUCTS ADVISORY COMMITTEE

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	MONDAY

	MARCH 5, 20001

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		The Advisory Committee met in Versailles I and II Rooms in the Holiday Inn Bethesda, 8120 Wisconsin Avenue, Bethesda, Maryland, at 8:30 a.m., Dennis R. Ownby, Chairman, presiding.

PRESENT:

DENNIS R. OWNBY, M.D.	            Chairman
HENRY N. CLAMAN, M.D.             Member
SAMUEL B. LEHRER, Ph.D.           Member
DOLORES C. LIBERA                 Member
ANDREW SAXON, M.D.                Member
MARIA C. SOTO-AGUILAR, M.D.       Member
DALE T. UMETSU, M.D., Ph.D.       Member
WILLIAM FREAS, Ph.D.              Executive Secretary

This transcript has not been edited or corrected, but appears as received from the commercial transcribing service. Accordingly the Food and Drug Administration makes no representation as to its accuracy. 

	I-N-D-E-X
	Page

Opening Remarks	8
  Dennis R. Ownby, Chairman

REPORTS:

  Laboratory of Immunobiochemistry 	9
  Personnel Update, Jay E. Slater, M.D.

  Lot Release Statistics	12
  
  New Guidance Documents	15

  Research Update	17

  Cockroach Standardization	44

  Compliance Report, Mary Anne Malarkey	77

REGULATORY UPDATES:

  TSE as it affects allergens, Jay E. Slater	91

  Committee Discussion	127

  Open Public Hearing	142

  Statistical Power of Clinical Studies	143

  Committee Discussion	158

  Particulates in Allergen Extracts	179

  Invited Comments from APMA	192

  Open Public Hearing	236

  Committee Discussion	236

ADJOURN	243

	P-R-O-C-E-E-D-I-N-G-S
	(8:34 a.m.)
		DR. FREAS:  Good morning.  I would like to welcome everybody here to this, our 17th meeting of the Allergenic Products Advisory Committee.
		At this time I normally would go around and introduce the participants on the Advisory Committee.  However, those of you who were following the weather over the last few days know that those who are here are not really participants.  They are heroes and heroines.  So I would like to go around and introduce them, because we sure are indebted to them for braving the weather predictions and arriving here on time.
		In the first seat -- I'm going to be starting around the edge of the table -- we have our consumer representative, Ms. Delores Libera, Director of Publications, Allergy and Asthma Networks, and Mothers of Asthmatics, Inc., from Fairfax, Virginia.
		In the next seat is Dr. Andrew Saxon, Professor of Medicine, UCLA School of Medicine.
		Coming around the table in front of the podium is Dr. Dale Umetsu, Chief, Division of Rheumatology and Clinical Immunology, Stanford University.
		Next we have Dr. Maria Soto-Aguilar, rheumatologist in private practice in Hudson, Florida.
		Next we have our Chairman, Dr. Dennis Ownby, Professor of Pediatrics, Medical College of Georgia.
		Next at the corner of the table we have Dr. Henry Claman, Distinguished Professor of Medicine and Immunology, University of Colorado Health Sciences Center.
		We have two FDA participants that are joining us, and they are Ms. Jennifer Bridgewater, actually at the end of the table, who is our consumer safety officer for FDA; and we have Dr. Jay Slater who is Chief, Laboratory of Immunology, again at FDA.
		I would now like to read into the official record the conflict of interest statement for this meeting.
		The following announcement addresses the conflict of interest issues associated with this meeting of the Allergenic Products Advisory Committee on March 5, 2001.  
		To determine if any conflicts of interest existed, the agency reviewed and submitted the agenda and all relevant financial interests reported by meeting participants.  As a result of this review, the following disclosures are being made related to the discussions to be held today.
		Doctors Gruchalla, Ownby and Saxon have associations with firms that could or appear to be affected by committee discussions.  However, in accordance with 18 U.S. Code and Section 2635.502 of the Standards of Conduct, it has been determined that none of these associations is sufficient to warrant the need for a waiver, a written appearance determination or an exclusion.
		In the event that the discussions involve other products or firms not already on the agenda for which FDA participants have a financial interest, the participants are aware of the need to exclude themselves from these discussions, and their exclusion will be noted for the public record.
		With respect to all other meeting participants, we ask, in the interest of fairness, that you state your name, affiliation, and any current or previous financial involvement with any firm whose product you may wish to comment upon.
		So ends the reading of the conflict of interest statement.  At this time, the Director of the Office of Vaccines at FDA would like to come and make a few announcements, Dr. Karen Midthune.
		Oh, we have just been joined by Dr. >Samuel Lehrer, who is the Research Professor of Medicine, Tulane University Medical Center, and he, I know firsthand, had a lot of trouble getting here.  So we really are appreciative that he made it.  Thank you very much.
		Dr. Midthune.
		DR. MIDTHUNE:  Good morning, and thank you for braving the predictions of bad weather.
		I would like to take this opportunity to mention that we have three exceptional members of our Advisory Committee whose terms ends this coming August.  Since we don't at this time plan to have another meeting between now and August that would be a face to face meeting, we would like to take this opportunity to express FDA's gratitude for the dedication and service that these members have provided to the Advisory Committee.
		Of course, it's our hope that we will continue to be able to work with these individuals of consultants to the Center in the future.  Would Doctors Henry Claman, Andrew Saxon and Dennis Ownby please come to the podium.
		First off, we are very thankful to Doctors Claman and Saxon for their four years of service on the Advisory Committee.  During that time, they provided us with meaningful and insightful advice regarding our research program, our grass standardization program, and also recommendations for standardization of other antigens.  They also provided strong support for FDA to maintain its standards and distribution program.
		You also provided us with advice on expanding our lot release limits and assisted us with implementing our proposed algorithm for the standardization of new antigens.  As a token of our appreciation, we've prepared some special plaques commemorating your service to FDA.
		First, Dr. Saxon.
		DR. SAXON:  Thank you very much.  
		DR. MIDTHUNE:  First, I would like to read the letter that Dr. Linda Sudaym, Senior Associate Commissioner, has written:
		"Dear Dr. Saxon:  I would like to express my deepest appreciation for your efforts and guidance during your term as a member of the Allergenic  Products Advisory Committee.  The success of this committee's work reinforces our conviction that responsible regulation of consumer products depends greatly on the participation and advice of the entire health community.
		"In recognition of your distinguished service to the Food and Drug Administration, I am pleased to present you with the enclosed certificate.  Sincerely yours, Linda Sudaym, Senior Associate Commissioner."
		DR. SLATER:  Thank you very much.  
		DR. MIDTHUNE:  And now Dr. Claman.  I won't read the letter again, but Dr. Claman, of course, got a similar letter from Dr. Linda Sudaym, and here's the plaque.  
		Now for Dr. Ownby, in addition to your service as a committee member, we are especially thankful to you for your willingness to serve as the Committee Chair and lead us through deliberations from 1998 through the present.  Thank you very much for your willingness to perform this public service and for working so closely with us over the years.  Thank you very much.
		(Applause.)
		CHAIRMAN OWNBY:  Now it's my turn to say something.  I would like to welcome you all here today to these deliberations.  We have a somewhat full agenda today, although I think we should be able to get through it without too much trouble, and we will try to keep everything on time.
		I believe our first report is going to be from Dr. Jay Slater, who is going to give us a review of what the laboratory of Immunochemistry has been doing.
		DR. SLATER:  Thank you, Dr. Ownby.  Again, I want to thank everybody for coming out, in spite of the threats of bad weather.
		The Laboratory of Immunobiochemistry had a very good year this year.  It's been a very busy year, especially over the last several months.  What I would do at this point is just review what our staffing is, and many of the staff members are here in the audience, and I am going to ask them to stand when I mention their names.
		I am Jay Slater.  I'm already standing.  I've been here for two and a half years as the Lab Chief.  Dr. Lyudmila Soldatova -- please stand -- is a visiting scientists.  She has been with the Lab for three and a half years, and we are going to be talking more about her work with bee venom and Hyaluronidase mutants later on.
		Melissa Patterson is our Senior Biologist.  She has been with us for a year and a half.  She has been very busy with cockroach studies over the last six months, and in addition, during a fairly lengthy period that we were pretty short staffed, she pretty much single handedly kept up the regulatory functions of the Laboratory.
		Mona Febus is a microbiologist who has been with us for a year.  She came from a different laboratory and has been learning our regulatory techniques very nicely, and really contributing actively to the Lab activities.
		William J., or Jonny, Finlay is one of the newest additions to our laboratory.  He is a post-doctoral fellow from Ireland, and he is going to be doing some further work with cockroaches that I'll make reference to later on.  He came with money that we obtained from the Asthma Initiative last year through the ERDA Fellowship Program.
		Marc Alston and Cherry Valerio are our two newest biologists.  They are standing back there.  Marc and Cherry just joined us a very short time ago, and actually in the short time that they have been with us, they have really picked up the protocols very, very quickly.  
		They are actively involved in our regulatory functions, and they are -- to my great excitement, they are just getting very much into the research activities as well.  So we are looking forward to them being major contributors to the Laboratory.
		Al Gam was a happy inheritance that we obtained from another laboratory.  Al is a very experienced biologist in the Division, and actually was closely involved with our laboratory during a period about three or four years ago, helping to get some of our assays into somewhat better shape.  Al is joining us, and will be involved with several special projects over the next several years.
		Finally, Ron Rabin indeed is our new Senior Staff Fellow.  He literally started one week ago today with our lab.  He comes from NIH, and will be doing both clinical reviews, medical activities and, in addition, is setting up his laboratory to continue some of the very exciting lymphocyte research that he has been doing as a part of his work at NIH for the past several years.
		Beth Cardinale is working part-time with us.  She is a biologist who has been working mostly on the Hev b 5 mutation studies.
		Gerry Poley is a physician at Children's Hospital who has been a Guest Worker with us doing research a day and a half a week, and Li Shan Hsieh, Ph.D., used to work in the lab full time.  She is now at CDER, but she's been continuing some of her projects in the lab part-time.
		This is a graph that I started showing two years ago, and this is a graph of the biologist staffing from 1998 to 2001.  You can see here that we are at a very happy state of really being, I think, at a full complement in terms of our biologist staffing.
		Just to note again that we went through several months this year, from August until just last month, of being fairly low in terms of our staffing, and this was a period in which Melissa and Mona really bore the full brunt of the regulatory activities on their shoulders, and I think they did a really, really good job, and we really didn't miss a beat during that period.
		Among the routine regulatory activities that we are involved in, many of them involved maintaining the U.S. references -- the U.S. standards of potency.  Our laboratory develops references, and we will be talking about that with reference to cockroaches shortly.
		We also distribute the reference standards to all of the manufacturers.  I just want to show you the volume that we do distribute.  In 1999 we distributed 1983 vials in 104 shipments sent to our allergen manufacturers.  
		That volume has increased.  In the year 2000 we sent our nearly 2400 vials in 146 shipments sent to manufacturers.  So the trend at least over the last couple of years is we are already sending out quite a large number of shipments and vials, and that has gone up.
		We are involved in reference maintenance, and this is actually a very time consuming activity.  We check all of our references every six months for potency and for appearance on SDS-PAGE, and we are involved in replacing those references that are either running out because of expiry or just not having enough reagent to continue.
		The reference replacement procedure is one that I've shown you before.  We identify the references to replace.  We select good candidates from recent submissions from the manufacturers.  We do initial testing.
		I think I sort of give short shrift to this initial testing.  We do a lot of initial testing of these products at this point.  Then we select a provisional reference replacement.
		We send it out to all of the manufacturers, and ask them to add to our body of information about reference replacement.  Then we review the results.  We either confirm our original selection or we go back to step 2, depending on the data that the manufacturers sent us.
		In the year 2000 we replaced four extracts:  sweet vernal, a dust mite serum, an anti Amb a 1 serum, and a dust mite extract.  We sent three out of four of these out to the manufacturers for testing.  For the sweet vernal, three manufacturers participated by sending us back data.  For the anti Amb a 1 five did, and for the dust mite four did.
		This is a comparison of the relative potencies of the new and old extracts of those that we have replaced, actually a slightly longer period, since October of 1999, and you can basically see that the potencies have really been maintained fairly tightly, which is what we are looking for.  
		It's certainly very disruptive to the manufacturers when we change references, and they are not really right on target as to the previous ones.  So we do invest quite a bit of effort to try to avoid that from going on.  
		We are going to be replacing more references in the year 2001.  On the basis of volume remaining, we absolutely have to replace these two, the Anti-Fel d 1 serum and the cat hair. 
		On the basis of the nominal expiry date, we have another six extracts that we should be replacing and, hopefully, we will be able to replace all of these over the course of the next year.
		We are trying to escape from the cycle of having to replace references on an ongoing and frequent basis by lyophilizing the extracts, as I reported to you two years ago, and last year we have started a study in which we are lyophilizing a portion of all of our extracts, and we are going to be following their stability performance over a period of several years, comparing it to the extracts that we have available, and I will be showing you some of that data in the next lecture.
		Before we get on to the presentation about the research activities, I just wanted to very briefly note that two guidance documents that you have seen as an Advisory Committee in their pre-draft stages and in their draft stages actually came out this year.
		I included those in your packet for you to see them.  I did not intend for there to be a lengthy discussion of them, but I do want to note that we do actually come out with guidance documents, and the good work that the Committee does to help us out does actually come to fruition.
		This is the document that we presented to you two years ago regarding the revision of the way we handle lot release for grass and mite allergen extracts in which we tested a slightly broader interval than the manufacturers actually test. That came out in November 2000.
		In fact, at the same electronic moment on the Web, this other guidance document came out.  This is a document on the stability protocols for standardized grass pollen extracts.  This is the document that introduced the concept into those stability studies of using the Bonferroni adjustment in order to correct for the errors that can be built into multiply looking at the same extract over a period of a stability protocol.
		Those two both came out in November 2002, and are now being used as our guidance documents for these kinds of features of our program.
		That ends the sort of introductory part of our presentation.  My next presentation will be the research review, and the next presentation after that will be the cockroach standardization studies that we have done.  But first I would be happy to answer any questions that anyone may have on what I've said so far.  
		DR. SAXON:  Jay, when you replaced like the cat Anti-Fel d 1 serum, how do you do that?
		DR. SLATER:  The Anti-Fel d 1 serum is a sheep anti-serum.
		DR. SAXON:  Oh, it's not human.
		DR. SLATER:  It's not human.  That's right.  The first thing you do is you obtain a good preparation of Fel d 1.  The second thing you do is you find a sheep, and you immunize it, and then having identified an adequate titer, you plasmapherese the animal.
		DR. SAXON:  It's sheep.  That wasn't clear to me, because if it was human, I was wondering where -- You don't go get one of the sheep from Britain, though.
		DR. SLATER:  I'm sorry?
		DR. SAXON:  It's another biologic issue we don't want to discuss, hoof and mouth disease.  But, okay, I got it.  So you just make it sheep.
		DR. SLATER:  Yes.  Any other questions?  Shall I proceed?  
		It's been a very busy year in terms of research in the laboratory.  Our drop in the number of biologists and the census of biologists between August and December did have an effect, though, on some of our research productivity during that period.  In spite of that, I hope you will agree that we've really accomplished quite a bit this year.
		I have very good, good hopes for next year.  This is scheduled to be a very brief presentation.  It's really going to be bullets from the research program.
		This is a slide that those of you that have been to these meetings before have seen.  This is the basic outline of what we are trying to do, the questions that we think it is of validity for a laboratory in our position to attempt to answer and to attempt to address.
		The first broad area of interest that we have is the issue of allergen structure and function, and the second broad area of interest that we have is the area of immunomodulation.  Everything that we do in the laboratory addresses these two broad areas.
		What I am going to do later on at the end of this particular sub-presentation is I am going to show you the specific regulatory purpose of each of those, just so you can have an idea of where we see our research fitting into the regulatory mission of the laboratory.
		So the first issue of allergen structure and function is the stability of lyophilized references, and this is something I referred to at the end of the first presentation.  
		This summarizes a lot of data, and the basic message I want you to get from this is that, when we lyophilize the references that we have lyophilized so far -- and that's D. pteronyssinus, D. farinae, red top, meadow fescue, and Bermuda grass, and we have also done cat but that's in a different slide -- when we look at it immediately and when we compare the glycerinated product with lyophilized product or with lyophilized product in the presence of mannitol, there is really no significant effect on the relative potency of these products.  These are potencies that are measured compared to the current standard.  
		Reassuringly enough, when we come back at four months, in some cases at eight months or ten months, or even 14 months, we continue to see no significant difference in terms of the overall potency nor do we see any consistent trends over the period of time that we have observed.
		Yes, Dr. Lehrer?
		DR. LEHRER:  I wondered -- It's something that hasn't been addressed generally with lyophilized materials, but it's been our experience that sometimes there are solubility issues after lyophilization, although I think you addressed that in that you showed you change in activity, which I would expect if there were solubility problems.  But I wondered, just visually, do you see any differences?
		DR. SLATER:  Yes.  I'm actually glad you brought that up, because I probably wouldn't have in this presentation.
		In terms of the -- Let me just go over this and view it another way.  One of the reasons to add mannitol to the lyophilized product is a bulking agent.  There's a fear that if you have a very small amount of protein, you might lose it in some deep, dark corner of the vial, and mannitol prevents that from happening.
		In fact, we have had significant problems with resolubilizing the mannitol containing vials, but we've had no problems at all resolubilizing the vials in which the product was lyophilized just in water.
		So far, with the ones that we have done, we have not had a problem with resolubilizing just the lyophilized product.
		DR. LEHRER:  How and for how long are the lyophilized materials stored?
		DR. SLATER:  We've been storing our lyophilized materials at minus 20, and we only started this project a little under a year and a half ago.  So the longest that we have is -- The longest data point we have is 14 months, but we are going to be carrying this out for years.  
		DR. LEHRER:  Are they stored under desiccation?
		DR. SLATER:  The vials themselves are packaged in a vacuum, and the vacuum -- One of the features is, is the vacuum maintained over the period of storage.  But, yes, they are supposed to be.  But, no, the vials themselves are not desiccated.  They are not in desiccator containers.
		DR. UMETSU:  Jay, what do these numbers represent? 
		DR. SLATER:  These numbers are relative potency numbers.  So a potency that is equal to the reference standard would be 1.0.  However, due to the standard deviation of the test itself, anything between .7 and 1.4 is statistically indistinguishable from 1.  So that range would bring your attention down to this vial here, which has a potency of 0.59, and that actually was a concern to me.  
		If you notice, this is the meadow fescue that was lyophilized in the presence of mannitol.  So I was very interested in what the follow-up potencies were going to be on that.  As you can see, they fell right back into line at 8 and 14 months.
		It makes you wonder whether, when this study was originally done, there was a problem getting the whole thing back into the solution.
		DR. UMETSU:  And are there standard deviations for each one?
		DR. SLATER:  Yes, there are.  I didn't put them on this, because I wanted to -- I actually originally had this sort of separated out into the whole -- into each extract preparation individually.
		The standard deviation for the test, regardless of the allergen that is being tested, is 0.1375, but that's in log space.  So the 95 percent confidence intervals tend to range about plus or minus .25 or something like that.
		DR. UMETSU:  Because I notice, for example, in the second line at four months it goes from .71 to 1.4.
		DR. SLATER:  Yes.  Now in fact, you know, with this particular test, these two numbers just about bracket one, one on the low end and one on the high end.
		CHAIRMAN OWNBY:  Jay, these are all ELISA inhibition potencies?
		DR. SLATER:  These are all competition ELISA potency tests.
		CHAIRMAN OWNBY:  And this is -- Again, it's a human pool or an animal?
		DR. SLATER:  This is with human pooled sera.  Now the cat pelt extracts that we lyophilized, we test for Fel d 1, again this time using a sheep anti-serum and the radioimmunodiffusion assay, and we have data from one month, four months, eight months and 15 months. 
		You can see again that the Fel d 1 content of the two different lyophilized preparations are certainly in range compared to the glycerinated product itself.
		We also ran SDS-PAGE gels on all of these.  I am not going to show all of the SDS-PAGE gels, simply to point out that in general we see a pattern somewhat similar to this.  This is with the red top pollen extract at one month and at eight months.  This is glycerinated, lyophilized, and lyophilized in the presence of mannitol, lyophilized glycerinated, lyophilized and lyophilized in the presence of mannitol.
		Almost invariably, what we see is that the major appearance, the overall appearance looks absolutely identical, and there certainly have been no differences in any bands that we could identify as major or important allergens.  But in two or three of the extracts we have the kind of pattern that we see here where there appears to be fairly small and subtle bands that are present in the glycerinated product.  In other words, the product had never been through the lyophilization, resolubilization process.
		That seemed to disappear right up front.  So it's not a stability issue.  This is an issue of how it handles lyophilization.  We have this in two or three of our other extracts as well, where there will be usually one small band of uncertain importance that appears to disappear in the lyophilized resolubilized product.
		Again, we will be following this, and certainly this is one of the things that's going to come into our thinking before we decide to adopt lyophilization.
		CHAIRMAN OWNBY:  Jay, relative to a band like that, if you just did a straight aqueous extract without the glycerol, does that band stay there or not?
		DR. SLATER:  I don't know the answer.  
		The next area of interest within the general topic of allergen structure and function is the question of glycosylation of allergens.  Dr. Soldatova has been actively pursuing her studies with bee venom hyaluronidase.  She has four mutants at  putative glycolation sites that she has been expressing.  She also has mutants at active site areas, and her studies really are continuing and going forward quite nicely.
		In addition, we are pursuing cockroach studies that I am going to discuss in great detail in the next talk, and those are really aimed toward the establishment of the U.S. standard, but in addition -- and this is the work that I have Jonny Finlay working on -- we are interested in trying to amplify on the work that's been done by other labs in terms of the identification of significant allergens, in view of the fact that most of the current data are based on seroprevalence studies.
		Although seroprevalence studies have great strength, they also have some weaknesses as well.  Furthermore, I am interested in pursuing with cockroach allergens a comparison of native and recombinant products, again thinking that down the line we are going to have to face the regulation of recombinant allergens, just as CBER is involved in the regulation of other recombinant proteins.
		Jonny is currently working on generating an IgE-specific Fab library, a combinatorial library.  He has actually just started on working on that, but our plan is to try to develop that library from several individuals who have demonstrated cockroach allergy and to use this library as a tool for several subsequent studies that we think will be very important for us.
		One is for the affinity purification of native allergens.  As you know, several cockroach allergens are available now in recombinant form, but the purification of native allergens can be difficult, and we would like to attempt at least using this IgE-specific library to do some affinity purification of native allergens.
		We would also like to use the library to help identify important allergens.  Although there are quantitative problems with using this kind of library toward that end, we would like to try to identify which allergens are important in these individuals. 
		We certainly should be able to help identify important epitopes, and most importantly, we would like to use what we learn from these to compare the immunogenicity and allergenicity of native and recombinant cockroach products.  This is a project that has literally just gotten off the ground in the last two weeks, and I'm hoping next year to have much more to tell you about it.
		Last year I introduced -- Two years ago I introduced our interest in using physicochemical methods to identify and characterize allergens.  In particular, we were going to focus on MALDI-TOF, and MALDI-TOF, as those of you that have been at this meeting before heard, is a matrix assisted laser desorption/ionization time-of-flight mass spec.
		MALDI-TOF is a technique that can give very precise prints, very precise profiles, of protein mixtures.  The protein mixture is embedded in a matrix on the sample plate.  The matrix and the protein mixture are bombarded with laser energy that vaporizes the protein and the matrix.
		The protein molecules are drawn into a tube by an electric field, and the particles then fly down the tube in free flight, down the tube toward a detector.  The time of flight through the flight tube is a direction function of molecular mass and an inverse function of the ionic charge.
		So by measuring the time of flight from ionization to the detector, we can determine the mass/ charge ratio with great accuracy.  
		Last year what I showed you were several MALDI-TOF profiles of bee venom allergens, and the lesson that we learned from the bee venom allergens is that we probably could make a pretty good profile of the bee venom allergens.  
		I was able to show you several different proteins that were supposed to be present in one venom and  not the other, and you could clearly see there the profile was rather different.
		This year we looked at ragweed extracts, and we showed once again that you could see Amb a 1 pretty clearly here in the SDS-PAGE, and this is a representative MALDI-TOF of several ragweed MALDIs that we did.  You can see very clearly that there is a peak here.
		You can also see that there are several other peaks at lower molecular weights, as you can on the SDS-PAGE.  But we get a very good degree of precision here, and we are very optimistic about what we will continue to find as we look at some of these extracts.
		So far, we know that we can do this with venoms.  We think we can do it with ragweed as well, and we've had quite a bit of difficulty with cat, which was our next target, and we have really, surprisingly, failed to detect Fel d 1 in MALDIs, and we think that we may need to do some further purifications prior to MALDI mass spec analysis with cat, but we will be continuing our studies with cat, and we will be extending our studies to some of the pollens as well.
		Again, our objective here is to try to develop some non-immunologically based, some physicochemical methods that are highly reproducible to profile the allergens that we are charged with regulating.
		Under immunomodulation, I presented last year some of our LPS work, some of our epitope work.  I am actually going to pause for a moment to talk about Ron Rabin's work.
		Now Ron Rabin, as I said, just joined the lab a week ago, and he's been involved in important issues such as getting his computers hooked up and getting the phone lines hooked up, and all of these things take a great deal of time.  But when he starts doing his research, which, hopefully, will be very soon, Ron's work has been to look at the role of the chembakine receptors, CXCR-3 and its ligands in secondary lymphoid tissue and in Th-1, Th-2 cd4 T-cell polarization.
		He is preparing a manuscript to describe the work that he's been doing over the last several years at NIH, and he is going to continue that work, and his work is really very exciting, as far as our lab is concerned because, first of all, it's integrally related to the immunomodulation work that we do, and yet will really add a completely new dimension to our capabilities and to our expertise.  I'm really very happy that he is going to be working with us.
		The LPS and the allergen response, you will remember, is a project that I became involved in when I was still at Children's, and it really has continued here, largely because the results have been so interesting, but also because, while I was interested in LPS for one reason while I was at Children's, I'm now interested in another reason being here at FDA.
		We know that several allergen extracts, most notably house dust extract but many others, have endotoxin in them, and one of the questions that really needs to be answered is what is the immunomodulatory role of those components, if any.
		We know that LPS is ubiquitous.  We know that it elicits broad immunologic effects in mice and in humans.  It has some adjuvant activity.  It can accentuate both Th1 and Th2 responses.  
		I won't go through all the data that I presented last year.   What we've done this year is we have expanded the study to look at the anatomic specificity of the effects that we have observed.  So we studied -- and this was a fairly large and lengthy study in which we looked at four groups of mice that were all given antigen and LPS.  So there was no negative control group in this group.
		They were all given allergen and LPS.  One group was given both of them by the nasal route.  Another group was given antigen by the tracheal route and LPS by the nasal route.  Another group was given antigen nasally and LPS tracheally, and finally group four received both of them tracheally.
		Obviously, they were immunized on a specific regimen.  There were several bleeds.  We did plethysmography on the mice, and we harvested their organs at the end of the study.
		I'm only going to show the really interesting result, and the really interesting result is the plethysmographic data.  Plethysmography, for those of you that haven't done it, can be done on mice now by a noninvasive technique.  You don't have to cannulate the mice and intubate the mice.  You can actually just put them in a chamber.
		What you get out of that is a parameter called penh, which is the enhanced pause in their expiration curve.  By measuring their enhanced pause, you can get a good measure of what their degree of bronchospasm is.  So the higher the penh, the more the bronchospasm.
		These were mice that had completed their immunization schedule several weeks before.  At time zero they were each given an injection intraperineally of ovalbumin, and then they were put into the box and measured for many hours.  This is a six and a half experiment.
		All of the mice did very, very well up until just about two hours, and at two hours one group of mice, the group 4 mice that received both allergen and LPS by the tracheal route, began to have some moderate amounts of bronchospasm that worsened over the next half-hour, and then plateaued and remained so until they were taken out of the box and put back into their cages.
		Now all of the mice recovered, and you can see here that the mice in groups 2 and 3 had some slight bronchospasm that developed later, but the bronchospasm that appeared in group 4 was really significantly different from the other mice, especially from that interval of about 120 to 150 minutes out.
		Now the other data were also collected, antibody, spleen cell proliferation, all those things.  They weren't different in any of the groups.  In other words, all of these groups received antigen and LPS.  They all had indistinguishable immune responses otherwise except for this, and we are going to be expanding on these studies this year.
		DR. CLAMAN:  Do you have any idea what the responsible immunoglobulin isotype might be, assuming that it is antibody mediated?
		DR. SLATER:  We found no differences in the isotype profiles among the four groups.  So the answer is no.  That doesn't mean that there isn't a difference, but we looked at IgE.  We looked at IgG-1, IgG-2A, total IgG.  We found no differences in those groups that were measurable, but we were measuring serum antibody.  There may have been local antibody differences.
		Beth Cardinale has continued her work on the site directed mutagenesis of Hev b 5.  There are three areas based on our previous studies that we are interested in studying in detail.  She has successfully obtained three mutants, one mutant at each of these locations.
		She has successfully expressed those mutants.  So we actually have three working mutant proteins of Hev b 5 that we are going to be continuing studies with.
		During the past several months we have expanded on our collaboration with Robyn O'Hehir's group in Melbourne, Australia.  We started collaborating with Robyn over two years ago with our studies with Hev b 5, and we have actually published two papers with that group together.  So it's been a very productive collaboration so far.
		We are now going to expand it around these Hev b 5 mutants.  Robyn is going to be splicing our mutants into her expression vector, which is a His-tag vector, and she is going to be expressing them, and we are going to perform the following antibody studies in parallel.
		We have a series of human antisera to Hev b 5, a series of mouse monoclonal antibodies to Hev b 5, some rabbit hyperimmune sera that we obtained from Don Beezhold, and Robyn especially will be doing some T-cell stimulation studies on the individuals that she has recruited in Melbourne to study with Hev b 5 allergy.
		Well, let me summarize now quickly and just go back over each of the items and tell you what the specific regulatory part of -- role in the program is.
	Our studies on the stability of lyophilized extracts, obviously, is tightly bound to our ability to maintain a U.S. reference standard in a timely manner.  
		Our glycosylation studies, both of the bee venom proteins and of the cockroach proteins, is very closely involved in our guess that we are going to be heavily involved in the regulation of recombinant allergens over the next five to six years.
		Our studies with MALDI-TOF and the identification methods is part of our effort to improve lot release for current and future products.
		Our epitope studies are intended to help open new immunotherapy options, and our LPS studies are very important in terms of adjuvant safety and efficacy.
		We submitted five abstracts to the Academy meeting that is going to be happening in March, actually in two weeks, in New Orleans.  Three of them are being presented as posters, two of them as oral presentations.  We also have two other abstracts that appeared in collaboration with other laboratories.
		We have six publications from authors in our laboratory in refereed journals from this past year.  Two of them that are bolded in top are primarily from our laboratory.  This is a paper by Dr. Soldatova and the rest of us on the stability of dust mite allergens in glycerinated extracts, which appeared early in the year in the JACI, and a paper by Rich Pastor and myself on statistical methods for determining equivalent doses of standardized allergens that appeared also early in the year in JACI.
		These other four papers are the product of collaborations, both with Dr. O'Hehir's group and with other groups around the country.
		We published three reviews this year,  I published an article with Jerry Poley in JACI on latent allergy, and then Lyudmila and I published our presentations at the last Paul Ehrlich Symposium as  review articles in those proceedings.
		Are there any questions about the research program before I go on to cockroaches?
		DR. SAXON:  Jay, you used a phased display library to do what?
		DR. SLATER:  To generate a combinatorial IgE library.  So the idea is to develop a library of Fab --
		DR. SAXON:  IgE Fab?
		DR. SLATER:  Well, it's going to be -- Yes, that's the idea.  There are some questions as to whether you really can do that, but that's what --
		DR. SAXON:  It's been done twice.
		DR. SLATER:  Right.
		DR. SAXON:  One I know in Australia, and I was very concerned when looking at the results with those at what they may have picked up.  They think they have picked up -- Basically, they are all excited about what they think are B 1 type antibodies from humans, and I pointed out it looks to me what they have picked up is carbohydrate reactivity and not the clinically relevant antibodies.  So it's very tricky.
		DR. SLATER:  Well, we are aware of the trickiness of that.
		DR. SAXON:  Good.
		DR. SLATER:  And in fact, I think it's from Robyn O'Hehir's group that that was done.  
		DR. SAXON:  Well, it's a guy named Collins in Sydney.  He's not in Melbourne.  But I just think it's very tricky when you -- to find out when you're talking about clinically relevant, because all of this culture activity against carbohydrates, which you are very, very aware of anyway --
		DR. SLATER:  I agree.
		DR. SAXON:  I'm sure you will knock it on the head, but it's going to be tough.
		DR. SOTO-AGUILAR:  I have a question.
		DR. SLATER:  Yes?
		DR. SOTO-AGUILAR:  In the LPS study with the mice, you said you harvested the lungs and the spleen at the end of the study.
		DR. SLATER:  Yes.
		DR. SOTO-AGUILAR:  Did you see lymphocyte infiltrating the lung or those particularly synthesized with the trachea?
		DR. SLATER:  Yes.  Yes, and actually, the mice that received especially LPS through the trachea had significantly more inflammatory responses when their lungs were harvested.  Mind you, the lungs were harvested several weeks after the immunization program.  So these were chronic changes.  These were not acute changes.
		The mice that received just antigen in the lung with the LPS nasally did not have as much inflammation as the two groups of mice that received LPS in the lung.
		DR. SOTO-AGUILAR:  So was it a picture of hypersensitivity pneumonitis?
		DR. SLATER:  No, it was not a picture of hypersensitivity pneumonitis, but it might have been burned out hypersensitivity pneumonitis.  It might have been early on hat they had hypersensitivity pneumonitis.
		DR. SAXON:  Jay, what is your feeling about using LPS in a mouse?  It's so different than the human.  When she brings an issue up like that, people are getting concerned.  I think it's pretty far different.  I mean, humans don't have the md-1 comolecule for the tol system, and they are just different.  We're not mice.
		In many ways, we are, but not in the -- when it comes to the LPS, I'm not sure you are getting a good look at human disease.
		DR. SLATER:  Well, I think you raise a good point, if you were talking about a B-cell, T-cell response purely.  Clearly, mice have a different B-cell response in this, by a lot, to LPS from humans.  But if you look at -- If you look at the effect in vivo in mice on the generation of IgE, this is not a B-cell driven process.  This is clearly a T-cell driven process.  It doesn't happen in athymic mice.
You can't do it.  And so --
		DR. SAXON:  Yes, but that is probably for another reason.  I mean, all the works done by Honjo, they take IL-4, LPS and pure B-cells, and they get antibody.  It does not happen in humans.
		DR. SLATER:  So if there were good reasons to believe that the effect that we see with the adjuvancy were a pure B-cell effect, your point would be 100 percent correct.  But there's actually a fair amount of evidence that it's not, and --
		DR. SAXON:  Dave Peden does it in humans and gets interesting results.  I'm just not sure they are the same system.  
		DR. SLATER:  Yes.  Dave Peden's study of administering it nasally to humans was actually very interesting, and actually correlated pretty neatly with what we are doing.
		DR. SAXON:  I think as a model it's interesting, because you get away from those questions of what's relevant in the human, because he does it in the human.
		DR. SLATER:  Right.  I agree with you up to a point.
		DR. LEHRER:  Jay, not to kick a dead horse, but I just want to touch upon lyophilization again, because I have some real concerns about it, just based on my own experience.  I think one has to be cautious when considering adopting that for storage of standards.
		Keep in mind that there can be variability in lyophilized vials, too.  For example, our experience has been that, if you -- when you are lyophilizing a large batch of material, if you have any differential in temperature, and you might have some slight thawing and then refreezing which can occur, you will see the material looks very different.
		You can have white, fluffy material as opposed to brown, crusty material even apart of that, and that material is very insoluble.  So, clearly, there can be significant changes.  If this occurs, then there can be subtle changes that one may not necessarily pick up that could alter activity.
		Lyophilized material picks up water very readily.  Maybe it's the high humidity in New Orleans, but we've actually weighed out lyophilized material on a scale and actually just seen it increasing pretty rapidly.
		Now I realize that's not going to be your approach, but nevertheless, one should be careful in terms of someone taking out a sample and then putting it back, taking it out at room temperature and putting it back in the refrigerator or freezer, because those small differences can significant changes.
		My last point is that, in my opinion, lyophilized materials can vary with regard to their solubility according to the source.  I think that highly soluble extracts from pollens and so on generally will probably work well, but some of the foods, for example, could be a big problem in terms of solubility.
		I know we've seen this, and I don't remember what extracts they were.  I just can't remember at this point.  But it's just a word of caution, that if you are thinking of adopting this for all types of allergenic preparations, you, I think, should try a variety of them first before adopting this.
		DR. SLATER:  Well, I don't think we are going to adopt it for any one without having demonstrated with that one particular allergen that it works well, and I think everything that you've said is something that we need to keep in mind as we go forward.  In addition, we probably are going to need to replicate our results on multiple lots of the same allergen, just to make sure we get the same kind of consistency.  I think every point you made is very important, and we will look at it very carefully.
		This is not a change that's going to happen between now and the next Advisory Committee meeting.  This is something that's going to take a while to generate enough data.  But if we don't start now, we're never going to generate the data.
		DR. LEHRER:  You're exactly right, and I think that it just -- One of the reasons why I'm making these remarks is because these are long term experiments, and I think -- and I'm sure you are cognizant of all of these issues.  Then it won't be a year from now or two years from now that you say, oh, well, we should have tried this.  
		I think your point is well made that probably it's going to work well for some extracts and for others it may not.
		DR. SLATE:  And it may not be an option for others.  Right.
		Cockroach:  At last year's Advisory Committee meeting, we talked at great length about how we are going to go forward with standardizing new and different allergens.  We talked about some of the standards that we were going to use, some of the criteria we were going to use, and I proposed three possible broad areas that we might standardize, cockroach and two different molds.
		There was a considerable amount of very constructive concern by the Committee at that time about how difficult, how inadvisable it might be to go down the road of trying to standardize molds at this point.  
		In contrast, there was enthusiasm about trying to standardize cockroach allergens.  So that's what we have actually gone forward with, and this is work that has gone on since the last Advisory Committee meeting.
		Now let's just review very quickly on the standardization of new allergens how we choose new allergens.  What are the impact criteria that we are looking for with a candidate allergen?
		We want to make sure that, when we standardize an allergen, that we at least have somewhere a stable, preferably lyophilized material, if possible, for use as a long term reference extract.
		We want to have a good idea of what the consistency is of the currently marketed product.  We want to -- Ideally, if we have a choice between products, we would like to standardize one that is in widespread use, again just a matter of conserving your resources.  If you want to have as much impact as possible, you want to standardize a product that is currently in great use.
		Take into consideration the number of manufacturers producing a product.  Take into consideration whether it's used just for diagnostics or whether it's used for diagnostics and immunotherapy.
		Finally, in a specific evaluation of a specific product, what the public health impact is of the correct diagnosis and/or of adequate treatment.
		So this slide is a shameless attempt to get into the record horrible pictures of cockroaches.  In fact, the cockroaches at the bottom are the cockroaches that we will be concerned about, German, American and Oriental cockroaches.  But these creatures, although they look like they are out of B-movies, really are -- 
		These are the Madagascar hissing cockroaches, and they really are consuming a real child-size piece of birthday cake here.  These are big things, and they actually had them at the Smithsonian some years ago, and the workers would take them out and put them on your shoulder.  It was really a wonderful event.  
		They are called the hissing cockroaches.  They do hiss, and if you want have fun at your Website, go ahead in and search for Madagascar hissing cockroaches.  There's one site where, if you click a real audio button, you can hear them hissing.  I don't suggest that if you have trouble sleeping at night.
		Why is cockroach allergy important?  Well, cockroaches are ubiquitous.  They are all over.   Wherever humans live, there are cockroaches.  There are sometimes cockroaches in smaller numbers, in greater numbers, but they are there, and they are extremely difficult to control.
		If there is one convincing lesson that those of us that have heard the presentations from Peyton Eggleston and others, part of the inner city asthma study, it is that it doesn't matter what environmentally safe or environmentally toxic weapons you throw at cockroaches, the cockroaches come out of it.  It seems just fine.
		It's very, very difficult to decrease the exposure to cockroach allergens in the human environment.
		Thirdly, there are data, especially over the past five or six years, that suggest that cockroaches may, in fact, be connected to inner city asthma.
		Finally, due to the good work of several laboratories, there appear to be several cloned allergens for us to work with and for us to study.
		So Phase I of the standardization approach that I proposed last year is the laboratory phase, and that's what I am going to report to you now on, in which we develop or adapt preexisting methods for allergen determination, compare the allergen content of different lots.
		In many cases, what we are doing here is we are reproducing work that's already been done.  Dr. Lehrer did many of these studies several years ago, but we needed to repeat them and to expand them for the purposes of standardizing.
		If the commercial products are highly consistent -- in other words, if looking at what's available commercially they all look pretty much alike, then we may wish to reconsider the impact.  We may find that we want to go to other allergens to standardize rather than to standardize these.
		Likewise, if the commercial products are all comparable to the best available material -- and of course, best is in quotation marks, because that's a loaded question -- then we may also wish to reconsider impact.
		In other words, if we want to get our maximum impact for standardization, we would like to standardize allergens that seem to be in trouble in terms of either their consistency or their overall potency.
		So for our preliminary studies we obtained  multiple lots of cockroach from all of the manufacturers.  The idea was to characterize and compare the extracts and to identify target allergens that we might be interested in.
		The immediate goals were to determine the consistency of available U.S. products in terms of protein content, specific allergen content, and overall allergenicity and, hopefully, eventually to determine the best lot release measures.
		What do we use as our reference?  You have to pick a reference before you can start studies like this, and the reference that you choose is not necessarily going to be the reference that you end up with.  But it turns out that we had large quantities of references that were cleverly named E2-Cg for cockroach German and E2-Ca.  
		These were lyophilized extracts that CBER had obtained many years ago from one of our manufacturers, and the CBER lab had actually done several studies on them many years ago.  So we had a body of data. We had a large quantity of lyophilized product, and we thought we would start studying that.
		Now before you ask the question, there was an E1-Ca, and there was an E1-Cg.  That was a glycerinated product that we also studied and turned out to be less potent than the E1-Ca and Cg.  You will be seeing E1 in some of the other studies, but it's not the standard that we used.
		Very attractive about E1-Ca and Cg is that we even had limited skin test data on them from studies that were done at several centers.  Let me talk about that.
		You are all familiar with the ID50 method of determining the potency of a new allergen.  This is basically a method in which serial dilution intradermal skin tests are done on a highly sensitive population.  
		The orthogonal diameters of the erythema responses are measured for each of the dilutions.  Some of the erythema responses are plotted against the log of the allergen dose, and these lines in log space are characterized by a slope and an intercept.
		Now when you are doing the parallel line bioassay study to compare to extracts, you actually can measure the relative potency as a function of the difference in the intercept.  But when you have a new allergen, an uncharacterized allergen, you use the ID50 method in which you determine the mean dose at which a 50 millimeter response occurs, and you can average those and then go backwards and determine what the potency is.
		Now when these initial studies were done by Dr. Turkeltaub and colleagues many years ago in our laboratory, they found that for several allergens the model D50 was about 14.  So allergens with a D50 of 14 were defined as having 100,000 BAU per ml.  By rearranging the numbers, you can actually calculate the BAU per ml as 100,000 x 3(D50-14).
		Now before I show you the clinical data, these are very limited clinical data, and we are going to actually give a talk later on that will give you an idea of just how many study subjects you actually need to do a statistically valid study, but based on the limited data that we have, we can draw some limited conclusions.
		These are studies that were done between 1995 and 1998 in four different centers in the United States.  Some 20 to 25 individuals were recruited, and out of them we only really got valid data on ten or 11 of them, depending on the extract.
		To you look at the German cockroach extract, there were 11 individuals on whom there was valid data.  Now these data are stratified in two different ways.  On line 1 of each of these two tables, there is a sigma E of zero.  
		That means that all these individuals were screened with a prick test, and that means that this is all of the study subjects in whom valid data was obtained without regard to what their initial level of sensitivity is.  In other words, all comers that came to the study are included in line one.
		In line 2, however, we apply a certain exclusion standard.  If your initial sigma E is not above 50 millimeters, you are excluded.  So this second line would tend to be the more hypersensitive individuals.
		Nonetheless, if you look at the D50 that was calculated for these groups, for the German cockroach it's 11.2 for all comers, and for the more allergic individuals it's slightly higher, say 11.9.  If you convert that into BAU/mL, you get something between 4,000 and 9,000 BAU per mL.
		Now look at the 95 percent confidence interval.  Okay.  It's huge.  Why is it huge?  Well, it's huge because n is very small.  You need more study subjects in this kind of study in order to get a tighter interval.  We are heading in that direction, but we're not there yet.
		What you can say is that this German extract product is not super potent.  This is not a 100,000 BAU per mL product or at least it doesn't seem to be, based on these initial studies.
		The American cockroach, likewise, interestingly, whether we used all comers or whether we excluded the bottom three individuals and only looked at the seven most hypersensitive individuals, we came out with essentially the same D50, again 16-1700 BAU per mL.
		So the take-home message from this is we don't know a lot about these extracts, but we don't think they are very hot.
		We obtained cockroach extracts from all the manufacturers.  We obtained 26 extracts.  Six of them were aqueous.  Twenty of them were glycerinated.  They are widely varied in their appearance.  The three on the left are aqueous extracts.  The one on the right is a glycerinated extract.
		I show this really to show you the vast difference in color and appearance.  You won't be surprised to learn that vial number 14 is a one to 1,000 aqueous extract, but I am also showing this as a sort of a preamble to a talk that Jennifer Bridgewater is going to be giving this afternoon.
		This little darkness at the bottom here, this orange color, is not a photograph artifact nor is the cloudiness in this extract.  These extracts both have precipitates in them, which was a fairly common finding among our cockroach extracts, the aqueous cockroach extracts in our study.
		Interestingly, even though this glycerinated material looks much darker and richer and you could easily imagine a lot more cockroach stuff in this, this was completely in solution.  There was no precipitate in this one.
		We looked at all of these extracts using various protein assays, SDS-PAGE, various kinds of ELISAs and Western Blots.  So here's the protein concentration data using the ninhydrin method.
		The bottom line on this is that the concentration of the glycerinated American cockroach extracts was about 9 milligrams per mL with a range of about 3.5 to 21, German glycerinated extracts about 4.5 with a range of 2.5 to 10.5.
		Now the aqueous data looked horrible in terms of the range.  It's about 6.8 mean, but the range is 0.09 to 24.82.  This is a little bit of an unfair representation of the aqueous extracts, because while the glycerinated extracts are either 1 to 10 or 1 to 20, the aqueous ones go down to 1 to 1,000.  
		So don't get too -- There are lots of reasons to say things about aqueous extracts that suggest that they are not as good as glycerinated extracts, but this isn't one of them.  This is simply a matter of the labeled dilution of the extract.
		DR. LEHRER:  Jay, just a quick question.  For your protein assay, what do you use as the standard?
		DR. SLATER:  Bovine albumin.  So it's an estimate.  Is there a standard you would think would be more appropriate for insect proteins?
		DR. LEHRER:  No.  It's always a concern, because any value is really artificial based on the protein that you use, and there are just variabilities.  But you're right.  I think there are going to be issues with any standard, and it's really just a reference to give you an idea.
		DR. SLATER:  But one thing to remember then -- So remember our numbers were about 4 to 8 milligrams per mL for the glycerinated extracts.  Now the E2 standards that we used were considerably higher.  They were 25 to 30 by the ninhydrin assay and, therefore, we scaled up the allergen levels and the relative potencies to the reference protein concentration.
		I didn't want to just measure this protein difference over and over and over again.  So we scaled up the numbers for the allergen levels and the relative potencies.  You'll see that in the data.  You'll be able to see that we've done that, but I just want to explain why we're doing that.
		We revisited the question of which protein assay is best and which ones are interfered with.  We came up with very familiar answers.  When we look at the correlation of the protein assays to each other, we find a fairly good correlation among the aqueous extracts among all the different assays, r2 of 99, 95 and 92.  So not too bad.
		When we look at the glycerinated extracts, things fall apart, and the correlation especially between the ninhydrin and the Bradford assay and the BCA and Bradford assay, and even the ninhydrin versus the BCA assay is really not well correlated at all.  Again, this is not particularly surprising.  
		We know from work that Paul Richman did when he was part of the allergenic products laboratory that glycerine interferes with the Bradford assay and, to a lesser degree, with the BCA assay; and it doesn't appear to interfere with the ninhydrin assay.
		So, really, this part of our study really validated the value of the ninhydrin assay as being a good protein assay when you are looking at glycerinated extracts.  It's not interfered with.  So for the rest of this study, we used the  ninhydrin protein concentrations for the remainder of our analysis.
		Again, our conclusions were that the protein concentrations of the commercial extracts vary widely, and are lower than the protein concentrations of our references, and again that glycerin interferes with the Bradford and, to a lesser degree, with the BCA assays.
		So now we're going to start to look at some of the specific allergens, and listed here is a listing of the different relevant allergens in cockroach, Bla g 1, 2, 4, 5, and 6, and an American cockroach Per a 1, Per a 3, and Per a 7.
		This is an SDS polyacrylamide gel of our various reference extracts.  Shown on the left are the German cockroach extracts and on the right are the American cockroach extracts.  You can identify several bands that you might think, based on their molecular mobility, might be some of the identified bands.
		Needless to say, this pattern is a little bit too complicated to identify this band specifically as Bla g 2 or this band specifically as Bla g 1, but you do get a good sort of rich pattern of specific bands that you can further study.
		Likewise when we look at -- I'm sorry.  Oh, when we look at the specific commercial products of German cockroach, we come up with some interesting observations.  The way this is organized is that multiple lots from manufacturers are grouped together.  So the first two columns are two different lots from one manufacturer.  The next two lanes are two different lots from another manufacturer.
		This fifth lane is from one manufacturer, and these next two lanes are again two different lots from one manufacturer.  What you see is within a manufacturer you have a pretty decent amount of consistency between the two products, but among the manufacturers you see a lot of heterogeneity.
		Again, this is an observation that, I think, has been made before.  Again, Dr. Lehrer's studies from the past looked at comparisons of different manufacturers' products.  But you can see specific bands that are present, major bands that are present for some manufacturers that seem to be almost completely absent for others, and vice versa.
		Looking at the commercial glycerinated American cockroach products -- Incidentally, the reason I am not showing the aqueous products is that there's not much to look at.  When you do the aqueous products on SDS-PAGE, you don't see much at all.  So you do see something.  It's not completely gone, but there's just not that much there.
		The glycerinated American cockroach products:  Again, there's a lot of heterogeneity between the manufacturers.  But for instance, these two -- and actually, even these two lanes, which are from the same manufacturer, there's really a big difference here in these bands.  
		Again, are these specific bands important?  We don't know, but there's certainly evidence here of heterogeneity.
		Now one of the things that bothered us was that all of these commercial products and our re-reference standards had a significant number of bands up in the region above 75 kilo-Daltons.  One of the things you always worry about with allergen extracts is whether you have aggregates present in those or whether you are really looking at well defined individual proteins.
		So we did some studies looking at these gels in the presence of 4 mole urea.  Now you all know that SDS, which is used as our denaturant in SDS polyacrylamide gels, is a strong denaturant, and also it has the additional benefit of conferring uniform charge density on the proteins so that they can be separated according to their molecular mass rather than mass and charge.
		Four mole urea appears to be a stronger denaturant than SDS, but in our case we used the urea in addition to SDS, because we still wanted to have that charge density uniformity so that we could read the gels more easily.
		What you see here on the left is a repeat of the German and American cockroach standard that we had using a regular SDS-PAGE gel.  The same products run concurrently in an SDS-PAGE gel that contained a significant amount of urea, 4 mole urea in the gel, and actually I think there were six or eight mole urea in the sample buffer itself.
		What you see here -- and since the markers migrated differently as well, I put these blue lines in to sort of guide you through it.  The lower blue line represents the 35 kilo-Dalton standard, and the upper blue line represents the 75 kilo-Dalton standard.
		What you see here is that, when you go to the urea gels, the products -- the bands that are above 75 kilo-Daltons disappear entirely, and the bands that are -- even the bands that are between 35 and 75 kilo-Daltons, while they don't disappear entirely, they go away a lot, and you see a real enrichment of the bands that appear below 35 kilo-Daltons.
		So we certainly seem to see some evidence here that a lot of the higher molecular weight bands that have been observed before may well be aggregates of lower molecular weight proteins.  Well, that's actually good news and bad news.
		It's good news, because it suggests that the list of allergens that we are looking at, which are, for the most part, lower molecular weight proteins, may in fact be a list that is, if not complete, at least close to complete.  At least we have a reasonable number of those.
		It's bad news, because urea gels are harder to run, but I suspect we are going to have to end up doing that for a good portion of our studies.
		Just for interest's sake, since we were pouring urea gels, we actually ran some recombinant cockroach proteins on urea gels, just to see if they migrated substantially differently.  So this is a regular SDS-PAGE gel, and this is a urea gel run concurrently.
		What you see here-- this is recombinant Bla g 5, recombinant Bla g 4 and recombinant Bla g 2, in the same order, recombinant Bla g 5, Bla g 4, and Bla g 2.  So if you look at recombinant Bla g 2, it migrates at about 50 kilo-Daltons in the SDS-PAGE gel, and it migrates just at about 50 kilo-Daltons in the urea gel.  So that's reassuring.
		Let's skip over to Bla g 5.  It migrates at about 25 kD in the SDS-PAGE gel.  It migrates a little bit faster in the urea gel, say at about 23 or 22 kilo-Daltons by apparent molecular mass.
		So that's not wildly different, and that's not terribly surprising.  But, gee, look what happens to the recombinant Bla g 4 that we got.  This migrated at 30 kilo-Daltons as, for the most part, a nomomer.  There was a small looked like a contaminant peptide here.  This is in the SDS-PAGE gel.  But it almost completely disappears in the urea gel, but if you look carefully, you see there's a band at 16 and a band at 14 kilo-Daltons.  It almost looks as though it's a heterodimer.
		Now that's not what is described, and I'm not about to take this and publish it as evidence that recombinant Bla g 4 is a heterodimer, but it certainly is something that we were surprised to see, and we are going to be looking at it some more.
		Back to our commercial products, we performed immunoblots using pooled allergic sera of our different products, of both American and German products.  For the purpose of time and efficiency, I am only showing the German immunoblot here.
		You can just see that there are a lot of different proteins that seem to light up, which suggests to us, at least pooled allergic sera does recognize several peptides in both of these products, and again with specific anti-Bla g 1 and specific anti-Bla g 2 that we obtained from Endo Biotechnologies, you see a sort of a different picture, depending on what you are looking at.
		In the blot using the anti-Bla g 1 monoclonal antibody, we see a band, a prominent band, at less than 10 kilo-Daltons and another band that's somewhere between 35 and 50 kilo-Daltons.  Now Bla g 1 is supposed to be between 20 and 25.  So we're not really sure what we are seeing here, but the migration of some of these does go differently, depending on the conditions under which they are run.
		Now with the anti-Bla g 2 monoclonal antibody, that's somewhat more reassuring.  The predominant band is at 36 kilo-Dalton right here, which is what you would expect to see in terms of the migration of Bla g 2.
		There is another -- a larger protein of about 80 kilo-Daltons, and we are not sure what that is, whether that is a dimer of Bla g 2 or whether that's another protein that cross-reacts.  
		Okay.  Let's get down to the major business, and that is looking at the relative potency of the cockroach extracts.  This was measured by competition ELISA using our pooled allergic serum, which consists of serum from 16 individuals allergic to cockroaches, both clinically and by skin testing.
		What you see here in this column is the relative potency data of the glycerinated American cockroach, glycerinated German cockroach and the aqueous cockroach.  And they are all pretty low, .07, .05 and zero.
		So just to orient you, a relative potency of .05 means that within the statistical deviation of the study it has about 120th of the potency of the reference material.  Remember, too, our reference material is not all that potent.  Okay?  Skin testing that I showed early on.
		Now when we scale it for the amount of protein there, interestingly, the American cockroach doesn't change all that much.  The German cockroach mean does increase to .22 with a range of .01 to .46.  So one or two of the German cockroach products actually came to something within 30 to 50 percent of the potency of the reference German product, E2-Cg.  The aqueous products are all just about at zero by this testing.
		We also did specific allergen assays for Bla g 1 and Bla g 2 using monoclonal antibodies.  These were actually done for us by Endo Biotechnologies, and the results there were sort of interesting.
		Again, this would only be done for German cockroach.  You wouldn't expect American cockroach to have much Bla g 1 or Bla g 2.  The mean scaled up Bla g 1 level for the various commercial products that we had was about 3500 units per mL with a range of 2200 to 4800. 
		Now just by way of comparison, E2-Cg had significantly more Bla g 1, 13,800 units per mL.  Interestingly, Bla g 2 -- it was just the opposite story.  There was a wide variation in the amount of Bla g 2 present in our commercial products with a mean of about 43,000 units per ml, but the variation was from 8000 to 66000 units, depending on the particular product.  But look at where our E2-Cg came out with the Bla g 2 level.  It was really on the low end.
		So, interestingly, although the products seemed to have significantly lower potency and, certainly, at least a moderately lower amount of Bla g 1, they don't have very much Bla g 2.  They have more Bla g 2 than our reference product does, in spite of those other findings.
		Just to look at the relationship of all of the different studies that we did, we found a very good correlation of protein concentration and relative potency, a very good correlation of protein concentration of Bla g 1 levels, and an excellent correlation of Bla g 1 levels and relative potency.  And Bla g 2 levels are sort of left out in the dark here.  They really don't correlate well with either potency or Bla g 1 levels or protein concentration.
		Now one word of warning about interpreting these data.  The lack of correlation between Bla g 2 levels and the relative potency could be completely artifactual.  Remember, I showed you that our reference standard doesn't have very much Bla g 2 in it.
		So even if the individuals that constitute our pool, our serum pool, have a lot of antibody directed to Bla g 2, we are not going to put Bla g 2 in the relative potency equation, because our standard doesn't contain much of it.  In other words, the stuff that we are coating with doesn't contain very much Bla g 2.
		Therefore, the absence of this arrow here, the absence of a correlation between the potency and the Bla g 2 level, may be an indication of a problem that we have with our reference standard.  That being said, the lack of correlation between Bla g 1 and Bla g 2 levels and protein concentration and Bla g 2 levels is not a victim of that particular artifact.
		So our conclusions:  Commercially available cockroach allergen extracts vary widely in protein content, Bla g 2 content, SDS-PAGE banding patterns, and overall allergenicity.  They appear to be less potent and to contain less Bla g 1 than the candidate reference extracts.  
		So where do we go from here?  Clearly, our next stage is to go back into study subjects that are cockroach allergic to determine by extensive IDEAL skin testing the best in vitro potency measures, and presumably the selection of a reference standard, which as I said at the beginning, may not be what we started out with so far.  We may go with something else.
		Just finally, as a way of acknowledgment, all of the experiments done here were done by Melissa Patterson.  Again, during that period in which we were relatively short staffed, she really did a terrific job.  Jonny Finlay, who just came on board a short time ago, has contributed to these studies as well, and we are hoping that all three of us will be continuing to do good studies in cockroach over the next year.
		Thank you.  Any questions about cockroach?
		DR. LEHRER:  Jay, you mentioned that these were standards that had been in storage for sometime that you were using for the study.
		DR. SLATER:  Yes.
		DR. LEHRER:  Do you have any concerns about the stability of your standards?
		DR. SLATER:  Well, I have concerns, but I can't address the concerns.  In other words --
		DR. LEHRER:  But could you have a fresh extract, for example?
		DR. SLATER:  Yes.  I think that's something that we are going to need to do.  Absolutely.  That's one of the things that Jonny is actually going to be helping us with, is making fresh extracts.  
		We have actually obtained some fresh cockroach material which we are going to start working with very shortly.
		DR. LEHRER:  The other point is with cockroach a major problem, as with a lot of fungal extracts that Bob Esch demonstrated, I think, very elegantly, is the high levels of protease enzymes.
		DR. SLATER:  Yes.
		DR. LEHRER:  And this can have substantial impact on stability, not only relative to the issues that you are addressing here, but also a concern I would have is use in the clinic, you know, of taking these extracts out and putting them back in.  
		I don't know if that's something that you can address, but it might be a consideration as you look into stability studies later on.
		DR. SLATER:  Thank you.
		DR. SOTO-AGUILAR:  I understand they used to prepare them in formaldehyde precipitated products.
		DR. SLATER:  I'm sorry?
		DR. SOTO-AGUILAR:  I don't know if they are still doing those, you know, with formaldehyde precipitation.  That does contain enzymes, while the right products are freer of enzymes and contaminants.  Which ones are being produced right now?
		DR. SLATER:  Which allergen extracts are being produced?
		DR. SOTO-AGUILAR:  Right.
		DR. SLATER:  They are not formaldehyde precipitated products.  These are either aqueous products that are just extracted in aqueous solutions of various sorts or they are glycerinated products in somewhere close to 30 to 50 percent glycerine.
		DR. SOTO-AGUILAR:  Have you recognized any enzymatic products in there?
		DR. SLATER:  I'm sorry?
		DR. SOTO-AGUILAR:  Have you recognized any enzymatic products?
		DR. SLATER:  I haven't looked.  
		MS. LIBERA:  When extracts are finally sent out for commercial use, how are they labeled?  Are they labeled with the actual potency or are they labeled with -- I mean, how are they labeled?
		DR. SLATER:  These are non-standardized products.  So these are products that bear the label that there is no U.S. standard of potency.  The labeling of non-standardized product can be one of -- typically is one of two ways.
		One way is by the dilution, which simply indicates really the manufacturing technique, a one to 10 dilution, one to 20 dilution, one to 1,000 dilution, or it can have a measure of the protein content on it by PNUs or protein-nitrogen units per ml.
		When those measures have been compared to actual potency measures, which is what we are trying to aim for here, actual potency measures, they have fallen short.  In other words, within a narrow dilution range, there will be a very broad range of potencies or within a relatively narrow PNU range, there will be a broad range of potencies.
		So the correlation between the labeled measures of the non-standardized extracts -- the quantification of those doesn't correlate well with the quantification of actual potency measures.
		So these products were all labeled by dilution, one to 10, one to 20, one to 1,000.  And, you know, between the one to 10 and one to 1,000, there's actually a pretty good correlation.  One to 1,000 has a lot less stuff in it than the one to 10.
		The real question is, among products that are labeled one to 10, how close are they to each other in terms of potency?  The answer is they don't seem to be all that close.
		MS. LIBERA:  Okay.  
		DR. SLATER:  Did I answer the question?
		MS. LIBERA:  Is it confusing for a physician to know then if something is actually working with which patients?
		DR. SLATER:  Well, one of the advantages of standardization is to give some kind of comprehendible, uniform unitage that a physician can use, not only to compare the same product from different manufacturers, but also to compare different products.
		In other words, the unitage is really based on the size of the skin test reaction that it will illicit in a highly sensitive population of individuals.  So there is at least some -- One of the great advantages of standardized products is that the unitage, once you get used to it, is really fairly straightforward to understand.  The unitage of non-standardized products is not quite as informative and can be confusing.  That's one of the problems.
		DR. CLAMAN:  Yes, it is confusing.  And also, if your one to 1,000 doesn't have less than your one to 100, you're in real trouble.
		What are the practical -- this is in the same line.  What are the practical consequences of your findings?  What should the practicing physician do with regard to cockroach diagnostics and cockroach immunotherapy in the light of what you've just told us?
		I mean, this is, obviously, a very difficult area of biochemistry dealing with complex materials and having to ultimately rely on bioassays as well as biochemical tests.  What should the practicing physician do, if anything, with regard to your findings, if the practicing physician knows about them?
		DR. SLATER:  The practicing physician should immediately try to encourage the FDA to go forward with its standardization efforts of cockroaches, should encourage the manufacturers to fully participate in that process, and to help us go forward with standardization of cockroach allergens.
		I think to give the practicing physician guidelines on dosing or appropriate usage of a non-standardized group of extracts is simply not possible.  I can't give guidelines based on this, because this is a study based on looking at 26 different lots.
		We don't have reason to believe that our specific quantitative conclusions are applicable to the far greater population of lots of German and American cockroach that are out there.  So I can't give specific applied conclusions for practicing physicians other than the practicing physician should understand that standardization is what really makes their job substantially easier in terms of knowing what dose is to be used and what a label means.
		CHAIRMAN OWNBY:  But, Jay, against your initial criteria that you set out as you started this pilot, you said, one, you wanted to know whether the current materials were consistent between manufacturers.  I think you would suggest that that's not true.  There's wide variability between current manufacturers.
		DR. SLATER:  Right.
		CHAIRMAN OWNBY:  And the second was whether the current materials are potent or not.  By the admittedly limited skin test data you currently have with your standard compared to these materials, you would say they are not very potent, far less potent than we would like to have.
		DR. SLATER:  Right.  So in case I wasn't clear, by the impact criteria that we discussed last year and that I laid out at the beginning of this talk, it's clear that cockroach is a good target for standardization, and we should be going forward with it, by all criteria.  
		DR. SOTO-AGUILAR:  With the test method, the ID50 EAL, you are measuring basically the erythema reaction, while the Europeans used the wheal measurement.  Are you looking for work comparing the result on the same study population and see how they correlate -- the two methods will correlate?
		DR. SLATER:  Well, we know how the two methods correlate.  We know how intradermal and erythema reactions correlate.  They do correlate.  The reason that erythema is advantageous over intradermal is that you get a better slope.
		In other words, with intradermal you'll get a flatter slope.  You'll get a more consistent --
		CHAIRMAN OWNBY:  You said intradermal, meaning wheal.
		DR. SLATER:  What I meant was -- thank you -- with the wheal reaction you get a flatter slope, and it's harder to get good comparative data among products, and it's harder to identify where the right ID50 is, because there's an error associated with all of these measurements.
		So if you are working in a flat slope and you've got this big error, it's impossible to know whether the ID50 is here or here; whereas, if you have a steeper slope, which is what you have with the erythema, even with the same error you can make a pretty guess at where the D50 actually is.
		DR. SOTO-AGUILAR:  I'd like to make a little comment.  I'm sorry about the time.  But I was involved in the studies with cockroach in '95 and '96, and as I remember, this is in a highly atopic population in Mobile, Alabama.  We saw differences between the Caucasian and the African American population where there were different epitopes apparently recognized with the two different populations.  So are you looking at that possibility, too?
		DR. SLATER:  Well, we have to look at that possibility.  One of the reasons that you need to do a large ID50 study is you need to get a population that is both geographically and ethnically diverse.  Certainly, from what you said, it makes it especially important for cockroach, but we would do that in any case, just because that's one of the things that we need to find out.
		DR. LEHRER  Can you get an ethnically diverse population with the erythema assay?
		DR. SLATER:  Well, there are limits on how ethnically -- You know, there certainly are some limits with the erythema assay, but you can.  You can.
I think it makes it more difficult, but it doesn't make it impossible.
		CHAIRMAN OWNBY:  Okay.  Jay, why don't we move on, and we'll try to take the next report and just have our break a little late.
		DR. SLATER:  Okay.  Let me introduce -- Mary Malarkey is the Director of the Division of Case Management in the Office of Compliance and Biologics Quality, and she will be giving us a compliance update on allergenic products.
		MS. MALARKEY:  Can everyone hear me?  All right.
		Well, good morning.  It's a pleasure to be here to present the Committee an update of the compliance status of the allergenics industry.  As Jay said, I am with the Division of Case Management in our Office of Compliance and Biologics Quality.
		When an inspection is performed of an allergenics manufacturer or any of our manufacturers and it is felt that significant deviations from the law and applicable regulations have been recognized, the case comes to our Division for further evaluation to determine whether additional action needs to be taken.
		So today, briefly, I would like to go over some of the enforcement actions that we have done, kind of go through what we mean by enforcement actions, and which have been taken against this industry, what we call biological product deviation reports, used to be called errors and accidents, recalls, issues leading to the enforcement actions that Ill mention, and finally some forward thinking in this area.
		Please, if you have any questions at anytime, don't hesitate to interrupt me.
		In addition, there are a couple of mistakes on my slides.  I apologize, but I'll point those out as we go along.
		So starting with enforcement actions, this is a listing of those that we normally use.  There are others, but these are what are used by all the FDA.  The first two are legal actions, meaning that we would have to go in front of a court to take them.  
		Seizures are when we feel a product is a danger to health.  We will ask a judge to allow us to seize the product.  U.S. Marshals are actually sent in to do so.  Injunctions are taken against firms that have a continuing history of compliance problems.  They are again a legal action, and I'm happy to report that neither of these actions have been taken in the allergenics industry.
		The rest of these actions are what we call administrative.  That is, FDA can take these actions on our own with just legal counsel, internal legal counsel.  Warning letters are exactly what they sound like.  They are a warning to the manufacturer that they are out of compliance with the applicable regulations and the law, and it's an opportunity for them to address the issues and move on, basically, without us taking any further action.
		Unique to biologics is the fact that we have a license.  Each one of these products, the standardized extracts, are all separately licensed.  The non-standardized fall under one license.  But with this we have tools that we can use.
		If we really feel there is a danger to health, we can suspend the license, and this means that the manufacturer can no longer distribute the product and is basically shut down.  Unfortunately, some of these actions have been taken against this industry, but I'm happy to report that none in recent years.
		License revocation is just that.  It's actually taking the license away.  We don't usually go to this unless a firm requests it under suspension.  Normally, we send what we call a notice of intent to revoke, and this is where, for example, after we've sent a warning letter and we go back out and the firm has to taken things seriously, if you will, or hasn't been able to correct the deficiencies, we will proceed to the notice of intent to revoke.
		This actually puts the firm under a corrective action plan and gives them an opportunity to come back into compliance.  We have other actions such as sending letters after further inspections under the notice of intent or often we will ask the firm to come in and speak to us face to face, because often that helps get them on the right track.
		This is kind of a rundown of the enforcement actions that we have taken with regard to this industry from 1997 to the present time.  I'll highlight the mistake in this slide.  In 1999 there were actually two notices of intent to revoke that were issued that are not reflected on this slide.
		It's interesting to note, if you've heard of Team Biologics -- and I think most people have these days -- Team Biologics took over the inspections of this industry in 1999.  So as you can see, many of the issues were first identified by CBER inspectors as well as those from the ORE district offices, and we have seen what we hope to continue to see, a decline.
		Now this kind of breaks it off into enforcement versus the inspections that were done.  So it gives you a better idea of percentages.  I've included here our other major industries, the plasma derivatives, the in vitro diagnostics -- that is, the viral marker test kits for our blood supply, for example -- therapeutics which are namely biotech, our vaccines, and finally the allergenics.
		So in fiscal year 1999, 25 percent of the inspections of the allergenics industry resulted in -- Oh, I'm sorry.  That's a mistake as well.  It's 37.5 percent because of the additional notices of intent to revoke on my first slide.
		Unfortunately, in fiscal year 2000 there was an increase from the number of inspections to the number of enforcement actions, up to 42 percent.  Finally, for this year I need to make a couple of notes.  Our fiscal years, of course, begin in October.  The numbers I have taken are from October to the middle of February.
		Given that, if an inspection occurred in fiscal year 2000 but action was taken this year, I included the inspection number under this year's numbers to make the numbers more meaningful.  Also, we have other inspections that are either under review in our office or have not been classified.
		Now what we mean by classified is, again, when the investigator feels that there's a problem, he will make it official action indicated, he or she, and this will then come to us.  But there are other classifications as well:  No action indicated, when the compliance looks good, and voluntary action indicated when there are some misuse, but it's felt they can be resolved without further action taken.
		So given those caveats, this is this year so far.  As you can see, we've done seven inspections and taken two actions, which comes out now to 28.5 percent.  That is a decrease, but again it's preliminary data.  So we really can't -- I wouldn't want to say anything.  I hope this holds out as the year progresses, but this is about five months in.
		Moving to biological product deviations, under Title 21 of the Code of Federal Regulations, Section 600.14, it is required that manufacturers report deviations that occur during manufacturing that may affect product that is already in distribution.
		These used to be called errors and accidents, but now we have a new regulation.  We call them BPDs or BPDRs for reporting, and this final rule, we hope, will be effective in July.  Most firms are already adopting it.  It's a much better system.  There's a form that can be used.  It can be submitted electronically.  So we are trying to streamline the process.
		In regard to this industry, the problems that we have seen:  Of course, we have talked about precipitates, and I think Jennifer will be speaking about them later, and there's also stability failures that may result in the need to report.
		This is again a three-year history of all of our manufacturers.  As you can see, there is a steady increase in allergenics.  There's also a bit of an increase in the year 2000 for just about everyone, and this is mainly because our manufacturers weren't reporting under the regulation.
		The blood and plasma industry was used to it, but our drug and device manufacturers were not.  So this is why -- partially why we have seen an increase.  But in the area of allergenics, really, the precipitates account for the high numbers that we see, and this year I believe there's been 81 -- there have ben 81 reports so far.
		Now in October we had discussions with APMA on this issue, and industry asked if there was some way they could combine these reports to make it less burdensome.  As it stood, or as it stands, they are reporting each lot that is affected as a separate report.
		We concur that this is burdensome.  It's burdensome both on the industry and on FDA.  So we have determined that the reports may be combined.  There is a 45-day reporting requirement.  So 45 days after a BPDR is discovered, it must be reported, but again these can be combined now.  So, hopefully, that will help matters and burden to industry and FDA.
		Now with regard to precipitates, there are interim measures in effect.  We recognize that firms may still be cited for this by an investigator.  However, as long as the firm is working toward ways to resolve this issue and have procedures in place to not allow product with precipitates to get out to the consumer, then we would not take further action at this time.
		Moving on to recalls:  Recalls, some people don't understand, are really voluntary.  Manufacturers choose to do this themselves based on potential for harm to the public.  FDA mandated recalls are very rare.  I can't remember us having done one.  This would be an imminent danger to health situation.  So what I am going to present are the numbers that the manufacturers themselves have done voluntary recalls on.
		In allergenics the main issue, at least for this year, has ben sterility assurance.  That is, the manufacturer is unsure about the sterility of the product on the market, and as a prudent measure has decided to recall that product.  
		As you can see again, there was some increase from '98 to 2000.  Thus far this year, we have nine on record.  Again, one recall may involve several lots or several different extracts.  So each extract was treated as one.
		What are the most frequent issues that result in these enforcement actions that I mentioned earlier?  Well, these aren't unique to this industry, I must say.  This is pretty much commonplace throughout industry.
		Probably the main issue is inadequate investigations when failures occur and what we call CAPA, which stands for corrective and preventive action taken to prevent those recurrences.  These can entail failures during production and testing, handling of complaints, and again the precipitates.
		In this industry we were seeing a lot of refiltration, reprocessing, and reworking.  I won't define those terms unless you would like me to.  I think refiltration is pretty self-evident.
		There isn't anything that precludes manufacturers from doing these things, but if they do them, there has to be validation to support their use, and when there is a final product sterility failure, we do not feel that refiltration and revialing of the product is in the best interest of the public.  So we are discouraging that in all cases.
		The container/closure issues:  These are the actual vial and stoppers that are used for the product.  These need to be shown to be integral when they are in place over the life span of the product, and they need to be processed in such a way to guaranty that they are sterile and pyrogen free.  These were some issues that were raised in earlier years.
		Validation:  The gentleman mentioned lyophilization.  That is certainly one, but all the processes, the systems, the assays, as well as the equipment used to manufacture the products.  There are some specific CFR regulations that were not being followed.  We have a requirement for retention samples to be kept and inspected on a yearly basis to see that the product is still okay.  
		Again, the biological product deviation reporting:  This is no longer a real issue, with one exception that I'll get to in a moment.
		SOPs are standard operating procedures.  It's an expectation that for everything a manufacturer does, they will have procedures in place to follow.  We found that there weren't procedures in place for all processes or all testing.
		Finally, we require an annual review of each product to look at trends, see if there are problems that are starting to crop up, and this again is a CFR requirement.
		Now where have we seen improvement?  I like to report positive where I can.  Certainly, with the container closure issue, manufacturers took that very seriously and we're no longer seeing a problem in that area.  SOPs are in place.  Some still need a little tweaking.  They are there quite yet, but they are in place and being followed.
		Validation is being addressed.  Of course, that can be a long term action.  It's not something that you can do in a day or a week or even a month at times.  Refiltration, where being done, has been validated.  Annual reviews and retention samples, those are being addressed. 
		Finally, the biological product deviation reporting has improved dramatically, as can be seen by the numbers, with the exception of the areas not related to precipitates.  So if there is a stability failure, for example, that needs to be reported, and it's not -- we're not quite there yet.
		Looking at kind of the future, the precipitate issue, we are very aware of it now in the Office of Compliance, and we've made the investigators aware of it.  
		We are hoping to continue work with the manufacturers, and I'm sure the Office of Vaccines is really the most involved in this, but we are trying to keep in the loop so we know what progress is being made.  We have made the BPDs allowance so that they can be reported en masse to, hopefully, help the industry as well as ourselves.  
		One issue I'd like to mention:  We have a sterility test requirement under 610.12 of our regs, and it's actually a sterility method that is derived from the USP-22, United States Pharmacopoeia.  We recognize that this is a very outdated regulation.  We are currently in USP-24.  All drug products -- that is, those regulated by CDER, Center for Drugs -- are required to follow the USP.  
		The CFR requirement allows for automatic retest when there is a failure and actually a repeat retest.  In addition to that, there is no need for investigation into these failures to find out why they may have occurred.  This is not in keeping with current USP.
		So we are now presently discussing what to do about the situation, whether we are going to revise or eliminate 610.12(b).  I think probably in the best interest of everyone, it would be best to eliminate the reg and refer to USP.  USP is always changing.  There are revisions, updates all the time.  As many of us know, regulations aren't quite that easy to change.
		Before any decision is made, a policy statement will certainly be disseminated to the public, and we hope to move on this later this year.
		So finally, just some contact information: If you would like to get some more information, the CBER home page is, of course, a wealth of that, not just in my area but in all areas affecting the biological products.
		The BPD final rule that I mentioned earlier is available on the Web, and if you have any questions after today regarding compliance issues, please don't hesitate to call us in the Division of Case Management.
		So I've tried to really condense this to try to keep everybody on somewhat of a schedule.  So if you have any questions, I'd be happy to answer them.
		DR. SOTO-AGUILAR:  About the containers, are the stoppers latex-free?
		MS. MALARKEY:  I'm sorry?
		DR. SOTO-AGUILAR:  About the containers, the vials, are the stoppers latex-free?
		MS. MALARKEY:  Latex-free --
		DR. SOTO-AGUILAR:  Is there any regulation about it?
		MS. MALARKEY:  We are currently working on that.  I can't say that all of our closures are presently latex-free.  Studies have to be done.  I don't know in this industry.  I'm talking in general terms, but it's a very big undertaking to replace the closures with latex-free.  
		Stability work has to be done.  Integrity studies have to be done, and we are moving in that direction.  I don't know if anyone from OBRR has any sense for the allergenics industry where we are.
		MS. BRIDGEWATER:  Yes.  Some of the manufacturers are moving in that direction to change to latex-free stoppers.  I don't recall exactly how many, which ones might be done, but it is a very time consuming process.
		DR. UMETSU:  With regard to the precipitate issues in the extracts, is that a new issue or it's just that it hasn't been reported in the past?
		MS. MALARKEY:  It hasn't -- My impression is it hasn't been reported in the past, but this has been an issue that has been going on or has been identified for sometime.  I think it probably came more to light during inspections, seeing it, you know, actually seeing it.  So this now in the last couple of years has really become a very large issue.
		CHAIRMAN OWNBY:  Okay.  Why don't we go ahead and take a 15 minute break.  So we'll reconvene at 10:45.
		(Whereupon, the foregoing matter went off the record at 10:28 a.m. and went back on the record at 10:46 a.m.)
		CHAIRMAN OWNBY:  Are there questions right now?  Otherwise, we will move on tot he discussion of TSE and the questions of whether we need to be concerned about this with the allergenic extracts.  Dr. Slater, I believe, is going to start that.
		DR. FREAS:  While he is getting set up, those of you who have been complaining that it's been cold, we apologize for that.  The thermostat is set all the way up.  Apparently, they are doing work down below. They have told us the heat will be on shortly, but it will take a little while to warm up.
		CHAIRMAN OWNBY:  You mean spring will come eventually.
		DR. SLATER:  We are now going to talk about a topic that we have not discussed with reference to allergens before.  That is the transmissible spongiform encephalopathies and allergen vaccines.  I'm about to embark on a fairly lengthy excursion about TSEs before I get back to allergens.
		So what I'd like to do before I do that is give my summary in advance, so you know what direction we are heading in, in terms of the allergenic products themselves.
		In summary, most allergen extracts are produced without any bovine components other than glycerol.  Glycerol, just again to give you a punchline in advance, is not a problem in terms of TSE for a number of reasons.  However, among allergen extracts, mold extracts are stored and propagated in culture media, some of which contain bovine components, and some of these are of uncertain origin.  However, the risks associated with these contaminations are minimal, and I will show you these calculations as well.
		So what are the transmissible spongiform encephalopathies?  Well, it's a group of fatal neurodegenerative diseases that are transmissible.  They are known to be transmitted from one organism to the other.  
		They are encephalopathies.  They are conditions characterized by abnormal brain function, and the spongiform refers to the microscopic appearance of the brain tissue in affected patients with the formation of holes or vacuoles and scarring or gliosis.  So the name itself includes both evidence about the epidemiology of the disease, the clinical appearance of the disease, and the pathological appearance of the disease.
		Well, what's the clinical appearance of the disease?  It's somewhat variable, but it almost invariably contains some cerebral symptoms, including dementia, behavior changes, visual disturbances.  There are often cerebellar symptoms, ataxia, nystagmus and speech abnormalities; pyramidal signs and symptoms, spasticity, weakness, and hyperreflexia; extrapyramidal signs and symptoms such as tremor, myoclonus, rigidity and akinesia; and invariably, death.  These are degenerative, fatal diseases.
		Pathologically, they are characterized by gliosis or proliferation of glial cells, neuronal loss, vacuoles, and amyloid plaques, and the amyloid plaques are variable.  They are not all associated with -- They are not all present uniformly in all of the diseases.  They are variable in CJD, but they are universal, for instance, in the new variant CJD.
		Now the first transmissible encephalopathies were described in animals, and of course, they weren't always known to be transmissible spongiform encephalopathies.  These were known in the past as the sort of sloe viral diseases.  When I was in medical school, that's how we studied them.
		Scrapie was the first to be described.  It's a fatal, progressive disease of sheep, and it was described in 1932 -- I'm sorry, in 1732.  Scrapie affects sheep and goats naturally and can be transmitted experimentally to several other animal species.
		Now chronic wasting disease is a rare disease of elk and deer, primarily in the United States.  It happens both in captive populations and in wild populations, and it appears to be a disease that is pathologically very similar to the other TSEs that have been described.
		Transmissible mink encephalopathy takes the form of a rapidly developing epidemic, usually involving single mink farms.  Again, the disease is uniformly fatal.
		The bovine spongiform encephalopathy is the one that, of course, concerns us most.  It was recognized as a potential member of the scrapie family as soon as it was first discovered in 1986 and shows all the pathological characteristics of TSE.
		Now feline spongiform encephalopathy, which appears in cats, is clearly a derivative disease of the bovine encephalopathies.  In other words, clearly, it is transmitted to cats who are fed contaminated materials from cows, and the epidemiology follows fairly precisely the epidemiology of the bovine encephalopathies.
		Of the human TSEs, the first described was kuru, which was described in the 1950s in isolated tribes in New Guinea.  It was spread by the ingestion of brain tissue of deceased relatives, and the prevalence of this disease decreased rapidly after the cause of it was identified and the practice discouraged.
		Kreutzfeldt-Jakob disease, CJD, occurs in about one case per million individuals per year.  This is the sporadic form of CJD.  It also appears in a familial form and in an iatrogenic form, by the transplantation of neural tissue contaminated with CJD, and in addition there is a new variant form that is associated with transmission of bovine TSEs.
		Now these other human TSEs are much rarer, and I am not going to discuss them, but simply note that there have been other human TSEs described.  But there is a key point that has to do with the pathogenesis of these diseases, that the diseases occur both in what appears to be endogenous cases -- in other words, the familial or genetic cases of human TSEs -- and what appear to exogenous cases, the clearly infectious ones.
		What's important to note is that, whether you have a so called endogenous type or an exogenous type, your tissue is infectious.  So that's a key point to remember when we get to talking about the pathogenesis of the disease.
		This is a slide to show the epidemiology of bovine TSE, and the point of the slide is to show that bovine TSE was isolated in both time and place.  The epidemic of bovine TSE was fairly well defined.  It started in 1986, and it peaked in around 1991-92, and it has been dropping rapidly since that time.
		So the interval between 1986 and, certainly, 1995 to 1997 were certainly the peak periods.  It hasn't dropped off to zero quite yet, but it has been dropping quite rapidly and, clearly, this is a well identified interval in time in which bovine TSE seemed to be occurring quite a great deal.
		Now this is a little harder to read, and these are the different countries in Europe and the number of cases of bovine spongiform encephalopathy detected.  This is cumulative detected through the year 2000, and you can see here that the epidemic is localized in terms of place as well, nearly 180,000 cases in Britain.  The next nearest country is Ireland which had 540 cases, Portugal 473, and on down from there.  So the epidemic is localized in terms of time and relatively localized in terms of geography, although as you can see, there have been cases reported in Europe as well.
		What you have here in the lower righthand corner are the cases of new variant CJD, which appeared in '95, '96, and '97, all at that time in the United Kingdom.  
		Now what about this new variant CJD?  The new variant CJD can be distinguished on clinical grounds from the classic CJD, on the basis of the differences that are demonstrated here.
		The time course of the new variant from diagnosis to death is somewhat slower, from eight months to 16 months.  The EEG changes that are very common in the sporadic form of CJD don't appear in the new variant CJD.  
		The clinical presentation tends to be more behavioral for a longer period of time in the new variant than it is in the sporadic form, and the age at presentation is significantly younger.  The sporadic, the mean age is 60, whereas the new variant has a mean age of 28 with a range of 16 to 52.
		Note here that the incidence of the sporadic is, as I said, one case per million per year.  Even at its peak in the United Kingdom, there were 22 cases over a period of 1994 to 1997 or for an incidence of 1.5 times 10-7 per year.  
		So even at the peak of the new variant CJD epidemic, if you had CJD and weren't clinically distinguished between the two, you had more of a chance of having the sporadic than the new variant form.  But, clearly, this was an epidemic, because the new variant doesn't occur elsewhere.
		So what are the pathogenesis clues?  CJD occurs in both familial and infectious forms.  The infectious material is insensitive to UV and ionizing radiation, has a low ratio of nucleic acid to protein, is partially susceptible to treatments that are known to destroy proteins.
		Hence, in 1982 Dr. Pruzner formulated the word prion for proteinaceous infectious particles that he proposed to be the infectious particle associated with the spongiform encephalopathies.  This is a group of 33 to 35 kilo-Dalton sialoglycoproteins.  
		It's important to note that these are normal proteins.  They are present in CNS tissue.  they are present in non-CNS tissue, and there is a normal cellular isoform so called PrPc.  However, the prion proteins appear to be transmissible agents for TSEs.
		The conversion, the infection with a TSE is associated with the appearance of protease resistant prions, which are designed either PrPres for protease resistant, or PrPsc, short for scrapie.  
		Infection with PrPres or PrPsc induces conformational changes in the normal cellular isoforms that are associated with a switch from an alpha helical formulation to a beta pleated confirmation, and associated with increased insolubility and aggregation of the proteins.
		Now susceptibility is a function of the primary sequence of the PrPs that are involved.  There is a species barrier associated with infection with TSE.  That appears to be a function of the differences between the PrP sequences among different species.
		There even are intraspecies polymorphisms which may in part account for differences in susceptibility of individual organisms.  And some of these susceptibility factors can be overcome with a transgene.  In other words, the experiment is, if you try to infect a mouse with a bovine TSE, you can overcome the species barrier by giving the mouse a transgene with bovine normal prions, and you can especially do so if you ablate the original murine PrP gene.
		Now this doesn't account for all infectivity factors.  So you still can be infected.  Knock-out mice, for instance, that have no prion protein at all have quite a significant barrier to infection.
		This is simply a three-dimensional model of the mouse and hamster PrPc.  You can see the alpha-helical confirmations, and what you can see is that there's -- in the normal confirmation there is a minimal beta-pleated sheet in this region, but this expands when the confirmation is changed by the appearance and aggregation with a mutant PrP.
		Well, how do you get from a inoculation to disease?  Typically, inoculation is by GI absorption.  The abnormal prion proteins replicate, and they replicate in the sense of inducing conformational changes in the normal endogenous prion proteins, and this happens in the Peyer's patches, in the spleen and in the dendritic cells and in the B cells.
		Now you can also get peripheral replication in peripheral nerves.  We know from the days that we used to think about this as slow viruses that neurosurgeons and neuropathologists would sometimes get infected by a percutaneous inoculation with infected tissue, and peripheral replication can occur in peripheral nerves and go centrally to the CNS.  But the final common pathway of these different replication methods is that you get aggregation in the CNS of abnormal prion proteins and the phenotypic changes associated with the disease.
		Can PrPsc be inactivated?  That's the hard part, and that's one of the reasons that we need to pay so much attention to prion proteins and to the TSEs, because classic microbicidal methods are completely ineffective.  Irradiation is ineffective.  Heat inactivation can be accomplished but is incomplete.  In other words, you can heat this and relatively inactivate them, but you get incomplete inactivation, and chemical inactivation is highly variable.
		CBER's role in the control of this problem touches on several different products.  Clearly, we are concerned about the blood supply, therapeutics, the vaccines, and now allergenics.
		This is a highly selective timeline about TSE and the FDA in the United States.  The first time that a letter was sent to the manufacturers from CBER was in May 1991.  This alerted the manufacturers that there was a potential problem with bovine products.
		In December 1993, the FDA sent a letter to all the manufacturers.  In December 1997 the U.S. Department of Agriculture expanded its list of banned countries to include all of Europe.  In April 2000 CBER sent another letter to the manufacturers, and as a follow-up to that letter, we in our Division sent a memo in May 2000 to the allergen manufacturers.
		We also sent another memo as a follow-up in August 2000, and in the interim in July 2000 the TSE Advisory Committee and the Vaccine Related Biological Products Advisory Committee had a meeting in July that also touched on several of the issues that we are going to talk about today.  
		The rest of my talk at this point about TSE is going to cover those memos that we sent to the allergen manufacturers and the data that came back and our analysis of those data.
		Well, how do you do a risk assessment on a biological product for the possibility of transmission of TSE?  Well, you have to quantify or estimate the risk based on the animal source.  Where did the animal come from, and when was that animal coming from that place?
		Remember that slide that I showed sometime back that the peak periods are confined both geographically and temporally. So we need information about time and place to quantify the risk.
		We also need to know about what the particular tissue is.  I'll talk about this in a few minutes, but certain tissues are higher risk than others.  We need to know what processing or dilution methods were gone through and how they might have impacted the infectivity.
		Specifically, as we get to immunotherapy, we need to know the protein doses associated with immunotherapy prions or proteins, and they co-purify with proteins.  So we need to have some estimate of what the total protein doses are.
		Finally, we need to know whether are any route specific risks, whether subcutaneous inoculation has a greater or lesser risk than other forms of inoculation.
		Well, let's talk a bit about the infectivity of various tissues.  The European scheme is presented here with some adaptations.  The scheme involves going from category I tissues to category IV  tissues, where category I tissues have the highest infectivity, and category IV tissues have no detected infectivity.
		The highest tissues, obviously, are brain, spinal cord and eye.  Those will have the largest amount of abnormal prion proteins in them.  Tissues in category II are called medium infectivity.  They include a variety of other largely lymphoid tissue that has a lesser degree of infectivity.
		Now the ratings of these two classifications are actually based on infectivity studies either from cattle to other cattle or from cattle to mice.
		In category III and IV, there's really no detected infectivity for the most part in those particular studies, but category III tissues, for the most part, ended up in this grouping because there were good data from scrapie studies that had been done in the past that these tissues were, in fact, infective, although at a relatively lower level.
		So even though the more recent infectivity studies with the bovine TSEs suggested that these tissues were in this group, the scrapie studies suggest that they were moderately infective and should be in a somewhat higher group.
		Again just one final comment about even the group IV is that the sensitivity of these kinds of studies is not infinitely low.  So that's why we use the term no detected infectivity, because there are limits to how sensitive the techniques are.  Yes?
		CHAIRMAN OWNBY:  Jay, in that category IV, is all blood and blood products contained in that blood clot you list there?
		DR. SLATER:  Yes, it is, but again I don't think we can be fully reassured necessarily by that.  There was a recent study from Lancet that clearly showed that sheep could be infected through a blood transfusion.  So there is some infectivity.
		Remember, when most of these studies are done by intracerebral injection into calves or mice, you can inject up to 50 microliters of material into a mouse's brain without killing the mouse immediately.  So that certainly limits your ability to detect the infectivity.  Even into calves, there's a limit to the volume that you can inject; whereas, with a blood transfusion, you will obviously have a much greater volume that you can inject.
		So we have to approach all of this with a proper sense of reserve or perhaps even skepticism.
		DR. SOTO-AGUILAR:  Would cornea be included in the eye?
		DR. SLATER:  I'm sorry?
		DR. SOTO-AGUILAR:  The cornea?
		DR. SLATER:  The cornea, I believe, is included in the eye.  Yes.  It's the way that CJD is transmitted iatrogenically is by corneal dura mater transplants.
		Now some special categories of interest.  Glycerol is, obviously, of major interest for people that are interested in allergens.  Well, glycerol can be obtained from both plant and animal sources, and in fact, for the most part, when we ask our allergen manufacturers, we found that they were derived from plant sources, not animal sources.  So that was reassuring right from the beginning.
		It turns out that, even when it's of animal origin, because glycerol is a distilled product, that the studies and the Advisory Committees have concluded that, even if glycerol is of animal origin, it's not considered to be infectious.
		Likewise, milk, by virtue of studies and other data, is not considered to be infectious.  Gelatin is an additive for some of our products that I'll talk about in a few minutes.
		It was originally not considered to be infectious in the first deliberations.  However, as a result of some spiking studies that were done in which prions that were spiked into gelatin before its processing and taken through the entire process actually made it out through the end, there was some concern about the use of gelatin, especially in parenteral use, and in a 1997 guidance document CBER recommends against parenteral use of gelatin unless it is from certified origin.
		MS. LIBERA:  Can the distilling process itself remove TSE from the glycerol?
		DR. SLATER:  I think that's the thinking, is that the distilling process separates the prions from the glycerol.  
		MS. LIBERA:  That's why it's done?
		DR. SLATER:  I don't think that's why it's done.  I think it's done in order to purify the glycerol from the tallow.
		MS. LIBERA:  So it can be removed?  So it can be in there, but it is removed in the distilling process?
		DR. SLATER:  Right.  Right.
		MS. LIBERA:  A hundred percent?
		DR. EGAN:  Bill Egan.  To answer your question a little bit with regard to the glycerol, I think it's first treated with sodium hydroxide to liberate the glycerol, and then the distillation itself, the glycerol being more volatile, would come over any proteinaceous material that stay, but also the temperature that is involved in the distillation would again inactivate protein.  
		So I think there are sort of three processes that are involved that would help to deactivate or eliminate any prions.  So I think that's why there was -- the tallow derivatives were not considered to be problematic.
		DR. SLATER:  Thank you.
		In May 2000 we sent our first memo to the allergen manufacturers.  We asked for four different types of information in that memo.  We asked for the specific animal sources that might have been used in any of the allergenic products.  
		We specifically asked if any neural tissue was used in any of the products.  We asked for the origin and the residence of the cattle, and we asked for the dates that the cattle were obtained.  
		Why did we ask for each of these?  Well, we asked for the country, because the risk is geographic.  The risk is significantly larger from U.K. cattle, greater than EU cattle and much, much greater than from anywhere else.
		We asked the dates, because the risk of TSE, especially prior to 1980, appears to be negligible.  We asked for the specific tissue, because there is a risk associated with specific organs.
		What did we learn?  Well, for the most part -- Yes, Dr. Claman?
		DR. CLAMAN:  It may be a minor point.  Are we sure that the geographic distribution is not the result of ascertainment bias?  The countries that presumably have little or none of it, have enough samples been tested or is it based on -- Is it based on clinical evidence of diseased animals?
		DR. SLATER:  No.  There are surveillance programs in place to collect pathological tissue.  Now if you are asking what the sensitivity of the surveillance programs is in each country, I can't tell you that, but there definitely are surveillance programs in place.  Is that correct?  No, it's not.
		DR. MIDTHUNE:  Karen Midthune.  I think in instances where the U.S. Department of Agriculture felt that the surveillance practices were such that they might not be sensitive enough, those countries were also put on the USDA list as countries where, although perhaps BSE might not have been identified, that there was a risk for BSE.
		DR. SLATER:  Thank you.  So the results of our first screen were somewhat reassuring.  We were somewhat concerned that our manufacturers had specific beef extracts, but we were reassured to learn that they all obtained them domestic sources, in some cases the local supermarket.
		There are some deer and deer hair and pelt and venison extracts out there.  These are obtained from domestic kills, and in the particular manufacturers' case, it was from greater than 20 years ago.
		Our area of concern focused on mold extracts.  Several of our manufacturers use media supplements that were of bovine origin.  So as a follow-up we asked for more information in our august 2000 memo, asking for lot-specific mold origin and culture information, asking for the number of lots possibly affected.
		For suspect lots, we asked for certification that the material was obtained from approved countries.  We asked for quantitative flow charts so that we could calculate the risk for each product, and we ask the manufacturers to actually do their own risk assessments.
		Why did we ask for all of this?  Well, the lot-specific mold origin and culture information -- we really wanted to start excluding lots from consideration.  What we got in the initial screen was a large number of lots in which bovine materials are used, and what we were looking for was to sort of narrow down this field of concern to products that were in the wrong geography, in the wrong time, as opposed to in the right geography, in the right time.
		So we were trying to exclude lots from consideration based on known dates and culture conditions.  
		Why did we ask for certification?  Clearly, to ascertain that bovine materials were sourced from approved countries.  We asked for quantitative flow charts to help us in assessing lot-specific risks, and we were asking them to do their risk assessments so that we could both do the risk assessments and at least compare them and see where we went.
		So this again is our selective timeline, and this is where we were when we got these data.  We did eventually get the data from the manufacturers, and what we are going to discuss now is how we analyzed those data and what our analysis showed.
		Well, what we were looking for first is to estimate the infectivity of the media itself.  So there are two stages in this process.  One is to go from the cow to the media supplement, and then the next stage is going to be from the media supplement to the actual final mold product and what the risk is associated with that.
		So let's take this one step at a time.  The first thing we are interested in is the tissue LD50s per gram.  That depends on what the specific organ is of the tissue.  Now, remember, we talked about category I, II, III and IV.  There actually are estimated LD50s associated with this based on transmission experiments that have been done.
		For category I, which is central nervous system tissue, the estimated LD50s is 107 LD50s per gram.  If you go down to category II, it's something under two and a half times 104.  If you go to categories III and IV, it's between .1 and 100 LD50s per gram.
		The next part of the process is to determine how much stuff goes into the lot,and we could do that in two ways.  We could determine the number of grams of tissue per cow that go into each product, and then how many cows go into each lot. 
		Well, it turns out those data are really available with any precision only for a product that's made with a specific organ, such as BHI, which is brain, heart, infusion media.  Now for BHI, or brain, heart, infusion media, we know that the cow brain is about three-quarters of a kilogram in weight.
		We know, by talking to some manufacturers, that they use about 2,000 cattle per lot or something to the order of 1.5 x 106 grams of cow tissue put into each lot.
		Now for other media components that are derived from skeletal muscle and mixed tissues, the specifics are really less certain.  We really don't know how many cows' skeletal muscle went into this product, and certainly for gelatin we don't have a clue.  But we have used this estimate of 1.5 x 106 grams per lot as an overall estimate of the number of cow tissue that goes into each lot for the purposes of our calculations.
		Next we are concerned about what the risk -- regional risk is per cow of being infected with TSE.  As I said, that risk is geographic.  Now at its peak the risk of a random cow being infected in the United Kingdom -- this is at the peak of the epidemic -- was about one in 100.  So one in 100 cattle had abnormal prion proteins and was infected during the peak of the epidemic.
		That has come down.  The estimate that is currently used for the European Union is that the risk is about 10-4 or one in every 10,000 cattle may be infected.  
		For the purposes of our calculations, since none of our manufacturers use UK specific suppliers, we have used the 10-4 number as our regional risk.
		DR. LEHRER:  Jay, when you say none of your manufacturers have used UK specific suppliers, is that the same thing as saying that none of your manufacturers have used suppliers obtaining material from the UK?
		DR. SLATER:  Well, I think that's a very good point.  None of our manufacturers are UK based.  So there is no a priori reason.  You see, we'll skip a little bit down the line just so I can answer your question.
		What we are dealing with here in all of the cases that I am going to calculate out is uncertainty.  We have no supplier.  We have no manufacturer that has come forward to us and say we know that we used a supplement that came from the Netherlands or that came from Germany.  We just don't have that information.
		That wasn't really the case with the vaccines deliberations back in July.  There were manufacturers in which they had specific origins that were of concern.  In our case, we don't really know that.  So we're making a guess based on uncertainty.
		Now for all we know, the manufacturers, for all their uncertainty, may have domestic products, and for the domestic product the risk is considered to be essentially zero.  So we think that using the EU estimate is a good mean estimate or actually it's a good worst case estimate, we think, of what the risk would be, what the regional risk would be.  But it is a guess in the midst of uncertainty.  Yes?
		CHAIRMAN OWNBY:  Wouldn't the worst case scenario be assume that they all came from the UK?
		DR. SLATER:  Well, that would be a very worst case scenario.  We really have no reason to believe that that's the case.  I think that, in fact, when we've done digging, we have found that some of our manufacturers have started being uncertain, ended up having domestic supplies anyway.  
		CHAIRMAN OWNBY:  But when the UK removed so many cattle from their herds, what happened to all those cattle?  Were they just summarily all destroyed or were any of them processed?
		DR. SLATER:  No.  They were destroyed.  That's a good question, but they were destroyed.
		Is there horizontal infectivity among cattle?  I don't believe so.  I haven't seen anything about horizontal infectivity among cattle.
		DR. LEHRER:  Relative to that point, Jay, when you talked about wild animals being infected, I wondered if that might be the case, because with deer, for example, wild deer, I couldn't imagine how else they could be affected.  Maybe you have some thoughts on that.
		DR. SLATER:  We don't know.  We don't know what the transmission is among -- for the chronic wasting disease.  This may be a spontaneous mutation.  In other words, even though all organisms that have TSEs generate infectious material, some of them do develop the disease spontaneously.  At least we think that that's the case.  
		So we don't know what the origin and transmission of the disease in the deer and elk is.
		DR. SAXON:  But it's not the exact same disease as is the bovine disease.  Many species have these spongiform encephalopathies, like humans.  
		DR. SLATER:  No, it's not a virus.  
		DR. SAXON:  Unless the Nobel Prize Committee is wrong twice and going for a third time, it's not a virus.
		DR. SLATER:  The next thing that we look at is process reductions in the course of making the media itself.  There almost certainly are some process reductions in the making of the media, but for the purposes of our discussion today, we are considering there to be no process reductions in making the media itself.  So we take that as a factor of one.
		Based on these numbers, we calculate what the LD50/lot are, and then we calculate the LD50/ml of the media supplement.  Not, actually, in making that conversion from the lot to the milliliter, we actually incorporate the use of the supplement in the final growth media product.  So we incorporate -- You know, if you added peptone up to a 30 percent dilution, then we would incorporate that into this number here.
		So this actually gives us the LD50 of the medium that is used to either store or grow the molds.
		So what did we learn about the different media supplements that we were told about?  What I'm listing here is all the media supplements that our manufacturers alerted us to the fact that they had bovine components.
		Well, it turns out for five of them there were no bovine components at all.  For proteose peptone number 2, proteose peptone number 3, peptamin, neurospora culture agar, and malt extract broth, there were no bovine components. 
		Now this isn't because the manufacturers  were telling us misinformation.  They were given ambiguous information by their suppliers.  In other words, the suppliers would sort of say, well, you know, it has this category.  Well, when we dug into it, we found out that these were components that were derived from other animals, not from cattle.
		For three of the products, peptone, malt extract agar, and YM agar and broth,there is a bovine component, and it was gelatin.  We're going to discuss those products in just a couple of minutes.
		For seven products -- one of them is a broad group of products, the saborauds media, they contain bovine milk, and those were excluded from consideration as well.
		Finally, when we narrowed it down, we found four products that actually contained either bovine muscle, organ tissue, or in one case neural tissue, the brain/heart/infusion media.  So what we're going to focus on for the rest of this analysis is these four media and the three media that contained bovine gelatin.
		Now having identified the infectivity of the media supplement, we now go from the media supplement to our final product, and the way we are going to determine that is ultimately we are going to take it through these process reductions, indicate whether we think there's a species barrier or not, a root barrier, estimate the annual U.S. dose of the product, give the LD50 per year then that's administered to the U.S. public, and by inverting that come to the number of years that we would have to go by to come to a case.
		What are the process reductions?  Well, the process reductions in making the mold product:  You have to look at how these molds are made, and this is a very generic slide.  The manufacturers all have differences among their processing, some of them subtle, some of them not so subtle.  
		Basically, you go from a master seed to a working stock to a production lot.  You harvest the molds, and there are various ways of harvesting the molds.  Sometimes the mold mat is just taken off the top.  Sometimes it is ground up with the media.  It really is highly variable, and then finally there is an extract preparation.
		Now we talk about process reductions and process dilutions.  But really, there are some steps that lead to concentration of protein.  After all, the manufacturers are trying to isolate the mold proteins and concentrate them for administration.  So some of the steps, such as precipitation, drying and lyophilization, actually lead to concentration of protein.
		So the process reductions were all individual for the products, and we calculated them out based on the flow charts that we got from the manufacturers.  
		What about a species barrier?  Well, there are no data on species barriers from any animal to humans.  There are data on species barriers from cows to mice.  There are data on species barriers between other animals, and the species barrier appears to be something on the order of one to 1,000.  But in our ignorance about species barriers to humans, we take the worst case assumption that there is no species barrier.  We think there probably is, but since we don't know what it is, we can't factor that in.
		What about route barriers?  Well, that there is a fair amount of data on from both cow to cow transmission and cow to mouse transmission.  Using intracerebral inoculation as the 1.0 in terms of efficiency of infection, we find that subcutaneous injection has an efficiency of infection of about one to 24,500, and we use that factor in our calculations.
		Now this is a very hard calculation, and  that is the annual U.S. dose, because we really don't know what the annual U.S. dose of any one of these particular products is.  We have, based on manufacturer data, that there are about 30 million doses of immunotherapy given every year, and now we have absolutely no data beyond this.  So the rest of this is fairly, I think, conservative hand waving. 		Assuming that about ten percent of the injections that are given contain some mold product or another, assuming that about ten percent of those are from one particular manufacturer, given the volume, the part of that a volume that we think would probably contain molds, we're estimating -- Now this is not for total mold immunotherapy in the United States.  This is for a particular product, a particular product or a particular product line -- about 30,000 milliliters dose of each individual product annually to the United States.
		Then based on that, we go through a calculation LD50 per year and number of years to go through for each case.  When all the dust settled, there were three different scenarios that we were worried about.
		The first scenario is the use of an uncertified media from Category IV tissue in mold propagation.  This was one manufacturer, one small portion of its product line.  
		Scenario number two:  Use of uncertified media containing gelatin in mold seed stocks.  So the gelatin was not in mold propagation.  It was in the  seed stocks that they started with initially.  This was three manufacturers, several products in their lines.
		Finally, we have the use of an uncertified media from Category I tissue -- that's brain/heart/infusion media -- used in mold seed stocks, one manufacturer, several parts of their product line.
		Let's go through the first calculation.  This is the use of uncertified media derived from Category IV tissue in mold propagation, one manufacturer, one portion of their product line.  I'm going to go through this particular spreadsheet in detail.  The other ones will sort of follow after this.
		Category IV tissue is the tissue that has .1 LD50 per gram.  This is the no detectable infectivity.  So this is the detection limits.  Again, the grams of tissue per cow, 750; the cows per lot, 2000; the regional risk we're taking as the EU risk or 10-4.  We assume no process reductions.  We come out with these numbers for the lot LD50 and for the milliliter LD50.
		Manufacturer process reductions of this particular product really only reduced the potential infectivity by .3.  Again, remember, some steps dilute.  Some steps concentrate the protein.  So in the end, when we analyzed this manufacturer's data, we came out with a dilution factor of about .3.  
		Again, we assumed no species barrier. This is our assumption throughout this.  We assume a root barrier of one to 25,000.  Again the annual U.S. dose gives us 1.1 x 10-7 LD50 per year or the number of years that we would have to wait to see a case is 18,500,000 years.  
		A little perspective:  If we were going backwards, humans were thought to arrive about a million goes ago.  So this is 18 times that interval.  So this is a good big number.  However, there was one assumption in that calculation that is highly questionable, and that is that the assumption that the material was derived with exclusively Category IV tissue.
		What if, as probably was the case with butchering practices sometime ago, there was contamination of our Category IV tissue with Category I tissue, spinal cord that made its way into this allegedly Category IV tissue?  
		Well, remember, Category I tissue has a very, very high LD50 of 107 LD50 per gram.  If there were a .01 percent contamination -- and this is the amount that was used in the vaccines estimates over the summer -- that would lead to LD50 of 1,000 LD50 per gram for this so called contaminated Category IV tissue.
		The numbers here are all the same except we start out with 1,000 LD50 per gram instead of .1 LD50 per gram.  We still come up with a comfortably large number of 1,850 years before we would expect to encounter a case of transmission, given these numbers.  Remember as well, we assume no process reductions.  We assume no species barrier.  
		Let's go to the next scenario.  This is the one where mold seed stocks are using uncertified media containing gelatin.  This was three manufacturers, and this is the worst case of those three manufacturers.  So we did the calculations for all three, and we came up with larger numbers, if anything, for all three of those.
		We start with the tissue LD50 per gram of 1,000 gelatin.  It's really uncertain what the LD50 are, but this is a good worst case scenario for gelatin.  Probably it has less.  All the numbers are the same.  The manufacturer process reductions in this particular case were .08, since this product started out in the seed stocks, and we come out with LD50 per year of 4 x 1004 or a 5,000 year interval.  
		Finally, the use of uncertified media derived from Category I tissue in mold seed stocks.  In this case -- this is the BHI case -- we have 107 LD50 per gram, but again because this was in seed stocks, this particular manufacturer had a lot of dilution factors that went in, and the dilution factor was .00018.  Here we come out with 4.2 x 10-3 LD50 per year or 470 years per case.
		So what's our summary?  This is what I showed you before.  Most allergen extracts are produced without any bovine components other than glycerol.  Mold extracts are stored and propagated in culture media, some of which contain bovine components of uncertain origin.
		The risks associated with these contaminations are minimal, and this I didn't have on the slide before:  Manufacturers have been directed to assure that henceforth all bovine components be certified to be from approved sources.
		Here's the question that we would like to ask the Advisory Committee at this point.
		In July 2000 the TSE/VRBPAC combined committee suggested that the master seed stocks of vaccines need not be rederived to reduce the likelihood of TSE transmission.  This joint committee came to this conclusion after agreeing that the risk of TSE transmission was remote, and the risks associated with the rederivation of the master seed stocks of bacterial vaccines were substantial.
		In contrast, CBER does not believe that there are any risks to product efficacy or safety associated with the rederivation of the master stocks of mold strains used for allergenic extracts.
		So our question for the committee is:  Does the Committee agree with CBER that the master stocks of mold strains used for allergenic extracts should be rederived to reduce the theoretical possibility of TSE transmission?
		Now, remember, of the three scenarios that I showed you, this affects two of them.  The first one where the manufacturers are using a questionable media supplement in the propagation -- that is not being asked now.  What we are asking is about those two scenarios, one involving the gelatin, and the other involving the BHI where there is going to have to be a change in the mold seed stocks themselves to get to products that are from certified origins.  That's our question.
		DR. UMETSU:  What were the risks that were thought to be a problem by the TSE Advisory Committee?
		DR. SAXON:  The bacterial, you're talking about?
		DR. UMETSU:  Right.  In the statement they said they didn't think it was necessary to rederive the stocks, because there are risks.
		DR. SLATER:  The risk is --
		DR. UMETSU:  Rederivation was high.
		DR. SLATER:  The risk associated with it is that, when you rederive a master stock for a vaccine, you essentially have a new product of uncertain immunogenicity that has to be retested, revalidated, and that was considered to be compared to the risk of TSE transmission, which was quite low, quite remote.  That was not considered to be worth the risk associated with trying to essentially come up with a new vaccine.
		DR. SAXON:  I don't quite understand. When you say you are going to ask them to rederive the stocks to get away from this risk, why can't one just replace the uncertified with certified materials in the growth of those materials versus rederive the seed stocks?  
		I mean, I don't understand why you need to rederive them.  You think the actual mold is going to contain within it these prions?  Is that the concern?  Versus just replacing the media with certified, why are you going to make them do that?
		DR. SLATER:  Well, there would probably have to passage it to get rid of the -- You know, even if you were just trying to take the mold and put it into -- There is good reason for manufacturers not to use Category I tissues, period.  There's good reason not to use BHI.  There are alternatives to BHI that they can use for the storage of these products and not lose any potency of their stored masters.
		To rederive the master would basically mean to take the master and, instead of using it the way they use it as the source for all of their working materials, to actually take the master and make a new master out of it.
		DR. SAXON:  They are just going to grow it up, insert it and refreeze it, you're saying?
		DR. SLATER:  Yes.
		DR. LEHRER:  So is that one of the choices we are discussing?
		DR. SAXON:  Yes, that's what he's saying.  Why shouldn't they do that, because in the bacterial vaccines they were concerned when they did that they may actually alter the basic structure in a way where you lose immunogenicity. Here it's not such a big issue.  I don't know.  What do the manufacturers think?
		DR. LEHRER:  Did you answer Andy's question, though, about is there any evidence that prions are contained within the molds or reproduce in molds?
		DR. SLATER:  I don't know that it's ever been looked at in molds.
		DR. LEHRER:  How do prions reproduce?  
		DR. SAXON:  They fold other normal prions. Unless the molds have a normal prion structure, which I've never heard of, they couldn't fold the protein in a mold, and it's extremely unlikely.  So they probably aren't.  I just was wondering what the issue was.
		You really just want to change the media they are kept in and stick them over there.
		DR. LEHRER:  I agree with you, absolutely.  It would essentially be several transfers.  Not a big deal.
		DR. UMETSU:  So that should be one of the choices that we should be assessing.
		DR. SAXON:  Is that what he's asking?
		DR. SLATER:  That is the question.  Is there a concern on the Committee between -- of our asking the manufacturers to do that?
		DR. SAXON:  What does it entail?  Maybe someone can tell us.  I mean, my sense, it's not a lot of work.  You make a couple of stabs, but are we asking -- What is involved?  Is someone here going to address it or will you?
		DR. CLAMAN:  I am sure it's more complicated  than that, but I don't know how.
		DR. SAXON:  Well, that's what I was trying to get.  Sam, you know how to do this, don't you?  
		DR. LEHRER:  Well, I would think it's like any microbial agent, that you would streak it out on a plate.  You want to get a single colony isolate and do it, you know, several times, and that would be it.  Then you would grow up your master stock again.
		The only thing is that the growing of fungi has a lot more requirements than bacteria.  So it's not as easy as streaking out strep or staph.  Nevertheless, if it can -- Obviously, it can grow on these synthetic media.  So I would think that it should be able to be done.
		DR. SAXON:  Did the manufacturers object?  Did anyone have a problem with this?
		DR. SLATER:  No.  
		DR. SAXON:  Makes them look good.
		DR. CLAMAN:  You may have answered this.  What were the risks felt to be for rederiving the vaccines?
		DR. SLATER:  Perhaps somebody from the vaccine side would like to address that.
		DR. MIDTHUNE:  Again, as Dr. Slater has pointed out, the risk calculations are based on a lot of assumptions, but in the Advisory Committee a number of different scenarios were presented.
		For example, with one of the viral vaccine scenarios that was considered, there had been use of fetal calf serum in the working seeds. For that particular scenario, it was estimated that the risk was approximately one in 40 billion vaccines being potentially contaminated.  
		That's when you have the issue at the working seed level.  So if you remove it at the master seed level where you have many full dilutions in between, you are at even much lower risk.  Thus, the Committee then considered the issue of what would be involved in actually rederiving the master seeds.
		That could potentially be extremely difficult, because as Dr. Slater was saying, that could actually alter the vaccine that you ultimately end up with at the end of the process, and this would mean that you would have to redo large scale safety and efficacy studies, because this is, clearly, if you will, a standardized product.  I mean, that is to say you really have very rigid demands on performance out of that product.
		So in the face of a very, very remote risk based on just having an issue with the master seed, it was the recommendation of the Advisory Committee that that not be undertaken because of the concerns over changing the vaccine at the end and, of course, having to go through extensive retesting of virtually what would be a new product.  
		DR. SAXON:  That is very different than what we are talking about.  We are talking about an allergen vaccine that's not got efficacy studies ever done anyway, and virus has got to be cultured out and dah, dah, dah.
		So this -- If the manufacturers -- You know, they could look good.  It's not hard.  Sounds good.
		DR. CLAMAN:  Besides, molds change all the time anyway, don't they?
		DR. SAXON:  Well, that is why they keep frozen in stocks.
		MS. LIBERA:  This is probably is a very basic question.  How often is a stock tested for TSE along the line?
		MS. BRIDGEWATER:  There is no test for TSE.  There is no diagnostic test at this point.
		DR. SAXON:  You couldn't test --
		MS. BRIDGEWATER:  If there was a diagnostic test, we wouldn't have testing.
		DR. SAXON:  You could inject it into mouse brain, I mean, the standard TSE test.  But I guess it's not been done.  There's no other way --
		DR. UMETSU:  The tests aren't sensitive enough to pick it up for most things other than cow's brain.  So injecting this allergen extract, you would probably get nothing.
		CHAIRMAN OWNBY:  It would depend on how many mice you want to inject.  You know, a few hundred, we probably wouldn't find it.
		DR. SAXON:  In millions, it may not.
		DR. CLAMAN:  There is no in vitro test.
		DR. SOTO-AGUILAR:  How is the surveillance done in other countries that have not been affected so far with mad cow disease?  Do they have to wait until the animals get sick?
		DR. EGAN:  In the United States the U.S. Department of Agriculture has a very active surveillance, and they look at cows with neurologic symptoms particularly and, you know, post mortem they examine the brain, and they look for the spongiform encephalopathy.
		In other countries there are also -- besides the morphological changes in the brain, there are antibody based diagnostics, but they are not as sensitive as trying to transmit, say,to mice, and waiting a year or so and then looking at which mice then die of the TSE.
		I think in the U.S. so far they have probably done around 12,000 animals.  USDA has done about 12,000 animals, and they have all been negative.  Then in other countries there's varying surveillances, and in most of Europe the stringency of that surveillance has picked up in recent years.  It's, again, histological examination and antibody.
		DR. CLAMAN:  I agree with Andy Saxon.  The risk of not rederiving seems to be very small.  If the manufacturers don't object, and we have been told that they don't -- I haven't heard them say so -- it would seem that the conservative thing to do is to rederive the stock.
		DR. LEHRER:  Absolutely.  I agree as well.  I think we have to consider that a lot of the figures that Jay was presenting are guesstimates anyway, and even though we are trying to weigh on the side of the worst case scenario, but I mean it's like living in Southern California knowing that the major earthquake is going to come every 200 years or something, and it's really not an issue unless you're there at the time of the major earthquake.
		I think it would be the same thing here.  It's very unlikely to be a problem.  Nevertheless, if there is even one case that it occurs, I think it would be terrible, and particularly since the remedy does to seem to be that much of an issue.
		DR. SAXON:  I'll give you another way, interesting.  In spite of the facts, they say there have been a case of variant CJD in this country in California.  It has been pushed away, but the person came here.  They didn't catch it here.
		So it's more likely we will get a case of variant CJD over here unrelated to anything here, but then the person will have been on mold shots and then someone will point the finger there, probably when they ate a hamburger in Britain, you know, ten years ago.
		So if it's real easy to go, we should do it, because otherwise people will start looking for scapegoats, you know.  
		DR. CLAMAN:  Scape-cows.
		DR. SAXON:  I want to pick on Washington where they pick on scapegoats, since I'm here for my last time.  So I think it's a great idea to do, so they don't turn around and start looking, you know, and it was probably something they did in Britain ten years ago and wasn't their mold shots.
		DR. UMETSU:  I would agree also that, since the risks seem to be low, that they should be rederived.  But the other thing you can certainly do is, if you have a manufacturer's label the ones that are rederived as rederived, and the ones that are not rederived, I'm sure that the ones that are rederived will be the ones that will be chosen to be used.
		DR. SAXON:  But they only need to rederive a few.  It's not all companies.  Right?  It's only a few.
		MS. BRIDGEWATER:  Right.  Let me also point out, too, we're talking about non-standardized products, obviously, with the mold.  There's some degree of -- I don't know if you would call it rederivation that goes on anyway, like many times when they start a new -- or manufacturers have to get a new mold seed stock, they will get it from the ATCC, and it's not that necessarily they would get the same lot every time.
		So there's some variability in the process already.
		DR. LEHRER:  But I thought they would rederive it from their master stock.
		MS. BRIDGEWATER:  Yes.  My point was that there's some variability in the process as they do it -- as it's normally done anyway.  So we're not introducing something new, but yes, we want them to rederive from what they have, if possible.
		CHAIRMAN OWNBY:  But when they rederive -- and my understanding was a lot of people don't maintain these real long term, that they often purchase from ATCC -- the question is then do they have to seek certification from whomever they are purchasing from that this has been propagated in materials that don't contain bovine materials?
		MS. BRIDGEWATER:  Yes, they do have to have that certification.  They get it from ATCC.  Now some of the manufacturers have stocks that they have had that go back to the Seventies and Eighties that they have maintained for a long time.
		CHAIRMAN OWNBY:  I think there's a consensus of the committee to request the manufacturers to rederive these, unless there's some reason to consider them exceptionally onerous compared to what we've heard so far.
		DR. SLATER:  Let me just ask for a slight wrinkle in the discussion, if you don't mind.
		What we have been using -- What I have been using as rederivation is taking the existing master and passaging it several times to get it into -- to make a new master.
		What that really does -- and this is something you discussed before -- is that adds several dilution steps.  Now it's several multi-log dilution steps.  So this is definitely a good step, and really would -- because with each passaging there will be some small amount of carryover, but this adds several dilution steps to the process.
		That is the question that I was asking.  There is another way to interpret rederivation, and that is actually a thing, you have to go back out and find new mold and find new mold isolates.  I just want to make it clear that that was not what I was asking  at this point.  
		I would like to clarify that what we are talking today for rederivation is really introducing several dilution steps to reduce the infectivity of this starting product by several logs.
		DR. SAXON:  It would be many logs, Jay.  I mean, not several.  If you are going to pick a colony, how much contamination by the time you grow it up several times is going to be huge.  It's not just -- You said -- I mean, 106.
		DR. SLATER:  I'm comfortable with what you are saying.  I just want it to be clear in the record that this is what we are talking about.
		DR. SLATER:  It would be an enormous change.
		CHAIRMAN OWNBY:  And you are going to provide the manufacturers some guidance as to how many steps are going to be required in this?  It seems to me, if I was a manufacturer, the first thing I would ask is how many times do I have to repropagate this before I can now say this is a certified, safe master?
		DR. SLATER:  Yes.
		DR. SAXON:  Two times will be at least a millionfold.
		DR. CLAMAN:  Is this a recommendation or a directive?  What are you doing, talking to your lawyer?
		DR. SLATER:  I am talking to my best friend at the moment.  
		This will be a recommendation from the committee that will be considered by us, and that we will discuss and go from that recommendation from the committee.
		DR. UMETSU:  Are these also organisms that the ATCC carries?  Is it something that, if you have a derived stock, that the ATCC can supply to the manufacturers?
		DR. SLATER:  What I didn't explain in great detail when I was talking about how different the different manufacturers handle this:  Only some manufacturers actually maintain their masters internally.  Many of the manufacturers get their masters from outside sources.  
		In some cases, it will be the outside sources that they will have to go to, to get them rederived.
		DR. UMETSU:  But can those be standardized in some way?
		DR. SLATER:  Well, standardized is a loaded word, especially --
		DR. UMETSU:  Or certified?
		DR. SLATER:  -- as we discussed at great length last year.  The outside sources, the sources that are used, do to a greater or lesser degree certify the identity of the mold strains, if that's what you are asking.
		DR. UMETSU:  Well, but now can they be certified in terms of TSE?
		DR. SLATER:  Well, they will have to be.  That will be --
		DR. UMETSU:  So that would help in your directive or recommendation to manufacturers?
		DR. SLATER:  Yes.
		CHAIRMAN OWNBY:  Anymore burning comments?  We were directed to have 30 minutes here for public comment.  Bill, have we received any requests for public comment?
		DR. FREAS:  I have received no responses to the Federal Register announcement, but if there's anybody here who would like to make a comment at this time, we welcome comments from the public.  
		MS. WILLIAMSON:  Can I just ask a question?  My name is Shirley Williamson, and I'm with Holister Stier Laboratories.
		Listening to the conversation just to add a couple of points, we currently have 32 master stocks that we maintain at our facility.  I think we have pretty much reached the conclusion that we will be rederiving and have started that process.
		At our current schedule, it will take us about a year.  It will take us the entire year.  So from our standpoint, that's what we are talking about.
		CHAIRMAN OWNBY:  Thank you.  Any other comments?  That answered your question?   Good.  Any others?
		Okay, I think we can move on then with our agenda.  The next was the presentation on the statistical power of clinical studies comparing allergen extracts by Dr. Lachenbruch and Jay Slater.  Jay, you are going to introduce this?
		DR. SLATER:  Thank you.  I am Jay Slater.
		Dr. Lachenbruch is going to give the main body of this discussion, but what I wanted to do was I wanted to introduce his remarks, and I am actually going to come back and say something briefly after his remarks.
		The source of this topic for presentation to the Advisory Committee is that it has come up a number of times in the last couple of years that we have had bilateral discussions with manufacturers about just these specific issues that we are going to introduce now, and since there has been some misunderstandings from the manufacturers, we wanted to discuss the issue of clinical bioequivalence as fully as possible in a public format for the Advisory Committee's discussion as well, so that these concepts could be discussed and aired.
		I am going to give here a generic, fictional example in which this kind of question comes up.  A manufacturer wishes to change its approved production method for standardized fedweed pollen extract.  Fedweed is a joke, okay?  I was trying to think of some funny name for a pollen, and no matter what funny name you think of, they are all there already.  So this is the only one that I could think of that hadn't been taken by nature yet.
		CBER acknowledges that existing in vitro potency tests will be applicable to the new preparation.  After all, it's a new preparation of an existing product.  However, CBER requests that the manufacturer demonstrate compositional similarity by the parallel line bioassay.
		Now what does this mean?  Well, again we are going to go back to the intradermal skin testing protocol.  This was the original report.  It was in November 1982 in JACI from Paul Turkeltaub, Dr. Rastogi, Dr. Baer, Chris Anderson and Phil Normal from Johns Hopkins.
		This is the study method in which you do serial dilution intradermal skin testing looking at the erythema, and looking at both potency and compositional similarity using this.  This paper was published in 1982.
		Now about a decade later, this method was incorporated into the methods of the Allergenic Products Testing Laboratory which was actually announced for release in an FR notice in November 1994.  I just want to quote a small section of that protocol for you to understand where the problem now comes in.
		This is part of that protocol, Quantitative Intradermal Procedures for Determining the Relative Potency and Compositional Differences of Allergenic Extracts, quote:  "At least four subjects are required per assay.  One or more test extracts are to be compared against a reference in each subject.  For evaluating compositional differences, subjects selectively sensitive to specific allergens in the crude mix should be selected.  In order to enhance the detection of compositional differences, subjects can be selected who differ widely, 10-10,000-fold, in their allergen skin test sensitivity to the test of reference extracts."
		So according to the protocol, applied correctly you can adequately look for compositional differences between two products by testing four study subjects.  
		DR. SAXON:  Jay, what does that mean, specific allergens in the crude mix?  
		DR. SLATER:  Yes, because the --
		DR. SAXON:  You're going to have to know their dura-p 1 -- I mean, the actual allergens in there, because there's multiple, ragweed, you know.
		DR. SLATER:  Right.  So to evaluate compositional differences -- in other words, whether allergen mix contains allergens A, B, and C -- you need to look for individuals who are selectively sensitive to those in order to detect the differences among them.
		It's hard.  I mean this is hard, but the problem that we've been getting hung up on is the question of four study subjects.  
		Now since 1993 and 1994, FDA has evolved, has refined its approached toward this issue of compositional similarity.  One example is in April 1996 a guidance document appeared concerning the demonstration of comparability of human biological products, including therapeutic biotechnology derived products, and more recently in 1998, an international conference on harmonization document appeared -- this is the E-9 document that is included in your pre-package -- entitled "Statistical Principles for Clinical Trials."
		One particular portion of this addresses the kinds of statistical considerations that would drive this kind of analysis.  
		Now before I turn the podium over to Dr. Lachenbruch, let me just make a brief statement about what this is not about.  We are not talking about lot release.  Lot to lot consistency within a given product is established by accepted in vitro equivalence testing.
		In these examples, we are addressing the bioequivalence among different products for which there are proposed changes in source materials, manufacturing or stabilizers or diluents.  
		In fact, one of the things that we showed in our analysis of the currently available standardized product is that within a standardized product, there is a tremendous amount of consistency, both in composition and in potency.  But here we are talking about situations in which the manufacturer has asked to change part of the process.
		Once you start changing either the source materials or the manufacturing or the packaging or the stabilizers, you have to demonstrate that the new product is compositionally similar to the licensed product, and that's where this discussion is coming from.
		Now I would like to introduce Dr. Tony Lachenbruch who is the Director of the Division of Biostatistics at CBER.
		DR. LACHENBRUCH:  Speaking as somebody who went through the 1994 January 16th earthquake in Los Angeles, I don't want to have another one like that.  It's rather exciting.  I don't think I've ever been quite so scared.
		Okay.  Well, in abstracting this to the simplest possible situation, we are really looking at -- let's say we are looking at a single response, and what we are trying to say is are these similar?  Are the responses similar?
		One of the most common mistakes that is made, and I have made it myself, is oh, I'm going to test a hypothesis that the means are the same, and failing to reject that, concluding that the two responses are the same.  This is a problem, because failing to reject, failing to see evidence of a difference is not evidence of no difference.
		So what you do is you set up your hypotheses so that, when you reject it, you are forced to the conclusion that they are the same.  So the null  hypothesis is going to be a little bit difference, which says the products are different by a certain amount, and therein lies a tough situation.
		So let's say we think of applying an antigen to the back of a subject.  The application should be randomly determined, and this assumes that there are no systemic effects that could muddy the comparison.
		So after a period of time, we look at the size of the wheals and measure them, and we need to show that the wheals are within some small limit of each other.  The question is, first of all, what is small?  That's a real headache.  And what's the correct method?  I've already hinted at that.
		So we might set the margin of difference to be ten percent of the mean of the standard.  so if you typically would see a three centimeter wheal, you would want it to be no more -- the differences in size of wheals to be no more than .3 apart.  So we are setting this -- we can set the margin as a fraction or sometimes we could set an absolute value for the margin, which might be based on a lot of previous experience.  So it may not differ very much from the fraction method.
		So we are requiring the difference of the means to be within a margin of zero.  That is this margin here.  So, for example, here is our picture, and we want to show that we are between -.3 and +.3. 
		The heavy lines would indicate a difference in the mean level.  So what we are saying is the heavy lines here are basically what the null hypothesis is that we are either more than -- that the new is different from standard by at least +.3 of a centimeter or -.3.  So we want to show that the true mean difference is in this lightly shaded area.
		Well, let's supposed we found that the mean difference of the pairs and their standard deviation were as follows.  We found the average difference.  This is a sample quantity.  It's .2, and we found the standard deviation of 2.5 for this difference.  We had 100 pairs.  so we found a t was 0.8.
		Ah, okay.  If we did the standard null hypothesis, no rejection, we would say there's no rejection.  We'll conclude that these are the same.  However, we can always choose a small sample size and find that there is -- have no power to detect a difference. 
		So we said in this case, let's suppose the historically known wheal size was three centimeters and ten percent would be three-tenths.  So we can compute a 90 percent confidence interval, and note that if it is entirely contained in the equivalence region -- it has to lie between -.3 and +.3 -- then we can conclude safely that we do have equivalence.
		So in this case we compute a confidence interval on this basis, and we see that it runs from -0.215 to +0.615.  So we could not conclude that the new and old are equivalent.
		So here, for example, we see it in pictures.  From -.3 to +.3 is our acceptance region, and our observed confidence interval goes from -0.215 to +0.615, and they don't overlap.  We can't conclude equivalence in this case.
		So either another study would have to be done or the product might, in fact, need to be reformulated.
		Another method that is sometimes used, and I think this is the method that is referred to in the material you received -- it was proposed originally by Don Sherman at CDER -- and it says let's test a joint null hypothesis.  The first part is that the mean difference is greater than +delta.  Delta in this case is the .3.  Or that it's less than -delta.  That's the negative .3.
		If we reject both of those hypotheses, then we conclude that the mean difference is less than delta.  So notice, what we are saying is the null hypothesis is the difference is bigger than delta.  The alternative is that it's the absolute difference is less.
		So let's just talk briefly about the conclusions.  In any situation when we deal with a sponsor, we need to discuss the selection of the margin, ten percent, an absolute figure, whatever it is.  This does need to come in, and I think sponsors should also do themselves a favor.  In case somehow we don't mention it, then they should ask us what's going to be okay.
		The sample size is going to be based on a null hypothesis that they aren't equivalent.  You can either use a confidence interval or the two one-sided tests.  There are lots of headaches in doing these studies, because typically in a short-a-difference type of thing, the null hypothesis is that they are the same.  
		If you have imperfect experimental treatment -- imperfect execution of your study, they tend to push the means from both groups toward one another; whereas, if you do that when you are looking at the equivalence studies, bad execution pushes the study in the wrong direction.  It's favoring the alternative hypothesis, and you want to be very cautions about that.
		So you should use both intent to treat and per protocol analyses and examine them, and you need to provide evidence of trial validity, both in looking at the design and in the efficacy of the control.  For example, if you normally saw a three centimeter wheal and instead you ended up seeing a 10 centimeter wheal, you would be concerned that somehow things weren't behaving properly.
		Is this the last one, Jay?  You've got all the other slides in there, too?  Okay.
		In your handout, I believe, there are many other slides, but I wanted to stick with these 11 slides, because the others are basically going through a calculation.  So I thank you.  I'm available, and Jay is available to answer questions.  But you are going to say a few words.
		DR. SLATER:  Thank you, Tony.  
		So what I wanted to do in the next few slides was just to come back to our specific example and give some specific number calculations that might give you an idea of what this means in terms of the specific proposal.  So let's go back to our fictional manufacturer.
		Based on the 1993 protocol, the manufacturer proposes to enlist between four and six study subjects of varying levels of sensitivity to fedweed pollen, and the manufacturer says that if the slopes of the two preparations are not significantly different in these study subjects, the manufacturer will then conclude that the two preparations are compositionally similar.
		Now as Tony has said and as I'll repeat, the failure to demonstrate a difference does not mean the demonstration of similarity, and that's an important point to continue.  But just for the purposes of putting numbers in this, let's say that the mean slope is about ten, and let's say that our acceptable differences between slopes is about ten percent or one of a difference in the slope.
		Again, let's say that the standard deviation of the slope is one.  We have an alpha of 0.05 and a beta of 0.2.  Now you will recall that Dr. Lachenbruch provided you with these formulas, or were those in the slides that you didn't show?  Oh, okay.  He didn't provide you with these formulas, but these formulas can help determine the significance using the two one-sided test approach.
		To give you a sense of what this approach might mean in terms of the study size, what we are really interested in is n, and now this specific rearrangement is one that I think we have to put a caveat in here, that this may not be applicable in terms of all of the analyses; but if you rearrange the numbers that we have, we can come up with a formula for n that is this one to determine the number of study subjects, n, required to detect a difference, delta, with a confidence of alpha and a power of one minus beta using the two one-sided test approach.
		This is one formula for n.  It's 2 x the Sigma-squared, which is the standard deviation, times the square of the sum of the two z values over delta squared.  Now if you solve this for alpha of .05 and a beta of 0.2 and reduce it, what you find is n is a constant factor 12 times the square of Sigma over delta.
		Now, remember, Sigma is the standard deviation of the test that you are using, and the broader it is-- Yes, Andy?
		DR. SAXON:  A beta of .2 is an 80 percent confidence of finding it, if it's there.  Is that right?
		DR. SLATER:  It's 80 percent power.
		DR. SAXON:  Oh, right.  That's the 80 percent power.  Okay.  
		DR. SLATER:  Right.  But the important thing to note here is that what's important in determining n is the ratio of Sigma to delta, of the standard deviation of your test to the acceptable differences, and in fact those as the square of those two.
		So we go through some number crunching to give you an idea of what we are actually talking about here.  Let's plug in some of the numbers.  In the specific example that I showed before, we -- For all of these, we accept an alpha of .05 and a beta of .2.  These are all fairly conservative assumptions and default positions.
		Let's say that the delta is 1, and let's say that the Sigma is 1.  In other words, for this test the acceptable difference is equal to the standard deviation between the two. 
		Well, if you plug that in, it doesn't take much arithmetic to show, if Sigma equals Delta, then n is about 13.  Now let's say that the Sigma is larger than that.  Let's say that the Sigma is twice what the Delta is.  Well, in that case Sigma over Delta is 2.  That squared is four, and we come up with an n of 50.
		Well, given an acceptable delta of 1, how low would your standard deviation have to be to get to that four to six study subjects?  The answer is you have to have a pretty low Sigma.  You would have to have a Sigma that was just a little bit over half of what your Delta was.
		Now, really, those are all the numbers that you need to look at this, but we can actually take it a step further.  Let's say that we decide that a Delta of ten percent is too restrictive, and makes very little sense.  So we are going to increase the Delta to 20 percent.
		Well, if the Sigma is 2, well, then you have the same situation you have up here, and you need 13 study subjects.  
		Well, with a Sigma of 2, how liberal would you have to be with your Delta to get down to that level of n of four to five, and again you would have to have a delta that was pretty substantial, 35 percent of what you were looking for.
		DR. SAXON:  In terms of erythema, it turns out it would be 2 millimeters?
		DR. SLATER:  No.  This is a slope.  Remember, when you're looking at compositional similarity of differences, it's actually a slope.  So it's a ratio of the erythema over the log dose.
		So in conclusion -- and again this is something I know we've repeated a couple of times, but it's important to know that the failure to demonstrate a difference is not sufficient to demonstrate equivalence.
		Given that, for the parallel line bioassay, in general, the Sigma is at least equal to but greater than Delta usually, the number of study subjects required to demonstrate equivalence will usually exceed the four to six.  
		The bottom line is that the four to six that was shown in the '93-94 document will only be sufficient if it leads to a sufficiently powered study, and that the subsequent documents give good guidance as to how to sufficiently power the study.
		We do ask that the Advisory Committee discuss this.  In particular, we request that the Committee discuss CBER's current approach to clinical bioequivalence studies as it applies to allergen extract studies.
		Are there any specific questions for me or Dr. Lachenbruch?
		DR. SAXON:  When they did the original study with Turkeltaub and Baer and Norman, did they actually do a power analysis like this?  Where did the four to six come from?  The back pocket?
		DR. SLATER:  I wouldn't say that.  They did a large number of these studies.  They did a lot of intradermal skin testing.  They had a significant amount of data.  I don't know the basis of the choice of those numbers.
		DR. SAXON:  Because today you would get your grant rejected without a power analysis.  You couldn't get it through.  So I mean, you're basically saying you need to do the basic power analysis to test the hypothesis.  Right?
		DR. SLATER:  What we are saying is the study has to be sufficiently powered to demonstrate that they are not different.
		DR. SAXON:  You couldn't get a grant funded today without that in your grant.  You can't just say I'm going to do 12 subjects.  You've got to have the analysis.
		DR. SLATER:  Dr. Lachenbruch?
		DR. LACHENBRUCH:  I believe Dr. Rastogi was involved with this and would have done the power analysis.  That original study, the 1982 paper, I think, was more directed toward showing a difference rather than an equivalence study, because around 1982 was the beginning of our interest in showing no difference, and then about 1987 was the Sherman paper, and there's been a very, very active interest in that in later years.
		DR. SAXON: So you're saying the original study with looking at the hypothesis being one side being different could be powered with four to six.
		DR. LACHENBRUCH:  Possibly, yes.  
		DR. SAXON:  But you've just shown us that it won't work with --
		DR. LACHENBRUCH:  In each instance, you have to go through this analysis and see where you go.
		CHAIRMAN OWNBY:  So I guess the real question that you would like some guidance on is the expense to manufacturers if they did bring a new process of showing equivalence with a much larger number of subjects than we have previously had versus the concern that with such a small number of subjects we might not -- we would be woefully underpowered to detect differences.  Is that correct?
		DR. LACHENBRUCH:  I am sorry.  I didn't hear the beginning of the question.  I was trying to answer another question over there.
		CHAIRMAN OWNBY:  Well, it seems like this is trying to balance the time and expense that it takes to do a study with a large number of subjects versus the current practice which we think is woefully underpowered to detect problems or to really establish that there is bioequivalence.
		DR. LACHENBRUCH:  It's answering the wrong question, I think.  It's saying are they different and, if you don't say they are different, you can't conclude they are the same.  Whereas, if you test are they different and you reject that, you can say that they are basically the same.
		One of the things that I would point out is that Dr. Slater's sample size calculations were for unpaired data, whereas often, if you are painting two sides of the same back, you can take account of the pairing, and that will usually reduce the variance or the standard deviation quite a bit.  So you are then less interested in saying it's within a certain tiny amount, but you might be able to take a larger margin relative to the standard deviation.
		Your study will be much more sensitive, because your standard deviation of the slopes would be much smaller.
		DR. SLATER:  I think the key thing from my point of view in terms of the discussion is that a manufacturer -- and manufacturers have done this.  They have come to us with the '94 methods manual and said, well, here we are following it A, B, C and D. 		We simply wanted to alert the committee to the changes that have happened since 1994, to the changes in thinking that are not being, by any means, specifically applied to allergenics and, in fact, you know, are -- but we are definitely interested in applying them to allergenics now, and we wanted to air that thinking for the Committee and, if possible, get some feedback from the Committee and some discussion about the concepts that we have introduced here.
		DR. UMETSU:  I think that the idea of proving nonequivalence versus equivalence is a very clear one, and it sounds like it should be implemented.  
		Could you give me an idea of the number of patients or subjects it would take?  Clearly, the old standard was four subjects.  In your estimation, how many subjects would be required to prove equivalence?
		DR. SLATER:  It depends on the slope, and it depends on the standard deviation, and the standard deviation could easily change, depending on the population that the manufacturer chooses.
		DR. UMETSU:  But are we talking about 50 subjects or 100 subjects?
		DR. SLATER:  Yes.  
		DR. SAXON:  Do you think that is reasonable?
		DR. SLATER:  I am not being evasive.  Yes, it could be 50.  it could be 100.
		DR. SAXON:  That's the concern.  It may become unreasonable.  How do you get 100 subjects?  I mean to do this, it seems unreasonable at that point.
		DR. SLATER:  Unreasonable in what sense?
		DR. SAXON:  Unreasonable to get 100 subjects who are -- Let me figure something that's not that common -- who are saltgrass or saltbush positive, and they are going to have to titer 100 people when they make a change.  It would certainly keep me from making a change.
		DR. SLATER:  Well --
		DR. LACHENBRUCH:  Our answer may, in fact, depend on the prevalence of the condition, too.
		DR. SAXON:  Prevalence isn't going to let you be anymore accurate.
		DR. LACHENBRUCH;  No, but if you have a condition in which you have hundreds of thousands of subjects who are affected versus something that may be 1,000 in the country are affected, your answer may be different.
		DR. SAXON;  Well, let's take even a common one, cat, and you were going to change our cat thing.  You're going to have to take 100 subjects and do these titered back responses on 100 subjects?  Have you done 100 subjects?  Has anyone here?  I mean, Turkeltaub started this stuff, I guess.  I don't know if he did, but has anyone actually taken 50 or done a big study?
		DR. SLATER:  We are budgeting our estimates for cockroach at about 200 study subjects per American and 200 for German.  
		DR. SAXON:  Two hundred?
		DR. SLATER:  That's just based on our initial estimates.
		DR. SAXON:  And you're going to do it -- What are you going to do with the 200?
		DR. SLATER:  Well, we are going to be determining the potency and the -- the relative potency and the potency of different extracts.  This is not the same as determining compositional similarity and differences, but I think in order to do an adequately powered study, you need to do the calculations of what the standard deviation is, and you need to power it adequately.  Otherwise, you just can't learn the information that you are trying to learn.
		DR. UMETSU:  So you estimate that it will take 200 subjects?
		DR. SLATER:  I'm starting -- In fact, what you need to determine is what this Sigma is for the study in the study population, and we don't really have that yet.  This is just a starting estimate.
		The other point is that this wouldn't come up with saltgrass.  This would only come up with a standardized allergen extract at this point.
		DR. SAXON:  But aren't they all going to be at some point?  Isn't that where we are going?
		DR. SLATER:  That is the direction.
		DR. SAXON:  It just strikes me that -- I mean, I understand the science.  You know, it's great science, but then, you know, there's got to be a better way.  If you're going to come up and tell me that I've got to test 100 people for cat -- and again, I'm not a manufacturer, but I can't imagine they would have as quality controlled people as you do.
		I mean, they are going to be less tightly controlled internally, I suspect, than your lab, Jay.  Your la should be the highest quality, and it's tough.  I mean, hiring people, getting good people.  I think it's going to be --
		DR. CLAMAN:  I think so, too.  I think that with a larger --
		DR. SAXON:  The delta is going to be huge.
		DR. CLAMAN:  The larger the number of subjects, you may be building in an extra amount of biological variability, and the variability won't be in the patient.
		DR. SAXON: We should -- Do the manufacturers actually do this test or isn't there a company that they farm it out to?
		CHAIRMAN OWNBY:  Well, isn't that, though, a business decision for the company as to how to approach this problem.  But it seems to me there's two issues we've brought up.  
		One is that under the -- that ideally here, the smaller number of subjects that can be achieved, the smaller your variation.  Isn't that true?
		DR. LACHENBRUCH:  Right.
		CHAIRMAN OWNBY:  That the smaller the standard deviation, the fewer subjects you would have to have.
		DR. LACHENBRUCH:  Right.
		CHAIRMAN OWNBY:  So therefore, the more precisely you do the study, the more economical it becomes.  So that, I think, would be a good principle that we would have.
		The other thing we've talked about is biological diversity, that skin testing people from different ethnic groups might produce different results, and you would like to have a sample large enough to incorporate some of that variation.  With, obviously, four or six subjects, that becomes very difficult to do.
		DR. CLAMAN:  But one of the handouts we got had some very good discussion of sequential analysis.  Can't you -- If you're asking whether the number of subjects ought to be increased, I'd say yes, that's a great idea.  But that's open-ended.  What do you mean by increase, 100, 200?
		DR. LACHENBRUCH:  Is this one of those that I handed out?  I don't remember talking about sequential analysis.
		DR. CLAMAN:  Well, can't you determine as you go along how many you need by, let's say, doing five or ten and, if the standard deviation is very small, then you say to yourself that's enough?
		DR. LACHENBRUCH:  You can do something like this, if it's in a formal plan.  You can't do it if you say, well, I'm going to start out and then I'm going to look at it this time, and then I'm going to look at it again.  You can't do it on an ad hoc basis there.
		DR. CLAMAN:  Well, I understand that.  But you could build it into your protocol.
		DR. LACHENBRUCH:  There are procedures.
		DR. CLAMAN:  Of course, there is.
		DR. LACHENBRUCH:  Sure.  Yes.
		DR. SAXON:  Dennis, this is our last chance here, you, me and Henry.  So I would --
		CHAIRMAN OWNBY:  They will have it easy after this, aren't they?
		DR. SAXON:  This is great.  One thing is, you know, I think Den made a great point.  If you could, number one, try to get the technique with better precision, because that will cut your delta down.  The other is I think this technique started in 1982.  I think it's time to look for another method.
		I won't suggest one, but this has been going on since 1982.  It's almost 20 years later.  Let's find a better.  We have a new Chief of Allergy here now and lab.  Find a better assay than this method measuring wheal flares.  I would strongly recommend that, and I see my colleague over there shaking his head yes, too.
		DR. SLATER:  I agree, and actually, this method was not without controversy when it was introduced.  I mean, there was tremendous debate about it, and it was implemented in spite of that, and unto this day even, to be honest with you, my question about biodiversity in this method, I think, I'm still not satisfied with the answer, and particularly in today's environment.
		Even if we didn't think that it was important, I think it's extremely important politically.  Be that as it may, I think it's extremely important, period, and I'm not convinced that it's being done, particularly in very dark pigment individuals.  It's just very difficult to read flares, or impossible.
		Many of these are the target population, particularly cockroach.  I think -- I mean, we've been skirting around the issue.  You know, Andy has a way of hitting it.  I think he's absolutely right.
		DR. SAXON:  As we end this panel, so to speak, I think one of the things this Committee should do is maybe get together with the FDA and get together a group of people who are interested in this with the Academies, whatever, under the FDA to have a go at this again after 20 years.
		Should there be a new method using transgenic mice, some method?  It's not going to cover -- That won't cover the biodiversity, but at least it will allow you to get the standardization in vivo system.  No one likes in vitro -- some in vivo system, but give it a day or day and a half of think tank and get rid of this.
		CHAIRMAN OWNBY:  No other comments?  I'm not sure we've given them the answer.
		DR. SAXON:  Dennis, remember, they do whatever they want, no matter what we say anyway.  But I think the answer is, you know -- right, Jay?  Jay, I think the answer that -- we all appreciate the need to power studies appropriately.  Every scientist knows that today.  Your point is very well made, right, that they are underpowered.
		They need to make -- You got to make it more precise.  Henry's got at least -- Then as you get the deltas, you go through the study, you'll know what is reasonable.  But you need to find ultimately a better way at this, because you are going to find ones where it's 200, and manufacturers aren't going to be able to do 200, are they?  Not really.  Probably not, not accurately like you will.
		DR. SLATER:  Was that a question for me?
		DR. SAXON:  Yes.  What do you think? Can you imagine saying, you know, you need to do 200 cat, and then they are going to have technicians who aren't as good as yours, who haven't really got the --
		DR. SLATER:  Well, remember that the 1993-94 manual has in it a qualification step for anyone performing this assay with sequential histamine skin tests.  So if one is following the '93-94 protocol, one has already narrowed the range in terms of the technical proficiency of the individuals performing the test.
		I appreciate the comment about the high quality of my lab, and I can accept that without further comment.  But I also accept that these are difficult studies to do.  
		There is, however, probably an irreducible sigma associated with these kinds of measurements in human skin, regardless of the pigment.  That, I think, is where the numbers come from, and I think the numbers are driven by the imprecision in the human skin response to injected allergens and by the difficulty in measuring it with reproducible accuracy.  But we do try to get around that with the proficiency testing for the technicians, which, quite frankly, some people have to repeat several times before they are demonstrated to be proficient. 
		It's not a trivial step.  So there is sort of a training step built in that, I think, tries to get around that problem and tries to bring everyone up to a certain proficiency level.
		DR. SOTO-AGUILAR:  Could CBER conduct the studies or each manufacturing company would seek patients who are very allergic to their products and then go forward with their protocol?
		DR. SLATER:  I'm sorry.  You are asking whether this is centralized?
		DR. SOTO-AGUILAR:  You are trying to find bioequivalent potency for standardization of extract.  Would CBER conduct the study, given products from all these manufacturing companies to multi-center to conduct the studies with two or three samples from three different companies and your own standard, or each company is going to be invited to do it on their own and then send the results to CBER?
		DR. SLATER:  The model is for CBER to coordinate the studies, and the data and the data analysis would be done by CBER.  Within our current constraints CBER itself, meaning me, could only do testing in Bethesda, Maryland, in an area that would attract individuals from around that area.
		It's important that we have both a  geographically and ethnically diverse population.  So at a minimum we would need to contract to other sites around the country.  
		I assume you are asking now about cockroach.  Is that correct?
		CHAIRMAN OWNBY:  No.  I think we've got at least two different questions here that are not very clear.  The question is, if a manufacturer wishes to change, for example, their source materials, then this is entirely incumbent upon the manufacturer, isn't it, to do the testing; whereas, with standardization of cockroach, you are talking about coordinating it through CBER.  
		DR. SLATER:  Right.  These are two separate issues.  These are unrelated issues.
		CHAIRMAN OWNBY:  It seems to me that there is -- One of the things we haven't asked, we are just presupposing that this is the only way to get a bioequivalence, and Andy was saying, you know, can't we come up with a different system?  Is it worth discussing that or is that just not an issue currently?
		DR. CLAMAN:  I think it would be difficult between now and lunch to define the new system that we think ought to be put into place.
		DR. SAXON:  That is why I suggested that the FDA -- someone like Jay pull together a -- You know, it's an opportunity.
		DR. CLAMAN:  Do you think we need a better system?
		DR. SLATER:  Cockroach will be my first firsthand experience with the existing system.  I think, as with any system, I think there are strengths and weaknesses associated with it.
		Certainly, it is a cumbersome system, and certainly, our discussion today suggests --
		DR. CLAMAN: And it may get more cumbersome.
		DR. SLATER:  -- a more cumbersome than we originally thought.  That being said, the strength of the system is that it is a clinical measure of overall allergenicity and, if done properly, it can give you a good mean assessment of what the consequences are of injecting this material into a population -- into an individual who is highly sensitive.
		Remember that the default position is to pick highly sensitive individuals for study in terms of standardization.  Now in terms of assessing compositional similarity and differences, you are supposed to take a range of sensitivities.
		I must say, it does have a fairly compelling strength, and that is that it does seem to give us what the potency of a product is in an allergic individual and in an allergic population.  But I'm very open to further discussion, both with the members of the Advisory Committee and with others, about alternative approaches that can be used.
		There are weaknesses, as you all know, to the purely in vitro approaches that really preclude adopting them as our gold standard for allergenicity, and I don't think anything Dr. Saxon said was aimed at pushing that idea.  Is that correct?
		DR. SAXON:  No, but I can give you one idea right here.  If you take the FCER or alpha transgenic mice, they express FCER receptors just like a human on dendritic cells.  The mouse receptor is knocked out.  You take serum from people.  You take the serum from the allergic people, and you inject those mice.  You sensitize them to human antigen.  You could then skin test those mice.  You can do PCA on those mice.
		The only shortcoming in that system:  The mice are small.  We need giant mice.  They don't have enough skin.  But I mean, conceptually we use that for other things, and we use it with human IgE antibody.  So you could get a pool.  You could have a pool of human Ig serum for ragweed, and you give it to -- you know, there's many things that could be done which would allow an in vivo system that you could put large numbers through, mice instead of people, just breed them.  Just an idea.
		CHAIRMAN OWNBY:  Are there any other questions that FDA was interested in pertaining to this?
		DR. SLATER:  Let me just clarify what I've  been hearing from the Committee, and please correct me if I'm wrong.
		It sounds like the Committee acknowledges the validity of adequately powering studies in order to demonstrate the kinds of bioequivalence that we've been talking about.
		It sounds to me like the Committee is somewhat concerned about the consequences of this in terms of how cumbersome the studies might be, and is suggesting that we think about other appropriate measures of overall allergenicity that might escape some of those.  But given that we haven't had this workshop yet, and given that we haven't found an alternative method that's been appropriately validated and as a good surrogate for this testing, the Committee is comfortable with the analysis that Dr. Lachenbruch and I discussed.
		DR. SAXON:  I am comfortable with it, but I must say, I feel some sensitivity.  If this was set up in 1982 -- When did it go in the Federal Register the first time, '94 you said, Jay?
		DR. SLATER:  November 1994.
		DR. SAXON:  Yes.  I mean, with the concept that it was four to six, and then all of a sudden, you come back eight years later and say, hey, guys, four to six has turned into 100.  It don't seem fair, to be frank.  It was set up, you know, by good people thinking they had done a good thing, and you may turn it into something burdensome.  That's all.  You know, that's the concern.
		DR. SOTO-AGUILAR:  Could 20 individuals per center be all right for a maximum of 100?
		DR. SLATER:  It's hard to answer that question specifically.  It would depend on how many centers, how diverse the centers were from each other, what the population was.  I really couldn't answer that specifically.  But there's certainly nothing a priori that says that that would not be adequate.  We would just have to look at how we are setting it up.
		CHAIRMAN OWNBY:  Any other comments or discussion on this item?  I get the distinct sense there is a consensus that it's lunchtime.
		So we will adjourn until 1:50, and we will take up the cloudy issue of particulates in the allergen extracts.  
		(Whereupon, the foregoing matter went off the record at 12:48 p.m.)

	A-F-T-E-R-N-O-O-N  S-E-S-S-I-O-N
	(1:51 p.m.)
		CHAIRMAN OWNBY:  We will reconvene the Committee meeting, please.
		The first item on this afternoon's agenda is a discussion of particulates in allergen extracts, and Ms. Bridgewater is going to present this.
		MS. BRIDGEWATER:  Thanks.  Okay.  Good afternoon.  I am going to speak to you today about a topic that you have heard about a couple of times this morning, and that is precipitates in allergenic extract.
		Before I discuss the precipitates, I will briefly review the product line.  There's currently 19 standardized products.  The short ragweed product comes in both glycerinated and aqueous formulations. The standardized cat, grasses and mites are generally glycerinated only.  There's a few exceptions.  Some of the cat pelt extracts come in an aqueous form and the intradermal tests have less than 50 percent glycerine.
		Standardized venoms don't enter into this.  They are freeze dried with an HSH -- 
		Now the unstandardized products account for the majority of the product line, and they come in both glycerinated and aqueous formulations.  I think -- I was talking to one of our manufacturers this morning, and I think this number is a little high these days.  Several of the manufacturers have cut down their product line.  Next slide, please.
		So historically, precipitates have been recognized for many years, and they are an intrinsic phenomenon.  In other words, the precipitates develop within the extract itself as opposed to an external contaminate which would more properly be defined as a particulate.
		Now there were early efforts by industry to characterize the precipitates such as trying to describe their physical appearance and look at their solubility properties.  Now there were also some early efforts by industry to remove the precipitates.
		An example of this would be a manufacturing change, for example, a bulk settling step. This is a procedure where the bulk is allowed to sit for a period of time to allow the precipitant to settle out of solution and then it's filtered, and the manufacturing process continues.
		There are also some early efforts in formulation changes, for example, changing the extraction fluids.  But these were generally unsuccessful.
		So considering that I've told you that precipitates were recognized for many years, how did this issue arise recently?  Well, the appearance of precipitates were noted during several Team Biologics inspections in the last few years.  
		The inspectors observed, physically observed, the precipitates in bulk containers, final containers and retention samples, and they were also found to be a primary cause of customer complaints and product returns.
		Can we go back?  Sorry.  I want to point out from a strictly current good manufacturing practice standpoint that particulates are an undesirable attribute of a parenteral product.  For example, the USP sets limits on particulates in parenteral products, and I'm making a distinction here, because the USP specifically talks about particulates.  It doesn't really distinguish between particulates and precipitates, as I've defined them.
		So here's some of the terms that industry uses to describe the appearance of the precipitate:  Crystalline, flaky, cloudy, powdery, tarry, fibrous, clumpy.  Next slide, please.
		Continuing, they may also describe the color of the precipitate, the ability to resuspend it in solution or not, the amount of precipitate at the bottom of the vial or perhaps the percentage of extract that contains precipitate.
		So I'd like to show you an example of a precipitated short ragweed extract.  This is something that was taken from our lab, and you can see here on the bottom of the vial there's clearly a ring of precipitate at the bottom with the clear extract solution on top.  Next slide.
		This is another example from a different manufacturer, and again this is a short ragweed.  This is aqueous.  Let me point that out.  You can see, this is a little bit different in appearance.  It's cloudier, but you can still see this amount of precipitate here at the bottom.  
		As these two examples illustrate, the appearance can vary widely between extracts, even of the same type.   Next slide, please.
		So currently the manufacturers -- we've  asked them to take some efforts to characterize their precipitates.  The first example here, microscopic examination to determine what their physical appearance is; sterility testing to rule out that the precipitates are not a result of microbial contamination; and finally, some of the manufacturers are doing isoelectric focusing or SDS-PAGE examination of the extracts.
		What we've seen from that -- and this is just very initial data -- that in some cases the profiles are similar, and in some cases bands are missing or lose definition.  But again, these are for unstandardized extracts.  So the relevance is not known yet.
		DR. SAXON:  Those are incredible examples.  I mean, I've been around a long time.  I have never seen a vial that had even that ring at the bottom.  That other one looked like 80 percent flocculent material. 
		Do you have any kind of -- I mean, every vial has a little bit of dust, if you shake it.  Do you have any idea of like a range of what's really been seen?  I mean, so we can get all our ideas around what are we talking about.
		MS. BRIDGEWATER:  Well, yes, that's a good question.  The short ragweed is by far the west offender.
		DR. SAXON;  At what concentration, for example?  When you say is this something at the highest concentration that might be sent out or is this something that we would never see?  Do you know?
		MS. BRIDGEWATER:  No.  I mean, this is a standard short ragweed extract.  That's a final container vial.  So that's how it could be shipped.
		DR. SAXON:  Incredible.  Has anyone ever seen -- Dennis, have you seen any?
		CHAIRMAN OWNBY:  Yes, I think running a large extract lab, you'll see these occasionally.  The question I would have on some of these -- I've seen some that look about as bad as that vial.  Though once you bring them to room temperature, it all disappears.
		Some of these seem to be very temperature labile.  Some of them aren't. 
		DR. SAXON;  I've just never seen any.  I mean, I see lots of them a little bit, you know.  Actually, it's like a cryocrit.  You know, what percent is -- But that one was incredible.  Okay.
		MS. BRIDGEWATER:  Well, I think it really varies.  In some extracts you will only see a few flakes in them perhaps, but the short ragweed, like I said, is the worst offender.
		DR. LEHRER:  Just to pursue that, is this -- do these occur after a certain period of time?  Do they occur immediately, and do you see similar amounts with the same preparations or does that vary?
		MS. BRIDGEWATER:  That is probably a better question for one of the manufacturers.  But, Shirley, did you want to address that?  I mean, I think we're going to get to some of that and, if I don't answer your question, remind me.  Next slide, please.
		Where am I?  Okay.  So here's some -- There's also some current industry efforts to characterize the precipitates in terms of, for example, looking at the potency of the short ragweed, which we just saw, looking at the potency of the final container extract to the initial value to see if there is a change in potency, and for the nonstandardized extracts looking at the protein nitrogen unit pH and phenol also over the shelf life of the product to see what the effect is.  Next slide, please.
		So now I'd like to show you some preliminary data that we've gotten.  As I said before, with the exception of the short ragweed, all the precipitated extracts are nonstandardized.
		So we have to look at some of the other attributes, and these are some preliminary data that we've gotten from manufacturers.  So you can see from the first example the penicillium extract, the comparison between the release and retest of the precipitated extract.  There's a decrease in the pH and the PNU.  
		Then if you look at another example, here this three-weed mix, there's actually an increase in the pH and the PNU.  In some cases -- and I picked this example specifically -- the pH dropped actually to where it was out of specs -- excuse me, out of specifications, and that's why this is in red.  But again -- and here in this final example, you have a decrease in the pH but an actual increase in the PNU.
		So at this point, we don't have a clear picture of what's happening, and we don't have enough data to really say with certainty what the long term effects are.  Next slide, please.
		So here are some data on potency of standardized short ragweed before and after precipitation.  These data were taken from pooled datasets, two sets of 19 lots each.  
		You can see in this example that for lots A, B and C there was a decrease in the antigen E, which is what we measure for potency, while in the last example -- this is D -- there was an increase.
		DR. SAXON:  What is pre and post, pre what and post what?
		MS. BRIDGEWATER:  Pre and post is -- and again this is how the manufacturer sent it to us.
		DR. SAXON:  Oh, I see.  So the manufacturer did this test?
		MS. BRIDGEWATER:  Right.  So the pre is before, I guess, the precipitate either occurred or was removed.  That was the value before, and this is the value after.	
		DR. SAXON:  Okay.  I thought you did it.
		MS. BRIDGEWATER:  No, sorry.  Let me point out, this is just a snapshot in time also.  This is not over the whole shelf life of the product.
		So when we looked at the whole dataset of all 38 values, we did notice that there was a five percent drop in antigen E potency, but the difference was not statistically significant.
		DR. CLAMAN:  These are four different lots from four different manufacturers?
		MS. BRIDGEWATER:  No.  These are actually from the same manufacturer, but they are four different lots.
		DR. CLAMAN:  So I don't see how you can say it's not statistically -- I mean, it's not fair to pool them.
		MS. BRIDGEWATER:  These are just -- Well, would you repeat that?
		DR. CLAMAN:  Well, in Lot C you've got a drop of 30 percent.  
		DR. SAXON:  An increase.
		DR. CLAMAN:  I'm sorry.  No, see, you've got a decrease of 30 percent.
		DR. SAXON:  113 percent.
		DR. CLAMAN:  No, that's a number.
		MS. BRIDGEWATER: Sorry.  That's just the absolute.  That's not the absolute.  That's the value, the antigen E value.  That's not a percentage.  
		DR. CLAMAN:  So I don't think it's fair to pool them and say there was no significant change.
		MS. BRIDGEWATER:  Jay, did you want to respond to that?
		DR. SLATER:  I think Dr. Claman is saying that he thinks there are significant changes.  But on the other hand, this is just four lots.  We need to actually look at more.
		MS. BRIDGEWATER:  Right.  And these are four lots that came from 38 values.  So it's not a complete picture, obviously.  I can't put all 38 up here.
		CHAIRMAN OWNBY:  Well, it really depends on what we're talking about as a significant change.  I think Henry's point is that for extract C it looks like there's certainly a significant loss of potency in that particular lot, whereas overall you haven't achieved statistical significance across all those lots.
		DR. SLATER:  Well, I think that's correct, and I think, you know, there are a couple of different ways to analyze it, and that is how many lots lose a significant amount of potency.  That's a different way of analyzing it.
		MS. BRIDGEWATER:  So what's our current state of knowledge about precipitates in extracts?  Well, generally we can say that the aqueous extracts precipitate far more than their glycerinated counterparts.  As you saw from those pictures, the aqueous short ragweed commonly precipitates.
		We know from data we have collected on inspection that precipitates are a primary cause of physician complaints, particularly as a visual appearance complaint, and product returns.
		They do not appear to be caused by microbial contamination.  On this last point, I think this is something we heard from industry, actually, that the extraction ratio or the concentration of the extracts and possibly the phenol may contribute to the precipitation problem.  Next slide, please.
		So here's what our current knowledge gaps are, and these are areas in which we would really like to develop some additional data:  The physicochemical composition of the precipitates themselves; the long term effect on the potency of the extracts, and again we are only talking about short ragweed here.
		Here's an area that we could really use some discussion on:  How precipitated extracts are treated in clinical practice?  Are they recognized by the people that are administering the shot, and how are they treated?  Are they administered as is?  Are they decanted and administered or just returned?
		Continuing:  Is there a higher risk for adverse events if the precipitate is injected?  Now we need to remember, most of our adverse events, of course, come in via the MedWatch system, which is voluntary for physicians and passive.  In fact, we receive a very limited number of adverse event reports on allergenic extracts, considering the total number of shots that are administered per year.
		We need to remember also that, because they are passively collected, they can't be used to link cause and effect.  Rather, the MedWatch system is used as a signal system to identify medical events or areas that need to be actively studied. 
		Finally, this is a problem frequently for allergenic extracts.  The information submitted is usually incomplete.  Many times you don't even have identification of the manufacturer of the product or the lot numbers.  
		So the current research area that industry is undergoing at the moment:  Again, physicochemical composition of the precipitate; some potency instability studies of precipitated standardized; and evaluation of pH, phenol and PNU of precipitated unstandardized extracts over the shelf life of the product.
		So our current regulatory position with respect to precipitated extracts is we have asked the manufacturers not to ship final containers in which the precipitates are visible.  We have asked them to develop an in-house quality control program to identify and describe the precipitates, validation  of any reprocessing procedures that they may perform to remove the precipitate, and right now we are also working on modification of the labeling to address this issue.
		Finally, as Mary Malarkey said earlier this morning, we've asked them to submit the biological product deviation reports, formerly known as error and accident reports, on all precipitated lots to CBER.
		Before we move on to the discussion and to the next presentation, let me just summarize the issues.  Again, we do know that precipitates occur in allergenic extracts.  We know that the aqueous extracts precipitate more than the glycerinated extracts, and following that, almost all of the precipitated extracts are for unstandardized products.
		We do have some knowledge gaps on precipitated products, and we are collaborating with industry to fill these gaps.  Really, in the absence of data, we've tried to take a prudent regulatory approach to this.
		My next slide is the committee discussion points.  I wonder -- It might be better if we hear from industry's perspective first before we come back to this.  Shirley?
		CHAIRMAN OWNBY:  Any burning questions, since we asked Ms. Williamson to present a discussion from the APMA point of view?  Okay.
		MS. WILLIAMSON:  Again, I think I introduced myself a little bit earlier, but I'm Shirley Williamson, and I'm representing actually the Allergen Products Manufacturers Association as the president of that organization.  
		I'd like to take this opportunity to thank Dr. Slater and whoever else was involved for giving us this opportunity to make this presentation.
		The first thing, I want to start by saying, as I go through this, you will be sitting there thinking, well, wait a minute, I just heard this.  I want to tell you from the outset, Jennifer and I did not know what each other was going to say in our presentations. 
		You will probably also figure that, if we had collaborated, we would have done a better job of not repeating each other.  But I think it does point to the fact that, in fact, industry and CBER, FDA personnel are having discussions about this, working together, and I think this will show we are pretty much working with the same information base as a starting point.
		Precipitation for our definition, as Jennifer pointed out, in allergenic extracts refers to the phenomena whereby intrinsic and initially soluble components from the solution become insoluble and form a visibly evident sediment over time.  
		This contrast was what we refer to as a particulate, which is an extrinsic particle that is present in the solution, generally, I think, of like a fiber, a piece of paper fiber, a small glass particle, something like that being a particulate.  
		The history, another review of history:  Precipitation has been recognized by the industry as product characteristic for over 30 years.  The reason I picked 30 years is that, actually, within the industry there are a couple of individuals that have been with their companies for 30 years, and they remember precipitate being a problem when they started their careers.  It probably existed long before that, but that's why we kind of picked the 30 year time point.
		Over these years, manufacturers have attempted to characterize or solve the problem of precipitate with very limited progress and even less success.
		As you heard earlier, precipitation was raised to a compliance level beginning with the observations that were made on the 483 Forms presented to several manufacturers during their inspections.  Resulting from this and concern about what was happening, representatives from several of the manufacturers met with CBER personnel in October of 200 to discuss the issues that were involved.
		Types of products:  Again, you have heard some of this, but essentially all types of allergenic extracts reportedly will form precipitate. However, we do tend to see precipitation most commonly in the aqueous or the non-glycerinated extracts, more highly concentrated extracts, the one to 10, the one to 20, 40,000 PNU.  But it's also been observed and reported in more dilute forms.
		Many pollen extracts will form a precipitate, but it has been noted by some manufacturers anyway that genera do seem to be more prone than others.
		Visual appearance:  I think Jennifer actually found one more or two more categories than I did, but the precipitate appearance will vary oftentimes, depending upon the product.  In some cases it is described as filamentous or like small fibers, stringlike particles in the extract; granular, sandlike, little particles that tend to more settle on the bottom; crystalline which are more angular, and they are generally clear and often they will actually be suspended within the extract; cloudy, which is diffused rather than distinct particles with kind of a hazy appearance; flaky, flat pieces, often have some color or sometimes it's a film, either at the top of the extract or on the bottom.
		An interesting thing that has been observed by most manufacturers is that precipitate may appear in one lot of product manufactured from a given lot of source material, and may not be seen in another lot of product manufactured from that same lot of source material, but manufactured under essentially the same manufacturing conditions.
		DR. SAXON:  If you get it one lot, you get it in all the vials, more or less.
		DR. WILLIAMSON:  Yes.  Now you will see it develop faster.  I mean, for some reason, you might see it in one vial and --
		DR. SAXON:  You might get 1,000 vials, and then you -- and lot -- they will have it, and then the next run-through may not, you are saying?
		DR. WILLIAMSON:  Correct.  One lot of source material may result in several lots of extract that don't form a precipitate, but the next lot of source material, those extracts may.  
		I think what is important with this is the description, the varied appearance that the precipitate will take on, that sometimes you see it, sometimes you don't, tells me more than probably anything else that -- at least my belief, and I think shared by many of us in the industry -- is it's not a single phenomenon.  There's not a single causative factor involved here.
		So that makes the attempt to solve the problem much more difficult, because it probably is a multitude of causative agents.
		So characterization analysis of the precipitate:  Again, as Jennifer pointed out, one of the things the manufacturers have done is examine these for any relationship to microbial contamination, that being our first and foremost concern.
		Some manufacturers use just a microscopic exam of the material.  Others actually do sterility tests.  But in no instance has a manufacturer reported an association of the precipitate with microbial contamination.  It's not to say you couldn't have a precipitated extract become contaminated, but when we have done this direct look-see, it is not microbial contamination.
		Some of the major difficulties in characterizing these precipitates result from the fact that, even though -- and you saw the example there that Jennifer had of this gross amount.  When you try to collect that material, you are actually surprised at how little you get to be able to do anything with.
		Then even if you were able to collect an amount of this, obviously, you want the precipitate clean if you are going to do any characterization studies because if it is, quote, "contaminated" with the extract, you may be reaching a wrong conclusion.  You may say, oh, it contains protein, and in fact you are looking or seeing the protein from the extract.
		A number of manufacturers that have attempted to do this have actually found that as you try to clean the extract to -- or the precipitate to remove it from the extract, it redissolves in your cleaning solvent.  Then there you are.
		A few limited characterization results have been obtained.  What little data there are suggest that the precipitates consist of chemicals derived from the source material, such as some polyphenols or flavinoids that will agglomerate proteins or agglomerated protein carbohydrates that form sediment.  
		In one instance, it was reported that some crystalline appearing precipitate was identified as calcium oxalate.  But that pretty much sums it up.  There is very little data.
		So again a concern for us is what is the effect of precipitate on the suitability of use of these products.  Foremost in all of our minds, be it the industry, be it the FDA, be it the consumer, is the safety.
		Again, no controlled studies have been performed, and I'm not even sure exactly how you would want or could do them.  But none of the manufacturers have reported any adverse events or patient safety issues associated with the presence of precipitate in extracts.
		Jennifer pointed out very clearly in what she said about this that it's not a very good system for saying there is no problem, because oftentimes again the reports are coming from physicians' offices.  They may not even be paying attention to the visual characteristics of the extract or in some cases I may be more concerned about the extract that I was using prior to the one that caused the adverse event.  That may be where the problem was.
		So the point of this is just that we don't have any evidence that says there is a safety issue, but we also acknowledge that the database here is also quite limited.
		We have also looked at the potency composition and protein content, as Jennifer mentioned.  At the meeting in October, there were some data presented, and there's been a little more data generated since then.  
		This again just reiterates -- This information was presented at this meeting in October.  Then go to the next one.  Standardized products, obviously, would be the best ones to kind of give us a better handle on what is happening to these extracts as they precipitate.  
		The ironic thing is it's the standardized extracts, at least the grasses and the mites, seem to be the least likely to precipitate.  However, one manufacturer did have two lots of Timothy that had precipitated some one to two years after the date of manufacture.  
		They took a look at the ELISA potency.  This was at the time of release.  This was -- They reassayed the extract with the precipitate still present.  They assayed the supernatant after they centrifuged it to remove the precipitate, and they also assayed the supernatant when the precipitate just settled by gravity.  
		Again, you can talk and are these numbers different or not.  However, all of these values do fall within the limit for labeled potency that these products were labeled at.
		Then there's some data on nonstandardized extracts, which again were presented at the October meeting, some data generated by both Greer Laboratories and Hollister Stier.
		This one is very busy.  I don't expect you to get much from the exact values.  However, this was a look-see at all the different extracts that were looked at.  They looked at the SDS-PAGE and concluded that before and after removal of precipitate, the profiles were comparable.
		The resulting phenol content was still within the release limit.  pH didn't seem to take any major shifts.  PNU -- again, some went up; some went down.  Typically, I would say the variability of a NU assay is somewhere in the 15 to 20 percent range.  So none of these really feel outside of what you just would see from normal variability.
		Another company, and again this was Hollister Stier -- took a look at some vial container products that had precipitated.  Again, all negative for the presence of microbial contamination.  SDS-PAGE profile did find in two products, a bottle brush and an English Plantain, that there was perhaps a loss of a band up at the 180 kiloDalton range there, and then loss of band definition of 40 kiloDalton.
		Also, they reported on a lot of AP Dog that we were able to quantitate the can F I.  With the precipitate two, there were 251 units per mil.  After the precipitate was removed by filtration, there were 253.
		These are just some examples of the SDS-PAGE profiles that, again, were shown at the October meeting, the p indicating the precipitate was still  present, the n meaning it had been removed, no precipitate.  Again, removal in this case was done via filtration, sterile filtration for sterilizing membrane.
		Since that time, since the October meeting, a little bit more data have been presented or provided by manufacturers.  This is an example, a company that looked at the protein content before reprocessing and after.  Again, you see some -- a couple go up, a couple go down.  This one goes up.
		We will be the first to acknowledge the database are quite limited, but I think the point is that overall everything that we are seeing is suggesting there is no major shift in protein, protein profiles or anything as a result of precip.
		Some additional data from Greer Laboratories, again looking at some reserve samples, some product lots.  In summary, again their conclusion being that in the overall aggregate, the presence of precipitate doesn't affect the overall characteristics of the extract.
		Some additional data since October on comparing PNU, pre-filtration,  post-filtration:  Again, a few higher, no change, a little lower, a little higher, all again within what I would consider the range of normal variability.
		Again just some more examples of different SDS-PAGE profiles on different products, again visually similar.  Again, if you look closely, there may be a few bands that tend to lose definition, but I think your eye will tell you that the precipitated and the nonprecipitated look pretty similar.
		Just some more examples, some with heavier, more predominant protein bands than others.
		Then the last one.  There were several agreements that came out of the meeting with CBER personnel in October.  One of them was that the manufacturers agreed that extracts with any visible precipitate would not be shipped to the customers.
		The second was that the manufacturers agreed to include common verbiage in their product instructions regarding precipitate.
		Third, manufacturers and CBER personnel agreed that attempts to characterize and look at these precipitates would continue and, fourth, to try to standardize the error and accident reporting, which as you heard earlier, is now the biological product deviation report.  
		So current initiatives by the industry resulting from these agreements that were reached in the October meeting:  To inspect the product just prior to shipping to customers, and do not ship the product is precipitate is visible.
		The effects of this initiative on both the industry and the patients include:  There will be product shortages when the manufacturer must discard the product.  They've got their product ready to ship.  They collect the vials to fill an order.  There's precip in it.  The lot will be discarded.  That patient will not receive that order.
		So these shortages will result in a disruption of product supply, which will ultimately affect the treatment regimes that the patients are undergoing who are receiving immunotherapy with these products.
		We all agree, I think, that there will be reduction in product lines as manufacturers will discontinue making these products, the ones that tend to precipitate almost all the time.
		There will be increased cost to the customers to offset the increased losses to the manufacturer, and a move toward the use of glycerinated extracts or more dilute non-glycerinated products.
		The agreement to add the verbiage regarding the presence of precipitate:  There have  been, my understanding is, some discussions between some manufacturers and CBER.  One of the proposals is to add wording in the dosage administration section of the instruction, and there is verbiage given in 21 CFR, Section 201.57 regarding the instructions to look at any product for the presence of discoloration or for particulate or precipitate.
		This will require submission and approval of each insert used by each manufacturer, although I understand there may be ways to get approval of the wording in one, and then provide annual updates or something.  So it may not be every insert needs to be approved, but certainly, the initial one will require preapproval.
		Continued evaluation of products with the existing technology, again including protein content, protein profiles, potency assays when it's a standardized product that precipitates.  That may ultimately expand to include immunoblot so that we can actually look at the allogenic proteins that are being affected as opposed to just the total protein profile.
		One of the other things we talked about was attempting to work with CBER personnel to expand use to new technologies.  This is really in its preliminary discussion stages, hasn't progressed anywhere.
		Then the last one, the ultimate goal is to develop alternate manufacturing methods that would prevent the formation of precipitate.  I feel this is a very long term project as, number one, we would have to do the extensive studies just to determine what we could do to keep the precipitate from forming.  
		Then once we were able to do that, there would still be the studies to show the equivalency, that we haven't affected the product in any way.  In light of the earlier presentation and the number of patients involved in that, that could get quite expensive.  
		That's where we're at.  Again, like I said at the outset, I apologize for repeating so much of what Jennifer said, but again I think it demonstrates that we are all pretty much working with the same knowledge base.
		So there was a couple of questions.  I don't know if they were, in fact, addressed.  
		DR. SAXON:  If I was approaching this scientifically, the first thing I would want to know is how common are precipitates that are visible out of the thousands of lots that are made.  I still don't understand.  Is this one percent, 50 percent?  How big is the issue in that regard?  Can you give us any idea?
		DR. WILLIAMSON:  Yes.  We did a survey -- Again, I can speak on behalf of my company.  I can't on the total industry, but I don't think we are probably that different.
		DR. SAXON: What's the number?
		DR. WILLIAMSON:  It was about 7.5 percent of the products.
		DR. SAXON:  So let's say ten percent.  Ten percent of extracts have a visible precipitate.
		Now the second question to ask:  What is the amount of that precipitate?  That's easy as pie.  If you haven't done it, I'm stunned.  You take a little capillary tube, and you spin it, and you measure the amount.	
		We don't know -- It looks like 80 percent.  I understand what you're saying.  It could be one percent.  So someone should take the ten percent of lots, get a little capillary tube, just spin them, and you measure the quantity so we know how frequent it is, the quantity it occurs; because this whole thing may be a non-issue.  
		I mean, the whole thing may be a non-issue.  If it turns out that the potency is no different, the quantity turns out to be one percent of material that when you spin it down, or less, and there's no difference, and you could prospectively before you throw this whole thing out -- We don't see short ragweed.  We don't have it California.  That's why, I guess, I've never seen those.  
		Yes, we don't have short ragweed in California.  No.  it's another reason to move there, in spite of the earthquakes.
		What you can say is -- So then before you say you can't send this stuff out -- You know, you have now said, basically, this stuff is bad.  May well be.  But it would be more interesting as a scientist to say, okay, we know this lot has precipitate.  We've measured it.  It's two percent.  We spun it down, and prospectively look what happens to that lot, versus prospectively to its cousin lot.  
		Ask the manufacturer to do a prospective, active surveillance.  You could actually solve the issue.  What you're going to do now is say the issue is bad, we must get rid of it.  And it's okay, but you're going to add extra expense, and it may be a waste of time.  You may be chasing nothing.
		So I think getting some data would be better than chasing something that may turn out to be irrelevant.
		DR. SLATER:  May I comment?  I certainly don't think anyone is going to disagree that we need a considerably larger body of data on this question.  I'm not really sure that the point was clearly made, and that is that we are actually rather ignorant about the effect that this might have on potency.
		The only extracts for which this happens with any frequency at all for which we have any valid potency measure is short ragweed.
		DR. SAXON:  You mean her data is invalid.  She just showed us a bunch of data.  I just -- I heard  what you said, but you just showed me a whole bunch of other data. 
		DR. SLATER:  Sure.  There is a bunch of other data about PNUs per ml.  There is data bout pH.
		DR. SAXON:  It was a huge -- It was 20 percent of the protein was precipitating.  It should have been going down.
		DR. SLATER:  Well, but we know very well that the correlation between PNUs per ml and potency is extremely poor.  In fact, you could have a dramatic decrease in the actual allergen content and preserve PNUs per ml.  
		DR. SAXON:  Fine, and you could do that with or without precipitates.
		DR. SLATER:  That's exactly correct.  But for a large portion of our extracts that are precipitating, which is, you know, according to industry data, something on the order of one in ten to one in 20 vials, it is actually quite remarkable -- It's not remarkable.  It is a testimony to the importance of standardization to study problems such as these that we are essentially having a problem because only one of the allergens that is standardized -- only one of the allergens that has this problem is standardized for which we have a measure.
		Furthermore, Dr. Claman pointed out that, even though as you pool the data you may not have a statistically significant difference, for some extracts it may be more significant than for others.
		DR. SAXON:  Yes, but again, you didn't do the controls of all the other lots that may have gone down that weren't precipitated.  I mean --
		DR. SLATER:  I just must differ with you about the concept of not worrying about extracts that have a relatively small precipitate crit.  I actually think that I worry a lot less about that slide that Jennifer showed that two-thirds of the bottle was full of precipitate, because -- let's be honest about it -- nobody would inject that into anybody.  So that would be one that I'm not worried about patient safety, because I don't think that vial would have ever gotten near a patient.
		I am much more concerned about the relatively more subtle precipitates, the kinds that we don't really know what the impact is and that might, in fact, be getting injected.  
		One of the things that I would really like to ask the Committee to spend at least a few minutes on is I would really like to find out what the sense is of how often you have actually seen this.  
		I must tell you that, when I first heard about it, having just come from clinical practice a short time before, I had never seen one of these, and I was horrified at the numbers that I was seeing, because it was clear that there were extracts that I was not aware that these actually had precipitates in them.
		So I would really be interested in finding out what the Committee thinks of this.
		DR. UMETSU:  Actually, we don't use ragweed either, because I'm in California.  I'm interested in finding out from -- The question is what percent of the ragweed extracts at Hollister Stier have precipitates?
		You gave us seven percent of all extracts, but what about ragweed?  What percent of short ragweed has precipitates?
		DR. WILLIAMSON:  I am trying to think here if I can -- drawing back on what I could say for experience.  One of the things with the ragweed is we do have to do stability study on ragweed, and formation of precipitate is one of the parameters that are examined throughout these stability studies.
		In reviewing that, I was in some ways surprised to note that at least the products that we have been studying on stability -- and we do one lot a year of glycerinated, one lot of non-glyce -- that again the glycerinated in our stability studies we hadn't see any.  
		The non-glyce will, but oftentimes -- Now the non-glyce short ragweed also has a very short shelf life.  It only gets 18 months total from the date the antigen E assay is initiated.
		So by the time we get the product manufactured, through the process, tested, released by CBER, there is really only several months that we have left to be able to get that product out the door, and then to give the end user at least a year's dating.
		Now we have seen in our stability studies occasions where that precipitate was present and showed up right at about that 18 month time point, and then beyond.  But I was surprised that in our stability studies we didn't see it that often.  However, we are now, again as agreed to in that October meeting, doing the visual inspection of all the extracts prior to shipment, and we are seeing a higher incidence of the products right out of our stock.
		I don't know the exact number, but I don't sense that it is much different than the approximate ten percent that we were talking about.
		DR. UMETSU:  But you mentioned that it's much higher with ragweed versus, say, other types of -- other antigens, or somebody said that.
		DR. WILLIAMSON:  I think again one of the questions that was asked was, well, how many do you see.  what we looked at was we looked at all the lots that we had in our inventory at the time we did this survey.
		We had approximately 1900 lots of product.  Now that could have been six lots of short ragweed and ten lots of this and so on.  Out of those 1900 lots, that's where we had the 7.5 percent of them showed precipitate, presence of precipitate when we did our survey.
		So how many lots of short ragweed out of all the lots that we would make in a given time -- and again, the non-glycerinated is the one that is going to precipitate.  Jennifer, I don't know.  You picture, I assume, was non-glyce, and really our observations of precip in glycerinated short ragweed is virtually none.
		MS. BRIDGEWATER:  Right.
		DR. SAXON:  I want to correct one thing, Jay.  I don't care if it's a lot or a little.  I'm not worried.  What I object to is no data.  It's sitting there in the vial.  Tell me how much is in there, and then other than spinning it, I bet if you go to a non-visual technique like a big light scatter, you could actually quantitate it.  
		You could quantitate it in another 20 percent, because there's things you can't see with your eye.  So I'm just saying you should get data.  Just looking at vials and guessing -- get data on what percent of vials have it by light scatter.  I mean, that's where I would start, with data.
		I think at the moment it sounds to me like anecdotes and anecdotes.
		DR. SOTO-AGUILAR:  I would like to ask, are they light sensitive or just temperature sensitive?  What makes them precipitate?
		DR. WILLIAMSON:  That was one of the things I pointed out.  That's a question we just don't yet.  For the most part, our extracts, once they are packaged -- and we don't package them unless they are precipitate free -- they are put in the boxes.  they are put in shipper boxes.  So they are protected from light at that time, but then we do see precip down the road.
		Temperature: Again, from start to finish there are processing.  Our extracts are maintained at one to five except for short durations when they are out actually being filled into final container.  So two to five or one to five refrigerated temperature certainly results in the formation of this precipitate, although some of the studies we have done where we have put product at room temperature as an accelerated condition will precipitate sooner.
		So I think there's some temperature dependence, but refrigeration certainly doesn't keep it from forming.
		DR. SOTO-AGUILAR:  And how about pH?  Is there any optimal pH?
		DR. WILLIAMSON:  Not that I'm aware of that has been developed.  That's probably one of the first things that could be looked at, at least in the short term, although I think a couple of manufacturers have done some preliminary studies that haven't suggested that that's the answer.
		DR. CLAMAN:  One would suspect -- As a non-physical chemist, one would suspect that these are like-like aggregates and homodimers or multimers, and that the presence of the glycerine just decreases the chance for two or more molecules to get together.
		Now what about -- Aside from warming them up, which I suspect would do something to decrease the precipitate -- maybe not eliminate it -- what about the use of detergents?  What about doing Western blots on precipitates versus soluble material from the same vial, etcetera, etcetera?
		You know, like gamma globulin -- Gamma globulin is a sticky molecule.  It sticks to itself.  Maybe short ragweed is a sticky molecule; it sticks to itself.  
		DR. WILLIAMSON:  And I think those are potential things to look at, but as I had pointed out earlier, one of the biggest difficulties is, even when it looks like you have a lot, by the time you try to get it clean so that you're comfortable you're not getting anything still there from the extract itself, and then try to analyze it, either you've lost it all through the cleaning or it will actually at that concentration redissolve.
		DR. CLAMAN:  That's good news, too.
		CHAIRMAN OWNBY:  Well, maybe the idea shouldn't be to clean it, because if this was really an aggregate of some component, you ought to see merit concentration shift, even if you didn't have -- you know, even if there was still some contamination left from the rest of the extract.
		I think that we are all pleading with the idea that we need some very basic data.  I mean, I think it would be interesting to know just whether this is a time phenomenon, whether now that you are examining all of your lots before you ship them, how many of them still come back, because I think most of us, the first thing when you saw a vial that was precipitated, you just called up the company and said, hey, you know, I'm sending you this one back because it's got a precipitate.
		You know, are you still getting those back that were clear when they shipped, and can you learn anything about the shipping conditions?  You know, these all come back for winter shipments or all come back from summer shipments or some of those other basic things.  Then just the physicochemical properties of the material that's there.
		DR. WILLIAMSON:  In answer to your question, he said, well, you still get them back.  At Hollister-Stier we implemented our visual check at the time of shipment right at the first of the year, January 2.  So we haven't been doing this that long, but that is certainly a parameter we will need to examine and see if there is a correlation, if in fact  we do see a reduction in returns now that we are sending them out clear or if the level stays the same, which means they are now precipitating once they leave our hands.
		DR. SAXON:  The real issue is safety.  I mean, Jay is after safety.  Right?  One way you look at that is, if it's lost potency, it's not as good anymore.  You can answer that pretty straightforward.  You should be able to, by looking at -- 
		The other issue which you're going to lose the opportunity to look at is I guess you stop -- you used to send them out when they were -- or has the industry sent them out previously when they had precipitate in them?  Okay.  Nothing wrong with that, and I'm not horrified.  I don't care.
		My point is it may be no difference.  But when you send them out, you still have an opportunity to look prospectively at what happens.  The alert system will never work.  No one reports allergy adverse events, because they are part of the natural history of the problem.  So we don't report every time a patient gets a swollen arm.  Right?  But if you took a lot that you knew had precipitate in it or had lots of returns, you should have an active surveillance.
		If it's not a problem, then it's not -- That's the only way you're going to get safety data on precipitate versus non-precipitated lots.  So you've had lots of it gone out, but you're going to have to actively surveil, not passively surveil.
		If it was my experiment, I would do the experiment.  I would get a lot that had precipitate or maybe if you can't do precipitate, you do a light test now, because you know it's got more material that you can't see, but you know it's got more than a cleaner lot.  If there's no difference, there's no difference.  The data will be the data.
		DR. UMETSU:  Clearly, there needs to be more data.  I would also suggest that perhaps the precipitated lots would be actually more efficacious.
		DR. CLAMAN:  Absolutely.  
		DR. UMETSU:  So you may actually get a better product when it precipitates.
		DR. SLATER:  I'm sorry.  Are you proposing that as a possibility?
		DR. SAXON:  Sure.
		DR. UMETSU:  I think that, if you look at it immunologically, if it's homodimers or homomultimers, those precipitated antigens would be more immunogenic, if that's what you are trying to do.  If you're trying to immunize, that's what would happen.
		DR. CLAMAN:  You don't agree?
		DR. SLATER:  I am not saying it is.  I'm just saying we need more data.  
		DR. CLAMAN:  there's a long history of increased potency of aggregated immunogens versus soluble immunogens.  This may not be what you're after.  We're not really sure.  But I agree with Dale 100 percent.
		DR. SAXON:  Before you throw the whole thing out --
		DR. CLAMAN:  That's right.  That's what I'm saying.  You need data.
		MR. SAUSVILLE:  Can I say something here?  I'm Bob Sausville with the Office of Compliance at Center for Biologics.
		I think our concern would be that a particulate is not -- the precipitate is not a particulate.  I mean, can you tell by looking at it that it's not microbial contamination and that it's only something that's some out of solution?  
		I mean, you've made reference to the fact that it could be microbial contamination or have microbial contamination with it.  I mean, I think our concern would be, just because you have this particulate or -- precipitate -- Anyway, what I'm trying to say is you can't be sure that it's only one thing and it's not a combination of things just by looking at it.
		DR. WILLIAMSON:  And I'll have to acknowledge that's true.  Just visually, can a nonexperienced -- well, even experienced look at it and say, oh, no, that's normal particulate -- precipitate.
		In some instances, I would venture to say you probably could, but not in all.  However, I will also say that from experience I have had the occasion to see a couple of lots that were contaminated microbially that were perfectly clear.
		So, you know, a clinic could have a perfectly clear extract that is microbially contaminated.  So whether or not it's fair to say, well, they have been using these extracts for years -- doctors have -- that have this precipitate, and again I am not aware of any instance ever that was reported for a bacterial infection associated with an injection from an allergenic extract and then let alone associated with precip.  But that is a point, that visually you wouldn't be able to tell.
		MR. SAUSVILLE:  Well, I think from a safety standpoint, you know, from my perspective, that's one of the most important.  If you can't make that distinction, then you can't just send it out without any concern.  
		DR. CLAMAN:  I have a really dumb question.  You have a bottle of extract, short ragweed.  If there were some way to --I guess you could on a column -- remove all the immunoreactive material, all the short ragweed antigens on a column and analyze what was left, what's left?  What percentage of what's in the bottle are, in fact, bioreactive ragweed antigens, and what percentage in the bottle are plant proteins that have nothing to do with allergenicity?
		I have no idea.  I never thought about it before.
		DR. SLATER:  No, I don't know.  
		CHAIRMAN OWNBY:  Please use the microphone, Dr. Pasteur.
		PARTICIPANT:  Yes.  We actually do have measurements of the protein content versus the potency, and we've seen that for extracts that have the same relative potency, the protein content can vary by over a factor of ten.
		So that's not a total answer to your question, but the fact is easily a factor of ten.  
		As long as I'm standing up, a little earlier you had made the point that, if you gave someone the precipitate, that would be sort of super-potent or, you know, extra good, so to speak.
		Well, the problem is, if it's -- it could also be super-potent, and in that sense it could actually be a very dangerous thing to actually give.  So it doesn't necessarily mean that it's sort of good if it's more potent.
		DR. SAXON:  It's going to be less than ten percent.  I mean, at least for things like -- when they looked at Amb a 1 -- Was it Amb a 1?  The studies on the graph -- so that's why you get this huge protein change.  But if they were aggregated, the allergen were aggregated, it wouldn't be more dangerous.  Right, Dale?  It would be safer, because it would be aggregated anyway.  So it wouldn't make it more dangerous to have aggregated immunogens.
		I just think you don't know, and I --
		DR. CLAMAN:  So what you are saying is it's likely that 90 percent of what's in the bottle is irrelevant to our subspecialty.
		DR. SAXON:  Yes.
		DR. CLAMAN:  And therefore, all other things being equal, which they never are, the chances are nine out of ten that the precipitate doesn't have anything to do with its immunogenicity either.
		DR. SAXON:  Right.  And the data will be the data.  But, Henry -- I mean, that's why people wanted to then clone antigens, but then it became, hell, you got to clone ten antigens or 12 and then reconstitute.  It ain't worth the trouble.  Use the natural stuff.  It has a bunch of carrier protein with it.
		I just think it's an unknown issue in making any kind of decision.  I mean, it looks cleaner.  Is it better?  Is it worse?  I don't know.
		CHAIRMAN OWNBY:  It just seems like there's so many other components in here that we normally don't think about.  We think about the allergens as proteins, but there are a lot of pigments.  There are a lot of phenols.  There are a lot of other compounds that potentially could be part of this.
		I wonder about some of these secondary effects we don't normally think about, like the pH of the glass that this is going into and the surface properties that you get into play.  It seems to me that this would drive a physical chemist wild for a while to try to figure out where these are coming from.
		DR. SAXON:  Wouldn't you be more interested to know if they were even worth worrying about?  See, that's my concern, because if they are not worth worrying about, I wouldn't waste my physical chemist's time and money worrying about them.  If they are a problem, then they shouldn't be used.
		CHAIRMAN OWNBY:  Well, if you are throwing away whole batches of extract, it becomes a pricy problem.  It needs a solution.
		The one thing we haven't touched on is what do physicians do in practice when they get these?  I mean, if they see it initially, I think most people send it back.  The question is what happens after it's in a treatment set and you see precipitates, what do physicians do?
		MS. BRIDGEWATER:  Shirley, I'm wondering if you could comment on that a little, about percentage of returns you get versus how much product you send out.
		DR. WILLIAMSON:  Again, we track our returns.  Anytime we get a return for a precipitate, we have a procedure that we follow.  One of them is looking at how many other vials from that lot were manufactured and have been shipped, and especially now with the biological product deviation report that's a part of that report.
		To be honest, what I've observed in evaluating these reports is we may have had a lot of 50 vials from a given extract and gotten two of them back for precip, and never heard on the other 48.  Yet in my experience, again I have seen that it's very unlikely that out of a lot of 50 you would only have two precipitate.  If two precipitate, they have all gone.
		So to be honest, I think in the majority of the cases -- and I think it's a matter of -- they are used to it.  It's a problem, as I said, we as an industry -- and not just us but the medical community also that uses these products -- have seen and recognized.  
		It used to be that, you know, people comment -- or the gal that handled the technical service reports would say, oh, must have a new nurse in Dr. So and So's office, because I'm getting complaints on precip.  And it would be -- yes, it would be cyclic.  But you kind of, oh, they're not used to seeing this, and so they are returning it.  Once they get used to it, we won't hear from them.
		DR. SLATER:  So, Shirley, would you conclude form this that the physicians even know?
		DR. SAXON:  Why don't you ask at the AAII meeting?  The FDA can get in touch with people that order them, and find out.  
		See, the other thing that concerns me, Jay, about this is that the concept at the human eye level of precip means something.  I tell you, if you use optical techniques, you'll find 30 or 40 percent.  Then what are you to say?  Well, we've regulated at the level of the human eye?
		You know, maybe you should regulate at a much more objective level, but you can set it by flocculation.  So before you get into that whole mess, I think you ought to figure out what's really going on.
		DR. CLAMAN:  See, you missed the point, Andy.
		DR. SAXON:  Yes, I always do.
		DR. CLAMAN:  Our Chairman said it and didn't realize that he said it.  He said this is something that needs a solution.
		DR. SAXON:  Right.  
		DR. SLATER:  If I could just come back to a point that was made a few minutes ago about the aggregates perhaps eliciting better immune responses, just recall that unless the aggregate happens to be made of the target allergen you're interested in, you may now be generating a good antigen for an immune response that is undesirable or perhaps competitive.
		DR. CLAMAN:  I said it mostly to be provocative.  I think we ought to find out what these precipitates are made of.
		DR. SLATER:  but I think most critically is that the appearance of the precipitates is an uncontrolled phenomenon.  It's a phenomenon on which, even within a given product line at a given manufacturer, may be relatively inconstant in terms of its degree and in terms of its composition.
		I mean, I think it should be stated explicitly that the precipitate in product A is almost certainly a different molecular entity than the precipitate in product B and, for all we know, precipitates within product A may be variable in terms of their precise composition.
		So I think it has to get into the record that we can't sort of accept this degree of uncertainty as a neutral event without knowing more about it.
		DR. CLAMAN;  I agree.  You could be on very weak ground if you ignored it.  
		DR. UMETSU:  Yes, I would agree.  When I mentioned that it might be more immunogenic, I'm just saying that we need more data.  We need a lot more data.  We need to find out what is in the precipitates and what causes them to precipitate in some lots, some vials, and not others.
		DR. SAXON:  I am uncomfortable.  I agree, but it's been five months, October, November, December, January, February, March.  This is six months, and this is what we know.  It's nothing.
		I mean, again, you could go and start measuring the amounts of this in ways and have data within a month.  It seems like this has been going on for six months.  I'll be gone and this will be going on in four years, and you still won't know anything.  There's no plan.
		CHAIRMAN OWNBY:  Are you going to propose a plan for us, Andy?
		DR. SAXON:  No.  I think Jay raised this thing.  They ought to have -- He should come back next time with a plan and some data.  I'd tell the manufacturers, spin it down, give me numbers, get some data together that they think is believable, and see if it is an issue or not an issue before they start making decisions based on anecdotes -- anyone makes decisions.  It's definitely of concern to all of us.
		CHAIRMAN OWNBY:  Jay, it would also seem not that oppressive to ask the manufacturers at one of the professional meetings to ask physicians and nurses who handle these what they do with them.  How many of them look at their extracts.  If they see something, what do make a decision?
		I think we've heard suggestions that, oh, well, once they are used to it, they just go ahead and use the stuff, even though they see it's a precipitate, because they don't consider it a problem, and others that may well be discarding all of this or sending it back to the manufacturer in some variable level.
		DR. SAXON:  Now you've got the manufacturers throwing it out.  Right?  So maybe they should send it to you instead of throwing it out, and you can measure the precipitate amounts.  I mean, that would be the very first thing to do.
		DR. CLAMAN:  I think this is appropriate both for the Practice Standards Committee or, as far as the Academy goes, for the State and Regional Societies.  They love this kind of thing, and it's very practical, and they could come up with some information probably pretty fast.
		DR. UMETSU:  One could also do some mouse studies to compare the precipitate version versus the unprecipitated version to get an idea of immunogenicity.
		DR. SOTO-AGUILAR:  I have just one question.  You said that the precipitate might look large, but when you measure it, the volume is negligible or is very small.
		Is that because the cloudiness is occurring at the glass side, in contact with the glass?  So it makes it look larger, but in fact the volume is not that large.
		DR. WILLIAMSON:  I think it may be related to several things.  Number one, as you're looking through a volume, even though it looks like there's a lot there, it may be that there's some distortion through the class, but also especially the flocculent ones are the ones that are the most surprising.
		You look at them and you think, oh, my goodness, there's a whole bunch of stuff in there.  We're going to get, you know, layer when we centrifuge this down.  But once you get it compacted down, the liquid is removed from it, it just isn't that much there.  And whether it's illusion or just the fact that when it becomes diffuse, scattered throughout the solution, it appears to be more there than there actually is.
		Just one question, if I may.  The suggestion was put forth for the industry to poll physicians such as at the Academy meeting.  I don't think the industry would have any objection.  We could certainly set up something like that.  But I guess my mind goes to the next step.  What would you do with that information?  How would that impact where we're at right now and how we would assess potential impact or what do we do with these products in the future?
		DR. SAXON:  I don't think just polling them like in March is going to help, because it's tremendously biased.  I think better is you've stopped sending precipitate out -- precipitated visually in January?  I don't know what the -- But if that's happened, then you -- what you want to do is prospectively.
		You've got to have a good study, not anecdote.  You've got to prospectively tell your customers to look for it over the year prospectively and see what you're getting, because otherwise it's what do I remember.  But if you're going to actually set it up to prospectively look at it, say we will be sending vials; please notify us if you see this.
		You don't have to say send it back, because some of them -- you know, you'll find out what their custom is.  I mean, tell us what you do with it.  Then you'll find out who keeps it and who doesn't.  But it needs to be done prospectively.  Just sort of a questionnaire is anecdote.
		MS. LIBERA:  Do I understand that there's no -- you haven't made any correlation between adverse effects on patients?
		DR. SLATER:  No.
		DR. SAXON:  They don't get enough reports on that to make that meaningful, I understand.  Right?  People get swollen arms all the time.  So who would know.
		MS. LIBERA:  Maybe that's why immunotherapy takes so long.
		DR. SOTO-AGUILAR:  Another possibility:  If you use a vial that's been used, is open already, has been interfered at times with needles, could air, entry of air into the vial confer some chemical property that changes into a physical property with flocculation or precipitation?
		DR. WILLIAMSON:  Again, that's always a possibility.  That may have some impact on the rate or what exactly forms.  However, we see it in vials that we have filled that have been sealed, that have never been entered.  In fact, when it's still at our facility, that's the form that's it's been in when we see it, and we'll discard the product at that time.
		So I don't -- The mere introduction of air or the mere reduction of volume within the vial is not the sole cause.  As I said earlier, again in the years that I've been involved with this industry and one of my first projects when I started with this company was to look at precip, we have a product that's unique to Hollister-Stier, which is AP Dog.
		I think the studies on the Can f 1 content and a number of other things have certainly demonstrated it is the most potent, but it is very prone to precipitation.
		The only study I think I ever found out was that the only way I ever stopped precip from happening was to make an extract just to study the precip.  It wouldn't precip.  But the characterizations, the differences that you see are so varied.  
		That's why I guess -- and I'll carry a bias with me from the years of experience that I've had, but I truly believe that there's a multitude of factors that are involved of which we haven't identified probably a single one, other than -- I believe concentration is certainly an impact, and in some instances it may be a function of just the solubility.  You've literally reached a saturation point, but I don't believe that's the case with all of them.
		So it is really a difficult problem to wrestle with, to try to solve it, when you -- at least I don't believe there is a single causative factor.
		CHAIRMAN OWNBY:  When you say concentration, also whether or not it's in glycerine.
		DR. WILLIAMSON:  Right.  
		CHAIRMAN OWNBY:  If it's in glycerine, it's much less likely to do it.
		DR. WILLIAMSON:  Correct.  Although there are glycerinated extracts that we will see precip in.
		CHAIRMAN OWNBY:  Okay.  We also have a period here for public comment on this issue, and I was wondering if there is anyone else who wanted to make a comment concerning this.  No one else wants to speak up on precipitates?  
		Okay, any other questions or comments from members of the Committee?
		DR. SLATER:  If I can just summarize this discussion.
		DR. SLATER:  We've been waiting for this, Jay.
		DR. SLATER:  No, I just want to make sure that I'm getting all the messages loud and clear.
		It seemed to me that there was some consensus that we need more data.  Am I correct with that?  And among the data that we are lacking are data on the prevalence of the problem, data on the sort of quantitative severity of the problem on an extract to extract basis, perhaps by somehow quantifying the amount of precipitate either by simple centrifugation or perhaps by other light scattering techniques.
		There is some sense that we could benefit from some animal immunogenicity data comparing extracts that are precipitated and non-precipitated.  I think I heard that there was some need for better physicochemical characterization of the precipitates themselves.
		Then in terms of further data, there was some interest in obtaining at least prospectively some idea of how often physicians encounter this and what they do with it when they do encounter it.
		DR. SAXON:  After January, just because they sort of changed after January, and you might get a confused picture.  Right?
		DR. SLATER:  Is there any other message about studies, because I think one of things we were looking for were kinds of studies and the kind of information that we would need.
		DR. UMETSU:  There might be some usefulness for safety data that correlates with the precipitate or absence of precipitate in terms of reactions.  But how I see the problem is how you're going to get that.  But at least that would help.  That kind of data would help.
		DR. SAXON:  I have a suggestion of how you could get that.  One of the other things you skipped was your work on the potency.  So the manufacturers are going to pull lots with precipitate now.  Right?  So what you've got now, what do you do with them?  Throw them out?  Send them to Jay.  No, no, I'm just kidding.
		What I meant is the concept is you have very limited data that you were saying about potency changes.  I mean, if it doesn't change the potency and doesn't change the safety, then it's not a very great concern.  If it does either of those others, it's of great concern.  Right?
		So you have the opportunity to look -- continue your data on precipitates.  Don't you think that should be done?  You didn't have that on your list is why I mentioned that, Jay.
		DR. SLATER:  It's on my list right now.
		DR. SAXON:  Okay.  The other is, if you are going to send it out to docs and say, okay, we are sending people out -- or materials -- and did you see a precipitate.  Then if you didn't return it, you can then also then go back and look at prospectively the reaction rate in those people versus the people where they, you know, didn't get -- who got another lot that didn't have precipitate or didn't see precipitate.
		I mean, it's not a simple study, by any means, and it's going to take effort and ascertainment and someone to follow up.  It's not a passive system, but that's the only way I could think of.
		The other way was to send out lots with precipitate and lots without precipitate, which you stopped doing now, and then prospectively just canvas all the doctors and find out, and you'll know which ones got precipitate and not.  But you stopped it.
		CHAIRMAN OWNBY:  Shirley, is it my understanding that when you -- Normally, you manufacture lots that are fairly large, several hundred vials at a time.  Is that correct?
		DR. WILLIAMSON:  It depends on the manufacturer.  In our case, we tend to -- after the product has been sterile filtered -- hold it in what we call a stock concentrate form, which are in larger containers, and then dispense into vials.
		CHAIRMAN OWNBY:  Your stock containers are what, 500 ml or several liters?  I mean, what kind of volume are you talking about?
		DR. WILLIAMSON:  They are either 150 or 500 ml bottles that hold the stock concentrate, and our lots size for our final container product will range from 18 to several hundred.
		CHAIRMAN OWNBY:  Okay.  And you are saying that usually, if one of those bottles develops a precipitate, all of them will develop a precipitate?
		DR. WILLIAMSON:  Yes.
		CHAIRMAN OWNBY:  So this seems to be a manufacturing lot phenomenon rather than after the extraction, the filling or handling after that point?
		DR. WILLIAMSON:  That's correct.
		CHAIRMAN OWNBY:  Okay.  And the one thing you've never been able to figure out is what's different between one lot to the next that allows this lot to precipitate and the previous lot didn't?
		DR. WILLIAMSON:  That's correct.
		CHAIRMAN OWNBY:  And most of the time, at least with large things like grasses or rag where you handle a large volume, you're -- probably blending isn't the right term, but you're using multiple lots of mixed pollen so that you try to maintain some stability of your initial material that you then use for extraction.  Is that correct?
		DR. WILLIAMSON:  Okay.
		CHAIRMAN OWNBY:  Well, I mean, you don't use just ragweed from one field one year to produce a lot of extract.  Usually, you have multiple years together.  You don't?
		DR. WILLIAMS:  No.  Again, generally, we'll hold to the first in, first out.  So we will be using up our inventory based on the oldest material first, and collection sizes for pollens can be fairly extensive, and we may get 10,000 grams from a given collection lot.  That in itself will make several lots.
		Then what happens is we get to the point where we have some left over, and it's not enough to make a full size lot.  Then we will go to the next oldest lot, combine those to make the extract.  But it actually is probably more common to see only one or two raw material sources in a given extract lot than it is to see multiples.  
		DR. SAXON:  I have a suggestion for the manufacturers.  It will be interesting to take your material and just do an optical density on them, a light scatter.  You may be able to predict, you know, before they precipitate.  You may have data in there when it comes out, you can say this one's got an optical density of this, it looks clear.  And you say, hey, this lot is going to precipitate.
		Then you will at least follow that.  So if I was in the business, I would do an optical density as soon as they got off that lot.  You may then actually have -- you don't know what's in there yet, but you at least have a predictive test, and it's almost free to run light source through something and measure the light scatter.
		CHAIRMAN OWNBY:  Except you have to document it, keep track of it.
		DR. SAXON:  Just don't tell the FDA.
		DR. WILLIAMSON:  I didn't listen to that.
		CHAIRMAN OWNBY:  Okay.  Are there any other more comments?  Any other items of business or for discussion today?  Jay, did you have anything else you wanted some non-advice on?
		DR. SLATER:  No, I think I've gotten very good advice and lots of it.
		CHAIRMAN OWNBY:  Okay, then I believe we are adjourned.
		DR. FREAS:  I would just like to thank the Committee members again for coming today and, Dr. Ownby, for your brilliant leadership.
		We're going to miss our three departing members, and we'll see you at our next meeting.  Thank you.
		(Whereupon, the foregoing matter went off the record at 3:13 p.m.)