FOOD AND DRUG ADMINISTRATION

Center for Biologics Evaluation and Research

ALLERGENIC PRODUCTS ADVISORY

COMMITTEE MEETING

February 10, 2000

Holiday Inn Bethesda
8120 Wisconsin Avenue
Bethesda, Maryland 20184

		

Proceedings By:

CASET Associates, Ltd.
10201 Lee Highway, Suite 160
Fairfax, Virginia 22030
(703) 352-0091

PARTICIPANTS:

Committee Members:
Dennis R. Ownby, M.D., Chairman
William Freas, Ph.D., Executive Secretary
Gail G. Shapiro, M.D.
Betty B. Wray, M.D.
Henry N. Claman, M.D.
Andrew Saxon, M.D.
Dale T. Umetsu, M.D., Ph.D.
Samuel B. Lehrer, Ph.D.
Dolores Libera
Maria C. Soto-Aguilar, M.D.

Temporary Voting Members:
Daniel Ein, M.D.
Te Piao King, Ph.D.
Nancy Sander

TABLE OF CONTENTS
Page
Opening Remarks	1
Laboratory of Immunobiochemistry	4
   Jay E. Slater, M.D.
Research Report	19
   Jay E. Slater, M.D.
Regulatory Proposals
Potency Limits for Standardized Allergen Vaccines	56
   Richard Pastor, Ph.D.
Allergen Standardization	82
   Jay E. Slater, M.D.
Open Public Hearing	109
Committee Discussion	123

P R O C E E D I N G S	[8:39 a.m.]
	Agenda Item:  Opening Remarks
	DR. FREAS:  Good morning.  I am Bill Freas, the executive secretary for the Allergenic Products Advisory Committee.  I would like to welcome everybody to this, the 16th meeting of this committee.
	Today's entire meeting will be open to the public.  At this time, I would like to go around and introduce the members of the head table to those in the audience.
	I will be starting on the right hand side of the table and I would ask the members to raise their hand not that there is anybody in the back of the room, but if there was somebody in the back in the room, so they could see who you were.
	I will start with Dr. Betty Wray, committee member.  She is professor of pediatrics and medicine, Medical College of Georgia.
	Coming around the table is Dr. Sam Lehrer.  Dr. Lehrer is a new committee member.  Welcome to our committee.  Dr. Lehrer is research professor of medicine, Tulane University Medical School.
	Coming around the table is a former committee member, who is participating as a temporary voting member for today, Dr. T. P. King, associate professor, Rockefeller University.
	Our next committee member is Dr. Gail Shapiro, clinical professor of pediatrics, Northwest Asthma and Allergy Center.
	In front of the podium is Dr. Andrew Saxon, professor of medicine, UCLA School of Medicine.
	Next is our chairman, Dr. Dennis Ownby, professor of pediatrics, Medical College of Georgia.
	At the corner is Dr. Maria Soto-Aguilar, a rheumatologist, allergy and immunologist in private practice from Florida.
	Next is Dr. Henry Claman, Distinguished Professor of Medicine and Immunology, University of Colorado Health Sciences Center.
	Next is another new committee representative, our consumer representative, Dr. Dolores Libera, director of publications, Allergy and Asthma Network and Mothers of Asthmatics, Incorporated, Fairfax, Virginia.
	At the end of the table is Dr. Dale Umetsu, Chief, Division of Allergy and Clinical Immunology, Stanford University.
	Again, thanks to everybody for coming this morning.  I would also like to thank the committee management specialist, Pearline Muckelvene, who is at the table outside, who was responsible for organizing this meeting, the administrative aspect of this meeting.
	I would now like to read into the public record the official conflict of interest statement for this meeting.
	"The following announcement addresses conflict of interest issues associated with this meeting of the Allergenic Products Advisory Committee on February 10, 2000.  Pursuant to the authority granted under the committee charter, the director of the Center for Biologics Evaluation and Research has appointed the following participants as temporary members:  Dr. Daniel Ein, Dr. T. P. King and Ms. Nancy Sander.
	"To determine if any conflicts of interest existed, the agency reviewed the submitted agenda and all relevant financial interests reported by the meeting participants.  As a result of this review, there are no COI disclosures for the public record at this time.
	"In the event 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 so 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.
	Dr. Ownby, I turn the meeting over to you.
	DR. OWNBY:  Thank you.
	I would like to welcome everyone, especially our new members and all of you attending for the first time and for those members, who are staying on as temporary voting members, thank you for all your time and effort on this.
	Hopefully, we can have an open and free discussion.  I don't intend to force any major time constraints on this meeting.  I think we have enough time to adequately cover the agenda.  So, for the committee members, if there are questions or things that come up, please feel free to ask them at the time.
	Our first speaker today is Dr. Jay Slater, chief of the Laboratory of Immunobiochemistry.  We will try to follow the agenda that you already have from here.
	Jay.
	Agenda Item:  Laboratory of Immunobiochemistry
	DR. SLATER:  Dennis, thank you very much.  Welcome to all the committee members.  I appreciate everybody coming from such a long distance to hear us present our work and our questions to you today.
	As you can see from the agenda, we are going to have several parts of this presentation.  In the first part we are going to be talking to you about the operations at the Laboratory of Immunobiochemistry, what we have done operationally over the past year, what we have done in terms of our research activities over the last year.
	In the second part, Dr. Richard Pastor and I will present to you some regulatory proposals that we have that we would like the committee to discuss and give us the committee's opinion on after the break.  These proposals involve expanding our definition of the release limits for lots that are sent to us for certain allergen vaccines, in addition to a formalization of the allergen standardization procedure that we have been working on that we would like to move forward with.
	Finally, we will be talking about some specific new standardization targets that we would like to propose in view of the Department of Health and Human Services recent asthma initiative.  What Dr. Ownby said goes for me as well.  Certainly, if there are questions that come up during the presentation, we can entertain them.  I will also be happy to entertain questions after the presentation as well.
	The Laboratory of Immunobiochemistry has had a very good year.  It has really been a very solid year for the lab in many ways.  Certainly operationally and from a research point of view, we are getting off to a very good start again, building on what has been done before and really expanding in terms of the research program that I described to the committee last year.
	We now have what I would consider to be, at least for the work that we currently have, a full complement of individuals.  I am the lab chief.  I have been in this position for the last year and a half.  Dr. Lyudmila Soldatova is a visiting scientist.  She has been with the laboratory for the past two and a half years.  She actually had been a Orise(?) Fellow before that time, but just since the summer, she was converted to a visiting scientist position.
	We have four biologists now working in the laboratory.  Our senior biologist is Maneesha Solanki.  She has been with the lab for about four years.  Clearly, she is the most experienced of all of us and has continued to be a terrific team member and a terrific research for the rest of us.
	We have two very new biologists in the lab.  Kristin Morrow came to us in August from the University of South Florida.  Kristin has a master's degree in microbiology with a special emphasis in molecular genetics and we are really planning on using her expertise extensively.
	In addition, Melissa Catena came to us just a few weeks later.  She has a master's degree and came to us from the University of Maryland.  Her area of study had been in animal and avian studies, with a special interest in endocrinology.  Again, she has been heavily involved in the scientific research in the laboratory as well.
	Mona Febus is a microbiologist, who actually has been with the Center of Biologics for many years, most recently working in the Laboratory of Biophysics, but she has transferred over to the Laboratory of Immunobiochemistry and will be assuming large amount of our lot release activity responsibilities.
	Beth Paupore, who had been full time when I spoke to you last year and had come to FDA with me from Children's Hospital, has now decided that she wanted to learn more about immunology and, in spite of all my best efforts, decided to go back to school half time.  She is in the Hopkins master's program of immunology and public health, but she is continuing to work with us part time and is devoting her activities to some of the research that I will be talking to you later.
	Gerry Poley was my fellow at Children's Hospital before I left.  He followed me to FDA as a guest worker in order to keep doing his research.  He is now a full attending at Children's Hospital and is continuing to do his research about half time in our laboratory.
	Now, looking at the staffing in a static way doesn't give the full picture.  So, I put together this slide indicating some of the fluctuations that we have had in our staffing.  And the bottom line take home is in spite of the fact that things are looking very well now, we have had some down periods where we have really only had two rather than four or five people working in the laboratory.
	In spite of that, we have been able to build up and we are now at, I think, a very good level of staffing, which I hope to maintain for most of the next year.  
	In terms of operational improvements within the laboratory, I presented last year to you the validation studies that we had done with the competition ELISA.  These validation studies will be referred to a little bit more in one of the later talks.  But basically we made some minor improvements in the competition ELISA.  We revalidated its accuracy and we distributed that information to all of the manufacturers so that they could at their discretion, institute some of the changes that we instituted in the competition ELISA.
	We, I think, have improved the speed of feedback to specific inquiries from the manufacturers.  We try very hard to get back to the manufacturers within hours, if possible.  I have really tried to have as much of an open door policy for all representatives and the manufacturers to talk to me about any specific questions that they have.
	In addition, as we discussed last year, I have instituted an aggressive proactive reference replacement program that I will be talking to you about in just a few minutes.
	One of the important administrative features of the past year for all of us has been the merger of the Division of Allergenic Products into the Division of Bacterial Products.  You heard about this last year from Dr. Thomas Hoffman, who introduced the concept to the committee and that merger actually did take place on September 1st, 1999.
	We are now part of a larger division that is called the Division of Bacterial, Parasitic and Allergenic Products or DBPAP.  Our acting director is Drusilla Burns.  Our deputy director is Carolyn Deal.  All change is anxiety producing and I will not lie to you and tell you that I had nothing in my mind that caused me any concern beforehand.
	But I can honestly say that really within -- let's see, the merger happened by Friday -- by mid-week the next week, I was thoroughly reassured about the merger.  We were clearly the beneficiaries of a very highly developed regulatory division that, if anything, has really contributed incredibly to our ability to do our regulatory job better.
	So, in fact, this was a merger of unequals.  We were a smaller division being merged into a larger division.  There are lots of concerns about that.  I am here to tell you six months later it has worked out better than I would have hoped, spectacularly well.  We have clearly had increased access to regulatory resources.  We have had increased access to administrative resources to help us do our job better.
	We have continued to have a very high level of program support.  The Laboratory of Immunobiochemistry Allergenics is in a very strong position at this point.  So, I would not be -- I really think that any concerns that we might have had beforehand have been amply allayed by our experience so far.  And furthermore, the research regulatory balance that I have and that my people have in the lab has been maintained and we are doing quite well with that as well.
	So, what are the regulatory activities of the Laboratory of Immunobiochemistry?  We have a large number of very routine regulatory activities.  These are our bread and butter activities that we do day in and day out all the time and they include what is on this list.  Protocol review, this is when lots that are -- of standardized allergen extracts that need to be released.  The protocols and samples are sent to us for us to review and approve before the lots can actually be released.
	Now, our lab is not the only unit that reviews the protocols.  There are other units that review the protocols as well.  The protocols have to go through checking and signing off by several different units within CBER and a certain number of them actually are tested by us and by some of the other labs as well to confirm the manufacturer's data.
	We also were involved on a frequent basis with reference development.  We certainly are involved with reference distribution and reference maintenance, including semi-annual checks and replacement.  In terms of protocol review, in 1999, we reviewed 477 protocols.  That means 477 lots of new standardized allergen vaccines were sent to us for approval prior to release. 
	One of them was withdrawn simply on review.  In other words, we just looked at the protocol.  We identified some irregularities.  We called the manufacturer and they withdrew the product. 
	Four of them failed on potency testing.  This was testing where we tested them.  They were outside of limits and they were failed.  Two lots failed on glycerol testing.  In other words, when our laboratory -- not our laboratory, but when the laboratory that tests for glycerol content, and that lot was withdrawn as well.
	In terms of reference distribution, in 1999, we distributed 1,983.  We distributed nearly 2,000 vials to the manufacturers of reference material in 104 separate shipments to the manufacturers.  So, the take-home message is we do a lot of shipping or reference to the manufacturers, that we are the source of these materials and we send them out.
	And our reference materials are maintained with semi-annual checks.  We go through all of them.  We do gels, potency assays, just to make sure that they are up to the requirements.
	Last year when I spoke to you, I introduced our aggressive reference replacement program and the genesis of that was that when I arrived at the lab, we found that of the references that we had in stock, the 24 references that we had in stock, 20 of them appeared to be beyond the three year dating period that had been provided by the manufacturer, based on stability studies.
	So, formally speaking, they were out of date.  In fact, there is no particular reason to believe that there is anything wrong with using those references because we do do six months checks on them.  We were monitoring them.  But from an administrative point of view, I certainly wanted our references to be as current as possible.
	So, we began a reference replacement program, the idea to bring the full inventory to within the stated date and our target completion date for that program was August 2001.  The procedures were fairly straightforward and obvious.  We identified the references to be replaced.  We select candidates from recent submissions by manufacturers to replace the references.  We do our own initial testing in the laboratory.
	We then select based on our extensive testing a provisional reference.  We then ship that provisional reference out to all of the manufacturers for them to actually review and test and compare to the previous reference.
	We usually give the manufacturers about 60 days to respond, send us back their data and then with a really large amount of data, not only our extensive testing but the testing from the manufacturers, we can decide either to go ahead and select that as our replacement reference or as happens sometimes to go back to Step No. 2 and try again.  It is a very time-consuming process, but it is a process that we need in order to keep our materials up to date.
	So, in 1998 --
	DR. OWNBY:  Jay, on that question, do you actively submit -- ask the manufacturers for extracts to be candidates for replacement or are these lots that come in under protocol for routine review.
	DR. SLATER:  Second.  In other words, we like to pick lots that have been fairly recently manufactured so they will have a good long life as a reference if we select it and we go back and look over, say, the last six months of products that we have screened and identify candidate materials that would be a good reference.
	The first thing we actually do is call the manufacturer and find out if they much of this stuff left because sometimes they have sold out of it by the time we indicate an interest.
	So, no, we don't actually actively solicit specific products for reference.
	DR. OWNBY:  Thank you.
	DR. SLATER:  So, in 1998-1999, we replaced four references, the pooled serum for D.pteronyssinus and D.farinae, cat, cat hair and D.pteronyssinus references and we are currently working on a series of grass references, red top, orchard, meadow fescue, sweet vernal -- actually these three are in the midst of the process already.  This one is just starting on the process.  We are also going to be replacing our short ragweed serum, which is a sheep serum, simply because it is running out.
	DR. SAXON:  Why do you have cat and cat hair?  I mean, it may be historic or is that for some intellectual reason for having those two different --
	DR. SLATER:  Cat pelt and cat hair.  So, the cat pelt contains albumin.
	DR. SAXON:  So, manufacturers will either make -- have made one of two kinds of products then?
	DR. SLATER:  They will make both products.
	DR. SAXON:  Is there any scientific reason?  Is one different from the other or better?
	DR. SLATER:  The scientific reason is that a certain small percentage of cat allergic individuals have significant cat albumin sensitivity in addition to their Fel d 1 sensitivity.
	DR. WRAY:  I can confirm that some patients react to one and not the other.
	DR. SAXON:  You have people who react to cat hair and not to cat pelt?  Dr. Shapiro, is that your experience?  I just was wondering.  I didn't realize you had both standards as one --
	DR. SHAPIRO:  I think of cat hair as an inert sort of substance rather than active stuff that was attached to the hair.  Maybe that is where the issue is.  What do you mean by cat hair?  Just the keratinized material or all the stuff attached to the keratinized material?
	DR. SLATER:  Both of the extracts are standardized according to their Fel d 1 content, but the difference is that on the immunoelectrophoresis, the cat pelt, one would expect to see an albumin band in addition to a Fel d 1 band.  We don't standardize it according to the cat albumin content, but because there is a minority of individuals, who are significantly allergic to the cat albumin, you want to make sure that that product is available for use.
	DR. SAXON:  I understand.
	DR. LEHRER:  Jay, the question is are there any patients that are positive to the cat hair and not the cat pelt?
	DR. SLATER:  You mean, why not just make cat pelt as the product?  You know, in essence, I don't know the answer to the question and I am not going to attempt to answer it, other than --
	DR. WRAY:  It seems logical but yet we have seen some that react only to the hair and not to the pelt.
	DR. SAXON:  That wouldn't surprise me if, in fact, what is on the hair is a little dust mite and a bunch of other things that the cats have picked up.  I was just interested.  I didn't realize they both existed.  I don't want to make a big deal of it.  I just am interested, didn't know about it.
	DR. UMETSU:  There is still a question, though, what do you use to treat patients with?
	DR. SAXON:  It is pretty obvious at UCLA we don't use cat hair.
	DR. UMETSU:  But if it based on the content of Fel d 1, isn't that what you want to treat patients with?
	DR. SAXON:  They both are standardized by Fel d 1.  So --
	DR. UMETSU:  They are both standardized on the basis of their Fel d 1 content.  But the cat pelt contains cat albumin as well.
	DR. SAXON:  And the other may contain other things.
	DR. UMETSU:  They are both the same in terms of their content of cat and the Fel d 1 dominates, but if you look at these on IEF, you know, the pelt has a huge amount of albumin compared to the Fel d 1 content.
	DR. SLATER:  We are actually going to see a gel of a cat pelt preparation.
	DR. SAXON:  I know.  I saw Olman's(?) work many years ago showing it and they were concerned it would make people sick but it didn't.  Have you ever done the opposite, take the cat hair and measure it for dust mite?
	DR. SLATER:  I don't know whether that was done in the initial --
	DR. SAXON:  I was just asking because Dr. Wray says she sees people react to the hair and not the pelt and I was wondering, gee, if it was something special about the hair, which I find antithecal to my thinking, but I assume it could pick up other things.  Curious.
	DR. LEHRER:  Are there some allergens in the air that are greater concentration than the pelts?  That would explain it.
	DR. SLATER:  You mean aside from Fel d 1?
	MS. BRIDGEWATER:  Jay, Jennifer Bridgewater.  Part of the issue, I think, is the way that they are collected and the manufacturers can correct me if I am wrong, but the cat hair they actually get, I think, by shaving the hair off the pelt.  So, you really wouldn't have a lot of albumin in there where the pelt is.  They actually cut strips of the pelt.
	DR. SAXON:  Great.  Understood.
	DR. SLATER:  I don't know the answer about other cat allergies that may be in higher concentrations.
	One of the lessons of our reference replacement program is not that only that our current activities to bring it up to date have been very resource consuming in terms of our time, but that even future efforts to keep it up date, if we are continuing to deal with 36 month expiration dates, are actually going to be very costly in terms of our time.
	Therefore, one of the things that I talked to you about last year was to initiate a reference, a lyophilize program whereby for the next few years, we are actually going to purchase about 20 percent more reference material from the manufacturers than we actually think we will need, lyophilize that material, study the potency of that material at time zero and then follow that material out, hopefully, beyond the three year dating period to see whether it is comparable to the glycerine aided material that we use as our current references.
	Our assumption is that this material will be more stable and if we can develop references that will have a ten year dating period as opposed to a three year dating period, we will considerably reduce our resources that we need to develop to this problem.
	So, the plan was this year and next year, to lyophilize a portion of the new reference extracts and then for the next three years, to assess the stability and reliability of the lyophilized products compared to the current references.  Once we have the results, we will distribute the results and samples to APMA membership for their comment prior to instituting any changes.
	Agenda Item:  Research Report
	That leads me into the next part of my talk, which is the research report because that is actually going to be the first research project I am going to tell you about.  
	We have had a good year in the lab in terms of research activities.  We have had a good year in terms of beginning several projects and many of the projects that I am going to talk to you about have just started over the past three or four months.
	So, we are not going to have any conclusive publication of quality data for a number of them.  But some of them have really come along quite well.  We have done well in terms of abstracts submitted.  We have done well in terms of papers accepted in refereed journals.  So, you have seen this research program summary before.  It hasn't changed since last year. 
	The two areas of major interest that we have are the areas of allergen structure and function and, in addition, the area of immunomodulation.  Within allergen structure and function, we have been studying the stability of lyophilized references, which is the first part that I will talk to you about.  We have also looked at these other questions that I will go to in order as we come to them.
	So, the first allergen that we attempted to lyophilize was a D.pteronyssinus allergen vaccine.  What you see here is an STS polyacrylamide gel of the glycerinated material in the right hand column, compared to lyophilized materials, compared to material that was lyophilized in the presence of mannitol.  Mannitol is a bulking agent for lyophilization.  It is thought to confer some added stability to the products.
	We should note, of course, that before lyophilizing the product, we have to dialyze the glycerol out of it, which adds a level of concern that you might be losing some essential allergens.  So, our plan was to do SDS polyacrylamide gel electrophoresis on these and you can see here pretty clearly that there is no visible difference in this gel between the glycerinated material, the lyophilized reconstituted material and the material that had been lyophilized in the presence of mannitol.
	You can also see clearly the bands that accordingly to their molecular weights, we are guessing are Der p 1 and Der p 2 and those bands do not appear to be changed in terms of their density after the lyophilization process.
	Yes, sir.
	DR. LEHRER:  I wanted to ask you a question about the lyophilized material in terms of the amount of material that you place on the gel because some believe that there are some denaturation with lyophilization and that you are -- you may not be able to solubilize all of the material.  So, I think it is really crucial in terms of how -- the amounts that you place on the gel.  If you redissolve the lyophilized material and then get a protein concentration and then adjust them so they are all the same or if you calculate how much material you have initially in the lyophilized material and you place an equivalent amounts, that would account for any loss due to lyophilization.
	DR. SLATER:  Right.  It wouldn't compensate for any loss.  The first metric would compensate for loss.  Right.  So, we use the second method.  In other words, we really wanted to see what happened when we took the original material that we had and in the end reconstituted the same amount of stuff in the equivalent volume.
	So, we did not do a protein concentration.  We didn't compensate for losses along the way.  So, if there were losses -- so, not only is there an issue of precipitance that you can't resolubilize, but there is an issue of selected protein loss on the dialysis tubing and through the process.
	So, no, we did it the second way because we really wanted to see what kind of losses you actually experienced.  Remember, even if we had seen losses initially, it wouldn't have invalidated the approach because really what we are looking at now is sort of the zero point in terms of the potency.  Let's just say that by lyophilizing it, we lost 20 percent of the protein across the board.
	If you still had a material that was 80 percent of potency that stayed completely stable for ten years, you would still have a reagent that was of value.  What we are more concerned about is do we lose -- does potency deteriorate over time in the lyophilized state relative to the glycerinated product?
	In addition, we are concerned about the possibility of losing minor allergens; in other words, changing the composition of the product by the processing.  And, again, this is basically the zero point.  This is not a stability study at all.  These relative potencies were done within just a few days of the lyophilization process.
	What you see here is that the relative potency of the glycerinated product, the lyophilized products, were all statistically indistinguishable from one, if you at the 95 percent confidence intervals, you can see that they bracket one and not only that, they bracket each other in terms of their content.
	So, this was reassuring in terms of the competition ELISA and suggests that the lyophilization process itself doesn't appear to cause any visible compositional changes or relative potency changes in the product.  The plan now is to follow these lyophilized products out over the next three years, along with the glycerinated product that is currently the standard.
	DR. LEHRER:  Could I ask you another question about lyophilization?
	DR. SLATER:  Sure.  Of course.
	DR. LEHRER:  Do you plan to look at other allergenic extracts?
	DR. SLATER:  Oh, absolutely.  Absolutely.  The plan is to serially just go through everyone that we do.  It is just a matter of getting started.
	We also did this on cat pelt and you can see here quite clearly that the cat albumin band, at about 68 kilo daltons is really quite prominent.  You can see the fel d 1 band here.  It is definite.  It is clear.  But it is not prominent.  It is really clearly a minority of the allergen in there.  But, again, the product is standardized for its fel d 1 content.
	Now, what is interesting here is you actually can see a difference between the glycerinated product, which is here and the lyophilized product and you can see it -- there is a triplet band here and the heaviest of the triplet disappears completely in the lyophilized products.  We don't know what allergen that is, but we definitely can see that something in our lyophilization product doesn't agree with this particular extract.
	Now, is this an allergen band that we need to be very concerned about?  We are not sure yet but we have to study that.
	DR. KING:  When you have a protein mix, you lyophilize it and it is usually stable, but when you have a purifier protein, you lyophilize it and it is the end of it.  The best one I myself personally know is this antigen and ragweed pollen.  If you have purified protein lyophilized, you store that and it gradually goes to pot.  It is true in many other enzymes, too.  Once they are purified, they cannot be lyophilized.  When you have a mixture extract, it is perfectly fine.  You lyophilize stores or lyophilize power.
	DR. SLATER:  So, we should be in luck with these.
	DR. KING:  I would just like you to be aware of the fact that lyophilization would not -- may, in fact, in this particular case, this protein may be not stable on lyophilization.  That is why you see glycerinated farther but you don't see it in lyophilization.
	DR. SLATER:  Good point.  Thank you.
	DR. KING:  The other question is you do all these analyses by comparing SDS gel electrophoresis.  I know I would do the same thing, too, because that has been our best resolution, but as you know very well, in fact, they are inactivated in the SDS gel electrophoresis.
	So, yes, the protein band is there, but is it really the immunoreactive band is another question.
	DR. SLATER:  I think that is a very good question.  I think with some of these extracts, we are going to have to do some allergen-specific measures as well, as we move along with the study.
	Again, just following up with the cat pelt lyophilization study, here is a comparison.  We measure Fel d 1 by radioimmunodiffusion.  So, we measure the radius of diffusion.  As you can see here, these are indistinguishable and, in fact, the unitage(?) of Fel d 1 was indistinguishable among those three as well.
	So, in terms of the Fel d 1 content, not the rest of the composition, we did not see any deterioration at all.  Another very important part of -- and I should say that the work that we are doing with the stability of the lyophilized references is being handled by Kristin Morrow, who is one of the biologists that really just started with us a few months ago and she has been involved in this and in other projects, but this has been one of the projects that she has been working on.
	In terms of glycosylation of allergens, this is a very important part of our laboratory's activity.  We are trying to address several important questions that really impact very heavily on the regulatory work that we do and that we are going to be doing.  
	Is the decreased antibody binding of the non-glycosylated antigen primarily a function of impaired folding?  What is the biochemical anatomy of glycosylation requirements?  Can non-glycosylated allergens equal native allergens in immunotherapy and most importantly, how can non-glycosylated products be evaluated by us for diagnosis and therapy.
	Our model of looking at the role of glycosylation allergen structure and function continues to be looking at bee venom hyaluronidase and bee venom acid phosphatase.  These are studies that have been run mostly by Lyudmila Soldatova, who has published in this field already and I discussed some of her results last year.  I will show you some of them again now.  
	She is moving forward with this project and, in addition, Kristin Morrow, who, as I said, just joined the lab in August, is going to be taking a chunk of this project and working with it herself.  It is a big project.  There is a lot of work to do.
	Our approach is, I think, fairly ambitious.  First of all, we are going to tool up the current enzyme assays because we really do need to have very good, precise, quantitative measurements of these enzyme activities.  We need to express glycosylation mutants in insect cells and we need to study the antibody binding and enzyme activity of the native materials, the recombinant materials and the mutant proteins.
	You are aware of the important allergens in honey bee venom phospholipase A2, hyaluronidase, acid phosphatase.  Our focus has been for the most part on hyaluronidase, which as part of Dr. Soldatova's studies, she expressed in E.coli, as well as in the bacula(?) virus system.
	Just to summarize her previous published work, she was able to show that the material that was expressed in E.coli, here in the open squares, was significantly lower in terms of RAST in addition with pooled human sera, than the bacula virus expressed material, which is in the open -- yes, in the filled squares here.  
	In fact, the bacula virus-expressed material was indistinguishable from the native material, which was in the open circles.  So, this was indicating that in terms of antibody binding to human antibodies, it was really a dramatic improvement when the recombinant material was expressed in the cell system that was capable of supporting glycosylation.  And in terms of enzyme activity, she found that the E.coli material had a low specific activity in units per milligram of material compared to the bacula virus expressed hyaluronidase, which as we saw in the previous slide was fully comparable with the native hyaluronidase in terms of enzyme activity.
	Yes, sir.
	DR. SAXON:  Jay, let me just make sure I understand.  I think I understand but I put it on the table.  The concern is that when people make recombinant antigens, are they going to be equivalent to native, right?  So, appropriately the concern is if you take the sugars off, will they be less potent.  Right?
	I just want to add there is another twist to that, I see now, and that is, in fact, when using in vitro tests, a lot of the cross reactivity against glyco -- sugars, is probably epi phenomenon and does not have biologic activity in vivo.  That was best worked out with nuts and seeds where, in fact, a lot of the cross reactivity, which is in RAST testing, does not show up on skin testing and a lot of the cross reactivity with latex in vitro assays is probably an epi phenomenon.
	So, just as another side to it so that, in fact, things without sugars, just because the in vitro test doesn't bind as well, doesn't mean in vivo they might not be biologically potent.  Right?
	I just put that on the table.
	DR. SLATER:  Absolutely.  And, of course, when we are working allergen vaccines, we are not only concerned about skin test reactivity -- in fact, it might be desirable to have one that has less skin test reactivity, but if it is fully active as an immunomodulator, it might be perfectly acceptable.  So, we really need to explore all of those features as the native and recombinant materials.
	In collaboration with colleagues, Dr. Soldatova has obtained several mutated clones of hyaluronidase.  She has obtained clones that are mutated in the four putative endoglycosylation sites, which are shown in aqua and, in addition, she has mutants that have been mutated in the active site.  She and Kristin Morrow are now in the process of expressing these clones.  They are going to develop systems for expressing them and for studying them and characterizing them and hopefully through these site-directed mutants, we will be able to come up with some real answers as to what the specific requirements are for these particular glycosylation sites, both in antibody binding and enzyme activity.
	Another important enzyme in acid phosphatase, which Dr. Soldatova has been studying over the past year to year and a half, her studies were initiated as an attempt to sequence the CDNA obtained from dissected honey bee glands.  Unfortunately her first attempt using degenerate primers to obtain a sequence from the messenger or from the CDNA was unsuccessful.  So, she went back to the genomic DNA, was able to get out a very nice segment from which she could then develop non-degenerate specific primers, went back to the CDNA and was able to come up with about a 70 percent  sequence for this material.
	She is continuing to extend the ends of these CDNA fragments and continuing to sequence the acid phosphatase message.  What was interesting is even from her preliminary results, we now know something about the event of acid phosphatase that we didn't know before.
	From the earlier tryptic digests and the limited sequencing that had been done, it had been assumed that the bee venom acid phosphatase was homologous with acid phosphatase from prostate, which is the best studied form of acid phosphatase.  It turns out that from this more complete sequence information, we can see that it is not homologous with prostatic acid phosphatase, but rather with Drosophila acid phosphatase and with the human lysosomal phosphatase precursors. 
	It is interesting because we know a lot about prostatic acid phosphatase and we know very little about the lysosomal acid phosphatase precursors.  So, this sequencing and characterization work has an opportunity not only to teach us something about a bee venom allergen, but also to teach us something about an enzyme that hasn't been particularly well studied up until this point.
	Another area of interest is the area of enzyme activity in allergens.  Enzyme activity is extremely important in several allergens.  We are concerned about the relationship between allergenicity and enzyme activity, in terms of the antibody binding, bioavailability and antigen processing.  The specific regulatory applications, of course, high minoptera(?) for which several of the important allergens, of course, have enzyme activity.  In fact, for high minoptera, the enzyme activity is our standardization method. 
	In other words, that is what we actually measure in terms of lot release.  But for dust mites and latex, as well, several of the allergens have enzyme activity.  Our model -- and this is a study that is being conducted by Melissa Catana, who started with us in September and she has been working very hard at this, but a lot of the methods have needed to be developed from the start.  So, she is getting a good experience in methods development right now.
	But our model for this is to look at the event of hyaluronidase and phospholipase A2, again, to validate and refine the current enzyme assays to develop good immunoassays for hyaluronidase and phospholipase A2 and to compare the enzyme  activity and antibody binding in fresh, old and inactivated materials.  This is just one of her preliminary studies in which she took serum that we had obtained for phospholipase A2 and was able in relatively short order to develop a good competition ELISA, comparing a current venom standard to two manufacturers products and to obtain a relative potency that is statistically valid based on that antibody.
	DR. CLAMAN:  Is your aim eventually to replace the standardization method so that you don't rely on enzyme activity?  Enzyme activity may be convenient, but perhaps not biologically the best.
	DR. SLATER:  I would be open to the results going either way.  I think that it is certainly plausible to hypothesize that enzyme activity is convenient, but probably irrelevant in terms of most immunologic functions that we want an allergen to fulfill.  On the other hand, we might find that enzyme activity correlates really well, in which case enzyme assays are awfully easy to do.  So, it might -- really the science could take us in either direction, but I think it is very important, especially since we have this dichotomy in the way we approach the extracts.
	One group of extracts, most of them we do immunologic evaluations.  The other group we do these enzymatic evaluations.  I think it is important to compare them head to head and this is a very good model for doing it because we have the antibodies.  We have animal models that we can use and we have measures of enzyme activity that seem to work.  So, it is a nice model and what I am hoping to do is to be able to draw some conclusions as to the validity of this in terms of following allergens.
	If it looks great, we might really want to explore looking at that for future standardization procedures.  But I am open for the data taking me either way on that.  
	We are concerned with identification methods.  We spend a lot of time identifying allergens, using mostly immunoelectrophoresis.  We have been looking at exploring the possibility of using physical chemical methods for measuring allergen content.  Again, as you heard, we use immunologic methods mostly.  We use some enzyme activity methods, but we wanted to explore the possibility of using physical chemical methods.  And this is a project that Beth Paupore, along with Bob Boykins, who really runs our MALDI-TOF mass spectroscopy unit in the division, has been moving forward with.  
	I talked about MALDI-TOF last year.  Our model is to take ultimately all of our standardized allergens and to evaluate these allergens in sequence with SDS-PAGE and with MALDI-TOF mass spectroscopy.  
	In the initial phase and we are still very much in the initial phase of the study -- it is really what I call the normative phase -- I really just want to look at what they look like to get an idea of what kind of diversity there is among the products that come out, what we can identify, what are bands that you always see, what are bands that you never see.
	That is definitely where we are right now.  Ultimately, I would like to identify specific allergens, correlate it with the SDS and with the MALDI-TOF and see what kind of results we get.  This is just by way of showing a standard SDS-PAGE of venoms.  This is a hornet venom, a wasp venom.  You can see the allergens that I have identified.  This is probably antigen 5, possible lipase A1.  This is a fairly faint band for hyaluronidase in these.
	This is good data but it is sort of the data that we have been looking at all along and there are limits to the resolution that you can get by doing these kinds of gels.  In contrast -- and this is fully to the credit of Bob Boykin's excellent work and mastery of a very, very costly piece of equipment -- the MALDI-TOF analysis of two venoms -- this is a honey bee venom; this is a vespid(?) venom -- really shows the power of this particular technique. 
	Not only can you clearly see phospholipase A2 in this honey bee venom, you can see the hyaluronidase peaks, which are low.  In both of the venoms, you can see the antigen 5 peak in this venom.  You could also see -- notice I have an arrow here and there is nothing at the bottom of it and there is nothing at the top.
	You can see that this particular vespid venom doesn't appear to have phospholipase A1 in it and we don't measure that, but there is no phospholipase A1 in this particular vespid venom.  What you can also see -- well, maybe you can't see, but I am going to try to convince you that this is true, is that you can see not only phospholipase A2, but you can see the multiple glycosylation isoforms of phospholipase A2 here.
	So, not only with MALDI-TOF can you get a fairly nice, specific fingerprint, if you will, of these particular products, but you can actually look at the fine detail of their glycosylation patterns in terms of their molecular weight.  You really don't know what the glycosylation units are, but you really have the opportunity to develop a quantitative profile of the lateral materials, but you also have an opportunity to use MALDI-TOF to carefully access the recombinant products that we know we are going to be receiving.
	When the recombinant products come in, we need to know how glycosylated they are.  We need to know what the consistency of manufacture is.  And this is a tool that I am now convinced we are going to be able to use to look at those products as they come in and make sure that they have the consistency that we require.
	I introduced you last year to studies that we had done with lipopolysaccharides.  This is work that had begun previously while I was at Children's, looking at latex and lipopolysaccharides.  We have extended it since then.  Maneesha Solanki has really taken hold of this project and has done a large amount of work.  
	Why are we interested in LPS and allergen responses?  We are interested because LPS is clearly ubiquitous.  It is not only present throughout the environment but it is also present in the allergen extracts that we market.  So, it is important for us to understand what the immunomodulatory consequences of co-injected LPS might be.  We know that LPS elicits broad immunologic effects in all systems that we look at.
	Now, in mice, as we had a discussion last year, in mice, LPS has a peculiar in vitro feature of being a very strong B cell mitogen.  That feature is not present in humans.  But in both humans and mice, LPS appears to ask as a strong adjuvant.  It seems to elicit both TH-1 and TH-2 responses in both humans and mice and it is clear from old studies that were done actually in the early 1970s, late 1960s, that the effect of LPS on antibody production in mice appears to be independent of this B cell mitogenesis feature.  
	It may not be fully independent but the bottom line is it doesn't happen in nude athymic mice.  If you take nude athymic mice, whose B cells when you take them out will actually respond to LPS and you inject them with LPS and antigen, it does not function as an adjuvant in those mice.
	We, of course, know that LPS has multiple immunologic consequences that really are complicated and depend on how you model the system.  In our previous work that I had talked to you about last year, we were able to show that LPS co-administered nasally with the latex allergen Hev b 5 led to accentuated IgE and IgG responses to Hev b 5 and to spleen cell responses to both Hev b 5 and the carrier protein, maltose(?)-binding protein.
	Our current model is to continue with airway immunization in mice and to verify the Hev b 5 results using ovalbumin.  I would like to continue studying Hev b 5, but it is kind of a limited product and once we decided to go ahead and study the LPS phenomenon, we were just as interested in starting with a more readily available allergen as well.
	It turns out this was a very good choice and I will show you why in a few minutes.  We also wanted to begin to assess functional responses and we have wanted to identify, if we could, the anatomic specificity and the anatomic requirements of this sensitization.  As you will see, we have really accomplished some of this, but by no means all of it.
	Once again, we see that LPS added to the ovalbumin clearly accentuates the IgG and IgG 1 responses.  In this picture, time after immunization is indicated on the abscissa, the normalized antibody titer that is the comparison to a hyperimmunized animal is indicated on the ordinate.  The large open arrows indicating the immunization periods -- we immunized the mice for 12 days, initially nasally and then about seven weeks later we repeated the immunization with three more immunizations at that point.
	As you can see, the dark brown line, there is a significant increase in the IgG against ovalbumin in the animals that received the ovalbumin in the presence of lipopolysaccharide.  There is no response in the other animals.  Now, I can't expect you to remember last year's slide with the Hev b 5, but with Hev b 5, which I can only interpret by the fact that Hev b 5 is such a strong allergen, that Hev b 5 actually elicited responses in all animals after the first immunization, but it was significantly greater with the LPS.  Once we gave a second immunization, all the animals have the same immune response anyway.  
	So, we could see a difference between the LPS-primed animals and the ones who just received Hev b 5.  The Hev b 5 mice had a significant response without the presence of lipopolysaccharide.  Likewise, we see a response in the specific IgG 1 and in addition, we see a definite response with ovalbumin specific IgE in these mice.  So, again, the mice that received ovalbumin in the presence of LPS have a strong response, specific IgE response to the ovalbumin; whereas, the other mice, including the one that received ovalbumin alone, don't appear to have any response at all.
	We had less good fortune in measuring airway changes.  We are using the Buxsco(?) whole body plethysmograph and our model was to do methacholine challenges in the various groups of mice and what you can see here is a general tendency for the mice that received LPS in the presence of ovalbumin to have greater methacholine responses, but these pesky error bars get in the way and the data really are truly not statistically significant at all.
	So, we are going to address that in the next phase of the study in a couple of different ways.  One is in terms of the plethysmography, we are going to measure responses both to methacholine and to antigen, which I think probably give us different results.
	We are also going to attempt to separate upper and lower airway delivery of the antigen.  I would like to see whether what is really important here is concurrent antigen delivery or not.  We are going to be looking at cellular responses, using bronchoalveolar lavage, measuring both cellular responses and cytokine responses to the product.
	DR. CLAMAN:  Question.  Sorry I wasn't here last year.  I was at the moment in an endotoxin-rich environment; namely, India.  There is a little bit of deja vu.  This subject brings me back to some experiments I did about the time you were in kindergarten, which I will not bore you with.  But is the implication that there is sufficient endotoxin in allergenic extracts to modulate the immune response itself?  I don't know what the endotoxin LPS content is.  Is it biologically active and relevant or is that one of the objectives of your research?
	DR. SLATER:  That is one of the objectives.  One of our objectives is to try to develop where we can see how much endotoxin is really necessary.  Now, Dave Piedon(?) has published an article in the last two or three months in the JACI in which he has been introducing 1 microgram of LPS into the noses of volunteer subjects and -- humans, I am sorry -- and demonstrated that in the atopic individuals, the 1 microgram of LPS is sufficient to cause measurable increase in the insinital(?) influx to the nose; whereas, in the non-atopics, it makes no difference.  So, clearly, I think, what you are saying is the dose response is key here.  I couldn't agree with you more.  We are heading in the direction of doing dose responsing, but we are not there yet.
	DR. SAXON:  How much endotoxin is there, are there, in extract?  I remember the old days when they worried about dust mite was basically endotoxin, you know, the mixture.  But today in the cat hair, how much endotoxin do those bacteria on the -- is that something that is known for all your standards?
	DR. SLATER:  No, it is not measured.  It is not determined.
	DR. LEHRER:  There must be some information on it because you have alluded to the fact that endotoxin is in there.
	DR. SLATER:  We know that it is in there, but I don't know what the recent data are.  I don't know what the --
	DR. SAXON:  So, manufacturers or the agency does not monitor any endotoxin levels in the product?
	DR. UMETSU:  It seems like it should be.
	MS. BRIDGEWATER:  Bridgewater, FDA.
	They are exempted from endotoxin testing under the regulations.
	DR. SAXON:  Why is that?
	MS. BRIDGEWATER:  Well, considering those were probably written in the sixties, I couldn't tell you that exactly but I --
	DR. SAXON:  It was bad in the sixties.  I don't want Henry to tell everybody how old he is, but even in my memory, there was that whole business where dust mite allergens were at, you know, zero to huge amounts and what people were reacting to was the endotoxin in it.  There were enormous differences.  It may be the agency --
	[Multiple discussions.]
	That is what I meant.  House dust.  I am sorry.  I meant house dust.  House dust was endotoxin.  I mean, basically what you were injecting was endotoxin.  So, it seems like maybe the agency should just think about, you know, is it worth looking?
	DR. OWNBY:  I thought there was a requirement that there was a pyrogenicity in rabbits for extracts or is that something that we were doing that was totally out?
	DR. TURKELTAUB:  There was an old paper looking at pyrogenicity of extracts.  There is no requirement, obviously, for FDA.  And they are all pyrogenic, the ones that were tested way back when.
	DR. LEHRER:  But has anything been done since the sixties on that?
	DR. TURKELTAUB:  Not by FDA.
	DR. SAXON:  Would it be difficult to do?
	DR. LEHRER:  The problem with endotoxin is how do you assay it and, although most people are using the limulus(?) lysate assay, I believe, that is still the method of choice because it is in a kit and it is easy to do, I myself question the specificity of that assay and certainly it doesn't demonstrate endotoxin biological activities as we know them, such as pyrogenicity or some of the other activities that we see in man.
	So, I think in addition to, you know, concerns about endotoxin, there would be concerns about the assay.  Probably most people would support the limulus assay, but I am not so sure that other carbohydrates or like materials that may have activity in that --
	DR. SAXON:  Would it make you feel better, though, if it was very low in the limulus assay?  If it is high, that doesn't mean it is a big problem.  But should there be some kind of at least screening to see what the situation is?
	DR. LEHRER:  I tend to agree that it would be useful to have some information about these extracts.  I would tend to feel that at least biologically we haven't heard a lot of information.  I mean, actually the clinicians could tell us more about that in terms of the adverse reactions to -- endotoxin-like reactions.  So, it may not be a serious problem, but I think we need to know.  I think it was much more of a problem when I was in kindergarten -- I wasn't in kindergarten.  I was probably in college with house dust because house dust was such a crude material and yet it was so active and nobody knew --
	DR. SAXON:  I understand.  I can think of -- I mean, in the vaccine story, people are now coming back and saying in the 1940s there were things in vaccines.  They knew they were there.  You know, this is unrelated to allergenic products.  It seems like maybe at least a little scan of the situation to see if it is enormous or not.
	I mean, if they came out enormous on the limulus assay, then you might want to have a more formal look.  If they are quite low, probably everybody would say you have shown due diligence.
	DR. UMETSU:  And there also may be significant differences between lots and so you might want to standardize it so that the amount of endotoxin is at least pretty stable between lots.
	DR. HAUCK:  I am Pete Hauck.  I am with Center Laboratories.
	A couple of things from a manufacturer's perspective.  What a manufacturer is obligated to do is make sure that they are not adding to the endotoxin load that is in the source material that we are obligated to do.  The source material does, even including pollens, does seem to be rich in endotoxins.
	Another concern -- and Dr. Lehrer alluded to it -- actually there was some work done about 20 years ago by Darrell Lu of FDA, suggested that a lot of the endotoxin tests would give you false positives if you have a lot of enzyme activity.  We know there is a lot of enzyme activities in these extracts. 
	So, before you run off and try and measure these, you have to look at enzyme interference with these kinds of tests.  That is probably the reason there are no endotoxin limits on these products.
	DR. OWNBY:  Thank you.
	DR. EIN:  Dan Ein.  Just speaking as a clinician, clinicians tend to be sort of bottom line types.  Sam said we ought to know what the clinical implications are and speaking as somebody who does a reasonable amount of immunotherapy, I can't say that we observe a lot of endotoxin-like reactions.  From time to time you get a lot of extract that seems a little problematical. 
	I don't know what individual differences there are in reactivity to endotoxin.  We talk about an endotoxin problem, but from the data that was suggested here, it may not be a problem.  A little bit of endotoxin may be a good thing.
	DR. SHAPIRO:  Is there a correlative between measurement of Der p 1, for example, and skin test reactivity that is done through the agency?  I mean, when you standardize for the active antigen, do you also do some skin test assays to see if there is comparability between products that way?
	DR. SLATER:  Well, the standardization is based on the skin test originally, the skin testing assay originally, and then at that point, a surrogate is chosen that correlates well to the skin test reactivity.  In the case of dust mites, the surrogate that was chosen was the competition ELISA, which looks globally at reactivity to all allergens.  We didn't select one particular allergen.
	DR. SHAPIRO:  Well, if would look at something like Fel d 1, where there is one antigen that you are using for standardization.
	DR. SLATER:  The exceptions to that were the ragweed allergens and the cat allergens in which the surrogate is, in fact, the specific allergen measurement in terms of Amb 1 or Fel d 1.  But for the grasses and the mites, the assay is based on the aggregate reactivity to cooled sera of highly allergic individuals as measured in the competition ELISA.
	DR. SHAPIRO:  So, what I am trying to get at is whether the endotoxin piece can be sorted out by looking at a correlation between the biologic reactivity and the actual molecular identity in the case of the situations where you are looking at just Fel d 1 or --
	DR. SLATER:  Right.  I think what you are saying is if there were substances among lots of endotoxin level, you might -- and endotoxin itself had its own skin test activity, you would pick that up by correlating the competition ELISA, by failing to correlate the competition ELISA value to the skin test.
	DR. SHAPIRO:  And you don't see that -- I mean, I am assuming that --
	DR. SLATER:  That is not an ongoing activity.  That was done with the initial standardization --
	DR. SHAPIRO:  So, that is not done on a yearly basis for antigens.  
	DR. SLATER:  No.
	DR. SHAPIRO:  Okay.
	DR. SOTO-AGUILAR:  I would like to know from the clinical standpoint the content of endotoxin in any particular extract.  Would it be more likely to cause an ateest(?) type phenomenon due to IgG reactivity rather than IgE when we see these big swollen arms or should we expect either IgE type reactivity or IgG?
	DR. SLATER:  I don't know the answer to that.
	Okay.  We are working our way rapidly towards the end.  
	The last area of interest in terms of our immunomodulatory part of the lab's activities is looking at epitopes of specific allergens.  Specifically, we are continuing to look at the epitopes of Hev b 5.  This is also a project that Beth Paupore had carried over with her from Children's.  
	This is our initial study that we performed that showed the various T cell and B cell epitopes of the latex allergen Hev b 5.  This work was not only confirmed in our laboratory in mice.  It was confirmed in Don Beeshold's(?) laboratory in humans and rabbits and confirmed by our collaborator, Robert O'Hare, in Australia in her human studies. 
	So, we have a pretty good idea of what the T cell and B cell epitopes of Hev b 5 are.  To define that further, what Beth is doing is she is attempting site-directed mutagenesis at three specific sites, three specific motifs that we think may be the important antibody-binding site. 
	She is attempting to mutate the lysine to proline in three different locations, 15, 23 and 28, and then is going to express those products and look at their antibody binding in the model that we have developed.  What you can see here is she has been successful at producing the p28 mutant of Hev b 5.  We have sequenced it.  We know it has the insert just in the right location and it seems to be in frame and we have been able to express the p28 mutant in E.coli.
	So, we are going to be moving forward with these studies, which, again, are really just getting off the ground in many ways.  But hopefully, we are going to be developing a series of mutants of Hev b 5 and we will be able to identify the critical antibody binding sites, again, looking toward the possibility of immunotherapeutic approaches either with DNA vaccines or with epitope specific immunotherapy.
	DR. LEHRER:  How were the initial epitopes identified?
	DR. SLATER:  We identified them with hyperimmunized mice, looking at their serologic response.  In addition, we identified them with mouse screen cell responses, looking at fragments.
	DR. LEHRER:  [Comment off microphone.] 
	DR. SLATER:  Actually, it was both.  We did it both ways.  We had synthetic peptides and fragments of the molecules.
	DR. LEHRER:  You were looking at IgG reactivity in the mice.  Now, Don Beeshole using the same fragments was looking at IgE reactivity in humans and Robin O'Hare, using our material, was looking at T cell reactivity in allergic humans in Melbourne, Australia.
	So, the studies really come from geographic diversity and species diversity as well.  We have some pretty good ideas where we are.
	So, that is the summary of the research program.  I just wanted to go down each of the research activities and identify for you what the sort of functional objectives are.  It has been my position since taking this job that I wanted the lab to do good basic research but that all of the research that we did had to have something to do with our regulatory function and I think you can see it pretty clearly, but just to drive the nail in, certainly the initial study that I talked to you about, Kristin's study on the stability of lyophilized extract, this is of critical importance to us in being able to maintain good U.S. standards of reference in a reasonable manner.
	The glycosylation studies that Dr. Soldatova and Melissa Catena are -- I am sorry -- Dr. Soldatova and Kristin Morrow are working on, again, will be critical as we start to evaluate these recombinant allergens that are coming through.  We need to have ways of determining how to measure the allergenicity and immunogenicity of these allergens.
	Those studies that Lyudmila and Kristin are doing are absolutely critical for us.
	The enzyme activity assays that Melissa Catena is doing, again, as we said before, we use enzyme assays for some of our lot release activities.  We need to know whether that makes sense.  We need to know whether it makes more sense than the immunologic assays.  Potentially, it is an important lot release tool.  Potentially it is a lot release tool we should abandon.  This is the kind of study that we need to do in order to do that.
	In terms of the identification methods, using the physical chemical identification methods, it would be wonderful if we could use physical chemical methods in order to do our lot release, especially as we get to allergens that have engineered or modified allergenicity.  So, certainly in terms or current products, it would be nice, but for future products, I think it is essential that we explore these methods and work them out well and figure out what we can identify using the MALDI-TOF and other methods.
	The epitope study, clearly one of the directions in which immunotherapy is going is to do epitope specific immunotherapy.  Hev b 5 is increasingly obvious it is an important latex allergen.  So, this work is critical in terms of moving forward in this important field.  
	Finally, the lipopolysaccharide studies, we know that LPS has adjuvant activities.  We know that it is an immunomodulator.  We know it is in the products that we regulate.  We need to understand how that happens in order to do a better job of assuring the safety and efficacy of the products that we regulate.
	We have had a very successful year in terms of abstracts and publications.  We have five abstracts in at the academy meeting in San Diego.  We are going to have a very busy time there covering the full range of our activities.  I won't go through each of them now because time is short.
	Dr.  Soldatova and I attended the Paul Erlich(?) Symposium in September and both of our papers will be published in the proceedings of the Paul Erlich Symposium, which I am optimistically indicating might be published in the year 2000.
	Finally, we have had four papers published or accepted in refereed journals.  The first is the paper that Beth and I and Robert O'Hare authored about the B cell and T cell epitopes of Hev b 5.  That appeared in Molecular Immunology towards the end of last year.
	The house dust stability that I am going to be talking to your about in about an hour, authored by Dr. Soldatova, Dr. Pastor and I.  It is going to be appearing in the JACI within the next month or two.  The work on the novel methods of determining equivalent doses of allergens is also going to appear in publication, the JACI probably in March, and finally a paper from Robert O'Hare's lab, in which I was a collaborator on the human T cell epitopes of Hev b 5 will be appearing in the spring as well.
	Thank you very much for your attention.  I apologize for running over.  I will turn it back to Dr. Ownby.
	DR. OWNBY:  Are there any further questions?
	Dale.
	DR. UMETSU:  I just had a question about the T cell epitopes with Hev b 5.  Are there single major determinants for that for many humans or how does that work?
	DR. SLATER:  Yes.  There are two major determinants that Robin identified in Hev b 5.  Her study was, I believe, of 13 or 14 latex allergic health care workers.  She didn't study any spina bifida patients.  These were all adult health care workers at medical centers.
	DR. UMETSU:  But does that go across many different HLA types?  I mean, getting back to the same problem that -- when people initially identified major epitopes for Fel d 1, it probably wasn't really the major determinants.
	DR. SLATER:  Right.  There was definitely heterogeneity of HLA types among the individuals that she studied.  I don't remember exactly what the breakdown was.
	DR. OWNBY:  Any other questions right now?
	We are scheduled for a break.  Why don't we go ahead and take a ten minute break and we will try not to run too far over for lunchtime.
	Thank you.
	[Brief recess.] 
	Agenda Item:  Regulatory Proposals -- Potency Limits for Standardized Allergen Vaccines
	DR. OWNBY:  Our next speaker -- I finally figured out what he is doing here today -- is Dr. Richard Pastor, who is chief of the Lab of Biophysics.  That seemed a little removed from allergy, but Jay was just informing me that he was the previous interim chief of the Laboratory of Immunobiochemistry.  So that he has been dealing with these issues and thinking about them.  
	We are happy to have him here.  Thank you.
	DR. PASTOR:  Thank you.
	I will be speaking about the limits that we set for these products.  Basically, you got part of this talk last year but in the course of the year we learned more stuff and had a broader picture of the whole issue.  So, I think it is worthwhile to go through it again.  So, here goes.
	So, in the first part of my talk, I will just talk about the general issues we have here and then I will actually speak about these new limits, which bottom line -- which we are going to make the manufacturers keep to their old limits and then the receiver will do the lot release test and if they are in between a relative potency of .5 and 2, they will actually get -- they won't fail.  So, it is a broad thing, as you will see.
	After I have gone through that, I will speak very briefly about some of the -- now, with this data, we can look at the non-standardized products, but what is the rationale for not having a protein content any longer.  The underlying theme of this may be a little new to say in the FDA, but it has been around for awhile and now we want to make it more formal.
	What we really want to do when we set a lot release specification is to make a balance between the manufacturer's risk and the consumer's risk.  What the manufacturer's risk is that is the risk that the manufacturer will make a perfectly acceptable product but because of the variability of testing and consistency of manufacturing, that acceptable product will fail.
	That is something you don't want to happen a lot, but it is part of the process in which there is the converse of that, a product that is on the borderline because of the variability of testing will like sometimes pass.  So, you can always ensure that there will never be a product on the borderline by basically failing every product you get.
	That is ludicrous, but that is an extreme.  So, one has to balance these things and as we go out of the research lab, where you get gel, you get MALDI-TOF, you get ELISA results.  At some point you have to make a decision to say, well, here is the spec.  This fails; this passes and this is the process by which we are trying to do it.
	I am a biophysicist, so I don't use power points, slides and that stuff.  I just like draw my curves, but now I am kind of caving in a little bit.  So, now I do them like this.
	So, what are the like three issues that we use in general and then more specifically for these extracts.  One is that does the stuff work.  Presumably, there is an efficacy range versus potency and at very low potencies, you don't get anything.  At very, very high potencies, it may keep working, but it may drop off for reasons we don't know.  We are going to presume somewhere in the middle there is some range at which the stuff works.
	So, if one were to take a range for ELISA limit, say, you might consider using here to here or, you know, here to here.  So, that is one way to get a range.  Another range would be to for a given potency, to just look at the reaction rate, anaphylaxis and so on.  
	Now, obviously, at very low doses, there won't be any.  At very high doses, you will get way too much.  If that is somewhere in the middle, but, in fact, we are going to rephrase that slightly and say, well, with allergen extracts, one of the big issues is if you switch a bottle.  So, if you get a -- because a bottle runs out, you actually get a new bottle.  Obviously a patient is going to get a dose from bottle 1 and then a dose from bottle 2.
	We can ask, well, what is the maximum dose change one can tolerate.  Obviously maximum is also something that you can work at, but, you know, you don't want the adverse reaction rate to be too high for a given change.
	Lastly, there is just the issue of manufacturing, of consistency.  Again, on just writing potency and now here my x axis is time and as you go along, you see everything is more or less, you know, the same and then all the sudden you get this guy here and then you might go back and then you might want to say, well, you don't quite know what happened wrong.  You just know it is really different and you want it out.
	So, you might imagine setting a limit something around here so that you won't allow this product in.  The bottom line on this is that the safety requirements are actually not that flexible.  We can't go above a certain reg.  This, like manufacturing practices is more flexible.  It is assumed that the products are safe, you know, once you have passed the clinical trials and so on.
	Now, these limits, where you will pick can actually vary.  For instance, say you do a bunch of these tests and you reject like 1 percent of a lot at each time.  If you keep on doing them, you will keep on like rejecting lots until you do a hundred of them.  You might get rid of almost all of them.
	So, once again, you have to be a little cautious and you have to think about how to balance stuff like manufacturer's risk as you set these limits, especially for here.
	With these extracts as Jay didn't get into yet, but you can see where the 95 percent confidence was of some of the numbers, they are actually quite broad, something you have to take into account.
	So, with our current ELISA method, if you just do a single replicate and you measure like relative potency of the reference itself.  So, you know that has to be 1 right, but as you measure it, it could be quite broad.  In fact, it does turn out to be quite broad because of the variability assay.  The 95 percent confidence intervals for this are, you know, basically between .5 and 2, not exactly, but, you know, in other words, you know, in 95 out of 100 cases, something that is 1.0 can be anywhere from .5 to 2.
	Now, that is quite broad so, in fact, what -- the way one narrows that down is to take more replicates.  We use 3.  That is a convenient number if it fits on an ELISA plate, sensible and the 95 percent confidence interval is .7 to 1.4; so, about a factor of 2.  You see where your assay is, a factor of 2.
	This is a big improvement over the RAST method, where the standard deviation was about twice as large.  So, we have really like narrowed it down by switching from RAST to ELISA.  It was done a little while ago.  How do we accept or reject lots now.  Well, we actually rejected at the 98 percent confidence because we were doing the retest and it worked out that it was 98 after we did the analysis.  So, that means any lot that is like outside of the range, .654 to 1.53, fails lot release.
	Curiously, I mean, this follows that because it is 98 percent confidence, about 2 percent of lots that is the relative potency is 1.000.  In other words, it is the reference itself actually fell.  So, that is the manufacturer's risk that is just thrown right there.  There is actually somewhat more risk -- as I said, a lot that was perfectly acceptable, well, is .9 acceptable, is, you know, 1.1 acceptable.  Well then it is broadened.
	So, basically you just have to take into account that the manufacturer lots are actually not 1.000.  Rather they have a distribution themselves and, in fact, this is a famous formula for those who know it well and, so, basically the standard deviation squared of the observed values that you get is actually equal to the variability of the assess plus that of the sample.  So, it is broad.
	So, now the situation is we have this broadened sample that we are working with and then the problem 
is -- sort of can be stated in kind of a down home way, what if a lot was really .7, you know, and the way you ascertain that is by doing a thousand replicates.  You know they are doing something with .7 and the manufacturer tested at .8, passes it, seems reasonable.  CBER gets it.  It tests at .6 and rails it.  And one of the things I asked when I started this -- I am a biophysicist and, you know, I didn't have any -- I hadn't thought about a lot of allergenic extracts.  I said, well, is that okay?  
	You know, people said, well, that is how we are doing it now.  We can't change it.  You know, one has to think, well, you know, maybe you have to think about changing it.  So, how do you go about changing the fact that this would happen, that this would fail?  You can't just say make it .5.  You know, you have to do some thinking here.
	So, in fact, I am going to take you through in the next little bit just sort of our thought process by which we went through these four things.
	First, you actually have to analyze by clinical data.  If you recall like CBER files, which were done with skin testing Paul Turkeltaub, were mostly to establish the reference standard.  There wasn't that much on what the safety range was.  It was just to say this is 100,000 BAUs.  
	The next thing we have to do is to revalidate our testing method again because the original validation was just testing multiple lots of reference to reference.  So, we never tested those extremes.  You want to know that, right?
	Next, we actually looked at the variability of the manufacturers' lots after the standardization.  We had a lot of data.  So, we could actually do that.  Then, lastly, we had a little math problem that was kind of cute.  You say, well, if you change bottles, what is the sort of relative -- the change in the relative potency and you can actually work that out.  We will show you how to do it.
	So, you heard this last year a little bit.  It is now an official draft guidance document.  We have tried to cut it a little closer, but we weren't able to.  So, it got out on the 8th.  You can find it on the CBER Web site under Guidelines.ACM or the actual file is this one here.  
	This is also -- the scientific reasoning for this was actually -- is in this paper, which will be published, hopefully, in a couple of weeks.  In the validation and the protein work is in this book chapter with Jay, et.al.
	Jay was the one who looked at the clinical data.  There is way to much on this slide to read right now.  I just want to point out that it is in your handout.  Basically, Jay looked at a number of studies done by a number of groups and got some results.
	So, let me tie in specific sets.  This is an easy-to-read slide.  So, first, let's look at efficacy.  Remember, the first thing we want to see is what is the range for potency and next, what is the range for safety.  So, it is efficacy.
	So, with this -- verbally, it is just right here, is that apparently in this study doses were worked for a thousand range.  Here, this person only did -- this group only did two that worked, 6 and 12.  Of course, so -- so, it is possible that if they had done three it would have worked as well.  But we can just say if 6 and 12 work, your range here is at least a factor of 2 and if you do the same sort of thing for all the other ones, you get factors of 14, 12 and like 30.  So, you see there is like a wide range of like potency appears to work for efficacy.
	To summarize that, my little slide here, so we can say, well, we aren't sure if this goes down or up or down, but we can say that it is like a plateau that is roughly a factor of 10.  So, that is point No. 1.  Now, the next thing we have to do is to say, well, how about potency change.  Is this factor of 10, if you work back and is it 5 percent, is it 10 percent or, you know, is it all the way up here?  We have to find out where this is.
	Alternatively, you can say, well, if we only want a 5 percent change, we can work backwards and find it here.  So, we don't know yet.
	So, we isolated the studies of that, you know, big table, which emphasized -- which we looked at safety.  I am not going to go into this, but these are the three and, you know, you can read the sort of stuff that happened.
	I will just show you one example of the analysis that we did.  Because it is at the low end of the curve, you can -- it is reasonable -- it is always good when you do this sort of analysis to do a couple of different kinds.  So, we did a log linear.  We looked at the log dose, looked at the percent of adverse reactions and you can see that this data for here gives, you know, reasonably straight; .91 is not so bad.  We did logarithm of dose versus the logarithm of the probably of adverse reaction over 1 minus that probability.
	That his a more standard way to look at adverse reactions and the line is better.  So, again, we are -- we did that for like each of the studies.  We combined them we could.
	Now, there are two points to make about this.  You know, one is that the lines are reasonable.  The other one is there is only three points.  You would like to have a lot more.  You would like to have a lot more studies with a lot of different allergens, but, you know, you have to start somewhere.  Hopefully, this will spur it on more this sort of data.
	In any case, let me just -- for purposes of time, let me just concentrate on this last line here, this third column.  So, we wanted to ask the question, which we could do, once you have that bit of data.  It is what dose increase is associated with a 5 percent increase in adverse reactions.  We felt that was a reasonable way to say it and the reason why we used these sort of studies is to say, well, if a person is using an extract of a certain bottle, that person is presumably every time he gets a shot, he is not having adverse reactions.  So, you say, all right, we are going to -- now, we will like raise this dose a certain amount.  What is the probability that then he will get an adverse reaction?
	Obviously, that is a spread.  Some might get it.  Some might not.  So, you say, well, if you analyze all the data and say, well, we want to keep that probability under 5 percent.  That will set an outside limit.  You might want it less, but 5 percent is what we pick.  When you do that, you can see that effectively from the different data you get different results, but it is about -- you know, if you average these things, about a factor of 4.
	Now, different data -- we use per patient data, so, it is a little tighter.  We don't necessarily think that is, you know, the optimum way to do it.  If you use different allergens, you might get different numbers.  But at this first stab, this is how we get that factor of 4.  
	So, now, we were -- so, so far, we got the factor of 10, which is, you know, larger than a factor of 4.  So, we say, okay, we are going to pick the smaller one.  It has got to be a factor of 4.  It can't be larger than a factor of 4.  Even though a factor of 10 works in terms of efficacy, it implies that there is like extra associated with that factor of 10, which we don't want to do.
	Now that we have the factor of 4, you know, these things are kind of happening at the same time, but then we want to say, okay, now let's see what our ELISA does when the relative potency varies by a factor of 4.  It is not obvious.  You hope it does.
	The real good news is that it works quite well.  So, let me just do just this one grass.  I mean, if you take the -- extract this .5, after 24 observations, we got, you know, .516.  One is still working.  Two -- you know, it is 2.00.  We get, you know, 2.01.  So, basically, you know, our tool is working.
	The original assay had a standard deviation of this .1375 and you can see that it is in the same ball park.  So, we have got the range.  We have got the tools.  Now let's see what the distribution of these lots actually is.  This is a little complicated.  So, I will try to, you know, kind of sketch it.  
	But after you look at -- we did both grasses and, like, mice.  We looked at 412 lots which were submitted by the manufacturers.  Of those, 12.4 percent failed, 29 and like 22 low.  So, that -- using that, we can work backwards and say, ha, ha, now let's work out what the variability of that lot is.  Now we just assume that, you know, half are high and half are low.  Pretty reasonable.
	So, in other words, if 12.4 percent, you know, fail, that would mean that under this assumption, 93.8 percent were actually -- were like below the upper limit.  Therefore, you can now write the distribution of the observed relative potencies, which consist both, remember, the assay and the manufacturing.  If you integrate that, it is .938.
	Therefore, from that equation -- therefore, you can just work out and it is not so bad that the standard deviation of these observed is .120.  Nw, remember, your variability of your assay is -- you know, is .1375 divided by the square root of 3.  We would use three replicates.  We got this.  We plug this into this equation and now we know that the variability of the sample is, you know, .09.  
	Now, these are just numbers flying around, but, oh, like tie them  into stuff real soon, but I think it is a really great thing that we could just work this out.  I found it very satisfying.
	We actually see that the variability of that sample is actually smaller than the grass.  That actually makes sense.  Remember, the lots of grass were actually lots which were in support of the PLA.  So, there were some ones that weren't so good; whereas, the lots of the grass were actually standardized products so the manufacturer really had the thing down right.
	So, it is really also nice that you actually got this slight change here.  These guys are doing a good job.  
	This is just a plot of the way that looks now.  So, you know, here is the .5 to minus .5 and -- well, log relative.  So, our -- this is in log RP.  A relative potency of .5 as a minus log RP and minus .3.  So, you can see this is through the mite, that the reserve for the mite is well within the factor of 4 and if you actually work backwards and say what is the sample for the mite, which is the green guy, this is well within that.
	So, you see where now we feel safer that these manufacturers are turning out lots that are well within this factor of 4.  We need to go a step further.  This is the last of this quartet.
	It is solvable.  So, now let's just think of a little problem you might give your kid or something.  It depends on the kid.  But if you have distribution and you want to pick two samples from that, what is the range of samples that you expect to get?
	Well, qualitatively, you kind of expect to get, you know, one kind of in the middle and another one off to one side.  So, you know, expect something like the standard deviation of samples, about.  What is less likely is, you know, one way or one end and one here and what is not likely at all is to get one from the far left and like one from the far right.
	No, how do you solve that problem?  You know, you just do it.  It is like you are trying to crank basically.  So, what the result ends up being is that your average range, which one calculates as an average time to density itself is given by this and then we actually define a maximum range that is worth just spending a second more on.
	This is like saying, okay, what is the range such that 95 percent of the changes are smaller than this are prime.  So, now we say all right, we want it so that 95 percent of these bottle changes are less than this number.  Then you can work it out in terms of the -- you have very simple expressions for the range, where this average range is, you know, .8 sigma, like I said, about a standard deviation and this maximum range is about 3 sigma.  
	So, it should be kind of comforting.  It is inherently reasonable.  You would have guessed that by yourselves.
	Now we can look at what are the ranges of our extracts.  If we actually look at the sample range now, just plugging in the numbers from the grass pollens, remember, we had this .09 in log relative potency.  You can now calculate your average range and average R prime in terms of log RP, but, you know, I think it is good to think of it in terms of what kind of change in like of relative potency you have got.
	But the grass pollen is -- you know, you send like two bottles out to the population and ask for what is the change in that, your average change is around like a 20 percent, just like maximum.  Ninety-five percent change is like less than a factor of 2.  If you look at mites, it is even smaller, only 12 percent, and about 50 percent.  So, in other words, the only -- I realize often it is hard to listen to math, but the bottom line is that the change in the relative potency for all these x axes is much less than a factor of 4.  That factor of 4 is what we determined was like good for safety.
	So, now we are track.  So, now let's go back to that original question.  If you had a relative potency of a lot that was really .7.  The manufacturer tested it at .8 and passed it.  We get .6 failing.  Now we can answer the question was this all right.  The answer is "no."  There was overly much risk.
	So, we have to do something because we already show this factor of 4 in terms of safety is fine.  Now we are failing lots of the manufacturers.  So, we need to do something.  So, we came up with this.  There are various things one can do.  We thought this was pretty reasonable.
	Basically, we have the manufacturers maintain their limits because they are already doing it anyway and we have shown that with these limits, we get a nice tight distribution.  Because their distribution is tight, CBER can like widen it.  So, now we would say, okay, forget about that .654 stuff.  You get .52.  So, we haven't compromised public safety and we have reduced the manufacturer's risk.
	The advantage of this is that you will actually still get the every once in awhile lot that, you know, something slips through.  The darn stuff is .3.  You have to sift that.  But, basically, we can act more of as a sieve to the really extreme lots and actually -- as opposed to like rejecting a lot of stuff, you know, it is probably okay.  We haven't compromised safety.
	We will actually certainly reduce the number of lots we reject, you know, the acceptable lots.  Then this one last one is kind of interesting because if a manufacturer does a better job at making the stuff, there is even a lower probability that it will failure CBER release.  And here is how.
	So, a manufacturer's test with n equals 3 on the ELISA assay and say he gets .699, which is a 95 percent confidence interval of that, if that lot is passed over CBER, there is only about a 10 percent chance that that lot will like fail.  That is reasonable, I think, because, remember, if it is tested at .699, it sort of could be lower.  Right?  So, you want to weed out  stuff.
	However, he tests n equals 6 and if it turns out to be .7, then the chance of failing it is only like 7 percent.  You can look at the various numbers.  They are in the guidance document.  Then there is some fine print explaining how we do this, you know, why that is true.
	So, just a couple of slides and sort of last comments and, hopefully, look at it now from a bigger picture.  So, what have we done?  Well, we like placed our limits so that this red one gets like filtered out.  See, these limits now are .5 to 2, which in log relative potency is plus .3 to minus .3.  Now, just look back for a second.  Remember, the observed relative potency for the graph was .12.  The observed relative potency for the mite is .1.  So, effectively we say we are going to set our manufacturing goals at 2.5 sigma observed for the grass and a 3 sigma of the mites.
	So, that is, you know, fairly FDA-wide policy anyway.  So, it is sort of interesting that it worked out that way.  We came through it with a different way.  I would also like to say, though, this is science driven.  We weren't set on widening these limits.
	If, in fact, the -- or for a new case, not just a new standardized product -- say if the relative potency of this material had been very broad -- I just made something up here, where this is, you know, a good chance to be .5, a good chance to be 4, what do you do then?  You can't try this thing of anything from .5 to 2 makes it because something that tests at .5 could be .3.  Something that tests at 2 could be 2.5.
	So, what we would do instead is do what we were originally doing.  We would draw a line between .7 and 1.4 and we would sort of sift it that way.  So, there are different ways you handle different materials.  You could say, well, gee, how come you like did that, did this equivalence now, you know, then and how come you just didn't do the .5 to 2 earlier?  Because we didn't have the data.  So, now because we have the data, we can widen it.
	Lastly, I just think it was interesting.  We actually looked at the labels of the manufacturers just to see what the range of potency for the unstandardized things were.  This is just on the labels.  You can read them yourselves.  Go buy a couple of bottles.  Open them up.  There we can actually look at that safety range again.  So, it just lets us do one quickly.  You can see that here for the water extracts, the range of like relative potencies is actually a factor of 9.
	Now, for the glycerinated, it is 5.  As you go down this road, you can see there was a factor of very high, 83.  So, here is this like range.  So, you can basically see this is a vindication of actually what we did.  We actually reduced this in a significant manner.
	It is almost reasonable after you have done something to say did we really have to do this.  We could ask now the question, this ELISA, could we just have used the protein content, which is, you know, pretty easy as opposed to the immunological assay, which is, you know, actually not more about the site and a lot of work.
	So, one of the ways you can look at it, again, now that we have the standardized results, we can actually plot the value of the relative potency.  Now, these all had a relative potency, which were in the confidence limits of like 1.  So, these are all equivalent to 1.0.  So, we can see that for lots of this relative potency was 1.  The protein content, you know, goes from, you know, something like .3 to 12, a huge number for -- that is 172 lots of grass, but 188 lots of mites, we have the same sort of result, you know, about a factor of 10.
	So, in other words, it is shown pretty clearly for grass and mites.  It might not be true for something else.  In fact, you really do have to use ELISA not protein content.
	In conclusion, I hope I have shown you where our thinking is in terms of these risks.  By widening limits, we have lowered this without changing this.  Last, I think I have -- you know, we have tried to tell you that the variability is unstandardized and then this is the way we will do things for each of these new products and put it through the same kind of thinking and, you know, hopefully, we will get things right.
	Thank you very much.
	DR. OWNBY:  Thank you.
	Are there any questions for Dr. Pastor at this time?
	Dale.
	DR. UMETSU:  It seems if I understand this correctly is one of the problems is due to the fact that the ELISA assay is not as reproducible as we would it to be.  So, you are saying that instead of doing the assay three times or in triplicate, you can do it in six duplicates to get a better value.  Why not just do it nine times to get a much more precise value so that you can still maintain relatively small specifications for each lot?
	DR. PASTOR:  Because to do it nine times is a lot more work.  With every ELISA you do, you actually have to use reference standards.  If you do nine, it is actually really like three times as much work as three because, you know, the way the plates are is you get kind of three per plate.
	So, you can say do you add significantly to the safety of the product by doing nine as opposed to doing three.  The answer was "yes" and you really -- then you say, yes, we really have to do it but if doing nine doesn't really change much, then you don't have to do it.  It is really a balance all the time.  When one does testing and lot release testing in manufacture, you have to say what is the benefit.  
	DR. UMETSU:  Well, I guess I can understand that.  If your main assumption is safety, you are saying that you can tolerate a fourfold difference in lot variation, but if you are really trying to say we want a product that is standardized so that every lot is as close to the last lot as possible, you might want to sort of use a different parameter rather than safety.  Safety is important, of course, but --
	Remember, we actually did look at two.  We said in terms of efficacy, the first analysis can be a factor of 10.  So, safety was a factor of 4.  If efficacy had been a factor of 2, then we would have chosen that.  You really have to always say -- you know, it is a freshman chemistry kind of thing.  When someone gives a result in significant figures, you have to say, well, first of all -- usually with freshmen chemistry you say the answer is "no," but with this you say do you have to do the experiment such as you have six significant figures?  The answer is "no."
	On your car, if you are driving -- if the speed limit is 30, 35, you have to have, you know, 34.99968.  35 or nothing.  You know, I had another analogy with cars.  If you are setting a limit, what do you need?  Do you need a speed bump, a stop light or a toll booth.  You know, so you have to work these things out so that there is a balance.  N equals three is plenty.
	Jay might want to add to that.  It looks like he might.
	DR. SLATER:  -- higher level of precision and that is when you are replacing your reference.  In fact, when we replace the reference, we end up doing between 24 and 50 tests if you accumulate all the data that we get from manufacturers in order to achieve as great a precision as possible and, in fact, we can achieve a high level of precision because if when you are switching your reference, which is your standard material that you are going to use.  If you allow drift that would be clinically acceptable to occur, it could keep occurring in the same direction and then you might enter an unacceptable zone pretty quickly.
	But I think one of the key things here is the therapeutic index of the product is broad enough so that even though a higher level of precision would be satisfying from a measurement science point of view, it is really not justifiable from the point of view of the products themselves.
	DR. CLAMAN:  That was a good presentation.  I get stuck with the sigmas and the stuff like that.  I am debating whether I would buy a used car from you or not and at the moment that jury is out.
	I would like to ask manufacturers' representatives how important changing these limits really is.
	MR. HAUCK:  I can say, as far as Center Laboratories is concerned, I think it is a pretty good idea.  We have kind of been in support of it.  In fact, most manufacturers, I believe, are now using the n of 6 at some point during their testing.  So, I think the fourfold limit is very reasonable from their perspective.  It is not reasonable from our perspective.  We can't use it, but it makes sense when they are reviewing our data and our products.
	DR. OWNBY:  If there are no other burning questions, I would like to move on.  I would remind the committee we have plenty of time this afternoon to come back and rediscuss the ins and outs of this and I think there are some other ramifications of it that we may want to discuss.  We would also like to hear from the manufacturers' representatives, their position and any public comment on this before we try to come to any conclusions.
	I believe next on the agenda is Dr. Slater again.  Not quite a one-man show today.
	Agenda Item:  Regulatory Proposals -- Allergen Standardization
	DR. SLATER:  Thank you.
	In your hands you have a package entitled "Allergen Standardization Algorithm, January 2000."  I suggest you pull that out.  That will be helpful as we go through this.  
	I am going to make some preliminary remarks about allergen standardization and I am going to take you through this new algorithm,, which is our effort to take allergen standardization forward.  But before we can take it forward, we need to look at where we have been.  That is part of what is a process that really started nearly a year ago in terms of a critical review of the allergen standardization process.
	I think that it is safe to say that allergen standardization in the United States has been a major public health success.  We have, as you know, many hundreds of allergen extracts marketed in the United States about which little if anything is known about their actual true biological potency.  
	As a result of CBER's Allergen Standardization Program, which just for the record entirely preceded by many years my appearance in this lab.  It is clear that we now have 19 products that have been well-standardized, that are safer for which their potency is assured.  As I will come back to over and over and over again, the standardization process was essential not only for the clinician and the patients; it was essential for the scientists as well.  You can't do good science with these extracts until you know what is in them.
	The potency, the allergen standardization process has been critical for this.  This is a listing of the currently standardized allergens and the tests that are currently used to assess their potency.
	When we began to consider going forward with allergen standardization into the future, we wanted to look backwards carefully and see where we had been.  By way of organizing ourselves, we decided to as a group in the division look at the scientific results of allergen standardization, the so-called QC/QA results of standardization to decide where we were going to go in terms of building our experience and how to move forward with planning next steps.
	Clearly, the most important lesson that was learned scientifically from allergen standardization is that protein does not equal potency.  If there is a single take-home message of the standardization effort, it is that the unitives that have been used for decades prior to this was not a good indication of biological potency. 
	This is a slide that Rich Pastor showed you just a few minutes ago, in which we actually look at standardized grass pollen and mite allergens; the grass pollen at a hundred thousand BAU per ml potency, the mites at 10,000 AUs per ml potency and we looked at the protein content of these and we saw a tremendous scatter of protein content.
	If you eliminate these outliers, the mite situation is definitely tighter than the grass situation, but both of them clearly indicate that there is no question that we shouldn't go back to the system of looking at the protein as a measure of potency.  It simply isn't.
	Furthermore, again, this is a slide of data taken from the manufacturer's package insert that clearly shows that in the prestandardization days that there was a tremendous variation of potency, especially among the aqueous extract, but also among the glycerinated extracts that you can see for a wide range of products.
	One of the other lessons that we learned is that clinical testing is essential.  You can't construct an allergen standardization program unless you introduce material into humans and see what is going on.  You have to do something that relates to clinical activity in order to standardize product.  The prime example of this is the Bermuda grass pollen extracts, which prior to the clinical studies no one really understood that the maximal potency of these products was significantly lower than it is for the other grass pollen extracts.
	That labeling has now been incorporated into the labeling for the standardized Bermuda grass pollens.  We also learned that in vitro tests can serve as surrogates for skin tests and this seems sort of obvious now.  It is clear that you can do this, but it wasn't necessarily obvious that these tests, the last inhibition tests, the competition ELISA test would really serve as good surrogates for biological testing, that they have been.
	We also learned that different in vitro tests may be optimal for different allergens.  For some allergens, for cat and for short ragweed it is okay to look at a single allergen.  That is a good measure of potency.  It is a good surrogate for potency measures; whereas, for the others, for mites and for grasses, you need to look at a more global approach, using cooled allergic sera in the competition ELISA assay, looking at multiple allergens at the same time.
	So, we know that we can't prejudge the situation.  We don't know what tests are going to be best.  We have to study each specific allergen extract and draw conclusions from that.  Again, this has been known for decades that glycerol improves the stability of extracts.  Standardization confirmed that the potency was clearly maintained better in glycerol solutions.
	Another more recent study that we actually did in our laboratory suggests that the stability assessments may actually be method dependent.  In other words, you have to be careful what methods you use to look at stability.  This comes from a study that was done in the lab prior to my arrival, by Dr. Lin(?), along with Teresa Lu(?) and a follow-up study that was started by Dr. Soldatova in collaboration with several people in the division to look at the stability of mite extracts in solution.
	The original study that was done looked at this but it was noticed shortly after it was completed that the control that was used, the 4 degree control, was not really the optimal control, that we really wanted something that was more stable than 4 degrees to see whether the 4 degree specimens were stable as well.
	So, the design of this study, which was begun before I arrived on the scene was to take lyophilized mite extract products at time 03, reconstitute them in 50 percent glycerol, to store them for up to 12 months at minus 70, minus 24 degrees and, believe it or not, at 37 degrees, as well.  And then to check the relative potency specific allergen content measures and immunoblocks at 6 and 12 months.
	These are data that I presented in part last year and I am going to go through them relatively quickly.  The bottom line was that for all of the extracts that we examined and if we looked at the relative potency, using the standard competition ELISA, what we found was that at minus 20 and at 4 degrees, the extracts were indistinguishable from the reference. 
	In other words, there didn't seem to be any deterioration at minus 20 and at 4 degrees in any of the extracts that we reconstituted after 6 and 12 months, compared to the lyophilized material, which was freshly dissolved at the time of the assay.  So, these data were very reassuring and clearly suggested that the materials were stable.
	However -- and I am sorry for the way this comes out.  This is the one failure of power point to be able to take a scanned blot, but, however, when we looked at it by two other methods, we found different results and I am going to take you through these shadows and this is going to be an indication of how much you trust me after this whole morning.
	What we see here is that the -- this is the minus 70 sample.  This is the minus 20 sample and these two are the 4 degree samples.  What you can see here is the loss of these bands and of these bands as well in the 4 degree specimens.  The 37 degree specimen you lose everything, but nobody here is particularly surprised by that.  Likewise, this is another batch.  We also lost this allergen completely.  This is the Der P 1 allergen in both cases -- Der f 1 allergen -- I am sorry -- in both cases by immunoblock.
	Mind you, by relative potency we didn't lose any activity in these extracts.  So, this is a divergence of the answers that you get depending on how you look.  Then when we used monoclonal antibodies to measure Group 1 and Group 2 allergen content at 12 months, we also found some heterogeneity in that especially in the Der f specimens, there seemed to be a decrease of 40 percent, 50 percent, again 40 percent, in both the minus 20 and the 4 degree specimens, compared to the lyophilized material.
	Remember, the lyophilized material is not shown on this graph.  The lyophilized material is the hundred percent one.  However, in the Der p 1s, these were much closer to a hundred percent and less of a problem with deterioration.
	We actually plotted this in a somewhat novel way in order to try to explain a lot of data that was a little contradictory, to look at the relative potency using pooled allergic sera plotted against the relative potency that was allergen specific.  That is a new parameter.  Okay?  Normally, when we talk about relative potency, we are talking about pooled allergic sera, the standard way that we do this.
	But we also took the allergen specific potency, which was simply in one example the Der P 1 content of the stored material divided by the Der P 1 content of the lyophilized material that was freshly dissolved for assay at that moment.
	So, it is simply a ratio of the potency of the test material versus the standards.  And there are a couple of observations to notice here.  First of all, the Der f, the D.farinae specimens, the closed figures are the D.pteronyssinus specimens.  The first thing to note is this is a true scatter plot.  There is no relationship here.  The slope of these lines if you tried to draw one is not statistically different from zero.  There is no correlation between these two values.  That is observation No. 1.
	Observation No. 2, though, is that, gee, it is interesting that all these Der f 1s are down here and all these Der p 1s are up here.  There is, in fact, a significant difference between the two populations.  What does that suggest?  Well, it is not earth shattering from a regulatory point of view yet.  We haven't really worked this out yet, but it suggests that in D.farinae extracts, that specific allergens appear to degrade relative to the relative potency of the material at a faster rate than the Der p 1s, the Der p extracts.
	Conversely, it may simply mean that there is a better correlation in D.pteronyssinus between the content of these two specific allergens and the overall relative potency, than there is in D.farinae.  We don't really know that yet.
	But the take home message from our point of view is that there is a discrepancy between the results that we get, depending on how we look.  The relative potencies look perfectly stable at minus 20 and 4 degrees, but there were specific allergens that appeared to degrade significantly at 4 degrees and some of them even at minus 20 degrees.  These are other results of the study.
	Protease inhibitors did not appear to contribute to stability.  We know now that lyophilized extracts appear to be more stable in these particular examples than even the glycerinated ones.  But it is clear that the stability of glycerinated extracts is enhanced at lower temperatures.  
	Again, looking backwards at allergen standardization, what have we learned?  Okay.  From a QA/QC point of view, clearly the most important result that we have from the standardization effort is the development of U.S. standards of potency.  We do not have in-house references.  This is not consistency monitoring.  There is a single U.S. standard of potency with a common, industry-wide unitage for each allergen extract.
	What are the advantages of that?  Well, it clearly increases safety and consistency of the products.  It clearly facilitates scientific studies.  If you look at the scientific studies that we were able to cite when we were trying to plot out the dose responses, it is no accident that most of them are Amb(?) a 1.  Those are the only ones you could do it with for awhile, because if you don't have a standardized material to work with, you simply can't do dose response by these.  You can't learn about the extracts in an organized quantitative way, unless you have a U.S. standard or some standard that you are comparing these with.
	Certainly, having a U.S. standard, facilitates scientific studies that can be done in Seattle and replicated in Bethesda with reasonable assurance.  Furthermore, having a single U.S. standard clearly enhances product choices for individual practitioners to go among different manufacturers.  In addition, QA/QC results include the development of standard operating procedures for testing methods.  We now have SOPs for testing all of these allergens and that allows us to have true quality assurance and quality control of the development of the potency measures.
	In addition, we now have stability monitoring programs in place for these allergens.  So, looking back on allergen standardization, what is it that we clearly want to incorporate as we build on our experience and move forward.  Well, clearly, we want to continue the standardization effort.  I am sure that is not a shock to anyone in the audience that I would say that.
	But, clearly, we want to move forward with allergen standardization and we want to move forward with the development of U.S. standards of potency.  In addition, we clearly want to keep clinical testing as the gold standard by which we measure all other tests.  How do we move forward?  
	Well, the first step is to try to build consensus, to construct a transparent process, which I hope you will feel what you have in your hands is a transparent process.  We want to identify specific decision points at which we would be able to change our minds.  In other words, as we embark down the road of standardization of product, one of the things that we wanted after much discussion to build into it was an intrinsic way in which we might decide for either good reasons or bad reasons to abort a particular standardization campaign.
	I will go into that in more detail later.  We certainly want to consider an increased role for industry and for other collaborations.  We want to make sure we choose the most stable references possible for reasons that I shared with you early on.  Given the studies that Rich and I have done, the analyses that Rich and I have done that you have already heard about both this year and last year, we certainly want to begin the process of considering clinical data in setting rational release limits.  
	Finally, we would like to consider for future allergens to consider enlisting the help of the U.S. Pharmacopeia in handling and distributing references.  So, the purpose of the algorithm that you have in front of you is to build upon our prior successes, which are substantial.  We want to establish priorities and procedures by which we can go forward as quickly and as efficiently as possible.
	If I had to identify some new aspects for this algorithm -- I mean, a lot of what you will see in the algorithm is just what was done already, but if I had to identify something new in the algorithm, I would say that we are making an attempt to state clearly at the outset what our criteria are for allergy selection.  We are going to attempt to delineate responsibilities for different parts of the allergen standardization process.
	Finally, we are going to be able to set exit points for reasons that I will outline momentarily.  Let's talk about the impact criteria.  The impact criteria were the subject of a great deal of discussion and the important thing that I want to state about the impact criteria is that they are not meant to be exclusionary.  The idea here is to give us an organized way of talking and thinking about allergens and I suppose the most obvious way to think about this is if you want to look at one impact criterion, think about two extracts about which everything else was equal.  We would like to focus on that one in which they unequal and make a decision based on that.
	What are the impact criteria that we agreed upon?  First of all, the availability of stable, preferably lyophilized materials for use as long range reference extracts.  Second, the consistency of currently marketed products needs to be taken into consideration.  Again, that probably requires a little explanation.  
	All other things being equal, if one product is remarkably consistent across the market, as we observe it in the unstandardized state and another product is dreadfully inconsistent, such that switching from bottle to bottle would pose a true safety risk to individuals that might get it, we would clearly want to focus on the inconsistent products.  But that doesn't mean that is the only thing we would look at.  It is certainly possible that a fully consistent product across the market might be, in fact, mediocre and we might need to standardize it to bring the level of the product up.
	We certainly want to consider how widespread the product is used.  Clearly, if we are establishing priorities, we want to standardize products that are used a lot as diagnostic and/or therapeutic reagents in the United States.  We want to know about the number of manufacturers producing a product.  We want to consider the potential use in immunotherapy for which we would give it a higher priority or perhaps just a diagnostic reagent which it would have a somewhat lower priority.
	We want to know what the public health impact of the correct diagnosis and/or adequate treatment would be.  Again, just to emphasize the point, there is nothing in here that is intended to be excluded.  For instance, one could easily imagine with peanut, for instance, which is not currently a therapeutic reagent, but just a diagnostic agent, the decision might be made that it was of such importance to go ahead and standardize it, that we would do that in spite of the fact that it is not currently used for any other therapy.
	Furthermore, we are very sensitive to the issue that when you standardize a product, you can encourage the science that can lead to further studies, that can lead to expanded usage of the product under appropriate procedures.  So, I just want to make sure it is quite clear that these are -- that none of these is intended to exclude anything.
	Theoretically, we could even decide to standardize a product that nobody makes, but that we know that it is of such public health importance that if we standardize it, manufacturers would come in and would be willing to produce the product.  So, there is a short form of the algorithm and a long form of the algorithm.  The short form is a lot easier to look at.
	Basically, it starts out with the preliminary region of identifying target allergens, using the impact criteria.  We go into a laboratory phase, a clinical phase, a manufacturing phase and a post approval phase.  At both the laboratory phase and the clinical phase, there are ways that we can decide that we don't want to proceed with this allergen that I will go into in greater detail.
	This is completely useless to put up except to say that I am now going to talk about the first part of the algorithm.  In the preliminary stage, we identify the target allergens using the criteria that I discussed.  At that point, CBER would ask to be sent multiple lots of the target allergen by each manufacturer.  Three lots is arbitrary, but we would certainly like to get an idea across the industry as to what was going on with this particular product.
	We would also be interested at that point in receiving products from other sources, from academic laboratories that study the particular product, from the NIH, from industry R&D departments and we also reserve the right to make our own product if, for some reason, we are not fully satisfied with what we are seeing.
	We would then do a series of comparisons among the products.  We would have to develop and adapt methods for allergen determination.  We would compare the allergen content of the different lots and then this part sort of then feeds back into the impact criteria.  If, for instance, we find that everything is remarkably consistent within the industry, we might wish to reconsider the impact or, better yet, if the commercial products are somehow comparable to the best material that we could get from, say, an academic lab or from other sources, then, again, we might reconsider the impact. 
	But, again, this decision would have to be taken fairly carefully based on some analysis and understanding of what is actually going on with the allergen and how good the allergen is out in the market.  In the clinical phase, Phase 2, we would initiate some clinical testing.  Very likely, the predominant testing would be intradermal skin testing by the model that has been used extensively, although it is possible that histamine release data might be used and, furthermore, it is also possible that some limited immunotherapy data might be sought in terms of helping us to establish biological unitage and ideal dosing ranges.
	One important dropout point here is that let's just say that we discover, based on the clinical testing that there is almost no evidence that this material is related to IgE mediated disease.  It is a possibility, after all, that we have to entertain.
	So, not only would we be willing to reconsider the impact of standardizing and stop the standardization effort, but we really need to take it a step further and consider getting this product off the market entirely.  So, withdrawing our standardization effort is sort of a 
-- you know, a little bit of a carrot and a little bit of a stick as well.  But we need to be open to the possibility that we will learn as a consequence of our studies that Product X really shouldn't be offered in either standardized or under unstandardized form.
	Now, moving on to the bottom part, the manufacturing phase, the idea here is to move into the real guts of how we are going to regulate the product and that is to develop in vitro surrogate testing.  As you can imagine from the first hour or so of my talking this morning, I am open to any kind of testing that would be a good surrogate.
	Based on previous experience, it is likely that immunologic testing is going to end up being the best surrogate, but it may be, if we are very lucky, that we could use some physical, chemical methods that would be a good surrogate.  By "surrogate," I mean, it would have to correlate to the clinical testing that had been done in the previous phase.  We would then select the provisional reference standard.
	This would be validated for testing and the standard would be validated by CBER and industry and then finally at the end of this phase, we would select the reference, establish release limits and initiate stability testing.  In the post approval stage, we will continue the stability testing, initiate safety monitoring of the products and then we would start to look at equivalent dosing.
	Remember, we were talking about earlier -- what Rich Pastor was talking about earlier, about using clinical data to establish release limits.  Certainly, in the absence of clinical data, we would want to use the best, most accurate testing available.  But once clinical data start to accrue with particular allergens, we would definitely be open to the possibility of looking at equivalent doses and establishing the release limits or modifying release limits based on that knowledge.
	So, that is the algorithm.  Let me just make another general comment about the algorithm itself before we go on to specific allergen standardization targets.  The algorithm is not contract precise.  It doesn't tell us exactly what we would do in every single situation.  The function of the algorithm, as far as I am concerned, is to enshrine what is essential about standardization and not let go of it.  And, yet, to be flexible enough to deal with a whole variety of standardization issues that may well come up as we look at a new generation of products to standardize.
	It is not clear what surrogates we are going to use.  We won't know that until we actually look.  It is not even clear necessarily what best clinical tests are going to be to use, although certainly our initial bias will be to use the intradermal skin testing, since we have so much experience with it.
	Yes, sir.
	DR. CLAMAN:  On a crass commercial level, who is going to do the clinical testing and who is going to pay for it?
	DR. SLATER:  That is not a crass commercial question at all.  It is a very good question.
	I am actually going to defer the answer to that question a little bit until I finish the next part of the talk because I think it will come in relevantly.  The short answer is we could do the testing.  We could pay for the testing or we could contract out and have the testing done elsewhere.  The advantage of contracting out is that we do want to make sure we have some geographical diversity in the individuals that we test.  So, we would have to do at least some contracting out and we are prepared to do that.
	I will put off the who is paying for it for just a minute if you don't mind.  So, what we really want to do is really want to enshrine the best about standardization without fossilizing the process.  I really wanted this to be -- and the consensus among the group that we wanted this to be as flexible a process as possible, but, in addition, giving us guidelines as to where to go.
	In terms of specific next standardization targets, I just want to review briefly what this committee -- the discussion this committee had in the 1998 meeting at which potential next standardization targets were discussed.  At that time, the allergens that I have up on this slide were prioritized roughly from high to low in terms of next possible targets.  They included latex, cockroach, tree pollen, peanut mold and dog.
	A new element has come into our discussion of standardization however and that is the strategic plan of the Department of Health and Human Services, dated March 1999, entitled "Action Against Asthma."  Now, this is a strategic plan by DHHS that was initiated sometime before at the request of Secretary Shalala, who had identified asthma as a major public health concern of the people of the United States.
	Toward that end, a large group of people, among them, CBER was represented by Dr. Paul Turkeltaub, a large group of people from DHHS got together to formulate a plan to go forward and see how the Department was going to deal with the problem of asthma in the United States.  Clearly, NIH, FDA and CDC are playing lead roles in this strategic plan.
	Under Dr. Turkeltaub's guidance, the group recognized explicitly that allergens played an important role in asthma and that allergen standardization clearly played a role in the improved treatment of asthma in terms of the improved diagnosis, allergen avoidance, better safety and immunotherapy and,  lest I beat on this horse anymore, better science with the effort of standardization. 
	We know that allergens and asthma are connected.  The case is strongest among the indoor allergens.  The case is strongest still among two indoor allergens that have already been standardized, the dust mite allergens and the cat allergens, but there are very compelling arguments to be made for the role of cockroach, molds, dog and even some outdoor molds in the etiology of asthma in certain children and adults.
	We were asked to come forward with a standardization proposal in response to the asthma initiative and this is the proposal that we have put together subject to the comments of the advisory committee.  That proposal is over the next three to five years to standardize cockroach, aspergillus fumigatus and alternaria alternata as three potential choices in response to the asthma initiative.
	DR. SAXON:  Which cockroach is it?  There are more than one of them.  Are you talking about several species?
	DR. SLATER:  Yes.  I am talking about several species.
	I am sorry?
	DR. SAXON:  Two or three?
	DR. SLATER:  Well, two or three.  So, the wateladramatica(?) and the paraplanata(?) mericana(?) are the two major ones.
	DR. SAXON:  The list is actually a little bit longer because --
	DR. SLATER:  That is right.
	But certainly cockroach is ubiquitous.  It clearly is associated with asthma, especially inner center asthma.  If there is one lesson that I have learned from the inner city asthma study it is that you can't eradicate cockroaches.  It is one of the most dramatic scientific results of the inner city asthma study is that you can kill them but you can't reduce the allergen exposure.
	So, I don't know how many of you heard Payton Eggleston(?) make his presentation last year about their efforts to eradicate cockroaches, but it was rather strikingly depressing.  So, if you are going to control the problem of the immune response to cockroach as a possible etiology for asthma, it seems to me that immunotherapy is an important way to go and if we are really going to learn anything about it, we need to standardize these allergens.
	As you said, there are several species that are known and several cloned allergens as well.  So, it seems to me to be a reasonable target.  Aspergillus fumigatus is a fungus that grows in both and the outdoor environment.  It is clearly involved in several allergic or hypersensitivity diseases.  It is involved in certain forms of allergic asthma, allergic sinusitis, allergic bronchopulmonary aspergillosis, as well as hypersensitivity pneumonitis, particular molt(?) worker's lung.  
	So, clearly it is an allergen that is important.  Several allergens from aspergillus have been cloned and, again, it seems like a reasonable target in terms of asthma.  
	Alternaria alternata is mainly an outdoor soil allergen.  It is highest in the grain-producing states.  There have been striking reports of fatal asthma associated with exposure to alternaria allergens.  Again, alternaria has been -- many of the allergens have been well characterized and it seems like a reasonable next target for allergen standardization in response to the asthma initiative.
	So, again, in response to the asthma initiative, which as you can imagine has some funding attached to it and so it is something that can help us more forward rather quickly with this, we have tentatively proposed to go forward with the standardization of cockroach, aspergillus fumigatus and alternaria alternata.
	We are now a minute late for our break.  But I am just going to take about 30 seconds more just to review the three questions that we are going to ask the committee to discuss in the discussion session this afternoon.
	Question No. 1:  Should CBER expand the lot release limits for standardized mite and grass pollen allergen vaccines to 0.5 to 2.0, as described in the draft "Guidance for Reviewers, Potency Limits for Standardized Dust Mite and Grass Allergen Vaccines:  A Revised Protocol"?
	Question No. 2:  Should CBER implement the proposed algorithm for the standardization of new allergens?
	Finally, Question No. 3:  Should cockroach, alternaria alternata and aspergillus fumigatus be selected as standardization targets, given the support of the DHHS asthma initiative?
	DR. OWNBY:  Are there any immediate questions for Jay before we take our lunch break?
	DR. SAXON:  Are we going to have a chance to question him in the afternoon?
	DR. OWNBY:  We can grill him all afternoon, yes.  We will also have time for comments from other interested parties that may pertain.
	Okay.  Then I think we have an hour for lunch.
	[Whereupon, at 11:38 a.m., the meeting was recessed, to reconvene at 12:40 p.m., the same day, Thursday, February 10, 2000.]

A F T E R N O O N   S E S S I O N	[12:40 p.m.]
	Agenda Item:  Open Public Hearing
	DR. OWNBY:  The committee is now open for public comment on any of the matters that were on today's agenda and I would encourage any members of the public or industry that would like to address the committee to let me know and we will take each of you in turn.
	I am hoping we don't have to worry about any major time constraints on that.  The committee does have, as proposed previously, three other items to discuss that are related, but let's have public input first.
	Yes.
	MR. HAUCK:  Thank you, Dr. Freas, for the opportunity to speak today.  I will be very brief.
	I am Peter Hauck and I am the president of the APMA.  The APMA is the Allergen Products Manufacturers Association.  The APMA is a non-profit trade association.  It consists of nine member companies, as well as eight associate members.  That is, the nine member companies are all of the nine U.S. licensed manufacturers of allergenic extracts.
	The associate members are other North American and European manufacturers and distributors of allergenic extracts, as well as raw material suppliers.  I provided the committee a list of our members, which is this document here and the last page are those of us who are foolish enough to be officers of this organization.
	Our association was started in 1987, recognizing the need to cooperate on issues that affect our industry, primarily dealing with scientific and regulatory concerns.  Our members meet regularly with FDA to participate in workshops and meetings, to exchange data, information and perspectives on various issues associated with these allergenic products.
	We have also provided speakers and sponsored workshops in seminars on immunotherapy and allergen standardization at both the academy and college meetings.  My sole purpose in being here today is to just tell the committee, let you know that we are a resource for you. Understanding that there are two sides to every coin, we encourage you to contact us on issues that may come before this committee.
	In that regard, I have also made a list of the various interest committees that we have in our organization with the individual chairpersons of those committees, along with their affiliations.  Feel free to contact them directly or any member of the executive committee.
	That is really all I had.  I am prepared to answer any questions that the committee may have on our organization or any particular position that we may have on any issue.  That is all.
	DR. OWNBY:  Are there any questions for Mr. Hauck?
	DR. SAXON:  How long have you been involved with this committee, the APMA?
	MR. HAUCK:  Since it is -- me personally?
	DR. SAXON:  Yes.
	MR. HAUCK:  Since its inception.
	DR. SAXON:  So, what do you think -- I mean, there have been some change now.  I have only been a member of this committee for a couple of years.  What do you think of this change?  How do you think it is going and what --
	MR. HAUCK:  I think as far as the allergenic -- the Laboratory of Immunobiochemistry -- those folks over thee, I think they have -- the last three or four years have made really remarkable progress.  They are a very good group of scientists, good people, very open dialogues.
	We support most of their work.  We may have issues, some regulatory issues that we take issue with.  We may have some logistics problems, but by and large we are very supportive of it.
	DR. SAXON:  You think the industry is pretty much on board on this standardization stuff?
	MR. HAUCK:  Yes.  We absolutely support the standardization of allergenic extracts.  On the other hand, there may be logistical issues or regulatory issues with that that we may not necessarily agree with.
	DR. CLAMAN:  Would you like to give me an example of a regulatory issue on which you don't agree?
	MR. HAUCK:  Sure.  How standardization is implemented can be an issue.  In other words, if it is considered a change in the regulations, there should be ample time for notice and comment.  And there are issues, regulatory issues that surround this that I would rather not go into that are part of the citizen's petitions.  That is an example of a regulatory issue.
	A logistical issue might be something like, well, we don't have enough of this reagent or we keep changing reagents and how does that affect our stability studies?  So, those are some examples.  Does that answer your question?
	DR. CLAMAN:  I guess.
	DR. OWNBY:  Do you have a follow-up question, Henry, or --
	DR. CLAMAN:  No.
	DR. OWNBY:  Sam.
	DR. LEHRER:  With regard to the standardization of allergenic products, Jay had mentioned a couple of products that they were proposing for standardization.  From your perspective, from the industry's perspective, do you have -- I think you have a pulse on what the public uses the most and what is not standardized.
	MR. HAUCK:  Yes.  I think we should be part of this equation, part of the algorithm.  Again, we are a resource not only to the committee but also a resource to FDA as far as maybe getting some input on what are widely used allergens.
	Beyond that, I think, not necessarily speaking for my organization, but in the general sense, looking at these group of three allergens, there was an initiative four or five years ago with cockroach, where a couple of manufacturers got together with FDA, developed some serum pools, started the standardization initiative and that kind of fell short for a number of reasons.
	So, I think most of the manufacturers, since they have -- many of them have developed serum pools have maybe internal reference preparations.  This might be an easy one to standardize.  As far as the fungi go, that might be biting off a bit more than you can chew.  I would encourage you to come up with a backup plan in case the alternaria and the aspergillus don't work or if it seems to be a little tough.
	I know a lot of the manufacturers, for example, have birch(?) systems.  Certainly, Sam, you have got a shrimp system and there is probably, you know, some kind of good reagents for standardizing peanut.  So, those are all things that maybe you want to -- you know, that you really talked about, Jay, as part of the equation.
	I just get concerned when I see molds up there for standardization.  That is a tough one.
	DR. SHAPIRO:  I hear rumors being spread about companies dropping out of the antigen manufacturing business and also about the withdrawal or the lack of availability of more and more allergens.  Again, I don't have any specific source but there is some concern in the allergy community.  Could you tell me something about what that means?
	MR. HAUCK:  Sure.  It is not a rumor.  It is true.  It becomes increasingly more expensive to produce these allergens.  That is one part of the equation, to keep them in compliance with what you would do with making an injectable drug.  I think it is clear that we have to manufacture these products to a higher standard than we have in the past.
	So, that takes a bit more money.  In the sense that there are fewer allergens, yes, manufacturers have looked at their product line and say, well, I can't make night blooming jasmine because I sell three vials a year and it costs me $2,000 to make it.
	So, sure, we are all trying to limit our product line to those items that are more common.  Another problem, which is a little more complex is there are fewer and fewer collectors of these source materials.  Because the price of these products has been depressed, it is very difficult to convince somebody to go out and collect pollen because it is a very tedious job and it involves some art as well as some science.
	So, the pollen collectors that are out there, the major firms that are pollen collecting companies are going to concentrate on those products and those source materials they collect well and that they can collect and make a buck on.  So, there are -- it is going to be more and more difficult or perhaps more and more expensive to purchase some of the more less commonly used items.
	Now, it doesn't mean that they are important.  They could be very important to a few of your patients and certainly a hundred percent of one patient is very important to that one individual.  But option may not be available anymore.
	I have some colleagues -- am I speaking correctly?
	PARTICIPANT:  Yes.
	DR. SOTO-AGUILAR:  I would like to know the process of development of any allergen extract in the manufacturer -- I was involved with the skin testing for standardization of one of the extracts a few years ago.  So, I became familiar with, you know, the skin testing method by Dr. Turkeltaub and I found it extremely interesting and very effective, but I would like to know what is the time line that you have.
	How do you come to have the ideal solution for certain allergen?  Then at which time is that allergen presented to the FDA for skin testing?  At which time is that lot selected and then sent for review and how much volume do you have of any particular lot that eventually if it is approved, will be available.  I mean, what is the process?  Is there any way to know about it?
	MR. HAUCK:  I think the process of standardization in general is more or less left to FDA and it is a consensus.  They more or less -- we get together and see -- you know, they tell us what the allergens are and then we see if we have the reagents to develop and it is a collaborative effort.
	Skin testing is certainly part of that.  That is the in vivo part.  But the initiative can come from industry.  It can come from FDA.  It can come from the allergist community.  So, I don't know --
	DR. SOTO-AGUILAR:  And then once a lot is approved, how much volume do you have at least a minimum in order to be feasible to be marketed, a lot in volume and, you know --
	MR. HAUCK:  Well, a lot could be -- it could be -- after the testing is done, it could be one vial.  I am not sure exactly what you were getting at.
	DR. SOTO-AGUILAR:  No, I am talking of the manufacturer, how much volume of that particular lot or batch will be available if it is approved, if it passes all the standards?  
	MR. HAUCK:  The amount of product that is sent out for lot release is minimal.  It is 20 or 30 mls.  So, it is whatever the manufacturer's batch size is, based on his projection or his market forecast for his percent of the market.  So, it could be a 5 liter batch.  It could be a 10 liter batch.  It could be a 20 liter batch.  It could be a 20 liter batch.  It could be 10 vials, a hundred vials or a thousand vials or maybe 2,000 vials.
	DR. SOTO-AGUILAR:  There are no set limits for that or minimum?
	MR. HAUCK:  No.  There is no minimum.  There is, obviously, an economic minimum.  I mean, I am not -- our company is not -- my company is not going to make two or three vials of something because by the time we are done with all the testing, those vials are going to be very dear to us because of the amount of labor and overhead into that.
	So, I guess I am not answering your question.
	DR. SOTO-AGUILAR:  Maybe I am not expressing myself well.  
	We talked about how many lots were tested.  Seven --
	MS. BLOOMER:  My name is Diane Bloomer.  I am with ALK-Abello.  Maybe I can give some clarification here.
	I think, Peter, you are talking about existing standardized products and what we have to go through.  And you are talking about how we would get a new standardized product or even bring a new product that is neither standardized nor non-standardized to market.
	DR. SOTO-AGUILAR:  Just a new product.  I mean, or a new batch or whatever is being tested, like the 477 lots that were tested in 1999.
	MS. BLOOMER:  Those were standardized products.
	DR. SOTO-AGUILAR:  They were standardized, but do they represent large volumes of batches?
	MR. HAUCK:  Generally, they do.
	MS. BLOOMER:  Yes.  They usually are fairly large size because we send in a specific amount to the FDA for their testing and then the remainder of it is for our stability studies and then for marketing.
	DR. SOTO-AGUILAR:  Which is the minimum that you have to have for any particular --
	DR. SAXON:  There is no rule about that and no one has suggested there ought to be a rule.
	MS. BLOOMER:  We don't want a rule.
	DR. SAXON:  It is the manufacturer's business to, you know, take care of how many -- whether they think there is going to be 10,000 vials of fahia(?) grass or 1 vial of this.  I mean, that is a marketing decision.
	PARTICIPANT:  -- straining the resources of FDA by the lot release --
	DR. SOTO-AGUILAR:  I am just thinking on the physician using the lot, buying the lot and having comparable lots to using the patients.  Like if I buy it now and then I buy it in six months and then you have got to -- we are always going to be talking of different lots.
	DR. SAXON:  That is why the FDA standardization program exists, so you don't have to worry about that, so they don't have to make one huge lot to last ten years.
	MR. HAUCK:  If you have a standardized product and if we look at the data we saw today from Dr. Pastor, it shouldn't really matter whether it is lot A or lot B.  If you do some, you know, maybe some minor dosage accommodations, going from lot A to lot B, but essentially when you prescribe a medication to a patient, you don't worry about what particular lot that patient is getting.
	So, I think the idea with standardization is -- at least the philosophy is to have some confidence that the product you are getting meets a certain standard of potency and it is more or less interchangeable from lot to lot, at least from the same manufacturer.
	DR. SOTO-AGUILAR:  Right.  I have that very clear in my mind and that is the reason we are here.  It was just for -- I was talking in the long term basis.  We are talking of lots that, hopefully, will be stable for ten years.
	MR. HAUCK:  Those are references.  Those are not --
	DR. SOTO-AGUILAR:  Those are the reference.  Okay.  All right.  Thank you.
	DR. WRAY:  I would just like to raise the question of latex again.  I understand it is not one of the priorities with asthma, but still is an important allergen that we need in clinical practice.  I wondered if any of the manufacturers are close to having a standardized product that might mean that we really should push on with that.  That may be --
	MR. HAUCK:  I can only tell you what I know.  There is one manufacturer that is involved with latex and they have done clinical trials.  They have done a lot of work with it.  If it is licensed and approved, it should be commercially available.
	DR. SAXON:  I don't think the manufacturers -- I think we need to address those questions to the FDA personnel if you want.
	DR. WRAY:  Well, it has to do with the priorities, though, as to what is going to be next on the plate.
	DR. SAXON:  I don't think this gentleman is in the loop on that.
	MR. HAUCK:  I don't think I should be.
	MS. BRIDGEWATER:  [Comment off microphone.] 
	MS. LIBERA:  Have you projected the type of savings that manufacturers would have with reduced lots that were thrown away?
	MR. HAUCK:  With Dr. Pastor's suggested changes, certainly there would be some savings.  It just -- but I personally or my company were -- as far as I know, the industry has not projected that.  But it just makes such imminent good sense in my mind.  I haven't really looked at the economic end.
	DR. OWNBY:  Do you think you could give us an estimate of the economics?  Are we talking hundreds, thousands or millions of dollars?
	MR. HAUCK:  If you throw out a batch of mite, it definitely gets the attention of the accounting department.  The source material per mite is about four times the price of gold.  You might remind your patients of that, too.
	DR. OWNBY:  Dr. Claman.
	DR. CLAMAN:  Mr. Hauck, you mentioned something about a citizen's initiative.  Is that the asthma initiative that you were talking about?
	MR. HAUCK:  No.  This is a legal thing.  This is what is called a citizen's petition.
	DR. CLAMAN:  And what does it say?
	MR. HAUCK:  It is a rather document drawn up by a lawyer.  It deals with two issues, the logistics of standardization and it deals also with the way standardization is done.
	DR. CLAMAN:  Does it deal with matters that we are discussing at this meeting?
	MR. HAUCK:  I don't know that it is terribly relevant to what is going on at this meeting.  It deals with more of the regulatory concern about notice and comment when you make a change in the regulation.  It is the perception of the majority of the membership of the APMA that when you standardize a product, how it is standardized and so forth, it should be in the Federal Register, but that is not why I am here today to discuss this -- that topic.
	DR. CLAMAN:  I gather that you as representing the manufacturers are not overly enthused about a mandate to do molds and to standardize molds.  What I would 
say --
	MR. HAUCK:  I would love to do it.  I am just saying I just think it is very difficult.  
	DR. CLAMAN:  Well, I would say -- I am not a chemist and I have forgotten most of the formal talks that I have listened to in the past x, y, z years, but I remember one a few years ago about mold allergens.  I came away from that talk, which was a very good one, and said to myself, my God, this is impossible.
	DR. OWNBY:  Other questions?
	Thank you very much.
	MR. HAUCK:  Thank you very much.
	DR. OWNBY:  Are there any other persons who would like to make comments to the committee on any of the issues for today?  No one?  Okay.
	Agenda Item:  Committee Discussion
	We were asked earlier today to make recommendations on three different issues.  Those are in your agenda.  The first one specifically is whether CBER should expand the lot release limits for standardized mite and graphs, pollen allergen vaccines, to 0.5 to 2 as described in the draft Guidance for Reviewers, Potency Limits for Standardized Dust Mite and Grass Allergen Vaccines:  A Revised Protocol.
	Is there discussion on the committee of any of the other issues or any questions you would like to address to Dr. Slater or other -- the people who are here about that draft or the rationale behind it or the implications?
	I was asked that we actually take a vote on this issue at the conclusion of this as a committee, but remembering that we are an advisory committee to the FDA.
	Dr. King.
	DR. KING:  Dr. Pastor said it doesn't matter whether the relative potency varies by a factor of 4 because you will not detect an adverse reactions or affect its immunogenic efficacy.  That very well is true; that is, whether you immunize somebody with 1 microgram or 3 microgram, you would probably get the same result.
	But still from the point of view of regulation, wouldn't you try to say that this batch of insulin has 5,000 units instead of 5,000 to 20,000 units?  I mean, that we should try to be able to state that in a little bit narrower range.  I understand where he also pointed out the amount of work involved.  To me, it is just sort of making it too -- perhaps ELISA inhibitions kind of draw all these curves to make it parallel and everything, but with some -- let's say if you are talking about some allergen has an enzymatic activity, you can easily measure very reliably.
	One should be able to cut down the standard deviation.
	MS. DEAL:  My name is Carolyn Deal, for those of you who I haven't met before.  Maybe I could put what we are talking about in a bit of a perspective compared to all of the other biological products that we regulate.  One of the things I think we are trying to do with this is this is not in the situation where we are evaluating, say, patients in a clinical trial or not.  Now we are looking at evaluating manufacturing and consistency of manufacturing.
	I don't come from an allergenics background but from a vaccine manufacturing background and this is very similar to the types of problems we deal with in that arena.  Ideally, correctly, you would like to be able to manufacture something exactly the same from lot to lot to lot.  But as you know, that is impossible in a manufacturing facility.  So, what we are trying to do is balance the inherent variability so that we minimize as much as possible in manufacturing.  Yet at the same time, maintaining the potency and safety of products within a limit.
	What I would say is the perspective is in all biological products we have limits.  There is nothing -- we can't say an absolute every time.  And if you go back, say, and look at the things that you look at for vaccines, all of those also have limits. 
	So, I think part of this is in the perspective of treating these products the same as all of our other biological products and maintaining the public health, safety and efficacy of the products; yet, at the same time not putting undue burdens on the manufacturing.  As we look at all of these with biological assays, there is inherent variability in a biological assay, that the more repetitions you get, you can get to the point of diminishing returns.
	So, just as a perspective for this discussion for these products, compared to all of our other products.
	DR. PASTOR:  Really that is -- don't forget, their actually was a sequence of events where there was the skin  testing.  After the skin testing got the standard of 100,000 BAU, the next step was to validate the ELISA, such that it gave the equivalent potency.
	So, then, therefore, because the ELISA is a test, we had to live with the variability of that test.  Now, if, in fact, we had then done a protein test and the average value of the protein test was exactly the same as the value of the ELISA test and the protein, as you know, is a much more precise test, then we would do exactly as you suggest.  We would base the limits on the protein test and, therefore, make it like narrower.  So, continuing with the protein example, as you saw, for the same value of the ELISA, the protein varied by a factor of 10.  So, that like didn't work.  If you were to find another test such that it gave the same average value as the ELISA and was much more precise, sure, we actually would switch.
	But since we don't have that, yet, we have to do something and as Mr. Hauck said, every time they like reject a lot of mite, it is actually quite a lot of money.  So, we have to ask very carefully is it appropriate that the FDA put that burden on the industry that they have to reject of mite.
	So, coming back to it, I think, in the future we will have tests that are more precise and when we have such a test and it is validated, we will switch to the more precise test.
	Does that answer your question or maybe Jay would like to add something.
	DR. SAXON:  I accept the variability in ELISA's.  I unfortunately have to live with them and they are very uncomfortable and that leads to some of that imprecision, which isn't a manufacturing issue, I feel.  And I understand that.
	What happens -- I don't quite understand.  If you get a lot that is submitted as a 1, right, and it comes out as a .6, which is fine, right, and you get another lot that is submitted as a 1 and it comes out as a 1.8, which again it is fine.  When they actually labeled by the manufacturer, does this label it as a 1?  Does it label it as a .6 or does it label it as a 1.8, meaning do they actually go by the number?  I just want to know the answer.  There has got to be an answer.
	Do they label them as a 1?  They get labeled as a 1.  Okay.  So, within that limit, they get labeled as a 1.  I understand.  Fine.
	You don't need to explain it.  I understand.  I just wondered how it worked.  It is fine.
	MS. LIBERA:  So, the lots are labeled whether they are high or low?
	[Multiple discussions.]
	DR. OWNBY:  Let me state this.  I think I am correct.  You can correct me if I am wrong, that if they fall within the acceptable limits, they are labeled as 1 and that is usually a hundred thousand or 20,000.
	MS. LIBERA:  So, they can fall in the larger range and still be labeled --
	DR. OWNBY:  They all receive the same limit as long as they are within the range.
	MS. LIBERA:  Does it matter to a patient who is on therapy whether they --
	DR. SAXON:  The problem is the variability of the assay is such, you are not really sure that your .6 is really not a 1.
	DR. CLAMAN:  And then the variability of the patient's response is shown by those dose response curves, which are rather flat.  Now, we are concerned with safety.  Obviously, that is the problem.  And we all know on a practical basis that the main safety problems in the delivery of allergenic extracts have nothing to do with the manufacturers at all.
	It is the nurse who picked up the wrong bottle or the pharmacist who mixed it wrong.  Now, we are not discussing those things and there isn't anything we can do about it here, but neither can the manufacturers and neither can the FA.  So, I think the fact that the dose response is rather flat is lucky for us and lucky for the patients that there is a built in safety factor, which includes some of this variability.
	Dr. King is coming right to the microphone because he is a chemist and he is going to kill me.
	DR. SLATER:  I would just like to recall for the committee two fundamental parts of the analysis.  One is we are doing absolutely nothing to change how the manufacturers select and screen the materials that they send to us.  The materials still have to observe the old release limits, which are basically statistical identity to reference.  Okay?  Those release limits are statistically indistinguishable, based on the test to reference.
	The proposal merely changes the way we handle materials that are truly at those statistical limits and how we are going to handle them; whereas, in the previous system, the current system, a material at the reference limit would have essentially a 50 percent change of failure, was the example that Rich gave.
	Under the proposed system, it would have something less than a 50 percent chance of failure, depending on the number of replicates, in other words, the reliability that the manufacturer gave us with the number.  The second important part of the analysis to recall is our analysis of the ranges, of the predictable ranges of potencies of two randomly selected materials taken out of the distribution of a particular product.
	Remember that that predicted range, either the mean range or the 95 percent maximum range, is purely a function of the sigma.  It has nothing to do with the limits that we pick or that we set.  It is a function of the distribution, i.e., the narrowness of that curve.  The narrower the curve, the less likely there will be much of a variation.
	In fact, based on the data that we were able to collect, really in the end from nearly 200 lots of material, it was fairly reassuring in that sense.  It said that over 95 percent of those random changes are going to be less than 80 percent off from each other, well within the fourfold, actually within the current limits as they currently exist.
	So, in fact, my estimate is, although this is, I think, conceptually an important change, it is going to be practically of limited significance, except that we are going to be treating those extracts that are on the edge in a statistically valid way.  Whereas, before, we really weren't.
	DR. OWNBY:  Dale, go ahead.
	DR. UMETSU:  So, my understanding is that if there -- the manufacturers are not going to be changing their procedure.  So, when they do ELISA assays for each lot, they do it six times.  Is that correct?
	DR. SLATER:  Some 3, some 6.
	DR. UMETSU:  And there is no way to standardize that.  Why are some 3 and why are some 6?
	DR. OWNBY:  Isn't that the manufacturer's gamble, that the more times they do it, the more it costs them, but the less likely they are to get a lot rejected; whereas, if they cut back on the QA, they are more likely to get a rejection.  So, it is a statistical gamble one way or the other.
	That is a manufacturing choice.  That is how you design your business.
	DR. UMETSU:  But isn't that this new proposal, though, assuming that the manufacturers have a fairly good quality assurance?  So, wouldn't 6 be better than 3?  It doesn't make a difference?
	DR. SLATER:  It is part of the license application.
	MR. HAUCK:  Most manufacturers are using 6 now and maybe it is not mandated, but certainly when FDA reviews a license amendment or in the standardized grass program, the suggestion was made by FDA during reviewing of these licenses, hey, guys, you know, your numbers were kind of all over the place, as Richard pointed out earlier.  How about more replicates during a point in manufacture, either in final containers or in bulk.  That is what manufacturers have done to reduce -- to be more consistent, not only from an economic point of view that you don't throw product out, but even a manufacturer is going to have tighter limits than its release limits.
	If I have an experience of .8 with a specific raw material in my method of manufacture and all of the sudden I come up with 1.3s, that is within the limits, but certainly as a manufacturer, you get a look at that.  Okay?  So, in essence, FDA has kind of guided the manufacturers into more replicates and, in fact, most of us do use 6.
	DR. OWNBY:  Other questions from the committee?
	DR. WRAY:  I was just going to make a comment that I think we need to keep in mind the importance of allergy causing asthma and that there are so many patients, who cannot afford the benefit of allergy extracts and that unless we have some overwhelming reason to think safety is an issue, that we should try to remove any obstacles to providing more extracts to people.
	DR. OWNBY:  Jay, it seems to me that if I was a manufacturer in business, I would try to optimize my profit and if I was extracting material that was four times the price of gold, I could have someone calculate for me the odds on rejection and I could add excess water to my extract and deliberately produce an extract at .8 reference or .75 reference and figure out what the probability is that I am going to get a rejection from the FDA, when I am deliberately at that level.  With this broadened limit, it is going to allow me more freedom to do that.
	DR. SLATER:  We actually can look at that and we can continue to look at that.  It is part of our analysis of the standard distribution of the products.  We looked at the failure rates of the products and we were pleased and reassured that the distribution of failures was roughly even high and low.  Had they all been low, I think we would have been suspicious about what what you said, but the fact that the distribution of failures was evenly distributed -- and, again, there weren't that many failures but the distribution of failures was evenly distributed -- suggests that the current manufacturing practice is exactly what we intend it to be and that to aim at identity to reference for a relative potency of 1.  
	I think we would --
	DR. OWNBY:  What about next year if the relative potency on the average lot of mite is .8 instead of 1?
	DR. SLATER:  Right.  We will know.
	DR. OWNBY:  But would that give you -- is there any regulatory way to say to manufacturers that something has changed and we are concerned about it?  I mean, there are still -- the majority of their extract is going to meet specification.
	DR. SLATER:  Right.  I think first of all, even though we will have broadened the limits that we use for failure, they still will be increasing their chances of failure by going low.  If you look at that table that is included, the failure rates are lowest at a relative potency of 1.  So, if a manufacturer really wants to minimize its chance of failing, they will continue to aim for a relative potency of 1 and we fully expect that that practice will continue.
	If, however, they make an economic decision to balance it off and aim for something lower, the failure rates will go up even by our method and, furthermore, their failure rates will go up asymmetrically and we will be tracking that and looking at it.
	Whether we have an explicit preexisting mechanism to jump in and try to rectify that, I have no idea, but I certainly would look for hard for one.
	MR. HAUCK:  First of all, you have given us a great idea on that.  The other side of the coin, why a manufacturer wouldn't do that, we put one lot -- you have to put these on stability.  So, if your products at or near the limit during its shelf life, it is going to fall out.  If it falls out, you have got all kinds of issues going on.  So, the manufacturer, trust me, wants to be as close to 1 as possible.
	DR. PASTOR:  It is also pretty hard to nail it down.
	MS. BRIDGEWATER:  I wanted to point out, Peter, to even say this, but the formulation is not that precise.  I think the way that I have seen -- when you are formulating, you always target 1.  It is not so precise that you can kind of monkey with the numbers to shoot for an 01.  It is too difficult.  I don't think you could even do it practically.  It would be very difficult.
	DR. OWNBY:  I think they may be creative, but, Henry, you had a -- 
	DR. CLAMAN:  Several comments.  One, you are a cynic.  All right?  
	No. 2, this reminds me of what goes on in Vermont on the dairy farms, where milk is sold by weight and which sometimes the weight is increased with water, which led Henry David Thoreau to say there is some circumstantial evidence, which is very strong, such as when you find a trout in the milk.
	DR. UMETSU:  I just have a question regarding what happens when a lot fails because it is too high.  Can you just dilute it out a little bit more?
	MR. HAUCK:  It all depends on your license and what you have negotiated with FDA.  In general, you may have to do an investigation and if the investigation suggests that it is maybe a raw material variability possibly, then if your license permits it, yes, you can dilute it.  
	If it is something else, it may be your assay system is a little off.  There could be something wrong with the reference.  It is possible, but it is not always the case.  So, it is theoretically possible, but it depends on the individual manufacturer and how their license reads.
	DR. OWNBY:  Are there any other general comments or discussion on this?
	DR. SOTO-AGUILAR:  I would like to ask a question.
	In what other ways does the FDA have an input in the manufacturing process of the allergens?  This is based on a comment that I heard recently, actually earlier this week, that the FDA would do actual inspections of the plant.  Is that correct or not?  That is what I was told.
	So, besides testing any particular lot, does the FDA go and visit the site?
	MS. BRIDGEWATER:  Yes.  Actually, I am glad you brought that up.  Yes, we do have an aggressive inspection program, as manufacturers can comment on that.  But when somebody is licensed, we do go out on biennial inspections and inspect the facilities and if the CBER inspectors are on the inspection, this is also something that we would be looking for; failure rates and failure investigations are always something that are part of an inspection.
	MS. LIBERA:  Why wouldn't both the manufacturers and the FDA have the same system?  Wouldn't that ensure the consistency and, you know, all the other things.  I guess I am having a hard time understanding why you have one system for a manufacturer and then another for the FDA.
	DR. OWNBY:  I think Jay is going to answer that, but they are, in fact, the same system with standardized protocols.  The only question is the limits on what is acceptable within that.
	Is that correct, Jay?
	DR. SLATER:  Sure.  The systems are identical and have been identical and will continue to be identical.  The problem that we are trying to escape is the problem of an extract that comes in at the edges of that curve.  We have accepted that an extract at the edge of the curve, say just inside the limits is okay, we have to also acknowledge the fact that an extract that is at that okay level is so close to the border that when we test it, we have close to a 50 percent chance of failing it, even though more extensive testing, as Rich said -- if you tested it a thousand times, you would know it was .75, which is within the limits.
	But you don't test it a thousand times.  You test it three times or six times and that lower number of tests leads to a spread, just as throwing the dart at the target, even if you are really good, you are going to have a spread around the target.
	So, it is that spread that we are trying to deal with with this.  What we are doing is we are -- based on the clinical data, we are accepting that the limits could be somewhat broader than they are.  We are requiring the manufacturers to do nothing to broaden it.
	Remember, their licenses say they still have to aim at reference.  If we have evidence that they are not aiming at reference, such as a drift downward or a failure rate greater on one end than on the other, we have some statistical evidence that there is a problem in terms of their executing what they have said they would do in their license.  
	So, the manufacturers need to give us the same products they have been giving us since their licenses were approved.  What we are simply acknowledging is the statistical limitations of our ability to analyze finally those products that are at the edge.  If we had a perfect way of measuring those products that were at the edge, either by having a better assay or deciding that we could have our biologists do a thousand tests instead of 3 or 6 tests, then we probably wouldn't have to have this discussion because we would be able to agree with great certainty as to where it was.
	We would have to figure out a way to deal rationally with the uncertainty and protocol until now, rational as it was, the idea -- they have the same limits, we have the same limits -- led to irrational results at the edge.
	MS. LIBERA:  I guess I am trying to figure out which patients that are on the edges that those will get that --
	DR. SLATER:  Right.  So, the first part of the analysis then is to see whether patients would be hurt at the edge, whether patients would be injured by falling over one side than another.  There are a couple ways to approach found that there was minimal likelihood of injury at the edge, we can say, okay, we can acknowledge that there is some imperfection in our methods and broaden the CBER-imposed limits beyond what the manufacturers need to impose when they send us product.
	The other approach would be, well, if we decided that the safety considerations were overwhelming, then we might say to the manufacturers, do a hundred tests.  Give us a real hard number.  Narrow the sigma.
	DR. OWNBY:  Gail.
	DR. SHAPIRO:  I understand the plateau in the patient response.  So, I feel comfortable about the safety issue because of that.  But I don't understand -- and I am no chemist and this is probably very naive, but why can't FDA provide a phantom of identity to pharmaceutical, to the manufacturers, so that they can all aim to the level that is 1?
	Also then is there a problem with licensing in terms of just adding solvent or solute to get to that level?  Is it like wine, where you are not supposed to fortify it or is it something where you could do it that way?
	DR. SLATER:  Let me answer the first question because it is easy.  We do.  That is the reference that we send out.  So, when we shipped nearly 2,000 vials of reference in 1999 out to the manufacturers in 104 separate shipments, that is what we were shipping them.  We were shipping them the reference material that they are using as their standard and that we use as our standard.  So, in fact, we are duplicating exactly what they do in our lab.  That is the whole point --
	DR. SHAPIRO:  I think it is hard for those of us who aren't in the lab to realize there can be such a discrepancy when systems are supposed to be the same.  It is sort of like stepping on the scale and having it say 150 and 170 the next day.
	DR. SLATER:  And there was a time when scales did exactly that.  Okay?  Or worse than that.
	DR. SHAPIRO:  I know.  Obviously, that happens and it is kind of mind boggling.
	DR. SLATER:  When you first switch from, you know, a balanced scale to a spring scale, the old spring scales had exactly that kind of horrible accuracy and they have improved substantially.
	We can anticipate improvements as well and a whole part of the presentation this morning was aiming towards types of assays that might be more precise.  But we are -- you know, I don't want to convey the feeling that a competition ELISA is a rotten test.  It is actually a great test.  It really works very well, but these are the limitations of the test.  The sigma that we have found for the test is based on hundreds of tests that have been evaluated, initially, ten years ago and then more recently a year and a half ago.
	A lot of testing has gone into this and these are the limitations of the test as it currently exists.  So, we are sort of stuck with that.
	DR. SHAPIRO:  So, if somebody sends in a lot that is rejected because it is low, can they just spike it with more and send it back?  I mean, that would seem to be a legitimate way to get to the right level.  It doesn't sound like you would have to throw anything out or --
	DR. SLATER:  It is actually not that simple --
	DR. SHAPIRO:  I figured it wasn't, but it just sounds like it should be.
	DR. SLATER:  And the manufacturers need to submit, need to have submitted in their license applications ways of handling these kinds of materials and their SOPs, their standard operating procedures, for handling materials that have not passed lot release needed to be removed and approved.  I mean, it is not trivial and it can't be done after the fact.  It has to be done with the standing operating procedure in place.
	DR. LEHRER:  What are the ways of handling that situation.
	DR. SLATER:  I am sorry?
	DR. LEHRER:  You said the manufacturers have -- there are several ways that they can handle that situation if a lot is rejected.  Gail asked if they actually can combine it with a higher potency law.  You really didn't answer what ways there are to handle that.
	So, I was curious to know what can they do?
	DR. SLATER:  Yes.  I don't like to give  answers that I am not exactly sure what the --
	MR. HAUCK:  Let me give you an example.  If it is something that may have a single standard of potency, like mite, you sell 10,000 or you sell -- generally, if you are a manufacturer or -- or cat, you are selling 10,000 and that is all you are licensed for.
	If your cat extract or your mite extract doesn't meet that minimal standard of potency, by and large, most manufacturers do not have a provision for doing anything with that except throwing it away.
	If it is something on the other hand like a grass extract, where you are selling two potencies, a hundred thousand and 10,000, there may be a provision in your license.  If when you make your 10,000, which is generally a tenfold dilution of your hundred thousand, but nonetheless has to be tested, if it comes up a little low and you have the hundred thousand, you may just fortify with more hundred thousand and that may be permitted, depending on the individual license.
	So, in most cases with the exception of possibly the grasses, there is not much you can do except throw it out for most manufacturers.  Is that --
	DR. PASTOR:  I would like to just add one thing because it is a great opportunity to get to speak with you all this stuff, is that I just want to go back to that scale analogy one more time.  The ELISA is like a scale.  If you weight 100 pounds, on any given reading, you step on that darn scale, it will read between about 53 pounds to like 197 about.  So, now, you say, oh, my God, that is terrible.  What you do then, you step on the scale a bunch of times and take a bunch of readings and after many, it will gradually end up being like one of his readings.  But because we can only take three, you get some idea, just in a way that we are dealing with something, that with three, that scale will read between 70 and 150 pounds.
	So, therefore, it gives you a better sense of when the manufacturer comes in with something that is like 80 pounds and we measure it with something that really within a factor of 2, why we have this problem.  I don't want to pursue something that really sounds kind of childish, but that is the problem.  The scale has a huge variability.  So, it is hard to solve this problem and that is why we came up with this way of solving it.  It is not an analytical method.  It is physical chemistry.  It is biochemistry.
	DR. UMETSU:  I understand the sigma and the statistical analysis of this but I still don't understand why the manufacturers do 6 in some instances, 6 determinations versus 3.  It seems to me that the sigma will be reduced quite significantly by doing 6 rather than 3.  If you could just sort of standardize the number of determinations, I think that might help reduce some of the variability.
	DR. PASTOR:  Almost all the manufacturers use sets.  There are a couple that actually use 3.  It was just in the process of the licenses that some of them had results that were varying over a wide range.  So, we said, well, you are failing these tests, the boundaries.  You guys ought to use 6.  Others, maybe for just blind luck, maybe because they had been processing it really well -- we don't know -- they are licensed -- their lots and their shelf life studies are all very close, around 1.
	So, we said, okay, it is just like sigma 3, but all the large manufacturers are actually with 6.  It is only some of the smaller ones that are using 3.  It is just the way it played out.  We don't have the -- I mean, you might argue to us -- I mean, you as a group could say, no, you should make all those other guys do 6, too.  I don't know what we would do, but you could ask us to and we could find out.  It is just what happened, just the history of the thing.
	Is that a fair way to say what happened?
	MR. LANKOW:  I am Richard Lankow.  I am with ALK.
	I think one of the things that isn't really clear here is that we are not talking about variability of the product that comes out our pipe at all.  We are just talking about the variability in the measurement method.  A lot of that just has to do, are we confident in our operators in the way we do it and how tight the product is that we make.  So, that kind of kind of influence the way people do it.  
	So, it is really looking at the measurement method and not the product.  So, it is our risk.  It is our risk if we or somebody else does 3 or 6 or 9 is, you know, what is the chance that when it comes to FDA and is repeated -- and, again, we are just talking about repeating the same assay on different days in a different laboratory, what are the chances it is going to fall outside of that?
	So, it really is kind of an internal operating decision that gets made and analyzed.
	DR. OWNBY:  Anyone else which to make any comments or an opinion on this or still discomfort with trying to make a decision?  I think everyone except maybe Dan hasn't made a comment on this.
	DR. EIN:  Yes.  I thought it was clear enough to me.
	DR. OWNBY:  Any other questions?  I don't want to cut people off, but I get a sense that people have pretty well covered their questions and concerns about this.
	I think the one thing that gives me some reassurance is the fact that this isn't the only quality control.  I mean, the FDA is investigating, is checking the records, is following the source of material from the time it is originally derived all the way through this manufacturing.  This is just one little bit, if you will, of a total quality program.  It is not the only thing that is done to try to make sure that these are adequate products.
	DR. EIN:  I just want to make one comment.  I think it is really instructive to go to a manufacturing facility and see how these products are made and the kinds of controls that now exist because of tightening of the FDA processes.  It is really very impressive.
	I had the privilege of going about a year ago to one of the plants and it is really very high tech, high sterility, quality control, from beginning to end, which gives me -- it doesn't speak to this particular issue, but in general, following up on what Dennis said, it gives you a lot of confidence in the product.
	DR. OWNBY:  Okay.  Well, I think we are ready to vote on this issue then.  
	Bill, you had a --
	DR. FREAS:  Before we do vote, I just want to for the purpose of the record state that there are 11 voting members sitting at the table and for the new members, I want to make sure that you are aware that you can either vote CBER expanding the lot limits.  You can vote against CBER expanding the lot limits or you can abstain.
	DR. OWNBY:  Should we take a show of hands on this or -- 
	DR. FREAS:  That would be fine.
	DR. OWNBY:  A show of hands would be adequate here?  Okay.
	All those in favor of the proposal to expand the limits as we have discussed, raise your hand.  
	DR. FREAS:  Eleven.
	DR. OWNBY:  I take it then there are no people opposed to it or abstaining.  None.  Okay.  That item is out of the way.
	Now, these next two items, I don't think we have to have a formal vote on, but we have been asked to give our opinion, the first one being whether CBER should implement the proposed algorithm for standardization of new allergens.  I think Jay adequately outlined that.  The purpose of the algorithm, the other comment I have heard is that perhaps manufacturing concerns should be also included within the algorithm.
	Are there other questions, comments, concerns, discussion?
	DR. WRAY:  My question is how different is this from what happens now?  This sounds very good, but I just didn't know -- is it really different or is it just really putting in print what happens now.
	DR. SLATER:  Great question.  It is fundamentally not terribly different from what has happened so far.  It has a few differences.  The exit points are different.  The fact that it is formalized into an algorithm that we can sit and discuss and argue about, perhaps, I think is different as well.  We wanted to make a process that was transparent so this discussion could be -- could focus on different points.
	Also, we anticipate standardizing several different products at the same time.  We are going to be at different points in the process with all of them.  This is simply almost an administrative way to make sure we are keeping track of the process and make sure we know where we are and where people are going.
	In terms of -- does that answer your -- 
	DR. WRAY:  Yes.  Thank you.
	DR. SLATER:  In terms of your point about getting manufacturing and manufacturers more involved, I glossed over that.  In fact, on the algorithm that you have written out, on the right hand column, there are boxes for potential collaborators to be involved with us.  APMA is involved in one form or another at almost every step of the process.
	Again, this is a process that we are announcing in advance in this way and our intention as we move forward with the standardization of allergens is for the entire process to be transparent and as inclusionary as possible.
	But, again, I don't think that is a radical difference.  I think we are simply formalizing the process.  If there are differences in terms of collaborators, I think we explicitly would attempt to enlist help from NIAID, for instance, but I don't think that is a radical change either.  I think that is always a possibility.
	DR. CLAMAN:  A small technical problem on the expanded algorithm.  You have up at the top and about a third of the way down initial testing for IgE binding, assess human IgE antibody binding Western blot.  Then about equal way down, you have clinical efficacy measures, skin tests, bronchial hyper reactivity, immunotherapy trials, evidence for IgE binding again.
	I assume that if there was no evidence for IgE binding at the top, you would never get down to the bottom.  It is a little hard to imagine having gotten down to the bottom with no evidence of IgE binding.  I suppose this stuff should turn out to be negative on skin tests and people known to be sensitive, which is --
	DR. SLATER:  No, I quite agree.  I think if there were no evidence of IgE binding on laboratory evaluation, I think we would be hard pressed to abort the process right then and there.  But I think what I was trying to say is that what we are looking for in the clinical studies is not only dosing assessments of biological unitage, but looking for clinical verification that this is an IgE-driven process.
	DR. CLAMAN:  I have no objection at all.  However, from the logistical point of view, there is an enormous difference in the time and effort required to do (a) skin test, (b) -- which take 20 minutes once you get it going -- bronchial reactivity, which is a pain in the neck and immunotherapy trials, which take years.  Who is going to make these decisions as to which of these or all of these are going to be used in a given case?
	DR. SLATER:  Well, I think because of the reasoning that you just laid out perfectly, the default position would be to do skin testing.  It would be the fastest and it clearly would be the most appropriate for most of the allergens that we would possibly be looking at.  There are currently going on at NIAID in the intramural program, rush immunotherapy trials, looking at lymphocyte transformation.  These are certainly not projects that are finished in an afternoon.  They take eight weeks to complete, but potentially if we had a product for which skin testing was somehow for some reason either engineered or natural in inappropriate or inadequate test, we could attempt to collaborate with the investigators at NIH.
	There are other possibilities as well.  I think, again, this falls into the line of trying to incorporate enough vagueness into the process, so that we had -- nonetheless that we had a process, we had a step at which we had to start making clinical relevance and dosing and unitage decisions.  
	We have to some method by which we are going to establish biologically-based unitage.  But I didn't want the process to be boxed into a particular model, in case, as we moved along, we found allergens for which that model --
	DR. CLAMAN:  I agree entirely.  I am a big -- in spite of the fact, I am supposed to be a scientist, I am greatly in favor of vagueness in some cases because you don't get your hands tied by your own procedural methods, which I have seen happen rather often.
	DR. WRAY:  There continue to be some little hints in papers suggesting that oral allergen immunotherapy works.  Is there anything in the works or any interest in that?  What if somebody came to you with a proposal on that?  Would the same sort of standardization be required or is that just so way out that we haven't thought about that yet?
	The decision as to whether oral immunotherapy works would be based on good science to demonstrate that it works.  In order to have good science, we would need to have a product that was standardized somehow.  It would be hard to attempt to standardize the product based on the study verifying that the method works.  So, we would need to have some independent method of verifying the potency of the product.
	But the actual -- that is actually a little bit outside of what this algorithm is proposing.  What you are talking about is novel methods of immunotherapy, novel delivery methods of immunotherapy.  That would be more appropriate to be studied by investigational new drug applications.
	DR. OWNBY:  Other comments?
	Yes.
	DR. SOTO-AGUILAR:  I would like to go back to the skin testing part, please.  I don't know if I am confused on this or not, but I understand that as new antigens are being developed by the manufacturers, at the same time the FDA is interested in identifying those that have the best quality to use a reference for future use and to compare new products coming in.  Is that correct?  Is that something you said earlier this morning?
	DR. SLATER:  It is correct, except for the tense in which you said it.  There currently aren't any skin testing protocols going on with -- well, if you are talking about new products, yes, if a new product came along that was under IND, very likely the sponsor would be required to do some intradermal skin testing to establish the potency of the material.
	Is that what you are asking?
	DR. SOTO-AGUILAR:  Right.  And once you have a material that has 100,000 BAUs and you think is going to be stable and excellent product and then other manufacturers bring similar allergens and the same type of allergen, are they going to be required to be skin tested, too, all of them or only in the process of selecting reference product?
	DR. SLATER:  Well, if another manufacturer is coming along with a product that has the same name, but may not be identical, they would probably be required to produce skin test data with that product as well.
	DR. SOTO-AGUILAR:  And that applies to your algorithm.  I just wondered if there was any other place in the algorithm to include the skin testing again later on?
	DR. SLATER:  It is not formally included in the algorithm.  It certainly is a very good thought.  In other words, several years later, coming back and validating that the reference material still is on target with skin testing.  That would probably fall under the post approval stage.  I would think that that would be a very important ongoing part of the evaluation of the reference materials as you get several changes out.
	DR. SHAPIRO:  Jay, I have a question about new antigens.  If something were to come along, as Betty mentioned, if some developer put together an oral vaccine or a new super antigen that they felt would be excellent for immunotherapy, does the lab ever get in a position of having users come or manufacturers come to you and offer user fees to promote the standardization or the proper pushing through the algorithm faster for their particular product?  Is that a possibility, if some venture group were to say we have got a new super duper vaccine and we want your group to help validate it?  Does that make no sense at all?
	DR. SLATER:  It sounds like it takes a conference call over here.
	MS. BRIDGEWATER:  Are you talking about user fees --
	DR. SHAPIRO:  If there was a food immunotherapy issue and somebody isolated the peanut allergen.  They felt that the potential was fantastic for immunization with peanut and they wanted to be the standardized allergen and have FDA approval for their product.  For other divisions of the FDA, there are user fees and people can get fast tracked.  Do you have anything like that?  Or would it be even conceivable that you would?
	MS. BRIDGEWATER:  Well, first, on the issue of user fees, once again, allergenic extracts are exempted from user fees under the law.  So, they don't pay user fees.  So, that would not be an issue.
	Your question about fast track, it is something that somebody could come -- there is a process for coming in and trying to get fast track approval.  So, they would be eligible for applying under that method, but they would not have to pay user fees.
	DR. SHAPIRO:  So, there is a way to bump your priorities.  I mean, there might be a way to bump the priorities.
	MS. BRIDGEWATER:  Sure.  If something comes in that is for a serious or life-threatening condition and a manufacturer can make a good argument that it is, you know, a significant public health benefit or it has indications for a serious or life-threatening condition, then, yes, there is a way to get yourself in what we call fast track approval that does push the priority up.
	DR. LEHRER:  I just wanted to make a comment expanding on what Gail mentioned and something that was said earlier with regard to new ways of using established allergenic extracts that haven't been approved as yet or even making available or standardizing new allergenic extracts that had not been available or not been standardized.  Jay had indicated at one point that a lot of this would be based on research that has been performed or information that has been provided, based on other studies, which I think has been fine and has served us reasonably well through the seventies and eighties, but as we went into the nineties and certainly now, I think there is really minimal funding from traditional sources for allergenic extracts.  You know, such as the NIH, I think, if you want to definitely kiss a grant goodbye, put in a study of an allergenic extract.
	Certainly, industry has cut back on funding of allergenic extracts as well.  The FDA has very limited resources.  So, I just wanted to make the point that it is something that I think everyone should think about in terms of where we are going with allergenic extracts.  If we are relying on information that has been funded by traditional sources, that information isn't going to be there.  It will carry you through for some of these, you know, cockroach, for example, that there has been a lot of studies up until now, but it is not going to in my opinion in the future and it is something that we will have to deal with and I think relates in part to what you raised.
	DR. OWNBY:  Do I have a sense of the committee concerning this particular issue of whether we should recommend acceptance of the algorithm at this point?  Is the general feeling that people are in favor of it and that we don't see any major fatal flaws or big problems with pursuing it at that point?
	Okay.  I think we are probably done with that.  Is that sufficient then?
	The last item is whether the next three items targeted for standardization should be cockroach -- and I understand from Jay that would be at least two species of cockroach, perhaps three; alternaria alternata and aspergillus fumigatus.
	DR. EIN:  I would like to ask Jay to expand a bit on the rational, not for the cockroach because I think there would be consensus about that, maybe aspergillus, but certainly alternaria.  On what data was that decision made and why that rather than dog, for example?  Is there any quantitative data about which are more important allergens in terms of asthma?  So, if you can talk about that, I would appreciate it.
	DR. SLATER:  I think when I was asked to develop a plan in terms of responding to the asthma initiative, as you can imagine, the first choice was obvious.  I think choosing cockroach was very easy.  I think the idea of going after aspergillus fumigatus and alternaria, I think, was clearly less clear.  I didn't feel that I could propose those two and dog.  
	I do think that there is some rationale for standardizing dog extracts.  I think the science is there as well.  I think the data are less convincing that dog is really that important, that dog allergen is actually that important in terms of the etiology of asthma.
	What I was looking for were allergens that were reasonably characterized, not completely characterized and in which the relationship to bronchial hyper reactivity had been reasonably well studied, at least in some controlled situations.  Cockroach, aspergillus and alternaria seemed to fit that bill.
	The data are clearly the weakest for alternaria.  I agree with you on that.  But I think, in fact, the data on alternaria are probably pretty comparable to dog, for which the connection is weak, as well.
	I am very open to suggestions about other allergens and I am open to the possibility as well -- and I would like to open the possibility that you may simply wish to expand the list.
	DR. LEHRER:  I must say that I was somewhat taken back by the remarks that Peter Hauck made concerning fungal allergens initially having been one that has been both intrigued and frustrated by the study of fungi as zero allergens.  I think clearly I think we all can agree that there are certainly very important, but they are very, very difficult to study.
	Nevertheless, because a problem is more challenging -- I am raising the question -- should we just allow the extracts to be perhaps, you know, of less quality, at least some of them compared to others.  
	On the other hand, I think Peter's point was well made and that there are limited resources available for standardization of allergenic extracts.  The study of fungi as allergens can be an unending task and it is not related as much to the allergens themselves, I think, as to the variability of the fungi and the production of allergens in which, you know, you see tremendous variability within the same strain, grown in the same lab by the same people.
	I am still kind of struggling with this issue myself because there are other allergens, I think, that are probably equally important.  For example, I think in general an area of allergy and certainly in allergenic extracts that have not been as well-characterized and, you know, people die every year of food allergic reactions and also related to the FDA's mission with regard to GM foods, we certainly need to have more information on food allergens.  That would relate to that mission, although I know it is not the primary concern of this group.
	So, I am not sure.  I think one thing I -- I think a good point that Peter Hauck made was that, perhaps, one should have maybe some limited objectives with the fungi.  You know, as I said, there is certainly a great need to have better fungal extracts, but if it turns out that this is a much more difficult task than anticipated or requires greater resources, which could be spent better in other ways, then if you had a limited approach and if it became clear that this wasn't going to be reached, then perhaps these resources could be devoted to other allergens.
	I don't know if this makes any sense or not, but I am a bit concerned about that because one could put a lot into fungi and not come out with anything.
	DR. SLATER:  I think the comments make a lot of sense.  I think definitely the fungi are a daunting target.  I guess my one response to it would be that although clearly the objective here is to come out with  standardized allergen vaccines that will improve the diagnostics and therapy of fungal-induced allergic disease, I think on the way there we can actually, potentially, learn a great deal about even just in the preliminary evaluations, just learning about what the variability is of the products that are out there currently and perhaps by enlisting the help of some of the research labs that are doing a lot more work with fungal allergens at this point, really trying to do some good, if you will, descriptive or normative studies of what is out there to draw some conclusions about what the need might be.  
	I think even the initial evaluation process for alternaria and aspergillus would be very instructive.
	DR. LEHRER:  Just to expand upon that, one point that you made early on in the discussions today was that -- I forgot exactly how you phrased it, but it was, I think, talking about techniques and using different approaches for different allergens and I think this is a perfect example in that you may not be able to use the same approaches that work so well with grass pollen allergens or other pollen allergens in which you have large amounts of relatively stable sources of allergens.  	So, again, I am repeating myself, but you may want to have perhaps a little more limited approach and, hopefully, you know, what I say won't hold up, that it won't be as difficult, but if it is then you -- I think it would be a good idea to have an alternative approach so that the resources will be well spent.
	DR. SAXON:  Jay, last year, we actually made up a different list.  The list included peanut, tree nuts and latex.  Now, I am familiar with the assessment issue.  I spent all day yesterday chairing an institute session at the NIH for asthma.  What has happened?  It has been a year.  Are we going to just throw latex away, throwing peanut away, throwing tree nut away because of this initiative?  
	Though I think it is important that the FDA participate in the asthma initiative, what about the other things?  This is a big sea change all of the sudden, it seems to me.  I wanted to know what happened in the last year to those other three antigens that were given high priority a year ago.
	DR. SLATER:  What happened in the last year was the effort to try to formalize the process so that we could move forward at this point.  The DHHS initiative really appeared on our radar screen fairly recently in terms of coming to CBER and asking us to make a specific proposal regarding standardization in response to the asthma initiative.
	No, peanuts and latex are not off the radar screen, nor are tree nuts.  I think those were main valid targets.  At this point, the asthma initiative has by virtue of sort of encouraging resources in one particular direction that pushed us one way versus another.  They are not exclusive.  We had resources before that we could devote to standardization.  So, I think that I don't intend to ignore the other potential allergen targets and certainly I think that we can move forward concurrently with other ones as well.
	DR. OWNBY:  Jay, I have got a question and I think through the committee is whether there is a fixed amount of resources that can be devoted to standardization or whether you think that there will be that amount and additional resources through this asthma initiative that might allow you to look at other standardization targets.  That is, do we have to prioritize within a single budget or is there a chance that if we prioritize in a different direction, the budget expands?
	DR. SLATER:  There is a possibility for budget expansion.
	DR. OWNBY:  Okay.
	DR. LEHRER:  With the asthma initiative, are any monies being made available for the study of these allergens?
	DR. SLATER:  The asthma initiative is a strategic plan intended to have the member agencies within DHHS build into their budgets plans to advance the goals of the asthma initiative.  So, there is no amount that was committed to the asthma initiative to start with.  
	That being said, the budgets -- our budget for this fiscal year and next fiscal year are already there.  So, when we first started talking about the asthma, we asked whether money could be made available before FY 2002 to support moving forward aggressively with these and the answer was "yes."
	So, we potentially have additional funds that will be available effective very soon to support allergen standardization as part of this particular initiative for the next year and a half and then thereafter it will be built into the budget requests that we have been told would be supported as it went up the line.
	DR. CLAMAN:  I would like to support what Sam said about the difficulty with mold allergy and trying to remember whether he was the one that gave that lecture that I heard that convinced me that mold immunochemistry was a can of mold.  I think it was you.
	Very difficult and one wonders how much resources should be -- whether it should be done in an incremental way. 
	My second comment is a question, the answer to which I should very well know, but I don't.  It has to do with dog allergens.  Is there compelling evidence that there is a single major dog antigen, which crosses all species and strains and, therefore, that is the one that you would put your money on, like in Fel d 1.  Is Can f 1 really that predominant?  Because if it is not, then you have to deal with the problem of the other possible species or strain specific antigens.
	DR. SLATER:  I think it has been found in all strains.  I know it hasn't been found in all species.
	DR. CLAMAN:  I wouldn't be surprised if it was found in all strains, but is it the major allergen?  That is a different question.
	DR. SLATER:  I don't know the answer to that.
	DR. CLAMAN:  I don't know either.
	DR. OWNBY:  It doesn't have nearly the dominance.  If you take cat allergic people, you can absorb out almost 90 percent of the IgE across a pool with Fel d 1.  With dog, with Can f 1, I think it is more like 30 percent.  So, there are clearly a number of other allergens that have a fair amount of reactivity.
	DR. CLAMAN:  Which makes sense from clinical experience as well.  So, there is another problem with dog there, having standardized Can f 1 and everything like that.  You have only gone part way.
	DR. SOTO-AGUILAR:  Coming from the south, clinical experience shows that molds are extremely important allergens and we are not just talking of aspergillus and alternaria.  Alternaria is very prevalent in the south and immunotherapy results have been very efficacious actually in the patients.
	However, the problem is the impurities.  So, I guess it will be worthwhile doing this effort at separated dose enzymes that tend to make sure the grass is in the same extract mixture.  I think that is a major problem in immunotherapy.
	DR. WRAY:  Do you have the data on, for example, cockroach and the relative potency as produced by the manufacturers now of the glycerinated extracts?  Like, for example, with the grasses here, with the glycerinated extracts, it was really only the red top that was so way out from batch to batch.  Is cockroach very variable?
	DR. SLATER:  Well, many of them were beyond what we would consider to be the safe limits.  Only the red top was --
	DR. WRAY:  Only two were above 4.  But what cockroach, do we have that data?
	DR. SLATER:  No.  That is --
	DR. WRAY:  I just wondered how much the standardization is going to improve the extract.  So, we need to know how much it varies now.
	DR. SLATER:  That is a perfect question.  That is the process that we haven't started yet and that is the process that we hope to begin.
	DR. LEHRER:  Kind of related to that, though, cockroaches and molds were identified, I think, as the two extracts that they have the highest level of proteolytic enzymes in them and, hence, the potential for degradation and also for causing problems when you mix them with other allergenic extracts.  So, I think certainly any information that you gain on cockroach allergen is going to be very important.
	DR. OWNBY:  I think there is a consensus that the committee could easily agree on cockroach and there is a lot of concern about mold and that the other candidate would be dog.  Considering that, at least in my own studies, and I think national data, there are about twice as many dogs in houses as cats, and that the analogy should hold, I would suggest that you pick one of those molds, probably aspergillus, you have better data on and use dog as your third one in terms of asthma.  But I would be willing to have input of other members of the panel on that.
	DR. SAXON:  Again, I agree, but I just want to say we shouldn't drop the others off the radar screen because of the asthma initiative.  There was strong feelings about peanut, latex, tree nuts before and those should just be kept on the overall radar screen separate from the asthma initiative where they are not so well-known.
	DR. WRAY:  I think clinically we pediatricians also see foods that cause wheezing in children without necessarily full blown anaphylaxis, to make a case for foods being important also.
	DR. LEHRER:  It seems like what Jay was saying, though, was that there would be extra funding available through the asthma initiative for this standardization so that if that is the case, then I would be more favorably inclined to go along with the recommendation because I don't think it would bump these other allergenic extracts that we think are important to standardize.  Then in a year and a half from time from this -- during this period, if the approach is not fruitful, you know, I would think that the agency would have the not to pursue it.
	So, my concern was that this may take away from other -- from standardization of other allergens.  You are telling me that this will not.
	DR. SLATER:  The entire effort at putting together the algorithm for going forward with standardization originated long before I was personally aware of the asthma initiative at all.  The people that were involved in developing the consensus and developing the algorithm, most of that went forward before we were asked to make a proposal for the asthma initiative.  That is -- the idea of going forward with standardization, with picking rational targets, with moving forward aggressively to try to standardize new target allergens, such as peanut, was in the works before the asthma initiative came on board.
	Andy, I don't really think the asthma initiative has co-opted the process at all and the way that we phrased the question is we phrased the question 
-- if you look at the second part, it says "Given the support of the asthma initiative, we would like you to assume that the added money is there to support this."  And, therefore, what we are looking for is your guidance, assuming there is money to support the asthma initiative in our activities.
	Then, are these reasonable choices?
	DR. CLAMAN:  I think that the situation is complex.  I don't want to find myself in the position of telling a good scientist what to do.  Besides, it doesn't work.  He has, I think, heard our concerns.  We meet now and then.  He works with us on a daily basis.  I think he knows what funding is available.  He knows the complexity of the system.  He lives with it everyday.  I think we trust his judgment to do what seems to be best to do.
	MR. LANDOW:  Since you are close to having a consensus, let me throw one more thing in without making a commitment either way just to think about from a strategic point of view.  Are the things that you propose to standardize going to have a big impact in a lot of ways?  I mean, as a manufacturing company, as we go through our strategic planning, we have to ask questions that a number of you have touched on before.  How many people get treated?  Is it a treatment?  
	You talked earlier -- is it an immunotherapy product?  Is it a diagnostic product?  The market for diagnostics is that big.  The market for immunotherapy is a little bigger.  It is not big.  This entire industry we have is less than three weeks of Clariton marketing.  So, you know, there are some issues.
	When we look from a strategic point of view, we look at the treatment or the options that you as allergists have with your patients, avoidance, pharmacotherapy, immunotherapy.  If you can avoid it, avoid it.  So, again, that -- I am not sure what I am saying.  I just know that we are discussing a lot of these same kinds of issues and there are a lot of impacts and you have a real challenge in deciding how to have really good scientists spread themselves over more objectives than they have time for.
	DR. OWNBY:  I was just wondering from the industry's perspective, since you know your marketing numbers much better than we do, is there some other extract that would have a major impact on the health or at least from your idea of sales that we haven't been discussing here?
	MR. HAUCK:  I believe in the past we had given out some marketing information to FDA, but I think as an organization, since we are meeting at the academy meeting, maybe we will raise that among our membership and provide some guidance because that has probably changed.  The last time they asked for it, I think, No. 1 was house dust.
	DR. OWNBY:  Okay.  Are there any other -- is that an adequate answer in consensus from -- okay.  Are there other items or other questions that need to be brought up today?
	Bill, did you have any concluding remarks or items that we need to cover?
	DR. FREAS:  That is it.  I just want to thank you on behalf of FDA.  We truly appreciate all the committee members coming across country and from around the area to give us your suggestions, comments and recommendations.  These are very important to us and we will give serious consideration to everything.
	In the meantime, I will update you on the -- I believe we have a tentatively scheduled meeting for October 24, but it is too far in advance for me to give you anything definite on that meeting.  I will be updating you within a couple of months.  I just thank our chair and thank all of you for all your recommendations.
	Thank you.
	DR. OWNBY:  Thank you all for coming.
	We are adjourned.
	[Whereupon, at 2:20 p.m., the meeting was concluded.]