Scope and Intent . . Pg 1

Introduction . . Pg 1

Background . . Pg 2

Scope of the Risks . . Pg 2

Methods of Viral Clearance . . Pg 3

Validation of Viral Clearance . . Pg 4

Operations and Inspections . . Pg 6

Removal Methods . . Pg 7

Inactivation Methods. . .Pg 7

Additional Considerations. . .Pg 7

Conclusions. . .Pg 8

The intended audience for this document is

the ORA field investigator. The document's

origins stem from the transfer of primary

responsibility for GMP inspections of

fractionators from CBER to ORA in the latter

part of 1996. While FDA field investigators

are well versed in the application of GMP to

the drug industry, this document is intended to

communicate differences in the application of

these regulations occasioned by the uniqueness

of plasma derivative products.

In particular, GMP requires and ORA field

investigators determine through inspections,

that significant aspects of drug manufacturing

operations have been validated. Although the

fractionation industry shares many of the same

process validation concerns with the larger

sterile drug manufacturing community, the

validation of viral clearance processes presents

issues with which the field investigator may not

be familiar.

This document, which has been prepared

jointly by the Office of Regulatory Affairs

(ORA) and the Center for Biologics Evaluation

and Research (CBER), attempts to provide

basic information on viral clearance validation

concepts, to enable the investigator to

distinguish those areas of viral clearance

processes that are appropriate for GMP

inspectional coverage. It is not an

all-encompassing technical guide on viral

inactivation processes, but rather a document

that seeks to assist field investigators in

conducting fractionator inspections. If the need

arises, this document may be amended or

expanded to include other issues of interest to

the field investigator concerning viral clearance

processes.

This guidance represents the Agency's

current thinking regarding inspectional

considerations of viral clearance processes for

plasma derivatives.

Plasma-derived proteins are important

therapeutics for many patients in the U.S. every

year. Because they are manufactured from

human plasma, special precautions must be

taken during the production of these proteins

to assure against the possibility of the products

transmitting infectious diseases to the

recipients.

This document focuses on the possibility

that a small proportion of the individual units of

plasma used as source material may be

contaminated with human viruses and the

processes used to remove or inactivate those

viruses. Additionally, as is the case for any

pharmaceutical, there is a possibility of

introducing adventitious pathogens during

production. This risk is one specifically

addressed by adherence to current Good

Manufacturing Practices (GMP) and for this

reason it is a natural focus of any facility

inspection. This document is to be used in

conjunction with the Compliance Program for

the inspection of Plasma Derivatives of Human

Origin (7342.006), the Guideline on the

General Principles of Process Validation (May

1987), the Investigations Operations Manual

(IOM), and the Code of Federal Regulations,

Title 21 (21 CFR).

Addressing the risks that may be associated

with plasma derivatives begins with the plasma

from which the products are manufactured. A

number of precautions are taken to prevent

contaminated units from being collected and to

eliminate contaminated units when they are

collected. These now familiar precautions

include:

1. Screening donors by medical and behavioral

risk history;

2. Maintaining donor deferral registries to

prevent the use of units from unsuitable

donors;

3. Testing blood and plasma donations for

markers of disease;

4. Quarantining blood and plasma until tests

and control procedures establish its safety;

and,

5. Monitoring and investigating adverse

incidents to ensure that deficiencies are

corrected.

Many investigators will be familiar with these

procedures from their inspections of blood

banks and plasma collection facilities. When

the plasma is used as source material for the

manufacture of plasma derivatives, an additional

layer of safety may be achieved by including

effective viral clearance step(s) in the

manufacturing process.

The viruses of greatest concern for safety of

plasma derivatives include the hepatitis viruses

(hepatitis B and C, abbreviated HBV and HCV,

respectively) and the human immunodeficiency

viruses Types 1 and 2 (collectively HIV; the

causative agent of AIDS). Another virus,

human parvovirus B19 (B19), is a common

contaminant of plasma. Hepatitis A virus (HAV)

contaminates plasma products less frequently.

B19 and HAV pose some unique problems

because they are small (15-30 nm) and do not

possess an outer envelope composed of lipids

(both are termed "non-enveloped" viruses for

this reason). Both B19 and HAV are

extraordinarily resistant to heat and chemical

treatment and are difficult to remove by

nanofiltration. Furthermore, no licensed

screening kits exist in the U.S. for either virus.

Fortunately, the diseases caused by B19 and

HAV are less serious than for other viruses of

concern, although they may cause serious

disease in susceptable populations.

Nevertheless, there are intense efforts

underway to develop new methods for

removing or inactivating these viruses and some

of these methods may begin to appear in

manufacturing processes over the next few

years.

The most clinically significant viruses, HIV,

HBV and HCV, are all "enveloped" viruses,

which renders them more susceptible to

inactivation methods based on chemical

treatments or heat. These viruses are also

larger and more effectively removed by

nanofiltration. Nearly all plasma derivative

manufacturing processes now include effective

methods for their inactivation and/or removal.

It should be noted that certain viruses which

may pose serious risks when associated with

blood components (human T-lymphotropic

virus (HTLV-I & -II) or cytomegalovirus (CMV))

are not considered to be significant risks with

respect to plasma derivatives. These viruses

infect white cells and therefore are associated

with the cellular components of blood which

are mostly absent from plasma. The viruses

also tend to lose their viability over periods of

refrigerated storage and they do not tolerate

freezing and thawing. For this reason, HTLV-I

& -II and CMV are not risk factors for plasma

derivatives.

It should also be noted that the possibility

that bacteria or protozoa may contaminate a

unit of plasma or whole blood is not

considered a significant risk to recipients of

plasma derivatives. Manufacturing under GMP

should remove or inactivate any of these

endogenous agents, and any exogenous agents

introduced during manufacture of the drug

substance. Therefore, properly processed

plasma derivatives pose no additional risk of

bacterial or protozoal contamination beyond

those associated with any aseptically processed

parenteral. However, the processes must be

carefully evaluated to insure that they

consistently produce sterile products ( see

Compliance Program 7342.006 and the

"Guideline on Sterile Drug Products Produced

by Aseptic Processing" 1987).

This document also does not cover possible

risks posed by the poorly characterized agents

thought to be responsible for transmissible

spongiform encephalopathies. There are no

known effective methods for screening out

plasma units that contain the infectious agent,

and no known manufacturing methods that

would be effective in removing or inactivating

the infectious agent.

Finally, this document applies to human

plasma derived products only. Products

derived from animal plasma pose different risks

than human plasma derivatives. Bovine plasma,

for example, may not be sourced from

countries in which the disease, bovine

spongiform encephalopathy (BSE) exists (FR

94-21279). Otherwise, no formal

recommendations regarding manufacturing

safeguards for TSEs have been developed at this

time.

A number of different types of

manufacturing steps are capable of removing or

inactivating viruses that may be present in

plasma pools from source or recovered plasma

donations. These steps may be divided into

two broad categories: removal, or partitioning,

is the physical separation of the virus or viral

particles from the therapeutic component; and

inactivation, which destroys the virus so that

the remaining viral fragments lack the structure

and components needed to infect an individual

receiving the product.

Removal processes include filtration, affinity

chromatography, ion exchange chromatography,

and polyethylene glycol fractionation. Heating

and solvent detergent treatments are examples

of processes that inactivate viruses.

Additionally, some processes, such as ethanol

fractionation, both remove and inactivate

viruses. Finally, a number of new methods are

under development, such as irradiation,

photoinactivation and treatment with a variety

of chemicals. Several of these novel techniques

have been incorporated into the production of

investigational products. In order to be

effective, viral inactivation techniques must

destroy at least one viral element essential to

replication. Photosensitizing techniques use

light-activated dyes that are irradiated, causing

the dyes to convert to molecules that can alter

DNA or membrane lipoproteins. Heat

treatment denatures viral proteins and nucleic

acids, rendering viruses incapable of replication.

Irradiation processes may destroy viral nucleic

acids by inducing breaks and linkages. Solvent

detergent techniques destroy the viral envelope

in lipid-enveloped viruses.

All processes which claim to remove virus

must be fully validated by the manufacturer, as

there are no methods codified in the applicable

regulations. All of these methods are employed

during production of the drug substance.

A specific viral inactivation method that is

employed on the drug product in final

containers, is heat-treatment at an attained

temperature of 60 C ñ 0.5 C for 10-11

(continuous) hours. This process is mandated

for two products: Albumin (Human) and Plasma

Protein Fraction (Human) in 21 CFR 640.81

and 640.91, respectively. Because the

conditions of treatment are specified by

regulation, manufacturers are not required to

validate the effectiveness of the treatment itself,

but must nevertheless demonstrate that the

process operating parameters are met

consistently during production.

Some other products are heat treated, in

bulk tanks or containers, using similar

methodology (e.g. Antihemophilic Factor

(Human), Intravenous Immune Globulin

(Human)); alternative methods may also be

used, such as vapor heat treatment of

lyophilized intermediates. Another inactivation

method that may be encountered (e.g. Immune

Globulin Intravenous (Human) is treatment of

in-process material with solvent and detergent,

for a given time at a given temperature, and

subsequent removal of these additives. This

removal is generally accomplished by extraction

and/or the use of chromatographic methods. In

all of these cases, it is the responsibility of the

manufacturer to validate the effectiveness of

the method, as well as demonstrate that the

validated operating parameters are met

consistently during production.

Validation studies are performed when a

new product is under development or when a

manufacturer wishes to introduce a new step

into a pre-existing manufacturing process. In

these cases, the validation is designed along

with the process which may be optimized for

viral clearance. It is necessary to consider

scale-up problems and the possibility that

validation studies at the pilot scale may not be

relevant to the final manufacturing process. If

scale-up involves changes in the manufacturing

process that could adversely affect the viral

clearance validated at the pilot stage, then viral

clearance would have to be revalidated for the

altered process.

It is also possible that a manufacturer, with a

product made by a well established process,

identifies a step in that process which may be

effective in removing or inactivating viruses.

The manufacturer may then attempt to validate

this step without changing it. In this case, the

manufacturer should take the process as it

exists, without attempting to optimize it for

viral clearance, in order to avoid additional

studies to demonstrate that the product has

not been adversely affected. If the process has

changed, the manufacturer should demonstrate

that the final product has not been adversely

affected.

Regardless of the setting in which the

validation is performed, all viral clearance

validation studies share certain common

features. These can be summarized as follows.

1. Validation is performed on scaled down

laboratory models of the production

process. It is an unacceptable hazard to

introduce viruses into a production area in

order to validate a viral clearance step. It is

also impossible in most cases to produce

sufficient amounts of a virus to validate its

removal at the full manufacturing scale. The

necessity of working with scaled down

systems presents discreet challenges to

validation of viral clearance processes:

a. The scaled down model should

accurately represent the full scale

manufacturing process. This is insured

by controlling parameters such as

temperature, volume, flow rates, contact

times, relative geometries, and load. The

most important measure is the actual

performance of the scaled down process,

measured by parameters such as

capacity, yield and purification if

applicable.

b. The starting material (process

intermediate) should accurately reflect

that of the full scale manufacturing

process. This is often accomplished by

sampling process material from a full

scale batch.

c. Even though scaled down systems are

used for validation, the studies should be

designed to represent the worst case

conditions that could be encountered

during full-scale production, i.e., those

conditions under which the removal or

inactivation of viruses would be expected

to be the least effective. The viral

titrations should be designed with

adequate replicates to assure a

scientifically and statistically sound result.

Validation of the scale-down model

should involve multiple runs of the

model, the results of which are

compared to those from the full-scale

process.

d. Appropriate operational and

performance qualification with reference

to the critical operating parameters

should be performed for the full-scale

manufacturing system. Qualification

should include acquiring sufficient data to

verify that the full-scale system

consistently delivers all critical

performance conditions as was specified

in the successful validation studies with

the representative scale-down system.

e. Subsequent changes in the full scale

manufacturing process can affect the

validity of prior viral clearance studies.

Whenever a process and/or equipment

change is made, the possible effect of

that change on viral clearance should be

considered and evaluated, and the viral

clearance should be revalidated to the

extent necessary.

2. Viral clearance validation studies usually take

the form of "spiking" experiments, in which

large amounts of a virus is added to the test

article. It should be noted that the viral

load is far in excess of what would be

expected in a "contaminated" plasma pool.

The reduction in the amount of added virus

by the manufacturing step in question is then

measured. Appropriate controls are

included to insure that the measurement of

the amounts (titers) of the virus does not

change the performance of the scaled-down

manufacturing step. Additional controls are

included to confirm viability of the indicator

cells and the infectivity of the virus.

3. In vitro analyses are most commonly used to

quantify virus levels in the course of a

validation study. These may take the form

of "plaque assays" or assays that measure

"cytopathic effect", both of which are

performed in tissue culture. These assays

measure infectivity of the virus used in the

study. Biochemical assays may also be

encountered, such as antigen-based or

nucleic acid assays (e.g., PCR). These are

acceptable when predictive of infectivity.

Finally, some studies may use animal models

such as primates or ducks, but these have

become less frequent because they are

expensive and adequate alternative methods

are available.

4. Most validation studies are performed with

model viruses sharing characteristics of the

relevant human viruses. The selection of

appropriate models is of critical importance.

Among the human viruses of concern, only

HIV and HAV have appropriate in vitro

systems with which their titers can be

measured. The clearance of HBV and HCV,

by some manufacturing processes, has been

validated in primate models, but these

studies are not common today.

5. The extent of validation with respect to the

number of steps validated and the number of

different viruses studied varies greatly from

product to product and from manufacturer

to manufacturer. With the exception of

some IGIM products (see footnote 4, page

8), all plasma derivatives are subjected to at

least one viral clearance step, in addition to

the fractionation process itself which has

been shown to reduce viral load for some

agents.

6. A validation study may be acceptable even if

some detectable virus is found, as

these studies should be designed to add

many times more virus to the test article

than would be encountered in actual

practice. Large amounts of virus ("high

titers") permit more precise quantification

and provide safety margins to the

manufacturing process.

7. When more than one manufacturing step in

a process has been validated to clear a

particular virus, the overall clearance of the

process is the product of the two steps

(often calculated by adding log reduction

factors), provided that the two steps

operate by independent principles. For

instance, the results of a filtration step and a

heating step may be combined because they

operate by different mechanisms. On the

other hand, merely repeating a step would

not double the viral clearance because there

would be no second mechanism involved; an

additive effect cannot be presumed.

8. Validation of the process or processes

serves to establish that the operating

parameters, used during normal production,

are appropriate. Changes may be made

based on the validation study. It is therefore

important that the parameters established

during the study are those used in actual

manufacture.

The validation of viral clearance methods is a

highly sophisticated and scientifically complex

undertaking. From time to time, it is expected

that technical questions will arise during the

inspection. The investigator should keep in

mind that the scientific resources of the Agency

are at his/her disposal. Questions may be

referred at any time during the inspection to

the Inspections Task Force (ITF) in CBER's

Office of Compliance (301-827-6191). The ITF

representative will contact the appropriate

reviewer to provide information.

The following elements are suggested as

important aspects of inspecting a manufacturing

process from the point of view of viral safety.

1. In most cases, the validation studies have

undergone scientific review, or are in the

process of being reviewed by CBER staff, as

part of the license application. As such, a

detailed technical review by the

investigatorof the findings is generally not

necessary. However, the manufacturer

should be following the process exactly, as

approved or submitted for approval, and the

investigators should familiarize themselves

with the approved and pending procedures

in applications and supplements. As an

alternative, studies which support claims of

viral safety, either approved by or pending

approval by CBER should be available for

review in the manufacturer's license files,

on-site, for the product(s) in question.

2. Validation studies may have been conducted

by the manufacturer, but often the studies

are performed by a contract firm that

specializes in such studies. As such, the raw

data for the viral assays may not be available

at the licensed site. Available records may

consist of reports from the contractor.

Audits of these contract sites should be

performed periodically to review and verify

raw data generated during these, and other,

studies. Data available at the licensed site

should also be reviewed for integrity.

3. Examine the full-scale manufacturing step

itself to insure that it consistently meets the

critical validated operating parameters that

were defined in the viral validation study. In

addition, manufacturing records should be

reviewed to insure that the process is

carried out as intended. Some examples

follow:

Removal Methods:

a) Removal methods involving nanofiltration

are fairly new in the industry. The

membranes are generally single use

involving tangential flow. Insure that:

i. validated filtration parameters are

being followed, e.g. pressure and

flow rates; and

ii. the membrane is post-production

integrity tested. (NOTE: unlike

membranes used for microbial

retention, for some of these

membranes this is accomplished

using colloids such as dextran.

These tests may be destructive.

Forward flow and bubble point

methods are not applicable with

these membranes).

b) Chromatographic methods (e.g. affinity,

ion exchange) are used in purification

of many plasma derivatives. These

methods may also be validated for their

viral removal capabilities. Insure that:

i. validated parameters (e.g. flow

rates, column height, protein load)

are being consistently followed;

ii. if a validated number of uses has

been established for the column

resin(s), the firm is consistently

using the resin(s) for no more than

the validated number of uses

(NOTE: Often, concurrent

validation is allowed to establish an

acceptable number of times for use

of the resin. If this is the case,

in-process parameters should be

consistently monitored to insure

column performance. In addition,

the resins continued ability to

remove validated levels of virus

should be evaluated, periodically,

during the concurrent study); and

iii. regeneration procedures are

followed between each lot of

product.

Inactivation Methods:

a) Heat treatment processes, either

codified in the regulations or validated

by the manufacturer, should be

performed according to pre-established

parameters to insure effectiveness.

Insure that:

i. the proper time and temperature

parameters are met consistently;

ii. the equipment used is qualified,

maintained and monitored to

insure that these parameters are

met; and

iii. if problems occur, e.g., the process

is interrupted, the manufacturer

should not "repeat" the process

unless such repetition has been

validated and approved under the

license to demonstrate that the

additional heat treatment does not

adversely affect the properties of

the product.

b) Manufacturers who use

Solvent/Detergent methods must

demonstrate that the validated

parameters are met both during the

treatment and the subsequent removal

of the additives used. Insure that:

i. the proper concentrations of

additives are consistently used, and

treatment occurs for the validated

time.

ii. removal procedures are being

properly followed (e.g. If a column

is used, it is employed for the

validated number of uses or

monitored for performance (see

NOTE section 3 (b)(ii)); and

iii. the in-process drug substance is

tested to insure that the additives

have been removed to validated

levels.

Additional Considerations:

1. Inspect the Adverse Event Report/complaint

files and the procedures the company

has developed for handling them. Companies

are required to report incidents of

suspected transmission of viruses to FDA in

30 day periodic reports.

2. Establish that the firm has not changed steps

upstream of the validated viral removal step

without the proper reporting to FDA. This

may significantly change the characteristics of

the intermediate entering the viral

removal/inactivation step(s) and should be

submitted to the Agency per 21 CFR 601.12.

3. Insure that there has been no change in the

ordering of steps in the manufacturing

process. This may affect the effectiveness of

the viral inactivation/removal steps. These

changes also should be reported as specified

in 21 CFR 601.12.

For your information, sanitizing agents

(bacteriocidal and/or sporicidal) used in the

cleaning of the facility and equipment generally

serve to inactivate the viruses of concern for

plasma derivatives (refer to the Compliance

Program 7342.006 for further instruction on

inspection of cleaning of facilities and

equipment).

The establishment inspection provides a

critical element in assuring the viral safety of

plasma derivatives. Although scientifically

sound methods for removing or inactivating

viral contaminants may have been designed into

a production process, it is essential that these

methods be executed consistently and reliably

on a day-to-day and lot-to-lot basis in order to

provide assurance that the product is safe for

use. The establishment inspection is the

principle setting in which the operational

validity of viral clearance procedures may be