International Conference on Harmonisation; Draft Guideline on Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin; Availability

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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

[Docket No. 96D-0058]

International Conference on Harmonisation; Draft Guideline on

Viral Safety Evaluation of Biotechnology Products Derived From Cell

Lines of Human or Animal Origin; Availability

AGENCY: Food and Drug Administration, HHS.

ACTION: Notice.

-----------------------------------------------------------------------

SUMMARY: The Food and Drug Administration (FDA) is publishing a draft

guideline entitled ``Viral Safety Evaluation of Biotechnology Products

Derived From Cell Lines of Human or Animal Origin.'' The draft

guideline was prepared under the auspices of the International

Conference on Harmonisation of Technical Requirements for Registration

of Pharmaceuticals for Human Use (ICH). The draft guideline describes

viral safety testing and evaluation of biotechnology products derived

from characterized cell lines of human or animal origin, and outlines

data that should be submitted in marketing applications.

DATES: Written comments by August 8, 1996.

ADDRESSES: Submit written comments on the draft guideline to the

Dockets Management Branch (HFA-305), Food and Drug Administration,

12420 Parklawn Dr., rm. 1-23, Rockville, MD 20857. Copies of the draft

guideline are available from the Division of Communications Management

(HFD-210), Center for Drug Evaluation and Research, Food and Drug

Administration, 7500 Standish Pl., Rockville, MD 20855, 301-594-1012.

An electronic version of this guideline is also available via Internet

by connecting to the CDER file transfer protocol (FTP) server

(CDVS2.CDER.FDA.GOV).

FOR FURTHER INFORMATION CONTACT:

Regarding the guideline: Ruth Wolff, Center for Biologics

Evaluation and Research (HFM-30), Food and Drug Administration, 1401

Rockville Pike, Rockville, MD 20852, 301-594-5660.

Regarding ICH: Janet J. Showalter, Office of Health Affairs (HFY-

20), -Food and Drug Administration, 5600 Fishers Lane, Rockville, MD

20857, 301-827-0864.

SUPPLEMENTARY INFORMATION: In recent years, many important initiatives

have been undertaken by regulatory authorities and industry

associations to promote international harmonization of regulatory

requirements. FDA has participated in many meetings designed to enhance

harmonization and is committed to seeking scientifically based

harmonized technical procedures for pharmaceutical development. One of

the goals of harmonization is to identify and then reduce differences

in technical requirements for drug development among regulatory

agencies.

ICH was organized to provide an opportunity for tripartite

harmonization initiatives to be developed with input from both

regulatory and industry representatives. FDA also seeks input from

consumer representatives and others. ICH is concerned with

harmonization of technical requirements for the registration of

pharmaceutical products among three regions: The European Union, Japan,

and the United States. The six ICH sponsors are the European

Commission, the European Federation of Pharmaceutical Industries

Associations, the Japanese Ministry of Health and Welfare, the Japanese

Pharmaceutical Manufacturers Association, the Centers for Drug

Evaluation and Research and Biologics Evaluation and Research, FDA, and

the Pharmaceutical Research and Manufacturers of America. The ICH

Secretariat, which coordinates the preparation of documentation, is

provided by the International Federation of Pharmaceutical

Manufacturers Associations (IFPMA).

The ICH Steering Committee includes representatives from each of

the ICH sponsors and the IFPMA, as well as observers from the World

Health Organization, the Canadian Health Protection Branch, and the

European Free Trade Area.

At a meeting held on November 29, 1995, the ICH Steering Committee

agreed that a draft guideline entitled ``Viral Safety Evaluation of

Biotechnology Products Derived From Cell Lines of Human or Animal

Origin'' should be made available for public comment. The draft

guideline is the product of the Quality Expert Working Group of the

ICH. Comments about this draft will be considered by FDA and the

Quality Expert Working Group. Ultimately, FDA intends to adopt the ICH

Steering Committee's final guideline.

The draft guideline describes approaches for evaluating the risk of

viral contamination and for removing viruses from biotechnology

products derived from human or animal cell lines. The draft guideline

emphasizes the value of many strategies including: (1) Thorough

characterization/screening of the cell substrate starting material in

order to identify which, if any, viral contaminants are present; (2)

assessment of risk by a determination of the human tropism of the

contaminants; (3) incorporation of studies that assess virus

inactivation and removal steps in the production process; (4) careful

design of viral clearance studies to avoid pitfalls and provide

interpretable results; and (5) use of different methods of virus

inactivation or removal in the same production process in order to

achieve maximum viral clearance.

In the past, guidelines have generally been issued under

Sec. 10.90(b) (21 CFR 10.90(b)), which provides for the use of

guidelines to state procedures or standards of general applicability

that are not legal requirements but are acceptable to FDA. The agency

is now in the process of revising Sec. 10.90(b). Although this

guideline does not create or confer any rights for or on any person and

does not operate to bind FDA, it does represent the agency's current

thinking on viral safety evaluation of biotechnology products.

Interested persons may, on or before August 8, 1996, submit to the

Dockets Management Branch (address above) written comments on the draft

guideline. Two copies of any comments are to be submitted, except that

individuals may submit one copy. Comments are to be identified with the

docket number found in brackets in the heading of this document. The

draft guideline and received comments may be seen in the office above

between 9 a.m. and 4 p.m., Monday through Friday.

The text of the draft guideline follows:

Viral Safety Evaluation of Biotechnology Products Derived From Cell

Lines of Human or Animal Origin

I. Introduction

This document is concerned with testing and evaluation of the

viral safety of biotechnology products derived from characterized

cell lines of human or animal origin (i.e., mammalian, avian,

insect), and outlines data that should be submitted in the marketing

application/registration package. For the purposes of this document,

the term virus excludes nonconventional transmissible agents like

those associated with Bovine Spongiform Encephalopathy (BSE) and

scrapie. Applicants are encouraged to discuss issues associated with

BSE with the regulatory authorities.

The scope of the document covers products derived from cell

cultures initiated from characterized cell banks. It covers products

derived from in vitro cell culture, such as interferons, monoclonal

antibodies, and recombinant deoxyribonucleic acid (DNA)- derived

products including recombinant subunit vaccines, and also includes

products

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derived from hybridoma cells grown in vivo as ascites. In this

latter case, special considerations apply and additional information

on testing cells propagated in vivo is contained in Appendix 1.

Inactivated vaccines, all live vaccines containing self-replicating

agents, and genetically engineered live vectors are excluded from

the scope of this document.

The risk of viral contamination is a feature common to all

biotechnology products derived from cell lines. Such contamination

could have serious clinical consequences and can arise from the

contamination of the source cell lines themselves (cell substrates)

or from adventitious introduction of virus during production. To

date, however, biotechnology products derived from cell lines have

not been implicated in the transmission of viruses. Nevertheless, it

is expected that the safety of these products with regard to viral

contamination can be reasonably assured only by the application of a

virus testing program and assessment of virus removal and

inactivation achieved by the manufacturing process, as outlined

below.

Three principal, complementary approaches have evolved to

control the potential viral contamination of biotechnology products:

(1) Selecting and testing cell lines and other raw materials,

including media components, for the absence of undesirable viruses

which may be infectious and/or pathogenic for humans;

(2) Assessing the capacity of the production processes to clear

infectious viruses; and

(3) Testing the product at appropriate stages of production for

the absence of contaminating infectious viruses.

All testing suffers from the inherent limitation of quantitative

virus assays in that the ability to detect low viral concentrations

depends for statistical reasons on the size of the sample.

Therefore, no single approach will necessarily establish the safety

of a product. Confidence that infectious virus is absent from the

final product will in many instances not be derived solely from

direct testing for their presence, but also from a demonstration

that the purification regimen is capable of removing and/or

inactivating the viruses.

The type and extent of viral tests and viral clearance studies

required at different stages of production will depend on various

factors and should be considered on a case-by-case and step-by-step

basis. The factors that should be taken into account include the

extent of cell bank characterization and qualification, the nature

of any viruses detected, culture medium constituents, culture

methods, facility and equipment design, the results of viral tests

after cell culture, the ability of the process to clear viruses, and

the type of product and its intended clinical use.

The purpose of this document is to provide a general framework

for virus testing, experiments for the assessment of viral

clearance, and a recommended approach for the design of viral tests

and viral clearance studies. Related information is described in the

appendices and selected definitions are provided in the Glossary.

Manufacturers should adjust the recommendations presented here

to their specific product and its production process. The approach

used by manufacturers in their overall strategy for ensuring viral

safety should be explained and justified. In addition to the

detailed data which is provided, an overall summary of the viral

safety assessment would be useful in facilitating the review by

regulatory authorities. This summary should contain a brief

description of all aspects of the viral safety studies and

strategies used to prevent virus contamination as they pertain to

this document.

II. Potential Sources of Virus Contamination

Viral contamination of biotechnology products may arise from the

original source of the cell lines or from adventitious introduction

of virus during production processes.

A. Viruses That Could Occur in the Master Cell Bank (MCB)

Cells may have latent or persistent virus infection (e.g.,

herpesvirus) or endogenous retrovirus which may be transmitted

vertically from one cell generation to the next, since the viral

genome persists within the cell. Such viruses may be constitutively

expressed or may unexpectedly become expressed as an infectious

virus.

Viruses can be introduced into the MCB by several routes such

as: (1) Derivation of cell lines from infected animals; (2) use of

virus to establish the cell line; (3) use of contaminated biological

reagents such as animal serum components; and (4) contamination

during cell handling.

B. Adventitious Viruses That Could Be Introduced During Production

Adventitious viruses can be introduced into the final product by

several routes including, but not limited to, the following: (1) Use

of contaminated biological reagents such as animal serum components;

(2) use of a virus for the induction of expression of specific genes

encoding a desired protein; (3) use of a contaminated reagent, such

as a monoclonal antibody affinity column; and (4) use of a

contaminated excipient during formulation.

III. Cell Line Qualification: Testing for Viruses

An important part of qualifying a cell line for use in the

production of a biotechnology product is the appropriate testing for

the presence of virus.

A. Suggested Virus Tests for MCB, Working Cell Bank (WCB), and

Cells at the Limit of In Vitro Cell Age Used for Production

Table 1 shows an example of virus tests to be performed once

only at various cell levels, including MCB, WCB, and cells at the

limit of in vitro cell age used for production.

1. Master Cell Bank

Extensive screening for both endogenous and nonendogenous viral

contamination should be performed on the MCB. For heterohybrid cell

lines in which one or more partners are human or nonhuman primate in

origin, tests should be performed in order to detect viruses of

human or nonhuman primate origin because viral contamination arising

from these cells may pose a particular hazard.

Testing for nonendogenous viruses should include in vitro and in

vivo inoculation tests and any other specific tests, including

species-specific tests such as the mouse antibody production (MAP)

test, that are appropriate, based on the passage history of the cell

line, to detect possible contaminating viruses.

2. Working Cell Bank

Each WCB as a starting cell substrate for drug production should

be tested for adventitious virus either by direct testing or by

analysis of cells at the limit of in vitro cell age, initiated from

the WCB. When appropriate nonendogenous virus tests have been

performed on the MCB and cells cultured up to or beyond the limit of

in vitro cell age have been derived from the WCB and used for

testing for the presence of adventitious viruses, similar tests need

not be performed on the initial WCB. Antibody production tests are

usually not necessary for the WCB. An alternative approach in which

full tests are carried out on the WCB rather than on the MCB would

also be acceptable.

3. Cells at the Limit of In Vitro Cell Age Used for Production

The limit of in vitro cell age used for production should be

based on data derived from production cells expanded under pilot-

plant scale or commercial-scale conditions to the proposed in vitro

cell age or beyond. Generally, the production cells are obtained by

expansion of the WCB; the MCB could also be used to prepare the

production cells. Cells at the limit of in vitro cell age should be

evaluated once for those endogenous viruses that may have been

undetected in the MCB and WCB. The performance of suitable tests

(e.g., in vitro and in vivo) at least once on cells at the limit of

in vitro cell age used for production would provide further

assurance that the production process is not prone to contamination

by adventitious virus. If any adventitious viruses are detected at

this level, the process should be carefully checked in order to

determine the cause of the contamination and completely redesigned

if necessary.

B. Recommended Viral Detection and Identification Assays

Numerous assays can be used for the detection of endogenous and

adventitious viruses. Table 2 outlines examples for these assays.

They should be regarded as assay protocols recommended for the

present, but the list is not all-inclusive or definitive. Since the

most appropriate techniques may change with scientific progress,

proposals for alternative techniques, when accompanied by adequate

supporting data, may be acceptable. Manufacturers are encouraged to

discuss these alternatives with the regulatory authorities. Other

tests may be necessary depending on the individual case. Assays

should include appropriate controls to ensure adequate sensitivity

and specificity. Wherever a relatively high possibility of the

presence of a specific virus can be predicted from the species of

origin of the cell

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substrate, specific tests and/or approaches may be necessary. If the

cell line used for production is of human or nonhuman primate

origin, additional tests for human viruses, such as those causing

immunodeficiency diseases and hepatitis, should be performed unless

otherwise justified. The polymerase chain reaction (PCR) may be

appropriate for detection of sequences of these human viruses as

well as for other specific viruses. The following is a brief

description of a general framework and philosophical background

within which the manufacturer should justify what was done.

1. Tests for Retroviruses

For the MCB and for cells cultured up to or beyond the limit of

in vitro cell age used for production, tests for retroviruses,

including infectivity assays in sensitive cell cultures and electron

microscopy (EM) studies, should be carried out. If infectivity is

not detected and no retrovirus or retrovirus-like particles have

been observed by EM, reverse transcriptase (RT) or other appropriate

assays should be performed to detect retroviruses which may be

noninfectious. Induction studies have not been found to be useful.

2. In Vitro Assays

In vitro tests are carried out by the inoculation of a test

article (see Table 2) into various susceptible indicator cell

cultures capable of detecting a wide range of human and relevant

animal viruses. The choice of cells used in the test is governed by

the species of origin of the cell bank to be tested, but should

include a human and/or a nonhuman primate cell line susceptible to

human viruses. The nature of the assay and the sample to be tested

are governed by the type of virus which may possibly be present

based on the origin or handling of the cells. Both cytopathic and

hemadsorbing viruses should be sought.

3. In Vivo Assays

A test article (see Table 2) should be inoculated into animals,

including suckling and adult mice, and in embryonated eggs to reveal

viruses that cannot grow in cell cultures. Additional animal species

may be used depending on the nature and source of the cell lines

being tested. The health of the animals should be monitored and any

abnormality should be investigated to establish the cause of the

illness.

4. Antibody Production Tests

Species-specific viruses present in rodent cell lines may be

detected by inoculating test article (see Table 2) into virus-free

animals and examining the serum antibody level or enzyme activity

after a specified period. Examples of such tests are the MAP test,

rat antibody production (RAP) test, and hamster antibody production

(HAP) test. The viruses currently screened for in the antibody

production assays are discussed in Table 3.

C. Acceptability of Cell Lines

It is recognized that some cell lines used for the manufacture

of product will contain endogenous retroviruses, other viruses, or

viral sequences. In such circumstances, the action plan recommended

for manufacture is described in section V. of this document. The

acceptability of cell lines containing viruses other than endogenous

retroviruses will be considered on an individual basis by the

regulatory authorities, by taking into account a risk/benefit

analysis based on the benefit of the product and its intended

clinical use, the nature of the contaminating viruses, their

potential for infecting humans or for causing disease in humans, the

purification process for the product (e.g., viral clearance

evaluation data), and the extent of the virus tests conducted on the

purified bulk.

IV. Testing for Viruses in Unprocessed Bulk

The unprocessed bulk constitutes one or multiple pooled harvests

of cells and culture media. When cells are not readily accessible

(e.g., hollow fiber or similar systems), the unprocessed bulk would

constitute fluids harvested from the fermenter. A representative

sample of the unprocessed bulk, removed from the production reactor

prior to further processing, represents one of the most suitable

levels at which the possibility of adventitious virus contamination

can be determined with a high probability of detection. Appropriate

testing for viruses should be performed at the unprocessed bulk

level unless virus testing is made more sensitive by initial partial

processing (e.g., unprocessed bulk may be toxic in test cell

cultures, whereas partially processed bulk may not be toxic).

In certain instances it may be more appropriate to test a

mixture consisting of both intact and disrupted cells and their cell

culture supernatants removed from the production reactor prior to

further processing. Data from at least 3 lots of unprocessed bulk at

pilot-plant scale or commercial scale should be submitted as part of

the registration/marketing application package.

It is recommended that manufacturers develop programs for the

ongoing assessment of adventitious viruses in production batches.

The scope, extent, and frequency of virus testing on the unprocessed

bulk should be determined by taking several points into

consideration including the nature of the cell lines used to produce

the desired products, the results and extent of virus tests

performed during the qualification of the cell lines, the

cultivation method, raw material sources, and results of viral

clearance studies. In vitro screening tests, using one or several

cell lines, are generally employed to test unprocessed bulk. If

appropriate, a PCR test or other suitable methods may be used.

Generally, harvest material in which adventitious virus has been

detected should not be used to manufacture the product. If any

adventitious viruses are detected at this level, the process should

be carefully checked to determine the cause of the contamination,

and appropriate actions taken.

V. Rationale and Action Plan for Viral Clearance Studies and Virus

Tests on Purified Bulk

It is important to design the most relevant and rational

protocol for virus tests from the MCB level through the various

stages of drug production to the final product, including evaluation

and characterization of viral clearance from unprocessed bulk. The

evaluation and characterization of viral clearance plays a critical

role in this scheme. The goal should be to obtain the best

reasonable assurance that the product is free of virus

contamination.

In selecting viruses to use for a clearance study, it is useful

to distinguish between the need to evaluate processes for their

ability to clear viruses that are known to be present and the desire

to estimate the robustness of the process by characterizing the

clearance of nonspecific ``model'' viruses (described later).

Definitions of ``relevant'', specific and nonspecific ``model''

viruses are given in the Glossary. Process evaluation requires

knowledge of how much virus may be present in the process, such as

the unprocessed bulk, and how much can be cleared in order to assess

product safety. Knowledge of the time dependence for inactivation

procedures is helpful in assuring the effectiveness of the

inactivation process.

When a manufacturing process is characterized for robustness of

clearance using nonspecific ``model'' viruses, less extensive virus

removal/inactivation studies are appropriate. Indepth, time-

dependent inactivation studies, demonstration of reproducibility of

inactivation/removal, and evaluation of process parameters are not

required. These studies should be performed on the manufacturing

process in Cases A through E as described below.

Table 4 presents an example of an action plan in terms of

process evaluation and characterization of viral clearance as well

as virus tests on purified bulk, in response to the results of virus

tests on cells and/or the unprocessed bulk. Various cases are

considered. In all cases, characterization of clearance using

nonspecific ``model'' viruses should be performed. The most common

situations are Cases A and B. Production systems contaminated with a

virus other than a rodent retrovirus are normally not used. Where

there are convincing and well justified reasons for drug production

using a cell line from Cases C, D, or E, these should be discussed

with the regulatory authorities. With Cases C, D, and E, it is

important to have validated effective steps to inactivate/remove the

virus in question from the manufacturing process.

Case A: Where no virus, virus-like particle, or retrovirus-like

particle has been demonstrated in the cells or the unprocessed bulk,

virus removal and inactivation studies should be performed with

nonspecific ``model'' viruses as previously stated.

Case B: Where only a rodent retrovirus (or a retrovirus-like

particle which is believed to be nonpathogenic, such as rodent A-

and R-type particles) is present, process evaluation using a

specific ``model'' virus, such as a murine leukemia virus, should be

performed. Purified bulk should be tested using suitable methods

having high specificity and sensitivity for the detection of the

virus in question. For marketing authorization, data from at least 3

lots of purified bulk at pilot-plant scale or commercial scale

should be provided. Cell lines such as Chinese hamster ovary (CHO),

C127, baby hamster kidney (BHK), and murine hybridoma cell lines

have frequently been used as substrates for drug

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production with no reported safety problems related to viral

contamination of the products. For these cell lines in which the

endogenous particles have been extensively characterized and

clearance has been demonstrated, it is not usually necessary to

assay for the presence of the noninfectious particles in purified

bulk.

Case C: When the cells or unprocessed bulk are known to contain

a virus, other than a rodent retrovirus, for which there is no

evidence of capacity for infecting humans, (such as those identified

by footnote 2 in Table 3, except rodent retroviruses (Case B)),

virus removal and inactivation evaluation studies should use the

identified virus. If it is not possible to use the identified virus,

``relevant'' or specific ``model'' viruses should be used to

demonstrate acceptable clearance. Time-dependent inactivation for

identified (or ``relevant'' or specific ``model'') viruses at the

critical inactivation step(s) should be obtained as part of process

evaluation for these viruses. Purified bulk should be tested using

suitable methods having high specificity and sensitivity for the

detection of the virus in question. For the purpose of marketing

authorization, data from at least 3 lots of purified bulk

manufactured at pilot-plant scale or commercial scale should be

provided.

Case D: Where a known human pathogen, such as those indicated by

footnote 1 in Table 3, is identified, the product may be acceptable

only under exceptional circumstances. In this instance, it is

recommended that the identified virus be used for virus removal and

inactivation evaluation studies and specific methods with high

specificity and sensitivity for the detection of the virus in

question be employed. If it is not possible to use the identified

virus, ``relevant'' and/or specific ``model'' viruses (described

later) should be used. The process should be shown to achieve the

removal and inactivation of the selected viruses during the

purification and inactivation processes. Time-dependent inactivation

data for the critical inactivation step(s) should be obtained as

part of process evaluation. Purified bulk should be tested using

suitable methods having high specificity and sensitivity for the

detection of the virus in question. For the purpose of marketing

authorization, data from at least 3 lots of purified bulk

manufactured at pilot-plant scale or commercial scale should be

provided.

Case E: When a virus which cannot be classified by currently

available methodologies is detected in the cells or unprocessed

bulk, the product is usually considered unacceptable since the virus

may prove to be pathogenic. In the very rare case where there are

convincing and well justified reasons for drug production using such

a cell line, this should be discussed with the regulatory

authorities before proceeding further.

VI. Evaluation and Characterization of Viral Clearance Procedures

Evaluation and characterization of the virus removal and/or

inactivation procedures plays an important role in establishing the

safety of biotechnology products. Many instances of contamination in

the past have occurred with agents whose presence was not known or

even suspected, and though this happened to biological products

derived from various source materials other than fully characterized

cell lines, assessment of viral clearance will provide a measure of

confidence that any unknown, unsuspected, and harmful viruses may be

removed.

The objective of viral clearance studies is to assess process

step(s) that can be considered to be effective in inactivating/

removing viruses and to estimate quantitatively the overall level of

virus reduction obtained by the process. This should be achieved by

the deliberate addition (``spiking'') of significant amounts of a

virus to the crude material and/or to different fractions obtained

during the various process stages and demonstrating its removal or

inactivation during the subsequent stages. It is not necessary to

evaluate or characterize every stage of a manufacturing process if

adequate clearance is demonstrated by the use of fewer steps.

The reduction of virus infectivity may be achieved by removal of

virus particles or by inactivation of viral infectivity. For each

production stage assessed, the possible mechanism of loss of viral

infectivity should be described with regard to whether it is due to

inactivation or removal. For inactivation steps, the study should be

planned in such a way that samples are taken at different times and

an inactivation curve constructed. Studies should be carried out in

a manner that is well documented and controlled (see section

VI.B.5).

Viral clearance evaluation studies are performed to demonstrate

the clearance of a virus known to be present in the MCB and/or to

provide some level of assurance that adventitious viruses which

could not be detected, or might gain access to the production

process, would be cleared.

In contrast to viral clearance studies described above for

viruses known to be present, studies to characterize the ability to

remove and/or inactivate other viruses should be conducted. The

purpose of the studies with viruses that are not known or expected

to be present is to characterize the robustness of the procedure

rather than to achieve a specific inactivation or removal goal. They

are not performed to evaluate a specific safety risk. Therefore, a

specific clearance value need not be achieved.

A. The Choice of Viruses for the Evaluation and Characterization of

Viral Clearance

Viruses for clearance evaluation and process characterization

studies should be chosen to resemble viruses which may contaminate

the product and to represent a wide range of physico-chemical

properties in order to test the ability of the system to eliminate

viruses in general. The manufacturer should justify the choice of

viruses in accordance with the aims of the evaluation and

characterization study and the guidance provided in this guideline.

1. ``Relevant'' Viruses and ``Model'' Viruses

A major issue in performing a viral clearance study is to

determine which viruses should be used. Such viruses fall into three

categories: ``Relevant'' viruses, specific ``model'' viruses, and

nonspecific ``model'' viruses.

``Relevant'' viruses are viruses used in process evaluation of

viral clearance studies which are either the identified viruses, or

of the same species as the viruses that are known, or likely to

contaminate the cell substrate or any other reagents or materials

used in the production process. The purification and/or inactivation

process should demonstrate the capability to remove and/or

inactivate such viruses. When a ``relevant'' virus is not available

or when it is not well adapted to process evaluation of viral

clearance studies (e.g., it cannot be grown in vitro to sufficiently

high titers), a specific ``model'' virus should be used as a

substitute. An appropriate specific ``model'' virus may be a virus

which is closely related to the known or suspected virus (same genus

or family), having similar physical and chemical properties to the

observed or suspected virus.

Cell lines derived from rodents usually contain endogenous

retrovirus particles or retrovirus-like particles, which may be

infectious (C-type particles) or noninfectious (cytoplasmic A- and

R-type particles). The capacity of the manufacturing process to

remove and/or inactivate rodent retroviruses from products obtained

from such cells should be determined. This may be accomplished by

using a murine leukemia virus, a specific ``model'' virus in the

case of cells of murine origin. When human cell lines secreting

monoclonal antibodies have been obtained by the immortalization of B

lymphocytes by Epstein-Barr Virus (EBV), the ability of the

manufacturing process to remove and/or inactivate a herpes virus

should be determined. Pseudorabies virus may also be used as a

specific ``model'' virus.

When the purpose is to characterize the capacity of the

manufacturing process to remove and/or inactivate viruses in

general, i.e., to characterize the robustness of the clearance

process, viral clearance characterization studies should be

performed with nonspecific ``model'' viruses with differing

properties. Data obtained from studies with ``relevant'' and/or

specific ``model'' viruses may also contribute to this assessment.

It is not necessary to test all types of viruses. Preference should

be given to viruses that display a significant resistance to

physical and/or chemical treatments. The results obtained for such

viruses provide useful information about the ability of the

production process to remove and/or inactivate viruses in general.

The choice and number of viruses used will be influenced by the

quality and characterization of the cell lines and the production

process.

Examples of useful ``model'' viruses representing a range of

physico-chemical structures and examples of viruses which have been

used in viral clearance studies are given in Appendix 2 and Table A-

1.

2. Other Considerations

Additional points to be considered are as follows:

(a) Viruses which can be grown to high titer are desirable,

although this may not always be possible.

(b) There should be an efficient and reliable assay for the

detection of each virus

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used, for every stage of manufacturing that is tested.

(c) Consideration should be given to the health hazard which

certain viruses may pose to the personnel performing the clearance

studies.

B. Design and Implications of Viral Clearance Evaluation and

Characterization Studies

1. Facility and Staff

It is inappropriate to introduce any virus into a production

facility because of good manufacturing practice (GMP) constraints.

Therefore, viral clearance studies should be conducted in a separate

laboratory equipped for virological work and performed by staff with

virological expertise in conjunction with production personnel

involved in designing and preparing a scaled-down version of the

purification process.

2. Scaled-down Production System

The validity of the scaling-down should be demonstrated. The

level of purification of the scaled-down version should represent as

closely as possible the production procedure. For chromatographic

equipment, column bed-height, linear flow-rate, flow-rate-to-bed-

volume ratio (i.e., contact time), buffer and gel types, pH,

temperature, and concentration of protein, salt, and product should

all be shown to be representative of commercial-scale manufacturing.

For other procedures, similar considerations apply. Deviations which

cannot be avoided should be discussed with regard to their influence

on the results.

3. Analysis of Step-wise Elimination of Virus

When viral clearance studies are being performed, it is

desirable to assess the contribution of more than one production

step to virus elimination. Essential stages of the purification

process should be individually assessed for their ability to remove

and inactivate virus and careful consideration should be given to

the exact definition of an individual stage. Sufficient virus should

be present in the material of each stage to be tested so that an

adequate assessment of the effectiveness of each step is obtained.

Generally, virus should be added to in-process material of each

stage to be tested. In some cases, simply adding high titer virus to

unpurified bulk and testing its concentration between steps will be

sufficient. When virucidal buffers are used in multiple steps within

the manufacturing process, alternative strategies such as parallel

spiking in less virucidal buffers may be carried out as part of the

overall process assessment. The virus titer before and after each

step being tested should be determined. Quantitative infectivity

assays should have adequate sensitivity and reproducibility and

should be performed with sufficient replicates to ensure adequate

statistical validity of the result (see Appendix 3). Quantitative

assays not associated with infectivity may be used if justified.

Appropriate virus controls should be included in all infectivity

assays to ensure the sensitivity of the method. Also, the statistics

of sampling virus when at low concentrations should be considered

(Appendix 4).

4. Determining Physical Removal Versus Inactivation

Reduction in virus infectivity may be achieved by the removal or

inactivation of virus. For each production stage assessed, the

possible mechanism of loss of viral infectivity should be described

with regard to whether it is due to inactivation or removal. If

little clearance of infectivity is achieved by the production

process, and the clearance of virus is considered to be a major

factor in the safety of the product, specific or additional

inactivation/removal steps should be introduced. It may be necessary

to distinguish between removal and inactivation for a particular

step, e.g., when there is a possibility that a buffer used in more

than one clearance step may contribute to inactivation during each

step, i.e., the contribution to inactivation by a buffer shared by

several chromatographic steps and the removal achieved by each of

these chromatographic steps should be distinguished. Assurance

should be provided that any virus potentially retained by the

production system would be adequately destroyed or removed prior to

reuse of the system. For example, such evidence may be provided by

demonstrating that the cleaning and regeneration procedures do

inactivate or remove virus.

5. Inactivation Assessment

For assessment of viral inactivation, unprocessed crude material

or intermediate material should be spiked with infectious virus and

the reduction factor calculated. The determination of initial virus

load for assessing inactivation potential may be derived from the

titer of the spiking virus preparation. This may be of importance

when virucidal buffers are used in multiple steps within the

manufacturing process. Virus inactivation is not a simple, first

order reaction and is usually more complex, with a fast ``phase 1''

and a slow ``phase 2.'' The study should, therefore, be planned in

such a way that samples are taken at different times and an

inactivation curve constructed. It is recommended that studies for

inactivation include at least one time point less than the minimum

exposure time and greater than zero, in addition to the minimum

exposure time. These types of data are particularly important where

the virus is a ``relevant'' virus known to be a human pathogen and

an effective inactivation process is being designed.

6. Function and Regeneration of Columns

Over time and after repeated use, the ability of chromatography

columns and other devices used in the purification scheme to clear

virus may vary. Some estimate of the stability of the viral

clearance after several uses may provide support for repeated use of

such columns. Assurance should be provided that any virus

potentially retained by the production system would be adequately

destroyed or removed prior to reuse of the system. For example, such

evidence may be provided by demonstrating that the cleaning and

regeneration procedures do inactivate or remove virus.

7. Specific Precautions

(a) Care should be taken in preparing the high-titer virus to

avoid aggregation which may enhance physical removal and decrease

inactivation, thus distorting the correlation with actual

production.

(b) Consideration should be given to the minimum quantity of

virus which can be reliably assayed.

(c) The study should include parallel control assays to assess

the loss of infectivity of the virus due to such reasons as the

dilution, concentration, filtration, or storage of samples before

titration.

(d) The virus ``spike'' should be added to the product in a

small volume so as not to dilute or change the characteristics of

the product. Diluted, test-protein sample is no longer identical to

the product obtained at commercial scale.

(e) Small differences in, for example, buffers, media, or

reagents, can substantially affect viral clearance.

(f) Virus inactivation is time-dependent, therefore, the amount

of time a spiked product remains in a particular buffer solution or

on a particular chromatography column should reflect the conditions

of the commercial-scale process.

(g) Buffers and product should be evaluated independently for

toxicity or interference in assays used to determine the virus

titer, as these components may adversely affect the indicator cells.

If the solutions are toxic to the indicator cells, dilution,

adjustment of the pH, or dialysis of the buffer containing spiked

virus may be necessary. If the product itself has antiviral

activity, the clearance study may need to be performed without the

product in a ``mock'' run, although omitting the product or

substituting a similar protein that does not have antiviral activity

could affect the behavior of the virus in some production steps.

Sufficient controls to demonstrate the effect of procedures used

solely to prepare the sample for assay (e.g., dialysis, storage) on

the removal/inactivation of the spiking virus should be included.

(h) Many purification schemes use the same or similar buffers or

columns repetitively. The effects of this approach should be taken

into account when analyzing the data. The effectiveness of virus

elimination by a particular process may vary with the manufacturing

stage at which it is used.

(i) Overall reduction factors may be underestimated where

production conditions or buffers are too cytotoxic or virucidal and

should be discussed on a case-by-case basis.

C. Interpretation of Viral Clearance Studies; Acceptability

The objective of assessing virus inactivation/removal is to

evaluate and characterize process steps that can be considered to be

effective in inactivating/removing viruses and to estimate

quantitatively the overall level of virus reduction obtained by the

manufacturing process. For virus contaminants, as in Cases B through

E, it is important to show that not only is the virus eliminated or

inactivated, but that there is excess capacity for viral clearance

built into the purification process to assure an appropriate level

of safety for the final product. The amount of virus eliminated or

inactivated by the production

[[Page 21887]]

process should be compared to the amount of virus which may be

present in unprocessed bulk. However, for inactivation studies in

which nonspecific ``model'' viruses are used, or when specific

``model'' viruses are used as surrogates for virus particles such as

the CHO intracytoplasmic retrovirus-like particles, it is sufficient

to demonstrate reproducible clearance in at least two independent

experiments. It is recommended that these studies for inactivation

include at least one time-point less than the minimum exposure time

and greater than zero, in addition to the minimum exposure time.

To carry out this comparison, it is important to estimate the

amount of virus in the unprocessed bulk. This estimate should be

obtained using assays for infectivity or other methods such as

transmission electron microscopy (TEM). The entire purification

process should be able to eliminate substantially more virus than is

estimated to be present in a single-dose-equivalent of unprocessed

bulk. See Appendix 5 for calculation of virus reduction factors and

Appendix 6 for calculation of estimated particles per dose.

A combination of factors must be considered when judging the

data supporting the effectiveness of virus inactivation/removal

procedures. These include:

(i) The appropriateness of the test viruses used;

(ii) The design of the clearance studies;

(iii) The log reduction achieved;

(iv) The time dependence of inactivation;

(v) The potential effects of variation in process parameters on

virus inactivation/removal; and

(vi) The limits of assay sensitivities.

Effective clearance may be achieved by any of the following:

Multiple inactivation steps, multiple complementary separation

steps, or combinations of inactivation and separation steps. Since

separation methods may be dependent on the extremely specific

physico-chemical properties of a virus which influence its

interaction with gel matrices and precipitation properties,

``model'' viruses may be separated in a different manner than a

target virus. Differences may originate from changes in surface

properties such as glycosylation. However, despite these potential

variables, effective removal can be obtained by a combination of

complementary separation steps or combinations of inactivation and

separation steps. Therefore, well-designed separation steps, such as

chromatographic procedures, filtration steps, and extractions, can

be effective virus removal steps provided that they are performed

under appropriately controlled conditions.

An overall reduction factor is generally expressed as the sum of

the individual factors. However, reduction in virus titer of the

order of 1 log10 or less would be considered negligible and

would be ignored unless assay variability were shown to be below

that order of magnitude.

If little reduction of infectivity is achieved by the production

process, and the removal of virus is considered to be a major factor

in the safety of the product, a specific, additional inactivation/

removal step or steps should be introduced. For all viruses,

manufacturers should justify the acceptability of the reduction

factors obtained. Results will be evaluated on the basis of the

factors listed above.

D. Limitations of Viral Clearance Studies

Viral clearance studies are useful for contributing to the

assurance that an acceptable level of safety in the final product is

achieved, but do not by themselves establish safety. However, a

number of factors in the design and execution of viral clearance

studies may lead to an incorrect estimate of the ability of the

process to remove virus infectivity (see Appendices 2, 3, and 4).

These factors include the following:

1. Virus preparations used in clearance studies for a production

process are likely to be produced in tissue culture. The behavior of

a tissue culture virus in a production step may be different from

that of the native virus, for example, if native and cultured

viruses differ in purity or degree of aggregation.

2. Inactivation of virus infectivity frequently follows a

biphasic curve in which a rapid initial phase is followed by a

slower phase. It is possible that virus escaping a first

inactivation step may be more resistant to subsequent steps. For

example, if the resistant fraction takes the form of virus

aggregates, infectivity may be resistant to a range of different

chemical treatments and to heating.

3. The ability of the overall process to remove infectivity is

expressed as the sum of the logarithm of the reductions at each

step. The summation of the reduction factors of multiple steps,

particularly of steps with little reduction (e.g., below 1

log10), may overestimate the true potential for virus

elimination. Furthermore, reduction values achieved by repetition of

identical or near identical procedures should not be included unless

justified.

4. The expression of reduction factors as logarithmic reductions

in titer implies that, while residual virus infectivity may be

greatly reduced, it will never be reduced to zero. For example, a

reduction in the infectivity of a preparation containing 8

log10 infectious units per milliliter (mL) by a factor of 8

log10 leaves zero log10 per mL or one infectious unit per

mL, taking into consideration the limit of detection of the assay.

5. Pilot-plant scale processing may differ from commercial-scale

processing despite care taken to design the scaled-down process.

6. Addition of individual virus reduction factors resulting from

similar inactivation mechanisms along the manufacturing process may

overestimate overall viral clearance.

E. Statistics

The viral clearance studies should include the use of

statistical analysis of the data to evaluate the results. The study

results should be statistically valid to support the conclusions

reached (see Appendices 3 and 4).

F. Re-Evaluation of Viral Clearance

Whenever significant changes in the production or purification

process are made, the effect of that change on viral clearance

should be considered and the system re-evaluated as needed. For

example, changes in production processes may cause significant

changes in the amount of virus produced by the cell line; changes in

process steps may change the extent of viral clearance.

VII. Summary

This document suggests approaches for the evaluation of the risk

of viral contamination and for the removal of virus from the

product, thus contributing to the production of safe biotechnology

products derived from animal or human cell lines, and emphasizes the

value of many strategies, including:

A. Thorough characterization/screening of cell substrate

starting material in order to identify which, if any, viral

contaminants are present;

B. Assessment of risk by determination of the human tropism of

the contaminants;

C. Incorporation of studies which assess virus inactivation and

removal steps into the production process;

D. Careful design of the viral clearance studies to avoid

pitfalls and provide interpretable results; and

E. Use of different methods of virus inactivation or removal in

the same production process in order to achieve maximum viral

clearance.

Glossary

Adventitious Virus. See virus.

Cell Substrate. Cells used to manufacture product.

Endogenous Virus. See virus.

Inactivation. Reduction of virus infectivity caused by chemical

or physical modification.

In Vitro Cell Age. A measure of the period between thawing of

the MCB vial(s) and harvest of the production vessel measured by

elapsed chronological time in culture, population doubling level of

the cells, or passage level of the cells when subcultivated by a

defined procedure for dilution of the culture.

Master Cell Bank (MCB). An aliquot of a single pool of cells

which generally has been prepared from the selected cell clone under

defined conditions, dispensed into multiple containers, and stored

under defined conditions. The MCB is used to derive all working cell

banks. The testing performed on a new MCB (from a previous initial

cell clone, MCB, or WCB) should be the same as for the MCB, unless

justified.

Minimum Exposure Time. The shortest period for which a treatment

step will be maintained.

Nonendogenous Virus. See virus.

Process Characterization of Viral Clearance. Viral clearance

studies in which nonspecific ``model'' viruses are used to assess

the robustness of the manufacturing process to remove and/or

inactivate viruses.

Process Evaluation Studies of Viral Clearance. Viral clearance

studies in which ``relevant'' and/or specific ``model'' viruses are

used to determine the ability of the manufacturing process to remove

and/or inactivate these viruses.

Production Cells. Cell substrate used to manufacture product.

Unprocessed Bulk. One or multiple pooled harvests of cells and

culture media. When

[[Page 21888]]

cells are not readily accessible, the unprocessed bulk would

constitute fluid harvested from the fermenter.

Virus. Intracellularly replicating infectious agents that are

potentially pathogenic, possessing only a single type of nucleic

acid (either ribonucleic acid (RNA) or DNA), are unable to grow and

undergo binary fission, and multiply in the form of their genetic

material.

Adventitious Virus. Unintentionally introduced contaminant

viruses.

Endogenous Virus. Viral entity whose genome is part of the germ

line of the species of origin of the cell line and is covalently

integrated into the genome of animal from which the parental cell

line was derived. For the purposes of this document, intentionally

introduced, nonintegrated viruses such as EBV used to immortalize

cell substrates or Bovine Papilloma Virus fit in this category.

Nonendogenous Virus. Viruses from external sources present in

the Master Cell Bank.

Nonspecific Model Virus. A virus used for characterization of

viral clearance of the process when the purpose is to characterize

the capacity of the manufacturing process to remove and/or

inactivate viruses in general, i.e., to characterize the robustness

of the purification process.

Relevant Virus. Virus used in process evaluation studies which

is either the identified virus, or of the same species as the virus

that is known, or likely to contaminate the cell substrate or any

other reagents or materials used in the production process.

Specific Model Virus. Virus which is closely related to the

known or suspected virus (same genus or family), having similar

physical and chemical properties to those of the observed or

suspected virus.

Viral Clearance. Elimination of target virus by removal of viral

particles or inactivation of viral infectivity.

Virus-like Particles. Structures visible by electron microscopy

which morphologically appear to be related to known viruses.

Virus Removal. Physical separation of virus particles from the

intended product.

Working Cell Bank (WCB). The WCB is prepared from aliquots of a

homogeneous suspension of cells obtained from culturing the MCB

under defined culture conditions.

Table 1.--Virus Tests to Be Performed Once at Various Cell Levels

------------------------------------------------------------------------

Cells at the

MCB WCB\1\ limit\2\

------------------------------------------------------------------------

Tests for

Infectivity + - +

Electron +\3\ - +\3\

microscopy\3\

Reverse +\4\ - +\4\

transcriptase\

4\

Other virus- as appropriate\5\ - as

specific appropriate\5\

tests\5\

Tests for

Nonendogenous or

Adventitious

Viruses

In vitro Assays + -\6\ +

In vivo Assays + -\6\ +

Antibody +\7\ - -

production

tests\7\

Other virus- +\8\ - -

specific

tests\8\

------------------------------------------------------------------------

\1\ See text--section III.A.2.

\2\ Cells at the limit; cells at the limit of in vitro cell age used for

production (See text--section III.A.3.)

\3\ May also detect other agents.

\4\ Not necessary if positive by retrovirus infectivity test.

\5\ As appropriate for cell lines which are known to have been infected

by such agents.

\6\ For the first WCB, this test should be performed on cells at the

limit of in vitro cell age, generated from that WCB; for WCB's

subsequent to - the first WCB, a single in vitro and in vivo test can

be done either - directly on the WCB or on cells at the limit of in

vitro cell age.

\7\ e.g., MAP, RAP, HAP--usually applicable for rodent cell lines.

\8\ e.g, tests for cell lines derived from human, nonhuman primate, or

other cell lines as appropriate.

Table 2.--Examples of the Use and Limitations of Assays Which May Be

Used to Test for Virus

------------------------------------------------------------------------

Detection Detection

Test Test article capability limitation

------------------------------------------------------------------------

Anbibody Lysate of cells Specific viral Agents not

production and their antigens infectious for

culture medium animal test

system.

in vivo virus Lysate of cells Broad range of Agents failing

screen and their viruses to replicate or

culture medium pathogenic for produce

humans diseases in the

test system.

in vitro virus Broad range of Agents failing

screen for viruses to replicate or

pathogenic for produce

humans diseases in the

test system.

1. Cell bank 1. Lysate of

characterization cells and their

culture medium

(for

cocultivation,

intact cells

should be in the

test article)

2. Production 2. Unprocessed

screen bulk harvest or

lysate of cells

and their cell

culture medium

from the

production

reactor

TEM on: Virus and virus- Qualitative

like particles assay with

assessment of

identity.

1. Cell substrate 1. Viable cells

2. Cell culture 2. Concentrated

supernatant cell-free

supernatant

[[Page 21889]]

Reverse Cell-free culture Retroviruses and Only detects

transcriptase supernatant expressed enzymes with

(RT) retroviral RT optimal

activity under

preferred

conditions.

Interpretation

may be

difficult due

to presence of

cellular

enzymes;

background with

some

concentrated

samples.

Retrovirus (RV) Cell-free culture Infectious RV failing to

infectivity supernatant retroviruses replicate or

form discrete

foci or plaques

in the chosen

test system.

Cocultivation Viable cells Infectious RV failing to

infectivity retroviruses replicate. See

endpoint above under RV

infectivity.

TEM endpoint See above under

TEM.\1\

RT endpoint See above under

RT.

PCR (Polymerase Cells, culture Specific virus Primer sequences

chain reaction) fluid and other sequences must be

materials present. Does

not indicate

whether virus

is infectious.

------------------------------------------------------------------------

\1\ In addition, difficult to distinguish test article from indicator

cells.

Table 3.--Virus Detected in Antibody Production Tests

----------------------------------------------------------------------------------------------------------------

MAP HAP RAP

----------------------------------------------------------------------------------------------------------------

Ectromelia Virus2,3 Lymphocytic Choriomeningitis-Virus Hantaan Virus1,3

(LCM)1,3

Hantaan Virus1,3 Pneumonia Virus of Mice (PVM)2,3 Kilham Rat Virus (KRV)2,3

K Virus2 Reovirus Type 3 (Reo3)1,3 Mouse Encephalomyelitis Virus

(Theiler's, GDVII)2

Lactic Dehydrogenase Virus (LDH)\2\ Sendai Virus1,3 Pneumonia Virus of Mice (PVM)2,3

Lymmphocytic Choriomeningitis Virus SV5 Rat Coronavirus (RCV)\2\

(LCM)1,3

Minute Virus of Mice (MVM)2,3 Reovirus Type 3 (Reo3)1,3

Mouse Adenovirus (MAV)2,3 Sendai Virus1,3

Mouse Cytomegalovirus (MCMV)2,3 Sialoacryoadenitis Virus (SDAV)\2\

Mouse Encephalomyelitis Virus Toolan Virus (HI)2,3

(Theiler's, GDVII)\2\

Mouse Hepatitis Virus (MHV)\2\

Mouse Rotavirus (EDIM)2,3

Pneumonia Virus of Mice (PVM)2,3

Polyoma Virus\2\

Reovirus Type 3 (Reo3)1,3

Sendai Virus1,3

Thymic Virus\2\

----------------------------------------------------------------------------------------------------------------

\1\ Viruses for which there is evidence of capacity for infecting humans or -primates.

\2\ Viruses for which there is no evidence of capacity for infecting humans.

\3\ Virus capable of replicating in vitro in cells of human or primate origin.

Table 4.--Action Plan for Process Assessment of Viral Clearance and Virus Tests on Purified Bulk

----------------------------------------------------------------------------------------------------------------

Case A Case B Case C\2\ Case D\2\ Case E\2\

----------------------------------------------------------------------------------------------------------------

Status

Presence of virus\1\ - - + + (+)\3\

Virus-like particles\1\ - - - - (+)\3\

Retrovirus-like particles\1\ - + - - (+)\3\

Virus identified not

applicable + + + -

Virus pathogenic for humans not

applicable -\4\ -\4\ + unknown

Action

Process characterization of viral

clearance using nonspecific ``model''

viruses yes\5\ yes\5\ yes\5\ yes\5\ yes\7\

Process evaluation of viral clearance

using ``relevant'' or specific

``model'' viruses no yes\6\ yes\6\ yes\6\ yes\7\

Test for virus in purified bulk not

applicable yes\8\ yes\8\ yes\8\ yes\8\

----------------------------------------------------------------------------------------------------------------

\1\ Results of virus tests for the cell substrate and/or at the unprocessed bulk level. Cell cultures used for

production which are contaminated with viruses will generally not be acceptable. Endogenous viruses (such as

retroviruses) or viruses that are an integral part of the MCB may be acceptable if appropriate viral clearance

evaluation procesures are followed.

\2\ The use of source material which is contaminated with viruses, whether or not they are known to be

infectious and/or pathogenic in humans, will only be permitted under very exceptional circumstances.

[[Page 21890]]

\3\ Virus has been observed by either direct or indirect methods.

\4\ Believed to be nonpathogenic.

\5\ Characterization of clearance using nonspecific ``model'' viruses should be performed.

\6\ Process evaluation for ``relevant'' viruses or specific ``model'' viruses should be performed.

\7\ See text under Case E.

\8\ The absence of detectible virus should be confirmed for purified bulk by means of suitable methods having

high specificity and sensitivity for the detection of the virus in question. For the purpose of marketing

authorization, data from at least 3 lots of purified bulk manufactured at pilot-plant scale or commercial

scale should be provided. However, for cell lines such as CHO cells for which the endogenous particles have

been extensively characterized and adequate clearance has been demonstrated, it is not usually necessary to

assay for the presence of the noninfectious particles in purified bulk.

Appendix 1

Products Derived From Characterized Cell Banks Which Were

Subsequently Grown In Vivo

For products manufactured from fluids harvested from animals

inoculated with cells from characterized banks, additional

information regarding the animals should be provided.

Whenever possible, animals used in the manufacture of

biotechnological/biological products should be obtained from well

defined, specific pathogen-free colonies. Quarantine procedures for

newly arrived as well as diseased animals should be described, and

assurance provided that all containment, cleaning, and

decontamination methodologies employed within the facility are

adequate to contain the spread of adventitious agents. This may be

accomplished through the use of a sentinel program. A listing of

agents for which testing is performed should also be included.

Veterinary support services should be available on-site or within

easy access. The degree to which the vivarium is segregated from

other areas of the manufacturing facility should be described.

Personnel practices should be adequate to ensure safety.

Procedures for the maintenance of the animals should be fully

described. These would include diet, cleaning, and feeding

schedules, provisions for periodic veterinary care if applicable,

and details of special handling that the animals may require once

inoculated. A description of the priming regimen(s) for the animals,

the preparation of the inoculum, and the site and route of

inoculation should also be included.

The primary harvest material from animals may be considered an

equivalent stage of manufacture to unprocessed bulk harvest from a

bioreactor. Therefore, all testing considerations previously

outlined in section IV. of this document should apply. In addition,

the manufacturer should assess the bioburden of the unprocessed

bulk, determine whether the material is free of mycoplasma, and

perform species-specific assay(s) as well as in vivo testing in

adult and suckling mice.

Appendix 2

The Choice of Viruses for Viral Clearance Studies

A. Examples of Useful ``Model'' Viruses:

1. Nonspecific ``model'' viruses representing a range of

physico-chemical structures:

SV40 (Polyomavirus maccacae 1), human polio virus 1

(Sabin), animal parvovirus or some other small, nonenveloped

viruses;

a parainfluenza virus or influenza virus, Sindbis virus

or some other medium-to-large, enveloped, RNA viruses;

a herpes virus (e.g., HSV-1 or a pseudorabies virus),

or some other medium-to-large, DNA viruses.

These viruses are examples only and their use is not mandatory.

2. For rodent cell substrates, murine retroviruses are commonly

used as specific ``model'' viruses.

B. Examples of Viruses Which Have Been Used in Viral Clearance

Studies.

Several viruses which have been used in viral clearance studies.

are listed in Table A-1. However, since these are merely examples,

the use of any of the viruses in the table is not mandatory and

manufacturers are invited to consider other viruses, especially

those which may be more appropriate for their individual production

processes. Generally, the process should be assessed for its ability

to clear at least three different viruses with differing

characteristics.

Table A-1.--Examples of Viruses Which Have Been Used in Viral Clearance Studies

----------------------------------------------------------------------------------------------------------------

Natural

Virus Family Genus host Genome Envelope Size Shape Resistance\1\

----------------------------------------------------------------------------------------------------------------

Vesicular Rhabdo Vesiculov Equine RNA yes 70x175 nm Bullet Low

stomatitis irus Bovine

virus

Parainfluenza Paramyxo Paramyxov Various RNA yes 100-200+ Pleo/ Low

virus irus nm Spheric

al

MuLV Retro Type C Mouse RNA yes 80-110 nm Spherica Low

oncoviru l

s

Sindbis virus Toga Alphaviru Human RNA yes 60-70 nm Spherica Low

s l

BVDV Flavi Pestiviru Bovine RNA yes 50-70 nm Pleo- Low

s Spheric

al

Pseudorabies Herpes Swine DNA yes 120-200 Spherica Medium

virus nm l

Poliovirus Sabin Picorna Enterovir Human RNA no 25-30 nm Icosahed Medium

Type 1 us ral

Encephalomyocard Picorna Cardiovir Mouse RNA no 25-30 nm Icosahed Medium

itis virus us ral

(EMC)

Reovirus 3 Reo Orthoreov Various RNA no 60-80 nm Spherica Medium

irus l

SV40 Papova Polyomavi Monkey DNA no 40-50 nm Icosahed Very high

rus ral

Parvoviruses Parvo Parvoviru Canine DNA no 18-24 nm Icosahed Very high

(canine, s Porcine ral

porcine)

----------------------------------------------------------------------------------------------------------------

\1\ Resistance to physico-chemical treatments based on studies of production processes. Resistance is relative

to the specific treatment and it is used in the context of the understanding of the biology of the virus and

the nature of the manufacturing process. Actual results will vary according to the treatment. These viruses

are examples only and their use is not mandatory.

Appendix 3

Statistical Considerations for Assessing Virus Assays

Virus titrations suffer the problems of variation common to all

biological assay systems. Assessment of the accuracy of the virus

titrations and reduction factors derived from them and the validity

of the assays should be performed to define the reliability of a

study. The objective of statistical evaluation is to establish that

the study has been carried out to an acceptable level of virological

competence.

1. Variation may arise within an assay as a result of dilution

errors, statistical effects, and differences within the assay system

that are either unknown or difficult to control.

[[Page 21891]]

These effects are likely to be greater when different assay runs are

compared (between-assay variation) than when results within a single

assay run are compared (within-assay variation).

2. Assay methods may be either quantal or quantitative. Quantal

methods include infectivity assays in animals or in tissue-culture-

infectious-dose (TCID) assays, in which the animal or cell culture

is scored as either infected or not. Infectivity titers are then

measured by the proportion of animals or culture infected. In

quantitative methods, the infectivity measured varies continuously

with the virus input. Quantitative methods include plaque assays

where each plaque counted corresponds to a single infectious unit.

Both quantal and quantitative assays are amenable to statistical

evaluation.

3. The 95 percent confidence limits for results of within-assay

variation normally should be on the order of 0.5

log10 of the mean. Within-assay variation can be assessed by

standard textbook methods. Between-assay variation can be monitored

by the inclusion of a reference preparation, the estimate of whose

potency should be within approximately 0.5 log10 of the mean

estimate established in the laboratory for the assay to be

acceptable. Assays with lower precision may be acceptable with

appropriate justification.

4. The 95 percent confidence limits for the reduction factor

observed should be calculated wherever possible in studies of

clearance of ``relevant'' and specific ``model'' viruses. If the 95

percent confidence limits for the viral assays of the starting

material are +s, and for the viral assays of the material after the

step are +a, the 95 percent confidence limits for the reduction

factor are in the equation below.

[GRAPHIC] [TIFF OMITTED] TN10MY96.084

Appendix 4

Probability of Detection of Viruses at Low Concentrations

At low virus concentrations (e.g., in the range of 10 to 1000

infectious particles per liter), it is evident that a sample of a

few milliliters may or may not contain infectious particles. The

probability, p, that this sample does not contain infectious viruses

is:

p = ((V-v)/V)n

where V (liter) is the overall volume of the material to be tested,

v (liter) is the volume of the sample and n is the absolute number

of infectious particles statistically distributed in V.

If V >> v, this equation can be approximated by the Poisson

distribution:

p = e-cv

where c is the concentration of infectious particles per liter.

or, c = ln p /-v

As an example, if a sample volume of 1 mL is tested, the

probabilities p at virus concentrations ranging from 10 to 1000

infectious particles per liter are:

[GRAPHIC] [TIFF OMITTED] TN10MY96.085

This indicates that for a concentration of 1,000 viruses per liter,

in 37 percent of sampling, 1 mL will not contain a virus particle.

If only a portion of a sample is tested for virus and the test

is negative, the amount of virus which would have to be present in

the total sample in order to achieve a positive result should be

calculated and this value taken into account when calculating a

reduction factor. Confidence limits at 95 percent are desirable.

However, in some instances, this may not be practical due to

material limitations.

Appendix 5

Calculation of Reduction Factors in Studies to Determine Viral

Clearance

The virus reduction factor of an individual purification or

inactivation step is defined as the log10 of the ratio of the

virus load in the pre-purification material and the virus load in

the post-purification material which is ready for use in the next

step of the process. If the following abbreviations are used:

Starting material: vol v'; titer 10a';

virus load: v'.10a',

Final material: vol v"; titer 10a";

virus load: v".10a",

the individual reduction factors Ri are calculated according to

10Ri = v'.10a' / v".10a"

This formula takes into account both the titers and volumes of the

materials before and after the purification step.

Because of the inherent imprecision of some virus titrations, an

individual reduction factor used for the calculation of an overall

reduction factor should be greater than 1. The overall reduction

factor for a complete production process is the sum logarithm of the

reduction factors of the individual steps. It represents the

logarithm of the ratio of the virus load at the beginning of the

first process clearance step and at the end of the last process

clearance step.

Appendix 6

Calculation of Estimated Particles per Dose

This is applicable to those viruses for which an estimate of

starting numbers can be made, such as endogenous retroviruses.

Example:

I. Assumptions

Measured or estimated concentration of virus in cell culture harvest

= 106/mL

Calculated viral clearance factor = >1015

Volume of culture harvest needed to make a dose of product = 1 liter

(l03mL)

II. Calculation of Estimated Particles/Dose

[GRAPHIC] [TIFF OMITTED] TN10MY96.086

= -6 particles/dose

Therefore, less than one particle per million doses would be

expected.

Dated: May 1, 1996.

William K. Hubbard,

Associate Commissioner for Policy Coordination.

[FR Doc. 96-11641 Filed 5-9-96; 8:45 am]

BILLING CODE 4160-01-F