[Federal Register Volume 63, Number 185 (Thursday, September 24, 1998)]
[Notices]
[Pages 51074-51084]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 98-25569]


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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration
[Docket No. 96D-0058]


International Conference on Harmonisation; Guidance 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.

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SUMMARY: The Food and Drug Administration (FDA) is publishing a 
guidance entitled ``Q5A Viral Safety Evaluation of Biotechnology 
Products Derived From Cell Lines of Human or Animal Origin.'' The 
guidance was prepared under the auspices of the International 
Conference on Harmonisation of Technical Requirements for Registration 
of Pharmaceuticals for Human Use (ICH). The guidance describes the 
testing and evaluation of the viral safety of biotechnology products 
derived from characterized cell lines of human or animal origin, and 
outlines data that should be submitted in marketing applications.

DATES: Effective September 24, 1998. Submit written comments at any 
time.

ADDRESSES: Submit written comments on the guidance to the Dockets 
Management Branch (HFA-305), Food and Drug Administration, 5630 Fishers 
Lane, rm. 1061, Rockville, MD 20852. Copies of the guidance are 
available from the Drug Information Branch (HFD-210), Center for Drug 
Evaluation and Research, Food and Drug Administration, 5600 Fishers 
Lane,

[[Page 51075]]

Rockville, MD 20857, 301-827-4573. Single copies of the guidance may be 
obtained by mail from the Office of Communication, Training and 
Manufacturers Assistance (HFM-40), Center for Biologics Evaluation and 
Research (CBER), Food and Drug Administration, 1401 Rockville Pike, 
Rockville, MD 20852-1448, or by calling the CBER Voice Information 
System at 1-800-835-4709 or 301-827-1800. Copies may be obtained from 
CBER's FAX Information System at 1-888-CBER-FAX or 301-827-3844.

FOR FURTHER INFORMATION CONTACT:
    Regarding the guidance: Neil D. Goldman, Center for Biologics 
Evaluation and Research (HFM-20), Food and Drug Administration, 1401 
Rockville Pike, Rockville, MD 20852, 301-827-0377.
    Regarding the 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.
    In the Federal Register of May 10, 1996 (61 FR 21882), FDA 
published a draft tripartite guideline entitled ``Viral Safety 
Evaluation of Biotechnology Products Derived From Cell Lines of Human 
or Animal Origin'' (Q5A). The notice gave interested persons an 
opportunity to submit comments by August 8, 1996.
    After consideration of the comments received and revisions to the 
guidance, a final draft of the guidance was submitted to the ICH 
Steering Committee and endorsed by the three participating regulatory 
agencies on March 4, 1997.
    In accordance with FDA's good guidance practices (62 FR 8961, 
February 27, 1997), this document has been designated a guidance, 
rather than a guideline.
    The guidance describes approaches for evaluating the risk of viral 
contamination and the potential of the production process to remove 
viruses from biotechnology products derived from human or animal cell 
lines. The guidance 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 into the production process of 
studies that assess virus inactivation and removal steps; (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.
    This guidance represents the agency's current thinking on viral 
safety evaluation of biotechnology products. It does not create or 
confer any rights for or on any person and does not operate to bind FDA 
or the public. An alternative approach may be used if such approach 
satisfies the requirements of the applicable statute, regulations, or 
both.
    As with all of FDA's guidances, the public is encouraged to submit 
written comments with new data or other new information pertinent to 
this guidance. The comments in the docket will be periodically 
reviewed, and, where appropriate, the guidance will be amended. The 
public will be notified of any such amendments through a notice in the 
Federal Register.
    Interested persons may, at any time, submit written comments on the 
guidance to the Dockets Management Branch (address above). 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 guidance and received 
comments may be seen in the office above between 9 a.m. and 4 p.m., 
Monday through Friday. An electronic version of this guidance is 
available on the Internet at ``http://www.fda.gov/cder/index.htm'' or 
at CBER's World Wide Web site at ``http://www.fda.gov/cber/
guidelines.htm''.
    The text of the guidance follows:

Q5A 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 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

[[Page 51076]]

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;
    (3) Testing the product at appropriate steps of production for 
absence of contaminating infectious viruses.
    All testing suffers from the inherent limitation of quantitative 
virus assays, i.e., 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 
needed at different steps 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 describe 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 that 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; (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; (4) use of a contaminated 
excipient during formulation; and (5) contamination during cell and 
medium handling. Monitoring of cell culture parameters can be 
helpful in the early detection of potential adventitious viral 
contamination.

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 examples 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 considered 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 should be 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 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 thioe 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

[[Page 51077]]

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 that 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 mouse 
antibody production (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 three lots of unprocessed 
bulk at pilot-plant scale or commercial scale should be submitted as 
part of the marketing application/registration 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 
steps 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 evaluating clearance of known 
contaminants, indepth, time-dependent inactivation studies, 
demonstration of reproducibility of inactivation/removal, and 
evaluation of process parameters should be provided. When a 
manufacturing process is characterized for robustness of clearance 
using nonspecific ``model'' viruses, particular attention should be 
paid to nonenveloped viruses in the study design. The extent of 
viral clearance characterization studies may be influenced by the 
results of tests on cell lines and unprocessed bulk. These studies 
should be performed as described in section VI. 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 in 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 that 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 
three 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 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. Studies with 
nonspecific ``model'' viruses, as in Case A, are appropriate.
    Case C: When the cells or unprocessed bulk are known to contain 
a virus, other than

[[Page 51078]]

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 three 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 three lots of purified bulk 
manufactured at pilot-plant scale or commercial scale should be 
provided.
    Case E: When a virus that 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 due virus removal and/or 
inactivation procedures play 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. Studies should be carried out in a manner that is well 
documented and controlled.
    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 steps and demonstrating its removal or 
inactivation during the subsequent steps. It is not considered 
necessary to evaluate or characterize every step of a manufacturing 
process if adequate clearance is demonstrated by the use of fewer 
steps. It should be borne in mind that other steps in the process 
may have an indirect effect on the viral inactivation/removal 
achieved. Manufacturers should explain and justify the approach used 
in studies for evaluating virus clearance.
    The reduction of virus infectivity may be achieved by removal of 
virus particles or by inactivation of viral infectivity. For each 
production step 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 (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. Reduction factors are normally expressed 
on a logarithmic scale, which implies that, while residual virus 
infectivity will never be reduced to zero, it may be greatly reduced 
mathematically.
    In addition to clearance studies for viruses known to be 
present, studies to characterize the ability to remove and/or 
inactivate other viruses should be conducted. The purpose of studies 
with viruses exhibiting a range of biochemical and biophysical 
properties 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. A demonstration of the 
capacity of the production process to inactivate or remove viruses 
is desirable (see section VI.C.). Such studies 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 document.

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:

[[Page 51079]]

    (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 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. 
A similar elution profile should result. For other procedures, 
similar considerations apply. Deviations that 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. Steps which are likely to clear virus 
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 step. Sufficient virus should be 
present in the material of each step 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 step 
to be tested. In some cases, simply adding high titer virus to 
unpurified bulk and testing its concentration between steps will be 
sufficient. Where virus removal results from separation procedures, 
it is recommended that, if appropriate and if possible, the 
distribution of the virus load in the different fractions be 
investigated. 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. 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 3).

4. Determining Physical Removal Versus Inactivation

    Reduction in virus infectivity may be achieved by the removal or 
inactivation of virus. For each production step 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, for example, 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.

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. It should be recognized that 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. Additional 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. 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, reproducible clearance 
should be demonstrated in at least two independent studies. Whenever 
possible, the initial virus load should be determined from the virus 
that can be detected in the spiked starting material. If this is not 
possible, the initial virus load may be calculated from the titer of 
the spiking virus preparation. Where inactivation is too rapid to 
plot an inactivation curve using process conditions, appropriate 
controls should be performed to demonstrate that infectivity is 
indeed lost by inactivation.

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 might be necessary. If the product itself has anti-viral 
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 anti-viral 
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. Overall reduction 
factors may also be overestimated due to inherent limitations or 
inadequate design of viral clearance studies.

[[Page 51080]]

C. Interpretation of Viral Clearance Studies; Acceptability

    The object 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 process should be compared to the 
amount of virus which may be present in unprocessed bulk.
    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 4 for calculation of virus reduction factors and 
Appendix 5 for calculation of estimated particles per dose.
    Manufacturers should recognize that clearance mechanisms may 
differ between virus classes. A combination of factors should 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;
    (vi) The limits of assay sensitivities;
    (vii) The possible selectivity of inactivation/removal 
procedure(s) for certain classes of viruses.
    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. Manufacturing parameters influencing separation should 
be properly defined and controlled. 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 effective virus removal step should give reproducible 
reduction of virus load shown by at least two independent studies.
    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 justified.
    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 would 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. 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 Appendix 3).

F. Reevaluation of Viral Clearance

    Whenever significant changes in the production or purification 
process are made, the effect of that change, both direct and 
indirect, 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 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. Establishment of an appropriate program of testing for 
adventitious viruses in unprocessed bulk;
    D. Careful design of viral clearance studies using different 
methods of virus inactivation or removal in the same production 
process in order to achieve maximum viral clearance; and
    E. Performance of studies which assess virus inactivation and 
removal.
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 original 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.

[[Page 51081]]

    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 cells are not readily accessible, the 
unprocessed bulk would constitute fluid harvested from the 
fermenter.
    Virus. Intracellularly replicating infectious agents that are 
potentially pathogenic, possess 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 
virus.
    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. Virus from external sources present in the 
MCB.
    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.--Examples of Virus Tests to Be Performed Once at Various Cell Levels
----------------------------------------------------------------------------------------------------------------
                                                                                                  Cells at the
                                                                  MCB               WCB1             limit2
----------------------------------------------------------------------------------------------------------------
Tests for Retroviruses and Other Endogenous Viruses
  Infectivity                                              +                  -                 +
  Electron microscopy3                                     +3                 -                 +3
  Reverse transcriptase4                                   +4                 -                 +4
  Other virus-specific tests5                              as appropriate5    -                 as appropriate5
Tests for Nonendogenous or Adventitious Viruses
  In vitro Assays                                          +                  -6                +
  In vivo Assays                                           +                  -6                +
  Antibody production tests7                               +7                 -                 -
  Other virus-specific tests8                              +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
------------------------------------------------------------------------
Antibody           Lysate of cells    Specific viral    Antigens 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 co-
                    cultivation,
                    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. Cell-free
 supernatant        culture
                    supernatant

[[Page 51082]]

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
                                       retroviruses      replicate
1. Infectivity                                          1. See above
 endpoint                                                under RV
                                                         infectivity
2. TEM endpoint                                         2. See above
                                                         under TEM1
3. RT endpoint                                          3. 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 (LCM)1,3                      Hantaan Virus1,3
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 (Theilers, GDVII)2
Lactic Dehydrogenase Virus (LDM)1,3                              Sendai Virus1,3                                                  Pneumonia Virus of Mice (PVM)2,3
Lymphocytic Choriomeningitis Virus (LCM)1,3                      SV5                                                              Rat Coronavirus (RCV)2
Minute Virus of Mice2,3                                                                                                           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 (Theilers, GDVII)2                                                                                  Toolan Virus (HI)2,3
Mouse Hepatitis Virus (MHV)2
Mouse Rotavirus (EDIM)2,3
Pneumonia Virus of Mice (PVM)2,3
Polyoma Virus2
Reovirus Type 3 (Reo3)1,3
Sendai Virus1,3
Thymic Virus2
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
\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 C2           Case D2           Case E2
----------------------------------------------------------------------------------------------------------------
Status
Presence of virus1      -                 -                 +                 +                 (+)3
Virus-like particles1   -                 -                 -                 -                 (+)3
Retrovirus-like         -                 +                 -                 -                 (+)3
 particles1
Virus identified        not applicable    +                 +                 +                 -
Virus pathogenic for    not applicable    -4                -4                +                 unknown
 humans
Action
Process                 yes5              yes5              yes5              yes5              yes7
 characterization of
 viral clearance using
 nonspecific ``model''
 viruses
Process evaluation of   no                yes6              yes6              yes6              yes7
 viral clearance using
 ``relevant'' or
 specific ``model''
 viruses
Test for virus in       not applicable    yes8              yes8              yes8              yes8
 purified bulk
----------------------------------------------------------------------------------------------------------------
\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 procedures 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 acceptable under very exceptional circumstances.
\3\ Virus has been observed by either direct or indirect methods.
\4\ Believed to be nonpathogenic.

[[Page 51083]]

\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 detectable 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 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. Adequate testing for 
appropriate viruses, such as those listed in Table 3, should be 
performed. 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 That Have Been Used in Viral Clearance 
Studies

    Several viruses that 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 considered 
mandatory and manufacturers are invited to consider other viruses, 
especially those that 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      Env    Size (nm)    Shape      Resistance1
----------------------------------------------------------------------------------------------------------------
Vesicular        Rhabdo      Vesiculo-   Equine       RNA         yes      70 x 150   Bullet     Low
 Stomatitis                   virus       Bovine
 Virus
Parainfluenza    Paramyxo    Paramyxo-   Various      RNA         yes      100-200+   Pleo/      Low
 Virus                        virus                                                    Spher
MuLV             Retro       Type C      Mouse        RNA         yes      80-110     Spherical  Low
                              oncovirus
Sindbis Virus    Toga        Alphavirus  Human        RNA         yes      60-70      Spherical  Low
BVDV             Flavi       Pestivirus  Bovine       RNA         yes      50-70      Pleo/      Low
                                                                                       Spher
Pseudo-rabies    Herpes                  Swine        DNA         yes      120-200    Spherical  Med
 Virus
Poliovirus       Picorna     Entero-     Human        RNA         no       25-30      Icosa-     Med
 Sabin Type 1                 virus                                                    hedral
Encephalomyo-    Picorna     Cardio-     Mouse        RNA         no       25-30      Icosa-     Med
 carditis Virus               virus                                                    hedral
 (EMC)
Reovirus 3       Roe         Orthoreo-   Various      DNA         no       60-80      Spherical  Med
                              virus
SV40             Papova      Polyomavir  Monkey       DNA         no       40-50      Icosa-     Very high
                              us                                                       hedral
Parvoviruses     Parvo       Parvovirus  Canine       DNA         no       18-24      Icosa-     Very high
 (canine,                                 Porcine                                      hedral
 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 considered mandatory.

Appendix 3

A. 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. 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

[[Page 51084]]

include plaque assays where each plaque counted corresponds to a 
single infectious unit. Both quantal and quantitative assays are 
amenable to statistical evaluation.
    2. Variation can arise within an assay as a result of dilution 
errors, statistical effects, and differences within the assay system 
which are either unknown or difficult to control. 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).
    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
[GRAPHIC] [TIFF OMITTED] TN24SE98.022

B. Probability of Detection of Viruses at Low Concentrations

    At low virus concentrations (e.g., in the range of 10 to 1,000 
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 1,000 
infectious particles per liter are:
[GRAPHIC] [TIFF OMITTED] TN24SE98.023

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 4

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. Reduction factors are normally 
expressed on a logarithmic scale which implies that, while residual 
virus infectivity will never be reduced to zero, it may be greatly 
reduced mathematically.

Appendix 5

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] TN24SE98.024

Therefore, less than one particle per million doses would be 
expected.

    Dated: September 16, 1998.
 William K. Hubbard,
 Associate Commissioner for Policy Coordination.
[FR Doc. 98-25569 Filed 9-23-98; 8:45 am]
BILLING CODE 4160-01-F