[Federal Register Volume 61, Number 92 (Friday, May 10, 1996)]
[Notices]
[Pages 21882-21891]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: 96-11641]




[[Page 21881]]


_______________________________________________________________________

Part IV





Department of Health and Human Services





_______________________________________________________________________



Food and Drug Administration



_______________________________________________________________________



Harmonisation International Conference; Guidelines Availability: 
Biotechnological/Biological Pharmaceutical Products; Viral Safety 
Evaluation; Notice

  Federal Register / Vol. 61, No. 92 / Friday, May 10, 1996 / Notices  
=======================================================================
-----------------------------------------------------------------------

[[Page 21882]]

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

[[Page 21883]]

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

[[Page 21884]]

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

[[Page 21885]]

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

[[Page 21886]]

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

    = <10-6 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