Federal Register, Volume 63 Issue 185 (Thursday, September 24, 1998)

[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 WCB^{1 limit^{2
----------------------------------------------------------------------------------------------------------------
Tests for Retroviruses and Other Endogenous Viruses
Infectivity + - +
Electron microscopy^{3 +^{3 - +^{3
Reverse transcriptase^{4 +^{4 - +^{4
Other virus-specific tests^{5 as appropriate^{5 - as appropriate^{5
Tests for Nonendogenous or Adventitious Viruses
In vitro Assays + -^{6 +
In vivo Assays + -^{6 +
Antibody production tests^{7 +^{7 - -
Other virus-specific tests^{8 +^{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 TEM^{1
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 Virus^{2,3 Lymphocytic Choriomeningitis Virus (LCM)^{1,3 Hantaan Virus^{1,3
Hantaan Virus^{1,3 Pneumonia Virus of Mice (PVM)^{2,3 Kilham Rat Virus (KRV)^{2,3
K Virus^{2 Reovirus Type 3 (Reo3)^{1,3 Mouse Encephalomyelitis Virus (Theilers, GDVII)^{2
Lactic Dehydrogenase Virus (LDM)^{1,3 Sendai Virus^{1,3 Pneumonia Virus of Mice (PVM)^{2,3
Lymphocytic Choriomeningitis Virus (LCM)^{1,3 SV5 Rat Coronavirus (RCV)^{2
Minute Virus of Mice^{2,3 Reovirus Type 3 (Reo3)^{1,3
Mouse Adenovirus (MAV)^{2,3 Sendai Virus^{1,3
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 Virus^{2
Reovirus Type 3 (Reo3)^{1,3
Sendai Virus^{1,3
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 - + - - (+)^{3
particles^{1
Virus identified not applicable + + + -
Virus pathogenic for not applicable -^{4 -^{4 + unknown
humans
Action
Process yes^{5 yes^{5 yes^{5 yes^{5 yes^{7
characterization of
viral clearance using
nonspecific ``model''
viruses
Process evaluation of no yes^{6 yes^{6 yes^{6 yes^{7
viral clearance using
``relevant'' or
specific ``model''
viruses
Test for virus in not applicable yes^{8 yes^{8 yes^{8 yes^{8
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 Resistance^{1
----------------------------------------------------------------------------------------------------------------
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 10^a';
virus load: (v')(10^a),
Final material: vol v''; titer 10^a'';
virus load: (v'')(10^a''),
the individual reduction factors Ri are calculated according to
10^Ri = (v')(10^a') /
(v'')(10^a'')
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}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}}