21882 Federal Register / Vol. 61, No. 92 / Friday, May 10, 1996 / Notices

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 § 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 § 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
Federal Register / Vol. 61, No. 92 / Friday, May 10, 1996 / Notices 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 selfreplicating 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 pilotplant 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
21884 Federal Register / Vol. 61, No. 92 / Friday, May 10, 1996 / Notices

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 Rtype 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 pilotplant 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
Federal Register / Vol. 61, No. 92 / Friday, May 10, 1996 / Notices 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 pilotplant 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
21886 Federal Register / Vol. 61, No. 92 / Friday, May 10, 1996 / Notices

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-bedvolume 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 commercialscale 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
Federal Register / Vol. 61, No. 92 / Friday, May 10, 1996 / Notices 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 physicochemical 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 reevaluated 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
21888 Federal Register / Vol. 61, No. 92 / Friday, May 10, 1996 / Notices

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

MCB WCB1 Cells at the limit2

Tests for Retroviruses and

Other Endogenous Viruses

Infectivity + – +

Electron microscopy3 +3 – +3

Reverse transcriptase4 +4 – +4

Other virus-specific tests5 as appropriate5 – as appropriate5

Tests for Nonendogenous or

Adventitious Viruses

In vitro Assays + –6 +

In vivo Assays + –6 +

Antibody production tests7 +7 – –

Other virus-specific tests8 +8 – –

1See 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.

5As appropriate for cell lines which are known to have been infected by such agents.

6For 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

Test Test article Detection capability Detection limitation

Anbibody production Lysate of cells and their culture

medium

Specific viral antigens Agents not infectious for animal

test system.

in vivo virus screen Lysate of cells and their culture

medium

Broad range of viruses pathogenic

for humans

Agents failing to replicate or

produce diseases in the test

system.

in vitro virus screen for Broad range of viruses pathogenic

for humans

Agents failing to replicate or

produce diseases in the test

system.

1. Cell bank characterization 1. Lysate of cells and their culture

medium (for cocultivation, intact

cells should be in the test article)

2. Production screen 2. Unprocessed bulk harvest or

lysate of cells and their cell culture medium from the production reactor

TEM on: Virus and virus-like particles Qualitative assay with assessment

of identity.

1. Cell substrate 1. Viable cells

2. Cell culture supernatant 2. Concentrated cell-free supernatant
Federal Register / Vol. 61, No. 92 / Friday, May 10, 1996 / Notices 21889

TABLE 2.—EXAMPLES OF THE USE AND LIMITATIONS OF ASSAYS WHICH MAY BE USED TO TEST FOR VIRUS—Continued

Test Test article Detection capability Detection limitation

Reverse transcriptase (RT) Cell-free culture supernatant Retroviruses and expressed

retroviral RT

Only detects enzymes with optimal activity under preferred

conditions. Interpretation may

be difficult due to presence of

cellular enzymes; background

with some concentrated samples.

Retrovirus (RV) infectivity Cell-free culture supernatant Infectious retroviruses RV failing to replicate or form discrete foci or plaques in the chosen test system.

Cocultivation infectivity endpoint Viable cells Infectious retroviruses RV failing to replicate. See above

under RV infectivity.

TEM endpoint See above under TEM.1

RT endpoint See above under RT.

PCR (Polymerase chain reaction) Cells, culture fluid and other materialsSpecific virus sequences Primer sequences must be

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 ChoriomeningitisVirus (LCM)1,3 Hantaan Virus1,3

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

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

GDVII)2

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

Lymmphocytic Choriomeningitis Virus (LCM)1,3 SV5 Rat Coronavirus (RCV)2

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 (Theiler’s,

GDVII)2

Toolan Virus (HI)2,3

Mouse Hepatitis Virus (MHV)2

Mouse Rotavirus (EDIM)2,3

Pneumonia Virus of Mice (PVM)2,3

Polyoma Virus2

Reovirus Type 3 (Reo3)1,3

Sendai Virus1,3

Thymic Virus2

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

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

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

TABLE 4.—ACTION PLAN FOR PROCESS ASSESSMENT OF VIRAL CLEARANCE AND VIRUS TESTS ON PURIFIED BULK

Case A Case B Case C2 Case D2 Case E2

Status

Presence of virus1 – – + + (+)3

Virus-like particles1 – – – – (+)3

Retrovirus-like particles1 – + – – (+)3

Virus identified not

applicable

+ + + –

Virus pathogenic for humans not

applicable

–4 –4 + unknown

Action

Process characterization of viral clearance using nonspecific ‘‘model’’

viruses yes5 yes5 yes5 yes5 yes7

Process evaluation of viral clearance using ‘‘relevant’’ or specific

‘‘model’’ viruses no yes6 yes6 yes6 yes7

Test for virus in purified bulk not

applicable

yes8 yes8 yes8 yes8

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. 2The 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.
21890 Federal Register / Vol. 61, No. 92 / Friday, May 10, 1996 / Notices

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

4Believed to be nonpathogenic.

5 Characterization of clearance using nonspecific ‘‘model’’ viruses should be performed.

6Process evaluation for ‘‘relevant’’ viruses or specific ‘‘model’’ viruses should be performed.

7See text under Case E.

8The 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 pilotplant 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-tolarge, 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

Virus Family Genus Natural

host Genome Envelope Size Shape Resistance1

Vesicular stomatitis virus Rhabdo Vesiculovirus Equine

Bovine

RNA yes 70x175 nm Bullet Low

Parainfluenza virus Paramyxo Paramyxovirus Various RNA yes 100–200+ nm Pleo/SphericalLow

MuLV Retro Type C

oncovirus

Mouse RNA yes 80–110 nm Spherical Low

Sindbis virus Toga Alphavirus Human RNA yes 60–70 nm Spherical Low

BVDV Flavi Pestivirus Bovine RNA yes 50–70 nm Pleo-SphericalLow

Pseudorabies virus Herpes Swine DNA yes 120–200 nm Spherical Medium

Poliovirus Sabin Type 1 Picorna Enterovirus Human RNA no 25–30 nm Icosahedral Medium

Encephalomyocarditis virus

(EMC)

Picorna Cardiovirus Mouse RNA no 25–30 nm Icosahedral Medium

Reovirus 3 Reo Orthoreovirus Various RNA no 60–80 nm Spherical Medium

SV40 Papova Polyomavirus Monkey DNA no 40–50 nm Icosahedral Very high

Parvoviruses (canine, porcine)Parvo Parvovirus Canine

Porcine

DNA no 18–24 nm Icosahedral Very high

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.
Federal Register / Vol. 61, No. 92 / Friday, May 10, 1996 / Notices 21891

These effects are likely to be greater when

different assay runs are compared (betweenassay 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 tissueculture-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. Betweenassay 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.

± + S a 2 2

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:

c 10 100 1 000

0

,

p .99 0.90 0.37

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

10 10

10

10

10

6 3

15

9

15

virus units mL mL dose

Clearance factor

particles dose

Clearance factor

/ / ( ) / × ( )

> = >

= <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]

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