Evaluation of cell substrates for the production of …...2007 and 2008. The Committee welcomed the...

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Evaluation of cell substrates for the production of biologicals: revision of WHO recommendations Report of the WHO Study Group on Cell Substrates for the Production of Biologicals 22-23 April 2009, Bethesda, USA

Contents

Abstract ...............................................................................................................2

1. Background .....................................................................................................3

2. Introduction.....................................................................................................4

3. Progress made since June 2007......................................................................6

4. Revision of WHO recommendations for cell substrates..............................7

5. International Standard for Mycoplasma ....................................................18

6. Replacement of WHO Vero reference cell bank 10-87..............................19

7. Conclusions and next steps...........................................................................21

8. Acknowledgement .........................................................................................25

9. References ......................................................................................................25

Abstract

Evaluating cell substrates for producing vaccines and other biologicals is one of the

critical aspects in assuring quality and safety of these products. As part of its mission in

setting standards for biological products, WHO provides recommendations for

manufacturing and evaluating biologicals. Regular updates of the guidance documents are

important to manufacturers and regulators worldwide. WHO Expert Committee on

Biological Standardization (ECBS) identified a need for revising the requirements for cell

substrates (WHO TRS 878, annex 1). In response, WHO established a Study Group (SG) in

2006 that prepared an updated set of recommendations for using cell substrates for the

production of biologicals. A summary of the proposed changes that the SG made in 2007 is

available at WHO web site

http://www.who.int/biologicals/publications/meetings/areas/vaccines/cells/en/index.html).

Draft revised recommendations was circulated to regulators, manufacturers and other

experts for comments in April 2009.

The Study Group held its third meeting on 22-23 April 2009 to review progress in the

revision and to propose further improvements. In addition, the experts discussed the need

for reference preparations, reference cell banks, and standardization of testing

methodologies. The SG proposed clarifications of the rationale for in vivo testing as well as

the potential for applying new methods for in vitro testing for detecting microbial agents. In

line with this, WHO should conduct review of the current manufacturers' practice in using

tests for microbial agents and interpreting these results. Additionally, WHO should take a

lead in the developing an International Standard for nucleic acid amplification test (NAT)

for detecting mycoplasma contamination in cell substrates. WHO Collaborating Centers

will lead this initiative, involving other relevant institutions in this area. Finally, advice on

the replacement of the WHO Vero reference cell bank 10-87 with respect to the source of

cells and re-characterization of the bank was provided. The intended use of the replacement

cell bank would be the same as for the current cell bank, which is to serve as a source of

well characterized cells for establishing master cell banks for the production of biologicals.

The SG will report outcomes of its discussion to the ECBS at its next meeting in October

2009 for further considerations and advice regarding the proposed course of action.

1. Background

The development of new biologicals is raising a number of challenges in evaluating

novel cell substrates. In particular, the assessment of new cell lines derived from tumors

or new animal cells that have not been used previously for the production of biological

products for human use has been the subject of international expert reviews over the past

decades.

Current WHO requirements for the use of animal cells as substrates for the

production of vaccines and other biologicals were adopted by the ECBS in October 1996

and published in the WHO Technical Report Series (TRS) 878, annex 1 (1). Since then, a

number of vaccines and other biologicals have been developed using new cell substrates

as well as new technologies for production. The primary concerns regarding the use of

new cell substrates have mostly been raised in the context of safety assessment and

include: limitations of currently used methodologies for the detection of microbial agents;

oncogenicity and infectivity of DNA; as well as difficulty in interpreting results of new

assays. However, development of vaccines and other biologicals has become a rapidly

evolving field that requires a dynamic regulatory framework with the capacity for

evaluating new biologicals and the necessary expertise as a basis for decision making.

A two-day interactive workshop (Microbial Agents in Animal Cell Substrates),

organized by the National Institute of Allergy and Infectious Diseases (NIAID) and the

International Association for Biologicals (IABS) and held on 20-21 April 2009 in

Bethesda, Maryland, USA, provided an excellent review of methodological advances in

this area. The workshop set the scene for the third meeting of the Study Group, where a

review of revised WHO recommendations for detection of microbial agents in cell

substrates was one of the key topics.

2. Introduction

Following the above mentioned workshop, WHO convened the third meeting of the

Study Group on cell substrates. The objective of the meeting was to review draft WHO

recommendations in the forum of regulators and manufacturers of vaccines and other

biologicals and to propose further improvements of the document. Additionally, the

meeting was a good opportunity to discuss the need for standardization of testing

methodologies, reference preparations, reference cell banks, and other issues of practical

importance.

Input from regulators and manufacturers is very much needed to define principles

that would improve current practice in establishing and characterizing cell banks for the

production of biologicals. In particular, sharing the information and experience would

help identify limitations and difficulties with the assays in use. This is a critical step for

re-affirming the value of the tests that provide specific information about quality and

safety of cell substrates, on the one hand, and for opening doors to new methodologies

that would complement or replace existing testing approaches, on the other. Comments

received from reviewers before the meeting are very much appreciated and will be

carefully considered in further revisions of WHO recommendations.

Dr. Ivana Knezevic, Scientist, Quality, Safety and Standards Team of the

Immunization, Vaccines and Biologicals Department of the World Health Organization,

opened the meeting by emphasizing the importance of improving methods for evaluating

quality and safety of cell substrates and facilitating the use of biologicals of assured

quality. In addition to the novelties associated with new cell substrates, there are also

numerous challenges in the evaluation of cell substrates with a long history of use, their

characterization according to current scientific knowledge, and testing methodologies, all

of which need to be addressed in the revision of the WHO recommendations for cell

substrates.

She stressed the importance of scientific evidence as a basis for setting WHO

recommendations for quality, safety, and efficacy of biologicals. However, data are

usually limited, and gaps in knowledge need to be addressed. A consensus on the

conclusions from such data needs to be reached by WHO expert groups. Potential risk

associated with new cell substrates is one of the issues for which data in the public

domain are limited. A critical aspect of assessing the suitability of a cell substrate for a

given product is the risk/ benefit ratio. However, because public health authorities expect

to see new vaccines with minimal risk associated with their use, and because the

sensitivity of parents with respect to immunizing their children has increased in the last

decade, there has been pressure on manufacturers and regulators to increase efforts to

assure quality, safety, and efficacy of vaccines and other biologicals. Dr Knezevic invited

all participants of the meeting to contribute to the information and knowledge sharing and

to provide information that would contribute to the evaluation of cell substrates.

Finally, Dr Knezevic explained the concept of WHO recommendations and their

implications for the worldwide regulation of biologicals. In this context, National

Regulatory Authorities (NRAs) and National Control Laboratories (NCLs) play a central

role in implementing WHO recommendations and putting guidance into practice. NRAs

are responsible for setting up national requirements that should be based on WHO

recommendations. However, NRAs may provide more specific advice on certain issues

according to their regulation and experience. Similarly, manufacturers of biological

products are important in implementing WHO recommendations into their practice. In

this context, continuous and productive communication between regulators and

manufacturers is of critical importance for assuring quality and safety of biologicals.

Furthermore, WHO recommendations, guidelines, and measurement standards serve as

benchmarks for global acceptability of vaccines for the United Nations (UN) supply.

The Study Group appointed Dr. J. Petricciani as Chairman and Dr. Glyn Stacey as

Rapporteur.

3. Progress made since June 2007

Following advice provided by the SG in June 2007 (2), key issues in the revision of

WHO requirements for cell substrates (TRS 878, annex 1) were reported to the ECBS in

2007 and 2008. The Committee welcomed the idea of providing some recommendations

for new cell substrates such as insect cells and stem cells. The Committee made some

further suggestions regarding the issues to be considered and agreed with the proposed

plan for the revision of WHO recommendations for cell substrates.

Review of scientific evidence conducted from 2006 and 2007 related to the revision

of the recommendations was mainly focused on the evaluation of new cell lines,

tumorigenicity of continuous cells lines, oncogenicity and infectivity of cell DNA, and

tests for the quantification of residual cell DNA. In 2008, discussions with manufacturers

and regulators revealed a need for providing guidance on good cell-culture practice.

Additionally, the use of new methods for the testing for microbial agents in animal cell

substrates was recognized as one of the promising approaches in assessing the safety of

vaccines and other biologicals. All these aspects have been considered in the revision of

the recommendations for cell substrates, and the meeting in April 2009 was set up to

discuss proposed changes and to receive feedback from the users of WHO standards.

The main changes from the current requirements published in TRS 878, annex 1,

include:

1. general manufacturing recommendations applicable to all types of cell-culture

production have been updated;

2. some considerations for the evaluation of new cell substrates such as insect cells

and stem cells have been added;

3. definitions have been updated and expanded in number and scope;

4. the structure of the document has been modified to address applicability of the

principles for the evaluation of different types of cell substrates;

5. a section on Good Cell Culture Practice has been added;

6. recommendations for tumorigenicity testing has been updated and a model

protocol for the nude mouse model was added as an appendix;

7. oncogenicity of DNA has been addressed;

8. recommendations for testing for microbial agents have been updated;

9. a summary of testing to be performed at different stages of use of cell substrates

based on a cell-banking system has been included in a tabulated form; and

10. a new section on risk reduction strategies during the manufacture of biological

products has been added.

4. Revision of WHO recommendations on the evaluation of cell substrates

4.1. Current approaches and recent developments in testing microbial agents in cell

substrates

Since the publication of TRS 878, annex 1, significant advances and improvements

have been made in methods for detecting microbial agents, and a growing body of

information has been recognized that can help inform and refine approaches to safety

testing of biological medicines. In response to these developments, a number of aspects

of detecting microbial agents in cell substrates were considered to be ready for review. In

particular, the rationale for the use of animals in testing had been brought into question,

and clarification was required.

4.1.1. Testing in animals for microbial agents

Use of animals for detecting microbial agents in cell cultures has a long history. In

the light of new methodologies for in vitro testing, the need of animal testing and the use

of these results in the overall evaluation of cell substrates was discussed.

4.1.1.1. Value of in vivo testing

The meeting participants concurred that there are now large data sets showing that

animal studies of recombinant cell products (primarily using CHO as the cell substrate)

have never revealed adventitious agents that were not also detected by other in vitro

methods. This also pertains to the testing of cell banks. However, there is no repository of

directly comparable data for in vivo vs. in vitro testing in terms of breadth (capacity to

detect a wide range of organisms) and sensitivity (quantitative limits of detection) to

various agents, and it was agreed that if information could be shared to shed light on this

gap in our knowledge, it might be possible to recommend refinements in the WHO

recommendations on animal testing and enable manufacturers to better focus their efforts

in assuring the safety of biological medicines. There are a number of reasons that limiting

the use of animals for testing is desirable, and these are articulated by bodies that are

leading the ‘3R’ initiative for reducing, replacing and refining animal use. Detailed

information about these initiatives is available at http://iccvam.niehs.nih.gov/ and

http://ecvam.jrc.it/index.htm.

The group supported a proposal to investigate current manufacturers' practice with

the use of in vivo and in vitro tests for detection of microbial agents. For this purpose, a

survey will be conducted over the coming months. The investigation will focus on the

following issues: 1) current use of animals and the value of in vivo testing for specific

purposes (e.g., characterizing cell banks, vaccine seed lot testing, lot release); 2)

perspective and experience on the value of use of in vitro tests for detection of microbial

agents as a replacement or complementary assay to in vivo tests; and 3) perceived value

and actual experience of the relative use of in vivo and in vitro tests for the

characterization of cell seeds (pre-master) vs. cell banks (master and working).

In the meantime, the group concurred that it would be helpful to consider what

recommendations might be made in the light of current knowledge. The SG agreed that a

wealth of experience serves as a solid basis for recommending that previously qualified

sources of “well established cell substrates”, such as CHO, WI-38, MRC-5 and Vero,

need not be tested repeatedly in animals (e.g., master cell banks established by

manufacturers from the WHO Vero reference cell bank). On the other hand, for cell

substrates for which there is relatively little manufacturing experience, appropriate animal

testing based on risk assessment should be performed. Furthermore, a number of species

currently incorporated in the list of animal tests had originally been included in testing

regimens for specific reasons that may not apply to all substrates. They apparently had

become part of the generic list for testing without due consideration for their relevance.

The group concluded that clarification of the rationale for their use could help to

eliminate unnecessary animal testing without any effect on the safety of the product. In

particular, some experts reminded the group that the use of fertilized hens’ eggs was

originally proposed for testing avian cell substrates, rabbits for detecting simian herpes B

virus from simian cell substrates, and guinea-pigs specifically for detecting mycobacteria

and lymphocytic choriomeningitis virus (LCMV). The SG therefore recommended that

the use of these species or others, beyond the conventional testing in adult and suckling

mice, should be justified on a case by case basis, following risk evaluation of the cell

substrate. In addition, other tests such as the mouse antibody production (MAP) test

should be performed where LCMV is identified as a risk. However, the SG group

questioned, if a “LCMV challenge” to detect inapparent infections is really necessary.

Similarly, an alternative method such as inoculation of special culture medium would be

appropriate for detecting potential mycobacterium contamination (i.e., where

environmental contamination is a special risk such as in primary cell cultures).

4.1.1.2 Route of Inoculation

The group reflected on the fact that testing regimens generally recommended intra-

peritoneal (IP) and intra-cranial (IC) inoculation and that at some time intramuscular

inoculation had become established in the WHO guidance as an additional route of

inoculation. There did not appear to be any clear rationale for this change based on the

detection of virus by intra-muscular (IM) inoculation, and the group recommended a

return to the use of only “IP and IC” inoculation in the revised WHO document.

4.1.1.3 Definitions for Maintenance of Animal Colonies

The principle of maintaining specific-pathogen-free groups of animals for testing

purposes is in part based on protecting them from the introduction of animals that might

bring infectious agents into the colony. The group agreed that the current use of the term

“closed” in relation to animal husbandry means that no animals are introduced from

outside the group. The use of a closed group prevents the introduction of genetically

transmittable viral elements (e.g., retroviral proviruses). Members of the group confirmed

that there are examples where this has been achieved for extensive periods. However, it

was also evident from discussions that in some systems of the management of animal

colonies there was also a need to avoid the adverse effects of inbreeding that can occur in

an outbred colony by the carefully controlled introduction of new animals following

rigorous screening for microbial agents. Such introduction of new animals would also

require assessment of the introduction of any divergent endogenous retroviruses from

those present in the original colony, herd, or flock.

4.1.1.4 Inoculation of in vitro cell cultures

Detection of adventitious agents by inoculation of different cell cultures susceptible

to a broad range of viruses is an important element in safety testing that could help to

justify a reduction in the use of animals for this purpose. The group concurred that two or

three cell types would be appropriate as a minimum for all cell substrates. For human or

non-human primate cell substrates, the use of human diploid cells and monkey kidney

cells would be appropriate, whereas for other species, in addition to those two types of

cultures, a third culture representing the same species and tissue as the production

substrate should be added. It was also noted by a number of individuals that many

manufacturers would, as a matter of routine, use a larger number of test cell lines for this

purpose to ensure adequate coverage of different viral groups. It was concluded that one

of these test cultures should originate from the same species as the production cell

substrate, and that the cell panel should assure detection of a broad range of human

viruses, usually achieved by inclusion of MRC-5 cells or another human diploid cell as

one member of the panel. However, it was agreed that additional cell lines may be

required depending on the potential contaminants of the cell substrate. The group also

recommended that the rationale for the choice of such additional cell lines should be

clearly stated. For instance, in the case of insect cell substrates, certain insect cell lines

may be used for detection of insect viruses, and BHK cells may serve for the detection of

arboviruses.

4.1.1.5 Retrovirus detection

Significant advances have been made over the last ten years in the detection of

retroviruses. In particular, the widely used product-enhanced reverse transcriptase

(PERT) assays provide a high level of sensitivity. The group confirmed that, in the light

of this, there was little justification for continuing to use the traditional reverse

transcriptase assays, and it was agreed that these could be eliminated if higher sensitivity

assays are used. This is possible because the logarithmic amplification that PCR affords

in the PERT assay negates any differences in the signal achieved in the initial cDNA

synthesis step with reverse transcriptases that require either manganese or magnesium.

However, certain members of the group emphasized that complications arise with

otherwise “acceptable” cell substrates that are known to be PERT positive due to

expression of endogenous retroviral sequences. In such cases, there is a risk that the

endogenous PERT activity might mask an infection with an exogenous retrovirus. It was

clear that this scenario required further consideration, and the development of a

recommendation for dealing with this situation was delegated to a subgroup of the SG.

The group agreed that, in general, it was important to know the status of the cell

substrate with respect to both endogenous and exogenous retroviruses. However, this

condition of the cell substrate should be considered in the context of the capacity of the

manufacturing process to remove and/or inactivate retroviral contaminants.

4.1.1.6 Rationale for traditional testing

It is important to periodically re-evaluate the relevance of tests performed on cell

substrates. It was therefore proposed that in the revised WHO guidance the “purpose” of

a particular test should be given formally alongside the “applicability” of each test

already proposed in the revised guidance.

In order to establish accurate purpose statements, information would be sought from

the WHO ‘BS’ document archive and from old reviews and documents from various

expert scientists with access to documents going back to the 1960s or earlier, when

vaccines were first being manufactured in cell cultures.

4.1.1.7 Testing for bovine and porcine adventitious agents in reagents such as

serum and trypsin

In the history of well established cell substrates, it is not unusual for them to have

had exposure to uncharacterized bovine serum and porcine trypsin. The group agreed that

where a cell substrate has been exposed to undefined bovine or porcine materials in the

past, it is important to test the cell substrate for those bovine and porcine viruses to which

it could have been exposed. However, they also acknowledged that convenient cell-

culture methods for viral isolation are not yet available for certain viruses e.g., bovine

polyomavirus; in such cases, direct testing of the cell line should be performed by

molecular methods.

4.2. Cell banking

4.2.1 “Control” Cells

The group agreed that “control cells” are parallel cultures propagated alongside

production cells to or beyond the production passage level and are specifically relevant to

vaccines only and are not needed for biotherapeutics, where the actual production

cultures can usually be used for testing. Since there were product-specific

recommendations to deal with this issue, and it is not relevant to the characterization of

cell banks but is a production issue, it was concluded that it was not an appropriate

subject for the revised WHO recommendations on cell substrates, and the discussion of

this topic should be removed from the document. However, data from “End of

Production” cells remain relevant to the WHO recommendations as they relate to general

characterization and suitability of the cell banks.

4.2.2 Characterization of MCB and WCB

Exhaustive characterization is normally performed on the MCB with reduced

testing on the WCB. However, the group concurred that the banking strategy of

individual manufacturers may vary, and that it may be acceptable to perform more

limited testing on the MCB provided that the exhaustive testing is done on each and every

WCB. In some circumstances, the MCB may be used directly for production, but in this

case, it must have been subjected to significant evaluation.

The current draft of the revised WHO recommendations contains a table

summarizing the tests performed at different levels of cell-substrate development. The

group agreed that this table could provide greater clarity on the tests required at each

stage if it were broken down into separate tables addressing different banking strategies.

Guidance was requested from experts on how to address the need for retesting of

established banks in the light of the identification of significant new risks. It was

proposed to draft a new section in the WHO recommendation to establish that retesting

should be instigated on a case-by-case basis when justified from a perspective of risk to

recipients of the product, but that generally re-testing of released materials would not be

necessary.

One of the more recent developments in cell biology is the recognition of the role of

microRNAs (miRNAs) in the modulation of various cellular processes. In the area of cell

substrates, their impact as product contaminants and as indicators of cell culture genetic

and phenotypic stability are of interest. The group concluded that whilst miRNAs have

great significance at the level of an individual cell, their wider implications for cell

substrates was unclear and that there were insufficient data to identify a clear hazard that

could be addressed. It was concluded that the knowledge available was too limited to

enable clear guidance, and the WHO recommendations would indicate that testing for

such molecules is premature as the scientific knowledge and applicable technology were

not yet ready.

4.2.3 Specific issues for biotherapeutic production substrates

Whilst uncloned cell cultures are often used in vaccine manufacture, the process of

developing a cell line for production of biotherapeutics very often involves a complex

process of cloning and selection. The group proposed that the section of the

recommendation dealing with this area should be developed (including preparation of

“seed stocks”) to give a more accurate reflection of the general strategy that

manufacturers adopt.

It became evident during discussion that there was a need to clarify what is meant

by "genetic stability" so as to be able to delineate the differences between the cell

substrates used for vaccines and biotherapeutics (e.g., stability of the engineered gene

insert vs. maintenance of a diploid nature). It was agreed that the recommendations

should be reviewed to ensure that there is clarity on this important aspect of the use of

cells for manufacturing.

4.2.4 Nomenclature for cell banking

A number of terms had been introduced into the current version of the draft

recommendations that needed modification.

4.2.4.1 Documentation on the cell bank

The SG recognized that the establishment and the characterization of cell banks

should be properly documented. However, the term “Cell Line Master File”, which was

proposed in the revised recommendations is not a commonly used term. It was agreed

that a more appropriate term should be used to emphasize the importance of the best

practice to assure traceability and well structured documentation on the cell bank.

Where a product is produced in primary cells, it is not unusual for the cultures to be

passaged a very limited number of times to provide sufficient cells for use. The revised

document should contain the recommendation that, wherever possible, cells that had been

passaged should be cryopreserved to enable testing to be performed prior to use in

manufacture. The group agreed that the term ‘Early Passage Working Cell Bank’ is not

appropriate for naming this frozen stock. The word “working” might cause confusion,

and the use of an alternative such as ‘low passage bank’ should be used.

4.2.4.2 “End of production” cells

Testing of cells at the end of production is an important element in the evaluation of

a cell substrate. The group considered that for the purposes of cell-substrate evaluation,

data of similar relevance can also be obtained from “Extended Cell Banks” (cells

passaged to or beyond the maximal end of production passage level or population

doubling and frozen and “banked” for purposes of characterization and testing) and “Post

Production Cells” (residual cells remaining at production harvest), and the

recommendations should carry some reference to the evaluation of such cells. The

difference between the two arises from the differences between vaccines and

biotherapeutics. For many viral vaccines, there are no intact cells remaining at the end of

production / harvest due to lysis by the vaccine virus. Thus, in order to test cells

equivalent to “post production” or “end of production”, cells from the MCB (or WCB)

must be passaged as though they were to be used for production but without infecting

with the vaccine virus; thus, such cultures are analogous to control cells.

4.3. Tumorigenicity of cell lines

4.3.1 Selection of animal model

In order to recommend any particular methodology, the availability of the tools to

use it on a broad international basis is vital. It was clear from discussion with members of

the group that the nude mouse system was widely, though not universally, available.

Alternatives to the nude mouse may be acceptable, and should be considered on a case-

by-case basis by NRAs. A protocol for the anti-thymocyte globulin (ATG)-treated

newborn rat method is being prepared for inclusion in the recommendations, and it is

hoped this will be released soon.

It was pointed out that microbial infection of the animals used for tumorigenicity

testing may have a significant influence on tumorigenicity data. Provision of appropriate

facilities and conditions for husbandry of animal colonies is therefore important.

However, it was clear from discussion amongst the group that guidance for this important

supporting function was not uniformly prescribed in different countries, and there was a

need for reference to appropriate guidance in this area in the new WHO document.

4.3.2 Regressing tumors

The interpretation of tumours arising in animal assays is one of the parameters for

defining the tumorigenicity profile of a cell line. It was agreed that cell substrates that

give rise to regressing nodules that are no longer present at the end of the observation

period should not be considered to be tumorigenic. However, the development of a

regressing nodule should be recorded.

4.3.3 Assay inocula

It was generally agreed that 107

cells in a 0.1 mL inoculum was normally acceptable

for introduction of cells into a mouse or other animal model. However, this may be

technically difficult, and it was agreed that a 0.2 mL inoculum is also acceptable.

4.4. Host cell DNA

Discussion was focused on the infectivity and oncogenicity of cell DNA, on the one

hand, and on the determination of residual cell DNA, on the other.

4.4.1 DNA Infectivity and Oncogenicity, Dr K. Peden

The group discussed recent data from CBER on DNA infectivity (3, 4) and

concluded that whilst this was highly valuable, it represented emerging knowledge, yet to

be replicated, and could not at this stage be considered definitive. The relevant text in the

revised recommendations would reflect this conclusion. The group recommended that

safety evaluations in respect of such emerging issues are facilitated by the use of DNase

treatment in the production process to reduce host-cell DNA to a size range documented

to have significantly reduced biological activity.

It was broadly agreed that the document should not recommend a DNA

oncogenicity test, but that the possible need and rationale for a DNA oncogenicity test

based on risk assessment should be clearly identified. However, further consideration

should be given to guidance on oncogenicity of cell lysates, and that section of the draft

should be retained and revised as this assay is considered to be a test for oncogenic

viruses.

4.4.2 A proposal for providing guidance on determination of rcDNA in the revised

recommendations, Dr L. Mallet

Key considerations for application of techniques for the detection of residual DNA

were outlined. It was proposed to describe the available methods with a presentation of

their advantages and disadvantages including applicability to different product types in an

appendix of the recommendations. This suggestion was enthusiastically supported by the

group, and Dr Mallet kindly agreed to submit a draft for this appendix including a table

comparing the different methods. It was also agreed that this information should state that

the choice of DNA detection method may be influenced by the type of production process

and the method used for reducing DNA. Dr Mallet also raised a number of issues for

DNA detection that were discussed. A series of conclusions were arrived at as follows:

1. It may be considered that the best point at which to test residual DNA is in the bulk

rather than the final product, as the bulk is more concentrated.

2. It is important for those setting release specifications to understand that the method

to be used should be considered at an early stage when defining the rcDNA

specification.

3. Where DNA levels are extremely low, it might not be technically feasible to

determine DNA size. It was agreed that the recommendations would include a

sentence on this issue.

4. Reference materials and controls should be based on multicopy target genes to

enhance sensitivity. However, to avoid background due to the inevitable

environmental contamination with human DNA, the PCR assay should be specific

to the species of the host cell and should not cross react with human DNA.

5. International Standard for nucleic acid amplification tests for detection

of Mycoplasma contamination in cell substrates

Dr Laurent Mallet presented a proposal for the development of an International

Standard for detection of Mycoplasma DNA and RNA by NAT. Although NAT methods

have been mentioned as an alternative to currently used culture methods in the European

Pharmacopoeia since January 2007, they have not been implemented. Lack of

standardization and difficulties with the interpretation of the results are two main

obstacles for their use. To overcome this problem, the Mycoplasma Task Force of the

Parenteral Drug Association (PDA) proposed development of the WHO International

Standard for NAT. A single strain of Mycoplasma, namely Acholeplasma laidlawii, was

proposed for this purpose, since it has already been proposed as a positive control for the

culture method. The standard will be tested in a collaborative study, and International

Units will be assigned.

The SG agreed with the proposal and requested that WHO coordinate development

and establishment of this important standard through its Collaborating Centers.

Procedures for establishing WHO International Standards will be followed (5), including

a collaborating study. Further discussion on the source material, intended use of the

standard, and details of the collaborative study are of critical importance for NAT

standardization. Progress in this area should be reported to the SG as well as to the ECBS.

Focal points from the Mycoplasma Task Force of the PDA will ensure communication

with this group. Also, the European Pharmacopoeia and other relevant parties will be

involved in further discussions on this matter.

6. Replacement of WHO Vero reference cell bank 10-87

Dr Knezevic presented the current status of the WHO Vero 10-87 reference cell

bank and the rationale for the proposed replacement. She explained that the Vero cell

bank had been established in 1990 through the project coordinated by WHO in

collaboration with leading scientific experts in the field. Since then, the Vero cell bank

has been available to manufacturers for the production of vaccines. Depletion of this bank

and the demand to use Vero cells for other purposes (e.g., development of new

biologicals; diagnostic and quality-control testing applications) meant that there was a

need to consider future demand for this reference cell bank and its possible replacement.

Despite having had interactions with manufacturers using Vero cells, WHO had not been

able to identify available sources of existing stocks of Vero cells that could be made

available, like the 10-87 stock, and had come to the conclusion that a new bank should be

established. Dr Knezevic requested feedback on how to establish requirements for a

replacement for the 10-87 cell bank.

Key aspects of the replacement that were discussed at the meeting were: 1) source

of cells; 2) prerequisites for manufacturing a new bank and 3) testing to be done for

characterization of the new cell bank.

Potential sources that would sustain the same lineage with the 10-87 stock included

remaining vials of 10-87, the CCL-81 Vero cells currently available from the American

Type Culture Collection (ATCC), and original stocks in the originating laboratory in

Japan. It was felt that the latter source was a remote possibility, and that there were many

questions on the condition of vials stored since the 1960s and their relationship to the

ATCC stock. It seems that the 10-87 and CCL-81 stocks were each being used by

manufacturers to derive master cell banks. The key difference between these sources was

the higher passage number of the 10-87 stock and the unclear passage history of the

currently available stocks of CCL81 cells while at ATCC. A benefit of using the current

cell bank is its immediate availability and its well documented history and

characterization. However, the Study Group suggested exploring the availability of CCL-

81 cells, which is the parental cell line to the current cell bank 10-87. This option would

lead to a replacement bank with a lower passage number, which would be preferred by

manufacturers.

The group discussed the issue of passage level at length and concluded that, as

stated in the WHO report on reference cell banks in 2002, a passage level above 150 for

production cells was acceptable provided this was qualified by the manufacturer. In

addition, work at CBER had shown that there was no evidence for increased

tumorigenicity at P140, and manufacturers at the meeting confirmed they had validation

data for use of 10-87 cells at passage levels beyond the arbitrary limit of P150 established

historically. Given the need to progress with this work as a matter of urgency, it was

proposed to pursue both the 10-87 and CCL-81 routes in parallel and reconsider the final

source of the cells as negotiations and development of a specification for the Bank were

developed.

The second key item for discussion was the extent of characterization required for

the new Vero cell bank. It was generally agreed that as a minimum, identity, sterility and

freedom from mycoplasma should be determined. In addition, it was agreed that

tumorigenicity data should be developed. A clear strategy for what adventitious agent

testing should be performed was not resolved, although there was a call not to repeat all

NATs done on the 10-87 bank if the new bank were established directly from that source.

It was agreed that further discussion on testing should take place at a later stage.

In conclusion, the Study Group agreed that the intended use for the replacement cell

bank would be the same as for the current cell bank, which is to serve as a source of well

characterized and documented cells for the establishment of master cell banks for the

production of biologicals.

It was agreed that it would be important to specify the conditions for preparation of

the new bank. The new cell bank should be prepared in a clean room environment

(preferably ISO14644) under a fully documented QA system, consistent with the GMP

requirements. The WHO proposed to initiate further discussion on required testing by

circulating a questionnaire on the use of the 10-87 bank to all manufacturers present. In

the meantime, the original reference cell bank remained the source of Vero cells

recommended by WHO for manufacturing biologicals.

7. Conclusions and next steps

During the meeting, a number of conclusions were drawn and several proposals for

further improvements of WHO recommendations for the evaluation of cell substrates as

well as for further assistance to regulators and manufacturers were made and are

summarized below.

7.1. Revision of WHO recommendations for cell cultures

The Study Group will conduct a further revision of recommendations for cell

substrates, and an updated draft with incorporated comments after consultations in April

will be presented to the ECBS for information and advice. Generally, the SG concluded

that WHO should consider possibilities for recommending better safety assessment in the

revised recommendations (e.g., use of methods with defined sensitivity and specificity for

detection of microbial agents, and refinements to animal testing). In particular, the SG

will take the following actions from June to December 2009:

7.1.1 Detection of microbial agents in cell substrates

7.1.1.1 Investigate current practice in using in vivo and in vitro tests for detection of

microbial agents;

7.1.1.2 Explore current approaches in maintaining animal colonies and provide

information on good practice for animal care;

7.1.1.3 Address the issue of retroviral contamination and possibilities for removal or

inactivation of retroviruses. Investigate and report on detection of endogenous

and exogenous retroviruses as part of cell-substrate characterization;

7.1.1.4. Review rationale for the use of "traditional" tests and assess their applicability

in the current context of QC testing and evaluation of cell banks.

7.1.2 Cell banking

The SG agreed to make the following changes in the section on cell

banking in further revision of the recommendations:

7.1.2.1 Add special considerations for cell substrates for the production of

biotherapeutics;

7.1.2.2 Exclude points regarding the control cells from the recommendations;

these issues are product-specific and are not relevant to cell banks.

7.1.2.3 Provide summary of testing to be performed on the MCB and WCB in a

tabulated form and clarify the testing strategies at each step of cell banking;

7.1.2.4 Emphasize the importance of establishing retesting policy by each

laboratory as part of GMP and GLP. It will be clearly stated that the need

for retesting should be considered on a case-by-case basis, taking into

account potential risk for the recipients of biological products.

7.1.2.5 Explain that the role of miRNAs in the control of various cellular

processes is not yet clear. Due to limited knowledge about the implications

of miRNAs for cell substrates, it is premature to provide any guidance on

this matter.

7.1.2.6 Clarify the terms "Cell Line Master File" and "End of production cells".

7.1.3 Tumorigenicity testing

7.1.3.1 Revised recommendations provide comprehensive information on the

advantages and disadvantages of animal models for testing tumorigenicity

in vivo. Selection of the animal model suitable for the cell substrate in

question should be made by manufacturers and approved by respective

NRA.

7.1.3.2 The protocol for testing in athymic nude mice, described in the appendix

of the revised recommendations, is intended to assist laboratories where

the model for in vivo testing has not yet been established. Another

appendix to be added will provide details of the testing in ATG-treated

newborn rats, which was first described in 1982.

7.1.3.3 Further guidance regarding the interpretation of regressing tumours and of

metastasis should be provided.

7.1.4 Host cell DNA

7.1.4.1. DNA Infectivity and Oncogenicity

Data generated up to now suggest the importance of these two aspects in

defining risk of a cell substrate. However, studies performed at CBER, for

research purposes, need to be replicated and complemented with data that

would answer some specific questions regarding the implications of the

results for the evaluation of cell substrates. Data from other laboratories

would be helpful in confirming and extending the CBER findings.

Because no animal model for DNA oncogenicity assay has been

determined, a DNA oncogenicity test is not recommended as part of cell-

substrate characterization at this time. Guidance on DNA oncogenicity and

DNA infectivity issues should be obtained from the NRA.

7.1.4.2. Determination of residual cell DNA

Consensus on the key issues to be provided in the appendix on rcDNA

determination was reached. Details of available methods, their advantages

and disadvantages, as well as the suitability of each method for a given

product are intended to improve current practice with the selection of

methods and evaluation of the results. A Subgroup of the SG will

coordinate preparation of the appendix.

7.2. International Standards for NAT for Mycoplasma detection

WHO will establish a working group for the preparation of the International

Standard for NAT for mycoplasma detection, and the work will be conducted as a joint

effort through WHO Collaborating Centers and involve other leading institutions in this

area.

7.3. Replacement of the WHO reference cell bank 10-87

A proposal for the replacement of the WHO Vero reference bank 10-87 will be

further developed taking into account comments made by the Study Group. WHO

Collaborating Centers will play a critical role in this process. An updated proposal for the

replacement of bank 10-87 will be presented to the ECBS for considerations and advice.

7.4. Next steps

Summary of the Study Group meeting will be published on the WHO web site and

will be submitted to a scientific journal as well.

Consultations with a broad audience of regulators, manufacturers and other experts

in this field are planned in 2009 and 2010. They will be handled through emails,

teleconferences and online meetings in Q3 and Q4 2009. The main issues in the revised

recommendations for the evaluation of cell substrates will be reported to the ECBS in

October 2009. Taking into account comments received from reviewers and advice from

the ECBS, further improvements will be made, and a mature draft will be posted on the

WHO web site for public consultation in Q1 2010. The next face-to-face consultation will

take place in April 2010.

8. Acknowledgements

Financial and technical support of NIAID and IABS in organizing the Workshop on

Microbial Agents in Animal Cells on 20-21 April 2009 is gratefully acknowledged.

Review of current approaches and recent developments in the detection of microbial

contaminants of animal cells facilitated the discussion of cell substrates in the SG

meeting.

9. References

1. Requirements for the use of animal cells as in vitro substrates for the production

of biologicals. In: WHO Expert Committee on Biological Standardization. Forty-

seventh Report. Geneva, World Health Organization, 1998, (WHO Technical

Report Series, No. 878, annex 1).

2. WHO Study Group on cell substrates for production of biologicals, Geneva,

Switzerland, 11-12 June 2007. Conference Report. Biologicals, 36 (2008), 203-

211.

3. Sheng-Fowler L, Lewis A, Peden K. Quantitative determination of the infectivity

of the proviral DNA of a retrovirus in vitro: Evaluation of methods for DNA

inactivation. Biologicals (2009), doi:10.1016/j.biologicals.2009.04.002.

4. Sheng-Fowler L, Lewis A, Peden K. Issues associated with residual cell-substrate

DNA in viral vaccines. Biologicals 37 (2009) 190 - 195.

5. Recommendations for the preparation, characterization and establishment of

international and other biological reference standards (revised 2004). In: WHO

Expert Committee on Biological Standardization. Fifty-fifth Report. Geneva,

World Health Organization, 2005, (WHO Technical Report Series 932, annex 2).

Authors

Dr I. Knezevic a**, Dr G. Stacey

b , Dr J. Petricciani

c and Dr R. Sheets

d on behalf of

the WHO Study Group on Cell Substrates1

a World Health Organization, Department of Immunization, Vaccines and Biologicals,

Quality, Safety and Standards Team, Avenue Appia, CH-1211 Geneva, Switzerland

b National Institute for Biological Standards and Control, Potters Bar, Hertfordshire,

EN6 3QG, UK

c Palm Springs, CA, USA

d National Institutes of Health/ National Institute of Allergy and Infectious Diseases,

Bethesda, MD, USA

** Corresponding author. Tel : +41 22 791 3136

Fax : +41 22 791 4971

E-mail : [email protected]

1 The WHO Study Group on Cell Substrates:

Dr K.S. Ahn, Gene Therapy Product Team, Korea Food and Drug Administration

(KFDA), Seoul, Republic of Korea; Dr J.H. Blusch, Novartis, Basel, Switzerland,

Representative of the International Federation of Pharmaceutical Manufacturers and

Associations (IFPMA); Dr M. Deschamps, Regulatory Affairs HPV Vaccines,

GlaxoSmithKline, Wavre, Belgium, Representative of the International Federation

of Pharmaceutical Manufacturers and Associations (IFPMA); Dr G. Dong, First

Division of Viral Vaccines, National Institute for the Control of Pharmaceutical &

Biological Products (NICPBP), Beijing, People's Republic of China; G. Gauvin,

Corporate Quality Control, Amgen Inc., Thousand Oaks, USA, Representative of

the International Federation of Pharmaceutical Manufacturers and Associations

(IFPMA); Dr H. Kavermann, Pharmaceutical Biotech Production & Development,

Roch Diagnostics GmbH, Penzeberg, Germany, Representative of the International

Federation of Pharmaceutical Manufacturers and Associations (IFPMA); Dr K.

King, FDA Center for Drug Evaluation and Research, Bethesda, USA; Dr A. Lewis,

Office of Vaccines Research and Review, Center for Biologics Evaluation and

Research (CBER), Bethesda, USA; Dr L. Mallet, Analytical Science & Assay

Development, Sanofi Pasteur, France, Representative of the International

Federation of Pharmaceutical Manufacturers and Associations (IFPMA); Dr P.

Minor, Division of Virology, National Institute for Biological Standards and

Control (NIBSC), Potters Bar, UK; Dr P. Nandapalan, Immunobiology Section,

Therapeutic Goods Administration Ltd., Woden, Australia; Dr D. Onions,

Invitrogen, Carlsbad, USA; Dr K. Peden, Office of Vaccines Research and Review,

Center for Biologics Evaluation and Research (CBER), Bethesda, USA; Dr J.

Petricciani, Palm Springs, USA; Dr E.I. Prawahju, National Quality Control

Laboratory of Drug and Food (NQCL DF), Jakarta Pusat, Indonesia; Dr R. Sheets,

National Institutes of Health (NIH), Bethesda, USA; Dr D. da Silva Guedes Jr.,

Departamento de Controle de Qualidade, Bio-Manguinhos/ Fiocruz, Rio de Janeiro,

Brazil, Representative of the Developing Country Vaccine Manufacturing Network

(DCVMN); Dr G. Stacey, National Institute for Biological Standards and Control

(NIBSC), Potters Bar, UK; Dr. R. Wagner, Department of Viral Vaccines, Paul-

Ehrlich-Institut, Langen, Germany; Dr I. Knezevic, Scientist, Quality, Safety and

Standards Team, Immunization, Vaccines & Biologicals Department, World Health

Organization, Geneva, Switzerland; and Dr D. Wood, Coordinator, Quality, Safety

and Standards, Immunization, Vaccines & Biologicals Department, World Health

Organization, Geneva, Switzerland.

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