Preparation of Reference Influenza Viruses in Mammalian Cells:

FDA Perspective

Prepared for the Vaccines and Related Biological Products Advisory Committee

February 18-19, 2004

Zhiping Ye, M. D., Ph.D.

Center For Biologics Evaluation and Research,

Division of Viral Products

Objectives

• To review the issues associated with the source of reference influenza viruses

• To discuss possible strategies for using mammalian cell lines to prepare influenza reference viruses

Background

• Since the 1940’s, inactivated influenza virus vaccines have been prepared in embryonated eggs, which provide reasonable virus yields

• The candidate vaccine viruses are originally isolated in surveillance labs using eggs as opposed to mammalian cells (a global consensus within WHO, national regulatory authorities and manufacturers)

• Over time, many surveillance labs have come to rely on cell cultures for virus isolation, for reasons of robustness and convenience, even though such isolates are not used as reference viruses

Background (cont.)

Isolation of circulating viruses from clinical samples in National Influenza Centers (surveillance labs )

Cell culture (Not qualified)- MDCK cells (majority of surveillance labs)

- Vero cells (some labs)

- Chicken embryo kidney cells (few labs)

Embryonated eggs (Few labs)

Isolation Rates of Influenza Viruses From Clinical Samples

(Govorkova, et al., 1996, J. Virol., 70:5519)

0

20

40

60

80

100

MDCK VERO Eggs

Type A

Type B

Background (cont.)

• Yearly strain selection can be negatively affected by the requirement for an egg isolate as seed virus– Delayed recommendation for vaccine

composition– Altered recommendation for vaccine strain

component because of availability

• A delay in the availability of strain selection during a pandemic situation could have disastrous consequences.

Influenza Virus Vaccines(licensed in USA)

• Inactivated Influenza Virus Vaccines– Commercially available for more than 50 Years

in USA– Produced in eggs– Parenteral injection

• Live Influenza Virus Vaccine– Recently licensed - 2003

– Produced in SPF-eggs

– Intranasal inoculation

Influenza Virus Vaccines(in development)

• Cell based- inactivated vaccines– MDCK – VERO

• Others:– Subunit DNA (Plasmid/bacteria)

– Subunit Protein (Insect cells)

Reference and Seed Virus Development

• WHO global surveillance systems (National Influenza Centers)– Provide reference viruses isolated from

clinical samples using eggs

• WHO collaborating centers (Atlanta, London, Melbourne and Tokyo) – Characterize and provide reference

viruses (high growth reassortants for type A) to manufactures

Reference and Seed Virus Development (cont.)

• Manufacturers– Develop proprietary seed viruses from

egg-isolates recommended by WHO and local authorities

• Regulatory agencies– Accept reference viruses and approve

seed viruses for use in licensed vaccine preparation

Reference and Seed Virus Development (Summary)

NIC (Egg-isolate)

WHOCC (Vaccine candidate)

Regulatory Manufactures

Ref. Ref.Seed

Ref.

Seed

Ref.

Use of Eggs for Isolation of Influenza Seed Strains

• A safety track record of more than 50 years

• Possible exclusion of some adventitious agents from clinical specimens

• Leads to high yields in vaccine manufacturing

• Validated for inactivation of common chicken viruses in vaccine process

Pros and Cons for the Consideration of Mammalian Cell Lines for Isolation of Seed Strains

• Pros– More robust than eggs for primary

isolation of influenza A viruses from clinical specimens

– Less selective pressure than in eggs for isolation of influenza virus with alternative receptor specificity

– Ready availability in many WHO global surveillance centers (MDCK cell lines)

Pros and Cons for the Consideration of Mammalian Cell Lines for Isolation of Seed Strains• Cons

– Limited experience in influenza vaccine development and production (e.g. isolates may not necessarily grow well in eggs)

– Issues related to the use of mammalian cells for isolation of seed strains (e.g. the possible contamination of adventitious agents)

Regulatory Requirements and Guidance Related to the Use of

Mammalian Cell Lines• 21 CFR 610.18 (c) Cell lines used for

manufacturing biological products • (1) General requirements

– Identified by history– Described with respect to cytogenic

characteristics and tumorigenicity– Characterized with respect to in vitro

growth characteristics and life potential– Tested for the presence of detectable

microbial agents

Regulatory Requirements and Guidance Related to the Use of Mammalian Cell Lines (Cont.)

• Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals

– Published in 1993; currently under revision by Office of Vaccines Research and Review

– Provides additional guidance on appropriate standards for characterization and qualification of cell substrates and viral seeds

Additional Regulatory Requirements and Guidance Related to the Use of

Mammalian Cell Lines

• WHO Technical Report Series, #878 (Requirements for Biological Substances No 50-1998)

• ICH Guidance, Q5A (Viral Safety Evaluation of Biotechnology Products)

• EMEA Guidance, CPMP/BWP/2490/00 (Cell Culture Inactivated influenza Vaccine-2000)

Strategies to Consider for the Use of Mammalian Cell-Isolates as

Reference Viruses 1. Retain the clinical samples after initial

isolation in NIC and forward the samples to select WHOCC for re-isolation in a qualified cell line– Similar to the current procedure except for the

use of cell lines for re-isolation– An appropriate cell bank would need to be

produced and qualified for distribution to select centers

– Further purification or adaptation of cell-isolates for vaccine production in eggs could be considered if necessary

Strategies to Consider for the Use of Mammalian Cell-Isolates as

Reference Viruses (Cont.)

2. Use reference viruses directly isolated on cell lines not characterized or qualified– Would require purification of reference viruses

to be acceptable (e.g., plaque purification on SPF-CEK cells)

– Resources and infrastructure for purification would be necessary

Strategies to Consider for the Use of Mammalian Cell-Isolates as

Reference Viruses (Cont.)

3. Use reference viruses isolated on characterized and qualified cell lines– Would require production and qualification of an

appropriate cell bank for distribution to NIC• choice of cell lines (MDCK, Vero, others?)• maintenance of quality control after distribution

– Further purification or adaptation of cell-isolates for vaccine production in eggs could be considered if necessary

Strategies to Consider for the Use of Mammalian Cell-Isolates as

Reference Viruses (Cont.)

4. Use reverse genetics methodology and a qualified cell line to generate new reference viruses– Would require production and qualification of an

appropriate cell bank for reverse genetics– May eliminate concerns regarding contaminates

regardless of the source of the sample– Acceptance of technology and intellectual

property issues may not permit near-term implementation

Conclusions

• The use of influenza seeds obtained from non-egg sources may be acceptable if concerns regarding the presence of adventitious agents in the original isolate or in the cell lines used for isolation can be addressed

• An investment in resources and infrastructure will be necessary

• A global consensus (WHO, national regulatory authorities, manufacturers) regarding the issues to be addressed would facilitate implementation

Acknowledgments

Jerry Weir

Roland Levandowski

Andrew Lewis

Keith Peden

Arifa Khan

Philip Krause