# Presentation: 899361

## Laboratory of Methods Development

- January 9, 2003
- Site Visit

## Laboratory of Methods Development

- Created in 1991 from the group of Dr. Inessa Levenbook within DPQC
- In 1997 moved to the DVP / OVRR
- Dr. David Asher, Chief
- In 1999 split in two parts
- TSE group moved to DTTD / OBRR
- Dr. Konstantin Chumakov, chief

## Initial LMD Goal

- Creation of new in vitro and in vivo methods to replace primates in vaccines quality control
- Refinement
- Reduction
- Replacement

## LMD Mission

- New methods for evaluation and quality control of vaccines
- Development
- Validation
- Implementation
- Novel approaches and concepts advancing safety of biologicals
- Unique role of CBER regulatory science
- Application of the new methods in regulatory process
- Reference reagents
- Evaluation of new products
- Lot release

**Notes:**
Cannot do it all, too many issues, too few resources
Only proof of concept
Be a catalyst

## LMD Support

- Personnel

- Financing

## Grant Support

## LMD Staff

- Konstantin Chumakov, Ph.D., D.Sc.

- Chief

- Eugenia Dragunsky, M.D., Ph.D.

- Staff Scientist

- Leader of the Biological assays group

- Joan Enterline, B.A.

- Biologist

- Potency testing

- Monica Parker, B.S.

- Biologist

- Laboratory logistics, Molecular tests

- Gennady Rezapkin, Ph.D.

- Staff Fellow

- MAPREC, YFV, ELISA

- Vladimir Chizhikov, Ph.D.

- Visiting Scientist

- Leader of the Microarray group

- Majid Laassri, Ph.D.

- Visiting Associate

- Orthopoxvirus diagnostics, CT projects

- Georgios Amexis, Ph.D.

- Fogarty Fellow

- Mumps vaccine consistency, Prions

- Anna Ivshina, M.D., Ph.D.

- IRTA Fellow

- Influenza Microchip, Statistics

- Nazma Jahan, Ph.D.

- ORISE Fellow

- Cell substrate project, Expression microchips

- Alexander Ivanov, M.D., Ph.D., D.Sci.

- ORISE Fellow

- Immunological methods for new IPV

- Elena Cherkasova, Ph.D.

- ORISE Fellow

- Poliovirus microchips

- Svetlana Potapova, M.S.

- IRTA Fellow

- Biological assays

## Principal LMD Research Projects

- MAPREC

- Tg-mouse test

- In vitro test

**Notes:**
Microarrays started for Polio, then DARPA gave money
YFV and Mumps follow the same paradigm
New IPV: two aspects, animal rule
D-antigen ELISA - entirely new approach
VDPV - post-marketing surveillance

## MAPREC for OPV Consistency

- MAPREC  for type 3 OPV was approved by WHO as a routine QC test
- Tests for type 1 and type 2 OPV were developed
- WHO Collaborative Study to validate the tests and reference reagents
- Troubleshooting for OPV manufactureres and WHO

## Molecular consistency of Yellow Fever Virus vaccine production

- 17D strain of YFV
- Excellent safety record over 70 years of use
- Isolation of vaccine-derived strains from rare cases of vaccine associated complications
- Recently observer adverse reactions:
- 4 cases of multiple system organ failure in 1.55 million vaccinees
- The need to validate new seed viruses

## Genetic stability of YFV

- Dr. Karganova @ IPVE, the YFV vaccine manufacturer, performed virus passaging and neurovirulence tests in mice
- LMD was responsible for molecular part of the project
- 17D-213 seed stock was serially passaged in embryonated chicken eggs
- Passage six was sequenced (sequence heterogeneity assay)
- only one mutation in E protein (1795-CT)

## Consistency monitoring of Mumps vaccine live

- Leningrad 3 was withdrawn from market in 1970s
- Urabe AM9-based vaccine (SKB) was also withdrawn
- Rubini strain is poorly immunogenic
- Jeryl Lynn strain is safe and efficacious
- Need to validate new seed virus stocks

## Urabe AM9 strain

- Mutational profiles of acceptable and unacceptable lots differ
- Passaging in cell culture results in changes of the profile
- MAPREC method and MALDI-TOF mass-spectrometry can be used for analysis

## Jeryl Lynn strain

- Complete sequences of JL1 and JL2 sub-strains were determined (4% differences)
- MAPREC, MALDI-TOF, and microarray methods for quantification
- Growth in different substrates results in selection of eigther JL1 or JL2
- Consistency method is proposed

## Transgenic mouse test for neurovirulence of OPV

-  Research stage (1992 – 1998)
-  WHO Collaborative Study (1993-2000)
-  Implementation stage (2000 – current)

## Phases of the Tg-mouse project

- 2000 - Tg mouse test was approved by the WHO  ECBS as alternative to the monkey neurovirulence test for all three types OPV
- 2002 - Tg mouse test was included into revised WHO Recommendations for production and control of OPV
- 2002 - Tg mouse test was approved for implementation into routine practice in Europe. It was recommended to include Tg mouse test into Ph Eur monograph

## New methods for evaluation of immunogenicity and protectivity of new Inactivated Poliovirus Vaccine (sIPV) 

- New IPV products can not be directly evaluated for efficacy
- Validation of surrogate markers is needed
- Tg-mouse immunization-challenge model

## Comparison of conventional and Sabin IPV

- Sabin 2-derived vaccine is less protective
- Conventional IPV provides broader protection
- Sabin 2 and MEF-1 are very different (17%) and are not fully immunologically compatible

## ELISA test for IPV and immunological profiles

- New robust implementation of D-antigen ELISA test was created
- Block-ELISA test with monoclonal antibodies reveals fine epitope composition of vaccines and vaccine-derived poliovirus strains isolated from VAPP

## Microarray methods for genotyping of pathogens

- Oligonucleotide micorarrays to identify point mutations if vaccine lots
- Genotyping of Rotaviruses, Orthopoxviruses, pathogenicity factors in E.coli, antibiotic resistance factors, etc.
- Point muitations in VDPV
- Recombinants in VDPV

## Screening of reassortans of Influenza B

- Micorarray-basedgenotyping of each of the 8 segments
- Detection of mixtures
- High throughput
- Influenza A

## Cell Banks Consistency Issues

- Passage level: avoid tumorigenicity
- Cell cycle and metabolic status
- Contamination with adventitious agents

- Patterns of genomic activity may provide a tool (marker) for monitoring consistency of cell banks

## Risk of endogenous PrP in cell banks:Experimental assessment

- Spontaneous mutation in PrP may lead to *de novo* emergence of pathological form of prion protein
- FDA workshop on Cell Substrates
- Rockville, September 7-10, 1999

-   Can we find any mutations in extensively passages cell lines?
-   Will the mutation in PrP lead to infectious agent?

## HeLa cells PrP gene

- More than 700 passages
- No mutations detected in PrP gene
- Loss of one chromosome in some lineages
- Diversity of HeLa-derived cultures

## Overexpression of PrP in Human Neuroblastoma cells

- Cell lines were created to overproduce normal and mutant PrP
- Inoculation into squirrel monkeys
- Development of in vitro assays

## Conclusions

- Diverse projects are aimed at  creation  of new cutting edge QC methods and their introduction into regulatgory practice and industry
- Leadership in the field of molecular consistency monitoring
- Issues of critical regulatory and public health importance