Alternatives to In-Vivo Testing for Animal Biologics: North American Regulatory Perspective

Transcription

1 Alternatives to In-Vivo Testing for Animal Biologics: North American Regulatory Perspective Geetha B. Srinivas, DVM, PhD Section Leader, Virology Center for Veterinary Biologics USDA/APHIS/VS IABS 2018, NIH, Bethesda, Maryland, USA October 16, 2018

2 Virus-Serum Toxin Act 1913 & 1985 Purity: Ensure product is not contaminated Safety: Ensure product is not dangerous or harmful Potency/Efficacy: Ensure product is not worthless

3 CVB Regulatory Activities Complex and Diverse Products Veterinary Biologics - >200 different Products (antigens); >35 Species; ~100B doses New technologies - biotechnology-derived; synthetic peptides; RNA particles; production platforms; recombinant antibodies; serum-free culture systems, etc.

4 Veterinary Biologics Concept Master Seed concept. Establish and use the MS - pure, safe and efficacious against the intended pathogen. Potent serials or batches.

5 Potency Assays for Veterinary Biologics Live Product in lieu of animal testing Viral products by Titration (1969) Live bacterial products Counting (1984) Conventional serial potency testing for inactivated vaccines and bacterins - most are in-vivo tests involving host or lab animals.

6 Historical Perspective (Milestones) US 1985 Amendment to the 1966 Animal Welfare Act Alternatives to painful or stressful procedures in biomedical research IACUC

7 Historical Perspective (Reduction & Replacement) 1980s/90s Shift from Host/Lab animal vaccination/challenge to vaccination/serology 1980s/90s Correlation of potency test to protective antigen quantification and host efficacy for killed virus products Mab antigen quantification

8 Historical Perspective (Reduction & Replacement) Relative Potency with ELISA Exemption to 3-year Master Seed Immunogenicity Reference qualification & requalification by serology

9 Historical Perspective (Refinement) Humane Endpoints acceptance & regulatory change in US Use of analgesics/anesthesia for IC inoculations

10 Leptospira In-vitro Potency Test

11 Leptospirosis Causes disease in animals and human (Zoonotic Disease) 11

12 Leptospira Serovars L. pomona, canicola, grippotyphosa and icterohemorrhagiae L. hardjo L. bratislava 12

13 Regulatory Oversight Potency Test Conducted on every serial (numbered lot) marketed in the United States Potency Test Options Codified Potency Assays o Hamster vaccination challenge assays o Title 9 Code of Federal Regulations (9 CFR) : Canicola, Grippotyphosa, Icterohaemorrhagiae, and Pomona

14 Overview 9 CFR Potency Tests for Leptospira Vaccines Wait Observe days 14 days Vaccinate Challenge w/leptospira Count survivors Animal Numbers Vaccinates Challenge Controls Concurrent Challenge (LD50 Titration)

15 Codified Potency Test for Leptospira Bacterins Vaccine Disposition Satisfactory: 80% Vaccinates Survive Validity Requirements 80% Death in Challenge Controls LD50:10 10,000

16 Leptospira Project Goals Leptospira in-vitro assay development Leptospira backtitration Cryopreservation of Leptospira challenge culture

17 Leptospira In-vitro Potency Assay Replace hamster test with in-vitro test Validate assay for each named antigen Establish national reference bacterins

18 Test Development Identification of relevant and specific antigen; monoclonal antibody and ascites production Supplemental Assay Methods for in-vitro potency testing Qualification of Reference Bacterins

19 Enzyme-linked Immunosorbent Assay (ELISA)

20 Supplemental Assay Methods Standardization/development of in-vitro assay SAM L. pomona SAM L. canicola SAM L. grippotyphosa SAM 627 L. icterohemorrhagiae

21 Reagents for In Vitro Assay Standard Reference Bacterins (SRB) IRP 523 (05): L. grippotyphosa SRB IRP 524 (05): L. pomona SRB IRP 542 (06): L. icterohaemorrhagiae SRB IRP 555 (07): L. canicola SRB Monoclonal Antibodies L. canicola - 4DB-001 L. grippotyphosa - LGF L. icterohaemorrhagiae L. Pomona - 2D7 Polyclonal Antibodies

22 Guidance Documents CVB Notice No Qualification of Leptospira grippotyphosa and Leptospira icterohaemorrhagiae Reference Bacterins for Products Intended for Use in Dogs CVB Notice No Qualification of Leptospira pomona and Leptospira canicola Reference Bacterins for Products Intended for Use in Dogs CVB Notice No Qualification of Leptospira Canicola, Leptospira Grippotyphosa,Leptospira Icterohaemorrhagiae, and Leptospira Pomona Reference Bacterins for Products Intended for Use in Swine and/or Cattle VSM Guidelines for Validation of In Vitro Potency Assays

23 Requirements for Exemption of Codified Test Identification of a Reference Bacterin Standard reference bacterin Product-matched reference bacterin - qualify it via a host animal vaccinationchallenge Product-matched reference bacterin - qualify via the codified hamster potency assay

24 Requirements for Exemption of Codified Test Cont. In vitro Assay Validation Specificity Reproducibility Dose response Parallelism

25 CVB Notices & Option to Remove Back-titration Hamsters from In Vivo Potency Tests for Leptospira Serogroups Canicola and Icterohaemorrhagiae Option to Remove Back-titration Hamsters from In Vivo Potency Tests for Leptospira Serogroups Pomona and Grippotyphosa

26 Outcome of CVB s 2/2 Efforts

27 Rabies

28 NIH Rabies Potency Test In-Vivo Mouse Vaccination-Challenge Assay Developed Several Alternate Methods SN Antibody Titration, ELISAs, Single Radial Diffusion, Immunoprecipitation, Antigen Binding Assays Tried with Limited Success NIH Test - Mandatory Potency Assay for the Inactivated Rabies Vaccine

29 Recognition by World Health Organization

30 NIH Rabies Potency Test Antigen extinction method - test vaccine & reference vaccine - relative potency Reference vaccine in the US - BPL inactivated rabies vaccine distributed by the CVB Challenge virus Strain CVS-11 Animals Non-Swiss Albino Female Mice Days of Age; 200 Mice per Test

31 NIH Test Validity Criteria At least 70% of immunized mice receiving the lowest dilution (highest dose) of vaccine must survive At least 70% of mice receiving the highest dilution (lowest dose) of vaccine must die A serial which does not meet the minimum RP value is not released

32 NIH Potency Assay Drawbacks Require Large Number of Mice Causes Distress to Animals Time Consuming Safety Concerns to Workers Significant Variability in Results due to minor Variations in Mice Age, Vaccination Route, Dosage & Duration between Doses, and Challenge Process

33 Historical Perspective (Refinement) Humane Endpoints acceptance & regulatory change in US.

34 Historical Perspective (Refinement) Use of analgesics/anesthesia for IC inoculations (CVB Notice 12-12)

35 Historical Perspective (Reduction) Elimination of LD 50 Upper Limit for NIH (Rabies) Potency Test (CVB Notice 13-10) SAM 308 update - addition of human endpoints, euthanasia, & LD50 update

36 In-vitro Potency Assay Development Rabies virus glycoprotein (G), structural protein induces virus-neutralizing antibodies (confers immunity) Glycoprotein - conformational epitope, trimeric state CVB identified two ATCC hybridomas (509-6 and ) producing rabies neutralizing antibodies

37 In-vitro Potency Assay Development Capture Neutralizes live rabies virus (PV, CVS, Flury LEP, PM, SAD) Detection Neutralizes live rabies virus (PV, CVS, Flury LEP, PM, SAD) Rabies reference vaccine sourced from CVB

38 Requirements for Replacement of NIH Potency Test Universal assay Standard reference vaccine

39 Requirements for Replacement of NIH Potency Test In vitro Assay Validation Specificity/Sensitivity Reproducibility Dose response Parallelism

40 Acknowledgements Dr. Byron Rippke Dr. Paul Hauer Ms. Alethea Fry Dr. Jennifer Johnson Dr. Angela Walker Dr. Jay Srinivas

41 THANKS * National Centers for Animal Health, Ames, Iowa, USA