Peter C. Fusco, Ph.D. Vice President, Immunobiology & Assay Development PharmAthene, Inc.

1. Development of Stability-Indicating Assays for a Recombinant Protective Antigen Vaccine Peter C. Fusco, Ph.D. Vice President, Immunobiology & Assay Development PharmAthene, Inc. Workshop on the Biology of Anthrax 11-12 March 2014, Cardiff, Wales, UK

2. Bulk Drug Substance and Final Drug Product • rPA Bulk Drug Substance (BDS) • 82.8 kDa protein • Codon-optimized • Purified from E. coli inclusion bodies • 1.4 mg/mL protein, 0.5 mMPhosphate, pH 7.1, 137 mMNaCl, 3 mMKCl, 0.04% v/v Polysorbate 20 • rPA Final Drug Product (FDP) • 0.1 mg/mL rPA in 0.5 mL(50 mg/dose) • 1.3 mg Alhydrogel®, *4 mM NaPO4, pH 7.2, 154 NaCl, 0.02% v/v Polysorbate 20 • * Watkinson et al., Clin. Vaccine Immunol. 2013, 20(11):1659. • Increasing phosphate yields increased thermal stability of rPA on alhydrogel.

3. Stability: Function & Structure • Stability • Potency • Structure • Pathways of Degradation • Deamidation • Fragmentation • Oxidation • Aggregation • Loss of Function (immune protection) • Bio-availability • (e.g., tighter binding to Alhydrogel) • Epitope structure • (e.g., loss of secondary structure/conformation)

4. FDP Stability by Previous Mouse Challenge Assay Watkinson et al., Clin. Vaccine Immunol. 2013, 20(11):1659.

5. New Potency Assay: IPA • There is a requirement for a more practical and sensitive alternative to lethal challenge animal models for potency testing of anthrax vaccines • We propose a mouse immunopotency assay (IPA) as a stability indicating parallel line relative potency (RP) assay for recombinant protective antigen anthrax vaccine final drug product • IPA has two components • The in vivo phase: mice vaccinated on Day 1 and bled on Day 28 • The in vitro phase: sera tested in antibody detection assay • Three initial studies resulted in selection of • Mouse strain (A/J, CD-1, C57BL/6) • Antibody detection assay (toxin neutralization assay [TNA] vs. ELISA) • Dose preparation diluent (Saline vs. Alhydrogel) • Dose dilution series

6. In vitro Assays for Antibody Detection Mouse TNA TMB ColorReaction TMB ColorReaction HRP HRP Rabbit anti-mouse IgG HRP Rabbit anti-mouse IgG HRP Mouse anti-rPA Mouse anti-rPA Indirect ELISACapture ELISA ColorReaction MTT rPA rPA Taken up by active mitochondria Lethal Toxin LF PA Goat anti-PA PAb Mouse anti-rPA J774A.1 – Mouse macrophage cell line

7. Potency Study 1: Feasibility • Study 1 investigated • Three mouse strains: A/J, CD-1, C57BL/6 • Three antibody detection assays: Indirect ELISA, Capture ELISA, mouse Toxin Neutralization Assay (mTNA) • Two test materials: native vaccine, heat degraded vaccine (24 hours at 50⁰C) • Dose volume and route: 0.1mL i.p. • Dose preparation diluent: PBS containing Alhydrogel™

8. Potency Study 1: Conclusions • All strains of mice (A/J, CD-1, C57BL/6) and all assays (TNA and ELISAs) were capable of discriminating between native and degraded vaccine • Indirect and Capture ELISA generated similar results – neither superior in terms of performance • Further optimization of dose range was required

9. Potency Study 2: Mouse Strain & in vitro Assay • Study 2 investigated • Two mouse strains: CD-1, A/J • Two antibody detection assays: Capture ELISA, mTNA • Three test materials: • Native vaccine • Heat degraded vaccine 1 (4 hours at 50⁰C) • Heat degraded vaccine 2 (3 minutes at 100⁰C) • Dose volume and route: 0.1mL i.p. • Dose preparation diluent: PBS containing Alhydrogel™

10. Potency Study 2: Conclusions • TNA selected as the in vitro assay • TNA is more sensitive in detecting degradation • ELISA showed no powering advantage over TNA in detecting vaccine concentration differences • CD-1 selected as the mouse strain • Mouse strains respond differently by TNA, but not by ELISA • A/J TNA response maximum too low for adequate dose range

11. Potency Study 3: Diluent & Dose Selection • Study 3 investigated • mTNA dose response in CD-1 mice • Two native vaccines lots • Two dose preparation diluents • PBS containing Alhydrogel™ • Saline • Dose volume and route: 0.1mL i.p.

12. Potency Study 3: Conclusions • Saline improved capability of IPA to detect 2-fold differences in vaccine concentration, minimizing animal numbers • With precision factors around 2, the IPA is much less variable than the mouse challenge assay which had precision factors ranging from 9 to 16(precision factor is the ratio of the upper 95% confidence limit to the lower 95% confidence limit for the RP)

13. Principal Pathways of rPA degradation Deamidation pI BDS Powell et al., 1997

14. Imaging Capillary Electrophoresis (iCE) www.proteinsimple.com/ice_technology.html PharmAthene Confidential PharmAthene Confidential

15. rPA FDP Forced Degradation Study • Stress Treatment • 37 oC • 25 oC • Structural Analysis • iCE • LDS-PAGE • Functional Analysis • IPA • DPIA FDP

16. Summary • Stability-indicating assays showed strong correlations between functional and structural measurements for FDP under mild heat stress (25⁰C & 37⁰C) • Specifically, potency measured by IPA or DPIA is inversely correlated with deamidation measured by iCEand fragmentation measured by LDS-PAGE

17. Acknowledgments PharmAthene, Inc. Robin Sun Karie Hirst Samuel Moore Sherry Crowe Howard Seligsohn James Bourdage Bradford Powell Funding Agency Biomedical Advanced Research and Development Authority (BARDA) (Contract No. HHSO100200900103C) The views expressed are those of the authors and do not reflect the official policy or position of the U.S. Government Commercial Partners Baxter BioPharma Solutions (BPS) Pharmaceutical Product Development, Inc. (PPD)