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

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 mM Phosphate, pH 7.1, 137 mM NaCl, 3 mM KCl, 0.04% v/v Polysorbate 20 rPA Final Drug Product (FDP) 0.1 mg/mL rPA in 0.5 mL (50  g/dose) 1.3 mg Alhydrogel ®, *4 mM NaPO 4, 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 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) 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 rPA Mouse anti-rPA Rabbit anti-mouse IgG HRP Color Reaction TMB HRP Goat anti-PA PAb rPA Mouse anti-rPA Rabbit anti-mouse IgG HRP Color Reaction TMB HRP Indirect ELISACapture ELISA J774A.1 – Mouse macrophage cell line PA LF Lethal Toxin Mouse anti-rPA Color Reaction MTT Taken up by active mitochondria

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 50 ⁰ C) 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 50 ⁰ C) Heat degraded vaccine 2 (3 minutes at 100 ⁰ C) –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 pI Deamidation Powell et al., 1997 BDS

14 Imaging Capillary Electrophoresis (iCE) PharmAthene Confidential

15 rPA FDP Forced Degradation Study Stress Treatment 37 o C 25 o C 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 (25 ⁰ C & 37 ⁰ C) Specifically, potency measured by IPA or DPIA is inversely correlated with deamidation measured by iCE and fragmentation measured by LDS-PAGE

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