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

PharmAthene, Inc.

Workshop on the Biology of Anthrax11-12 March 2014, Cardiff, Wales, UK

Development of Stability-Indicating Assays for a Recombinant Protective Antigen Vaccine

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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 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.

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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)

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FDP Stability by Previous Mouse Challenge Assay

Watkinson et al., Clin. Vaccine Immunol. 2013, 20(11):1659.

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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

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In vitro Assays for Antibody Detection

Mouse TNA

rPA

Mouse anti-rPA

Rabbit anti-mouse IgG HRP

Color ReactionTMB

HRP

Goat anti-PA PAbrPA

Mouse anti-rPA

Rabbit anti-mouse IgG HRP

Color ReactionTMB

HRP

Indirect ELISA Capture ELISAJ774A.1 – Mouse macrophage cell line

PALF

Lethal Toxin

Mouse anti-rPA

Color ReactionMTT

Taken up by active mitochondria

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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™

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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

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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™

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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

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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.

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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)

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Principal Pathways of rPA degradation

pI

Deamidation

Powell et al., 1997

MEVKQENRLL NESESSSQGL LGYYFSDLNF QAPMVVTSST TGDLSIPSSE LENIPSENQY FQSAIWSGFI KVKKSDEYTF ATSADNHVTM WVDDQEVINK ASNSNKIRLE KGRLYQIKIQ YQRENPTEKG LDFKLYWTDS QNKKEVISSD NLQLPELKQK SSNSRKKRST SAGPTVPDRD NDGIPDSLEV EGYTVDVKNK RTFLSPWISN IHEKKGLTKY KSSPEKWSTA SDPYSDFEKV TGRIDKNVSP EARHPLVAAY PIVHVDMENI ILSKNEDQST QNTDSQTRTI SKNTSTSRTH TSEVHGNAEV HASFFDIGGS VSAGFSNSNS STVAIDHSLS LAGERTWAET MGLNTADTAR LNANIRYVNT GTAPIYNVLP TTSLVLGKNQ TLATIKAKEN QLSQILAPNN YYPSKNLAPI ALNAQDDFSS TPITMNYNQF LELEKTKQLR LDTDQVYGNI ATYNFENGRV RVDTGSNWSE VLPQIQETTA RIIFNGKDLN LVERRIAAVN PSDPLETTKP DMTLKEALKI AFGFNEPNGN LQYQGKDITE FDFNFDQQTS QNIKNQLAEL NATNIYTVLD KIKLNAKMNI LIRDKRFHYD RNNIAVGADE SVVKEAHREV INSSTEGLLL NIDKDIRKIL SGYIVEIEDT EGLKEVINDR YDMLNISSLR QDGKTFIDFK KYNDKLPLYI SNPNYKVNVY AVTKENTIIN PSENGDTSTN GIKKILIFSK KGYEIG

BDS

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Imaging Capillary Electrophoresis (iCE)

PharmAthene ConfidentialPharmAthene Confidentialwww.proteinsimple.com/ice_technology.html

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rPA FDP Forced Degradation Study

Stress Treatment

• 37 oC

• 25 oC

Structural Analysis

• iCE

• LDS-PAGE

Functional Analysis

• IPA

• DPIA

FDP

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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 iCE and fragmentation measured by LDS-PAGE

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Acknowledgments

Funding AgencyBiomedical 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

PharmAthene, Inc.Robin SunKarie Hirst

Samuel MooreSherry Crowe

Howard SeligsohnJames BourdageBradford Powell

Baxter BioPharma Solutions (BPS)Pharmaceutical Product Development, Inc. (PPD)