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
1
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.
2
Stability: Function & Structure
Stability
• Potency
• Structure
Loss of Function
(immune protection)
• Bio-availability
Pathways of Degradation
•
•
•
•
Deamidation
Fragmentation
Oxidation
Aggregation
(e.g., tighter binding to
Alhydrogel)
• Epitope structure
(e.g., loss of secondary
structure/conformation)
3
FDP Stability by Previous Mouse Challenge Assay
Watkinson et al., Clin. Vaccine Immunol. 2013, 20(11):1659.
4
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
5
In vitro Assays for Antibody Detection
Mouse TNA
Color Reaction
MTT
Taken up by active mitochondria
Lethal Toxin
PA
LF
Mouse anti-rPA
J774A.1 – Mouse macrophage cell line
Indirect ELISA
TMB
HRP
Capture ELISA
TMB
Color Reaction
HRP
Rabbit anti-mouse IgG HRP
Color Reaction
Rabbit anti-mouse IgG HRP
Mouse anti-rPA
Mouse anti-rPA
rPA
rPA
Goat anti-PA PAb
6
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™
7
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
8
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™
9
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
10
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.
11
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)
12
Principal Pathways of rPA degradation
Deamidation
pI
BDS
MEVKQENRLL
LENIPSENQY
ASNSNKIRLE
NLQLPELKQK
RTFLSPWISN
EARHPLVAAY
TSEVHGNAEV
MGLNTADTAR
QLSQILAPNN
LDTDQVYGNI
LVERRIAAVN
FDFNFDQQTS
RNNIAVGADE
EGLKEVINDR
AVTKENTIIN
NESESSSQGL
FQSAIWSGFI
KGRLYQIKIQ
SSNSRKKRST
IHEKKGLTKY
PIVHVDMENI
HASFFDIGGS
LNANIRYVNT
YYPSKNLAPI
ATYNFENGRV
PSDPLETTKP
QNIKNQLAEL
SVVKEAHREV
YDMLNISSLR
PSENGDTSTN
LGYYFSDLNF
KVKKSDEYTF
YQRENPTEKG
SAGPTVPDRD
KSSPEKWSTA
ILSKNEDQST
VSAGFSNSNS
GTAPIYNVLP
ALNAQDDFSS
RVDTGSNWSE
DMTLKEALKI
NATNIYTVLD
INSSTEGLLL
QDGKTFIDFK
GIKKILIFSK
QAPMVVTSST
ATSADNHVTM
LDFKLYWTDS
NDGIPDSLEV
SDPYSDFEKV
QNTDSQTRTI
STVAIDHSLS
TTSLVLGKNQ
TPITMNYNQF
VLPQIQETTA
AFGFNEPNGN
KIKLNAKMNI
NIDKDIRKIL
KYNDKLPLYI
KGYEIG
TGDLSIPSSE
WVDDQEVINK
QNKKEVISSD
EGYTVDVKNK
TGRIDKNVSP
SKNTSTSRTH
LAGERTWAET
TLATIKAKEN
LELEKTKQLR
RIIFNGKDLN
LQYQGKDITE
LIRDKRFHYD
SGYIVEIEDT
SNPNYKVNVY
Powell et al., 1997
13
Imaging Capillary Electrophoresis (iCE)
www.proteinsimple.com/ice_technology.html
PharmAthene Confidential
14
rPA FDP Forced Degradation Study
FDP
Stress Treatment
Structural Analysis
• 37 oC
• iCE
• 25 oC
• LDS-PAGE
Functional Analysis
• IPA
• DPIA
15
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
16
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)
17