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Frederick, MD, United States

Dux M.P.,Novartis Animal Health U.S. Inc | Dux M.P.,University of Nebraska - Lincoln | Huang J.,Nektar Therapeutics | Huang J.,University of Nebraska - Lincoln | And 12 more authors.
Protein Expression and Purification | Year: 2011

A purification process for the manufacture of a recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype C [rBoNTC(H c)], a potential vaccine candidate, has been defined and successfully scaled-up. The rBoNTC(Hc) was produced intracellularly in Pichia pastoris X-33 using a three step fermentation process, i.e., glycerol batch phase, a glycerol fed-batch phase to achieve high cell densities, followed by a methanol induction phase. The rBoNTC(Hc) was captured from the soluble protein fraction of cell lysate using hydrophobic charge induction chromatography (HCIC; MEP HyperCel™), and then further purified using a CM 650M ion exchange chromatography step followed by a polishing step using HCIC once again. Method development at the bench scale was achieved using 5-100 mL columns and the process was performed at the pilot scale using 0.6-1.6 L columns in preparation for technology transfer to cGMP manufacturing. The process yielded approximately 2.5 g of rBoNTC(Hc)/kg wet cell weight (WCW) at the bench scale and 1.6 g rBoNTC(Hc)/kg WCW at the pilot scale. The purified rBoNTC(Hc) was stable for at least 3 months at 5 and -80 °C as determined by reverse phase-HPLC and SDS-PAGE and was stable for 24 months at -80 °C based on mouse potency bioassay. N-Terminal amino acid sequencing confirmed that the N-terminus of the purified rBoNTC(Hc) was intact. © 2010 Elsevier Inc. All rights reserved. Source

Sanford D.C.,Battelle | Barnewall R.E.,Battelle | Vassar M.L.,Battelle | Niemuth N.,Battelle | And 5 more authors.
Clinical and Vaccine Immunology | Year: 2010

A recombinant botulinum vaccine (rBV A/B) is being developed for protection against inhalational intoxication with botulinum neurotoxin (BoNT) complex serotype A, subtype A1 (BoNT/A1), and BoNT serotype B, subtype B1 (BoNT/B1). A critical component for evaluating rBV A/B efficacy will be the use of animal models in which the pathophysiology and dose-response relationships following aerosol exposure to well-characterized BoNT are thoroughly understood and documented. This study was designed to estimate inhaled 50% lethal doses (LD50) and to estimate 50% lethal exposure concentrations relative to time (LCt50) in rhesus macaques exposed to well-characterized BoNT/A1 and BoNT/B1. During the course of this study, clinical observations, body weights, clinical hematology results, clinical chemistry results, circulating neurotoxin levels, and telemetric parameters were documented to aid in the understanding of disease progression. The inhaled LD50 and LCt50 for BoNT/A1 and BoNT/B1 in rhesus macaques were determined using well-characterized challenge material. Clinical observations were consistent with the recognized pattern of botulism disease progression. A dose response was demonstrated with regard to the onset of these clinical signs for both BoNT/A1 and BoNT/B1. Dose-related changes in physiologic parameters measured by telemetry were also observed. In contrast, notable changes in body weight, hematology, and clinical chemistry parameters were not observed. Circulating levels of BoNT/B1 were detected in animals exposed to the highest levels of BoNT/B1; however, BoNT/A1 was not detected in the circulation at any aerosol exposure level. The rhesus macaque aerosol challenge model will be used for future evaluations of rBV A/B efficacy against inhalational BoNT/A1 and BoNT/B1 intoxication. Copyright © 2010, American Society for Microbiology. All Rights Reserved. Source

Martin S.S.,DynPort Vaccine Company LLC DVC | Bakken R.R.,U.S. Army | Lind C.M.,U.S. Army | Garcia P.,U.S. Army | And 5 more authors.
Vaccine | Year: 2010

V3526, a genetically modified strain of Venezuelan equine encephalitis virus (VEEV), was formalin inactivated for evaluation as a next generation vaccine candidate for VEEV. In this study, we tested formalin-inactivated V3526 (fV3526) with and without adjuvant for immunogenicity and efficacy in BALB/c mice and results were compared to the existing inactivated VEEV vaccine, C84. Mice were vaccinated intramuscularly (IM) or subcutaneously (SC) with fV3526 formulations and challenged with VEEV IAB Trinidad donkey (VEEV TrD) strain by SC or aerosol exposure. Efficacy following SC or aerosol challenge was not significantly different between the fV3526 formulations or compared to C84 despite C84 being administered in more doses and higher concentration of viral protein per dose. These data support further evaluation of fV3526 formulations as a next generation VEEV vaccine. © 2010 Elsevier Ltd. Source

Martin S.S.,DynPort Vaccine Company LLC DVC | Bakken R.R.,U.S. Army | Lind C.M.,U.S. Army | Garcia P.,U.S. Army | And 5 more authors.
Vaccine | Year: 2010

We recently developed a gamma-irradiation method to inactivate V3526, a live-attenuated Venezuelan equine encephalitis virus (VEEV) vaccine candidate. Dosage and schedule studies were conducted to evaluate the immunogenicity and efficacy of gamma-irradiated V3526 (gV3526). Subcutaneous (SC) and low dosage intramuscular (IM) administration of gV3526 were highly effective in protecting mice against a SC challenge with VEEV IA/B Trinidad Donkey strain, but not against an equivalent aerosol challenge. More robust immune responses and increased protective efficacy were noted when the IM dosage of gV3526 was increased. IM administration of gV3526 formulated with either CpG or CpG plus Alhydrogel™ further augmented the immune response in mice and resulted in 100% protection against aerosol challenge. © 2009 Elsevier Ltd. Source

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