RESUMO
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(H(c)) 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(H(c)) 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 5-100mL columns and the process was performed at the pilot scale using 0.6-1.6L columns in preparation for technology transfer to cGMP manufacturing. The process yielded approximately 2.5 g of rBoNTC(H(c))/kg wet cell weight (WCW) at the bench scale and 1.6 g rBoNTC(H(c))/kg WCW at the pilot scale. The purified rBoNTC(H(c)) 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(H(c)) was intact.
Assuntos
Toxinas Botulínicas , Clostridium botulinum/química , Pichia , Proteínas Recombinantes , Sequência de Aminoácidos , Animais , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/metabolismo , Biotecnologia , Bioterrorismo/prevenção & controle , Toxinas Botulínicas/genética , Toxinas Botulínicas/imunologia , Toxinas Botulínicas/isolamento & purificação , Toxinas Botulínicas/metabolismo , Cromatografia , Clonagem Molecular , Feminino , Fermentação , Camundongos , Dados de Sequência Molecular , Projetos Piloto , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transferência de TecnologiaRESUMO
A gene homologous to Saccharomyces cerevisiae PMR1 has been cloned in the methylotrophic yeast Pichia pastoris. The entire P. pastoris PMR1 gene (PpPMR1) codes a protein of 924 amino acids. Sequence analysis of the PpPMR1 cDNA and the genomic DNA revealed that there is no intron in the coding region. The putative gene product contains all of the conserved regions observed in P-type ATPases and exhibits 66.2%, 60.3% and 50.6% identity to Pichia angusta (Hansenula polymorpha), Saccharomyces cerevisiae PMR1 and human ATP2C1 gene products, respectively. A pmr1 null mutant strain of P. pastoris exhibited growth defects in media with the addition of EGTA, but with supplementation of Ca2+ to a calcium-deficient media reversed the growth defects of the mutant strain. Manganese reversed the growth defects of the mutant strain; however, the cell growth was not as profound as the Ca2+ -supplemented media. The results demonstrated that the P. pastoris gene encodes the functional homologue of the S. cerevisiae PMR1 gene product, a P-type Ca2+/Mn2+ -ATPase. The DNA sequence of the P. pastoris PMR1 gene has been submitted to GenBank under Accession No. DQ239958.
Assuntos
ATPases Transportadoras de Cálcio/genética , Cálcio/metabolismo , Manganês/metabolismo , Pichia/enzimologia , Pichia/genética , Sequência de Aminoácidos , Sequência de Bases , ATPases Transportadoras de Cálcio/química , Clonagem Molecular , Dados de Sequência Molecular , Mutagênese Insercional , Mutação , Pichia/crescimento & desenvolvimento , RNA Fúngico/química , RNA Fúngico/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
A recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype E (BoNT/E) is proposed as a vaccine against the serotype E neurotoxin. This fragment, rBoNTE(Hc), was produced intracellular in Pichia pastoris GS115 by a three-step fermentation process, i.e., glycerol batch phase and a glycerol fed-batch phase to achieve high cell densities, followed by a methanol fed-batch induction phase. The rBoNTE(Hc) protein was purified from the soluble fraction of cell lysates using three ion-exchange chromatography steps (SP Sepharose Fast Flow, Q Sepharose Fast Flow, Sp Sepharose High Performance) and polished with a hydrophobic charge induction chromatography step (MEP HyperCel). Method development at the bench scale was achieved using 7-380 mL columns and the process was performed at the pilot scale using 0.5-3.1 L columns in preparation for technology transfer to cGMP manufacturing. The purification process resulted in greater than 98% pure rBoNTE(Hc) based on HPLC and yielded up to 1.01g of rBoNTE(Hc)/kg cells at the bench scale and 580mg vaccine/kg cells at the pilot scale. N-terminal sequencing showed that the purified rBoNTE(Hc) N-terminus is intact and was found to protect mice against a challenge of 1000 mouse intraperitoneal LD50's of BoNT/E.
