RESUMO
Structural conversion of the serotype A recombinant botulinum neurotoxin heavy chain fragment (rBoNTA(Hc)) produced intracellularly in Pichia pastoris yeast was observed and characterized during purification development efforts. A pH screening study captured the transformation stages of the original recovered species into its derived counterpart and a number of analytical tools such as peptide mapping by LC/MS confirmed the formation of a disulfide bond, especially in samples of neutral to basic pH. A cation exchange chromatographic method proved useful in following the incidence of the reaction in various rBoNTA(Hc) samples. The disulfide formation kinetics were characterized using a one-quarter quadratic factorial design, following the investigation and development of controlled oxidation conditions using cysteine and cystamine as the redox pair. Temperature, pH and concentration of the redox pair had a significant effect on the yield and rate of the disulfide formation. This controlled reaction was eventually introduced as a functional unit operation in the purification process. The summation of preliminary scale-up and potency data showed scalability and robustness in the production of an active disulfide-bonded form of a recombinant botulism vaccine candidate. The presence of the disulfide bond did not effect the vaccine potency and it enhanced the molecule's thermal stability.
Assuntos
Biofarmácia/métodos , Toxinas Botulínicas Tipo A/química , Dissulfetos/química , Pichia/química , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Vacinas Sintéticas/química , Sequência de Aminoácidos , Animais , Toxinas Botulínicas Tipo A/síntese química , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/uso terapêutico , Botulismo/prevenção & controle , Desenho de Fármacos , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Camundongos , Dados de Sequência Molecular , Oxirredução , Pichia/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Relação Estrutura-Atividade , Temperatura , Vacinas Sintéticas/isolamento & purificação , Vacinas Sintéticas/metabolismo , Vacinas Sintéticas/uso terapêuticoRESUMO
Initial purification of two serotypic variants of recombinant botulinum neurotoxin toxin heavy chain fragment [rBoNT(Hc)], produced intracellularly in the yeast Pichia pastoris, using hydrophobic charge induction chromatography (HCIC) is reported. HCIC employs a matrix containing a weakly ionizable ligand that binds proteins through hydrophobic interactions at neutral pH and elutes the proteins by charge repulsion at acidic pH. HCIC optimization led to different purification conditions for each of the proteins even though they have 58% sequence similarity. The HCIC resin has a higher affinity for the fragment of serotype A than that of serotype B. The 10% dynamic breakthrough capacity for the serotype A fragment is >12.5 mg per ml of resin and is approximately 3.5 mg or the serotype B fragment per ml of resin. Stable elution conditions are also different for the two serotypes. The serotype A fragment is unstable when citrate is used to elute the product. However the serotype B fragment is stable when eluted with citrate buffer, and it is further purified by a overnight precipitation caused by the citrate buffer. This paper reports the development strategy, dynamic capacity breakthrough curves, resin and separation reproducibility, and preliminary scale-up data. The summation of the data demonstrates that HCIC is a scaleable process step for biopharmaceutical production of rBoNT(Hc) proteins.
