Initial purification of recombinant botulinum neurotoxin fragments for pharmaceutical production using hydrophobic charge induction chromatography.
Initial purification of recombinant botulinum neurotoxin fragments for pharmaceutical production using hydrophobic charge induction chromatography.
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DOI：
10.1016/s0021-9673(02)00074-2
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发表时间：
2002-04
期刊：
影响因子：
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通讯作者：
G. Weatherly;A. Bouvier;Debra D Lydiard;Jaymi Chapline;I. Henderson;J. Schrimsher;S. Shepard
中科院分区：
文献类型：
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作者：
G. Weatherly;A. Bouvier;Debra D Lydiard;Jaymi Chapline;I. Henderson;J. Schrimsher;S. Shepard
据报道，使用疏水电荷诱导色谱 (HCIC) 对酵母毕赤酵母细胞内产生的重组肉毒杆菌神经毒素重链片段 [rBoNT(Hc)] 的两种血清型变体进行了初步纯化。 HCIC 采用含有弱电离配体的基质，该配体在中性 pH 下通过疏水相互作用结合蛋白质，并在酸性 pH 下通过电荷排斥洗脱蛋白质。 HCIC 优化导致每种蛋白质的纯化条件不同，尽管它们具有 58% 的序列相似性。 HCIC 树脂对血清型 A 片段的亲和力高于血清型 B 片段。血清型 A 片段的 10% 动态突破能力为 >12.5 mg/ml 树脂，而血清型 B 片段为 ~3.5 mg/ml 树脂。两种血清型的稳定洗脱条件也不同。当使用柠檬酸盐洗脱产物时，血清型 A 片段不稳定。然而，用柠檬酸盐缓冲液洗脱时，血清型 B 片段是稳定的，并且通过柠檬酸盐缓冲液引起的过夜沉淀进一步纯化。本文报告了开发策略、动态容量突破曲线、树脂和分离重现性以及初步放大数据。数据总结表明，HCIC 是 rBoNT(Hc) 蛋白生物制药生产的可扩展工艺步骤。
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 ∼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.