Process Development, Bristle-Myers Squibb, Devens, Massachusetts.
Molecular and Analytic Development, Bristol-Myers Squibb, Pennington, New Jersey.
Biotechnol Bioeng. 2018 Apr;115(4):900-909. doi: 10.1002/bit.26511. Epub 2017 Dec 19.
Process control for manufacturing biologics is critical for ensuring product quality, safety, and lot to lot consistency of therapeutic proteins. In this study, we investigated the root cause of the pink coloration observed for various in-process pools and drug substances in the antibody manufacturing process. Vitamin B is covalently bound to mAbs via a cobalt-sulfur coordinate bond via the cysteine residues. The vitamin B was identified to attach to an IgG4 molecule at cysteine residues on light chain (Cys-214), and heavy chain (Cys-134, Cys-321, Cys-367, and Cys-425). Prior to attachment to mAbs, the vitamin B needs to be in its active form of hydroxocobalamin. During culture media preparation, storage and cell culture processing, cyanocobalamin, the chemical form of vitamin B added to media, is converted to hydroxocobalamin by white fluorescence light (about 50% degradation in 11-14 days at room temperature and with room light intensity about 500-1,000 lux) and by short-wavelength visible light (400-550 nm). However, cyanocobalamin is stable under red light (wavelength >600 nm) exposure and does not convert to hydroxocobalamin. Our findings suggests that the intensity of pink color depends on concentrations of both free sulfhydryl groups on reduced mAb and hydroxocobalamin, the active form of vitamin B . Both reactants are necessary and neither one of them is sufficient to generate pink color, therefore process control strategy can consider limiting either one or both factors. A process control strategy to install red light (wavelength >600 nm) in culture media preparation, storage and culture processing areas is proposed to provide safe light for biologics and to prevent light-induced color variations in final products.
生物制品制造过程的控制对于确保产品质量、安全性以及治疗性蛋白质的批间一致性至关重要。在这项研究中，我们调查了抗体制造过程中各种中间池和原料药中观察到的粉红色的根本原因。维生素 B 通过钴-硫配位键与 mAbs 共价结合，通过半胱氨酸残基。维生素 B 被鉴定为附着在 IgG4 分子的轻链（Cys-214）和重链（Cys-134、Cys-321、Cys-367 和 Cys-425）上的半胱氨酸残基上。在与 mAbs 结合之前，维生素 B 需要处于其活性形式羟钴胺素。在培养基制备、储存和细胞培养过程中，添加到培养基中的维生素 B 的化学形式氰钴胺素会被白色荧光光（在室温下约 11-14 天降解 50%，在 500-1000 勒克斯的室内光照强度下）和短波长可见光（400-550nm）转化为羟钴胺素。然而，氰钴胺素在红光（波长>600nm）照射下稳定，不会转化为羟钴胺素。我们的研究结果表明，粉红色的强度取决于还原型 mAb 上游离巯基基团和维生素 B 的活性形式羟钴胺素的浓度。这两种反应物都是必需的，它们中的任何一种都不足以产生粉红色，因此过程控制策略可以考虑限制其中一种或两种因素。提出了一种在培养基制备、储存和培养处理区域安装红光（波长>600nm）的过程控制策略，为生物制品提供安全的光照，并防止最终产品中的光诱导颜色变化。
