半胱氨酸和胱氨酸是哺乳动物细胞培养物中存在的必需氨基酸。在促进生物质合成的同时,重组蛋白生产,和抗氧化防御机制,半胱氨酸由于其稳定性差和氧化为胱氨酸,在培养基配方中构成了重大挑战,半胱氨酸二聚体。由于其溶解性差,胱氨酸会导致饲料介质沉淀,形成不希望的产物,因此,降低半胱氨酸的生物利用度。在这项研究中,含有二肽二聚体的高度可溶性半胱氨酸,Ala-Cys-Cys-Ala(ACCA),被评估为CHO细胞培养物中半胱氨酸和胱氨酸的合适替代品。用ACCA代替基础培养基中的半胱氨酸和胱氨酸不能维持细胞生长。然而,在CHO-GS和CHO-K1分批培养物中,向含有半胱氨酸和胱氨酸的基础培养基中加入4mM和8mM的ACCA分别促进细胞生长达15%和27%,并导致IgG滴度成比例增加。13C-代谢通量分析揭示ACCA的补充使糖酵解通量减少20%,导致CHO-K1细胞中更有效的葡萄糖代谢。在补料分批培养中,ACCA能够替代饲料培养基中的半胱氨酸和胱氨酸。此外,在基础培养基中以高浓度补充ACCA消除了对补料培养基中任何半胱氨酸当量的需要,并且增加了补料分批培养中的细胞密度和活力,而对IgG电荷变体没有任何显著影响。一起来看,这项研究证明了ACCA改善CHO细胞生长的潜力,生产力,和代谢,同时也有利于不含半胱氨酸和胱氨酸的补料培养基的配制。半胱氨酸和胱氨酸的这种替代品将通过增加培养物中的细胞密度和延长高度浓缩的进料培养基的储存作为实现强化的生物生产过程的一部分而为增强的生物制造铺平道路。
Cysteine and cystine are essential amino acids present in mammalian cell cultures. While contributing to biomass synthesis, recombinant protein production, and antioxidant defense mechanisms, cysteine poses a major challenge in media formulations owing to its poor stability and oxidation to cystine, a cysteine dimer. Due to its poor solubility, cystine can cause precipitation of feed media, formation of undesired products, and consequently, reduce cysteine bioavailability. In this study, a highly soluble cysteine containing dipeptide dimer, Ala-Cys-Cys-Ala (ACCA), was evaluated as a suitable alternative to cysteine and cystine in CHO cell cultures. Replacing cysteine and cystine in basal medium with ACCA did not sustain cell growth. However, addition of ACCA at 4 mM and 8 mM to basal medium containing cysteine and cystine boosted cell growth up to 15% and 27% in CHO-GS and CHO-K1 batch cell cultures respectively and led to a proportionate increase in IgG titer. 13C-Metabolic flux analysis revealed that supplementation of ACCA reduced glycolytic fluxes by 20% leading to more efficient glucose metabolism in CHO-K1 cells. In fed-batch cultures, ACCA was able to replace cysteine and cystine in feed medium. Furthermore, supplementation of ACCA at high concentrations in basal medium eliminated the need for any cysteine equivalents in feed medium and increased cell densities and viabilities in fed-batch cultures without any significant impact on IgG charge variants. Taken together, this study demonstrates the potential of ACCA to improve CHO cell growth, productivity, and metabolism while also facilitating the formulation of cysteine- and cystine-free feed media. Such alternatives to cysteine and cystine will pave the way for enhanced biomanufacturing by increasing cell densities in culture and extending the storage of highly concentrated feed media as part of achieving intensified bioproduction processes.