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.

中文翻译：

---

Ala-Cys-Cys-Ala 二肽二聚体可缓解培养基配方中有问题的半胱氨酸和胱氨酸水平，并增强 CHO 细胞生长和代谢


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。半胱氨酸在促进生物质合成、重组蛋白生产和抗氧化防御机制的同时，由于其稳定性差和氧化成胱氨酸（一种半胱氨酸二聚体）而对培养基配方构成重大挑战。由于溶解度差，胱氨酸会导致饲料培养基沉淀，形成不需要的产物，从而降低半胱氨酸的生物利用度。在这项研究中，一种含有二肽二聚体的高可溶性半胱氨酸 Ala-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 替代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，在含有半胱氨酸和胱氨酸的基础培养基中添加 4 mM 和 8 mM 的 ACCA 可分别将 CHO-GS 和 CHO-K1 批量细胞培养物中的细胞生长提高 15% 和 27%，并导致 IgG 滴度成比例增加。13C-代谢通量分析显示，补充 ACCA 可将糖酵解通量降低 20%，从而提高 CHO-K1 细胞的葡萄糖代谢效率。在补料分批培养中，ACCA 能够替代补料培养基中的半胱氨酸和胱氨酸。此外，在基础培养基中添加高浓度 ACCA 消除了在补料培养基中添加任何半胱氨酸当量的需求，并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。综上所述，本研究证明了 ACCA 在改善 CHO 细胞生长、生产力和新陈代谢方面的潜力，同时也促进了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浓度饲料培养基的储存，作为实现强化生物生产过程的一部分，为增强生物制造铺平道路。