自2015年以来,FDA已经批准了超过34种生物仿制药。生物仿制药竞争的新时代刺激了专注于治疗性蛋白质或生物制造的新技术发展。生物类似物开发中的一个挑战是用于制造生物制品的宿主细胞系的遗传差异。例如,1994年至2011年批准的许多生物制剂在小鼠NS0和SP2/0细胞系中表达.中国仓鼠卵巢(CHO)细胞,然而,由于生产率的提高,已经成为首选的生产主机,易用性,和稳定性。已经在使用鼠和CHO细胞产生的生物制剂中鉴定了鼠和仓鼠糖基化之间的差异。在单克隆抗体(mAb)的情况下,聚糖结构可以显著影响关键的抗体效应子功能,结合活性,稳定性,功效,和体内半衰期。为了利用CHO表达系统的内在优势并匹配参考生物鼠糖基化,我们设计了一种表达抗体的CHO细胞,该抗体最初是在鼠细胞系中产生的,以产生鼠样聚糖。具体来说,我们过表达胞苷单磷酸-N-乙酰神经氨酸羟化酶(CMAH)和N-乙酰氨基乙酸α-1,3-半乳糖基转移酶(GGTA),以获得具有N-羟乙酰神经氨酸(Neu5Gc)和半乳糖-α-1,3-半乳糖(αgal)的聚糖。所得到的CHO细胞显示产生具有鼠聚糖的mAb,然后通过通常用于证明分析相似性的分析方法的光谱对它们进行分析,作为证明生物相似性的一部分。这包括高分辨率质谱,生物化学,以及基于细胞的检测。通过补料分批培养中的选择和优化,鉴定出两个具有与原始细胞系相似的生长和生产率标准的CHO细胞克隆。它们维持了65个群体倍增时间的稳定生产,同时匹配在鼠细胞中表达的参考产物的糖基化谱和功能。这项研究证明了改造CHO细胞以表达具有鼠聚糖的mAb的可行性,以促进与在鼠细胞中表达的市售参考产品高度相似的生物仿制药的开发。此外,这项技术可以潜在地减少关于生物相似性的剩余不确定性,导致获得监管批准的可能性更高,并可能降低开发成本和时间。
Since 2015 more than 34 biosimilars have been approved by the FDA. This new era of biosimilar competition has stimulated renewed technology development focused on therapeutic protein or biologic manufacturing. One challenge in biosimilar development is the genetic differences in the host cell lines used to manufacture the biologics. For example, many biologics approved between 1994 and 2011 were expressed in murine NS0 and SP2/0 cell lines. Chinese Hamster ovary (CHO) cells, however, have since become the preferred hosts for production due to their increased productivity, ease of use, and stability. Differences between murine and hamster glycosylation have been identified in biologics produced using murine and CHO cells. In the case of monoclonal antibodies (mAbs), glycan structure can significantly affect critical antibody effector function, binding activity, stability, efficacy, and in vivo half-life. In an attempt to leverage the intrinsic advantages of the CHO expression system and match the reference biologic murine glycosylation, we engineered a CHO cell expressing an antibody that was originally produced in a murine cell line to produce murine-like glycans. Specifically, we overexpressed cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) and N-acetyllactosaminide alpha-1,3-galactosyltransferase (GGTA) to obtain glycans with N-glycolylneuraminic acid (Neu5Gc) and galactose-α-1,3-galactose (alpha gal). The resulting CHO cells were shown to produce mAbs with murine glycans, and they were then analyzed by the spectrum of analytical methods typically used to demonstrate analytical similarity as a part of demonstrating biosimilarity. This included high-resolution mass spectrometry, biochemical, as well as cell-based assays. Through selection and optimization in fed-batch cultures, two CHO cell clones were identified with similar growth and productivity criteria to the original cell line. They maintained stable production for 65 population doubling times while matching the glycosylation profile and function of the reference product expressed in murine cells. This study demonstrates the feasibility of engineering CHO cells to express mAbs with murine glycans to facilitate the development of biosimilars that are highly similar to marketed reference products expressed in murine cells. Furthermore, this technology can potentially reduce the residual uncertainty regarding biosimilarity, resulting in a higher probability of regulatory approval and potentially reduced costs and time in development.