Pacis Efren, Yu Marcella, Autsen Jennifer, Bayer Robert, Li Feng
Oceanside Pharma Technical Development, 1 Antibody Way, Oceanside, CA 92056, US Biologics Pharma Technical Development, Genentech, Inc., telephone: 760-231-3046; fax: 760-231-2465.
Biotechnol Bioeng. 2011 Oct;108(10):2348-58. doi: 10.1002/bit.23200. Epub 2011 May 24.
The glycosylation profile of therapeutic antibodies is routinely analyzed throughout development to monitor the impact of process parameters and to ensure consistency, efficacy, and safety for clinical and commercial batches of therapeutic products. In this study, unusually high levels of the mannose-5 (Man5) glycoform were observed during the early development of a therapeutic antibody produced from a Chinese hamster ovary (CHO) cell line, model cell line A. Follow up studies indicated that the antibody Man5 level was increased throughout the course of cell culture production as a result of increasing cell culture medium osmolality levels and extending culture duration. With model cell line A, Man5 glycosylation increased more than twofold from 12% to 28% in the fed-batch process through a combination of high basal and feed media osmolality and increased run duration. The osmolality and culture duration effects were also observed for four other CHO antibody producing cell lines by adding NaCl in both basal and feed media and extending the culture duration of the cell culture process. Moreover, reduction of Man5 level from model cell line A was achieved by supplementing MnCl2 at appropriate concentrations. To further understand the role of glycosyltransferases in Man5 level, N-acetylglucosaminyltransferase I GnT-I mRNA levels at different osmolality conditions were measured. It has been hypothesized that specific enzyme activity in the glycosylation pathway could have been altered in this fed-batch process.
在治疗性抗体的整个研发过程中，通常会对其糖基化谱进行分析，以监测工艺参数的影响，并确保临床和商业批次治疗产品的一致性、有效性和安全性。在本研究中，在由中国仓鼠卵巢（CHO）细胞系（模型细胞系A）生产的治疗性抗体的早期研发过程中，观察到甘露糖-5（Man5）糖型水平异常高。后续研究表明，由于细胞培养基渗透压水平的升高和培养时间的延长，抗体Man5水平在细胞培养生产过程中持续增加。对于模型细胞系A，在补料分批培养过程中，通过高基础和补料培养基渗透压以及延长培养时间的组合，Man5糖基化增加了两倍多，从12%增加到28%。通过在基础培养基和补料培养基中添加NaCl并延长细胞培养过程的培养时间这两种方式，在其他四种CHO抗体生产细胞系中也观察到了渗透压和培养时间的影响。此外，通过添加适当浓度的MnCl2，实现了模型细胞系A中Man5水平的降低。为了进一步了解糖基转移酶在Man5水平中的作用，测量了不同渗透压条件下N-乙酰葡糖胺基转移酶I GnT-I的mRNA水平。据推测，在该补料分批培养过程中，糖基化途径中的特定酶活性可能发生了改变。
