Huang Chung-Jr, Lin Henry, Yang Jerry Xiaoming
Cell Science & Technology, Process and Product Development, Amgen Inc., One Amgen Center Drive, Thousand Oaks, California 91320.
Biotechnol Bioeng. 2015 Jun;112(6):1200-9. doi: 10.1002/bit.25534. Epub 2015 Apr 17.
High mannose (HM) glycoforms on antibody Fc glycan are recognized as critical quality attributes for therapeutic antibody products. Methods to control HM have been largely empirical, and it is challenging to target a desired HM level during antibody process development. A novel and robust method to increase antibody HM glycoforms is demonstrated in this study using multiple antibodies and cell lines without adversely impacting cell culture performance, including viable cell density, viability, and protein titer. This approach utilizes mannose as a carbon source and the ratio of mannose to total hexose (glucose and mannose) in feed media determines the extent of HM glycan content of an antibody expressed in cell culture. Scale-up of this strategy from 3 mL small scale plate to bioreactor (1.5 L) is also demonstrated with comparable results. Further full glycan map analysis shows that HM increase predominantly correlates with the decrease in G0F glycan, with minimum impact on other glycoforms. Possible hypotheses for the HM glycan modulation using mannose as carbon source are also discussed. Three pathways, including GDP-mannose biosynthesis, early protein glycosylation and UDP-N-acetylglucosamine biosynthesis, might be involved and contribute to this HM modulation.
抗体Fc聚糖上的高甘露糖（HM）糖型被认为是治疗性抗体产品的关键质量属性。控制HM的方法大多是经验性的，在抗体工艺开发过程中要达到理想的HM水平具有挑战性。本研究展示了一种新颖且稳健的方法，可增加多种抗体和细胞系的抗体HM糖型，且不会对细胞培养性能产生不利影响，包括活细胞密度、活力和蛋白滴度。该方法利用甘露糖作为碳源，补料培养基中甘露糖与总己糖（葡萄糖和甘露糖）的比例决定了细胞培养中表达的抗体HM聚糖含量的程度。还展示了该策略从3 mL小规模平板放大到生物反应器（1.5 L）的过程，结果相当。进一步的全聚糖图谱分析表明，HM增加主要与G0F聚糖的减少相关，对其他糖型的影响最小。还讨论了使用甘露糖作为碳源进行HM聚糖调节的可能假设。包括GDP-甘露糖生物合成、早期蛋白质糖基化和UDP-N-乙酰葡糖胺生物合成在内的三条途径可能参与并促成了这种HM调节。
