St Amand Melissa M, Radhakrishnan Devesh, Robinson Anne S, Ogunnaike Babatunde A
Department of Chemical and Biomolecular Engineering, University of Delaware, 150 Academy Street, Newark, Delaware, 19716.
Biotechnol Bioeng. 2014 Oct;111(10):1957-70. doi: 10.1002/bit.25251. Epub 2014 May 22.
N-linked glycan distribution affects important end-use characteristics such as the bioactivity and efficacy of many therapeutic proteins, (including monoclonal antibodies), in vivo. Yet, obtaining desired glycan distributions consistently during batch-to-batch production can be challenging for biopharmaceutical manufacturers. While an appropriately implemented on-line glycosylation control strategy during production can help to ensure a consistent glycan distribution, to date no such strategies have been reported. Our goal is to develop and validate a comprehensive strategy for effective on-line control of glycosylation, the successful achievement of which requires first identifying appropriate manipulated variables that can be used to direct the glycan distribution to a desired state. While various culture conditions such as bioreactor process variables, media type, and media supplements have been shown to affect the glycan distribution, in this study we focus on the latter. Specifically, we implemented a statistically designed series of experiments to determine the significant main effects (as well as interaction effects) of media supplementation with manganese, galactose, ammonia and found that each had significant effects on certain glycans. We also include data indicating the glycosylation enzyme gene transcript levels as well as the intracellular nucleotide sugar concentrations in the presence of the media supplements to provide insight into the intracellular conditions that may be contributing to the changes in glycan distribution. The acquired experimental data sets were then used to identify which glycans can be controlled by the media supplements and to what degree. We determined that MnCl2 can be used as a manipulated variable to increase the relative abundance of M51 and decrease FA2 simultaneously, and galactose can be used as a manipulated variable to increase the relative abundance of FA2G1 and decrease FA2 and A2 simultaneously.
N-连接聚糖分布会影响许多治疗性蛋白质（包括单克隆抗体）在体内的重要最终使用特性，如生物活性和功效。然而，对于生物制药制造商来说，在批次间生产过程中始终获得所需的聚糖分布可能具有挑战性。虽然在生产过程中适当实施在线糖基化控制策略有助于确保一致的聚糖分布，但迄今为止尚未有此类策略的报道。我们的目标是开发并验证一种有效的在线糖基化控制综合策略，要成功实现这一目标，首先需要确定可用于将聚糖分布引导至所需状态的合适操作变量。虽然各种培养条件，如生物反应器工艺变量、培养基类型和培养基补充剂已被证明会影响聚糖分布，但在本研究中我们关注的是后者。具体而言，我们实施了一系列经过统计设计的实验，以确定添加锰、半乳糖、氨对培养基的显著主效应（以及交互效应），并发现每种物质对某些聚糖都有显著影响。我们还纳入了数据，表明在添加培养基补充剂的情况下糖基化酶基因转录水平以及细胞内核苷糖浓度，以深入了解可能导致聚糖分布变化的细胞内条件。然后，利用获得的实验数据集来确定哪些聚糖可以由培养基补充剂控制以及控制程度如何。我们确定MnCl₂ 可作为操作变量，以同时提高M51的相对丰度并降低FA2，半乳糖可作为操作变量，以同时提高FA2G1的相对丰度并降低FA2和A2。
