The control of the glycosylation of recombinant proteins in mammalian cell culture

哺乳动物细胞用于大规模培养生物过程，以生产用于人类保健的具有商业价值的化合物。许多生物药品是通过这种生物过程生产的重组蛋白。大规模生产的最新技术是饲料分批培养。然而，在高生产率下，重要的是要确保蛋白质的生物活性和临床疗效不受损害。分泌蛋白的质量可以通过包括聚集或异常糖基化模式在内的许多因素而降低。我的实验室最近的工作表明，聚集是疏水蛋白（如β -干扰素）的一个特殊问题。低温灌注系统的设计使这种现象最小化。在目前的提议中，我们打算评估导致所选重组蛋白糖基化模式变化的培养条件，这些重组蛋白包括免疫球蛋白、组织型纤溶酶原激活剂、干扰素- β和干扰素- γ，这些重组蛋白是从中国仓鼠卵巢（CHO）细胞中产生的，主要用于工业。蛋白质的糖基化谱被定义为附着碳水化合物的类型和数量，即使在培养过程中也会发生变化。该轮廓对于蛋白质的生物活性非常重要，特别是当用作治疗药物时。我们将研究一些可能与蛋白质糖基化变异性有关的现象。当介质底物（如碳水化合物源）耗尽时，聚糖位点占用减少。这一现象将被调查与营养水平显示延长活细胞培养通过饲料分批策略。为维持最佳的低水平营养而饲喂的频率可能会影响位点占用和产生的聚糖谱。在模拟大规模间歇饲料培养生产的实验中，营养物质的供应将会变化，以努力了解它们在引起代谢组的任何可变性中的作用，这些可变性可能影响最终的糖蛋白产物。提出的研究目的是了解和控制大规模生物工艺中与生物药品糖基化相关的关键质量属性。

Mammalian cells are used in large-scale culture bioprocesses for the production of commercially valuable compounds used in human health-care. Many biopharmaceuticals are produced as recombinant proteins from such bioprocesses. The most current technology for large-scale production is by fed-batch cultures. However, at a high productivity it is important to ensure that the protein quality for bioactivity and clinical efficacy is not compromised. The quality of a secreted protein may be reduced through many factors that include aggregation or aberrant glycosylation patterns. Recent work in my laboratory has shown that aggregation is a particular problem for hydrophobic protein such as beta-interferon. This phenomenon was minimized by the design of a low-temperature perfusion system. In the present proposal we intend to evaluate the culture conditions that cause variability in glycosylation patterns of selected recombinant proteins that will include immunoglobulin, tissue-type plasminogen activator, interferon-beta and interferon-gamma produced from Chinese hamster ovary (CHO) cells which are used predominantly in industry. The glycosylation profile of the proteins is defined as the type and quantity of attached carbohydrates which can vary even during culture. The profile is important for the bioactivity of the proteins particularly when applied as therapeutics. We will investigate a number of phenomena that may be related to the variability of protein glycosylation. Glycan site occupancy is reduced when media substrates, such as a carbohydrate source, are depleted. This phenomenon will be investigated with nutrient levels shown to prolong viable cell cultures through fed-batch strategies. The frequency of feeding to maintain optimal low level nutrients may affect both site occupancy and the resulting glycan profile. In experiments to simulate large-scale fed-batch culture production the supply of nutrients will be varied in an effort to understand their role in causing any variability of the metabolome that might affect the final glycoprotein product. The objective of the proposed research is to understand and control the critical quality attributes associated with glycosylation of biopharmaceuticals in large-scale bioprocesses.