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工艺，以生产用于人类保健的有商业价值的化合物。 许多生物制药是从这样的生物过程中作为重组蛋白生产的。 目前大规模生产的最新技术是分批补料培养。 然而，在高生产率下，重要的是确保蛋白质的生物活性和临床功效不受损害。 分泌蛋白质的质量可通过包括聚集或异常糖基化模式的许多因素而降低。 最近在我的实验室的工作表明，聚集是一个特殊的问题，如β-干扰素的疏水蛋白。 通过低温灌注系统的设计，这种现象被最小化。 在本提案中，我们打算评价导致选定重组蛋白糖基化模式变异性的培养条件，所述重组蛋白将包括免疫球蛋白、组织型纤溶酶原激活剂、干扰素-β和干扰素-γ，其由主要用于工业的中国仓鼠卵巢（CHO）细胞产生。 蛋白质的糖基化谱被定义为即使在培养期间也可以变化的连接的碳水化合物的类型和数量。 该谱对于蛋白质的生物活性是重要的，特别是当用作治疗剂时。 我们将研究一些可能与蛋白质糖基化的变异性有关的现象。 当培养基底物（如碳水化合物源）耗尽时，聚糖位点占用减少。 将研究这种现象，营养水平显示通过分批补料策略延长活细胞培养。 维持最佳低营养水平的饲喂频率可能会影响位点占用率和所得聚糖谱。 在模拟大规模补料分批培养生产的实验中，将改变营养物的供应，以努力了解它们在引起可能影响最终糖蛋白产物的代谢组的任何变化中的作用。 拟议研究的目的是了解和控制与大规模生物工艺中生物制药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.