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

哺乳动物细胞被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。许多生物制药都是以重组蛋白质的形式从这种生物过程中生产出来的。最新的规模化生产技术是补料分批培养。然而，在高生产率的情况下，确保生物活性和临床疗效的蛋白质质量不受影响是很重要的。分泌蛋白质的质量可能会因许多因素而降低，这些因素包括聚集或异常糖基化模式。我的实验室最近的工作表明，聚集是疏水性蛋白质(如β-干扰素)的一个特殊问题。这种现象通过低温灌流系统的设计得以最小化。在本提案中，我们打算评估导致某些重组蛋白糖基化模式变化的培养条件，这些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，这些重组蛋白主要用于工业上。蛋白质的糖基化图谱被定义为附着的碳水化合物的类型和数量，即使在培养过程中也可能发生变化。这一特性对于蛋白质的生物活性很重要，尤其是在作为治疗药物应用时。我们将研究一些可能与蛋白质糖基化的可变性有关的现象。当碳水化合物来源等介质底物耗尽时，葡聚糖部位的占有率会减少。这一现象将通过补料批次策略来研究延长活细胞培养所需的营养水平。维持最佳低营养水平的摄食频率可能会影响场地占有率和由此产生的糖链分布。在模拟大规模补料分批培养生产的实验中，营养素的供应将发生变化，以努力了解它们在引起可能影响最终糖蛋白产品的代谢物的任何变异性方面所起的作用。这项拟议研究的目的是了解和控制大规模生物过程中与生物药物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.