Control strategies for mammalian cell bioprocesses in the production of glycosylated biopharmaceuticals

哺乳动物细胞技术是生产越来越多的生物药物的基础，这些生物药物用作未满足的医疗需求的治疗剂。 该提案旨在控制生产生物工艺，以实现这些糖蛋白的高产率，并具有确保高生物活性的关键质量属性。 该研究将在一个实验室进行，该实验室具有哺乳动物细胞培养设施以及用于分析重组产物及其糖基化谱的生物分析工具。该提案将通过一系列项目进行，每个项目将涉及到在该计划的过程中最多6名研究生的培训。 (a)将建立分泌抗体的中国仓鼠卵巢（CHO）细胞的恒化培养物，以评估培养参数对单克隆抗体（Mab）产量和糖基化特征的影响。 参数将包括pH值、溶解氧和营养物浓度，特别是葡萄糖、半乳糖和谷氨酰胺。 在这些参数的增量阶跃变化后，将建立细胞的稳态代谢。 将测量关键前体（如核苷酸糖）的营养摄取速率和稳态细胞内浓度，以获得可用于开发产物产率和糖基化谱预测模型的数据。 (b)将使用我们先前开发的固态技术在生物反应器中生产的单克隆抗体纯化期间实施酶重组。 将尝试通过使用刚刚商业上可获得的所选酶研究酶转化的动力学来获得高产率的单一糖型实体。 (c)将表征导致潜在免疫原性聚糖形式的培养条件。 这将包括羟乙酰神经氨酸和galα1-3gal表位。 与CHO细胞一样，该项目将使用小鼠细胞（NS 0），因为它们倾向于形成此类表位。 (d)将开发化学成分明确、无动物成分和无血清的培养基，用于困难细胞系（如病毒疫苗生产中使用的贴壁依赖性细胞）的生长和生产。 * 这些项目将使用现有的工业相关细胞系进行，并已用于初步研究。 这些包括分泌许多重组产物如人源化单克隆抗体、嵌合人-美洲驼单克隆抗体和组织特异性纤溶酶原激活剂（t-PA）的CHO细胞系。这些产物中的每一种都具有独特的结构，可以影响对其功能至关重要的糖基化谱。 在可能的情况下，研究中获得的结构特征将与功能测定相关。 这些研究对于实施用于工业生物过程的平台技术的战略发展非常重要。**

Mammalian cell technology is the basis for the production of an increasing number of biopharmaceuticals used as therapeutics for unmet medical needs. This proposal aims to control the production bioprocesses to enable high yield of these glycoproteins with the critical quality attributes that ensure high bioactivity. The research will be conducted in a laboratory which has facilities for mammalian cell culture as well as bioanalytical tools for the analysis of recombinant products and their glycosylation profiles.***The proposal will be conducted through a series of projects, each of which will be related to the training of up to 6 graduate students over the course of the program. (a) Chemostat cultures of antibody-secreting Chinese hamster ovary (CHO) cells will be established to assess the effect of culture parameters on the monoclonal antibody (Mab) yield and glycosylation profile. The parameters will include pH, dissolved oxygen and nutrient concentrations, particularly glucose, galactose and glutamine. Steady-state metabolism of the cells will be established following incremental step changes of these parameters. Nutrient uptake rates and steady state intracellular concentrations of critical precursors such as nucleotide sugars will be measured to obtain data that may be used to develop predictive models for product yields and glycosylation profiles. (b) Enzymic re-modelling will be implemented during the purification of Mabs produced in the bioreactor using a solid state technique that we have previously developed. It will be attempted to obtain high yields of single glycoform entities by studying the kinetics of enzymic conversions using selected enzymes that have just been made commercially available. (c) Culture conditions that result in potentially immunogenic glycan forms will be characterized. This will include the glycolyl neuraminic acid and galα1-3gal epitopes. As well as CHO cells, this project will use mouse cells (NS0) because of their propensity to form such epitopes. (d) Chemically-defined, animal-component-free and serum-free medium will be developed for the growth and productivity of difficult cell lines such as the anchorage-dependent cells used in viral vaccine production. ***These projects will be conducted with the use of industrially-relevant cell lines that are available and have been used in preliminary studies. These include CHO cell lines that secrete a number of recombinant products such as humanized Mabs, chimeric human-llama Mabs and tissue-specific plasminogen activator (t-PA). Each of these products has a distinctive structure that can affect glycosylation profiles that are critical for their function. Where possible the structural profiles obtained in the studies will be related to functional assays. These studies are important to strategic developments in the implementation of platform technologies to be used in industrial bioprocesses. **