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，这些药物用于治疗未得到满足的医疗需求。这项提议旨在控制生产生物过程，以使这些具有关键质量属性的糖蛋白能够获得高产量，从而确保高生物活性。这项研究将在一个实验室进行，该实验室拥有哺乳动物细胞培养设施以及用于分析重组产品及其糖基化特征的生物分析工具。 该提案将通过一系列项目进行，每个项目都与在整个项目过程中培训最多6名研究生有关。(A)建立分泌抗体的中国仓鼠卵巢(CHO)细胞的恒化培养，以评估培养参数对单抗产量和糖基化水平的影响。这些参数将包括pH值、溶解氧和营养浓度，特别是葡萄糖、半乳糖和谷氨酰胺。随着这些参数的递增阶跃变化，细胞的稳态代谢将被建立。将测量营养物质的摄取率和关键前体的稳态细胞内浓度，如核苷酸糖，以获得数据，这些数据可用于开发产品产量和糖基化情况的预测模型。(B)在生物反应器中产生的单抗的纯化过程中，将使用我们先前开发的固态技术进行酶重新建模。将试图通过研究使用刚刚上市的选定酶进行酶转化的动力学来获得高产率的单糖实体。(C)将描述可能导致免疫原性的葡聚糖形成的培养条件。这将包括羟基神经氨酸和Gala1-3半乳糖表位。除了CHO细胞，该项目还将使用小鼠细胞(NS0)，因为它们有形成这种表位的倾向。(D)将为困难细胞系的生长和生产力开发化学定义的、不含动物成分和无血清的培养液，例如用于病毒疫苗生产的锚定依赖细胞。 这些项目将利用与工业相关的细胞系进行，这些细胞系已在初步研究中使用。其中包括分泌大量重组产物的CHO细胞系，如人源化单抗、嵌合人骆驼单抗和组织特异性纤溶酶原激活剂(t-PA)。这些产物中的每一个都有一个独特的结构，可以影响对其功能至关重要的糖基化特征。在可能的情况下，在研究中获得的结构轮廓将与功能分析相关。这些研究对实施工业生物过程中使用的平台技术的战略发展很重要。

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 gala1-3gal 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.