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用作治疗未满足医疗需求的生物制药的基础。本提案旨在控制生产生物过程，以确保这些糖蛋白的高产量，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，该实验室拥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，每个项目将在该计划的过程中培训最多6名研究生。(a)建立分泌抗体的中国仓鼠卵巢（CHO）细胞的趋化培养，以评估培养参数对单克隆抗体（Mab）产量和糖基化谱的影响。这些参数将包括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。随着这些参数的逐步变化，将建立细胞的稳态代谢。将测量营养摄取率和关键前体（如核苷酸糖）的稳态细胞内浓度，以获得可用于开发产品产量和糖基化谱的预测模型的数据。(b)在纯化生物反应器中生产的单克隆抗体时，将使用我们先前开发的固态技术进行酶重建模。它将试图获得高产量的单糖型实体通过研究动力学的酶转化使用选定的酶，刚刚在商业上可用。(c)产生潜在免疫原性聚糖形式的培养条件将被描述。这将包括乙醇基神经氨酸和半乳糖1-3半乳糖表位。除了CHO细胞外，该项目还将使用小鼠细胞（NS0），因为它们倾向于形成这样的表位。(d)将开发具有化学定义的、不含动物成分和不含血清的培养基，用于培养难培养的细胞系，如用于生产病毒疫苗的依赖于锚定的细胞。***这些项目将使用现有并已用于初步研究的与工业有关的细胞系进行。这些包括分泌许多重组产品的CHO细胞系，如人源化单克隆抗体、嵌合人-羊驼单克隆抗体和组织特异性纤溶酶原激活剂（t-PA）。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，研究中获得的结构剖面将与功能分析相关。这些研究对于在工业生物过程中应用平台技术的战略发展是重要的。**

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 gal1-3gal 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. **