Defining and preventing the mechanisms responsible for disulphide bond reduction of monoclonal antibodies during bioprocessing

哺乳动物细胞（特别是中国仓鼠卵巢）由于其产生正确折叠和组装的异源蛋白的能力以及其翻译后修饰的能力而成为用于产生基于IgG的治疗性抗体的主要系统。此类IgG抗体的功能的先决条件是它们正确组装成限定的四级结构，所述四级结构由形成二硫键键合的HC 2LC 2分子的两个重链（HC）和两个轻链（LC）多肽组成。折叠、组装和翻译后修饰发生在ER内，其中分子在被运输到高尔基体之前经受严格的质量控制。IgG多肽依次与ER复合物内存在的一系列分子伴侣、折叠酶和氧化还原酶相互作用。LC多肽可以作为单体和二硫键二聚体分泌。相反，HC多肽分泌需要LC的存在以进行分泌，LC的缺乏导致HC多肽在被运输到蛋白酶体进行降解之前在ER内保持未折叠状态。据报道，生物化学和生理刺激对ER施加压力，并导致细胞内未折叠或错误折叠的蛋白质的积累。此外，与先前报告的结果相反，Lonza生成的数据显示，收获时暴露于低氧条件的培养液中存在HC单体和二聚体。游离LC和HC单体和二聚体的存在是不希望的，并且需要定义这些单体和二聚体的衍生机制和避免这些单体和二聚体存在的方法。还需要了解生物反应器中理化参数的变化与允许从ER分泌错误折叠/未折叠蛋白的分子机制之间的关系，以及细胞培养上清液对完全分泌的抗体的结构和稳定性的影响。拟议工作方案。学生将使用分子和细胞生物学技术的组合来开发方法，以防止在IgG 1和IgG 4亚型的二硫键还原。初步研究将使用产生二硫键还原产物的细胞系（和产物）。这些细胞系将用于建立实验室规模的试验系统，该系统可提供分析材料。然后学生将识别（a）在不同生理化学条件下导致错误折叠/未折叠蛋白质分泌的细胞内因子（即通过克服ERAD途径使错误折叠/未折叠蛋白分泌的因子）和（B）细胞培养过程中产生对二硫键还原/改组更敏感的细胞培养上清液的关键因子（即定义分泌后上清液中发生的那些减少/改组过程）。该信息将用于鉴定细胞培养上清液中的特异性标志物，该标志物可确定抗体在开始收获和下游处理前解离的倾向。博士学位的成果将是：1.建立在还原条件下导致错误折叠蛋白（游离HC/LC）分泌的关键细胞内因子的分子模型（IgG 1和IgG 4亚型之间是否存在差异，宿主细胞系之间有何差异？）。2.鉴定细胞培养上清液中催化二硫键还原的还原组分。3.确定细胞培养过程中产生的上清液对二硫键还原/改组更敏感的因素（例如培养基、溶解氧张力、温度、pH值、过滤、离心、总细胞蛋白）。4.鉴定细胞培养上清液中的标志物，确定抗体在收获和下游处理步骤开始前解离的倾向（例如细胞凋亡、还原酶活性）。

Mammalian cells (particularly Chinese hamster ovary) have become the dominant system for the production of IgG based therapeutic antibodies due to their ability to produce properly folded and assembled heterologous proteins as well as their capability for post translational modifications. A prerequisite for the function of such IgG antibodies is their correct assembly into defined quaternary structures, composed of two heavy chain (HC) and two light chain (LC) polypeptides that form disulphide bonded HC2LC2 molecules. Folding, assembly and post-translational modifications occur within the ER where the molecule is subjected to rigorous quality control before being transported to the Golgi. IgG polypeptides sequentially interact with a range of molecular chaperones, foldases and oxioreductases present within the ER complex. The LC polypeptide can be secreted as both monomer and disulphide bonded dimer. Conversely, HC polypeptide secretion requires the presence of LC for secretion, the absence of which results in the HC polypeptide being retained in an unfolded state within the ER before being trafficked to the proteasome for degradation. Biochemical and physiological stimuli have been reported to impose stress on the ER and lead to the accumulation of unfolded or misfolded proteins within the cell. In addition, contrary to previously reported results, data generated at Lonza have shown the presence of HC monomer and dimer in culture broths exposed to low oxygen conditions at harvest. The presence of free LC and HC monomers and dimers is undesirable and defining the mechanism(s) by which these are derived and approaches to avoid the presence of these is required. There is also a need to understand the relationship between changes in physiochemical parameters in the bioreactor and the molecular mechanisms allowing the secretion of misfolded/unfolded proteins from the ER and the influence of cell culture supernatant on the structure and stability of fully secreted antibody. Proposed work programme. The student will use a combination of molecular and cell biology techniques to develop methods to prevent disulphide bond reduction in both IgG1 and IgG4 subtypes. Initial studies will use cell lines (and product) that yield disulphide bond reduction products. These cell lines will be used to establish a laboratory scale test system that can provide material for analysis. The student will then identify (a) intra-cellular factors that cause the secretion of misfolded/unfolded proteins under different physiochemical conditions (i.e. factors that enable the secretion of misfolded/unfolded proteins by overcoming the ERAD pathway) and (b) key factors in cell culture processes that yield cell culture supernatant that is more susceptible to disulphide bond reduction/shuffling (i.e. define those reductions/shuffling processes that occur in the supernatant post-secretion). This information will be used to identify specific markers within cell culture supernatant that can determine the propensity of antibodies to dissociate prior to initiation of harvest and downstream processing. The outcomes of the PhD will be: 1.Establishment of a molecular model of the key intra-cellular factors that cause the secretion of mis-folded proteins (free HC/LC) under reducing conditions (are there differences between IgG1 and IgG4 subtypes, how does this differ between host cell lines?). 2.Identification of reducing components in cell culture supernatant that catalyze the reduction of disulphide bonds. 3.Identification of factors in cell culture processes that yield supernatant more susceptible to disulphide bond reduction/shuffling (e.g. media, dissolved oxygen tension, temperature, pH, filtration, centrifugation, total cellular protein). 4.Identification of markers within cell culture supernatant that determine the propensity of antibodies to dissociate prior to initiation of harvest and downstream processing steps (e.g. apoptosis, activity of reducing enzymes.