Integrating upstream host cell line selection and development with improved downstream bioprocessing

目前正在开发的许多新药都是基于蛋白质而不是传统的小分子(例如抗生素)。其中一种特别具有挑战性的蛋白质分子是抗体，例如赫赛汀。这些蛋白质药物是通过在特定条件下培养的哺乳动物细胞生产出来的，用于治疗癌症等疾病。这样做的一个问题是，我们用来制造这种蛋白质的细胞不仅将目标蛋白质分泌到细胞生长的介质中，还会分泌来自细胞的其他蛋白质，称为宿主细胞蛋白(Hcps)。此外，发酵或下游处理(如离心法)过程中的细胞破碎会导致细胞内蛋白质物质的释放。让事情更复杂的是，这些HCP是什么(污染物)，以及它们在整个细胞发酵过程中如何变化以及与目标产品的关系尚不清楚。这意味着目标药物必须从介质中的其余材料中提纯，然后才被认为是安全使用的，这被称为下游生物加工。下游生物加工现在是制造这类药物的总成本的主要部分(40%)，因此这一领域的改进将对制造商和最终患者都有重大好处。我们的目标是开始解决在培养中国仓鼠卵巢细胞的过程中对培养液中HCP缺乏了解的问题，这些细胞被设计成表达重组单抗。具体地说，我们将在整个培养过程和标准的单抗模板纯化过程中确定hCP的分布。我们打算利用这些信息通过细胞工程方法去除最麻烦的HCP，并确定这对产品产量、细胞生长和后续纯化过程有什么影响。最终，我们预计这些信息将使我们能够重新设计下游纯化程序，以删除或整合更好的当前色谱分析步骤，这些步骤既昂贵又耗时。这一信息具有很高的工业相关性，因为由于目前需要进行多个层析步骤来将目标蛋白纯化到可接受的水平，因此可能会阻碍具有商业价值的蛋白质(例如抗体)的生产，并且成本会急剧上升。更好地了解HCP的概况以及这如何影响下游加工是非常重要的，因为预计随着越来越多的蛋白质“药物”的开发，我们将无法以大多数人能够负担得起的成本生产足够大的数量来满足所需的需求。因此，这些产品对许多但非常有效的药物来说仍然昂贵得令人望而却步。

Many of the new drugs currently under development are based upon proteins rather than traditional small molecules (e.g. antibiotics). One of the type of protein molecules that is particularly challenging to make are antibodies e.g. herceptin. These protein drugs are produced for the treatment of diseases such as cancer by mammalian cells kept in culture under defined conditions. One problem with this is that the cells we use to make such proteins secrete not only the target protein into the medium in which the cells grow, but other proteins from the cell as well, called host cell proteins (HCPs). Further, cell breakage during fermentation or downstream handling (e.g. centrifugation) can result in the release of intracellular protein material. To complicate things further, what these HCPs are (the contaminants) and how they change throughout cell fermentation and with target products is not known. What this means is that the target drug must be purified from the rest of the material in the medium before it is deemed safe for use and this is referred to as downstream bioprocessing. Downstream bioprocessing is now a major part (>40%) of the total cost of manufacturing such drugs and as such improvements in this area would be of major benefit to both manufacturers and the end patient. We aim to begin addressing this lack of knowledge with respect to the HCPs in the medium during the culturing of Chinese hamster ovary cells engineered to express a recombinant monoclonal antibody. Specifically, we will determine the HCP profile throughout culture and throughout a standard template purification procedure for a monoclonal antibody. We intend to use this information to then remove the most troublesome HCPs by cell engineering approaches and determine what effect this has on product yield, cell growth and subsequent purification procedures. Ultimately we envisage that this information will allow us to redesign downstream purification procedures either to remove or to integrate better current chromatographic steps which are expensive and time consuming. This information is of high industrial relevance since the production of commercially valuable proteins (e.g. antibodies) can be hindered, and the cost dramatically escalated, as a result of the multiple chromatographic steps currently required to purify the target protein to acceptable levels. A better understanding of the HCP profile and how this influences downstream processing is very important as it is expected that with an increasing number of protein 'drugs' being developed we will lack the capability of producing large enough amounts to meet the required demand at a cost which can be affordable for the majority. Hence these products remain prohibitively expensive to many, but very effective, medicines.