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

目前正在开发的许多新药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。这些蛋白质药物是由在特定条件下培养的哺乳动物细胞生产的，用于治疗癌症等疾病。其中一个问题是，我们用来制造这种蛋白质的细胞不仅会将目标蛋白质分泌到细胞生长的培养基中，还会将细胞中的其他蛋白质分泌到培养基中，这些蛋白质被称为宿主细胞蛋白（HCPs）。此外，在发酵或下游处理（如离心）过程中，细胞破裂可能导致细胞内蛋白质物质的释放。更复杂的是，这些HCPs是什么（污染物），以及它们在细胞发酵过程中如何变化，以及与目标产物的变化，目前还不清楚。这意味着目标药物在被认为可以安全使用之前必须从培养基中的其余物质中纯化出来，这被称为下游生物处理。下游生物处理现在是制造这类药物总成本的主要部分（约40%），因为这一领域的改进将对制造商和最终患者都有重大好处。我们的目标是在培养中国仓鼠卵巢细胞以表达重组单克隆抗体的过程中，开始解决这方面的知识缺乏。具体来说，我们将在整个培养过程和单克隆抗体的标准模板纯化过程中确定HCP谱。我们打算利用这些信息，然后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程序的影响。最终，我们设想这些信息将使我们能够重新设计下游纯化程序，以消除或整合更好的当前昂贵且耗时的色谱步骤。这一信息具有很高的工业相关性，因为有商业价值的蛋白质（如抗体）的生产可能会受到阻碍，并且成本急剧上升，因为目前需要多个色谱步骤将目标蛋白质纯化到可接受的水平。更好地了解HCP概况及其对下游加工的影响是非常重要的，因为预计随着越来越多的蛋白质“药物”正在开发，我们将缺乏以大多数人可以负担得起的成本生产足够多的满足所需需求的能力。因此，这些产品对许多人来说仍然贵得令人望而却步，但却是非常有效的药物。

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.