Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly
Host Cell Proteins in Biologics Manufacturing: The Good, the Bad, and the Ugly
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DOI：
10.3390/antib6030013
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发表时间：
2017-09-01
期刊：
影响因子：
4.7
通讯作者：
Strube, Jochen
中科院分区：
文献类型：
--
作者：
Kornecki, Martin;Mestmaecker, Fabian;Strube, Jochen
通过提高USP（上游处理）滴度中的总体滴度，在不提高USP成本的情况下，生物制药生产取得了重大进展。此外，平台工艺的开发提高了工艺耐用性。尽管取得了这些成就，甚至由于取得了这些成就，新的挑战就在眼前。更高的上游滴度在质量和组成方面产生更复杂的杂质谱，这要求下游处理（DSP）具有更高的分离能力和选择性。这导致了成本从USP到DSP的重大转变。为了解决这个问题，USP和DSP集成方法可以被开发并用于整体工艺优化。本研究的重点是DSP的每个单元操作中宿主细胞蛋白（HCP）的表征和分类（即，含水两相萃取、集成逆流色谱法）。这些结果为USP创建了一个数据驱动的反馈，该反馈将用于培养基和工艺优化，以减少甚至消除新生的关键HCP。这将提高分离效率，并可能导致定量过程的理解。根据DSP分离参数的严格标准，使用2D-PAGE分析根据其在凝胶上的位置，将不同的HCP物质根据pI和MW分为“好的、坏的和丑的”。使用LC-MS/MS分析鉴定这些斑点。HCP特别难以去除并且在整个DSP中持续存在（即，“坏”或“丑”），必须通过其被分离的能力来评估。在这种方法中，HCP（被认为是“丑陋的”）代表分子量大于15 kDa且pI在7.30和9.30之间的蛋白质。“不良”HCP同样可以使用MW（>15 kDa）和pI（4.75-7.30和9.30-10.00）进行分类。分子量小于15 kDa且pI低于4.75且高于10.00的HCP被归类为“良好”，因为其理化性质与产品显著不同。为了评价这种分类方案，使用正交分析方法如IEX、HIC和SEC是至关重要的。
Significant progress in the manufacturing of biopharmaceuticals has been made by increasing the overall titers in the USP (upstream processing) titers without raising the cost of the USP. In addition, the development of platform processes led to a higher process robustness. Despite or even due to those achievements, novel challenges are in sight. The higher upstream titers created more complex impurity profiles, both in mass and composition, demanding higher separation capacities and selectivity in downstream processing (DSP). This creates a major shift of costs from USP to DSP. In order to solve this issue, USP and DSP integration approaches can be developed and used for overall process optimization. This study focuses on the characterization and classification of host cell proteins (HCPs) in each unit operation of the DSP (i.e., aqueous two-phase extraction, integrated countercurrent chromatography). The results create a data-driven feedback to the USP, which will serve for media and process optimizations in order to reduce, or even eliminate nascent critical HCPs. This will improve separation efficiency and may lead to a quantitative process understanding. Different HCP species were classified by stringent criteria with regard to DSP separation parameters into "The Good, the Bad, and the Ugly" in terms of pI and MW using 2D-PAGE analysis depending on their positions on the gels. Those spots were identified using LC-MS/MS analysis. HCPs, which are especially difficult to remove and persistent throughout the DSP (i.e., "Bad" or "Ugly"), have to be evaluated by their ability to be separated. In this approach, HCPs, considered "Ugly," represent proteins with a MW larger than 15 kDa and a pI between 7.30 and 9.30. "Bad" HCPs can likewise be classified using MW (>15 kDa) and pI (4.75-7.30 and 9.30-10.00). HCPs with a MW smaller than 15 kDa and a pI lower than 4.75 and higher than 10.00 are classified as "Good" since their physicochemical properties differ significantly from the product. In order to evaluate this classification scheme, it is of utmost importance to use orthogonal analytical methods such as IEX, HIC, and SEC.