Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth
Effectiveness of host cell protein removal using depth filtration with a filter containing diatomaceous earth
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DOI：
10.1002/btpr.3028
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发表时间：
2020-07-06
影响因子：
2.9
通讯作者：
Zydney, Andrew L.
中科院分区：
文献类型：
--
作者：
Nejatishahidein, Negin;Borujeni, Ehsan E.;Zydney, Andrew L.
生物制造中细胞密度和产品效价的增加导致更多地使用深度过滤作为细胞培养液初始澄清的一部分，无论是作为独立的单元操作还是在离心之后。最近的几项研究表明，深度过滤器还可以降低宿主细胞蛋白(HCP)和DNA等较小杂质的浓度，从而减轻后续层析操作的负担。本研究的目的是评估Pall PDH4深度滤料、含有硅藻土、纤维素纤维和粘结剂的模型深度滤料对HCP的去除性能。实验用细胞培养液(CCF)和一系列模型蛋白进行，这些蛋白具有确定的等电点、相对分子质量和疏水性，以匹配典型的HCP范围。利用共聚焦扫描激光显微镜确定被吸附(荧光标记的)蛋白质在深度滤光片中的位置。带正电且疏水性较强的蛋白质结合更强，这与带负电的硅藻土的吸附一致。结合程度最低的是等电点附近的蛋白质，这种过滤器去除的效果很差。这些结果对影响含硅藻土深度过滤器的过滤能力和性能特性的因素提供了新的机理见解，这些过滤器被广泛用于CCF的澄清。
The increased cell density and product titer in biomanufacturing have led to greater use of depth filtration as part of the initial clarification of cell culture fluid, either as a stand-alone unit operation or after centrifugation. Several recent studies have shown that depth filters can also reduce the concentration of smaller impurities like host cell proteins (HCP) and DNA, decreasing the burden on subsequent chromatographic operations. The objective of this study was to evaluate the HCP removal properties of the Pall PDH4 depth filter media, a model depth filter containing diatomaceous earth, cellulose fibers, and a binder. Experiments were performed with both cell culture fluid (CCF) and a series of model proteins with defined pI, molecular weight, and hydrophobicity chosen to match the range of typical HCP. The location of adsorbed (fluorescently labeled) proteins within the depth filters was determined using confocal scanning laser microscopy. Protein binding was greater for proteins that were positively charged and more hydrophobic, consistent with adsorption to the negatively charged diatomaceous earth. The lowest degree of binding was seen with proteins near their pI, which were poorly removed by this filter. These results provide new mechanistic insights into the factors governing the filter capacity and performance characteristics of depth filters containing diatomaceous earth that are widely used in the clarification of CCF.