Analysis of fouling and breakthrough of process related impurities during depth filtration using confocal microscopy.
Analysis of fouling and breakthrough of process related impurities during depth filtration using confocal microscopy.
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DOI：
10.1002/btpr.3233
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发表时间：
2022-03
影响因子：
2.9
通讯作者：
Bracewell, Daniel G.
中科院分区：
文献类型：
--
作者：
Parau, Maria;Johnson, Thomas F.;Pullen, James;Bracewell, Daniel G.
效价的提高推动了单抗生产过程的强化。然而，这伴随着高细胞密度和相关过程相关杂质的缺点，如细胞碎片、宿主细胞蛋白(HCP)和DNA。这会影响深度过滤器的能力，并可能导致杂质携带到蛋白A层析中，从而导致早期树脂结垢。新的深度过滤材料提供了在工艺的早期阶段去除更多工艺相关杂质的机会。因此，有必要了解这些过滤器中杂质去除的机理。在这项工作中，通过考察DNA和HCP的突破，将二级深度过滤器Milistak+X0HC(纤维素和硅藻土)与X0SP(合成)进行了比较。此外，还开发了一种新的方法，在共焦显微镜下对深度滤光片结构中的关键杂质位置进行成像。在75、100和250 lMH下测试的通量被发现影响基于30 psi的最大压力的最大吞吐量，但在Hcp和DNA突破中没有看到显著的变化。然而，细胞培养活力从87%下降到37%，导致X0HC的DNA突破率从81下降到55 L/m2，X0SP的DNA突破率从105下降到47 L/m2。HCP突破不受细胞培养活力和过滤器类型的影响。根据可行性的不同，X0SP过滤器的最大吞吐能力高出30%-50%，这可以通过共聚焦成像来解释，其中碎片和DNA在过滤器吊舱的各层中分布不同，X0SP中利用了更多的第二个更紧密的层。
Titer improvement has driven process intensification in mAb manufacture. However, this has come with the drawback of high cell densities and associated process related impurities such as cell debris, host cell protein (HCP), and DNA. This affects the capacity of depth filters and can lead to carryover of impurities to protein A chromatography leading to early resin fouling. New depth filter materials provide the opportunity to remove more process related impurities at this early stage in the process. Hence, there is a need to understand the mechanism of impurity removal within these filters. In this work, the secondary depth filter Millistak+ X0HC (cellulose and diatomaceous earth) is compared with the X0SP (synthetic), by examining the breakthrough of DNA and HCP. Additionally, a novel method was developed to image the location of key impurities within the depth filter structure under a confocal microscope. Flux, tested at 75, 100, and 250 LMH was found to affect the maximal throughput based on the max pressure of 30 psi, but no significant changes were seen in the HCP and DNA breakthrough. However, a drop in cell culture viability, from 87% to 37%, lead to the DNA breakthrough at 10% decreasing from 81 to 55 L/m2 for X0HC and from 105 to 47 L/m2 for X0SP. The HCP breakthrough was not affected by cell culture viability or filter type. The X0SP filter has a 30%–50% higher max throughput depending on viability, which can be explained by the confocal imaging where the debris and DNA are distributed differently in the layers of the filter pods, with more of the second tighter layer being utilized in the X0SP.
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DOI：
10.7171/jbt.15-2602-003
发表时间：
2015-07-01
期刊：
Journal of biomolecular techniques : JBT
影响因子：
--
作者：
Jonkman, James;Brown, Claire M
通讯作者：
Brown, Claire M
影响因子：
4.4
作者：
GERBA, CP;HOU, K
通讯作者：
HOU, K
影响因子：
4.5
作者：
Johnson, T. F.;Iacoviello, F.;Bracewell, D. G.
通讯作者：
Bracewell, D. G.
影响因子：
2.9
作者：
Lutz, Herb;Chefer, Kate;Zhao, Xiaoyang
通讯作者：
Zhao, Xiaoyang
影响因子：
2.9
作者：
Nejatishahidein, Negin;Borujeni, Ehsan E.;Zydney, Andrew L.
通讯作者：
Zydney, Andrew L.