Fluorescence dye-based detection of mAb aggregates in CHO culture supernatants
Fluorescence dye-based detection of mAb aggregates in CHO culture supernatants
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DOI：
10.1007/s00216-015-8672-8
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发表时间：
2015-06-01
影响因子：
4.3
通讯作者：
Hesse, Friedemann
中科院分区：
文献类型：
--
作者：
Paul, Albert Jesuran;Schwab, Karen;Hesse, Friedemann
在上游加工过程中形成较高分子量的聚集体会降低单克隆抗体（mAb）的产品产量、有效性和安全性。mAb聚集体形成的过程中表征是一个挑战，因为缺乏快速检测方法来鉴定细胞培养物中的mAb聚集体。在这项工作中，我们提出了一种快速的方法来表征单克隆抗体聚集含有中国仓鼠卵巢（CHO）细胞培养上清液。荧光染料硫磺素T（ThT）和4-4-双-1-苯基氨基-8-萘磺酸盐（Bis-ANS）使得能够检测可溶性以及大的mAb聚集体。偏最小二乘（PLS）回归模型用于评价缓冲液中基于染料的mAb聚集体检测的线性，直至mAb聚集体浓度为2.4 μ g mL（-1）。此外，使用Bis-ANS和ThT在生物过程培养基中检测到mAb聚集体。染料与聚集体的结合在60分钟内是稳定的，这使得该方法稳健可靠。最后，使用10 μ mol L-1 Bis-ANS开发的方法能够区分含有不同水平mAb聚集体的CHO细胞培养上清液。该方法可适用于高通量筛选，例如，筛选影响mAb产品质量的细胞培养条件，从而有助于改进哺乳动物细胞培养中的生物药物生产工艺。
Product yields, efficacy, and safety of monoclonal antibodies (mAbs) are reduced by the formation of higher molecular weight aggregates during upstream processing. In-process characterization of mAb aggregate formation is a challenge since there is a lack of a fast detection method to identify mAb aggregates in cell culture. In this work, we present a rapid method to characterize mAb aggregate-containing Chinese hamster ovary (CHO) cell culture supernatants. The fluorescence dyes thioflavin T (ThT) and 4-4-bis-1-phenylamino-8-naphthalene sulfonate (Bis-ANS) enabled the detection of soluble as well as large mAb aggregates. Partial least square (PLS) regression models were used to evaluate the linearity of the dye-based mAb aggregate detection in buffer down to a mAb aggregate concentration of 2.4 mu g mL(-1). Furthermore, mAb aggregates were detected in bioprocess medium using Bis-ANS and ThT. Dye binding to aggregates was stable for 60 min, making the method robust and reliable. Finally, the developed method using 10 mu mol L-1 Bis-ANS enabled discrimination between CHO cell culture supernatants containing different levels of mAb aggregates. The method can be adapted for high-throughput screening, e.g., to screen for cell culture conditions influencing mAb product quality, and hence can contribute to the improvement of production processes of biopharmaceuticals in mammalian cell culture.