Paul Albert Jesuran, Schwab Karen, Prokoph Nina, Haas Elena, Handrick René, Hesse Friedemann
Institute of Applied Biotechnology (IAB), Biberach University of Applied Sciences, Hubertus-Liebrecht-Strasse 35, 88400, Biberach, Germany,
Anal Bioanal Chem. 2015 Jun;407(16):4849-56. doi: 10.1007/s00216-015-8672-8. Epub 2015 Apr 14.
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 μg 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 μmol 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.
在上游加工过程中，单克隆抗体（mAb）的产物产量、效力和安全性会因更高分子量聚集体的形成而降低。由于缺乏一种快速检测方法来识别细胞培养物中的mAb聚集体，因此对mAb聚集体形成进行过程中的表征是一项挑战。在这项工作中，我们提出了一种快速方法来表征含有mAb聚集体的中国仓鼠卵巢（CHO）细胞培养上清液。荧光染料硫黄素T（ThT）和4-4-双-1-苯基氨基-8-萘磺酸盐（Bis-ANS）能够检测可溶性以及大的mAb聚集体。偏最小二乘（PLS）回归模型用于评估基于染料的mAb聚集体检测在缓冲液中低至2.4μg mL（-1）的mAb聚集体浓度时的线性。此外，使用Bis-ANS和ThT在生物工艺培养基中检测到了mAb聚集体。染料与聚集体的结合在60分钟内稳定，使得该方法稳健且可靠。最后，使用10μmol 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，因此有助于改善哺乳动物细胞培养中生物制药的生产过程。
