揭示生物制剂开发的挑战:对单克隆抗体制剂中发现的 CHO 细胞衍生水解酶进行系统表征。

IF 5.6 2区 医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL
mAbs Pub Date : 2024-01-01 Epub Date: 2024-07-10 DOI:10.1080/19420862.2024.2375798
Melanie Maier, Linus Weiß, Nikolas Zeh, Valerie Schmieder-Todtenhaupt, Alireza Dehghani, Marius Nicolaus Felix, Daniel Heinzelmann, Benjamin Lindner, Moritz Schmidt, Joey Studts, Patrick Schulz, Bernd Reisinger, Kerstin Otte, Matthias Franzreb, Daniel Lakatos, Simon Fischer
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引用次数: 0

摘要

单克隆抗体(mAb)和其他生物药物会受到酶促聚山梨醇酯(PS)降解的影响,从而降低产品的稳定性,危及创新药物的供应。聚山梨醇酯是一种重要的表面活性剂,可稳定由重组中国仓鼠卵巢(CHO)细胞系生产的活性药物成分。多年来,可能降解 PS 的 CHO 宿主细胞蛋白(HCP)越来越多,但与工业相关的 HCP 的实际数据仍然很少。通过高灵敏度的液相色谱-串联质谱方法,我们研究了七种不同的 mAb 产品,最终确定了 12 种潜在的 PS 降解水解酶,其中包括强 PS 降解脂蛋白脂肪酶(LPL)。利用 LPL 基因敲除的 CHO 宿主细胞系,我们通过正交亲和层析方法稳定过表达和纯化了其余候选水解酶,并对它们进行了详细的功能表征。通过 PS 降解试验,我们发现了九种主要为分泌型、具有不同水解活性的 PS 活性水解酶。所有活性水解酶都显示出丝氨酸-组氨酸-天冬氨酸/谷氨酸催化三元组。此外,我们还对这些活性水解酶进行了 pH 筛选,结果发现它们具有多种最佳活性,这有助于在生物工艺开发过程中识别残余水解酶。最后,我们根据 PAF-AH、LIPA、PPT1 和 LPLA2 的细胞表达、纯化抗体中的检测结果、活性分泌和 PS 降解活性,将数据集汇编成一个风险矩阵,确定它们是高度关键的水解酶。通过这项工作,我们为全面描述 PS 降解水解酶的功能特性铺平了道路,并为今后减少生物制药产品中的 PS 降解提供了依据。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Illuminating a biologics development challenge: systematic characterization of CHO cell-derived hydrolases identified in monoclonal antibody formulations.

Monoclonal antibodies (mAb) and other biological drugs are affected by enzymatic polysorbate (PS) degradation that reduces product stability and jeopardizes the supply of innovative medicines. PS represents a critical surfactant stabilizing the active pharmaceutical ingredients, which are produced by recombinant Chinese hamster ovary (CHO) cell lines. While the list of potential PS-degrading CHO host cell proteins (HCPs) has grown over the years, tangible data on industrially relevant HCPs are still scarce. By means of a highly sensitive liquid chromatography-tandem mass spectrometry method, we investigated seven different mAb products, resulting in the identification of 12 potentially PS-degrading hydrolases, including the strongly PS-degrading lipoprotein lipase (LPL). Using an LPL knockout CHO host cell line, we were able to stably overexpress and purify the remaining candidate hydrolases through orthogonal affinity chromatography methods, enabling their detailed functional characterization. Applying a PS degradation assay, we found nine mostly secreted, PS-active hydrolases with varying hydrolytic activity. All active hydrolases showed a serine-histidine-aspartate/glutamate catalytical triad. Further, we subjected the active hydrolases to pH-screenings and revealed a diverse range of activity optima, which can facilitate the identification of residual hydrolases during bioprocess development. Ultimately, we compiled our dataset in a risk matrix identifying PAF-AH, LIPA, PPT1, and LPLA2 as highly critical hydrolases based on their cellular expression, detection in purified antibodies, active secretion, and PS degradation activity. With this work, we pave the way toward a comprehensive functional characterization of PS-degrading hydrolases and provide a basis for a future reduction of PS degradation in biopharmaceutical drug products.

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来源期刊
mAbs
mAbs 工程技术-仪器仪表
CiteScore
10.70
自引率
11.30%
发文量
77
审稿时长
6-12 weeks
期刊介绍: mAbs is a multi-disciplinary journal dedicated to the art and science of antibody research and development. The journal has a strong scientific and medical focus, but also strives to serve a broader readership. The articles are thus of interest to scientists, clinical researchers, and physicians, as well as the wider mAb community, including our readers involved in technology transfer, legal issues, investment, strategic planning and the regulation of therapeutics.
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