Roche Diagnostics GmbH, Nonnenwald 2, 82377 Penzberg, Germany.
Pharma Technical Development, Genentech, 1 DNA Way, South San Francisco, California, USA.
J Pharm Sci. 2021 Nov;110(11):3558-3567. doi: 10.1016/j.xphs.2021.06.033. Epub 2021 Jul 2.
Degradation of polysorbate (PS) by hydrolytically active host cell proteins (HCPs) in drug products may impair the protein-stabilizing properties of PS and lead to the formation of particles due to the accumulation of poorly soluble free fatty acids upon long-term storage. The identification of the causative enzymes is challenging due to their low-abundance even when using state-of-the-art instrumentation and workflows. To overcome these challenges, we developed a rigorous enrichment strategy for HCPs, utilizing both Protein A and anti-HCP affinity chromatography, which facilitated the in-depth characterization of the HCP population in a monoclonal antibody formulation prone to PS hydrolysis. Based on the HCPs identified by liquid chromatography coupled to tandem mass spectrometry, a number of enzymes annotated as hydrolases were recombinantly expressed and characterized in terms of polysorbate degradation. Among the selected candidates, Lipoprotein Lipase, Lysosomal Acid Lipase (LIPA) and Palmitoyl-Protein Thioesterase 1 (PPT1) exhibited notable activity towards PS. To our knowledge, this is the first report to identify LIPA and PPT1 as residual HCPs that can contribute to PS degradation in a biological product.
聚山梨酯（PS）在药物产品中被水解活性宿主细胞蛋白（HCP）降解，可能会损害 PS 的蛋白质稳定特性，并由于长期储存时难溶性游离脂肪酸的积累而导致颗粒形成。由于即使使用最先进的仪器和工作流程，它们的丰度也很低，因此鉴定致病酶具有挑战性。为了克服这些挑战，我们开发了一种严格的 HCP 富集策略，同时利用 Protein A 和抗 HCP 亲和层析，这有助于深入表征易发生 PS 水解的单克隆抗体制剂中的 HCP 群体。基于液质联用鉴定的 HCP，许多注释为水解酶的酶被重组表达，并根据聚山梨酯降解进行了表征。在所选择的候选物中，脂蛋白脂肪酶、溶酶体酸性脂肪酶（LIPA）和棕榈酰蛋白硫酯酶 1（PPT1）对 PS 表现出显著的活性。据我们所知，这是首次报道鉴定 LIPA 和 PPT1 为残留 HCP，它们可导致生物制品中 PS 降解。
