Host cell proteins (HCPs) are process-related impurities derived from host organisms, which need to be controlled to ensure adequate product quality and safety. In this study, product quality attributes were tracked for several monoclonal antibodies (mAbs) under the intended storage and accelerated stability conditions. One product quality attribute not expected to be stability indicating is the N-glycan heterogeneity profile. However, significant N-glycan degradation was observed for one mAb under accelerated and stressed stability conditions. The root cause for this instability was attributed to hexosaminidase B (HEXB), an enzyme known to remove terminal N-acetylglucosamine (GlcNAc). HEXB was identified by liquid chromatography–mass spectrometry (LC–MS)-based proteomics approach to be enriched in the impacted stability batches from mAb-1. Subsequently, enzymatic and targeted multiple reaction monitoring (MRM) MS assays were developed to support process and product characterization. A potential interaction between HEXB and mAb-1 was initially observed from the analysis of process intermediates by proteomics among several mAbs and later supported by computational modeling. An improved bioprocess was developed to significantly reduce HEXB levels in the final drug substance. A risk assessment was conducted by evaluating the in silico immunogenicity risk and the impact on product quality. To the best of our knowledge, HEXB is the first residual HCP reported to have impact on the glycan profile of a formulated drug product. The combination of different analytical tools, mass spectrometry, and computational modeling provides a general strategy on how to study residual HCP for biotherapeutics development.

中文翻译：

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治疗性重组单克隆抗体产品稳定性研究中与 N-聚糖降解相关的残留宿主细胞蛋白己糖胺酶 B 的鉴定和表征

宿主细胞蛋白 (HCP) 是源自宿主生物的与工艺相关的杂质，需要对其进行控制以确保足够的产品质量和安全性。在这项研究中，在预期的储存和加速稳定性条件下，跟踪了几种单克隆抗体 (mAb) 的产品质量属性。一个产品质量属性预计不是稳定性指示是N-聚糖异质性概况。然而，在加速和压力稳定性条件下，观察到一种 mAb 的N-聚糖显着降解。这种不稳定性的根本原因归因于己糖胺酶 B (HEXB)，一种已知可去除末端 N-乙酰氨基葡萄糖( GlcNAc )的酶。十六进制通过基于液相色谱-质谱 (LC-MS) 的蛋白质组学方法鉴定，以富集来自 mAb-1 的受影响的稳定性批次。随后，开发了酶促和靶向多反应监测 (MRM) MS 测定以支持工艺和产品表征。HEXB 和 mAb-1 之间的潜在相互作用最初是通过蛋白质组学对几个 mAb 之间的过程中间体的分析观察到的，后来得到了计算建模的支持。开发了一种改进的生物工艺，以显着降低最终原料药中的 HEXB 水平。通过评估计算机免疫原性风险和对产品质量的影响进行风险评估。据我们所知，HEXB 是第一个据报道对制剂药物产品的聚糖谱有影响的残留 HCP。