Zhang Sisi, Zhao Bo, Adaniya Stephanie, Xiao Hui, Li Ning
Regeneron Pharmaceuticals Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591-6706, United States.
Anal Chem. 2023 Apr 11;95(14):6002-6008. doi: 10.1021/acs.analchem.3c00020. Epub 2023 Mar 28.
Certain host cell proteins (HCPs) in biotherapeutic drugs may be detrimental to drug product quality even when they are present at the subppm level. Therefore, an analytical method that can reliably quantify trace amounts of HCPs is desirable. This study demonstrates a novel strategy to quantify HCPs present at subppm levels with ProteoMiner enrichment coupled with limited digestion followed by targeted analysis with nano-liquid chromatography-parallel reaction monitoring. The method can achieve LLOQ values as low as 0.06 ppm, with an accuracy of 85%-111% of the theoretical value, and inter-run and intrarun precision within 12% and 25%, respectively. The approach was applied to the quantification of five high-risk HCPs in drug products. The results indicated that 2.5 ppm lysosomal acid lipase, 0.14 ppm liver carboxylesterase, 1.8 ppm palmitoyl-protein thioesterase 1, and 1 ppm cathepsin D affected the stability of drug products, whereas drug products could safely contain 1.5 ppm lipoprotein lipase, 0.1 ppm lysosomal acid lipase, or 0.3 ppm cathepsin D. In combination with lipase activity analysis, the accurate quantification of lipases/esterases in drug products enables better understanding and comparison of the enzymatic activity of polysorbate degradation from endogenous proteins.
生物治疗药物中的某些宿主细胞蛋白（HCPs）即使以亚ppm水平存在，也可能对药品质量有害。因此，需要一种能够可靠地定量痕量HCPs的分析方法。本研究展示了一种新策略，通过ProteoMiner富集结合有限酶切，然后用纳升液相色谱 - 平行反应监测进行靶向分析，来定量亚ppm水平的HCPs。该方法的LLOQ值可低至0.06 ppm，准确度为理论值的85% - 111%，批间和批内精密度分别在12%和25%以内。该方法应用于药品中五种高风险HCPs的定量。结果表明，2.5 ppm的溶酶体酸性脂肪酶、0.14 ppm的肝脏羧酸酯酶、1.8 ppm的棕榈酰蛋白硫酯酶1和1 ppm的组织蛋白酶D会影响药品稳定性，而药品可安全含有1.5 ppm的脂蛋白脂肪酶、0.1 ppm的溶酶体酸性脂肪酶或0.3 ppm的组织蛋白酶D。结合脂肪酶活性分析，准确测定药品中的脂肪酶/酯酶能够更好地理解和比较内源性蛋白对聚山梨酯降解的酶活性。
