Ahluwalia Deepti, Dhillon Harbhajan, Slaney Thomas, Song Hangtian, Boux Heather, Mehta Sheetal, Zhang Lei, Valdez Anulfo, Krishnamurthy Girija
Biologics Manufacturing and Process Development (BMPD), Bristol-Myers Squibb, 311 Pennington-rocky hill road, Pennington, NJ USA.
Biologics Manufacturing and Process Development (BMPD), Bristol-Myers Squibb, 311 Pennington-rocky hill road, Pennington, NJ USA.
J Pharm Biomed Anal. 2017 Jul 15;141:32-38. doi: 10.1016/j.jpba.2017.03.065. Epub 2017 Apr 9.
Residual host cell proteins (HCPs) are process-related impurities present in biotherapeutics that can pose safety health risks to patients. An adequate control of HCP levels in the final product, and demonstration of HCP clearance throughout a product manufacturing process is critical for all biotherapeutic products. Developing effective downstream purification processes can be challenging as HCPs and product proteins may possess an affinity for each other or have similar physicochemical properties, resulting in co-purification. In the current study, we identified the presence of CHO-catalase subunit protein as an impurity present in purified P1 protein. This previously unreported HCP impurity, was detected in P1 protein generated in Chinese hamster ovary (CHO) cells. Purified drug substance samples contained elevated CHO HCP levels when measured using a commercial anti-CHO HCP Enzyme-Linked Immunosorbent Assay (ELISA) kit. This finding, prompted further characterization of the HCP profile using 1D and 2D gels/ western blots using an anti-human IgG antibody as well as a commercial anti-CHO HCP antibody (Cygnus 813) for the detection of host cell proteins. The CHO-catalase protein has been characterized using a combination approach of one-dimensional (1D) and two-dimensional (2D) gels and western blotting techniques, and the identity confirmed using liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Western blot analyses using the anti-CHO HCP antibody detected a potential HCP band at ∼60 kDa and a pI of ∼8 in the purified P1 sample. The 60 kDa HCP band was excised from 1D SDS-PAGE gels and LC-MS/MS analysis identified it to be CHO-catalase subunit. The identity of catalase monomer was further confirmed by western blot analysis using a specific anti-catalase antibody.
残留宿主细胞蛋白（HCPs）是生物治疗药物中存在的与工艺相关的杂质，可能对患者的安全健康构成风险。对最终产品中HCP水平进行充分控制，并证明在整个产品制造过程中HCP的清除情况，对所有生物治疗产品而言都至关重要。开发有效的下游纯化工艺可能具有挑战性，因为HCP和产品蛋白可能相互具有亲和力或具有相似的物理化学性质，从而导致共纯化。在本研究中，我们鉴定出中国仓鼠卵巢（CHO）-过氧化氢酶亚基蛋白作为纯化的P1蛋白中存在的一种杂质。这种先前未报道的HCP杂质，在CHO细胞中产生的P1蛋白中被检测到。当使用商业抗CHO HCP酶联免疫吸附测定（ELISA）试剂盒进行测量时，纯化的原料药样品中CHO HCP水平升高。这一发现促使我们使用一维（1D）和二维（2D）凝胶/蛋白质印迹法，并使用抗人IgG抗体以及商业抗CHO HCP抗体（Cygnus 813）对HCP谱进行进一步表征，以检测宿主细胞蛋白。已使用一维（1D）和二维（2D）凝胶以及蛋白质印迹技术的组合方法对CHO-过氧化氢酶蛋白进行了表征，并使用液相色谱-质谱联用（LC-MS/MS）分析确认了其身份。使用抗CHO HCP抗体进行的蛋白质印迹分析在纯化后的P1样品中检测到一条约60 kDa、pI约为8的潜在HCP条带。从1D SDS-PAGE凝胶中切下60 kDa的HCP条带，LC-MS/MS分析确定其为CHO-过氧化氢酶亚基。使用特异性抗过氧化氢酶抗体进行的蛋白质印迹分析进一步确认了过氧化氢酶单体的身份。
