Analysis of anti-host cell protein antibody coverage rate by the immunomagnetic beads separation technique

DOU Min-hua; CHEN Qiu-yan; ZHANG Hong-sheng; SHAO Yu-hao; MA Quan-gang; YANG Zhi-xing

doi:10.3969/j.issn.1003-3734.2024.21.015

Abstract：Objective:To establish an immunomagnetic beads separation(IMBS)method for the application in the analysis of host cell protein(HCP)antibody coverage.Methods:Firstly,the IMBS method was developed and optimized,including key steps such as HCP antibody magnetic bead coupling,washing,and elution conditions.Secondly,for the two-dimensional electrophoresis experiments,isoelectric focusing and silver staining quality control materials were designed,and a sample pre-processing method was also established for IMBS-treated samples.The developed methods are applicable to LC-MS/MS to calculate the HCP antibody coverage.Results:The optimized IMBS method was developed and the highest capture efficiency of HCP antibody was achieved by coupling 500 pL magnetic beads with 0.5 mg antibody.Eight washing steps were employed to eliminate nonspecific binding of HCP,and three elution steps were performed to thoroughly harvest bound HCP.Two isoelectric focusing quality control samples based on fluorescently labeled peptides and a silver staining sensitivity control sample were developed to effectively monitor the two-dimensional electrophoresis experiment process.The MS is of high resolution and measurement accuracy.When combining MS with IMBS,more HCPs can be identified than the combination of IMBS with two-dimensional electrophoresis.Moreover,the coverage demonstrated by the IMBS combined with mass spectrometry method is higher than that of IMBS combined with two-dimensional electrophoresis.Additionally,the IMBS combined with MS can assess antibody performance by recognizing high-risk HCPs.Conclusion:IMBS-2DE method and IMBS-LC/MS/MS method,which are stable and reliable,analyzing HCP antibody coverage were established,respectively.As a result,the antibody coverage levels obtained from the two technological platforms showed significant differences due to variations in analytical sensitivity.