Objective: To explore and optimize enzyme digestion pretreatment and high-performance liquid chromatography for charge isomer analysis of monoclonal antibody. Methods: An ion-exchange chromatography method based on Thermo ProPacTM WCX-10 column was adopted and optimized. The phosphate buffer solution (pH 6.3) containing 10 mmol·L-1 phosphate and 1 mol·L-1 sodium chloride was used as mobile phase for gradient elution. The flow rate was 0.5 mL·min-1,and the detection wavelength for protein adsorbance was 280 nm. Charge isomers were detected before and after IdeS cutting, and the specificity, linearity, repeatability and precision of the method were investigated. Results: The method was applied to several commercial antibody products as examples of demonstrating the method application based on cation exchange chromatography (CEX) and enzyme digestion. Results of comparative analyses of the intact IgG versus F(ab’)2 and Fc sub-domains generated by IdeS digestion provide more specific information about the location of the charge differences. There was no interference peak in the blank solvent;the linear relationship between the sample volume and the absorption peak was good within the range of 10-80 μg, with R2 being 1.0;the RSD of the peak area for six injections was 0.52%;The daytime precision was 3.3%;the assay has good durability;the sample was stable within 24 h, and the RSD of the peak was 3.5%;the RSD of the peak area for the three batches of samples was 2.5%. Conclusion: The method could be applied to the whole process of antibody drug development, including molecular screening in the early stage, industrial process development in the middle stage, and product quality standards and stability studies.
SU Yang, WANG Yang, HU Ping
. Charge heterogeneity analysis of therapeutic antibody based on IgG-degrading enzyme of streptococcus pyogenes and cation exchange chromatography[J]. Chinese Journal of Pharmaceutical Analysis, 2021
, 41(12)
: 2078
-2086
.
DOI: 10.16155/j.0254-1793.2021.12.05
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