Title:Charge Variants Analysis of a Bispecific Antibody Using a Fully Automated
One-step Capillary Isoelectric Focusing - Mass Spectrometry
Method
Volume: 18
Issue: 9
Author(s): Gang Wu, Chuanfei Yu, Wenbo Wang, Jialiang Du, Gangling Xu, Zhihao Fu and Lan Wang*
Affiliation:
- Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products, National
Institutes for Food and Drug Control, Daxing District, Beijing, P.R. China
Keywords:
Capillary electrophoresis, capillary electrophoresis-mass spectrometry capillary, isoelectric focusing, capillary, isoelectric focusing-mass spectrometry, bispecific antibody, charge variants.
Abstract:
Background: Bispecific antibody (BsAb) therapeutics have emerged as the nextgeneration
immuno-oncology therapy. The architecture of bsabs is inherently more complex than
that of mAb therapeutics. As a result, prior knowledge of critical quality attributes (CQAs) assessment
of mAbs is no longer inclusive for bsabs.
Objective: The main objective of this work is to develop a fully automated one-step capillary
isoelectric focusing - mass spectrometry (cIEF-MS) workflow for the charge variant analysis of a
bispecific antibody molecule.
Methods: A number of critical factors for the method development are investigated: the performance
of two commonly used ampholytes compared; the impact of protein concentration for the
cIEF-MS assay is examined; as for sample preparation, off-line and on-line desalting are compared;
various combinations of Pharmalyte® 3-10 and 8-10.5 are considered.
Results: In this fully automated workflow, the charge variants of this BsAb molecule are clearly
separated and accurately identified. Based on six repeat injections, RSDs of the migration time of
the identified charge variants are between 3 and 6%. The identified masses of each charge variant
show a variation between 0.48 and 1.40 Da. The delta masses of the basic and acidic variants are
from the most basic to the most acidic, -58.59, 162.26, 453.44, -907.47, 1,563.60, and 1,566.98
Da, respectively.
Conclusion: Overall, the separation resolution, system sensitivity, robustness, and reproducibility
of this fully automated cIEF-MS workflow, as demonstrated using this BsAb example, proves it a
powerful assay for the quality assessment of recombinant protein therapeutics.