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Published on: October 20, 2021
Microheterogeneous monoclonal antibody subspecies with differential hepatitis C virus core antigen binding properties
A Scott Muerhoff1, Kevin Rupprecht, Qiaoqiao Ruan
1Infectious Diseases Research and Development, Abbott Diagnostics, Abbott Laboratories, Abbott Park, IL 60064-6015, USA. scott.muerhoff@abbott.com
Insights
This study reveals that a single monoclonal antibody (mAb) against hepatitis C virus (HCV) core protein comprises distinct subspecies. These subspecies display varied antigen-binding properties due to post-translational modifications, impacting immunoassay performance.
Area of Science:
- Biochemistry
- Immunology
- Analytical Chemistry
Background:
- Monoclonal antibodies (mAbs) are crucial in immunoassays.
- Hepatitis C virus (HCV) core protein detection is vital for diagnostics.
- Characterizing mAb heterogeneity is essential for assay optimization.
Purpose of the Study:
- To investigate the microheterogeneity of a monoclonal antibody targeting the HCV core protein.
- To determine the impact of identified subspecies on antigen-binding properties.
- To assess the utility of this mAb in a sandwich antigen immunoassay.
Main Methods:
- High-Performance Liquid Chromatography (HPLC) size exclusion chromatography (SEC) using different column matrices and buffer ionic strengths.
- Isoelectric focusing (IEF) gel electrophoresis.
- Fluorescence correlation spectroscopy (FCS) for dissociation constant (Kd) measurements.
- Electrospray Ionization Mass Spectrometry (ESI-MS) for analyzing post-translational modifications.
Main Results:
- Two distinct IgG subspecies were resolved using methacrylate-based SEC in low ionic strength buffer.
- These subspecies exhibited differential reactivity towards HCV core antigen.
- ESI-MS identified unique post-translational modifications (reduction-sensitive and insensitive) on the heavy chain variable regions of one subspecies.
- FCS revealed differences in Kd values between subspecies in low salt buffer, which diminished in high salt buffer.
Conclusions:
- The monoclonal antibody is microheterogeneous, composed of subspecies with varying antigen-binding affinities.
- Post-translational modifications of the heavy chain variable region are associated with these functional differences.
- The selection of SEC column matrix and buffer conditions is critical for resolving and characterizing mAb subspecies.
- Understanding mAb heterogeneity is essential for developing robust and reliable diagnostic immunoassays.
Abstract:
A monoclonal antibody directed against the core protein of hepatitis C virus was characterized for its utility in a sandwich antigen immunoassay wherein the mAb was used as the conjugate. Analysis of unconjugated and acridinium-conjugated monoclonal IgG using a silica-based HPLC size exclusion column revealed the existence of a single, symmetrical peak. Subsequent analysis of unconjugated IgG using a methacrylate-based HPLC size exclusion column revealed the presence of two species of IgG, but only by using a low ionic strength mobile phase buffer. Independent conjugation and testing of the two species showed significant differential reactivity towards HCV core antigen. Isoelectric focusing gels indicated subtle differences in the subspecies composition. Measurement of target peptide dissociation constants using fluorescence correlation spectroscopy indicated that the two HPLC column fractions exhibited a two-fold difference in Kd in low salt buffer that disappeared in high salt buffer. ESI-MS analysis of the fractionated IgG peaks revealed a reduction sensitive modification of the IgG and F(ab')2 of approximately 674 Da. In addition, both IgG and F(ab')2 contained two major heavy chain subspecies differing by about 1216 Da that was reduction insensitive. These modifications were present in only the one of the two SEC-HPLC peaks. These results suggest that this monoclonal antibody consists of microheterogeneous subspecies that exhibit different antigen binding properties associated with differences in post-translational modification of the heavy chain variable region. The choice of size exclusion column matrix and buffer composition was critical to the identification of these monoclonal IgG subspecies.
