Separation of Native Monoclonal Antibodies and Identification of Charge Variants: Teamwork of the Agilent 3100 OFFGEL Fractionator, Agilent 2100 Bioanalyzer and Agilent LC/MS Systems

Importance of the Topic

Monoclonal antibodies are a cornerstone of modern biotherapeutics. Their therapeutic efficacy, stability, and safety depend on precise structural attributes. Post-translational modifications introduce charge heterogeneity that can impact binding affinity, shelf life, and immunogenicity. Thorough characterization of these charge variants is critical for quality control, regulatory compliance, and product comparability in both drug development and manufacturing.

Study Objectives and Overview

This work presents an analytical workflow designed to separate, monitor, and identify charge variants of a therapeutic monoclonal antibody. By integrating three complementary Agilent platforms—an OFFGEL fractionator for isoelectric focusing in solution, a Bioanalyzer for rapid protein sizing, and a high-resolution LC/MS system—this study aims to deliver a robust solution for detailed charge variant profiling under native and reduced conditions.

Materials and Methods

The antibody sample was a proprietary therapeutic IgG1. Isoelectric focusing used Immobiline DryStrip IPG 6–9 in a 24-well OFFGEL device with pH 6–11 buffer and 5% glycerol. No denaturants were added to preserve native structure. Fractions (150 µL each) were collected and analyzed by microfluidic protein sizing.

Instrumental Setup

• Agilent 3100 OFFGEL Fractionator with 24-well frame, pI range 6–9
• Agilent 2100 Bioanalyzer with Protein 230 kit under nonreducing conditions
• Agilent 1290 Infinity LC System coupled to 6530 Accurate-Mass Quadrupole Time-of-Flight MS
• MassHunter Acquisition, Qualitative Analysis, BioConfirm, and pMod deconvolution software

Results and Discussion

OFFGEL fractionation yielded distinct zones: acidic variants in wells 3–7, the main species in well 8, and basic variants in wells 9–12. Bioanalyzer profiles confirmed intact ~150 kDa antibody across fractions. Reduced fractions analyzed by LC/MS revealed:

• Acidic fraction (well 6) carried a mono-sialylated, fucosylated biantennary glycan (+291 Da).
• Main fraction (well 8) displayed typical glycoforms G0F, G1F, and G2F.
• Basic fraction (well 12) included a +128 Da mass shift corresponding to C-terminal lysine variants.

This demonstrates the method’s capacity to enrich and identify low-abundance charge variants with high confidence.

Benefits and Practical Applications

• Preparative separation under native conditions preserves antibody integrity.
• Microfluidic QC enables rapid purity and size assessment.
• High-resolution LC/MS provides accurate mass assignment and structural interpretation of variants.
• The workflow supports comparability studies, biosimilar development, and routine QC in biopharmaceutical analysis.

Future Trends and Opportunities

Emerging directions include automated multidimensional fractionation, integration with peptide mapping for site-specific modifications, enhanced deconvolution algorithms, and high-throughput formats. Expansion to other biotherapeutic formats and coupling with advanced data analytics will further streamline variant characterization and regulatory submissions.

Conclusion

The combined use of the Agilent 3100 OFFGEL Fractionator, 2100 Bioanalyzer, and 6530 Q-TOF LC/MS establishes a versatile workflow for in-depth analysis of monoclonal antibody charge variants. This approach delivers preparative enrichment, rapid molecular weight screening, and precise mass measurement, meeting the rigorous demands of biopharmaceutical R&D and quality control.

References

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