A native multi-dimensional monitoring workflow for at-line characterization of mAb titer, size, charge, and glycoform heterogeneities in cell culture supernatant

Significance of the Topic

A robust analytical platform that characterizes mAb titer, size variants (aggregates, fragments), charge heterogeneity, and glycoforms directly in cell culture supernatant streamlines bioprocess monitoring and accelerates quality control.

Objectives and Study Overview

This study presents the development and demonstration of a native multi-dimensional multi-attribute monitoring (n-MD-MAM) workflow combining Protein A affinity (ProA), size exclusion chromatography (SEC), weak cation exchange (WCX), and Q-TOF mass spectrometry to enable at-line measurement of monoclonal antibody quality attributes without offline polishing steps.

Methodology and Instrumentation

The 1D ProA‐SEC dimension uses an Agilent 1290 Infinity II system with a ProA monolith column coupled inline to an SEC column under volatile, MS‐compatible ammonium acetate buffers. Heart cuts from SEC are directed via a 2D comprehensive valve into an Agilent BioMonolith NP5 WCX column for charge variant separation at 0.4 mL/min and 30 °C using a step gradient. Detection employs UV at 280 and 220 nm, and Agilent 6545XT Advance Bio LC/Q-TOF mass spectrometer. Data analysis is performed with Agilent MassHunter BioConfirm software.

Key Results and Discussion

The combined workflow resolves monomeric antibodies and size variants in the first dimension, and eight distinct charge variants in the second dimension. MS deconvolution identified glycoform distributions (G0/G0F, G0F/G1F, G1F/G1F) and modifications (deamidation, oxidation) across monomeric, high molecular weight, and low molecular weight species. The method achieved LOD of 0.50 µg and LOQ of 1.52 µg in 1D, and LOQ of 6.51 µg and 19.73 µg in the 2D for accurate titer estimation. Cell culture supernatant (DS) and purified drug product (DP) profiles showed comparable resolution and allowed direct comparison of product attributes.

Benefits and Practical Applications

• Direct at-line characterization in under 25 min without intermediate polishing steps
• Comprehensive monitoring of titer, aggregate content, charge heterogeneity, and glycoform profiles
• Compatibility with process analytical technology (PAT) frameworks for real-time decision making
• Reduced sample preparation and faster process development cycles

Future Trends and Opportunities

The integration of multi-dimensional chromatography with high-resolution MS paves the way for fully automated PAT solutions, expansion to other biotherapeutics (e.g., bispecifics, fusion proteins), and advanced real-time data analytics for predictive process control and accelerated commercial manufacturing.

Conclusion

The n-MD-MAM workflow demonstrates a rapid, robust approach for multi-attribute analysis of mAbs directly from cell culture supernatant, offering significant advantages for bioprocess monitoring and quality assurance, and laying a foundation for future real-time PAT implementations.

References

1. Bhattacharya S, Joshi S, Rathore AS. A native multi-dimensional monitoring workflow for at-line characterization of mAb titer, size, charge, and glycoform heterogeneities in cell culture supernatant. Journal of Chromatography A. 2023;1696:463983.
2. Jakes C, Füssl F, Zaborowska I, Bones J. Rapid analysis of biotherapeutics using Protein A chromatography coupled to Orbitrap mass spectrometry. Analytical Chemistry. 2021;93(40):13505-12.