A novel approach for multiple PQAs monitoring of mAbs from bioreactors using 2D-LC-MS

Significance of the Topic

The microheterogeneity of monoclonal antibodies (mAbs) influences critical quality attributes such as glycosylation, charge variants and aggregation. Robust, high-throughput monitoring of these attributes during bioreactor runs is essential for biopharmaceutical process control, regulatory compliance and consistent product efficacy.

Study Objectives and Overview

This study presents a novel two-dimensional liquid chromatography–mass spectrometry (2D-LC-MS) approach for simultaneous mAb titer determination and multi-attribute monitoring directly from harvested cell culture (HCC) samples over a ten-day bioreactor run. Key aims:

• Quantify mAb titer online.
• Resolve and identify major glycoforms and charge variants.
• Assess aggregation by size exclusion.

Methodology

A switchable, heart-cut 2D-LC platform was configured in two modes: ProA-SCX-MS for charge variant and glycoform analysis and ProA-SCX-SEC-MS for size variant profiling. Sample preparation and workflow:

• First dimension: Protein A affinity capture isolates mAb and provides titer data under native pH gradient elution.
• Heart-cut to second dimension: SCX column for charge variant separation via pH gradient.
• Alternate path: Trap mAb on SCX, then pulse-elute to SEC column for size exclusion analysis.
• High-resolution MS detection for accurate mass determination and deconvolution.

Used Instrumentation

• Thermo Scientific Vanquish Flex Simple Switch 2D-LC system (Quaternary Pump F, Binary Pump F, Dual Split Sampler FT, Column Compartments, Diode Array Detector)
• Thermo Scientific Orbitrap Exploris MX mass spectrometer with BioPharma option
• Thermo Scientific MAbPac Protein A HPLC column (4 × 35 mm, 12 µm)
• Thermo Scientific ProPac 3R SCX column (2 × 50 mm, 3 µm)
• Thermo Scientific MAbPac SEC-1 column (4 × 300 mm, 5 µm)

Main Results and Discussion

mAb titer rose from 7 µg/mL on day 1 to 52 µg/mL on day 10. Glycoform profiling revealed G0F_G1F as the dominant species early, with G0F_G0F becoming most abundant by day 9–10. Multiple unknown charge variant peaks were detected and tentatively identified based on mass deltas. The SCX-SEC trap/elute mode significantly sharpened SEC peak shapes, improving monomer vs. high-molecular-weight separation compared to direct ProA-SEC.

Benefits and Practical Applications

This multi-attribute 2D-LC-MS method delivers a comprehensive quality control package within one hour per sample. It enables:

• Real-time mAb titer assessment.
• Quantitative glycoform and charge variant monitoring.
• Aggregation level determination.
• Streamlined process monitoring for new modalities and established mAb products at the intact protein level.

Future Trends and Possibilities

Further developments may include automated data processing with machine learning for variant identification, integration with process analytical technologies (PAT), and extension to complex modalities such as bispecifics, ADCs and glycoengineered formats.

Conclusion

The switchable heart-cut 2D-LC-MS approach provides rapid, detailed insight into multiple critical quality attributes of mAb products directly from bioreactor samples, supporting accelerated process development and quality assurance in biomanufacturing.

Reference

1. Carillo S, Bones J, et al. European Journal of Pharmaceutics and Biopharmaceutics. 2022;241–248.