Monitoring of multiple quality attributes of intact monoclonal antibodies from bioreactors by switchable 2D-LC-MS

Importance of topic

Monoclonal antibodies (mAbs) represent a dominant class of biopharmaceuticals whose complex microheterogeneity—stemming from glycosylation, deamidation, oxidation, and aggregation—directly impacts product safety and efficacy. Comprehensive monitoring of multiple product quality attributes (PQAs) such as titer, charge variants, glycoforms, and aggregates is essential for process control and regulatory compliance. Integrating two-dimensional liquid chromatography with native mass spectrometry (2D-LC-MS) streamlines multi-attribute analysis and accelerates decision-making in bioproduction.

Objectives and study overview

This work presents a switchable 2D-LC-MS workflow to monitor intact mAb PQAs directly from Chinese hamster ovary (CHO) bioreactor supernatants over a 10-day culture. The method combines:

• Protein A (ProA) affinity capture for titer determination and sample cleanup (1D).
• Optional strong cation exchange (SCX) separation via pH gradient for charge variant analysis (2D-CVA).
• Optional SCX–size exclusion chromatography (SEC) trap-and-elute sequence for aggregate quantification (2D-aggregate).

Initially, the National Institute of Standards and Technology mAb reference material (NISTmAb RM 8671) was used for method development, calibration, and glycoform identification. The optimized workflow was then applied to a NISTmAb analogue from two parallel CHO bioreactors (cNISTmAb) sampled daily.

Methodology and instrumentation

A Thermo Scientific Vanquish Flex Simple Switch 2D-LC system enabled rapid heart-cutting from ProA to a short ProPac 3R SCX column and/or an inline MAbPac SEC-1 column. All mobile phases used volatile buffers (ammonium acetate, bicarbonate, hydroxide, acetic acid) compatible with native MS. Key instrumentation:

• 1D: MAbPac Protein A column; UV detection and time-triggered heart-cut loop (175 µL).
• 2D-CVA: ProPac 3R SCX column with pH gradient (5.3→10.9) at 0.2 mL/min.
• 2D-aggregate: SCX trap followed by SEC elution on MAbPac SEC-1 (4×300 mm).
• Mass detection: Thermo Scientific Orbitrap Exploris MX (BioPharma option) at m/z 2,500–8,000; resolution 60,000; native protein mode.
• Software: Chromeleon 7.3 CDS, BioPharma Finder 5.2.

Main results and discussion

Titer determination by ProA-UV showed mAb levels rising from ca. 7 µg/mL on day 1 to 52 µg/mL on day 10 with excellent calibration linearity (R2=0.9991). SCX-MS charge variant analysis resolved acidic, main, and basic peaks. Deconvolution identified five major glycoforms (G0F_G1F, G0F_G0F, G1F_G2F, etc.) and tracked their dynamics:

• G0F_G1F dominated early culture (35–40%), then declined as G0F_G0F rose (from 18% to 40%).
• G1F_G1F decreased steadily from 27% to 19%.
• Minor variants with mass shifts (+98 Da, +177 Da, –58 Da) and sialylated species (~+906 Da) were detected and quantified.

SEC-MS analysis of aggregated species revealed a low high-molecular-weight fraction (<0.01% day 1), increasing to ~0.12% by day 10. Native MS of monomer and HMW peaks provided intact mass confirmation.

Benefits and practical applications of the method

• One-hour end-to-end workflow delivers titer, glycoform profile, charge variant distribution, and aggregation level from raw bioreactor fluid.
• Volatile buffer systems enable direct native MS coupling, eliminating offline desalting.
• Switchable 2D-LC allows targeted analysis (charge or size variants) without system reconfiguration.
• Supports real-time process monitoring, quality control, and multifactorial attribute assessment (iMAM).

Future trends and potential applications

Emerging directions include:

• Extension to novel modalities (bispecifics, ADCs) with tailored 2D workflows.
• Automated data analytics and machine-learning–driven quality prediction.
• Integration with peptide mapping, released glycan, and higher-order structure assays for comprehensive deep characterization.
• Implementation of process analytical technology (PAT) for tighter bioprocess control.

Conclusion

The switchable 2D-LC-MS platform combining ProA capture, SCX, and SEC separations under native conditions provides rapid, multiplexed insight into intact mAb quality attributes. It enables robust titer measurement, dynamic glycoform and charge variant profiling, and aggregation monitoring directly from bioreactor samples, promising enhanced process understanding and accelerated biotherapeutic development.

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