Monitoring monoclonal antibody bioproduction processes with 2D-LC-UV

Significance of the topic

Monitoring monoclonal antibody (mAb) upstream production is critical for ensuring product quality, safety, and process control in biopharmaceutical manufacturing. Rapid at-line analytical methods that quantify titer and track critical quality attributes (CQAs) such as charge variants and size variants (aggregates/fragments) enable timely corrective actions during cell culture campaigns and reduce the need for off-line, time-consuming assays. The presented heart-cut two-dimensional liquid chromatography (2D-LC) workflow with UV detection provides a pragmatic balance between information content and operational speed for routine process monitoring.

Goals and study overview

This application study established and demonstrated a switchable heart-cut 2D-LC-UV method for at-line monitoring of multiple mAb quality attributes directly from harvested CHO cell culture samples. Specific objectives were:

• Rapid titer determination by protein A affinity chromatography (1D).
• Purification and transfer of the intact mAb to a 2D strong cation exchange (SCX) column for charge variant profiling.
• An alternate 2D path in which the SCX column traps and refocuses the mAb prior to size-exclusion chromatography (SEC) for size-variant (HMW/LMW) analysis.
• Validation of linearity, reproducibility, and the ability to monitor temporal trends in two parallel lab-scale bioreactor batches sampled over 10 days.

Methodology

The strategy used an affinity purification heart-cut from a 1D Protein A (ProA) column into a 2D configuration selectable between SCX and SCX-SEC workflows. Key elements:

• Sample input: filtered harvested cell culture (HCC) samples taken daily from two lab-scale CHO bioreactors expressing a NISTmAb analogue (cNISTmAb) over 10 days.
• 1D: MAbPac Protein A column captured mAb from culture media at neutral pH and eluted under acidic conditions; the mAb fraction was transferred via a 250 µL heart-cut loop (175 µL fraction set in software) to the 2D.
• 2D path a (ProA-SCX): ProPac 3R SCX column separated charge variants using a linear NaCl gradient (monitoring up to six acidic and 13 basic variants relative to the main peak).
• 2D path b (ProA-SCX-SEC): SCX acted as a trap/re-focusing stage followed by step elution into a MAbPac SEC-1 column for isocratic size separation, minimizing band dispersion and improving SEC peak shape.
• Detection: UV at 280 nm for titer and PQAs; chromatography methods were kept consistent across modes (20 mM MES with varying NaCl and pH adjustments) to speed sequential processing.
• Data handling: Thermo Chromeleon CDS controlled the Vanquish Flex Simple Switch 2D-LC system; PrepareNextInjection was used to overlap cycle preparation and shorten throughput time.

Instrumentation used

The experimental platform was a Thermo Scientific Vanquish Flex Simple Switch 2D-LC system configured for loop heart-cutting. Important modules and components included:

• Vanquish System Base (Horizon/Flex), Vanquish Quaternary Pump (1D), Vanquish Binary Pump (2D), and Vanquish Variable Wavelength Detectors (1D and 2D).
• Vanquish Dual Split Sampler FT (optional single sampler configuration noted).
• MAbPac Protein A Analytical/Purification column (1D), ProPac 3R SCX (short trap/analytical column) for 2D, and MAbPac SEC-1 (4 × 300 mm) for size analysis.
• Viper/MP35N capillaries, fractionation loop (250 µL), active pre-heaters, column compartments, and relevant Viper fittings and filters.

Main results and discussion

• Titer quantification: Calibration using NISTmAb RM 8671 yielded excellent linearity (R2 = 0.99965) across injected amounts; measured cNISTmAb titers in culture rose from ~15 µg/mL on day 1 to ~82 µg/mL on day 10. Injection volumes were adjusted over the campaign (1,000 µL down to 250 µL) to maintain signals within the calibrated range.
• Charge variant profiling (ProA-SCX-UV): The SCX 2D separated multiple acidic and basic variants. The main peak relative area showed reproducible trends across two parallel batches: starting near 46% relative area on day 1, increasing to ~55% by day 7 and then plateauing. Some charge variant peaks revealed batch-specific divergences on certain days, demonstrating the method’s sensitivity to process deviations.
• Size variant analysis (ProA-SCX-SEC-UV): Using SCX as an intermediate trap before SEC significantly improved SEC peak shape compared with direct ProA-SEC transfers. SEC resolved monomer, one low molecular weight (LMW) fragment, and two high molecular weight (HMW) aggregate peaks. Observed trends: LMW decreased from ≈10% to ≈4% over early days; HMW-1 rose from ≈2% to ≈3.5% then declined as HMW-2 emerged, indicating evolving aggregate species during culture.
• Operational performance: The heart-cut loop (fraction limited to 175 µL) and short SCX trap minimized sample breakthrough and band dispersion. Overlapping injection preparation reduced cycle time. UV detection provided sufficient actionable information for at-line decision making without the complexity of HRAM-MS when only gross PQAs are required.

Benefits and practical applications

• At-line capability: Direct injection of filtered HCC samples enabled rapid monitoring across the production campaign without extensive sample preparation.
• Flexible 2D switching: The same 2D configuration supports either charge-variant separation or trapping prior to SEC, allowing tailored analysis depending on the immediate analytical need.
• Improved SEC performance: SCX trapping before SEC reduced band dispersion and improved resolution of size variants, making SEC more robust in heart-cut workflows.
• Process control value: The workflow allows early detection of batch-to-batch deviations in charge and size profiles, enabling timely process interventions to maintain product quality.
• Resource efficiency: UV-based detection simplifies routine monitoring compared with full MS characterization while delivering the essential PQAs for control decisions.

Future trends and potential applications

• Integration with PAT: Combining 2D-LC at-line data streams with process analytical technology (PAT) frameworks and multivariate analytics will enable predictive control and automated interventions.
• Increased automation: Further automation of sampling and method switching can increase throughput and reduce operator intervention for real-time monitoring in GMP-like environments.
• Hybrid workflows: When deeper molecular information is required, the same 2D-LC platform can be coupled to HRAM-MS for orthogonal characterization while retaining UV-based modes for routine control.
• Higher-dimensional separations: Expanding to multi-dimensional LC (mD-LC) approaches with online digestion or reduction can deliver comprehensive attribute mapping when needed for development or investigational use.

Conclusion

A switchable heart-cut 2D-LC-UV approach built around ProA capture, SCX separation/trapping, and optional SEC provides an efficient, flexible at-line solution for monitoring mAb titer, charge variants, and size variants directly from bioreactor harvests. The workflow demonstrated high linearity for titer, robust separation of multiple charge species, improved SEC performance via SCX trapping, and sensitivity to batch-dependent attribute changes. For routine process control where rapid, actionable information is the priority, this UV-based 2D-LC strategy offers a practical compromise between speed and analytical depth.

Reference

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