Affinity titer analysis to enable scalable mAb and bispecific production

Significance of the topic

Monitoring monoclonal antibody (mAb) and bispecific production requires rapid, selective and multi-attribute analytics to guide upstream decisions and to ensure critical quality attributes (CQAs) are maintained during scale-up. Affinity-based titer measurement combined with orthogonal separations for charge variants and aggregate content provides a compact, high-information workflow that supports process development, control and regulatory compliance.

Objectives and overview of the study

The study aimed to demonstrate a 2D-LC workflow that links a Protein A affinity HPLC titer assay with downstream separations (strong cation exchange, SCX, and size exclusion chromatography, SEC) to deliver simultaneous information on productivity (titer) and product quality (charge variants and aggregation) from the same clarified bioreactor sample. The workflow was applied to a CHO-produced trastuzumab biosimilar over a 14-day culture period to illustrate applicability for upstream process monitoring.

Methods and sample preparation

Samples were taken from CHO-K1 cell culture producing a trastuzumab biosimilar. Harvests were clarified by centrifugation (5000 rpm, 5 min), transferred to autosampler vials and injected directly for Protein A analysis. IgG calibration standards were prepared by serial dilution of purified IgG and treated identically prior to injection. The Protein A affinity step provided a clean capture fraction (heart-cut) for second-dimension SCX or SEC analysis.

Used instrumentation

• Affinity column: Thermo Scientific POROS A 20 µm, 2.1 x 30 mm (Protein A affinity).
• SCX column: Thermo Scientific ProPac 3R SCX, 3 µm, 4 x 50 mm.
• SEC column: Thermo Scientific MabPac SEC-1, 7.8 x 300 mm.
• 2D-LC platform: Thermo Scientific Vanquish Flex Simple Switch 2D-LC system configured for loop heart-cutting with dual samplers and dual pumps (Flex Dual Sampler, Flex Dual Pump).
• Detector and data system: UV detection at 280 nm; Thermo Scientific Chromeleon Chromatography Data System for acquisition and analysis.
• Buffers and method conditions: Protein A buffers—50 mM ammonium acetate pH 7 (A) and 300 mM acetic acid pH 2.6 (B); gradient program with short affinity elution. SCX used CX-1 pH gradient buffers (A pH 5.6, B pH 10.2) with a 0–100% B gradient. SEC used 100 mM ammonium acetate pH 5 isocratic buffer.

Analytical methodology details

The Protein A affinity assay was run primarily at 0.5 mL/min (20 µL injection) to match downstream flow needs, and also evaluated at 2 mL/min to demonstrate rapid quantitation (<2 min). Affinity eluate fractions were transferred (heart-cut) into a second-dimension run: either SCX to resolve charge variants (acidic, neutral, basic species) or SEC to quantify high molecular weight (HMW) and low molecular weight (LMW) species. SCX gradients and SEC conditions were selected to provide robust separation of charge heterogeneity and aggregation, respectively.

Main results and discussion

• Titer measurement: Protein A affinity chromatography enabled rapid, selective quantitation of mAb concentration directly from clarified culture supernatant with minimal matrix interference. Measured titers exceeded 7 g/L across the 14-day run.
• Throughput vs compatibility: A higher-flow (2 mL/min) Protein A method produced equivalent quantitation and reduced analysis time (<2 min), while the lower-flow (0.5 mL/min) method was more compatible with 2D coupling to SCX/SEC.
• Charge variants: SCX analysis of heart-cut fractions revealed an increasing proportion of acidic variants over the culture duration (notably after day 8 onward), indicating time-dependent modifications that affect product heterogeneity and potentially potency or stability.
• Aggregation: SEC monitoring showed a rise in HMW species beginning after day 8, consistent with extended residence time or process stress causing aggregation. This trend underscores the need to balance titer gains with product quality control.
• Integrated insight: Combining Protein A titer with SCX and SEC in a single workflow allowed simultaneous assessment of productivity and multiple CQAs from one sample, enabling data-driven decisions during upstream optimization.

Benefits and practical applications

• Rapid decision-making: Fast affinity titer readouts support timely interventions in upstream cultures (e.g., harvest timing, feed adjustments).
• Reduced sample handling: Direct injection of clarified harvest and heart-cut transfer minimize sample prep, lowering variability and time-to-result.
• Comprehensive CQA coverage: Inline coupling permits monitoring of titer, charge heterogeneity and aggregation on the same material, improving correlation between productivity and quality.
• Scalability and throughput: The option to run a high-flow Protein A assay for rapid screening, or a lower-flow assay for 2D compatibility, offers flexibility for development and routine monitoring.
• Regulatory alignment: The approach supports ICH expectations for CQA monitoring and robust analytical control during development and manufacture.

Future trends and possibilities for use

• Deeper multi-attribute integration: Extending 2D-LC to include peptide mapping or MS detection downstream of affinity capture could enable site-specific modification analysis in-line.
• Automated process analytical technology (PAT): Embedding such linked workflows in automated sampling and control loops could enable closed-loop adjustments of culture conditions based on real-time CQA data.
• High-throughput process screens: Combining rapid affinity quantitation at high flow with parallel quality assays could accelerate clone selection and media/feed optimization campaigns.
• Standardization for regulatory submission: Harmonizing workflows, calibrants and data processing will help translate integrated 2D analytics into validated in-process controls for GMP environments.

Conclusion

Linking Protein A affinity titer analysis with orthogonal SCX and SEC separations via a 2D-LC heart-cutting strategy offers a practical, information-rich platform for upstream mAb process monitoring. The workflow provides rapid, selective titer measurement and simultaneous assessment of charge variants and aggregation from a single clarified sample. Observed increases in acidic variants and aggregates during extended culture emphasize the need to couple productivity monitoring with quality analytics to ensure robust, scalable production and maintain CQA control.

References

• ICH Q11: Development and Manufacture of Drug Substances.
• ICH Q6B: Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products.