NBC: Modification of Particle and Hardware Surfaces for Efficient Biotherapeutic Method Development

Importance of the topic

Analytical workflows for monoclonal antibody (mAb) biotherapeutics frequently encounter nonspecific binding of analytes to metal hardware surfaces, which compromises sensitivity, resolution and reproducibility. Tailoring both chromatographic particle and flow-path surfaces offers a practical strategy to mitigate metal-based adsorption, streamline mobile phase composition, and accelerate method development for critical quality attribute (CQA) analysis in biopharmaceutical research and manufacturing.

Objectives and overview of the study

The principal goal was to evaluate novel surface modifications on chromatographic particles and hardware to reduce metal-sensitive adsorption and assess their impact on two key CQA assays: size-exclusion chromatography (SEC) for aggregation and monomer profiling, and ion-exchange chromatography (IEX) for charge variant analysis of monoclonal antibodies.

Methods and instrumentation used

The study employed the following systems and materials:

• SEC Analysis: Waters ACQUITY UPLC H-Class Bio System with XBridge Premier Protein SEC 250 Å (2.5 µm, 4.6 × 150 mm) and BioResolve SEC mAb 200 Å columns; mobile phases: 200 mM sodium phosphate pH 6.8 (A), 1 M NaCl (B) and water (C); flow rate 0.35 mL/min; detection at 280 nm.
• IEX Analysis: Waters ACQUITY Premier System with BioResolve SCX mAb column (3 µm, 2.1 × 100 mm); IonHance CX-MS pH Concentrates A and B; RDa™ mass detector for MS-compatible charge variant profiling; temperature 40 °C; flow rate 0.1 mL/min.
• Surface modifications: Hydroxy-terminated polyethylene oxide bonded particles and modified hardware surfaces designed to minimize metal-based adsorption.

Main results and discussion

– SEC: Columns with modified particle and hardware surfaces maintained consistent monomer peak shape and aggregate recovery across 50–200 mM NaCl, without the need for elevated salt concentrations. Unmodified stainless-steel columns required >200 mM NaCl to achieve comparable performance.
– IEX: The novel pH buffer system delivered a linear pH response over a broad range compatible with both UV and MS detection. The ACQUITY Premier System with modified surfaces resolved three dominant charge variants of infliximab with retention time precision (%RSD) below 1% and enabled direct mass assignment of truncated and glycosylation variants.

Benefits and practical applications

Surface-modified chromatography offers:

• Improved sensitivity and resolution for mAb CQA assays using simpler mobile phase compositions.
• Elimination of extensive hardware passivation steps.
• Robust, reproducible SEC and IEX performance in QC, R&D and manufacturing environments.

Future trends and possibilities

Upcoming advancements may include extending surface modification technologies to a wider range of biotherapeutic modalities, integrating high-throughput MS workflows for comprehensive profiling, and engineering tailored chemistries to target specific adsorption mechanisms. Such innovations will further accelerate biologics development and support real-time quality monitoring.

Conclusion

Implementation of hydroxy-terminated polyethylene oxide bonded particles and hardware surface modifications effectively mitigates metal-based adsorption, delivering reproducible and robust chromatographic performance for size and charge variant analysis of mAbs. This technology streamlines method development, enhances data quality, and expedites biotherapeutic product workflows.

Reference

• Kizekai L, Shiner SJ, Lauber MA. Waters ACQUITY and XBridge Premier Protein SEC 250 Å Columns: A New Benchmark in Inert SEC Column Design. Waters Biosimilar Application Note. 2022. 720007493.
• Birdsall RE, Koshel B, Yu YQ. Accelerating Charge Variant Analysis of Biotherapeutics with the BioAccord™ System. Waters Application Note. 2022. 720007706.