Reducing Cycle Time for Charge Variant Analysis of Monoclonal Antibodies

Significance of the Topic

Charge variant analysis of monoclonal antibodies is a critical tool in biopharmaceutical development and quality control. Post-translational modifications such as deamidation or glycosylation produce charge heterogeneity, which can affect product efficacy, stability, and safety. Rapid and precise characterization of these variants supports regulatory compliance and process optimization.

Objectives and Study Overview

This application note demonstrates how to halve cycle time and nearly double throughput for mAb charge variant analysis by implementing alternating column regeneration. The study employs two Agilent Bio-inert Quaternary LC systems, a Quick-Change 2-position/10-port valve, and pH gradient elution to achieve both high resolution and low variability.

Methodology and Instrumentation

The separation is based on weak cation-exchange chromatography using a linear pH gradient (6.4 to 7.0) for narrow band focusing. Alternating regeneration of two 2.1 × 250 mm, 5 µm Bio MAb PEEK columns is realized with a second quaternary pump and a 2-position/10-port valve.

• Agilent 1260 Infinity Bio-inert Quaternary Pumps (2 × G5611A)
• Agilent 1260 Infinity Bio-inert High-Performance Autosampler (G5667A)
• Agilent 1290 Infinity Thermostat for sample cooling (G1330B)
• Agilent 1290 Infinity Thermostatted Column Compartment with bio-inert heat exchangers (G1316C, G5616-81000)
• Agilent 1200 Infinity Series Quick-Change Bio-inert 2-position/10-port Valve (G5632A)
• Agilent 1260 Infinity DAD VL, 10 mm bio-inert cell (G1315D)

Chromatographic conditions include a 30 mM phosphate buffer pH gradient, flow rate 0.3 mL/min, 7 µL injection volume, and UV detection at 280 nm.

Main Results and Discussion

Using alternating column regeneration, the total cycle time was reduced by 44 %, from 45 min in a one-column setup to 25 min per cycle. Six charge variants of the RAT Anti-DYKDDDDK mAb were resolved with high resolution. Intra-column precision (n=6) of retention time was < 0.31 % RSD and area < 2.8 % RSD (except one minor variant). Inter-column precision (n=12) remained below 0.29 % RSD for retention time and 3.7 % RSD for area. Throughput nearly doubled due to the simultaneous regeneration strategy.

Benefits and Practical Applications

• Reduced analysis time and increased sample throughput
• High reproducibility and precision for charge variant profiling
• Efficient column utilization with minimal downtime
• Scalable approach for QA/QC and stability studies in biomanufacturing

Future Trends and Applications

Further enhancements may include integration of online pH and conductivity monitoring, automated method development software, multi-column valves for continuous processing, and application of similar alternating regeneration strategies to other ion-exchange or affinity separations. Real-time data analytics and machine learning could optimize gradient profiles and further cut cycle times.

Conclusion

Alternating column regeneration using two Bio-inert quaternary pumps and a Quick-Change 2-position valve substantially elevates throughput for mAb charge variant analysis while maintaining high precision. This approach delivers a 44 % reduction in cycle time and nearly doubles analytical capacity, offering a robust solution for biotherapeutic quality control.

References

• Simple Method Optimization in mAb Charge Variant Analysis using pH Gradients Generated from Buffer Advisor with Online pH and Conductivity Monitoring, Agilent Technologies Application Note, publication number 5991-3365EN, 2013.
• Protein Separation with pH Gradients Using Composite Buffer Systems Calculated by the Agilent Buffer Advisor Software, Agilent Technologies Application Note, publication number 5991-1408EN, 2012.