Automated two step purification of mouse antibody IgG1

Importance of the topic

The purification of immunoglobulin G (IgG) antibodies is a fundamental step in biopharmaceutical and research workflows. Ensuring high purity and correct buffer conditions is vital for downstream applications such as diagnostics, therapeutics, and analytical assays.
A fully automated two-step approach reduces manual handling, lowers contamination risk, and accelerates the workflow.

Study objectives and overview

This application note presents the development of a hands-free, two-step purification protocol for mouse IgG1 using the AZURA Bio purification system. The process combines affinity capture on a protein A column with size-exclusion-based buffer exchange to deliver antibodies in the desired formulation.

Methodology and used instrumentation

• System components: AZURA P 6.1L HPG pump, ASM modules with feed pump and UV detector, six-port valves, column switching valve, conductivity monitor, and fraction collector.
• Columns: 1 mL Protein A affinity column and a desalting/gel filtration column.
• Buffers: TBS for equilibration and washing, 0.2 M sodium citrate (pH 3) for elution, PBS (pH 7.4) for final buffer exchange.
• Operational steps at 1 mL/min: column equilibration, sample loading, washing, elution and peak parking, re-injection onto desalting column, and fraction collection.

Main results and discussion

Chromatographic data show four distinct phases: equilibration, flow-through of unbound matrix, elution of IgG1, and buffer exchange. Conductivity monitoring confirmed efficient desalting.
SDS-PAGE analysis revealed clear heavy (≈55 kDa) and light (≈22 kDa) chains after both affinity and desalting steps, with no detectable higher-molecular-weight contaminants. Protein recovery remained consistent, indicating negligible losses during buffer exchange.

Benefits and practical applications

• Complete automation minimizes manual intervention and reduces process time.
• High-purity IgG1 in specified buffer suitable for storage, functional assays, or therapeutic formulation.
• Protocol adaptability to other antibody subclasses and protein targets.

Future trends and applications

• Integration of additional polishing steps such as ion-exchange or hydrophobic interaction chromatography.
• Development of continuous or high-throughput purification platforms.
• Real-time process analytics for in-line quality control and yield optimization.

Conclusion

The automated two-step purification using the AZURA Bio system successfully isolated mouse IgG1 from cell culture supernatant with high purity and efficient buffer exchange. This approach offers a scalable, time-saving solution for antibody preparation in research and industrial settings.