Comparison of IgG purification with two different protein A media

Importance of the Topic

Antibody purification is a cornerstone of biopharmaceutical research and quality control, enabling the isolation of immunoglobulins from complex biological mixtures such as human plasma. Protein A affinity chromatography delivers high purity and yield, supporting downstream applications in diagnostics, therapeutics and biochemical research.

Study Objectives and Overview

This study evaluated and compared two commercially available prepacked Protein A column media—Sepapure Protein A FF and Protein A HP from a second vendor—for their capacity and performance in purifying human IgG using an automated system. Key metrics included binding capacity, yield and purity of the recovered antibodies.

Methodology

The purification workflow employed an AZURA Bio purification system configured with dual injection valves, a diode array detector, conductivity monitor and fraction collector. Each 5 mL Protein A column was equilibrated with phosphate buffer (20 mM, pH 7.0) at 2 mL/min, loaded with 500 µL human plasma, washed and eluted with glycine–HCl buffer (0.1 M, pH 2.7). Eluted fractions (1 mL) were collected automatically. Three replicate runs per column type were performed. IgG concentration in each fraction was measured by UV absorbance at 280 nm using a NanoDrop spectrophotometer, and purity was assessed by SDS–PAGE detecting heavy (48 kDa) and light chains (25 kDa).

Instrumentation

• AZURA P 6.1L LPG metal-free pump
• AZURA ASM 2.1L assistant module with dual injection valves
• AZURA DAD 2.1L diode array detector (10 mm, 10 µL flow cell)
• AZURA CM 2.1 conductivity monitor
• Foxy R1 fraction collector
• NanoDrop 2000 UV spectrophotometer

Results and Discussion

Both columns achieved similar IgG recoveries of approximately 6 mg from 500 µL plasma per run, corresponding to average concentrations of 0.82–1.04 mg/mL in collected fractions. Chromatograms revealed consistent injection, wash and elution peaks for each medium. SDS–PAGE analysis confirmed high purity, with clear heavy and light chain bands and no detectable contaminants. No statistically significant differences in capacity or yield were observed between the two Protein A media.

Benefits and Practical Applications

• Interchangeable column media support procurement flexibility.
• Automated workflow reduces hands-on time and variability.
• High purity IgG isolate suitable for analytical assays, therapeutic candidate evaluation and quality control.
• Scalable method adaptable to larger volumes and different antibody isotypes.

Future Trends and Potential Applications

Emerging directions in antibody purification include development of high-throughput and continuous chromatographic platforms, novel ligand chemistries for improved binding selectivity, and integration with in-line analytical techniques. Monolithic supports and membrane adsorbers promise faster processing times and lower backpressure, facilitating rapid process development in biomanufacturing.

Conclusion

The comparative study demonstrated that Sepapure Protein A FF and the alternative Protein A HP columns deliver equivalent performance for human IgG purification under standardized, automated conditions. Researchers and process developers can confidently select either medium based on availability and cost considerations without compromising yield or purity.

References

[1] Janeway CA Jr, Travers P, Walport M, et al. Immunology: The Immune System in Health and Disease. 5th Edition. New York: Garland Science; 2001.