Comparison of two column sets for antibody purification in an automated two step purification process

Importance of the Topic

Antibodies are essential biopharmaceutical tools used across research, diagnostics and therapeutics. Ensuring high purity and reproducible yields is vital for downstream applications and regulatory compliance. Automating capture and buffer exchange steps reduces manual intervention, minimizes variability and enhances throughput in antibody purification workflows.

Objectives and Overview of the Study

The study aimed to evaluate two different column sets for automated two-step antibody purification on the AZURA® Bio purification 50 system by:

• Comparing yield and purity of antibodies captured on two protein A resins followed by desalting columns.
• Assessing chromatographic performance and process integration without manual transfers.

Materials and Instrumentation

The automated setup comprised the AZURA® Bio purification 50 platform with:

• AZURA P 6.1L pump with metal-free 50 mL head and assistant modules (ASM 2.1L) equipped with PEEK 6-port valves.
• UVD 2.1S UV detector (280 nm) and AZURA CM 2.1S conductivity monitor.
• Foxy R1 fraction collector for final eluate recovery.
• Columns evaluated:
  
  • Vendor X Protein A FF (1 mL) and Vendor X desalting (5 mL).
  • Sepapure Protein A FF (1 mL) and Sepapure desalting (5 mL).
• Buffers: 20 mM sodium phosphate pH 7.4 (equilibration) and 0.1 M glycine-HCl pH 2.7 (elution).
• Protein quantification by NanoDrop 2000 and purity assessment via SDS-PAGE.

Methodology

Human plasma (100 µL) was injected onto the Protein A column, non-binders were washed off, and antibodies were eluted under acidic conditions into a sample loop. The looped eluate was then transferred to the desalting column for buffer exchange back to pH 7.4. Flow rates were 1 mL/min for affinity capture and 5 mL/min for desalting. Each column set was tested in triplicate under identical conditions.

Main Results and Discussion

• Average yields were 0.37 ± 0.05 mg (Vendor X set) and 0.41 ± 0.10 mg (Sepapure set), showing no significant difference.
• Overlayed chromatograms demonstrated similar binding, elution and buffer exchange profiles for both column combinations.
• SDS-PAGE confirmed high antibody purity with distinct heavy and light chain bands and minimal contaminants.
• Automated parking and transfer eliminated manual sample handling, reducing risk of cross-contamination and improving reproducibility.

Benefits and Practical Applications

• Fully automated two-step purification streamlines antibody workflows in research and QC laboratories.
• Interchangeable column options provide flexibility in vendor sourcing without sacrificing performance.
• Scalable and adaptable for various sample volumes and different affinity media.

Future Trends and Applications

• Integration of real-time monitoring and inline analytics to further optimize process control.
• Extension to other affinity ligands (e.g., His-tag, lectins) and multimodal resins.
• Development of continuous flow purification platforms to enhance throughput and reduce buffer consumption.
• Multiplexed purification strategies enabling parallel processing of multiple targets.

Conclusion

The AZURA® Bio purification 50 two-step system delivers equivalent yields and purities across two distinct column sets, demonstrating reliable automation of antibody capture and desalting. Its modular design and vendor-agnostic compatibility make it a robust solution for diverse bioprocessing needs.

References

No external references were cited in this application note.