Two step purification with a basic set up and PurityChrom 6

Importance of the Topic

The two-step purification approach leverages automated multicolumn chromatography to streamline protein isolation. By coupling two distinct separation stages without manual intervention, this method improves purity, reproducibility, and throughput in bioanalytical workflows. Its simplicity and adaptability make it valuable for research, quality control, and industrial protein production.

Objectives and Study Overview

This application note describes the implementation of an automated two-step purification using a basic KNAUER AZURA® Bio Lab setup and PurityChrom 6 software. The goal is to demonstrate how an ion exchange step followed by desalting can be carried out sequentially in one uninterrupted run, maximizing yield and purity of the target protein.

Methodology and Instrumentation

Two independent chromatographic methods were written in PurityChrom 6: one for ion exchange and one for desalting. Valve switching, threshold detection, and fraction collection are orchestrated by:

• AZURA Bio Lab system with standard pump, UV detector, and conductivity monitor
• Biocompatible multiposition column selection valve (8-port)
• Multifunctional injection valve (sample loop for injection and intermediate storage)
• Smart outlet valve directing flow to fraction collector or waste
• Inline and dummy filters to protect pressure sensors and valves

Sample injection, gradient elution, threshold-triggered peak capture in the loop, and automated redirection to the second column are configured via valve state changes and execution delays to account for tubing volumes.

Key Results and Discussion

The basic setup successfully isolated a target protein on an ion exchange column, captured the peak above 100 mAU, and transferred it to a desalting column without user intervention. The two-step sequence improved sample purity and reduced hands-on time. Limitations include sample loop volume constraints; options to inject via the main pump and replace the pressure filter with a dummy cartridge were discussed to accommodate larger volumes. Inline filters are recommended to prevent blockages and maintain system performance.

Benefits and Practical Applications

• Fully automated workflow minimizes manual handling and contamination risk
• Consistent peak detection and fraction collection enhance reproducibility
• Modular setup allows rapid method development for diverse proteins
• Suitable for R&D labs, QC/QA environments, and small-scale manufacturing

Future Trends and Opportunities

Advancements may include integration of real-time analytics (e.g., mass spectrometry), adaptive method control using machine learning, and expanded valve architectures for multi-target purification. Scaling to higher throughput and coupling with downstream analytics will further streamline bioprocessing.

Conclusion

The basic two-step purification setup on the AZURA Bio Lab platform demonstrates an efficient, reproducible approach for automated protein purification. By combining ion exchange and desalting in a single run with minimal hardware additions, users can achieve high purity while reducing labor and potential errors.

References

1. VTN0013 – Guide for two-step purification principles and system setup
2. VTN0014 – Two-step purification with a basic setup
3. VTN0015 – Two-step purification with the sample pump setup
4. VTN0026 – Two-step purification with the sample pump setup and PurityChrom 6