A simple and efficient method for the purification of human gamma interferon.

Area of Science:

• Biochemistry
• Protein Purification
• Chromatography

Background:

• Human gamma interferon (HuIFN-gamma) is a crucial therapeutic protein.
• Efficient and scalable purification methods are essential for its clinical application.
• Existing purification techniques can be time-consuming and labor-intensive.

Purpose of the Study:

• To develop a preparative, sequential chromatographic procedure for high-purity HuIFN-gamma.
• To optimize the purification process for speed, ease of use, and amenability to automation.
• To demonstrate the efficiency and scalability of the developed method.

Main Methods:

• A four-step sequential chromatography procedure was employed: Controlled Pore Glass-adsorption, Concanavalin-A affinity, Heparin-Sepharose affinity, and gel-filtration.
• A coordinated effluent-affluent buffer scheme was developed to streamline column loading.
• The procedure was designed for semi-automation.

Main Results:

• The purification procedure achieved an overall purification factor greater than 75,000-fold.
• A specific activity of 5.2 x 10(7) units/mg was obtained with 95.5% recovery.
• The peak fraction showed a specific activity of 1.0 x 10(8) units/mg protein, representing >145,000-fold purification.
• SDS-PAGE analysis indicated approximately 40% of the final material was HuIFN-gamma.

Conclusions:

• The developed sequential chromatographic procedure provides a rapid, efficient, and scalable method for purifying HuIFN-gamma.
• The method's amenability to semi-automation simplifies the process and reduces hands-on time.
• High purity and specific activity of the purified HuIFN-gamma were achieved, suitable for therapeutic applications.