Purification of antibody Fab and F(ab')2 fragments using Gradiflow technology

Area of Science:

• Biochemistry
• Immunology
• Analytical Chemistry

Background:

• Antibody fragments like Fab and F(ab")2 are crucial in diagnostics and therapeutics.
• Existing purification methods, such as ion exchange and gel filtration chromatography, can be time-consuming and complex.
• Enzyme digestion of antibodies generates fragments with differing isoelectric points (pI), enabling charge-based separation.

Purpose of the Study:

• To evaluate the efficacy of the Gradiflow preparative electrophoresis instrument for purifying antibody Fab and F(ab")2 fragments.
• To demonstrate a charge-based purification strategy leveraging pI differences between antibody fragments.
• To assess the applicability of this method across diverse antibody types.

Main Methods:

• Utilizing the Gradiflow instrument, a preparative electrophoresis system.
• Employing a charge-based separation technique exploiting differences in isoelectric points (pI) between antibody fragments (Fab/F(ab")2) and Fc fragments.
• Applying the method to purified antibody fragments derived from enzyme digestion of whole antibodies.

Main Results:

• Successful purification of F(ab")2 fragments from mouse monoclonal antibodies (IgG1 and IgG2a isotypes).
• Successful purification of Fab fragments from egg yolk IgY polyclonal antibodies.
• Demonstrated rapid purification with advantages over conventional chromatography techniques.

Conclusions:

• The Gradiflow instrument provides a rapid and effective method for purifying antibody Fab and F(ab")2 fragments.
• The charge-based separation strategy is applicable to a wide range of monoclonal and polyclonal antibodies.
• This preparative electrophoresis technique presents a viable alternative to traditional purification methods for antibody digest preparations.