Purification of antibodies using protein L-binding framework structures in the light chain variable domain

Area of Science:

• Biochemistry
• Immunology
• Protein Engineering

Background:

• Protein L, derived from Peptostreptococcus magnus, exhibits specific binding to the variable domain of immunoglobulin (Ig) light chains.
• This interaction does not impede the antigen-binding site of the Ig molecule.

Purpose of the Study:

• To utilize a genetically engineered fragment of Protein L for the purification of immunoglobulins from diverse sources.
• To demonstrate the feasibility of engineering antibodies and antibody fragments for Protein L-based purification.

Main Methods:

• Affinity chromatography using Protein L-Sepharose was employed for Ig purification.
• Purification was performed on various Ig isotypes (IgG, IgM, IgA) from human and mouse serum.
• Protein L-Sepharose was also used to purify monoclonal antibodies, antibody fragments (Fab), and recombinant antibodies from cell cultures.

Main Results:

• Single-step purification of IgG, IgM, and IgA from human and mouse serum was achieved.
• Purification of monoclonal antibodies, Fab fragments, and chimeric antibodies from hybridoma and bacterial cell cultures was successful.
• Humanized antibodies with engineered Protein L-binding framework regions were purified using this method.

Conclusions:

• Protein L-Sepharose is an effective tool for the single-step purification of various immunoglobulins and antibody fragments.
• Antibodies and antibody fragments can be engineered to incorporate Protein L-binding regions.
• This engineering enables the application of Protein L-based purification protocols for a wider range of antibody formats.