Affinity ligands for immunoglobulins based on the multicomponent Ugi reaction

Area of Science:

• Biochemistry
• Chemical Engineering
• Protein Chemistry

Background:

• Affinity chromatography is crucial for purifying proteins like immunoglobulins (IgG).
• Existing methods may lack specificity or efficiency for isolating specific IgG fragments.
• The Ugi reaction offers a versatile platform for combinatorial chemistry and ligand development.

Purpose of the Study:

• To develop a novel solid-phase affinity ligand using the Ugi reaction for immunoglobulin purification.
• To create a combinatorial library of ligands for selective binding of IgG and its fragments.
• To optimize and characterize the performance of the developed affinity adsorbent.

Main Methods:

• Utilized a four-component Ugi reaction with an aldehyde-functionalized Sepharose support.
• Generated a combinatorial library of tri-substituted peptoidal scaffolds.
• Screened the ligand library against human IgG, Fc, and Fab fragments using affinity chromatography.
• Optimized ligand density and spacer-arm chemistry for enhanced binding capacity.
• Performed SDS-PAGE and densitometry for purity analysis.
• Determined binding capacity and affinity constants.
• Conducted in silico modeling and docking studies.

Main Results:

• A Fab-specific lead ligand was identified, showing differential binding to Fab over Fc.
• Achieved 100% IgG adsorption and 81.0% purity elution from human serum under non-optimized conditions.
• High purity isolation of Fab and IgG from yeast and E. coli host cell proteins was demonstrated.
• Optimized adsorbent exhibited a dynamic binding capacity of 73.06 mg IgG/ml and static binding capacity of 16.1 mg Fab/ml.
• Affinity constant K(d) was determined to be (2.6±0.3)x10⁻⁶ M for the lead ligand.
• In silico modeling suggested a binding interface involving hydrogen and hydrophobic interactions.

Conclusions:

• The Ugi reaction provides a powerful strategy for generating novel affinity ligands for protein purification.
• The developed Fab-specific ligand demonstrates high efficiency and selectivity for isolating IgG fragments.
• This method offers a robust and scalable approach for purifying immunoglobulins and their fragments in biopharmaceutical applications.