Structure-based engineering of a monoclonal antibody for improved solubility

Area of Science:

• Biochemistry
• Protein Engineering
• Pharmaceutical Sciences

Background:

• Protein aggregation poses significant challenges in pharmaceutical formulations.
• Aggregation is implicated in the pathogenesis of various diseases.
• Monoclonal antibodies require optimized solubility for therapeutic efficacy.

Purpose of the Study:

• To engineer an anti-IL-13 monoclonal antibody (CNTO607) with improved solubility.
• To investigate structure-based engineering strategies for enhancing antibody solubility.
• To assess the impact of modifications on antibody binding affinity and structural integrity.

Main Methods:

• Structure-based protein engineering approaches were employed.
• Strategies included modifying isoelectric point (pI), decreasing surface hydrophobicity, and reintroducing N-linked glycosylation.
• Binding affinity, solubility, and glycosylation occupancy were assessed using various biochemical and mass spectrometric techniques.

Main Results:

• Modification of pI resulted in a 2-fold increase in solubility.
• Decreased surface hydrophobicity led to moderate solubility improvements.
• Reintroducing an N-linked carbohydrate moiety in a complementarity-determining region (CDR) significantly enhanced solubility while maintaining antigen binding affinity.
• An aggregation 'hot spot' in H-CDR3 was identified but its residues were critical for binding.

Conclusions:

• Structure-based engineering effectively improves antibody solubility.
• Reintroducing N-linked glycosylation into the CDR is a highly effective strategy for enhancing antibody solubility.
• This approach offers a promising method for developing more stable and effective protein-based therapeutics.