Insights into ultra-low affinity lipase-antibody noncovalent complex binding mechanisms

Area of Science:

• Biochemistry and Biophysics
• Protein-Ligand Interactions
• Biopharmaceutical Manufacturing

Background:

• Host cell protein (HCP) impurities, particularly lipases, pose a safety risk in recombinant monoclonal antibody (mAb) drug products.
• Understanding the mechanisms of lipase-mAb interactions is crucial for optimizing downstream purification processes and ensuring drug safety.

Purpose of the Study:

• To elucidate the binding mechanisms between HCP lipases and monoclonal antibodies (mAbs).
• To identify specific protein domains and post-translational modifications involved in lipase-mAb interactions.
• To provide structural insights for developing strategies to reduce HCP impurities in biopharmaceuticals.

Main Methods:

• Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry (MS) to identify binding regions.
• Surface plasmon resonance (SPR) and native MS with collisional dissociation to characterize binding affinities and complex stability.
• Ion mobility (IM) and exoglycosidase experiments to determine complex stoichiometries and the role of glycosylation.

Main Results:

• The CH1 domain of IgG1 and IgG4 mAbs was identified as a conserved binding site for phospholipase B-like protein (PLBL2) and lysosomal phospholipase A2 (LPLA2).
• Mutations in the CH1 domain significantly reduced binding affinity (3- to 70-fold K_D reduction).
• Lipase glycosylation was found to be critical, with deglycosylated forms preventing binding and specific glycoforms promoting it.

Conclusions:

• This study provides the first in-depth structural report of lipase-mAb binding, highlighting the roles of the CH1 domain and lipase glycosylation.
• Integrated biophysical approaches are effective for characterizing low-affinity protein complexes.
• The findings offer novel strategies for bioengineering cells or mAbs to minimize HCP impurities in biopharmaceutical production.