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

Abstract

Detection of host cell protein (HCP) impurities is critical to ensuring that recombinant drug products, including monoclonal antibodies (mAbs), are safe. Mechanistic characterization as to how HCPs persist in drug products is important to refining downstream processing. It has been hypothesized that weak lipase–mAb interactions enable HCP lipases to evade drug purification processes. Here, we apply state-of-the-art methods to establish lipase-mAb binding mechanisms. First, the mass spectrometry (MS) approach of fast photochemical oxidation of proteins was used to elucidate putative binding regions. The CH1 domain was identified as a conserved interaction site for IgG1 and IgG4 mAbs against the HCPs phospholipase B-like protein (PLBL2) and lysosomal phospholipase A2 (LPLA2). Rationally designed mutations in the CH1 domain of the IgG4 mAb caused a 3- to 70-fold K D reduction against PLBL2 by surface plasmon resonance (SPR). LPLA2-IgG4 mutant complexes, undetected by SPR and studied using native MS collisional dissociation experiments, also showed significant complex disruption, from 16% to 100%. Native MS and ion mobility (IM) determined complex stoichiometries for four lipase-IgG4 complexes and directly interrogated the enrichment of specific lipase glycoforms. Confirmed with time-course and exoglycosidase experiments, deglycosylated lipases prevented binding, and low-molecular-weight glycoforms promoted binding, to mAbs. This work demonstrates the value of integrated biophysical approaches to characterize micromolar affinity complexes. It is the first in-depth structural report of lipase-mAb binding, finding roles for the CH1 domain and lipase glycosylation in mediating binding. The structural insights gained offer new approaches for the bioengineering of cells or mAbs to reduce HCP impurity levels. Abbreviations: CAN, Acetonitrile; AMAC, Ammonium acetate; BFGS, Broyden–Fletcher–Goldfarb–Shanno; CHO, Chinese Hamster Ovary; K D , Dissociation constant; DTT, Dithiothreitol; ELISA, Enzyme-linked immunosorbent assay; FPOP, Fast photochemical oxidation of proteins; FA, Formic acid; F(ab’), Fragment antibodies; HCP, Host cell protein; IgG, Immunoglobulin; IM, Ion mobility; LOD, Lower limit of detection; LPLA2, Lysosomal phospholipase A2; Man, Mannose; MS, Mass spectrometry; MeOH, Methanol; MST, Microscale thermophoresis; mAbs, Monoclonal antibodies; PPT1, Palmitoyl protein thioesterase; ppm, Parts per million; PLBL2, Phospholipase B-like protein; PLD3, Phospholipase D3; PS-20, Polysorbate-20; SP, Sphingomyelin phosphodiesterase; SPR, Surface plasmon resonance; TFA, Trifluoroacetic acid.

Technical advancement and practical considerations of LC-MS/MS-based methods for host cell protein identification and quantitation to support process development

“…Recently, non-covalent mAb-lipase complexes were studied by native MS to demonstrate the direct role of HCP lipase glycosylation in its binding to mAb drugs. 34 The study indicated purification strategies could be implemented to remove lipases, such as using lectins to remove high-mannose HCP glycoforms or specific washes to disrupt electrostatic or hydrophobic interactions. In addition, fast photochemical oxidation of proteins LC-MS provided higher order structure details to confirm the interaction sites between mAb and lipases, suggesting rationally designed mutations in the interaction domain could also decrease the co-purification.…”

“…Therefore, to study potentially high-risk enzymes, we need to take a holistic look and use orthogonal approaches, such as enzyme expression, activity assays, and PS content and free fatty acids assays, to study function, mechanism and potential risks of the hydrolases and ensure drug quality and safety. 12 , 34 , 115 …”

The Recent Advances in the Utility of Microbial Lipases: A Review

“…Initially, this was regarded as an expensive technique. However, new developments in antibody mass production have paved the way for the deployment of immunopurification at the industrial level by using different monoclonal antibodies [ 85 , 86 ]. A study by Rahimi et al [ 87 ] revealed two monoclonal antibodies (MoAbs), BF11 and VNH9, that immobilize lipase obtained from Candida rugosa , wherein both antibodies of the IgG1 isotype recognize specific antigenic determinants shared by different C. rugosa lipase isoforms.…”

Dissecting the structural heterogeneity of proteins by native mass spectrometry

“…Native MS and glycoproteomics methods were recently used to show the different selectivity of N‐glycans using two different lectins (Wu et al, 2019). The technique is also being used to directly study glycoprotein complexes to resolve the contribution of individual glycoforms to binding affinities (Hecht, Mehta, et al, 2022; Wu et al, 2018; Wu et al, 2022; Wu & Robinson, 2022). Figure 5 describes an application to define the composition of the biopharmaceutical follitropin, a heterodimer of two glycosylated subunits.…”