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

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 KD 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; KD, 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.

Understanding Loss of Soluble High Molecular Weight Species during Filtration of Low Concentration Therapeutic Monoclonal Antibodies.

Sterile filtration is an integral step in the manufacturing process of biological therapeutics. Protein adsorption to the surface of the filter is an unfortunate, common occurrence that can result in manufacturing difficulties, such as filter fouling or product loss. Although many filters have surface modifications to minimize adsorption, under certain conditions binding can still occur. We observed the loss of high molecular weight species (HMWS) during sterile filtration of eight different therapeutic monoclonal antibodies formulated at low protein concentrations across a commonly used hydrophilic polyvinylidene fluoride or polyvinylidene difluoride (PVDF) filter membrane. The protein absorption was specific to HMWS, and each antibody exhibited different degrees of filter adsorption. Debye screening length parameters of the solution (e.g. ionic strength) were adjusted, and influenced the amount of HMWS lost during filtration. Additionally, HMWS of a representative antibody (mAb1) were observed to be more positively charged than other size variants by ion-exchange chromatography. From these results, it is concluded that this HMWS loss is due to electrostatic interactions between HMWS and the filter surface. This adsorption can be reduced by increasing the ionic strength of the buffer matrix, demonstrating the influence of the Debye screening length in the filtration of low concentration proteins.