Adsorption Of Monoclonal Antibodies Research Articles

Mechanistic Insights into the Adsorption of Monoclonal Antibodies at the Water/Vapor Interface.

Monoclonal antibodies (mAbs) are active components of therapeutic formulations that interact with the water-vapor interface during manufacturing, storage, and administration. Surface adsorption has been demonstrated to mediate antibody aggregation, which leads to a loss of therapeutic efficacy. Controlling mAb adsorption at interfaces requires a deep understanding of the microscopic processes that lead to adsorption and identification of the protein regions that drive mAb surface activity. Here, we report all-atom molecular dynamics (MD) simulations of the adsorption behavior of a full IgG1-type antibody at the water/vapor interface. We demonstrate that small local changes in the protein structure play a crucial role in promoting adsorption. Also, interfacial adsorption triggers structural changes in the antibody, potentially contributing to the further enhancement of surface activity. Moreover, we identify key amino acid sequences that determine the adsorption of antibodies at the water-air interface and outline strategies to control the surface activity of these important therapeutic proteins.

Adsorption of non-ionic surfactant and monoclonal antibody on siliconized surface studied by neutron reflectometry

The adsorption of monoclonal antibodies (mAbs) on hydrophobic surfaces is known to cause protein aggregation and degradation. Therefore, surfactants, such as Poloxamer 188, are widely used in therapeutic formulations to stabilize mAbs and protect mAbs from interacting with liquid-solid interfaces. Here, the adsorption of Poloxamer 188, one mAb and their competitive adsorption on a model hydrophobic siliconized surface is investigated with neutron scattering coupled with contrast variation to determine the molecular structure of adsorbed layers for each case. Small angle neutron scattering measurements of the affinity of Poloxamer 188 to this mAb indicate that there is negligible binding at these solution conditions. Neutron reflectometry measurements of the mAb show irreversible adsorption on the siliconized surface, which cannot be washed off with neat buffer. Poloxamer 188 can be adsorbed on the surface already occupied by mAb, which enables partial removal of some adsorbed mAb by washing with buffer. The adsorption of the surfactant introduces significant conformational changes for mAb molecules that remain on the surface. In contrast, if the siliconized surface is first saturated with the surfactant, no adsorption of mAb is observed. Competitive adsorption of mAb and Poloxamer 188 from solution leads to a surface dominantly occupied with surfactant molecules, whereas only a minor amount of mAb absorbs. These findings clearly indicate that Poloxamer 188 can protect against mAb adsorption as well as modify the adsorbed conformation of previously adsorbed mAb.

Adsorption and recovery issues of recombinant monoclonal antibodies in reversed-phase liquid chromatography.

The poor recovery of large biomolecules is a well-known issue in reversed-phase liquid chromatography. Several papers have reported this problem, but the reasons behind this behavior are not yet fully understood. In the present study, state-of-the-art reversed-phase wide-pore stationary phases were used to evaluate the adsorption of therapeutic monoclonal antibodies. These biomolecules possess molar mass of approximately 150,000 g/mol and isoelectric points between 6.6 and 9.3. Two types of stationary phases were tested, the Phenomenex Aeris Widepore (silica based), with 3.6 μm superficially porous particles, and the Waters Acquity BEH300 (ethylene-bridged hybrid), with 1.7 μm fully porous particles. A systematic investigation was carried out using 11 immunoglobulin G1, G2, and G4 antibodies, namely, panitumumab, natalizumab, cetuximab, bevacizumab, trastuzumab, rituximab, palivizumab, belimumab, adalimumab, denosumab, and ofatumumab. All are approved by the Food and Drug Administration and the European Medicines Agency in various therapeutic indications and are considered as reference antibodies. Several test proteins, such as human serum albumin, transferrin, apoferritin, ovalbumin, and others, possessing a molar mass between 42,000 and 443,000 g/mol were also evaluated to draw reliable conclusions. The purpose of this study was to find a correlation between the adsorption of monoclonal antibodies and their physicochemical properties. Therefore, the impact of isoelectric point, molar mass, protein glycosylation, and hydrophobicity was investigated. The adsorption of intact antibodies on the stationary phase was significantly higher than that of proteins of similar size, isoelectric point, or hydrophobicity. The present study also demonstrates the unique behavior of monoclonal antibodies, contributing some unwanted and unpredictable strong secondary interactions.

AN ATOMIC FORCE MICROSCOPY STUDY IN THE DETECTION OF HEPATITIS B SURFACE ANTIGEN BY ENZYME-LINKED IMMUNOSORBENT ASSAY

Atomic force microscopy (AFM) was employed to study certain enzyme immunoassay steps for the detection of hepatitis B surface antigen (HBsAg). Physical adsorption of monoclonal antibodies (mAb), blocking of surface active sites free of antibodies by neutral proteins, and capture of HBsAg particles by sensitized surfaces were visualized successively in microplate wells of standard immunological plates from various manufacturers. The previously undescribed details such as “etching holes” up to 20 nm in depth were observed on the surface of plates some companies. The quantitative relationships between the optical density (OD) values obtained by enzyme immunoassay (EIA) and the number of antigen–antibody complexes in AFM were calculated.