Antibody Binding Heterogeneity of Protein A Resins (original) (raw)
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Journal of Chromatography A, 2009
Dynamic binding capacity (DBC) of a monoclonal antibody on agarose based strong cation exchange resins is determined as a function of resin ligand density, apparent pore size of the base matrix, and protein charge. The maximum DBC is found to be unaffected by resin ligand density, apparent pore size, or protein charge within the tested range. The critical conductivity (conductivity at maximum DBC) is seen to vary with ligand density. It is hypothesized that the maximum DBC is determined by the effective size of the proteins and the proximity to which they can approach one another. Once a certain minimum resin ligand density is supplied, additional ligand is not beneficial in terms of resin capacity. Additional ligand can provide flexibility in designing ion exchange resins for a particular application as the critical conductivity could be matched to the feedstock conductivity and it may also affect the selectivity.
… of Chromatography B, 2012
An integrated experimental and modeling approach for the design of high productivity protein A chromatography is presented to maximize productivity in bioproduct manufacture. The approach consists of four steps: (1) small-scale experimentation, (2) model parameter estimation, (3) productivity optimization and (4) model validation with process verification. The integrated use of process experimentation and modeling enables fewer experiments to be performed, and thus minimizes the time and materials required in order to gain process understanding, which is of key importance during process development. The application of the approach is demonstrated for the capture of antibody by a novel silica-based high performance protein A adsorbent named AbSolute. In the example, a series of pulse injections and breakthrough experiments were performed to develop a lumped parameter model, which was then used to find the best design that optimizes the productivity of a batch protein A chromatographic process for human IgG capture. An optimum productivity of 2.9 kg L −1 day −1 for a column of 5 mm diameter and 8.5 cm length was predicted, and subsequently verified experimentally, completing the whole process design approach in only 75 person-hours (or approximately 2 weeks).
Journal of Chromatography A, 2020
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… of Chromatography A, 2009
Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand-ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.
Journal of Chromatography A, 2020
A fast method for assessing the stability of monoclonal antibodies (mAbs) adsorbed on ion exchange resins has been developed. The method exploited a real time polymerase chain reaction equipment to determine the temperature of protein phase transition, i.e., the so called melting temperature, based on differential scanning fluorimetry. Changes to the melting temperature were screened under various adsorption conditions and correlated with the protein stability upon adsorption. The method was tested for two different mAbs bound to various types of strong cation exchangers at different pH and loading concentrations. The mAbs destabilized upon adsorption due to strong binding, which manifested itself in aggregate formation and recovery reduction. The phenomenon depended on the resin type and binding conditions. However, regardless of the process conditions and resins used, drop in the melting temperatures to a critical value of about 30 °could serve as an indicator of destructive changes in the protein structure in the adsorbed phase. The measurements were simultaneously accomplished for a number of samples with very small material consumption. Therefore, the method may be applied for screening resins and operating variables for a given mAb to exclude conditions that induce structure destabilization and aggregation.
In situ neutron scattering of antibody adsorption during protein A chromatography
Journal of Chromatography A
A deeper understanding of the nanoscale and mesoscale structure of chromatographic adsorbents and the distribution of proteins within the media, is critical to a mechanistic understanding of separation processes using these materials. Characterisation of the media's architecture at this scale and protein adsorption within, is challenging using conventional techniques. In this study, we propose a novel resin characterisation technique that enables in-situ measurement of the structure of the adsorbed protein layer within the resin, under typical chromatographic conditions. A quartz flow-through cell was designed and fabricated for use with Small Angle Neutron Scattering (SANS), in order to measure the nanoscale to mesoscale structures of a silica based protein A chromatography resin during the monoclonal antibody sorption process. We were able to examine the pore-to-pore (˜133 nm) and pore size (˜63 nm) correlations of the resin and the in-plane adsorbed antibody molecules (˜4.2 nm) correlation at different protein loadings and washing buffers, in real time using a contrast matching approach. When 0.03 M sodium phosphate with 1 M urea and 10 % isopropanol buffer, pH 8, was introduced into the system as a wash buffer, it disrupted the system's order by causing partial unfolding of the adsorbed antibody, as evidenced by a loss of the in-plane protein correlation. This method offers new ways to investigate the nanoscale structure and ligand immobilisation within chromatography resins; and perhaps most importantly understand the in-situ behaviour of adsorbed proteins within the media under different mobile phase conditions within a sample environment replicating that of a chromatography column.
Analytical Chemistry, 2012
The conserved nucleotide binding site (NBS), found within the Fab variable domain of antibodies, remains a not-so-widely known and underutilized site. Here we describe a novel affinity chromatography method that utilizes the NBS as a target for selectively purifying antibodies from complex mixtures. The affinity column was prepared by coupling indole butyric acid (IBA), which has a monovalent affinity for the NBS with a K d ranging between 1 and 8 μM, to ToyoPearl resin resulting in the NBS targeting affinity column (NBS IBA). The proof-of-concept studies performed using the chimeric pharmaceutical antibody rituximab demonstrated that antibodies were selectively captured and retained on the NBS IBA column and were successfully eluted by applying a mild NaCl gradient at pH 7.0. Furthermore, the NBS IBA column consistently yielded >95% antibody recovery with >98% purity, even when the antibody was purified from complex mixtures such as conditioned cell culture supernatant, hybridoma media, and mouse ascites fluid. The results presented in this study establish the NBS IBA column as a viable small-molecule-based affinity chromatography method for antibody purification with significant implications in industrial antibody production. Potential advantages of the NBS IBA platform are improved antibody batch quality, enhanced column durability, and reduced overall production cost.