Effects of Temperature, Flow Rate and Composition of Binding Buffer on Adsorption of Mouse Monoclonal IgG₁Antibodies to Protein a Sepharose 4 Fast Flow

Abstract

The binding capacity of protein A Sepharose 4 Fast Flow for mouse IgG1 monoclonal antibodies (mabs) appears to be highly dependent on the buffer composition with respect to both concentration and ion type. Depending on the particular mab dynamic binding capacities up to 20 mg mab per ml gel could be obtained, when these mabs were isolated from supernatants of protein free hollow fibre cell culture systems. Variation of linear flow rate from 10 up to 300 cm/h and temperature (4 degrees C versus 25 degrees C) had a slight effect on the dynamic binding capacity, when a high ionic strength buffer was used during adsorption. Applying optimum binding conditions, final IgG fractions with a purity of more than 95% monomeric IgG were obtained. However, as side effect of the use of binding buffers with high ionic strength, the binding of acid proteases was also promoted.

Evaluation of commercial chromatographic adsorbents for the direct capture of polyclonal rabbit antibodies from clarified antiserum

“…3), despite the doubling in Ig concentration and increased ionic strength of the latter feedstock ( Table 3). The strength and specificity of interaction between immunoglobulins and Protein A is reported to be dependent on ionic strength, where increasing the ionic strength of the feedstock is thought to simultaneously reduce electrostatic repulsion and enhance hydrophobic interaction between the ligand-target pair [7,33]. In this work we have found no evidence for enhanced Ig binding capacity at increased ionic strengths.…”

Possible Chemical Source of Discrepancy between in Vitro and in Vivo Tests in Nanotoxicology Caused by Strong Adsorption of Buffer Components

“…6 Despite the equivalent functional behavior, these two buffering agents are significantly different in chemical nature. [8][9][10] More recently, the effect of buffer on protein adsorption at the solid/liquid interface has been investigated on flat surfaces as TiO 2 films 11,12 and those of germanium and crystalline silica present into ATR/FTIR cells and quartz microbalance respectively. In turn this may influence the fate of the adsorption at the nanoparticle surface of other species present in the incubation media, typically proteins, through the formation of the so called "protein corona" which in some cases constitute the actual surface "seen" by cells.…”

“…The crucial role played by the buffer composition in protein adsorption has been recognized since the development of chromatographic protein separation. − More recently, the effect of buffer on protein adsorption at the solid/liquid interface has been investigated on flat surfaces such as TiO 2 films , and those of germanium and crystalline silica present in ATR/FTIR cells and quartz microbalance, respectively. , Focusing on nanoparticles, phosphate species in PBS were recognized to play a detrimental role against the antioxidant activity of CeO 2 NPs toward DNA, while silver NPs grown in HEPES buffer exerted cytoprotective activity toward HIV-1 infected cells . Both effects might be partly related to the interaction of the buffer with the particle surface.…”

Industrial Purification of Pharmaceutical Antibodies: Development, Operation, and Validation of Chromatography Processes

“…the antibody concentration in the load, the column temperature and column length, the buffer, conductivity) and pH of the load, and the flow rate (Katoh et aI., 1978;Tu et al, 1988;Fuglistaller, 1989;Kamiya et al, 1990;Kang and Ryu, 1991;Schuler and Reillacher, 1991;Van Sommeren et al, 1992). Of these variables~the simplest to control for production and the ones that will have the most significant impact on capacity are the column length, the flow rate.…”