Antibody purification via affinity membrane chromatography method utilizing nucleotide binding site targeting with a small molecule

Abstract

Here, we present an affinity membrane chromatography technique for purification of monoclonal and polyclonal antibodies from cell culture media of hybridomas and ascites fluids. The m-NBST method utilizes the nucleotide-binding site (NBS) that is located on the Fab variable domain of immunoglobulins to enable capturing of antibody molecules on a membrane affinity column via a small molecule, tryptamine, which has a moderate binding affinity to the NBS. Regenerated cellulose membrane was selected as a matrix due to multiple advantages over traditionally used resin-based affinity systems. Rituximab was used for proof of concept experiments. Antibody purification was accomplished by first capture of injected samples while running equilibration buffer (50 mM sodium phosphate pH 7.0), followed by elution achieved by running a gradient of mild elution buffer (3 M NaCl in 50 mM phosphate pH 7.0). The results indicate that the m-NBST column efficiency for Rituximab was >98%, with a purity level of >98%. The quality and the capacity of this small molecule membrane affinity purification method is further evaluated for a number of parameters such as: injection concentrations, volumes, wash/bind time, elution gradient, antibody/protein-contaminant combinations, effects of injection buffer, post-purification antigen binding activity of antibodies, and column reusability and stability.

Enhancement of Binding Affinity of Anti-Hapten Polyclonal IgG Recognizing Mitragynine using Affinity Purification

“…In Figure 3, there was an absence of additional bands in the SDS-PAGE and immunoblot, which verified the purity of the IgG from the rabbit of non-purified anti-sera. These results were according to the previous studies, in which the pure IgG was obtained from polyclonal antibodies through affinity chromatography (Kang et al, 2016;Sadeghi et al, 2018, Mustafaoglu et al, 2016.…”

“…However, the purification technique may and may not enhance the binding affinity of the antibody. As reported by Mustafaoglu et al (2016), purification of antibodies did not improve the binding activity of the antibody due to the antibody activity (including both antigen detection and Fc recognition) was utterly preserved after purification. Limited studies were reported on the difference of binding affinity of purified poly-IgG or non-purified anti-sera in immunoassay.…”

Protein adsorption and desorption behavior of a pH-responsive membrane based on ethylene vinyl alcohol copolymer

“…A predominantly convective transport mechanism of the solutes through the membrane pores to the adsorption sites is superior to the intraparticle diffusion transport mechanism of resinbased chromatography. Until now, various types of membranes have been investigated for protein purication, 1,2 i.e., membrane chromatography based on ion exchange, [3][4][5][6] affinity, [7][8][9] and hydrophobic interactions. 10,11 Stimuli-responsive membrane can change structural and functional properties (i.e.…”

Recent Developments of Liquid Chromatography Stationary Phases for Compound Separation: From Proteins to Small Organic Compounds

“…Sericin, a silkworm polypeptide, upon blending with chitosan membranes with a ratio of 4:1, was capable of recognizing serum albumin and led to a 45% increase in adsorption capacity compared to chitosan alone [ 65 ]. For antibody purification, an alternative for affinity ligands is tryptamine, which can bind the nucleotide-binding site on the Fab domain of the antibodies [ 66 ]. This strategy created both monoclonal and polyclonal antibodies purified from cell culture media of ascites fluids.…”