Design, synthesis and application of benzyl-sulfonate biomimetic affinity adsorbents for monoclonal antibody purification from transgenic corn

Abstract

The human anti-human immunodeficiency virus (HIV) antibody 2G12 (mAb 2G12) is one of the most broadly neutralizing antibodies against HIV that recognizes a unique epitope on the surface glycoprotein gp120. In the present work, a limited affinity-ligand library was synthesized and evaluated for its ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn. The affinity ligands were structural fragments of polysulfonate triazine dye Cibacron Blue 3GA (CB3GA) and represent novel lead scaffolds for designing synthetic affinity ligands. Solid phase chemistry was used to synthesize variants of CB3GA lead ligand. One immobilized ligand, bearing 4-aminobenzyl sulfonic acid (4ABS) linked on two chlorine atoms of the triazine ring (4ABS-Trz-4ABS), displayed high affinity for mAb 2G12. Absorption equilibrium, 3D molecular modelling and molecular dynamics simulation studies were carried out to provide a detailed picture of the 4ABS-Trz-4ABS interaction with mAb 2G12. This biomimetic affinity ligand was exploited for the development of a facile two-step purification protocol for mAb 2G12. In the first step of the procedure, mAb 2G12 was purified on an S-Sepharose FF cation exchanger, and in the second step, mAb 2G12 was purified using affinity chromatography on 4ABS-Trz-4ABS affinity adsorbent. Analysis of the antibody preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay showed that the mAb 2G12 was fully active and of sufficient purity suitable for analytical applications.

Protein A chromatography: Challenges and progress in the purification of monoclonal antibodies

“…The fragments ability to bind and purify recombinant mAb 2G12 expressed in transgenic corn was tested; 4‐aminobenzyl sulfonic acid linked on two chlorine atoms of triazine ring showed high affinity for mAbs. A two‐step purification protocol was developed; the first step consisted in purification of mAb 2G12 on a S‐Sepharose(R) FF cation exchanger and then, affinity chromatography on the triazine based affinity adsorbent .…”

Partitioning in aqueous two‐phase systems: Analysis of strengths, weaknesses, opportunities and threats

“…As noted herein cost comparisons can be quite similar for the two approaches. However in the near future there is some expectation that mAb processing costs may be reduced via use of high capacity resins comprising affinity peptide‐based ligands [114, 115] or, alternatively, new mixed mode [116, 117], mimetic ligand [118] or hydroxyapatite resins [117, 133] with improved chemical stability, capacity and selectivity. Insight to the expected reduction in chromatographic processing costs can be gained from protein A based process economy studies [119].…”

Purification of Anti-Interleukin-6 Monoclonal Antibody Using Precipitation and Immobilized Metal-Ion Affinity Chromatography

“…With regard to purification of antibodies, previous studies have used immobilized ligands (e.g. thiophilic and biomimetic ligands), peptides, amino acids, dyes and metal ions or multimodal agents (Boschetti 2001;Bresolin et al 2010;Ghose et al 2013;Gondim et al 2012;Maltezos et al 2014;Rajak et al 2012;Roque et al 2007;Sadavarte et al 2014;Sugita et al 2013;Wolfe et al 2014).…”