Combinatorial de novo design and application of a biomimetic affinity ligand for the purification of human anti‐HIV mAb 4E10 from transgenic tobacco

Abstract

Monoclonal anti-HIV antibody 4E10 (mAb 4E10) is one of the most broadly neutralizing antibodies against HIV, directed against a specific epitope on envelope protein gp41. In the present study, a combinatorial de novo design approach was used for the development of a biomimetic ligand for the affinity purification of mAb 4E10 from tobacco transgenic extract in a single chromatographic step. The biomimetic ligand (4E10lig) was based on a L-Phe/beta-Ala bi-substituted 1,3,5-triazine (Trz) scaffold (beta-Ala-Trz-L-Phe, 4E10lig) which potentially mimics the more pronounced electrostatic and hydrophobic interactions of mAb 4E10-binding sequence determined by screening of a random peptide library. This library was comprised of Escherichia coli cells harboring a plasmid (pFlitrx) engineered to express a fusion protein containing random dodecapeptides that were inserted into the active loop of thioredoxin, which itself was inserted into the dispensable region of the flagellin gene. Adsorption equilibrium studies with this biomimetic ligand and mAb 4E10 determined a dissociation constant (K(D)) of 0.41 +/- 0.05 microM. Molecular modeling studies of the biomimetic ligand revealed that it can potentially occupy the same binding site as the natural binding core peptide epitope. The biomimetic affinity adsorbent was exploited in the development of a facile mAb 4E10 purification protocol, affording mAb 4E10 of high purity (approximately 95%) with good overall yield (60-80%). Analysis of the antibody preparation by SDS-PAGE, enzyme-linked immunosorbent assays (ELISA), and western blot showed that the mAb 4E10 was fully active and free of degraded variants, polyphenols, and alkaloids.

Extracellular expression and affinity purification of L-asparaginase from E. chrysanthemi in E. coli

“…The calculated dissociation constant was estimated to be K D = 0.21 μM. This value falls within the range expected for a highly selective affinity adsorbent [35][36][37]. The maximum binding capacity q max was estimated 4.7 mg/g moist wet adsorbent.…”

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

“…However, it suffers from problems, such as ligand instability, leaching and high cost because Protein A and Protein G resins are very expensive and readily amenable to chemical and biological degradation (Jiang et al ., ; Ren et al ., ). On the other hand, alternative conventional chromatographic methods, such as ion exchange and biomimetic‐ligand affinity chromatography (Li et al ., ; Branco et al ., ) are suitable for large‐scale chromatography, because they are inexpensive, resistant to chemical or biological degradation, and display high protein‐binding capacity (Platis et al ., ; Platis and Labrou, ; Platis et al ., ; Liu et al ., ; Menegatti et al ., ).…”

“…One of the major challenges in the molecular pharming industry is the development of efficient purification protocols for the production of active recombinant mAbs from transgenic plants Platis et al, 2009;Barros et al, 2011;McLean et al, 2012). In order to satisfy this growing demand, to increase the production yields, and lower the production costs, alternative purification systems based on affinity chromatography are being actively developed (Horak et al, 2010;Arnold et al, 2011;Dinon et al, 2011;Naik et al, 2011;Lund et al, 2012).…”