Optimization of Sequence, Display, and Mode of Operation of IgG-Binding Peptide Ligands to Develop Robust, High-Capacity Affinity Adsorbents That Afford High IgG Product Quality

Abstract

This work presents the use of peptide ligand HWRGWV and its cognate sequences to develop affinity adsorbents that compete with Protein A in terms of binding capacity and quality of the eluted product. First, the peptide ligand was conjugated to crosslinked agarose resins (WorkBeads) at different densities and using different spacer arms. The optimization of ligand density and display resulted in values of static and dynamic binding capacity of 85 mg/mL and 65 mg/mL, respectively. A selected peptide-WorkBeads adsorbent was utilized for purifying Mabs from Chinese Hamster Ovary (CHO) cell culture supernatants. The peptide-WorkBeads adsorbent was found able to withstand sanitization with strong alkaline solutions (0.5 M NaOH). The purity of the eluted product was consistently higher than 95%, with logarithmic removal value (LRV) of 1.5 for host cell proteins (HCPs) and 4.0 for DNA. HCP clearance was significantly improved by adding a post-load washing step with either 0.1 M Tris HCl pH 9 or 1 M NaCl. The cognate peptide of HWRGWV, constructed by replacing arginine (R) with citrulline, further increased the HCP LRV to 2.15. The peptide-based adsorbent also showed a remarkable performance in terms of removal of Mab aggregates; unlike Protein A, in fact, HWRGWV was found to bind only monomeric IgG. Collectively, these results demonstrate the potential of peptide-based adsorbents as alternative to Protein A for the purification of therapeutic antibodies.

Peptides, Antibodies, Peptide Antibodies and More

“…Similar results have been obtained with many other MAbs, which verifies the stringent requirements for an optimal antibody-antigen interaction [33,46,96,97]. MAbs may be used as crude culture supernatants (which usually contain bovine Ig) or after purification, e.g., on protein A/G [94,98] or synthetic resins, as illustrated by Islam et al in this issue [90]. However, it is important to remember, that such purification methods may not remove the bovine IgG, which originates from the cell culture medium [99,100].…”

“…After immunization and boosting, PAbs are obtained, while recognizing different epitopes of an antigen. Upon prolonged immunization, specific clones may become dominant, which increases the specificity of the Abs, and immuno-affinity purification may be used to obtain monospecific PAbs [90]. In the case of a natural infection, the spectrum of PAbs can be characterised by proteomic methods, e.g., after isolation of IgG, as illustrated by Lundström et al in this special issue (SpotLight proteomics) [91] and used for production of recombinant Abs.…”

“…Other notable examples are recombinant MAbs to tumour necrosis factor, TNF, and programmed death-1, PD-1, used for treating rheumatoid arthritis and malignant melanoma, respectively [74,75]. Recombinant Abs may be produced in various systems, including mammalian cell lines, yeasts, and phage display [90,113]. For phage display, engineered single chain fragment variable (scFv) antibodies are a preferred format due to the reduced size, which makes expression and production easier.…”

A novel dextran‐grafted tetrapeptide resin for antibody purification

“…The results of Section 3.3 showed pH 9.0 was the optimal condition for the binding of the resin and hIgG. Moreover, the condition of pH 9.0 has commonly used in antibody separation [4,24,25]. For example, Tuhidul Islam et al.…”

“…Moreover, the most serious environmental (pH 9.0) chosen in this article was discovered as a common condition used in antibody purification. And this condition could separate protein that cannot be easily separated under neutral or acidic conditions without damaging its structure [4,24,25].…”