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.

Tuhidul Islam,Stefano Menegatti,Ruben G Carbonell,Yasuhiro Hashimoto,Amith D Naik

doi:10.3390/ijms20010161

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.

Highlights

Introduction140–200 billion dollars by 2022 [1]
With over twenty products approved by the FDA and hundreds in clinical trials, monoclonal antibodies (Mabs) represent the dominant class of biotherapeutics, with a global market estimated at140–200 billion dollars by 2022 [1]
The peptide ligands were subsequently immobilized to the aminated resin via iodoacetic acid (IAA) coupling chemistry as described in Section 3.2 (Figure 1)

Summary

Introduction

140–200 billion dollars by 2022 [1] These Mab products are very expensive and unaffordable for people in emerging markets. 50–80% of the cost of goods (COGS) for Mabs [2,3] there is an interest to look into new alternative technologies and process improvements that can reduce the costs [4,5]. Several chromatographic and non-chromatographic techniques have been proposed for the purification of Mabs, fc fusion proteins and bispecifics [6,7,8,9] including efforts towards identifying alternatives to Protein A-based chromatography [10,11]. While being regarded as the standard for antibody purification owing to their high affinity and selectivity, these ligands present limitations, including high cost and the potential release of immunogenic fragments. An additional issue with Protein A concerns the presence of antibody aggregates in eluted fraction and their poor selectivity towards monomeric and correctly folded versus misfolded and/or aggregated IgG [12,13]

Objectives

Methods

Results

Conclusion