The objective of this study was to develop an efficient peptide ligand affinity adsorbent for the large-scale purification of monoclonal antibodies (Mabs). A linear hexamer peptide HWRGWV that binds with high specificity to the Fc region of immunoglobulin G (IgG) was immobilized on a crosslinked agarose chromatographic support. Various linear and branched spacer arms such as Tris(2-aminoethyl) amine (TREN), 3,3′-Diaminodipropylamine (DADPA), 3,3′-Diamino-N-methyldipropylamine (mDADPA), Hexamethylenediamine (HMDA) and Ethylenediamine (EDA) were evaluated in this study. The resin with TREN spacer arm showed the highest equilibrium binding capacity of about 92 mg/ml of resin for pure polyclonal IgG. The dynamic binding capacity at 3 minute residence time was about 65 mg/ml. Peptide resins were employed to purify Mabs from CHO cell culture supernatants. Binding of Mabs was done under PBS conditions, elution of bound Mabs was achieved using pH 4.5. The regeneration and sanitization of the column was performed using 0.5 M NaOH. SDS-PAGE and SEC analysis showed that purity was &gt; 95% for all the spacer arms. All the resins gave an excellent DNA removal (LRV ~ 4). The host cell protein removal, with a simple PBS wash was in range of LRV 1-1.5. The HCP removal was significantly improved by using post-load washing conditions with either 0.1 M Tris-HCl, pH 9 or with 1 M NaCl. Moreover, the replacement of arginine (R) residue with an analogous non-natural amino acid, Citrulline further improved the HCP removal ability of the peptide adsorbent. The peptide resins also demonstrated an excellent ability to completely remove the Mab aggregates resulting in monomeric Mab species in the elution.

Development of peptide based affinity media for the purification of monoclonal antibodies: Effect of spacer arms, peptide variants and process conditions

251st National Meeting (2016)

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