Peptide-Based Affinity Adsorbents with High Binding Capacity for the Purification of Monoclonal Antibodies

Abstract

High binding capacity and selectivity are key features for the successful application of affinity adsorbents for antibody purification. This study presents the development of affinity resins based on hexapeptide ligand HWRGWV for recovering monoclonal antibodies from cell culture fluids. Methods are presented for the immobilization of the peptide ligand and its variants on polymethacrylate and agarose based chromatographic supports using two main coupling strategies. The first one involves the formation of a peptide bond between the amino groups on the substrate and the peptide C-terminus activated with the uronium coupling agent HATU. The second approach involves resin activation with iodoacetic acid, followed by coupling of a cysteine-terminated variant of the ligand to form a thioether bond. The reaction conditions of peptide coupling were optimized to maximize the binding capacity of the resulting adsorbents. The peptide resins were characterized by measuring their static IgG binding capacities. The measured static binding capacity ranged from 35 to 48 mg/mL. The dynamic binding capacities (DBC) of four selected adsorbents were also determined, and they ranged from of 35 to 42 mg/mL with a 5-min residence time. All the resins exhibited high selectivity toward the Fc fragment of IgG. The affinity resins were used to purify two MAbs, a chimeric IgG1 and a humanized IgG4, from commercial CHO cell culture fluids. The resulting yields and purities for both MAbs were found to be in the range of 87–93% and >94%, respectively, which compare well with the purity and yield values obtained using commercially available Protein A media. Finally, the peptide resin with the highest IgG binding capacity, HWRGWVC-WorkBeads, was tested for 20 DBC cycles which included cleaning in place with 0.1 M NaOH after every cycle. The resin showed a high degree of reusability and alkaline stability, as it maintained 90% of its initial capacity.

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

“…Briefly, IAA was activated with EDC and incubated with the WB resin, for 3 h, at room temperature, under mild stirring. The resulting iodoacetyl-WB resin was incubated with peptide HWRGWVC in DMF, overnight, at room temperature; the conjugation occurred by substitution between the thiol group displayed by the cysteine residue (C) on the HWRGWVC peptide and the iodoacetyl groups on the WB resin [32]. The supernatant was collected and analyzed by UV-Vis spectrophotometry at 280 nm to determine the amount of unconjugated peptide, from which the ligand density on the WB resin was derived.…”

“…There has been an increasing interest among the biotechnological industries to use carbohydrate chromatographic supports, such as agarose or cellulose, as these resins can be modified easily to attach ligand and exhibit low non-specific binding for host cell proteins [31]. Preliminary studies of coupling HWRGWV to an agarose base resin, WorkBeads ACT (Bio-Works, Uppsala, Sweden) had resulted in a higher IgG binding capacity as compared to polymethcarylate base resin [32]. In this work the peptide and peptide variants have been conjugated to Workbeads base resin at varying ligand densities and tested for binding capacity, selectivity and stability.…”

Rational design of peptide affinity ligands for the purification of therapeutic enzymes

“…Peptides were synthesized directly onto two chromatographic matrices, WorkBeads 40 ACT and Toyopearl Amino‐650M, which have been previously reported as suitable materials for affinity peptide ligands . Based on the limited yield from these initial synthesis reactions, resins were evaluated in batch chromatography studies with 5 μl of resin per well.…”

“…Lead peptides selected for batch and column studies were synthesized on two chromatography resin supports: WorkBeads 40 ACT (Bio‐Works Technologies AB, Sweden) and Toyopearl Amino‐650M (Tosoh Bioscience LLC, South San Francisco, CA). Toyopearl Amino‐650M resin was functionalized as described by Menegatti et al, while WorkBeads 40 ACT resin was functionalized as described by Kish et al Peptides were subsequently synthesized in situ on the functionalized resins using standard Fmoc chemistry on an automated parallel peptide synthesizer (Multiprep, Intavis Bioanalytical Instruments AG, Germany) as described before. After synthesis, peptide‐resin conjugates were treated with a standard workup procedure for side chain de‐protection as described before.…”

“…Peptides were synthesized directly onto two chromatographic matrices, WorkBeads 40 ACT and Toyopearl Amino-650M, which have been previously reported as suitable materials for affinity peptide ligands. 34 Based on the limited yield from these initial synthesis reactions, resins were evaluated in batch chromatography studies with 5 ll of resin per well. An initial challenge with pure target in PBS showed a significant amount of binding to both the low and high affinity peptide ligands, but minimal binding to the glycine-conjugated control ( Figure 8A).…”