@article{islam_naik_hashimoto_menegatti_carbonell_2019, title={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}, volume={20}, ISSN={["1422-0067"]}, DOI={10.3390/ijms20010161}, abstractNote={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.}, number={1}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Islam, Tuhidul and Naik, Amith D. and Hashimoto, Yasuhiro and Menegatti, Stefano and Carbonell, Ruben G.}, year={2019}, month={Jan} }
 @article{naika_islam_terasaka_ohara_hashimoto_menegatti_carbonell_2019, title={Silica resins and peptide ligands to develop disposable affinity adsorbents for antibody purification}, volume={145}, ISSN={["1873-295X"]}, DOI={10.1016/j.bej.2018.07.011}, abstractNote={A study is presented on the use of porous silica resins with tailored properties to develop affinity adsorbents for the purification of immunoglobulin G (IgG). Chromatorex® silica resins were utilized to study the dependence of IgG binding upon functional group density, pore size, and specific surface area. The IgG-binding peptide HWRGWV was chosen to demonstrate the potential of combining inexpensive substrates and ligands into efficient, yet disposable, adsorbents. The static binding capacity (SBC) of silica-peptide adsorbents depends significantly on surface area and pore size, yet minimally on ligand density. Chromatorex®-NH2 MB 800 HC (pore size 800 Å, surface area 31 m2/g) and MB 700 HC (700 Å, 44 m2/g) showed SBC of 55 and 75 mg IgG per mL resin, respectively. The dynamic binding capacity (DBC) reached values of up to 60 mg/mL at 5 min residence time, and was found to be almost independent of flow rate, thus offering a much higher productivity (capacity vs. residence time) than Sepharose resins. A selected adsorbent was utilized for purifying monoclonal antibodies from Chinese hamster ovary (CHO) cell culture supernatants, and polyclonal antibodies from llama and rabbit serum. Under optimized conditions, the silica-peptide adsorbent gave a Mab purity above 90%, 4 log removal of host cell DNA, 1.5 log removal of host cell proteins (HCPs) and Mab recovery of 89% and 92%. Similarly, llama and rabbit IgG were isolated at 80%–85% purity. These results demonstrate that porous silica, a non-traditional substrate for protein purification, shows great promise as potentially single-use affinity adsorbent for protein purification.}, journal={BIOCHEMICAL ENGINEERING JOURNAL}, author={Naika, Amith D. and Islam, Tuhidul and Terasaka, Takaaki and Ohara, Yuki and Hashimoto, Yasuhiro and Menegatti, Stefano and Carbonell, Ruben}, year={2019}, month={May}, pages={53–61} }
 @article{menegatti_bobay_ward_islam_kish_naik_carbonell_2016, title={Design of protease-resistant peptide ligands for the purification of antibodies from human plasma}, volume={1445}, DOI={10.1016/j.chroma.2016.03.087}, abstractNote={A strategy is presented for developing variants of peptide ligands with enhanced biochemical stability for the purification of antibodies from animal sera. Antibody-binding sequences HWRGWV, HYFKFD, and HFRRHL, previously discovered by our group, were modified with non-natural amino acids to gain resistance to proteolysis, while maintaining target affinity and selectivity. As trypsin and α-chymotrypsin were chosen as models of natural proteolytic enzymes, the basic (arginine and lysine) and aromatic (tryptophan, phenylalanine, and tyrosine) amino acids were replaced with non-natural analogs. Using the docking software HADDOCK, a virtual library of peptide variants was designed and screened in-silico against the known HWRGWV binding site on the pFc fragment of IgG. A pool of selected sequences with the highest predicted free energy of binding was synthesized on chromatographic resin, and the resulting adsorbents were tested for IgG binding and resistance to proteases. The ligand variants exhibited binding capacities and specificities comparable to the original sequences, yet with much higher proteolytic resistances. The sequences HWMetCitGWMetV and HFMetCitCitHL was used for purifying polyclonal IgG from IgG-rich fractions of human plasma, with yields and purity above 90%. Notably, due to electrical neutrality, the variant showed higher selectivity than the original sequence. Binding isotherms were also constructed, which confirmed the docking predictions. This method represents a general strategy for enhancing the biochemical stability as well as the affinity and selectivity of natural or synthetic peptide ligands for bioseparations.}, journal={Journal of Chromatography A}, author={Menegatti, S. and Bobay, B. G. and Ward, K. L. and Islam, T. and Kish, W. S. and Naik, A. D. and Carbonell, R. G.}, year={2016}, month={May}, pages={93–104} }