@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{kish_sachi_naik_roach_bobay_blackburn_menegatti_carbonell_2017, title={Design, selection, and development of cyclic peptide ligands for human erythropoietin}, volume={1500}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2017.04.019}, abstractNote={This work presents the selection and characterization of erythropoietin (EPO)-binding cyclic peptide ligands. The sequences were selected by screening a focused library of cyclic depsipeptides cyclo[(Nα-Ac)Dap(A)-X1-X6-AE], whose structure and amino acid compositions were tailored to mimic the EPO receptor. The sequences identified through library screening were synthesized on chromatographic resin and characterized via binding-and-elution studies against EPO to select a pool of candidate ligands. Sequences with higher hydrophobicity consistently showed stronger binding to EPO, with the exception of FSLLSH, which was noted for its lower hydrophobicity and high EPO binding. Mutagenesis studies performed on FSLLSH with natural and non-natural amino acid substitutions led to the identification of critical EPO-binding determinants, and the discovery of new peptide ligands. In particular, histidine-scanning mutagenesis performed on three lead sequences yielded the discovery of variants whose EPO-binding is more pH-sensitive, which facilitates EPO recovery. Selected ligands were studied to correlate the elution yield to the salinity of the binding buffer and the elution pH. Elution yields were consistently higher when EPO binding was performed at low ionic strength. The crystal structures of lead cyclic peptides were docked in silico against EPO to estimate the binding affinity in solution. Isotherm adsorption studies performed on FSLLSH indicated that the cyclic version of the ligand (KD = 0.46 μM) has a higher affinity for EPO than its corresponding linear variant (KD = 1.44 μM). Collectively, these studies set the stage for use of the cyclic peptide ligands as EPO purification and detection tools.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Kish, William S. and Sachi, Hiroyuki and Naik, Amith D. and Roach, Matthew K. and Bobay, Benjamin G. and Blackburn, Robert K. and Menegatti, Stefano and Carbonell, Ruben G.}, year={2017}, month={Jun}, pages={105–120} }
 @article{kish_naik_menegatti_carbonell_2013, title={Peptide-based affinity adsorbents with high binding capacity for the purification of monoclonal antibodies}, volume={52}, DOI={10.1021/ie302345w}, abstractNote={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 m...}, number={26}, journal={Industrial & Engineering Chemistry Research}, author={Kish, W. S. and Naik, A. D. and Menegatti, S. and Carbonell, R. G.}, year={2013}, pages={8800–8811} }
 @article{menegatti_ward_naik_kish_blackburn_carbonell_2013, title={Reversible cyclic peptide libraries for the discovery of affinity ligands}, volume={85}, DOI={10.1021/ac401954k}, abstractNote={A novel strategy is presented for the identification of cyclic peptide ligands from combinatorial libraries of reversible cyclic depsipeptides. A method for the solid-phase synthesis of individual cyclic depsipeptides and combinatorial libraries of these compounds is proposed, which employs lactic acid (Lact) and the dipeptide ester (Nα-Ac)-Ser(Ala)- as linkers for dilactonization. Upon alkaline treatment of the beads selected by screening a model library, the cyclic depsipeptides are linearized and released from the solid support to the liquid phase, to be sequenced via single-step tandem mass spectrometry (MS/MS). The protocol presented for library synthesis provides for wide structural diversity. Two model sequences, VVWVVK and AAWAAR, were chosen to present different structural examples for depsipeptide libraries and demonstrate the process of sequence determination by mass spectrometry. Further, a case study using the IgG binding cyclic depsipeptide cyclo[(Nα-Ac)-S(A)-RWHYFK-Lact-E] is presented to demonstrate the process of library screening and sequence determination on the selected beads. Finally, a method is shown for synthesis of the irreversible cyclic peptide corresponding to the proposed depsipeptide structure, to make the ligand stable to the aqueous acid and alkaline conditions encountered in affinity chromatographic applications. The cyclic peptide ligand was synthesized on a poly(methacrylate) resin and used for chromatographic binding of the target IgG.}, number={19}, journal={Analytical Chemistry}, author={Menegatti, S. and Ward, K. L. and Naik, A. D. and Kish, W. S. and Blackburn, R. K. and Carbonell, R. G.}, year={2013}, pages={9229–9237} }
 @article{menegatti_hussain_naik_carbonell_rao_2013, title={mRNA display selection and solid-phase synthesis of Fc-binding cyclic peptide affinity ligands}, volume={110}, DOI={10.1002/bit.24760}, abstractNote={Abstract}, number={3}, journal={Biotechnology and Bioengineering}, author={Menegatti, S. and Hussain, M. and Naik, A. D. and Carbonell, R. G. and Rao, B. M.}, year={2013}, pages={857–870} }
 @article{menegatti_naik_gurgel_carbonell_2012, title={Alkaline-stable peptide ligand affinity adsorbents for the purification of biomolecules}, volume={1245}, DOI={10.1016/j.chroma.2012.04.072}, abstractNote={A strategy of modification of resin surface chemistry is presented to produce hydrophilic peptide-based alkaline-stable affinity adsorbents for the purification of biopharmaceuticals from complex media. In this work, the peptide-based affinity adsorbent HWRGWV-Toyopearl resin for the purification of IgG is presented as an example. When prepared by direct peptide synthesis on the chromatographic matrix, the peptide-based resin showed lability under alkaline conditions. In fact, the regeneration with aqueous 0.1 M NaOH caused the leaching of 40% of the peptide ligand, resulting in a decrease of IgG yield from 85% to 23%. It was found that the ligand leaching was caused by the coupling of a significant amount of peptide by alkaline-labile ester bonds. A method was designed to prevent the formation of ester bonds and allow the synthesis of the ligand exclusively on alkaline-stable bonds. The method consists in activating the hydrophilic base resin, blocking the hydroxyl groups responsible for alkaline lability and performing the peptide synthesis exclusively via alkaline-stable amide bonds. Repeated cycles of IgG purification from a cell culture medium were performed, each followed by cleaning with aqueous NaOH (0.1 M, 0.5 M and 1 M). The IgG yield decreased from 91% to 85% after 200 purification cycles with 0.1 M NaOH. However, the IgG purity remained almost constant at around 95% based on SDS-PAGE analysis. The procedure presented is rapid, efficient and inexpensive and does not require any equipment other than the conventional instrumentation for peptide synthesis. The method also has a broad application since it is valid for any peptide ligand identified for the purification of a biopharmaceutical target.}, journal={Journal of Chromatography A}, author={Menegatti, S. and Naik, A. D. and Gurgel, P. V. and Carbonell, R. G.}, year={2012}, month={Jul}, pages={55–64} }
 @article{naik_menegatti_reese_gurgel_carbonell_2012, title={Process for purification of monoclonal antibody expressed in transgenic Lemna plant extract using dextran-coated charcoal and hexamer peptide affinity resin}, volume={1260}, DOI={10.1016/j.chroma.2012.08.043}, abstractNote={The production of therapeutic proteins using transgenic plants offers several advantages, including low production cost, absence of human pathogens, presence of glycosylation mechanisms, and the ability to fold complex therapeutic proteins into their proper conformation. However, impurities such as phenolic compounds and pigments encountered during purification are quite different from those faced during purification from mammalian cell culture supernatants. This paper deals with the development of a pretreatment and affinity separation process for the purification of a monoclonal antibody from transgenic Lemna plant extract. A pretreatment step is described using dextran-coated charcoal for the removal of pigments and phenolic compounds without reducing the antibody concentration. Then, the peptide affinity ligand HWRGWV coupled to a commercial polymethacrylate resin is used for the capture and purification of MAb from the pretreated plant extract. The final yield and purity of the MAb obtained were 90% and 96% respectively. The performance of the hexamer peptide resin after the pretreatment step was found to be similar to that obtained with a commercial Protein A resin.}, journal={Journal of Chromatography A}, author={Naik, A. D. and Menegatti, S. and Reese, H. R. and Gurgel, P. V. and Carbonell, R. G.}, year={2012}, pages={61–66} }
 @article{naik_menegatti_gurgel_carbonell_2011, title={Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media}, volume={1218}, DOI={10.1016/j.chroma.2010.11.071}, abstractNote={Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2 M guanidine–HCl or a combination of 0.85% phosphoric acid followed by 2 M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20 mg/mL.}, number={13}, journal={Journal of Chromatography A}, author={Naik, A. D. and Menegatti, S. and Gurgel, P. V. and Carbonell, R. G.}, year={2011}, month={Apr}, pages={1691–1700} }