@article{oh_becker_mendola_choe_min_lee_yigzaw_seay_bill_li_et al._2024, title={Factors affecting product association as a mechanism of host-cell protein persistence in bioprocessing}, volume={1}, ISSN={["1097-0290"]}, DOI={10.1002/bit.28658}, abstractNote={Abstract}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Oh, Young Hoon and Becker, Matthew L. and Mendola, Kerri M. and Choe, Leila H. and Min, Lie and Lee, Kelvin H. and Yigzaw, Yinges and Seay, Alexander and Bill, Jerome and Li, Xuanwen and et al.}, year={2024}, month={Jan} }
 @article{altern_kocot_lebarre_boi_phillips_roush_menegatti_cramer_2024, title={Mechanistic model-based characterization of size-exclusion-mixed-mode resins for removal of monoclonal antibody fragments}, volume={1718}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2024.464717}, abstractNote={Although antibody fragments are a critical impurity to remove from process streams, few platformable purification techniques have been developed to this end. In this work, a novel size-exclusion-mixed-mode (SEMM) resin was characterized with respect to its efficacy in mAb fragment removal. Inverse size-exclusion chromatography showed that the silica-based resin had a narrow pore size distribution and a median pore radius of roughly 6.2 nm. Model-based characterization was carried out with Chromatography Analysis and Design Toolkit (CADET), using the general rate model and the multicomponent Langmuir isotherm. Model parameters were obtained from fitting breakthrough curves, performed at multiple residence times, for a mixture of mAb, aggregates, and an array of fragments (varying in size). Accurate fits were obtained to the frontal chromatographic data across a range of residence times. Model validation was then performed with a scaled-up column, altering residence time and feed composition from the calibration run. Accurate predictions were obtained, thereby illustrating the model's interpolative and extrapolative capabilities. Additonally, the SEMM resin achieved 90% mAb yield, 37% aggregate removal, 29% F(ab′)2 removal, 54% Fab/Fc removal, 100% Fc fragments removal, and a productivity of 72.3 g mAbL×h. Model predictions for these statistics were all within 5%. Simulated batch uptake experiments showed that resin penetration depth was directly related to protein size, with the exception of the aggregate species, and that separation was governed by differential pore diffusion rates. Additional simulations were performed to characterize the dependence of fragment removal on column dimension, load density, and feed composition. Fragment removal was found to be highly dependent on column load density, where optimal purification was achieved below 100 mg protein/mL column. Furthermore, fragment removal was dependent on column volume (constant load mass), but agnostic to whether column length or diameter was changed. Lastly, the dependence on feed composition was shown to be complex. While fragment removal was inversely related to fragment mass fraction in the feed, the extent depended on fragment size. Overall, the results from this study illustrated the efficacy of the SEMM resin in fragment and aggregate removal and elucidated relationships with key operational parameters through model-based characterization.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Altern, Scott H. and Kocot, Andrew J. and Lebarre, Jacob P. and Boi, Cristiana and Phillips, Michael W. and Roush, David J. and Menegatti, Stefano and Cramer, Steven M.}, year={2024}, month={Mar} }
 @article{barbieri_mollica_moore_sripada_shastry_kilgore_loudermilk_whitacre_kilgour_wuestenhagen_et al._2024, title={Peptide ligands targeting the vesicular stomatitis virus G (VSV-G) protein for the affinity purification of lentivirus particles}, volume={121}, ISSN={["1097-0290"]}, DOI={10.1002/bit.28594}, abstractNote={Abstract}, number={2}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Barbieri, Eduardo and Mollica, Gina N. and Moore, Brandyn D. and Sripada, Sobhana A. and Shastry, Shriarjun and Kilgore, Ryan E. and Loudermilk, Casee M. and Whitacre, Zachary H. and Kilgour, Katie M. and Wuestenhagen, Elena and et al.}, year={2024}, month={Feb}, pages={618–639} }
 @article{turner_twiddy_wilkins_ramesh_kilgour_domingos_nasrallah_menegatti_daniele_2023, title={Biodegradable elastomeric circuit boards from citric acid-based polyesters}, volume={7}, ISSN={["2397-4621"]}, DOI={10.1038/s41528-023-00258-z}, abstractNote={Abstract}, number={1}, journal={NPJ FLEXIBLE ELECTRONICS}, author={Turner, Brendan L. and Twiddy, Jack and Wilkins, Michael D. and Ramesh, Srivatsan and Kilgour, Katie M. and Domingos, Eleo and Nasrallah, Olivia and Menegatti, Stefano and Daniele, Michael A.}, year={2023}, month={Jun} }
 @article{li_menegatti_crook_2023, title={Breakdown of polyethylene therepthalate microplastics under saltwater conditions using engineered Vibrio natriegens}, volume={9}, ISSN={["1547-5905"]}, url={https://doi.org/10.1002/aic.18228}, DOI={10.1002/aic.18228}, abstractNote={Abstract}, journal={AICHE JOURNAL}, author={Li, Tianyu and Menegatti, Stefano and Crook, Nathan}, year={2023}, month={Sep} }
 @article{sohail_pirzada_guenther_barbieri_sit_menegatti_crook_opperman_khan_2023, title={Cellulose Acetate-Stabilized Pickering Emulsions: Preparation, Rheology, and Incorporation of Agricultural Active Ingredients}, volume={11}, ISSN={["2168-0485"]}, url={https://doi.org/10.1021/acssuschemeng.3c02428}, DOI={10.1021/acssuschemeng.3c02428}, abstractNote={We report the use of cellulose acetate (CA) nanoparticles (NPs) to produce oil in water Pickering emulsions. The CA NP can emulsify various oils and form stable emulsions at concentrations as low as 0.5 wt %. Rheological and microscopic analyses show evidence of interconnected NP aggregate networks between droplets. Yield stress measurements display evidence of “double” yielding. We postulate that the presence of the NP aggregates provides a secondary network between droplet clusters resulting in such behavior. We demonstrate the suitability of the emulsions as agriculture formulations by incorporating an agrochemical, abamectin (Abm), and a plant-growth-promoting microbe (PGPM) in the emulsions. Release assays exhibit sustained Abm release, promising higher efficacy at lower usage volumes. Incorporation of nonsporulating PGPM Pseudomonas simiae in the emulsions shows significantly higher microbe viability compared to controls after 70 days of storage. By demonstrating the application of CA NPs as a sustainable Pickering emulsifier, this study introduces the use of CA as a platform technology for the delivery of diverse agriculture cargos. A comprehensive evaluation of the system is articulated in a fundamental microstructure analysis and a demonstration of practical on-site attributes, including shelf-life stability and functional performance, verified through bioassays and plant growth studies.}, number={42}, journal={ACS SUSTAINABLE CHEMISTRY & ENGINEERING}, author={Sohail, Mariam and Pirzada, Tahira and Guenther, Richard and Barbieri, Eduardo and Sit, Tim and Menegatti, Stefano and Crook, Nathan and Opperman, Charles H. and Khan, Saad A.}, year={2023}, month={Sep}, pages={15178–15191} }
 @article{oh_becker_mendola_choe_min_lee_yigzaw_seay_bill_li_et al._2023, title={Characterization and implications of host-cell protein aggregates in biopharmaceutical processing}, volume={1}, ISSN={["1097-0290"]}, DOI={10.1002/bit.28325}, abstractNote={Abstract}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Oh, Young Hoon and Becker, Matthew L. and Mendola, Kerri M. and Choe, Leila H. and Min, Lie and Lee, Kelvin H. H. and Yigzaw, Yinges and Seay, Alexander and Bill, Jerome and Li, Xuanwen and et al.}, year={2023}, month={Jan} }
 @article{sarma_catella_san pedro_xiao_durmusoglu_menegatti_crook_magness_hall_2023, title={Design of 8-mer peptides that block <i>Clostridioides difficile</i> toxin A in intestinal cells}, volume={6}, ISSN={["2399-3642"]}, url={https://doi.org/10.1038/s42003-023-05242-x}, DOI={10.1038/s42003-023-05242-x}, abstractNote={Abstract}, number={1}, journal={COMMUNICATIONS BIOLOGY}, author={Sarma, Sudeep and Catella, Carly M. and San Pedro, Ellyce T. and Xiao, Xingqing and Durmusoglu, Deniz and Menegatti, Stefano and Crook, Nathan and Magness, Scott T. and Hall, Carol K.}, year={2023}, month={Aug} }
 @article{kilgore_chu_bhandari_fischler_carbonell_crapanzano_menegatti_2023, title={Development of peptide affinity ligands for the purification of polyclonal and monoclonal Fabs from recombinant fluids}, volume={1687}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2022.463701}, abstractNote={Engineered multi-specific monoclonal antibodies (msAbs) and antibody fragments offer valuable therapeutic options against metabolic disorders, aggressive cancers, and viral infections. The advancement in molecular design and recombinant expression of these next-generation drugs, however, is not equaled by the progress in downstream bioprocess technology. The purification of msAbs and fragments requires affinity adsorbents with orthogonal biorecognition of different portions of the antibody structure, namely its Fc (fragment crystallizable) and Fab (fragment antigen-binding) regions or the CH1-3 and CL chains. Current adsorbents rely on protein ligands that, while featuring high binding capacity and selectivity, need harsh elution conditions and suffer from high cost, limited biochemical stability, and potential release of immunogenic fragments. Responding to these challenges, we undertook the de novo discovery of peptide ligands that target different regions of human Fab and enable product release under mild conditions. The ligands were discovered by screening a focused library of 12-mer peptides against a feedstock comprising human Fab and Chinese hamster ovary host cell proteins (CHO HCPs). The identified ligands were evaluated via binding studies as well as molecular docking simulations, returning excellent values of binding capacity (Qmax ∼ 20 mg of Fab per mL of resin) and dissociation constant (KD = 2.16·10-6 M). Selected ligand FRWNFHRNTFFP and commercial Protein L ligands were further characterized by measuring the dynamic binding capacity (DBC10%) at different residence times (RT) and performing the purification of polyclonal and monoclonal Fabs from CHO-K1 cell culture fluids. The peptide ligand featured DBC10% ∼ 6-16 mg/mL (RT of 2 min) and afforded values of yield (93-96%) and purity (89-96%) comparable to those provided by Protein L resins.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Kilgore, Ryan and Chu, Wenning and Bhandari, Dipendra and Fischler, David and Carbonell, Ruben G. and Crapanzano, Michael and Menegatti, Stefano}, year={2023}, month={Jan} }
 @article{oh_mendola_choe_min_lavoie_sripada_williams_lee_yigzaw_seay_et al._2023, title={Identification and characterization of CHO host-cell proteins in monoclonal antibody bioprocessing}, volume={10}, ISSN={["1097-0290"]}, DOI={10.1002/bit.28568}, abstractNote={Abstract}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Oh, Young Hoon and Mendola, Kerri M. and Choe, Leila H. and Min, Lie and Lavoie, Ashton R. and Sripada, Sobhana A. and Williams, Taufika Islam and Lee, Kelvin H. and Yigzaw, Yinges and Seay, Alexander and et al.}, year={2023}, month={Oct} }
 @article{mukherjee_yow_sarakbi_menegatti_v. gurgel_carbonell_bobay_2023, title={Integrated in silico and experimental discovery of trimeric peptide ligands targeting Butyrylcholinesterase}, volume={102}, ISSN={["1476-928X"]}, DOI={10.1016/j.compbiolchem.2022.107797}, abstractNote={Butyrylcholinesterase (BChE) is recognized as a high value biotherapeutic in the treatment of Alzheimer's disease and drug addiction. This study presents the rational design and screening of an in-silico library of trimeric peptides against BChE and the experimental characterization of peptide ligands for purification. The selected peptides consistently afforded high BChE recovery (> 90 %) and purity, yielding up to a 1000-fold purification factor. This study revealed a marked anti-correlated conformational movement governed by the ionic strength and pH of the aqueous environment, which ultimately controls BChE binding and release during chromatographic purification; and highlighted the role of residues within and allosteric to the catalytic triad of BChE in determining biorecognition, thus providing useful guidance for ligand design and affinity maturation.}, journal={COMPUTATIONAL BIOLOGY AND CHEMISTRY}, author={Mukherjee, Rudra Palash and Yow, Geok-Yong and Sarakbi, Samuel and Menegatti, Stefano and V. Gurgel, Patrick and Carbonell, Ruben G. and Bobay, Benjamin G.}, year={2023}, month={Feb} }
 @article{prodromou_moore_chu_deal_san miguel_brown_daniele_pozdin_menegatti_2023, title={Molecular Engineering of Cyclic Azobenzene-Peptide Hybrid Ligands for the Purification of Human Blood Factor VIII via Photo-Affinity Chromatography}, volume={1}, ISSN={["1616-3028"]}, url={http://dx.doi.org/10.1002/adfm.202213881}, DOI={10.1002/adfm.202213881}, abstractNote={Abstract}, journal={ADVANCED FUNCTIONAL MATERIALS}, publisher={Wiley}, author={Prodromou, Raphael and Moore, Brandyn David and Chu, Wenning and Deal, Halston and San Miguel, Adriana and Brown, Ashley Carson and Daniele, Michael Angelo-Anthony and Pozdin, Vladimir Aleksandrovich and Menegatti, Stefano}, year={2023}, month={Jan} }
 @article{kilgour_turner_daniele_menegatti_2023, title={One-Step Quantification of anti-Covid-19 Antibodies via Dual Affinity Ratiometric Quenching Assays}, volume={6}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.3c01266}, abstractNote={The global pandemic caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has affected millions of people and paralyzed healthcare systems worldwide. Developing rapid and accurate tests to detect and quantify anti-SARS-CoV-2 antibodies in complex fluids is critical to (i) track and address the spread of SARS-CoV-2 variants with different virulence and (ii) support the industrial manufacturing and clinical administration of anti-SARS-CoV-2 therapeutic antibodies. Conventional immunoassays, such as lateral flow, ELISA, and surface plasmon resonance (SPR), are either qualitative or, when quantitative, are laborious and expensive and suffer from high variability. Responding to these challenges, this study evaluates the performance of the Dual-Affinity Ratiometric Quenching (DARQ) assay for the quantification of anti-SARS-CoV-2 antibodies in bioprocess harvests and intermediate fractions (i.e., a Chinese hamster ovary (CHO) cell culture supernatant and a purified eluate) and human fluids (i.e., saliva and plasma). Monoclonal antibodies targeting the SARS-CoV-2 nucleocapsid as well as the spike protein of the delta and omicron variants are adopted as model analytes. Additionally, conjugate pads loaded with dried protein were studied as an at-line quantification method that can be used in clinical or manufacturing laboratories. Our results indicate that the DARQ assay is a highly reproducible (coefficient of variation ∼0.5-3%) and rapid (<10 min) test, whose sensitivity (∼0.23-2.5 ng/mL), limit of detection (23-250 ng/mL), and dynamic range (70-1300 ng/mL) are independent of sample complexity, thus representing a valuable tool for monitoring anti-SARS-CoV-2 antibodies.}, journal={ANALYTICAL CHEMISTRY}, author={Kilgour, Katie M. and Turner, Brendan L. and Daniele, Michael and Menegatti, Stefano}, year={2023}, month={Jun} }
 @article{wenning_shastry_barbieri_prodromou_greback-clarke_smith_moore_kilgore_cummings_pancorbo_et al._2023, title={Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates}, volume={2}, url={http://dx.doi.org/10.1101/2023.02.19.529155}, DOI={10.1101/2023.02.19.529155}, abstractNote={Abstract}, publisher={Cold Spring Harbor Laboratory}, author={WENNING, CHU and Shastry, Shriarjun and Barbieri, Eduardo and Prodromou, Raphael and Greback-Clarke, Paul and Smith, Will and Moore, Brandyn and Kilgore, Ryan and Cummings, Christopher and Pancorbo, Jennifer and et al.}, year={2023}, month={Feb} }
 @article{chu_shastry_barbieri_prodromou_greback-clarke_smith_moore_kilgore_cummings_pancorbo_et al._2023, title={Peptide ligands for the affinity purification of adeno-associated viruses from HEK 293 cell lysates}, volume={7}, ISSN={["1097-0290"]}, DOI={10.1002/bit.28495}, abstractNote={Abstract}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Chu, Wenning and Shastry, Shriarjun and Barbieri, Eduardo and Prodromou, Raphael and Greback-Clarke, Paul and Smith, Will and Moore, Brandyn and Kilgore, Ryan and Cummings, Christopher and Pancorbo, Jennifer and et al.}, year={2023}, month={Jul} }
 @article{zhang_barbieri_lebarre_rameez_mostafa_menegatti_2023, title={Peptonics: A new family of cell-protecting surfactants for the recombinant expression of therapeutic proteins in mammalian cell cultures}, volume={10}, ISSN={["1860-7314"]}, DOI={10.1002/biot.202300261}, abstractNote={Abstract}, journal={BIOTECHNOLOGY JOURNAL}, author={Zhang, Ka and Barbieri, Eduardo and Lebarre, Jacob and Rameez, Shahid and Mostafa, Sigma and Menegatti, Stefano}, year={2023}, month={Oct} }
 @article{sripada_elhanafi_collins_williams_linova_woodley_boi_menegatti_2023, title={Pseudo-affinity capture of <i>K. phaffii</i> host cell proteins in flow-through mode: Purification of protein therapeutics and proteomic study}, volume={326}, ISSN={["1873-3794"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85169830823&partnerID=MN8TOARS}, DOI={10.1016/j.seppur.2023.124777}, abstractNote={K. phaffii is a versatile expression system that is increasingly utilized to produce biological therapeutics – including enzymes, engineered antibodies, and gene-editing tools – that feature multiple subunits and complex post-translational modifications. Two major roadblocks limit the adoption of K. phaffii in industrial biomanufacturing: its proteome, while known, has not been linked to downstream process operations and detailed knowledge is missing on problematic host cell proteins (HCPs) that endanger patient safety or product stability. Furthermore, the purification toolbox has not evolved beyond the capture of monospecific antibodies, and few solutions are available for engineered antibody fragments and other protein therapeutics. To unlock the potential of yeast-based biopharmaceutical manufacturing, this study presents the development and performance validation of a novel adsorbent – PichiaGuard – functionalized with peptide ligands that target the whole spectrum of K. phaffii HCPs and designed for protein purification in flow-through mode. The PichiaGuard adsorbent features high HCP binding capacity (∼25 g per liter of resin) and successfully purified a monoclonal antibody and an ScFv fragment from clarified K. phaffii harvests, affording > 300-fold removal of HCPs and high product yields (70–80%). Notably, PichiaGuard outperformed commercial ion exchange and mixed-mode resins without salt gradients or optimization in removing high-risk HCPs – including aspartic proteases, ribosomal subunits, and other peptidases – thus demonstrating its value in modern biopharmaceutical processing.}, journal={SEPARATION AND PURIFICATION TECHNOLOGY}, author={Sripada, Sobhana A. and Elhanafi, Driss and Collins, Leonard B. and Williams, Taufika I. and Linova, Marina Y. and Woodley, John M. and Boi, Cristiana and Menegatti, Stefano}, year={2023}, month={Dec} }
 @article{fan_sripada_pham_linova_woodley_menegatti_boi_carbonell_2023, title={Purification of a monoclonal antibody using a novel high-capacity multimodal cation exchange nonwoven membrane}, volume={317}, ISSN={["1873-3794"]}, DOI={10.1016/j.seppur.2023.123920}, abstractNote={A high-capacity, multimodal cation exchange (MMC) chromatographic membrane was developed by conjugating a multimodal ligand – 2-mercaptopyridine-3-carboxylic acid (MPCA) – on a polybutylene terepthalate (PBT) nonwoven fabric. The membrane features an equilibrium binding capacity of ≈ 1000 mg of human polyclonal IgG (IgG) per g of membrane and dynamic binding capacities (DBC10%) ranging from 77.5 to 115.1 mg/mL (residence times of 1 and 5 min, respectively); these values are 2-to-3-fold higher than those of commercial MMC adsorbents. The effects of buffer composition, pH, conductivity on the binding behavior of the MMC-MPCA membrane were investigated in detail. As a moderate cation exchange binder, MPCA enables effective protein elution using buffers with mild pH (8.0–9.0) and conductivity (≈13 mS/cm), thus circumventing the harsh conditions often needed in multimodal chromatography. The MMC-MPCA membrane was evaluated for product capture in bind-and-elute mode on a Chinese hamster ovary (CHO) cell culture harvest containing therapeutic monoclonal antibodies, using commercial multimodal (Capto MMC and MX-Trp-650M) and affinity (AF-rProtein A HC-650F) resins as controls. The MMC-MPCA membrane outperformed the multimodal resins in terms of binding capacity as well as clearance of host cell proteins (HCPs) and aggregates. The membrane was then evaluated by polishing the mAb from a Protein A eluate in bind-and-elute mode. The MMC-MPCA membrane reduced the level of high molecular weight components from 11% to 4% and the HCP content from 1319.7 ppm to 48.7 ppm (LRV of 1.4). Most notably, proteomics analysis of the product demonstrated the clearance of a significant fraction of persistent, high-risk HCPs from the Protein A eluate.}, journal={SEPARATION AND PURIFICATION TECHNOLOGY}, author={Fan, Jinxin and Sripada, Sobhana A. and Pham, Dan N. and Linova, Marina Y. and Woodley, John M. and Menegatti, Stefano and Boi, Cristiana and Carbonell, Ruben G.}, year={2023}, month={Jul} }
 @article{shastry_chu_barbieri_greback-clarke_smith_cummings_minzoni_pancorbo_gilleskie_ritola_et al._2023, title={Rational design and experimental evaluation of peptide ligands for the purification of adeno-associated viruses via affinity chromatography}, volume={9}, ISSN={["1860-7314"]}, DOI={10.1002/biot.202300230}, abstractNote={Abstract}, journal={BIOTECHNOLOGY JOURNAL}, author={Shastry, Shriarjun and Chu, Wenning and Barbieri, Eduardo and Greback-Clarke, Paul and Smith, William K. and Cummings, Christopher and Minzoni, Arianna and Pancorbo, Jennifer and Gilleskie, Gary and Ritola, Kimberly and et al.}, year={2023}, month={Sep} }
 @article{kilgore_minzoni_shastry_smith_barbieri_wu_lebarre_chu_o'brien_menegatti_2023, title={The downstream bioprocess toolbox for therapeutic viral vectors}, volume={1709}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2023.464337}, abstractNote={Viral vectors are poised to acquire a prominent position in modern medicine and biotechnology owing to their role as delivery agents for gene therapies, oncolytic agents, vaccine platforms, and a gateway to engineer cell therapies as well as plants and animals for sustainable agriculture. The success of viral vectors will critically depend on the availability of flexible and affordable biomanufacturing strategies that can meet the growing demand by clinics and biotech companies worldwide. In this context, a key role will be played by downstream process technology: while initially adapted from protein purification media, the purification toolbox for viral vectors is currently undergoing a rapid expansion to fit the unique biomolecular characteristics of these products. Innovation efforts are articulated on two fronts, namely (i) the discovery of affinity ligands that target adeno-associated virus, lentivirus, adenovirus, etc.; (ii) the development of adsorbents with innovative morphologies, such as membranes and 3D printed monoliths, that fit the size of viral vectors. Complementing these efforts are the design of novel process layouts that capitalize on novel ligands and adsorbents to ensure high yield and purity of the product while safeguarding its therapeutic efficacy and safety; and a growing panel of analytical methods that monitor the complex array of critical quality attributes of viral vectors and correlate them to the purification strategies. To help explore this complex and evolving environment, this study presents a comprehensive overview of the downstream bioprocess toolbox for viral vectors established in the last decade, and discusses present efforts and future directions contributing to the success of this promising class of biological medicines.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Kilgore, Ryan and Minzoni, Arianna and Shastry, Shriarjun and Smith, Will and Barbieri, Eduardo and Wu, Yuxuan and Lebarre, Jacob P. and Chu, Wenning and O'Brien, Juliana and Menegatti, Stefano}, year={2023}, month={Oct} }
 @article{wang_hosseini_shastry_barbieri_chu_menegatti_daniele_2023, title={Toward the quantification of adeno-associated virus titer by electrochemical impedance spectroscopy}, DOI={10.1109/BioSensors58001.2023.10281105}, abstractNote={Gene therapies have shown great promise for the potential treatment of a broad range of diseases. Adeno-associated viruses (AAVs) are popular gene vectors because of their ability to target specific tissues, and they have demonstrated high transduction efficiencies in multiple neurological targets. While these therapeutics hold great promise, their biomanufacturing has limited potential cost-reduction and more widespread adoption. Herein, we report the preliminary development of an immunosensor for measuring the titer of adeno-associated virus 2 (AAV2), which may be deployed for rapid quantification of product yield during AAV biomanufacturing. We functionalized an interdigitated electrode array with anti-AAV2 antibodies, and electrochemical impedance spectroscopy was employed to investigate the response to AAV2 titer. A Faradaic sensing principle was utilized, in which the charge transfer resistance (Rct) of an electrochemical reporter was monitored after capture of AAV2 on the surface of the sensor. A linear response was measured over titers 1012 - 1013 capsids/mL.}, journal={2023 IEEE BIOSENSORS CONFERENCE, BIOSENSORS}, author={Wang, Junhyeong and Hosseini, Mahshid and Shastry, Shriarjun and Barbieri, Eduardo and Chu, Wenning and Menegatti, Stefano and Daniele, Michael A.}, year={2023} }
 @article{sharkey_twiddy_peterson_aroche_menegatti_daniele_2023, title={Towards electrochemical control of pH for regeneration of biosensors}, DOI={10.1109/BioSensors58001.2023.10281061}, abstractNote={Most affinity-based biosensors are designed to be single-use devices, based on the measurement of irreversible binding events, which makes longitudinal monitoring resource-intensive, and typically prohibits the measurement of analyte fluctuations over time using the same device. Selective reversal of biorecognition events, i.e., regeneration, may enable repeated and longitudinal use of affinity-based biosensors; however, typical regeneration methods utilize additional chemical reagents, requiring longer processing times and increasing the likelihood of operator error. The development of a “solid-state” regeneration method provides significant value for extending the utility of affinity-based biosensors, such as electrochemical immunosensors and aptasensors. Herein, we report the characterization of a method for electronically controlling pH without additional reagents. Palladium was used to induce pH swings in aqueous electrolytes and buffers by application of an electric potential. The developed system was able to affect acidic and basic pH changes of ± 4. The efficacy of this method was further demonstrated by reversing common affinity-binding complexes and compared to conventional glycine-based regeneration.}, journal={2023 IEEE BIOSENSORS CONFERENCE, BIOSENSORS}, author={Sharkey, Christopher and Twiddy, Jack and Peterson, Kaila L. and Aroche, Angelica F. and Menegatti, Stefano and Daniele, Michael A.}, year={2023} }
 @article{rahmanian_ebrahim_razavi_abdelmigeed_barbieri_menegatti_parsons_li_pirzada_khan_2023, title={Vapor phase synthesis of metal-organic frameworks on a nanofibrous aerogel creates enhanced functionality}, volume={11}, ISSN={["2050-7496"]}, url={https://doi.org/10.1039/D3TA05299K}, DOI={10.1039/d3ta05299k}, abstractNote={Vapor-phase synthesis of metal–organic frameworks (MOFs) on nanofibrous aerogels provides a hierarchically porous and mechanically robust material platform for use in a multitude of applications, from carbon dioxide capture to heavy metal removal.}, journal={JOURNAL OF MATERIALS CHEMISTRY A}, author={Rahmanian, Vahid and Ebrahim, Muhammed Ziauddin Ahmad and Razavi, Seyedamin and Abdelmigeed, Mai and Barbieri, Eduardo and Menegatti, Stefano and Parsons, Gregory N. and Li, Fanxing and Pirzada, Tahira and Khan, Saad A.}, year={2023}, month={Nov} }
 @article{xiao_kilgore_sarma_chu_menegatti_hall_2022, title={<p>De novo discovery of peptide-based affinity ligands for the fab fragment of human immunoglobulin G</p>}, volume={1669}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2022.462941}, abstractNote={Antibody fragments and their engineered variants show true potential as next-generation therapeutics as they combine excellent targeting with superior biodistribution and blood clearance. Unlike full antibodies, however, antibody fragments do not yet have a standard platform purification process for large-scale production. Short peptide ligands are viable alternatives to protein ligands in affinity chromatography. In this work, an integrated computational and experimental scheme is described to de novo design 9-mer peptides that bind to Fab fragments. The first cohort of designed sequences was tested experimentally using human polyclonal Fab, and the top performing sequence was selected as a prototype for a subsequent round of ligand refinement in silico. The resulting peptides were conjugated to chromatographic resins and evaluated via equilibrium and dynamic binding studies using human Fab-κ and Fab-λ. The equilibrium studies returned values of binding capacities up to 32 mg of Fab per mL of resin with mild affinity (KD ∼ 10-5 M) that are conducive to high product capture and recovery. Dynamic studies returned values of product yield up to ∼90%. Preliminary purification studies provided purities of 83-93% and yields of 11-89%. These results lay the groundwork for future development of these ligands towards biomanufacturing translation.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Xiao, Xingqing and Kilgore, Ryan and Sarma, Sudeep and Chu, Wenning and Menegatti, Stefano and Hall, Carol K.}, year={2022}, month={Apr} }
 @article{conner_mcandrew_menegatti_velev_2022, title={An accelerated antibody aggregation test based on time sequenced dynamic light scattering}, volume={653}, ISSN={["1873-4359"]}, DOI={10.1016/j.colsurfa.2022.129833}, abstractNote={A rapid method for determination of the colloidal stability of protein molecules in solution is reported as an efficient tool for evaluating the stability of antibody formulations. Using human polyclonal immunoglobulin G (IgG) as a model protein and dynamic light scattering (DLS) as a technique to determine the size of particles in solution, the rate of aggregation is investigated at different temperatures and antibody concentrations. To reduce the observation period while increasing precision, a new approach to DLS analysis is developed that comprises: (i) a distribution analysis of high-resolution data, and fitting for multiple particle sizes present in a solution, (ii) a temperature ramp to an intermediate temperature followed by a stress test at constant temperature over several hours, and (iii) 3-D plotting to reveal the time-dependent evolution of the particle size distribution at the selected temperature. The resulting 3-D plots enable robust identification of the onset of aggregation with different dispersion conditions. This method enables rapid evaluation of the effects of parameters such as temperature and concentration on the stability of antibody solutions.}, journal={COLLOIDS AND SURFACES A-PHYSICOCHEMICAL AND ENGINEERING ASPECTS}, author={Conner, Cathryn G. and McAndrew, James and Menegatti, Stefano and Velev, Orlin D.}, year={2022}, month={Nov} }
 @article{chu_prodromou_moore_elhanafi_kilgore_shastry_menegatti_2022, title={Development of peptide ligands for the purification of a-1 antitrypsin from cell culture fluids}, volume={1679}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2022.463363}, abstractNote={α-1 antitrypsin (AAT) deficiency, a major risk factor for chronic obstructive pulmonary disease, is one of the most prevalent and fatal hereditary diseases. The rising demand of AAT poses a defined need for new processes of AAT manufacturing from recombinant sources. Commercial affinity adsorbents for AAT purification present the intrinsic limitations of protein ligands - chiefly, the high cost and the lability towards the proteases in the feedstocks and the cleaning-in-place utilized in biomanufacturing - which limit their application despite their high capacity and selectivity. This work presents the development of small peptide affinity ligands for the purification of AAT from Chinese hamster ovary (CHO) cell culture harvests. An ensemble of ligand candidates identified via library screening were conjugated on Toyopearl resin and evaluated via experimental and in silico AAT-binding studies. Initial ranking based on equilibrium binding capacity indicated WHAKKSKFG- (12.9 mg of AAT per mL of resin), WHAKKSHFG- (16.3 mg/mL), and KWKHSHKWG- (15.8 mg/mL) Toyopearl resins as top performing adsorbents. Notably, the fitting of adsorption data to Langmuir isotherms concurred with molecular docking and dynamics in returning values of dissociation constant (KD) between 1 - 10 µM. These peptide-based adsorbents were thus selected for AAT purification from CHO fluids, affording values of AAT binding capacity up to 13 gram per liter of resin, and product yield and purity up to 77% and 97%. WHAKKSHFG-Toyopearl resin maintained its purification activity upon 20 consecutive uses, demonstrating its potential for AAT manufacturing from recombinant sources.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Chu, Wenning and Prodromou, Raphael and Moore, Brandyn and Elhanafi, Driss and Kilgore, Ryan and Shastry, Shriarjun and Menegatti, Stefano}, year={2022}, month={Aug} }
 @article{ramesh_davis_roros_zhou_he_gao_menegatti_khan_genzer_2022, title={Nonwoven Membranes with Infrared Light-Controlled Permeability}, volume={9}, ISSN={["1944-8252"]}, DOI={10.1021/acsami.2c13280}, abstractNote={This study presents the development of the first composite nonwoven fiber mats (NWFs) with infrared light-controlled permeability. The membranes were prepared by coating polypropylene NWFs with a photothermal layer of poly(N-isopropylacrylamide) (PNIPAm)-based microgels impregnated with graphene oxide nanoparticles (GONPs). This design enables "photothermal smart-gating" using light dosage as remote control of the membrane's permeability to electrolytes. Upon exposure to infrared light, the GONPs trigger a rapid local increase in temperature, which contracts the PNIPAm-based microgels lodged in the pore space of the NWFs. The contraction of the microgels can be reverted by cooling from the surrounding aqueous environment. The efficient conversion of infrared light into localized heat by GONPs coupled with the phase transition of the microgels above the lower critical solution temperature (LCST) of PNIPAm provide effective control over the effective porosity, and thus the permeability, of the membrane. The material design parameters, namely the monomer composition of the microgels and the GONP-to-microgel ratio, enable tuning the permeability shift in response to IR light; control NWFs coated with GONP-free microgels displayed thermal responsiveness only, whereas native NWFs showed no smart-gating behavior at all. This technology shows potential toward processing temperature-sensitive bioactive ingredients or remote-controlled bioreactors.}, journal={ACS APPLIED MATERIALS & INTERFACES}, author={Ramesh, Srivatsan and Davis, Jack and Roros, Alexandra and Zhou, Chuanzhen and He, Nanfei and Gao, Wei and Menegatti, Stefano and Khan, Saad and Genzer, Jan}, year={2022}, month={Sep} }
 @article{barbieri_cutright_ramesh_khan_efimenko_genzer_menegatti_2022, title={Potent Antibacterial Composite Nonwovens Functionalized with Bioactive Peptides and Polymers}, volume={8}, ISSN={["2196-7350"]}, url={https://doi.org/10.1002/admi.202201061}, DOI={10.1002/admi.202201061}, abstractNote={Abstract}, journal={ADVANCED MATERIALS INTERFACES}, author={Barbieri, Eduardo and Cutright, Camden C. and Ramesh, Srivatsan and Khan, Saad A. and Efimenko, Kirill and Genzer, Jan and Menegatti, Stefano}, year={2022}, month={Aug} }
 @article{fan_barbieri_shastry_menegatti_boi_carbonell_2022, title={Purification of Adeno-Associated Virus (AAV) Serotype 2 from Spodoptera frugiperda (Sf9) Lysate by Chromatographic Nonwoven Membranes}, volume={12}, ISSN={["2077-0375"]}, DOI={10.3390/membranes12100944}, abstractNote={The success of adeno-associated virus (AAV)-based therapeutics in gene therapy poses the need for rapid and efficient processes that can support the growing clinical demand. Nonwoven membranes represent an ideal tool for the future of virus purification: owing to their small fiber diameters and high porosity, they can operate at high flowrates while allowing full access to target viral particles without diffusional limitations. This study describes the development of nonwoven ion-exchange membrane adsorbents for the purification of AAV2 from an Sf9 cell lysate. A strong anion-exchange (AEX) membrane was developed by UV grafting glycidyl methacrylate on a polybutylene terephthalate nonwoven followed by functionalization with triethylamine (TEA), resulting in a quaternary amine ligand (AEX-TEA membrane). When operated in bind-and-elute mode at a pH higher than the pI of the capsids, this membrane exhibited a high AAV2 binding capacity (9.6 × 1013 vp·mL−1) at the residence time of 1 min, and outperformed commercial cast membranes by isolating AAV2 from an Sf9 lysate with high productivity (2.4 × 1013 capsids·mL−1·min−1) and logarithmic reduction value of host cell proteins (HCP LRV ~ 1.8). An iminodiacetic acid cation-exchange nonwoven (CEX-IDA membrane) was also prepared and utilized at a pH lower than the pI of capsids to purify AAV2 in a bind-and-elute mode, affording high capsid recovery and impurity removal by eluting with a salt gradient. To further increase purity, the CEX-IDA and AEX-TEA membranes were utilized in series to purify the AAV2 from the Sf9 cell lysate. This membrane-based chromatography process also achieved excellent DNA clearance and a recovery of infectivity higher that that reported using ion-exchange resin chromatography.}, number={10}, journal={MEMBRANES}, author={Fan, Jinxin and Barbieri, Eduardo and Shastry, Shriarjun and Menegatti, Stefano and Boi, Cristiana and Carbonell, Ruben G.}, year={2022}, month={Oct} }
 @misc{ramesh_khan_park_ford_menegatti_genzer_2022, title={Self-healing and repair of fabrics: A comprehensive review of the application toolkit}, volume={54}, ISSN={["1873-4103"]}, DOI={10.1016/j.mattod.2021.11.016}, abstractNote={Self-healing fabrics respond to chemical and physical damage by restoring functional, structural, and morphological features. We present a comprehensive review of textile hybrids or composites capable of self-healing and repairing fabrics against damages across the micro- (µm), meso- (µm – mm), and macro-scale (>mm). The reviewed literature is organized in three sections presenting (i) the chemistry and fabrication principles of designing self-healing fabrics against increasing size scales of repair, (ii) stimuli-driven and autonomous healing, and (iii) the methods to characterize the recovery of wettability, barrier, morphological, mechanical, and other properties. The discussion of mainstream methods for developing self-healing fabrics focuses on coatings, composites, and specialized fabrication techniques required as the damage size grows from µm to mm to >mm. The section on stimuli-driven repair and autonomous recovery discusses the time scales associated with different damage repair, showing how external stimuli provide a higher driving force towards healing and accelerate material restoration than autonomous recovery. Finally, an array of optical, mechanical, and functional characterization techniques is discussed to evaluate the recovery yield and understand the repair mechanisms of the various fabrics. This review demonstrates the virtually limitless uses of next-generation self-healing systems, from separations to protective clothing, anti-fouling, and self-cleaning.}, journal={MATERIALS TODAY}, author={Ramesh, Srivatsan and Khan, Saad and Park, Yaewon and Ford, Ericka and Menegatti, Stefano and Genzer, Jan}, year={2022}, month={Apr}, pages={90–109} }
 @article{lee_aroche_menegatti_daniele_2022, title={Toward an Aptasensor for Monitoring of Tacrolimus}, ISSN={["1930-0395"]}, DOI={10.1109/SENSORS52175.2022.9967265}, abstractNote={Developing a simple and accurate method for monitoring the levels of circulating tacrolimus (TAC) can inform dosing schedule and reduce the risk of rejection in solid organ transplantation. Thus, an aptasensor was designed, fabricated, and characterized for electrochemical quantification of TAC concentration. The TAC-aptasensor comprised a gold electrode functionalized with a thiolated TAC-binding aptamer (APT122) derivatized with methylene blue. Surface plasmon resonance (SPR) was used to characterize the binding strength and specificity of the TAC:APT122 complex. Square wave voltammetry (SWV) was used to quantify the aptasensor response to varying concentrations of TAC. A dose-response curve was observed at concentrations of TAC < 7 µM, when operated at 50 and 80 Hz. Typical sensitivity of 0.07 µA· µM-1 was observed at 50 Hz. Limited specificity for the TAC aptasensors was observed with SWV against cyclosporine, amoxicillin, and tetracycline. Future efforts will explore a multi-aptamer system to extend the dynamic range and improve surface functionality to limit non-specific binding.}, journal={2022 IEEE SENSORS}, author={Lee, Bang Hyun and Aroche, Angelica F. and Menegatti, Stefano and Daniele, Michael A.}, year={2022} }
 @article{sripada_chu_williams_teten_mosley_carbonell_lenhoff_cramer_bill_yigzaw_et al._2022, title={Towards continuous mAb purification: Clearance of host cell proteins from CHO cell culture harvests via "flow-through affinity chromatography" using peptide-based adsorbents}, volume={4}, ISSN={["1097-0290"]}, url={https://doi.org/10.1002/bit.28096}, DOI={10.1002/bit.28096}, abstractNote={Abstract}, number={7}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, publisher={Wiley}, author={Sripada, Sobhana Alekhya and Chu, Wenning and Williams, Taufika Islam and Teten, Matthew A. and Mosley, Brian J. and Carbonell, Ruben G. and Lenhoff, Abraham M. and Cramer, Steven M. and Bill, Jerome and Yigzaw, Yinges and et al.}, year={2022}, month={Apr} }
 @article{durmusoglu_catella_purnell_menegatti_crook_2021, title={Design and in situ biosynthesis of precision therapies against gastrointestinal pathogens}, volume={23}, ISSN={["2468-8673"]}, DOI={10.1016/j.cophys.2021.06.007}, abstractNote={Gastrointestinal pathogens employ a variety of mechanisms to damage host tissue, acquire nutrients, and evade treatment. To supplement broad-spectrum antimicrobials, there has been increasing interest in designing molecules that target specific taxa and virulence processes. Excitingly, these antivirulence therapies may be able to be synthesized by gut-resident microbes, thereby enabling delivery of these drugs directly to the spatial and temporal site of infection. In this review, we highlight recent progress in our understanding of small molecules that inhibit specific virulence mechanisms. We additionally discuss emerging methods to discover pathogen-specific and mechanism-specific peptides and small proteins. Finally, we cover recent demonstrations of probiotics engineered to produce antimicrobials in response to pathogen-specific cues in the gut. Collectively, these advances point to an emerging integrative approach to treatment of gastrointestinal diseases, comprising microbiologists, peptide chemists, and synthetic biologists.}, journal={CURRENT OPINION IN PHYSIOLOGY}, author={Durmusoglu, Deniz and Catella, Carly M. and Purnell, Ethan F. and Menegatti, Stefano and Crook, Nathan C.}, year={2021}, month={Oct} }
 @article{lavoie_islam_blackburn_carbonell_menegatti_2021, title={Development of Peptide Ligands for Targeted Capture of Host Cell Proteins from Cell Culture Production Harvests}, volume={2261}, ISBN={["978-1-0716-1185-2"]}, ISSN={["1940-6029"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85099721618&partnerID=MN8TOARS}, DOI={10.1007/978-1-0716-1186-9_31}, abstractNote={Capture of host cell proteins (HCPs) from cell culture production harvests is critical to ensure the maximum levels specified by international regulatory bodies of product purity for therapeutic monoclonal antibodies (mAbs). Peptide ligands that selectively target the whole spectrum of the HCPs, while letting the mAb product flow through unbound, are an ideal complement to the affinity-based capture step via Protein A chromatography. In this work, we describe the development of HCP-binding peptide ligands, especially focusing on the steps of (1) peptide selection via library screening and (2) quantification of HCP removal via proteomics by mass spectrometry.}, journal={PROTEOMIC PROFILING, 2 EDITION}, author={Lavoie, R. Ashton and Islam, Taufika and Blackburn, Williams R. Kevin and Carbonell, Ruben G. and Menegatti, Stefano}, year={2021}, pages={489–506} }
 @article{ramesh_davis_roros_eiben_fabiani_smith_reynolds_pourdeyhimi_khan_genzer_et al._2021, title={Dual-Responsive Microgels for Structural Repair and Recovery of Nonwoven Membranes for Liquid Filtration}, volume={3}, ISSN={["2637-6105"]}, url={https://doi.org/10.1021/acsapm.0c01360}, DOI={10.1021/acsapm.0c01360}, abstractNote={This study presents dual-responsive colloidal microgels to repair nonwoven fiber mats (NWFs) and recover their native morphological and functional properties. The formulation comprises poly(N-isopr...}, number={3}, journal={ACS APPLIED POLYMER MATERIALS}, publisher={American Chemical Society (ACS)}, author={Ramesh, Srivatsan and Davis, Jack and Roros, Alexandra and Eiben, Justin and Fabiani, Thomas and Smith, Ryan and Reynolds, Lewis and Pourdeyhimi, Behnam and Khan, Saad and Genzer, Jan and et al.}, year={2021}, month={Mar}, pages={1508–1517} }
 @article{prodromou_day_saberi-bosari_schneible_mabe_san miguel_daniele_pozdin_menegatti_2021, title={Engineering Next Generation Cyclized Peptide Ligands for Light-Controlled Capture and Release of Therapeutic Proteins}, volume={31}, ISSN={["1616-3028"]}, url={http://dx.doi.org/10.1002/adfm.202101410}, DOI={10.1002/adfm.202101410}, abstractNote={Abstract}, number={27}, journal={ADVANCED FUNCTIONAL MATERIALS}, publisher={Wiley}, author={Prodromou, Raphael and Day, Kevin N. and Saberi-Bosari, Sahand and Schneible, John D. and Mabe, Matthew D. and San Miguel, Adriana and Daniele, Michael A. and Pozdin, Vladimir and Menegatti, Stefano}, year={2021}, month={Jul} }
 @article{xiao_sarma_menegatti_crook_magness_hall_2021, title={In Silico Identification and Experimental Validation of Peptide-Based Inhibitors Targeting Clostridium difficile Toxin A}, volume={12}, ISSN={["1554-8937"]}, url={https://doi.org/10.1021/acschembio.1c00743}, DOI={10.1021/acschembio.1c00743}, abstractNote={Clostridium difficile infection is mediated by two major exotoxins: toxins A (TcdA) and B (TcdB). Inhibiting the biocatalytic activities of these toxins with targeted peptide-based drugs can reduce the risk of C. difficile infection. In this work, we used a computational strategy that integrates a peptide binding design (PepBD) algorithm and explicit-solvent atomistic molecular dynamics simulation to determine promising toxin A-targeting peptides that can recognize and bind to the catalytic site of the TcdA glucosyltransferase domain (GTD). Our simulation results revealed that two out of three in silico discovered peptides, viz. the neutralizing peptides A (NPA) and B (NPB), exhibit lower binding free energies when bound to the TcdA GTD than the phage-display discovered peptide, viz. the reference peptide (RP). These peptides may serve as potential inhibitors against C. difficile infection. The efficacy of the peptides RP, NPA, and NPB to neutralize the cytopathic effects of TcdA was tested in vitro in human jejunum cells. Both phage-display peptide RP and in silico peptide NPA were found to exhibit strong toxin-neutralizing properties, thereby preventing the TcdA toxicity. However, the in silico peptide NPB demonstrates a relatively low efficacy against TcdA.}, number={1}, journal={ACS CHEMICAL BIOLOGY}, publisher={American Chemical Society (ACS)}, author={Xiao, Xingqing and Sarma, Sudeep and Menegatti, Stefano and Crook, Nathan and Magness, Scott T. and Hall, Carol K.}, year={2021}, month={Dec} }
 @article{chu_prodromou_day_schneible_bacon_bowen_kilgore_catella_moore_mabe_et al._2021, title={Peptides and pseudopeptide ligands: a powerful toolbox for the affinity purification of current and next-generation biotherapeutics}, volume={1635}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2020.461632}, abstractNote={Following the consolidation of therapeutic proteins in the fight against cancer, autoimmune, and neurodegenerative diseases, recent advancements in biochemistry and biotechnology have introduced a host of next-generation biotherapeutics, such as CRISPR-Cas nucleases, stem and car-T cells, and viral vectors for gene therapy. With these drugs entering the clinical pipeline, a new challenge lies ahead: how to manufacture large quantities of high-purity biotherapeutics that meet the growing demand by clinics and biotech companies worldwide. The protein ligands employed by the industry are inadequate to confront this challenge: while featuring high binding affinity and selectivity, these ligands require laborious engineering and expensive manufacturing, are prone to biochemical degradation, and pose safety concerns related to their bacterial origin. Peptides and pseudopeptides make excellent candidates to form a new cohort of ligands for the purification of next-generation biotherapeutics. Peptide-based ligands feature excellent target biorecognition, low or no toxicity and immunogenicity, and can be manufactured affordably at large scale. This work presents a comprehensive and systematic review of the literature on peptide-based ligands and their use in the affinity purification of established and upcoming biological drugs. A comparative analysis is first presented on peptide engineering principles, the development of ligands targeting different biomolecular targets, and the promises and challenges connected to the industrial implementation of peptide ligands. The reviewed literature is organized in (i) conventional (α-)peptides targeting antibodies and other therapeutic proteins, gene therapy products, and therapeutic cells; (ii) cyclic peptides and pseudo-peptides for protein purification and capture of viral and bacterial pathogens; and (iii) the forefront of peptide mimetics, such as β-/γ-peptides, peptoids, foldamers, and stimuli-responsive peptides for advanced processing of biologics.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Chu, Wenning and Prodromou, Raphael and Day, Kevin N. and Schneible, John D. and Bacon, Kaitlyn B. and Bowen, John D. and Kilgore, Ryan E. and Catella, Carly M. and Moore, Brandyn D. and Mabe, Matthew D. and et al.}, year={2021}, month={Jan} }
 @article{chu_sripada_reese_bhandari_adams_sly_crapanzano_menegatti_2021, title={Purification of polyclonal immunoglobulin G from human serum using peptide-based adsorbents}, volume={10}, ISSN={["1547-5905"]}, DOI={10.1002/aic.17482}, abstractNote={Abstract}, journal={AICHE JOURNAL}, author={Chu, Wenning and Sripada, Sobhana A. and Reese, Hannah R. and Bhandari, Dipendra and Adams, Augustus and Sly, Jae and Crapanzano, Michael and Menegatti, Stefano}, year={2021}, month={Oct} }
 @article{bacon_blain_bowen_burroughs_mcarthur_menegatti_rao_2021, title={Quantitative Yeast-Yeast Two Hybrid for the Discovery and Binding Affinity Estimation of Protein-Protein Interactions}, volume={10}, ISSN={["2161-5063"]}, DOI={10.1021/acssynbio.0c00472}, abstractNote={Quantifying the binding affinity of protein-protein interactions is important for elucidating connections within biochemical signaling pathways, as well as characterization of binding proteins isolated from combinatorial libraries. We describe a quantitative yeast-yeast two-hybrid (qYY2H) system that not only enables the discovery of specific protein-protein interactions but also efficient, quantitative estimation of their binding affinities (KD). In qYY2H, the bait and prey proteins are expressed as yeast cell surface fusions using yeast surface display. We developed a semiempirical framework for estimating the KD of monovalent bait-prey interactions, using measurements of bait-prey yeast-yeast binding, which is mediated by multivalent interactions between yeast-displayed bait and prey. Using qYY2H, we identified interaction partners of SMAD3 and the tandem WW domains of YAP from a cDNA library and characterized their binding affinities. Finally, we showed that qYY2H could also quantitatively evaluate binding interactions mediated by post-translational modifications on the bait protein.}, number={3}, journal={ACS SYNTHETIC BIOLOGY}, author={Bacon, Kaitlyn and Blain, Abigail and Bowen, John and Burroughs, Matthew and McArthur, Nikki and Menegatti, Stefano and Rao, Balaji M.}, year={2021}, month={Mar}, pages={505–514} }
 @article{lavoie_chu_lavoie_hetzler_williams_carbonell_menegatti_2021, title={Removal of host cell proteins from cell culture fluids by weak partitioning chromatography using peptide-based adsorbents}, volume={257}, ISSN={["1873-3794"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85093930679&partnerID=MN8TOARS}, DOI={10.1016/j.seppur.2020.117890}, abstractNote={This work presents the removal of host cell proteins (HCPs) from a Chinese Hamster Ovary clarified cell culture fluid (CHO CCCF) containing a therapeutic monoclonal antibody (mAb) by weak partitioning chromatography (WPC). The chromatographic adsorbents were produced by functionalizing Toyopearl resin with HCP-binding tetrameric multipolar (4MP) or hexameric hydrophobic/cationic (6HP) peptides. The CCCF was loaded on columns packed with either 4MP-Toyopearl or 6HP-Toyopearl resin only, or a 4MP and 6HP resin mixture at different values of residence time (RT: 0.5, 1, 2, and 5 min). The temporal profiles of concentration of HCPs and mAb in the effluents confirmed the binding mechanism by WPC, where both HCPs and mAb are initially bound by the peptide ligands, but, as more CCCF is fed to the column, the incoming HCPs displace the bound mAbs. In particular, 4MP was shown to capture more selectively high molecular weight HCPs, while 6HP was more effective in binding low molecular weight HCPs. Under optimal loading conditions (~60–80 g of proteins per L of adsorbent; RT of 5 min), the 6HP+4MP-Toyopearl adsorbent provided mAb yield and purity of >80% and up to 90%, respectively. Conversely, the control resin Toyopearl SuperQ-650 M resulted in 70% yield and 75% purity under the same conditions. Proteomic analysis of the effluents demonstrated that 6HP+4MP-Toyopearl adsorbent removes HCPs known for their immunogenicity or IgG co-elution or degradation, demonstrating the potential of these peptide-based resins as HCP scrubbers in mAb purification processes.}, journal={SEPARATION AND PURIFICATION TECHNOLOGY}, author={Lavoie, R. Ashton and Chu, Wenning and Lavoie, Joseph H. and Hetzler, Zachary and Williams, Taufika Islam and Carbonell, Ruben and Menegatti, Stefano}, year={2021}, month={Feb} }
 @article{turner_ramesh_menegatti_daniele_2021, title={Resorbable elastomers for implantable medical devices: highlights and applications}, volume={12}, ISSN={["1097-0126"]}, DOI={10.1002/pi.6349}, abstractNote={Abstract}, journal={POLYMER INTERNATIONAL}, author={Turner, Brendan and Ramesh, Srivatsan and Menegatti, Stefano and Daniele, Michael}, year={2021}, month={Dec} }
 @article{bowen_schneible_bacon_labar_menegatti_rao_2021, title={Screening of Yeast Display Libraries of Enzymatically Treated Peptides to Discover Macrocyclic Peptide Ligands}, volume={22}, ISSN={["1422-0067"]}, url={https://www.mdpi.com/1422-0067/22/4/1634}, DOI={10.3390/ijms22041634}, abstractNote={We present the construction and screening of yeast display libraries of post-translationally modified peptides wherein site-selective enzymatic treatment of linear peptides is achieved using bacterial transglutaminase. To this end, we developed two alternative routes, namely (i) yeast display of linear peptides followed by treatment with recombinant transglutaminase in solution; or (ii) intracellular co-expression of linear peptides and transglutaminase to achieve peptide modification in the endoplasmic reticulum prior to yeast surface display. The efficiency of peptide modification was evaluated via orthogonal detection of epitope tags integrated in the yeast-displayed peptides by flow cytometry, and via comparative cleavage of putative cyclic vs. linear peptides by tobacco etch virus (TEV) protease. Subsequently, yeast display libraries of transglutaminase-treated peptides were screened to isolate binders to the N-terminal region of the Yes-Associated Protein (YAP) and its WW domains using magnetic selection and fluorescence activated cell sorting (FACS). The identified peptide cyclo[E-LYLAYPAH-K] featured a KD of 1.75 μM for YAP and 0.68 μM for the WW domains of YAP as well as high binding selectivity against albumin and lysozyme. These results demonstrate the usefulness of enzyme-mediated cyclization in screening combinatorial libraries to identify cyclic peptide binders.}, number={4}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Bowen, John and Schneible, John and Bacon, Kaitlyn and Labar, Collin and Menegatti, Stefano and Rao, Balaji M.}, year={2021}, month={Feb} }
 @article{cutright_harris_ramesh_khan_genzer_menegatti_2021, title={Surface-Bound Microgels for Separation, Sensing, and Biomedical Applications}, volume={8}, ISSN={["1616-3028"]}, url={https://doi.org/10.1002/adfm.202104164}, DOI={10.1002/adfm.202104164}, abstractNote={Abstract}, number={47}, journal={ADVANCED FUNCTIONAL MATERIALS}, publisher={Wiley}, author={Cutright, Camden C. and Harris, Jacob L. and Ramesh, Srivatsan and Khan, Saad A. and Genzer, Jan and Menegatti, Stefano}, year={2021}, month={Aug} }
 @article{nandi_mihalko_nellenbach_castaneda_schneible_harp_deal_daniele_menegatti_barker_et al._2021, title={Synthetic Platelet Microgels Containing Fibrin Knob B Mimetic Motifs Enhance Clotting Responses}, volume={4}, ISSN={["2366-3987"]}, DOI={10.1002/adtp.202100010}, abstractNote={Abstract}, number={5}, journal={ADVANCED THERAPEUTICS}, author={Nandi, Seema and Mihalko, Emily and Nellenbach, Kimberly and Castaneda, Mario and Schneible, John and Harp, Mary and Deal, Halston and Daniele, Michael and Menegatti, Stefano and Barker, Thomas H. and et al.}, year={2021}, month={May} }
 @article{turner_senevirathne_kilgour_mcart_biggs_menegatti_daniele_2021, title={Ultrasound-Powered Implants: A Critical Review of Piezoelectric Material Selection and Applications}, volume={7}, ISSN={["2192-2659"]}, DOI={10.1002/adhm.202100986}, abstractNote={Abstract}, journal={ADVANCED HEALTHCARE MATERIALS}, author={Turner, Brendan L. and Senevirathne, Seedevi and Kilgour, Katie and McArt, Darragh and Biggs, Manus and Menegatti, Stefano and Daniele, Michael A.}, year={2021}, month={Jul} }
 @article{singhal_schneible_lilova_hall_menegatti_grafmueller_2020, title={A multiscale coarse-grained model to predict the molecular architecture and drug transport properties of modified chitosan hydrogels}, volume={16}, ISSN={["1744-6848"]}, DOI={10.1039/d0sm01243b}, abstractNote={Hydrogels constructed with functionalized polysaccharides are of interest in a multitude of applications, especially in the design of therapeutic and regenerative formulations. Computational models can efficiently guide their design.}, number={47}, journal={SOFT MATTER}, author={Singhal, Ankush and Schneible, John D. and Lilova, Radina L. and Hall, Carol K. and Menegatti, Stefano and Grafmueller, Andrea}, year={2020}, month={Dec} }
 @article{barozzi_lavoie_day_prodromou_menegatti_2020, title={Affibody-Binding Ligands}, volume={21}, ISSN={["1422-0067"]}, url={https://www.mdpi.com/1422-0067/21/11/3769}, DOI={10.3390/ijms21113769}, abstractNote={While antibodies remain established therapeutic and diagnostic tools, other protein scaffolds are emerging as effective and safer alternatives. Affibodies in particular are a new class of small proteins marketed as bio-analytic reagents. They feature tailorable binding affinity, low immunogenicity, high tissue permeation, and high expression titer in bacterial hosts. This work presents the development of affibody-binding peptides to be utilized as ligands for their purification from bacterial lysates. Affibody-binding candidates were identified by screening a peptide library simultaneously against two model affibodies (anti-immunoglobulin G (IgG) and anti-albumin) with the aim of selecting peptides targeting the conserved domain of affibodies. An ensemble of homologous sequences identified from screening was synthesized on Toyopearl® resin and evaluated via binding studies to select sequences that afford high product binding and recovery. The affibody–peptide interaction was also evaluated by in silico docking, which corroborated the targeting of the conserved domain. Ligand IGKQRI was validated through purification of an anti-ErbB2 affibody from an Escherichia coli lysate. The values of binding capacity (~5 mg affibody per mL of resin), affinity (KD ~1 μM), recovery and purity (64–71% and 86–91%), and resin lifetime (100 cycles) demonstrate that IGKQRI can be employed as ligand in affibody purification processes.}, number={11}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Barozzi, Annalisa and Lavoie, R. Ashton and Day, Kevin N. and Prodromou, Raphael and Menegatti, Stefano}, year={2020}, month={Jun} }
 @article{schneible_young_daniele_menegatti_2020, title={Chitosan Hydrogels for Synergistic Delivery of Chemotherapeutics to Triple Negative Breast Cancer Cells and Spheroids}, volume={37}, ISBN={1573-904X}, DOI={10.1007/s11095-020-02864-2}, abstractNote={This study aimed to develop a hydrogel system for treating aggressive triple negative breast cancer (TNBC) via kinetically-controlled delivery of the synergistic drug pair doxorubicin (DOX) and gemcitabine (GEM). A 2D assay was adopted to evaluate therapeutic efficacy by determining combination index (CI), and a 3D assay using cancer spheroids was implemented to assess the potential for translation in vivo. The release of DOX and GEM from an acetylated-chitosan (ACS, degree of acetylation χAc = 40 ± 5%) was characterized to identify a combined drug loading that affords release kinetics and dose that are therapeutically synergistic. The selected DOX/GEM-ACS formulation was evaluated in vitro with 2-D and 3-D models of TNBC to determine the combination index (CI) and the tumor volume reduction, respectively. Therapeutically desired release dosages and kinetics of GEM and DOX were achieved. When evaluated with a 2-D model of TNBC, the hydrogel afforded a CI of 0.14, indicating a stronger synergism than concurrent administration of DOX and GEM (CI = 0.23). Finally, the therapeutic hydrogel accomplished a notable volume reduction of the cancer spheroids (up to 30%), whereas the corresponding dosages of free drugs only reduced growth rate. The ACS hydrogel delivery system accomplishes drug release kinetics and molar ratio that affords strong therapeutically synergism. These results, in combination with the choice of ACS as affordable and highly abundant source material, provide a strong pre-clinical demonstration of the potential of the proposed system for complementing surgical resection of aggressive solid tumors.}, number={7}, journal={PHARMACEUTICAL RESEARCH}, author={Schneible, John D. and Young, Ashlyn T. and Daniele, M. A. and Menegatti, S.}, year={2020} }
 @article{reese_shanahan_lembo_tsonev_hirsh_menegatti_2020, title={Chromatographic assay to probe the binding energy and mechanisms of homologous proteins to surface-bound ligands}, volume={1136}, ISSN={["1873-376X"]}, DOI={10.1016/j.jchromb.2019.121927}, abstractNote={Probing the affinity of a ligand for homologous protein targets currently relies on laborious assays that need special equipment and high amounts of isolated, highly pure proteins. Herein we present the use of pISep, an integrated buffer system and modeling package, as an analytical method to rapidly and accurately probe the binding strength and mechanisms of homologous proteins to surface-bound ligands. To demonstrate our method, we utilized the four subclasses of human immunoglobulin G (IgG) as model homologous protein targets and the IgG-binding peptide HWRGWV as model ligand. Following IgG adsorption on a HWRGWV-Toyopearl adsorbent, the pISep buffer system was used to run uncoupled dual elution gradients of pH (from pH 8.5 to 2.5) and either isocratic or time dependent salt concentration. Both the sequence and partial overlap of elution times (IgG4 > IgG3 ≥ IgG1 > IgG2) was found to match closely the values of binding strength (KD) determined with both in silico docking simulations and isothermal titration calorimetry experiments. pISep gradients performed at different values of ionic strengths provided a means to compare the contribution of hydrophobic vs. electrostatic interactions to the IgG-peptide affinity. The shifts in retention times indicated that, among the various components of the binding energy, the hydrophobic interaction dominates in the binding of IgG2 and IgG4, whereas the binding of IgG1 and IgG3 features a balance of electrostatic and hydrophobic modes. These findings were also confirmed by the in silico analysis of the complexes formed by HWRGWV and the Fc fragment of the IgG subclasses. Collectively, these results indicate that the retention times on pISep elution gradients – in particular peak max, overlap, and shift under different conditions – directly correlate to the strength and nature of protein-ligand interactions. This work demonstrates the effectiveness of the pISep toolbox for probing the differential binding of homologous proteins to a reference ligand and informing the optimization of platform processes for the purification and fractionation of biotherapeutics.}, journal={JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES}, author={Reese, Hannah R. and Shanahan, Calvin C. and Lembo, Jacopo and Tsonev, Latchezar and Hirsh, Allen and Menegatti, Stefano}, year={2020}, month={Jan} }
 @article{bowen_schloop_reeves_menegatti_rao_2020, title={Discovery of Membrane-Permeating Cyclic Peptides via mRNA Display}, volume={31}, ISSN={["1520-4812"]}, DOI={10.1021/acs.bioconjchem.0c00413}, abstractNote={Small synthetic peptides capable of crossing biological membranes represent valuable tools in cell biology and drug delivery. While several cell-penetrating peptides (CPPs) of natural or synthetic origin have been reported, no peptide is currently known to cross both cytoplasmic and outer embryonic membranes. Here, we describe a method to engineer membrane-permeating cyclic peptides (MPPs) with broad permeation activity by screening mRNA display libraries of cyclic peptides against embryos at different developmental stages. The proposed method was demonstrated by identifying peptides capable of permeating Drosophila melanogaster (fruit fly) embryos and mammalian cells. The selected peptide cyclo[Glut-MRKRHASRRE-K*] showed a strong permeation activity of embryos exposed to minimal permeabilization pretreatment, as well as human embryonic stem cells and a murine fibroblast cell line. Notably, in both embryos and mammalian cells, the cyclic peptide outperformed its linear counterpart and the control MPPs. Confocal microscopy and single cell flow cytometry analysis were utilized to assess the degree of permeation both qualitatively and quantitatively. These MPPs have potential application in studying and nondisruptively controlling intracellular or intraembryonic processes.}, number={10}, journal={BIOCONJUGATE CHEMISTRY}, author={Bowen, John and Schloop, Allison E. and Reeves, Gregory T. and Menegatti, Stefano and Rao, Balaji M.}, year={2020}, month={Oct}, pages={2325–2338} }
 @article{turner_kilgour_stine_daniele_menegatti_2020, title={Dual-Affinity Ratiometric Quenching (DARQ) Assay for the Quantification of Therapeutic Antibodies in CHO-S Cell Culture Fluids}, volume={92}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.0c04269}, abstractNote={More than 100 monoclonal antibodies (mAbs) are in industrial and clinical development to treat myriad diseases. Accurate quantification of mAbs in complex media, derived from industrial and patient samples, is vital to determine production efficiency or pharmacokinetic properties. To date, mAb quantification requires time and labor-intensive assays. Herein, we report a novel dual-affinity ratiometric quenching (DARQ) assay, which combines selective biorecognition and quenching of fluorescence signals for rapid and sensitive quantification of therapeutic monoclonal antibodies (mAbs). The reported assay relies on the affinity complexation of the target mAb by the corresponding antigens and Protein L (PrL, which targets the Fab region of the antibody), respectively, labeled with fluorescein and rhodamine. Within the affinity complex, the mAb acts as a scaffold framing the labeled affinity tags (PrL and antigen) in a molecular proximity that results in ratiometric quenching of their fluorescence emission. Notably, the decrease in fluorescence emission intensity is linearly dependent upon mAb concentration in solution. Control experiments conducted with one affinity tag only, two tags labeled with equal fluorophores, or two tags labeled with fluorophores of discrete absorbance and emission bands exhibited significantly reduced effect. The assay was evaluated in noncompetitive (pure mAb) and competitive conditions (mAb in a Chinese Hamster Ovary (CHO) cell culture harvest). The "DARQ" assay is highly reproducible (coefficient of variation ∼0.8-0.7%) and rapid (5 min), and its sensitivity (∼0.2-0.5 ng·mL-1), limit of detection (75-119 ng·mL-1), and dynamic range (300-1600 ng·mL-1) are independent of the presence of CHO host cell proteins.}, number={24}, journal={ANALYTICAL CHEMISTRY}, author={Turner, Brendan L. and Kilgour, Katie M. and Stine, Sydney J. and Daniele, Michael and Menegatti, Stefano}, year={2020}, month={Dec}, pages={16274–16283} }
 @article{smith_fabiani_wang_ramesh_khan_santiso_silva_gorman_menegatti_2020, title={Exploring the physicochemical and morphological properties of peptide‐hybridized dendrimers (
            DendriPeps
            ) and their aggregates}, volume={58}, ISSN={2642-4150 2642-4169}, url={http://dx.doi.org/10.1002/pol.20200277}, DOI={10.1002/pol.20200277}, abstractNote={Abstract}, number={16}, journal={Journal of Polymer Science}, publisher={Wiley}, author={Smith, Ryan J. and Fabiani, Thomas and Wang, Siyao and Ramesh, Srivatsan and Khan, Saad and Santiso, Erik and Silva, Fernando Luis Barroso and Gorman, Christopher and Menegatti, Stefano}, year={2020}, month={Jul}, pages={2234–2247} }
 @article{raghuvanshi_zhu_ramezani_menegatti_santiso_mason_rodgers_janka_abolhasani_2020, title={Highly Efficient 1-Octene Hydroformylation at Low Syngas Pressure: From Single-Droplet Screening to Continuous Flow Synthesis}, volume={10}, ISSN={["2155-5435"]}, DOI={10.1021/acscatal.0c01515}, abstractNote={We present a reconfigurable flow chemistry strategy for facile transition between accelerated screening and continuous synthesis of linear aldehydes through homogeneous rhodium-catalyzed hydroformy...}, number={14}, journal={ACS CATALYSIS}, author={Raghuvanshi, Keshav and Zhu, Cheng and Ramezani, Mahdi and Menegatti, Stefano and Santiso, Erik E. and Mason, Dawn and Rodgers, Jody and Janka, Mesfin E. and Abolhasani, Milad}, year={2020}, month={Jul}, pages={7535–7542} }
 @article{bacon_blain_burroughs_mcarthrur_rao_menegatti_2020, title={Isolation of Chemically Cyclized Peptide Binders Using Yeast Surface Display}, volume={22}, ISSN={["2156-8944"]}, DOI={10.1021/acscombsci.0c00076}, abstractNote={Cyclic peptides with engineered protein-binding activity have gained increasing attention for use in therapeutic and biotechnology applications. We describe the efficient isolation and characterization of cyclic peptide binders from genetically encoded combinatorial libraries using yeast surface display. Here, peptide cyclization is achieved by disuccinimidyl glutarate-mediated crosslinking of amine groups with-in a linear peptide sequence that is expressed as a yeast cell surface fusion. Using this approach, we first screened a library of cyclic heptapeptides by magnetic selection and fluorescence activated cell sorting (FACS), to isolate binders for a model target (lysozyme) with low micromolar binding affinity (KD ~ 1.2 - 3.7 M). The isolated peptides bound lysozyme selectively, and only when cyclized. Importantly, we showed that yeast surface displayed cyclic peptides could be used to efficiently obtain quantitative es-timates of binding affinity, without chemical synthesis of the selected peptides. Subsequently, to demonstrate broader applicability of our approach, we isolated cyclic heptapeptides that bind human in-terleukin-17 (IL-17) using yeast-displayed IL-17 as a target for magnetic selection, followed by FACS using recombinant IL-17. Molecular docking simulations and follow-up experimental analyses identi-fied a candidate cyclic peptide that binds IL-17 in its receptor binding region with moderate affinity (KD ~ 300 nM). Taken together, our results show that yeast surface display can be used to efficiently isolate and characterize cyclic peptides generated by chemical modification from combinatorial libraries.}, number={10}, journal={ACS COMBINATORIAL SCIENCE}, author={Bacon, Kaitlyn and Blain, Abigail and Burroughs, Matthew and McArthrur, Nikki and Rao, Balaji M. and Menegatti, Stefano}, year={2020}, month={Oct}, pages={519–532} }
 @article{schneible_shi_young_ramesh_he_dowdey_dubnansky_libya_gao_santiso_et al._2020, title={Modified gaphene oxide (GO) particles in peptide hydrogels: a hybrid system enabling scheduled delivery of synergistic combinations of chemotherapeutics}, volume={8}, ISSN={["2050-7518"]}, DOI={10.1039/d0tb00064g}, abstractNote={Composite material enabling the delivery of synergistic combination of doxorubicin and gemcitabine against breast cancer with molar and kinetic precision.}, number={17}, journal={JOURNAL OF MATERIALS CHEMISTRY B}, author={Schneible, John D. and Shi, Kaihang and Young, Ashlyn T. and Ramesh, Srivatsan and He, Nanfei and Dowdey, Clay E. and Dubnansky, Jean Marie and Libya, Radina L. and Gao, Wei and Santiso, Erik and et al.}, year={2020}, month={May}, pages={3852–3868} }
 @article{cutright_finkelstein_orlowski_mcintosh_brotherton_fabiani_khan_genzer_menegatti_2020, title={Nonwoven fiber mats with thermo-responsive permeability to inorganic and organic electrolytes}, volume={616}, ISSN={["1873-3123"]}, DOI={10.1016/j.memsci.2020.118439}, abstractNote={This study presents the development and characterization of nonwoven fiber mats (NWFs) with stimuli-controlled permeability. An ensemble of membranes was initially constructed by coating the fibers of polypropylene NWFs with a layer of poly ((N-isopropyl acrylamide)-co-(acrylic acid)) (PNIPAm-co-AA) hydrogel. Different coatings were produced by varying the PNIPAm/AA monomer ratio between 3.9 and 18.6. The thermo-responsive layer is expanded at room temperature and contracts when heated above its lower critical solution temperature (LCST). The resulting membranes were first characterized via laser scanning microscopy and fluorescence confocal microscopy to evaluate the thickness and morphology of the hydrogel layer. Microscopy shows uniform coating of the fibers, with a thickness comparable to the fiber diameter, and homogeneous filling of the pore space. The permeability of the NWFs was then evaluated using different solutes, namely an inorganic salt (sodium chloride), an organic acid (citric acid), and an amphiphilic drug (Doxorubicin). These tests consistently show that the flux of the solute is (1) higher at temperatures > LCST, where the hydrogel layer collapses and opens the pore space, and (2) decreases at room temperature (<LCST) in a composition-dependent manner (lower for hydrogels with higher AA content); and (3) the thermo-responsive change in permeability is fully reversible.}, journal={JOURNAL OF MEMBRANE SCIENCE}, author={Cutright, Camden and Finkelstein, Rachel and Orlowski, Elliot and McIntosh, Evan and Brotherton, Zach and Fabiani, Thomas and Khan, Saad and Genzer, Jan and Menegatti, Stefano}, year={2020}, month={Dec} }
 @article{reese_xiao_shanahan_driessche_fourches_carbonell_hall_menegatti_2020, title={Novel peptide ligands for antibody purification provide superior clearance of host cell protein impurities}, volume={1625}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2020.461237}, abstractNote={The quest for ligands alternative to Protein A for the purification of monoclonal antibodies (mAbs) has been pursued for almost three decades. Yet, the IgG-binding peptides known to date still fall short of the host cell protein (HCP) logarithmic removal value (LRV) set by Protein A media (2.5-3.1). In this study, we present an integrated computational-experimental approach leading to the discovery of peptide ligands that provide HCP LRVs on par with Protein A. First, the screening of 60,000 peptide variants was performed using a high-throughput search algorithm to identify sequences that ensure IgG affinity binding. Select sequences WQRHGI, MWRGWQ, RHLGWF, and GWLHQR were then negatively screened in silico against a panel of model HCPs to ensure the selection of peptides with high binding selectivity. Candidate ligands WQRHGI and MWRGWQ were conjugated to chromatographic resins and characterized by isothermal binding and breakthrough assays to quantify static and dynamic binding capacity (Qmax and DBC10%), respectively. The resulting Qmax were 52.6 mg of IgG per mL of adsorbent for WQRHGI and 57.48 mg/mL for MWRGWQ, while the DBC10% (2 minutes residence time) were 30.1 mg/mL for WQRHGI and 36.4 mg/mL for MWRGWQ. Evaluation of the peptides by isothermal titration calorimetry (ITC) confirmed the binding energy predicted in silico, and an amino acid scanning study corroborated the affinity-like binding activity of the peptides. WQRHGI-WorkBeads resin was finally characterized by purification of a monoclonal antibody from a Chinese Hamster Ovary (CHO) cell culture harvest, affording a remarkable HCP LRV of 2.7, and consistent product yield and purity over 100 chromatographic cycles. These results demonstrate the potential of WQRHGI as an effective alternative to Protein A for antibody purification.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Reese, Hannah R. and Xiao, Xingqing and Shanahan, Calvin C. and Driessche, George A. and Fourches, Denis and Carbonell, Ruben G. and Hall, Carol K. and Menegatti, Stefano}, year={2020}, month={Aug} }
 @article{reese_bordelon_shanahan_crapanzano_sly_menegatti_2020, title={Novel peptoid-based adsorbents for purifying IgM and IgG from polyclonal and recombinant sources}, volume={1137}, ISSN={["1873-376X"]}, DOI={10.1016/j.jchromb.2019.121909}, abstractNote={Polyclonal immunoglobulin therapeutics comprising dosed IgG and IgM combinations are powerful tools in fighting cancer and severe infections. The inability of protein ligands to produce polyclonal IgG- and IgM-enriched formulations and recover monoclonal IgM calls for novel ligands with superior biorecognition activity. In this study, a peptoid ligand discovered by our group, and integrated into affinity adsorbents LigaTrap Technologies’ “Human IgG” and “Human IgM”, were utilized to purify IgG and IgM from complex fluids. IgG purification from human serum using LigaTrap IgG afforded 94.6% purity and 62.9% yield, on par with Protein A/G resins. When challenged with CHO and HEK cell culture harvests with low IgG titer (<1 mg/mL), LigaTrap IgG returned values of yield and purity well above 60% and 90%. LigaTrap IgM was evaluated for purifying IgM in comparison with commercial adsorbents, and afforded a product purity of 93% from a CHO harvest (IgM titer of 1 mg/mL) and 75.1% yield from a HEK harvest (0.5 mg/mL). LigaTrap-M provided IgM enrichment up to 11-fold higher than HiTrap resin. The peptoid adsorbents separated IgG-depleted human serum into IgM- and IgA-enriched fractions. These results demonstrate the potential of the peptoid ligand for manufacturing polyclonal Ig formulations and monoclonal IgM therapeutics.}, journal={JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES}, author={Reese, Hannah and Bordelon, Tee and Shanahan, Calvin and Crapanzano, Michael and Sly, Jae and Menegatti, Stefano}, year={2020}, month={Jan} }
 @article{cutright_brotherton_alexander_harris_shi_khan_genzer_menegatti_2020, title={Packing density, homogeneity, and regularity: Quantitative correlations between topology and thermoresponsive morphology of PNIPAM-co-PAA microgel coatings}, volume={508}, ISSN={["1873-5584"]}, DOI={10.1016/j.apsusc.2019.145129}, abstractNote={This study investigates the formation of monolayers of microgel particles comprising poly[(N-isopropylacrylamide)-co-(acrylic acid)] on solid substrates, their surface morphology, and stimuli-responsiveness. Crosslinked microgels with different chemical composition were produced to show a broad range of responses in hydrodynamic radius and degree of self-assembly with temperature and pH. Microgels were deposited on silicon wafers primed with a bilayer of poly(octadecene-alt-maleic anhydride) and polyethyleneimine by either incubation or spin coating of aqueous suspension of microgels at different temperature and pH. The characterization of the microgel-coated wafers led to the identification of three metrics describing microgel arrangement: density (ρ); heterogeneity (H), which correlates strongly with ρ and depends on deposition temperature and pH with statistical significance, but not on microgel composition; and packing efficiency (PE), which portrays the regularity of microgel arrangement and exhibits no correlation with ρ nor H. The values of ρ, H, and PE calculated for in silico models of microgel coatings confirmed that these three metrics portray distinct characteristics of surface topology. Finally, profilometry analysis showed that microgel coatings respond to thermal stimuli with sensible variations in surface roughness; notably, the thermal variation of roughness correlates strongly with ρ and H, and to a lesser extent with PE.}, journal={APPLIED SURFACE SCIENCE}, author={Cutright, Camden and Brotherton, Zach and Alexander, Landon and Harris, Jacob and Shi, Kaihang and Khan, Saad and Genzer, Jan and Menegatti, Stefano}, year={2020}, month={Apr} }
 @misc{bacon_lavoie_rao_daniele_menegatti_2020, title={Past, Present, and Future of Affinity-based Cell Separation Technologies}, volume={112}, ISSN={["1878-7568"]}, DOI={10.1016/j.actbio.2020.05.004}, abstractNote={Progress in cell purification technology is critical to increase the availability of viable cells for therapeutic, diagnostic, and research applications. A variety of techniques are now available for cell separation, ranging from non-affinity methods such as density gradient centrifugation, dielectrophoresis, and filtration, to affinity methods such as chromatography, two-phase partitioning, and magnetic-/fluorescence-assisted cell sorting. For clinical and analytical procedures that require highly purified cells, the choice of cell purification method is crucial, since every method offers a different balance between yield, purity, and bioactivity of the cell product. For most applications, the requisite purity is only achievable through affinity methods, owing to the high target specificity that they grant. In this review, we discuss past and current methods for developing cell-targeting affinity ligands and their application in cell purification, along with the benefits and challenges associated with different purification formats. We further present new technologies, like stimuli-responsive ligands and parallelized microfluidic devices, towards improving the viability and throughput of cell products for tissue engineering and regenerative medicine. Our comparative analysis provides guidance in the multifarious landscape of cell separation techniques and highlights new technologies that are poised to play a key role in the future of cell purification in clinical settings and the biotech industry. STATEMENT OF SIGNIFICANCE: Technologies for cell purification have served science, medicine, and industrial biotechnology and biomanufacturing for decades. This review presents a comprehensive survey of this field by highlighting the scope and relevance of all known methods for cell isolation, old and new alike. The first section covers the main classes of target cells and compares traditional non-affinity and affinity-based purification techniques, focusing on established ligands and chromatographic formats. The second section presents an excursus of affinity-based pseudo-chromatographic and non-chromatographic technologies, especially focusing on magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). Finally, the third section presents an overview of new technologies and emerging trends, highlighting how the progress in chemical, material, and microfluidic sciences has opened new exciting avenues towards high-throughput and high-purity cell isolation processes. This review is designed to guide scientists and engineers in their choice of suitable cell purification techniques for research or bioprocessing needs.}, journal={ACTA BIOMATERIALIA}, author={Bacon, Kaitlyn and Lavoie, Ashton and Rao, Balaji M. and Daniele, Michael and Menegatti, Stefano}, year={2020}, month={Aug}, pages={29–51} }
 @misc{reese_shanahan_proulx_menegatti_2020, title={Peptide science: A "rule model" for new generations of peptidomimetics}, volume={102}, ISSN={["1878-7568"]}, DOI={10.1016/j.actbio.2019.10.045}, abstractNote={Peptides have been heavily investigated for their biocompatible and bioactive properties. Though a wide array of functionalities can be introduced by varying the amino acid sequence or by structural constraints, properties such as proteolytic stability, catalytic activity, and phase behavior in solution are difficult or impossible to impart upon naturally occurring α-L-peptides. To this end, sequence-controlled peptidomimetics exhibit new folds, morphologies, and chemical modifications that create new structures and functions. The study of these new classes of polymers, especially α-peptoids, has been highly influenced by the analysis, computational, and design techniques developed for peptides. This review examines techniques to determine primary, secondary, and tertiary structure of peptides, and how they have been adapted to investigate peptoid structure. Computational models developed for peptides have been modified to predict the morphologies of peptoids and have increased in accuracy in recent years. The combination of in vitro and in silico techniques have led to secondary and tertiary structure design principles that mirror those for peptides. We then examine several important developments in peptoid applications inspired by peptides such as pharmaceuticals, catalysis, and protein-binding. A brief survey of alternative backbone structures and research investigating these peptidomimetics shows how the advancement of peptide and peptoid science has influenced the growth of numerous fields of study. As peptide, peptoid, and other peptidomimetic studies continue to advance, we will expect to see higher throughput structural analyses, greater computational accuracy and functionality, and wider application space that can improve human health, solve environmental challenges, and meet industrial needs. Many historical, chemical, and functional relations draw a thread connecting peptides to their recent cognates, the “peptidomimetics”. This review presents a comprehensive survey of this field by highlighting the width and relevance of these familial connections. In the first section, we examine the experimental and computational techniques originally developed for peptides and their morphing into a broader analytical and predictive toolbox. The second section presents an excursus of the structures and properties of prominent peptidomimetics, and how the expansion of the chemical and structural diversity has returned new exciting properties. The third section presents an overview of technological applications and new families of peptidomimetics. As the field grows, new compounds emerge with clear potential in medicine and advanced manufacturing.}, journal={ACTA BIOMATERIALIA}, author={Reese, Hannah R. and Shanahan, Calvin C. and Proulx, Caroline and Menegatti, Stefano}, year={2020}, month={Jan}, pages={35–74} }
 @article{day_schneible_young_pozdin_driessche_gaffney_prodromou_freytes_fourches_daniele_et al._2020, title={Photoinduced reconfiguration to control the protein-binding affinity of azobenzene-cyclized peptides}, volume={8}, ISSN={["2050-7518"]}, DOI={10.1039/d0tb01189d}, abstractNote={Light-controlled switching of cell-binding activity of fluorescently-labeled peptides for on-demand cell labeling.}, number={33}, journal={JOURNAL OF MATERIALS CHEMISTRY B}, author={Day, Kevin and Schneible, John D. and Young, Ashlyn T. and Pozdin, Vladimir A. and Driessche, George and Gaffney, Lewis A. and Prodromou, Raphael and Freytes, Donald O. and Fourches, Denis and Daniele, Michael and et al.}, year={2020}, month={Sep}, pages={7413–7427} }
 @article{gilbertie_schaer_schubert_jacob_menegatti_lavoie_schnabel_2020, title={Platelet-rich plasma lysate displays antibiofilm properties and restores antimicrobial activity against synovial fluid biofilms in vitro}, volume={38}, ISSN={["1554-527X"]}, DOI={10.1002/jor.24584}, abstractNote={Abstract}, number={6}, journal={JOURNAL OF ORTHOPAEDIC RESEARCH}, author={Gilbertie, Jessica M. and Schaer, Thomas P. and Schubert, Alicia G. and Jacob, Megan E. and Menegatti, Stefano and Lavoie, R. Ashton and Schnabel, Lauren V}, year={2020}, month={Jun}, pages={1365–1374} }
 @article{reese_bordelon_odeh_broussard_kormos_murphy_shanahan_menegatti_2020, title={Purification of animal immunoglobulin G (IgG) using peptoid affinity ligands}, ISBN={1520-6033}, DOI={10.1002/btpr.2994}, abstractNote={Abstract}, journal={BIOTECHNOLOGY PROGRESS}, author={Reese, Hannah and Bordelon, Tee and Odeh, Fuad and Broussard, Amanda and Kormos, Chad and Murphy, Andrew and Shanahan, Calvin and Menegatti, Stefano}, year={2020} }
 @article{lavoie_fazio_williams_carbonell_menegatti_2020, title={Targeted capture of Chinese hamster ovary host cell proteins: Peptide ligand binding by proteomic analysis}, volume={117}, ISSN={["1097-0290"]}, url={http://europepmc.org/abstract/med/31654407}, DOI={10.1002/bit.27213}, abstractNote={Abstract}, number={2}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Lavoie, R. Ashton and Fazio, Alice and Williams, Taufika Islam and Carbonell, Ruben and Menegatti, Stefano}, year={2020}, month={Feb}, pages={438–452} }
 @article{bacon_bowen_reese_rao_menegatti_2020, title={Use of Target-Displaying Magnetized Yeast in Screening mRNA-Display Peptide Libraries to Identify Ligands}, volume={22}, ISSN={["2156-8944"]}, DOI={10.1021/acscombsci.0c00171}, abstractNote={This work presents the first use of yeast-displayed protein targets for screening mRNA-display libraries of cyclic and linear peptides. The WW domains of Yes-Associated Protein 1 (WW-YAP) and mitochondrial import receptor subunit TOM22 were adopted as protein targets. Yeast cells displaying WW-YAP or TOM22 were magnetized with iron oxide nanoparticles to enable the isolation of target-binding mRNA-peptide fusions. Equilibrium adsorption studies were conducted to estimate the binding affinity (KD) of select WW-YAP-binding peptides: KD values of 37 and 4 μM were obtained for cyclo[M-AFRLC-K] and its linear cognate, and 40 and 3 μM for cyclo[M-LDFVNHRSRG-K] and its linear cognate, respectively. TOM22-binding peptide cyclo[M-PELNRAI-K] was conjugated to magnetic beads and incubated with yeast cells expressing TOM22 and luciferase. A luciferase-based assay showed a 4.5-fold higher binding of TOM22+ yeast compared to control cells. This work demonstrates that integrating mRNA- and yeast-display accelerates the discovery of peptide ligands.}, number={12}, journal={ACS COMBINATORIAL SCIENCE}, author={Bacon, Kaitlyn and Bowen, John and Reese, Hannah and Rao, Balaji M. and Menegatti, Stefano}, year={2020}, month={Dec}, pages={738–744} }
 @article{saberi-bosari_omary_lavoie_prodromou_day_menegatti_san-miguel_2019, title={Affordable Microfluidic Bead-Sorting Platform for Automated Selection of Porous Particles Functionalized with Bioactive Compounds}, volume={9}, ISSN={["2045-2322"]}, url={http://dx.doi.org/10.1038/s41598-019-42869-5}, DOI={10.1038/s41598-019-42869-5}, abstractNote={Abstract}, number={1}, journal={SCIENTIFIC REPORTS}, publisher={Springer Science and Business Media LLC}, author={Saberi-Bosari, Sahand and Omary, Mohammad and Lavoie, Ashton and Prodromou, Raphael and Day, Kevin and Menegatti, Stefano and San-Miguel, Adriana}, year={2019}, month={May} }
 @article{smith_gorman_menegatti_2019, title={DendriPeps: Expanding Dendrimer Functionality by Hybridizing Poly(amidoamine) (PAMAM) Scaffolds with Peptide Segments}, volume={40}, ISSN={["1521-3927"]}, url={https://doi.org/10.1002/marc.201900325}, DOI={10.1002/marc.201900325}, abstractNote={Abstract}, number={22}, journal={MACROMOLECULAR RAPID COMMUNICATIONS}, publisher={Wiley}, author={Smith, Ryan J. and Gorman, Christopher B. and Menegatti, Stefano}, year={2019}, month={Nov} }
 @article{day_prodromou_bosari_lavoie_omary_market_san miguel_menegatti_2019, title={Discovery and Evaluation of Peptide Ligands for Selective Adsorption and Release of Cas9 Nuclease on Solid Substrates}, volume={30}, ISSN={["1520-4812"]}, url={http://dx.doi.org/10.1021/acs.bioconjchem.9b00703}, DOI={10.1021/acs.bioconjchem.9b00703}, abstractNote={The rapid expansion of CRISPR in biotechnology, medicine, and bioprocessing poses an urgent need for advanced manufacturing of Cas nucleases. The lack of Cas-targeting ligands, however, prevents the development of platform processes for purifying this class of molecules. This work represents the first effort at developing short synthetic Cas9-binding peptides and demonstrates their applicability as affinity ligands for the purification of a Cas nuclease. Candidate Cas9-targeting peptides were initially identified by screening a solid-phase peptide library against a model mixture of Streptococcus pyogenes Cas9 spiked in Escherichia coli cell lysate. An ensemble of homologous sequences were identified, conjugated on Toyopearl resin, and evaluated by Cas9 binding studies to identify sequences providing selective Cas9 capture and efficient release. In silico docking studies were also performed to evaluate the binding energy and site of the various peptides on Cas9. Notably, sequences GYYRYSEY and YYHRHGLQ were shown to target the RecII domain of Cas9, which is not involved in nuclease activity, and was targeted as ideal binding site. The peptide ligands were validated by purifying Cas9 from the E. coli lysate in dynamic conditions and through measurements of binding capacity and strength (Qmax and KD). The resulting values of Qmax = 4 - 5 mg Cas9 per mL of resin and KD ~ 0.1 - 0.3 μM, and product recovery (86 - 89%) and purity (91% - 93%) indicate that both peptides, and YYHRHGLQ in particular, can serve as capture ligands in a platform purification process of Cas9.}, number={12}, journal={BIOCONJUGATE CHEMISTRY}, publisher={American Chemical Society (ACS)}, author={Day, Kevin and Prodromou, Raphael and Bosari, Sahand Saberi and Lavoie, Ashton and Omary, Mohammad and Market, Connor and San Miguel, Adriana and Menegatti, Stefano}, year={2019}, month={Dec}, pages={3057–3068} }
 @article{lavoie_fazio_carbonell_menegatti_2019, title={Multiplexed Competitive Screening of One-Bead-One-Component Combinatorial Libraries Using a ClonePix 2 Colony Sorter}, volume={20}, ISSN={["1422-0067"]}, DOI={10.3390/ijms20205119}, abstractNote={Screening solid-phase combinatorial libraries of bioactive compounds against fluorescently labeled target biomolecules is an established technology in ligand and drug discovery. Rarely, however, do screening methods include comprehensive strategies—beyond mere library blocking and competitive screening—to ensure binding selectivity of selected leads. This work presents a method for multiplexed solid-phase peptide library screening using a ClonePix 2 Colony Picker that integrates (i) orthogonal fluorescent labeling for positive selection against a target protein and negative selection against competitor species with (ii) semi-quantitative tracking of target vs. competitor binding for every library bead. The ClonePix 2 technology enables global at-a-glance evaluation and customization of the parameters for bead selection to ensure high affinity and selectivity of the isolated leads. A case study is presented by screening a peptide library against green-labeled human immunoglobulin G (IgG) and red-labeled host cell proteins (HCPs) using ClonePix 2 to select HCP-binding ligands for flow-through chromatography applications. Using this approach, 79 peptide ligand candidates (6.6% of the total number of ligands screened) were identified as potential HCP-selective ligands, enabling a potential rate of >3,000 library beads screened per hour.}, number={20}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Lavoie, R. Ashton and Fazio, Alice and Carbonell, Ruben G. and Menegatti, Stefano}, year={2019}, month={Oct} }
 @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{bacon_burroughs_blain_menegatti_rao_2019, title={Screening Yeast Display Libraries against Magnetized Yeast Cell Targets Enables Efficient Isolation of Membrane Protein Binders}, volume={21}, ISSN={["2156-8944"]}, DOI={10.1021/acscombsci.9b00147}, abstractNote={When isolating binders from yeast displayed combinatorial libraries, a soluble, recombinantly expressed form of the target protein is typically utilized. As an alternative, we describe the use of target proteins displayed as surface fusions on magnetized yeast cells. In our strategy, the target protein is coexpressed on the yeast surface with an iron oxide binding protein; incubation of these yeast cells with iron oxide nanoparticles results in their magnetization. Subsequently, binder cells that interact with the magnetized target cells can be isolated using a magnet. Using a known binder–target pair with modest binding affinity (KD ≈ 400 nM), we showed that a binder present at low frequency (1 in 105) could be enriched more than 100-fold, in a single round of screening, suggesting feasibility of screening combinatorial libraries. Subsequently, we screened yeast display libraries of Sso7d and nanobody variants against yeast displayed targets to isolate binders specific to the cytosolic domain of the mitochondrial membrane protein TOM22 (KD ≈ 272–1934 nM) and the extracellular domain of the c-Kit receptor (KD ≈ 93 to KD > 2000 nM). Additional studies showed that the TOM22 binders identified using this approach could be used for the enrichment of mitochondria from cell lysates, thereby confirming binding to the native mitochondrial protein. The ease of expressing a membrane protein or a domain thereof as a yeast cell surface fusion—in contrast to recombinant soluble expression—makes the use of yeast-displayed targets particularly attractive. Therefore, we expect the use of magnetized yeast cell targets will enable efficient isolation of binders to membrane proteins.}, number={12}, journal={ACS COMBINATORIAL SCIENCE}, author={Bacon, Kaitlyn and Burroughs, Matthew and Blain, Abigail and Menegatti, Stefano and Rao, Balaji M.}, year={2019}, month={Dec}, pages={817–832} }
 @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{schneible_singhal_lilova_hall_grafmueller_menegatti_2019, title={Tailoring the Chemical Modification of Chitosan Hydrogels to Fine-Tune the Release of a Synergistic Combination of Chemotherapeutics}, volume={20}, ISSN={["1526-4602"]}, DOI={10.1021/acs.biomac.9b00707}, abstractNote={Combination chemotherapy with defined ratio and sequence of drug release is a clinically established and effective route to treat advanced solid tumors. In this context, a growing body of literature demonstrates the potential of hydrogels constructed with chemically modified polysaccharides as depots for controlled release of chemotherapeutics. Identifying the appropriate modification in terms of physicochemical properties of the functional group and its degree of substitution (χ) to achieve the desired release profile for multiple drugs is, however, a complex multivariate problem. To address this issue, we have developed a computational toolbox that models the migration of a drug pair through a hydrated network of polysaccharide chains modified with hydrophobic moieties. In this study, we chose Doxorubicin (DOX) and Gemcitabine (GEM) as model drugs, as their synergistic effect against breast cancer has been thoroughly investigated, and chitosan as model polymer. Our model describes how the modification of chitosan chains with acetyl, butanoyl, and heptanoyl moieties at different values χ governs both the structure of the hydrogel network and drug migration through it. Our experimental data confirm the in silico predictions for both single and dual-drug release, and, most notably, the counterintuitive inversion of release vs. χ that occurs when switching from a single to a dual-drug system. Consensus between predicted and experimental data indicates that acetyl modifications (χ = 32-42%) and butanoyl-modifications (χ = 19-24%) provide synergistic GEM/DOX release molar ratios (5-10 i.e.,). Collectively, these results demonstrate the potential of this model in guiding the design of chemotherapeutic hydrogels to combat cancer.}, number={8}, journal={BIOMACROMOLECULES}, author={Schneible, John D. and Singhal, Ankush and Lilova, Radina L. and Hall, Carol K. and Grafmueller, Andrea and Menegatti, Stefano}, year={2019}, month={Aug}, pages={3126–3141} }
 @article{lavoie_fazio_blackburn_goshe_carbonell_menegatti_2019, title={Targeted Capture of Chinese Hamster Ovary Host Cell Proteins: Peptide Ligand Discovery}, volume={20}, ISSN={["1422-0067"]}, DOI={10.3390/ijms20071729}, abstractNote={The growing integration of quality-by-design (QbD) concepts in biomanufacturing calls for a detailed and quantitative knowledge of the profile of impurities and their impact on the product safety and efficacy. Particularly valuable is the determination of the residual level of host cell proteins (HCPs) secreted, together with the product of interest, by the recombinant cells utilized for production. Though often referred to as a single impurity, HCPs comprise a variety of species with diverse abundance, size, function, and composition. The clearance of these impurities is a complex issue due to their cell line to cell line, product-to-product, and batch-to-batch variations. Improvements in HCP monitoring through proteomic-based methods have led to identification of a subset of “problematic” HCPs that are particularly challenging to remove, both at the product capture and product polishing steps, and compromise product stability and safety even at trace concentrations. This paper describes the development of synthetic peptide ligands capable of capturing a broad spectrum of Chinese hamster ovary (CHO) HCPs with a combination of peptide species that allow for advanced mixed-mode binding. Solid phase peptide libraries were screened for identification and characterization of peptides that capture CHO HCPs while showing minimal binding of human IgG, utilized here as a model product. Tetrameric and hexameric ligands featuring either multipolar or hydrophobic/positive amino acid compositions were found to be the most effective. Tetrameric multipolar ligands exhibited the highest targeted binding ratio (ratio of HCP clearance over IgG loss), more than double that of commercial mixed-mode and anion exchange resins utilized by industry for IgG polishing. All peptide resins tested showed preferential binding to HCPs compared to IgG, indicating potential uses in flow-through mode or weak-partitioning-mode chromatography.}, number={7}, journal={INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES}, author={Lavoie, R. Ashton and Fazio, Alice and Blackburn, R. Kevin and Goshe, Michael B. and Carbonell, Ruben G. and Menegatti, Stefano}, year={2019}, month={Apr} }
 @article{bordelon_bobay_murphy_reese_shanahan_odeh_broussard_kormos_menegatti_2019, title={Translating antibody-binding peptides into peptoid ligands with improved affinity and stability}, volume={1602}, ISSN={["1873-3778"]}, DOI={10.1016/j.chroma.2019.05.047}, abstractNote={A great number of protein-binding peptides are known and utilized as drugs, diagnostic reagents, and affinity ligands. Recently, however, peptide mimetics have been proposed as valuable alternative to peptides by virtue of their excellent biorecognition activity and higher biochemical stability. This poses the need to develop a strategy for translating known protein-binding peptides into peptoid analogues with comparable or better affinity. This work proposes a route for translation utilizing the IgG-binding peptide HWRGWV as reference sequence. An ensemble of peptoid analogues of HWRGWV were produced by adjusting the number and sequence arrangement of residues containing functional groups that resemble both natural and non-natural amino acids. The variants were initially screened via IgG binding tests in non-competitive mode to select candidate ligands. A set of selected peptoids were studied in silico by docking onto putative binding sites identified on the crystal structures of human IgG1, IgG2, IgG3, and IgG4 subclasses, returning values of predicted binding energy that aligned well with the binding data. Selected peptoids PL-16 and PL-22 were further characterized by binding isotherm analysis to determine maximum capacity (Qmax ˜ 48–57 mg of IgG per mL of adsorbent) and binding strength on solid phase (KD ˜ 5.4–7.8 10−7 M). Adsorbents PL-16-Workbeads and PL-22-Workbeads were used for purifying human IgG from a cell culture supernatant added with bovine serum, affording high values of IgG recovery (up to 85%) and purity (up to 98%) under optimized binding and elution conditions. Both peptoid ligands also proved to be stable against proteolytic enzymes and strong alkaline agents. Collectively, these studies form a method guiding the design of peptoid variants of cognate peptide ligands, and help addressing the challenges that, despite the structural similarity, the peptide-to-peptoid translation presents.}, journal={JOURNAL OF CHROMATOGRAPHY A}, author={Bordelon, Tee and Bobay, Benjamin and Murphy, Andrew and Reese, Hannah and Shanahan, Calvin and Odeh, Fuad and Broussard, Amanda and Kormos, Chad and Menegatti, Stefano}, year={2019}, month={Sep}, pages={284–299} }
 @article{pusuluri_krishnan_lensch_sarode_bunyan_vogus_menegatti_soh_mitragotri_2019, title={Treating Tumors at Low Drug Doses Using an Aptamer-Peptide Synergistic Drug Conjugate}, volume={58}, ISSN={["1521-3773"]}, DOI={10.1002/anie.201812650}, abstractNote={Abstract}, number={5}, journal={ANGEWANDTE CHEMIE-INTERNATIONAL EDITION}, author={Pusuluri, Anusha and Krishnan, Vinu and Lensch, Valerie and Sarode, Apoorva and Bunyan, Elaine and Vogus, Douglas R. and Menegatti, Stefano and Soh, H. Tom and Mitragotri, Samir}, year={2019}, month={Jan}, pages={1437–1441} }
 @article{kish_roach_sachi_naik_menegatti_carbonell_2018, title={Purification of human erythropoietin by affinity chromatography using cyclic peptide ligands}, volume={1085}, ISSN={["1873-376X"]}, DOI={10.1016/j.jchromb.2018.03.039}, abstractNote={Prior work described the identification and characterization of erythropoietin-binding cyclic peptides SLFFLH, VVFFVH, FSLLHH and FSLLSH (all of the form cyclo[(Nα-Ac)Dap(A)-X1-X6-AE], wherein X1-X6 is the listed sequences). In this work, the peptide ligands were synthesized on Toyopearl chromatographic resins and utilized for purifying recombinant human erythropoietin (rHuEPO) from complex sources. Elution buffer pH and composition were optimized to maximize the recovery of standard rHuEPO from the peptide resins. The peptide-based adsorbents were employed for separating rHuEPO from a mixture of albumin, myoglobin, and IgG to examine their selectivity. When using FSLLHH, the inclusion of low amounts of surfactants in the wash and elution buffers facilitated the recovery of rHuEPO with high yield and purity. Specifically, FSLLSH and VVFFVH afforded the most efficient separation of rHuEPO, with yield and purity of 85% and 95–97%, respectively. The affinity resins were also utilized to purify rHuEPO from spiked CHO cell culture fluid. In particular, FSLLSH provided the most successful separation from CHO, with yield and purity above 90%, and 1.0 log10 reduction of host cell proteins. The influence of conductivity and pH in the CHO-rHuEPO load was investigated. Finally, FSLLSH-based resins were used to purify rHuEPO spiked into a Pichia pastoris cell culture fluid, resulting in product yield and purity of 96% and 84%, respectively, and 1.3 log10 reduction of host DNA. These results compare well with values obtained using wheat germ agglutinin agarose and clearly indicate the potential of the cyclic peptide resins as a viable tool for rHuEPO purification.}, journal={JOURNAL OF CHROMATOGRAPHY B-ANALYTICAL TECHNOLOGIES IN THE BIOMEDICAL AND LIFE SCIENCES}, author={Kish, William S. and Roach, Matthew K. and Sachi, Hiroyuki and Naik, Amith D. and Menegatti, Stefano and Carbonell, Ruben G.}, year={2018}, month={May}, pages={1–12} }
 @article{wilkins_turner_rivera_menegatti_daniele_2018, title={Quantum dot enabled lateral flow immunoassay for detection of cardiac biomarker NT-proBNP}, volume={21}, ISSN={2214-1804}, url={http://dx.doi.org/10.1016/J.SBSR.2018.10.002}, DOI={10.1016/J.SBSR.2018.10.002}, abstractNote={B-type natriuretic peptide (BNP) is a cardiac-derived neurohormone regulated by intra-cardiac pressure. N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a bio-inert product of BNP production identified as a blood biomarker for diagnosing cardiac distress. Rapid, point-of-care quantification of NT-proBNP concentration is challenging because accurate recognition is often masked by interference of native BNP in blood samples. Recently, monoclonal antibodies have been engineered with high specificity to the tail, i.e. cleaved region, of NT-proBNP; herein, we present a case study using two monoclonal antibodies for NT-proBNP, respectively, as detector and capture antibodies. Using the selected antibodies, we fabricated and characterized a lateral flow immunoassay for NT-proBNP using antibody-labeled quantum dots to improve limit of detection. The reported lateral flow immunoassay exhibited clinically-relevant linear response range: 50–1200 pg mL−1, good stability and repeatability with pooled variance of 8.8% across concentrations of NT-proBNP—highlighting a method for high-resolution, low-cost quantification of NT-proBNP as a point-of-care, single-step diagnostic sensor.}, journal={Sensing and Bio-Sensing Research}, publisher={Elsevier BV}, author={Wilkins, Michael D. and Turner, Brendan L. and Rivera, Kristina R. and Menegatti, Stefano and Daniele, Michael}, year={2018}, month={Nov}, pages={46–53} }
 @article{vogus_evans_pusuluri_barajas_zhang_krishnan_nowak_menegatti_helgeson_squires_et al._2017, title={A hyaluronic acid conjugate engineered to synergistically and sequentially deliver gemcitabine and doxorubicin to treat triple negative breast cancer}, volume={267}, ISSN={0168-3659}, url={http://dx.doi.org/10.1016/J.JCONREL.2017.08.016}, DOI={10.1016/J.JCONREL.2017.08.016}, abstractNote={Combination chemotherapy is commonly used to treat advanced breast cancer. However, treatment success is often limited due to systemic toxicity. To improve therapeutic efficacy, polymer drug conjugates carrying synergistic pairs of chemotherapy drugs can be used to reduce drug administration dose. Here, we systematically evaluated the effect of temporal scheduling of doxorubicin (DOX) and gemcitabine (GEM) on drug synergy. Hyaluronic acid (HA) drug conjugates with distinct linkers conjugating both DOX and GEM were synthesized to control relative release kinetics of each drug. We show that polymer conjugates that release GEM faster than DOX are more effective at killing triple negative breast cancer cells in vitro. We further show that the optimal dual drug conjugate more effectively inhibits the growth of an aggressive, orthotopic 4T1 tumor model in vivo than free DOX and GEM and the single drug HA conjugates. The dual drug HA conjugate can inhibit 4T1 tumor growth in vivo during treatment through both intravenous and non-local subcutaneous injections. These results emphasize the importance of understanding the effect release rates have on the efficacy of synergistic drug carriers and motivate the use of HA as a delivery platform for multiple cancer types.}, journal={Journal of Controlled Release}, publisher={Elsevier BV}, author={Vogus, Douglas R. and Evans, Michael A. and Pusuluri, Anusha and Barajas, Alexandra and Zhang, Mengwen and Krishnan, Vinu and Nowak, Maksymilian and Menegatti, Stefano and Helgeson, Matthew E. and Squires, Todd M. and et al.}, year={2017}, month={Dec}, pages={191–202} }
 @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{wojnilowicz_tortora_bobay_santiso_caruso_micheli_venanzi_menegatti_cavalieri_2016, title={A combined approach for predicting the cytotoxic effect of drug-nanoaggregates}, volume={4}, ISSN={2050-750X 2050-7518}, url={http://dx.doi.org/10.1039/C6TB02105K}, DOI={10.1039/C6TB02105K}, abstractNote={Polymer carriers induce assembly of drugs into nanoaggregates and play a role in tuning the architecture and bioactivity of drug nanoaggregates.}, number={40}, journal={Journal of Materials Chemistry B}, publisher={Royal Society of Chemistry (RSC)}, author={Wojnilowicz, M. and Tortora, M. and Bobay, B. G. and Santiso, E. and Caruso, M. and Micheli, L. and Venanzi, M. and Menegatti, S. and Cavalieri, F.}, year={2016}, pages={6516–6523} }
 @article{camacho_menegatti_vogus_pusuluri_fuchs_jarvis_zakrewsky_evans_chen_mitragotri_2016, title={DAFODIL: A novel liposome-encapsulated synergistic combination of doxorubicin and 5FU for low dose chemotherapy}, volume={229}, ISSN={["1873-4995"]}, DOI={10.1016/j.jconrel.2016.03.027}, abstractNote={PEGylated liposomes have transformed chemotherapeutic use of doxorubicin by reducing its cardiotoxicity; however, it remains unclear whether liposomal doxorubicin is therapeutically superior to free doxorubicin. Here, we demonstrate a novel PEGylated liposome system, named DAFODIL (Doxorubicin And 5-Flurouracil Optimally Delivered In a Liposome) that inarguably offers superior therapeutic efficacies compared to free drug administrations. Delivery of synergistic ratios of this drug pair led to greater than 90% reduction in tumor growth of murine 4T1 mammary carcinoma in vivo. By exploiting synergistic ratios, the effect was achieved at remarkably low doses, far below the maximum tolerable drug doses. Our approach re-invents the use of liposomes for multi-drug delivery by providing a chemotherapy vehicle which can both reduce toxicity and improve therapeutic efficacy. This methodology is made feasible by the extension of the ammonium-sulfate gradient encapsulation method to nucleobase analogues, a liposomal entrapment method once conceived useful only for anthracyclines. Therefore, our strategy can be utilized to efficiently evaluate various chemotherapy combinations in an effort to translate more effective combinations into the clinic.}, journal={JOURNAL OF CONTROLLED RELEASE}, author={Camacho, Kathryn M. and Menegatti, Stefano and Vogus, Douglas R. and Pusuluri, Anusha and Fuchs, Zoe and Jarvis, Maria and Zakrewsky, Michael and Evans, Michael A. and Chen, Renwei and Mitragotri, Samir}, year={2016}, month={May}, pages={154–162} }
 @article{menegatti_zakrewsky_kumar_de oliveira_muraski_mitragotri_2016, title={De Novo Design of Skin-Penetrating Peptides for Enhanced Transdermal Delivery of Peptide Drugs}, volume={5}, ISSN={["2192-2659"]}, DOI={10.1002/adhm.201500634}, abstractNote={Skin‐penetrating peptides (SPPs) are attracting increasing attention as a non‐invasive strategy for transdermal delivery of therapeutics. The identification of SPP sequences, however, currently performed by experimental screening of peptide libraries, is very laborious. Recent studies have shown that, to be effective enhancers, SPPs must possess affinity for both skin keratin and the drug of interest. We therefore developed a computational process for generating and screening virtual libraries of disulfide‐cyclic peptides against keratin and cyclosporine A (CsA) to identify SPPs capable of enhancing transdermal CsA delivery. The selected sequences were experimentally tested and found to bind both CsA and keratin, as determined by mass spectrometry and affinity chromatography, and enhance transdermal permeation of CsA. Four heptameric sequences that emerged as leading candidates (ACSATLQHSCG, ACSLTVNWNCG, ACTSTGRNACG, and ACSASTNHNCG) were tested and yielded CsA permeation on par with previously identified SPP SPACE TM. An octameric peptide (ACNAHQARSTCG) yielded significantly higher delivery of CsA compared to heptameric SPPs. The safety profile of the selected sequences was also validated by incubation with skin keratinocytes. This method thus represents an effective procedure for the de novo design of skin‐penetrating peptides for the delivery of desired therapeutic or cosmetic agents.}, number={5}, journal={ADVANCED HEALTHCARE MATERIALS}, author={Menegatti, Stefano and Zakrewsky, Michael and Kumar, Sunny and De Oliveira, Joshua Sanchez and Muraski, John A. and Mitragotri, Samir}, year={2016}, month={Mar}, pages={602–609} }
 @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} }
 @article{camacho_menegatti_mitragotri_2016, title={Low-molecular-weight polymer-drug conjugates for synergistic anticancer activity of camptothecin and doxorubicin combinations}, volume={11}, DOI={10.2217/nnm.16.33}, abstractNote={ Background: High-molecular-weight (MW) polymers (>50,000 Da) can be conjugated to chemotherapy drugs in order to improve their tumor accumulation, while low MW polymers ≤10,000 Da are often overlooked due to faster plasma clearance. Small polymers, however, may facilitate deeper tumor penetration. Materials & methods: Here, we investigate the anticancer efficacy of 10 kDa hyaluronic acid or poly(vinyl alcohol) conjugated to synergistic combinations of camptothecin and doxorubicin, with emphasis on chemical linker impacts. Results: Our results emphasize drug hydrolyzability for synergy preservation, and also demonstrate superior cancer cell inhibition with low MW polymer–drug conjugates. Conclusion: This study shows the high therapeutic potential of low MW polymer–drug conjugates for polychemotherapy delivery, and provides further insight into the development of polymer-drug therapeutics. }, number={9}, journal={Nanomedicine}, author={Camacho, K. M. and Menegatti, S. and Mitragotri, S.}, year={2016}, pages={1139–1151} }
 @article{anselmo_zhang_kumar_vogus_menegatti_helgeson_mitragotri_2015, title={Elasticity of Nanoparticles Influences Their Blood Circulation, Phagocytosis, Endocytosis, and Targeting}, volume={9}, ISSN={1936-0851 1936-086X}, url={http://dx.doi.org/10.1021/ACSNANO.5B00147}, DOI={10.1021/ACSNANO.5B00147}, abstractNote={The impact of physical and chemical modifications of nanoparticles on their biological function has been systemically investigated and exploited to improve their circulation and targeting. However, the impact of nanoparticles' flexibility (i.e., elastic modulus) on their function has been explored to a far lesser extent, and the potential benefits of tuning nanoparticle elasticity are not clear. Here, we describe a method to synthesize polyethylene glycol (PEG)-based hydrogel nanoparticles of uniform size (200 nm) with elastic moduli ranging from 0.255 to 3000 kPa. These particles are used to investigate the role of particle elasticity on key functions including blood circulation time, biodistribution, antibody-mediated targeting, endocytosis, and phagocytosis. Our results demonstrate that softer nanoparticles (10 kPa) offer enhanced circulation and subsequently enhanced targeting compared to harder nanoparticles (3000 kPa) in vivo. Furthermore, in vitro experiments show that softer nanoparticles exhibit significantly reduced cellular uptake in immune cells (J774 macrophages), endothelial cells (bEnd.3), and cancer cells (4T1). Tuning nanoparticle elasticity potentially offers a method to improve the biological fate of nanoparticles by offering enhanced circulation, reduced immune system uptake, and improved targeting.}, number={3}, journal={ACS Nano}, publisher={American Chemical Society (ACS)}, author={Anselmo, Aaron C. and Zhang, Mengwen and Kumar, Sunny and Vogus, Douglas R. and Menegatti, Stefano and Helgeson, Matthew E. and Mitragotri, Samir}, year={2015}, month={Mar}, pages={3169–3177} }
 @article{kumar_zakrewsky_chen_menegatti_muraski_mitragotri_2015, title={Peptides as skin penetration enhancers: Mechanisms of action}, volume={199}, ISSN={0168-3659}, url={http://dx.doi.org/10.1016/J.JCONREL.2014.12.006}, DOI={10.1016/J.JCONREL.2014.12.006}, abstractNote={Skin penetrating peptides (SPPs) have garnered wide attention in recent years and emerged as a simple and effective noninvasive strategy for macromolecule delivery into the skin. Although SPPs have demonstrated their potential in enhancing skin delivery, they are still evolving as a new class of skin penetration enhancers. Detailed studies elucidating their mechanisms of action are still lacking. Using five SPPs (SPACE peptide, TD-1, polyarginine, a dermis-localizing peptide and a skin penetrating linear peptide) and a model hydrophobic macromolecule (Cyclosporine A, CsA), herein we provide a mechanistic understanding of SPPs. To evaluate the mechanism and safety of SPPs, their effects on skin lipids, proteins and keratinocyte cells were evaluated. Three SPPs (SPACE, Polyarginine and TD-1) significantly enhanced CsA penetration into the skin. SPPs did not alter the skin lipid barrier as measured by skin resistance, transepidermal water loss (TEWL) and Fourier transform infrared (FTIR) spectroscopic analysis. In contrast, SPPs interacted with skin proteins and induced changes in skin protein secondary structures (α-helices, β-sheet, random coils and turns), as evaluated by FTIR analysis and confirmed by in-silico docking. SPPs enhanced CsA skin penetration, via a transcellular pathway, enhancing its partitioning into keratin-rich corneocytes through concurrent binding of SPP with keratin and CsA. Interaction between SPP and keratin best correlated with measured CsA skin transport. Many SPPs appeared to be safe as shown by negligible effect on skin integrity, nominal skin irritation potential and cytotoxicity. Among the peptides tested, SPACE peptide was found to be least toxic to keratinocytes, and among the most effective at delivering CsA into the skin.}, journal={Journal of Controlled Release}, publisher={Elsevier BV}, author={Kumar, Sunny and Zakrewsky, Michael and Chen, Ming and Menegatti, Stefano and Muraski, John A. and Mitragotri, Samir}, year={2015}, month={Feb}, pages={168–178} }
 @article{camacho_kumar_menegatti_vogus_anselmo_mitragotri_2015, title={Synergistic antitumor activity of camptothecin–doxorubicin combinations and their conjugates with hyaluronic acid}, volume={210}, ISSN={0168-3659}, url={http://dx.doi.org/10.1016/J.JCONREL.2015.04.031}, DOI={10.1016/J.JCONREL.2015.04.031}, abstractNote={Combinations of topoisomerase inhibitors I and II have been found to synergistically inhibit cancer cell growth in vitro, yet clinical studies of these types of combinations have not progressed beyond phase II trials. The results of clinical combinations of topoisomerase (top) I and II inhibitors typically fall within one of two categories: little to no improvement in therapeutic efficacy, or augmented toxicity compared to the single drug counterparts. Hence, despite the promising activity of top I and II inhibitor combinations in vitro, their clinical applicability has not been realized. Here, we report the use of polymer-drug conjugates as a means to co-deliver synergistic doses of top I and II inhibitors camptothecin (CPT) and doxorubicin (DOX) to tumors in vivo in a 4T1 breast cancer model. At specific molar ratios, DOX and CPT were found to be among the most synergistic combinations reported to date, with combination indices between 0.01 and 0.1. The identified optimal ratios were controllably conjugated to hyaluronic acid, and elicited significant tumor reduction of murine 4T1 breast cancer model when administered intravenously. This study elucidates a method to identify synergistic drug combinations and translate them to in vivo by preserving the synergistic ratio via conjugation to a carrier polymer, thus opening a promising approach to translate drug combinations to clinically viable treatment regimens.}, journal={Journal of Controlled Release}, publisher={Elsevier BV}, author={Camacho, Kathryn M. and Kumar, Sunny and Menegatti, Stefano and Vogus, Douglas R. and Anselmo, Aaron C. and Mitragotri, Samir}, year={2015}, month={Jul}, pages={198–207} }
 @article{menegatti_ruocco_kumar_zakrewsky_sanchez de oliveira_helgeson_leal_mitragotri_2015, title={Synthesis and Characterization of a Self-Fluorescent Hyaluronic Acid-Based Gel for Dermal Applications}, volume={4}, ISSN={2192-2640}, url={http://dx.doi.org/10.1002/ADHM.201500619}, DOI={10.1002/ADHM.201500619}, abstractNote={Combinations of polymer conjugates affording in situ gelation hold promise for treatment of pathological cavities (e.g., arthritis) and sustained drug release. In particular, hyaluronic acid (HA) functionalized with reactive groups is regarded as an excellent biomaterial due to its tunable cross‐linking kinetics and mechanical properties. HA‐based reagents, however, can be irritating to surrounding tissues due to the reactivity of pendant groups, and their fast gelation kinetics can result in poor cavity filling. In this study, a biocompatible “click” reaction between cyanobenzothiazole (CBT) and d‐cysteine (d‐Cys) is employed to produce HA‐based conjugates for in situ gelation. Rheological studies conducted on a gel obtained from the combination of HA‐CBT and HA‐d‐Cys indicate optimal gelation time and mechanical properties. Further, in vitro studies on porcine skin demonstrate the ability of the gel to form in situ upon subcutaneous injection or topical application, and to act as a reservoir for sustained release of protein therapeutics. Finally, the safety of the HA‐based conjugates is demonstrated on human keratinocytes. The presented results demonstrate the applicability of the binary mixture for in situ gelation and the potential of the proposed system for a variety of biomedical applications.}, number={15}, journal={Advanced Healthcare Materials}, publisher={Wiley}, author={Menegatti, Stefano and Ruocco, Nino and Kumar, Sunny and Zakrewsky, Michael and Sanchez De Oliveira, Joshua and Helgeson, Matthew. E. and Leal, Gary L. and Mitragotri, Samir}, year={2015}, month={Sep}, pages={2297–2305} }
 @article{anselmo_modery-pawlowski_menegatti_kumar_vogus_tian_chen_squires_sen gupta_mitragotri_2014, title={Platelet-like Nanoparticles: Mimicking Shape, Flexibility, and Surface Biology of Platelets To Target Vascular Injuries}, volume={8}, ISSN={1936-0851 1936-086X}, url={http://dx.doi.org/10.1021/NN503732M}, DOI={10.1021/NN503732M}, abstractNote={Targeted delivery of therapeutic and imaging agents in the vascular compartment represents a significant hurdle in using nanomedicine for treating hemorrhage, thrombosis, and atherosclerosis. While several types of nanoparticles have been developed to meet this goal, their utility is limited by poor circulation, limited margination, and minimal targeting. Platelets have an innate ability to marginate to the vascular wall and specifically interact with vascular injury sites. These platelet functions are mediated by their shape, flexibility, and complex surface interactions. Inspired by this, we report the design and evaluation of nanoparticles that exhibit platelet-like functions including vascular injury site-directed margination, site-specific adhesion, and amplification of injury site-specific aggregation. Our nanoparticles mimic four key attributes of platelets, (i) discoidal morphology, (ii) mechanical flexibility, (iii) biophysically and biochemically mediated aggregation, and (iv) heteromultivalent presentation of ligands that mediate adhesion to both von Willebrand Factor and collagen, as well as specific clustering to activated platelets. Platelet-like nanoparticles (PLNs) exhibit enhanced surface-binding compared to spherical and rigid discoidal counterparts and site-selective adhesive and platelet-aggregatory properties under physiological flow conditions in vitro. In vivo studies in a mouse model demonstrated that PLNs accumulate at the wound site and induce ∼65% reduction in bleeding time, effectively mimicking and improving the hemostatic functions of natural platelets. We show that both the biochemical and biophysical design parameters of PLNs are essential in mimicking platelets and their hemostatic functions. PLNs offer a nanoscale technology that integrates platelet-mimetic biophysical and biochemical properties for potential applications in injectable synthetic hemostats and vascularly targeted payload delivery.}, number={11}, journal={ACS Nano}, publisher={American Chemical Society (ACS)}, author={Anselmo, Aaron C. and Modery-Pawlowski, Christa Lynn and Menegatti, Stefano and Kumar, Sunny and Vogus, Douglas R. and Tian, Lewis L. and Chen, Ming and Squires, Todd M. and Sen Gupta, Anirban and Mitragotri, Samir}, year={2014}, month={Oct}, pages={11243–11253} }
 @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{menegatti_naik_gurgel_carbonell_2012, title={Purification of polyclonal antibodies from Cohn fraction II + III, skim milk, and whey by affinity chromatography using a hexamer peptide ligand}, volume={35}, ISSN={1615-9306}, url={http://dx.doi.org/10.1002/jssc.201200199}, DOI={10.1002/jssc.201200199}, abstractNote={HWRGWV, a peptide that binds specifically to the Fc fragment of human immunoglobulin G (IgG), was used for the purification of IgG from Cohn fraction II + III of human plasma and from bovine skim milk and whey. The concentration of sodium chloride and sodium caprylate in the binding buffer as well as the pH of the elution buffer were optimized to achieve high IgG yield and purity. Under optimized conditions, IgG was recovered from plasma fractions with yield and purity up to 84% and 95%, respectively. IgG was also purified from skim milk with 74% yield and 92% purity and from whey with 85% yield and 93% purity. Purification experiments were also performed with Protein A resin and the results were found to be similar to those obtained with the peptide adsorbent.}, number={22}, journal={Journal of Separation Science}, publisher={Wiley}, author={Menegatti, Stefano and Naik, Amith D. and Gurgel, Patrick V. and Carbonell, Ruben G.}, year={2012}, month={Jul}, pages={3139–3148} }
 @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} }