@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} }
 @misc{overton_boi_shastry_smith-moore_balchunas_sambandan_gilleskie_2023, title={Development and Delivery of a Hands-On Short Course in Adeno-Associated Virus Manufacturing to Support Growing Workforce Needs in Gene Therapy}, volume={34}, ISSN={["1557-7422"]}, url={http://dx.doi.org/10.1089/hum.2022.235}, DOI={10.1089/hum.2022.235}, abstractNote={The manufacturing of gene therapy products is a rapidly growing industry bolstered by the tremendous potential of these therapies to provide lifesaving treatment for rare and complex genetic diseases. The industry's steep rise has resulted in a high demand for skilled staff required to manufacture gene therapy products of the expected high quality. To address this skill shortage, more opportunities for education and training in all aspects of gene therapy manufacturing are needed. The Biomanufacturing Training and Education Center (BTEC) at the North Carolina State University (NC State) has developed and delivered (and continues to deliver) a four-day, hands-on course titled Hands-On cGMP Biomanufacturing of Vectors for Gene Therapy. The course, which consists of 60% hands-on laboratory activities and 40% lectures, aims to provide a comprehensive understanding of the gene therapy production process, from vial thaw through the final formulation step, and analytical testing. This paper discusses the design of the course, the backgrounds of the nearly 80 students who have participated in the seven offerings held since March 2019, and feedback from the course participants.}, number={7-8}, journal={HUMAN GENE THERAPY}, publisher={Mary Ann Liebert Inc}, author={Overton, Laurie and Boi, Cristiana and Shastry, Shriarjun and Smith-Moore, Caroline and Balchunas, John and Sambandan, Deepa and Gilleskie, Gary}, year={2023}, month={Apr}, pages={259–272} }
 @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{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{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{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} }