@misc{sripada_hosseini_ramesh_wang_ritola_menegatti_daniele_2024, title={Advances and opportunities in process analytical technologies for viral vector manufacturing}, volume={74}, ISSN={["1873-1899"]}, DOI={10.1016/j.biotechadv.2024.108391}, abstractNote={Viral vectors are an emerging, exciting class of biologics whose application in vaccines, oncology, and gene therapy has grown exponentially in recent years. Following first regulatory approval, this class of therapeutics has been vigorously pursued to treat monogenic disorders including orphan diseases, entering hundreds of new products into pipelines. Viral vector manufacturing supporting clinical efforts has spurred the introduction of a broad swath of analytical techniques dedicated to assessing the diverse and evolving panel of Critical Quality Attributes (CQAs) of these products. Herein, we provide an overview of the current state of analytics enabling measurement of CQAs such as capsid and vector identities, product titer, transduction efficiency, impurity clearance etc. We highlight orthogonal methods and discuss the advantages and limitations of these techniques while evaluating their adaptation as process analytical technologies. Finally, we identify gaps and propose opportunities in enabling existing technologies for real-time monitoring from hardware, software, and data analysis viewpoints for technology development within viral vector biomanufacturing.}, journal={BIOTECHNOLOGY ADVANCES}, author={Sripada, Sobhana A. and Hosseini, Mahshid and Ramesh, Srivatsan and Wang, Junhyeong and Ritola, Kimberly and Menegatti, Stefano and Daniele, Michael A.}, year={2024}, month={Sep} }
 @article{sripada_barbieri_shastry_wuestenhagen_aldinger_rammo_schulte_daniele_menegatti_2024, title={Multiangle Light Scattering as a Lentivirus Purification Process Analytical Technology}, volume={5}, ISSN={["1520-6882"]}, DOI={10.1021/acs.analchem.4c01209}, abstractNote={The limited biomolecular and functional stability of lentiviral vectors (LVVs) for cell therapy poses the need for analytical tools that can monitor their titers and activity throughout the various steps of expression and purification. In this study, we describe a rapid (25 min) and reproducible (coefficient of variance ∼0.5–2%) method that leverages size exclusion chromatography coupled with multiangle light scattering detection (SEC-MALS) to determine size, purity, and particle count of LVVs purified from bioreactor harvests. The SEC-MALS data were corroborated by orthogonal methods, namely, dynamic light scattering (DLS) and transmission electron microscopy. The method was also evaluated for robustness in the range of 2.78 × 105–2.67 × 107 particles per sample. Notably, MALS-based particle counts correlated with the titer of infectious LVVs measured via transduction assays (R2 = 0.77). Using a combination of SEC-MALS and DLS, we discerned the effects of purification parameters on LVV quality, such as the separation between heterogeneous LV, which can facilitate critical decision-making in the biomanufacturing of gene and cell therapies.}, journal={ANALYTICAL CHEMISTRY}, author={Sripada, Sobhana A. and Barbieri, Eduardo and Shastry, Shriarjun and Wuestenhagen, Elena and Aldinger, Annika and Rammo, Oliver and Schulte, Michael M. and Daniele, Michael and Menegatti, Stefano}, year={2024}, month={May} }
 @article{kilgore_moore_sripada_chu_shastry_barbieri_hu_tian_petersen_mohammadifar_et al._2024, title={Peptide ligands for the universal purification of exosomes by affinity chromatography}, volume={8}, ISSN={["1097-0290"]}, DOI={10.1002/bit.28821}, abstractNote={Abstract Exosomes are gaining prominence as vectors for drug delivery, vaccination, and regenerative medicine. Owing to their surface biochemistry, which reflects the parent cell membrane, these nanoscale biologics feature low immunogenicity, tunable tissue tropism, and the ability to carry a variety of payloads across biological barriers. The heterogeneity of exosomes' size and composition, however, makes their purification challenging. Traditional techniques, like ultracentrifugation and filtration, afford low product yield and purity, and jeopardizes particle integrity. Affinity chromatography represents an excellent avenue for exosome purification. Yet, current affinity media rely on antibody ligands whose selectivity grants high product purity, but mandates the customization of adsorbents for exosomes with different surface biochemistry while their binding strength imposes elution conditions that may harm product's activity. Addressing these issues, this study introduces the first peptide affinity ligands for the universal purification of exosomes from recombinant feedstocks. The peptides were designed to (1) possess promiscuous biorecognition of exosome markers, without binding process‐related contaminants and (2) elute the product under conditions that safeguard product stability. Selected ligands SNGFKKHI and TAHFKKKH demonstrated the ability to capture of exosomes secreted by 14 cell sources and purified exosomes derived from HEK293, PC3, MM1, U87, and COLO1 cells with yields of up to 80% and up‐to 50‐fold reduction of host cell proteins (HCPs) upon eluting with pH gradient from 7.4 to 10.5, recommended for exosome stability. SNGFKKHI‐Toyopearl resin was finally employed in a two‐step purification process to isolate exosomes from HEK293 cell fluids, affording a yield of 68% and reducing the titer of HCPs to 68 ng/mL. The biomolecular and morphological features of the isolated exosomes were confirmed by analytical chromatography, Western blot analysis, transmission electron microscopy, nanoparticle tracking analysis.}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Kilgore, Ryan E. and Moore, Brandyn D. and Sripada, Sobhana A. and Chu, Wenning and Shastry, Shriarjun and Barbieri, Eduardo and Hu, Shiqi and Tian, Weihua and Petersen, Heidi and Mohammadifar, Mohammad and et al.}, year={2024}, month={Aug} }
 @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={AbstractHost‐cell proteins (HCPs) are the foremost class of process‐related impurities to be controlled and removed in downstream processing steps in monoclonal antibody (mAb) manufacturing. However, some HCPs may evade clearance in multiple purification steps and reach the final drug product, potentially threatening drug stability and patient safety. This study extends prior work on HCP characterization and persistence in mAb process streams by using mass spectrometry (MS)‐based methods to track HCPs through downstream processing steps for seven mAbs that were generated by five different cell lines. The results show considerable variability in HCP identities in the processing steps but extensive commonality in the identities and quantities of the most abundant HCPs in the harvests for different processes. Analysis of HCP abundance in the harvests shows a likely relationship between abundance and the reproducibility of quantification measurements and suggests that some groups of HCPs may hinder the characterization. Quantitative monitoring of HCPs persisting through purification steps coupled with the findings from the harvest analysis suggest that multiple factors, including HCP abundance and mAb‐HCP interactions, can contribute to the persistence of individual HCPs and the identification of groups of common, persistent HCPs in mAb manufacturing.}, 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{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={AbstractThe recent uptick in the approval of ex vivo cell therapies highlights the relevance of lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification—currently reliant on filtration and anion‐exchange or size‐exclusion chromatography—suffers from long process times and low yield of transducing particles, which translate into high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV‐G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual‐fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding‐and‐release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50%–60% of viral genomes; 40%–50% of HT1080 cell‐transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide‐based adsorbents also presented remarkable values of binding capacity (up to 3·109 TU per mL of resin, or 5·1011 vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 220‐fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning‐in‐place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality.}, 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{sripada_elhanafi_collins_williams_linova_woodley_boi_menegatti_2023, title={Pseudo-affinity capture of K. phaffii host cell proteins in flow-through mode: Purification of protein therapeutics and proteomic study}, volume={326}, ISSN={1383-5866}, url={http://dx.doi.org/10.1016/j.seppur.2023.124777}, 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}, publisher={Elsevier BV}, 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}, pages={124777} }
 @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={1383-5866}, url={http://dx.doi.org/10.1016/j.seppur.2023.123920}, 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}, publisher={Elsevier BV}, 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}, pages={123920} }
 @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={119}, ISSN={["1097-0290"]}, url={https://doi.org/10.1002/bit.28096}, DOI={10.1002/bit.28096}, abstractNote={AbstractThe growth of advanced analytics in manufacturing monoclonal antibodies (mAbs) has highlighted the challenges associated with the clearance of host cell proteins (HCPs). Of special concern is the removal of “persistent” HCPs, including immunogenic and mAb‐degrading proteins, that co‐elute from the Protein A resin and can escape the polishing steps. Responding to this challenge, we introduced an ensemble of peptide ligands that target the HCPs in Chinese hamster ovary (CHO) cell culture fluids and enable mAb purification via flow‐through affinity chromatography. This study describes their integration into LigaGuard™, an affinity adsorbent featuring an equilibrium binding capacity of ~30 mg of HCPs per mL of resin as well as dynamic capacities up to 16 and 22 mg/ml at 1‐ and 2‐min residence times, respectively. When evaluated against cell culture harvests with different mAb and HCP titers and properties, LigaGuard™ afforded high HCP clearance, with logarithmic removal values (LRVs) up to 1.5, and mAb yield above 90%. Proteomic analysis of the effluents confirmed the removal of high‐risk HCPs, including cathepsins, histones, glutathione‐S transferase, and lipoprotein lipases. Finally, combining LigaGuard™ for HCP removal with affinity adsorbents for product capture afforded a global mAb yield of 85%, and HCP and DNA LRVs > 4.}, 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{sternisha_mukherjee_alex_chaney_barkalifa_wan_lee_rico-jimenez_zurauskas_spillman_et al._2021, title={Longitudinal monitoring of cell metabolism in biopharmaceutical production using label-free fluorescence lifetime imaging microscopy}, ISSN={["1860-7314"]}, DOI={10.1002/biot.202000629}, abstractNote={AbstractChinese hamster ovary (CHO) cells are routinely used in the biopharmaceutical industry for production of therapeutic monoclonal antibodies (mAbs). Although multiple offline and time‐consuming measurements of spent media composition and cell viability assays are used to monitor the status of culture in biopharmaceutical manufacturing, the day‐to‐day changes in the cellular microenvironment need further in‐depth characterization. In this study, two‐photon fluorescence lifetime imaging microscopy (2P‐FLIM) was used as a tool to directly probe into the health of CHO cells from a bioreactor, exploiting the autofluorescence of intracellular nicotinamide adenine dinucleotide phosphate (NAD(P)H), an enzymatic cofactor that determines the redox state of the cells. A custom‐built multimodal microscope with two‐photon FLIM capability was utilized to monitor changes in NAD(P)H fluorescence for longitudinal characterization of a changing environment during cell culture processes. Three different cell lines were cultured in 0.5 L shake flasks and 3 L bioreactors. The resulting FLIM data revealed differences in the fluorescence lifetime parameters, which were an indicator of alterations in metabolic activity. In addition, a simple principal component analysis (PCA) of these optical parameters was able to identify differences in metabolic progression of two cell lines cultured in bioreactors. Improved understanding of cell health during antibody production processes can result in better streamlining of process development, thereby improving product titer and verification of scale‐up. To our knowledge, this is the first study to use FLIM as a label‐free measure of cellular metabolism in a biopharmaceutically relevant and clinically important CHO cell line.}, journal={BIOTECHNOLOGY JOURNAL}, author={Sternisha, Shawn M. and Mukherjee, Prabuddha and Alex, Aneesh and Chaney, Eric J. and Barkalifa, Ronit and Wan, Boyong and Lee, Jang Hyuk and Rico-Jimenez, Jose and Zurauskas, Mantas and Spillman, Darold R., Jr. and et al.}, year={2021}, month={Jun} }
 @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={AbstractThis study presents the chromatographic purification of immunoglobulin G (IgG) from human plasma using a two‐column process integrating the peptide‐based adsorbents LigaGuard™, which captures non‐Ig plasma proteins in flow‐through mode, and LigaTrap™, which isolates IgG in bind‐and‐elute. Buffer composition and column loading were optimized for both adsorbents. Two process configurations were evaluated. In the first design, plasma was fed to a LigaGuard™ column to capture plasma proteins, the effluent was loaded on the LigaTrap™ column, and the bound IgG was eluted with 63.8% global recovery and 99.7% purity; in comparison, Protein G agarose afforded approximately 67% recovery and 97.2% purity. In the alternative design, the LigaGuard™ column was utilized to polish the LigaTrap™ elution stream, affording 82.3% global recovery and 98.8% purity. Collectively, these results demonstrate the potential of a fully chromatographic process for purifying polyclonal IgG from plasma feedstocks.}, 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} }