TY - JOUR TI - Flexible sensor patch for continuous carbon dioxide monitoring AU - Hetzler, Zach AU - Wang, Yan AU - Krafft, Danny AU - Jamalzadegan, Sina AU - Overton, Laurie AU - Kudenov, Michael W. AU - Ligler, Frances S. AU - Wei, Qingshan T2 - FRONTIERS IN CHEMISTRY AB - Monitoring and measurement of carbon dioxide (CO2) is critical for many fields. The gold standard CO2 sensor, the Severinghaus electrode, has remained unchanged for decades. In recent years, many other CO2 sensor formats, such as detection based upon pH-sensitive dyes, have been demonstrated, opening the door for relatively simple optical detection schemes. However, a majority of these optochemical sensors require complex sensor preparation steps and are difficult to control and repeatably execute. Here, we report a facile CO2 sensor generation method that suffers from none of the typical fabrication issues. The method described here utilizes polydimethylsiloxane (PDMS) as the flexible sensor matrix and 1-hydroxypyrene-3,6,8-trisulfonate (HPTS), a pH-sensitive dye, as the sensing material. HPTS, a base (NaOH), and glycerol are loaded as dense droplets into a thin PDMS layer which is subsequently cured around the droplet. The fabrication process does not require prior knowledge in chemistry or device fabrication and can be completed as quickly as PDMS cures (∼2 h). We demonstrate the application of this thin-patch sensor for in-line CO2 quantification in cell culture media. To this end, we optimized the sensing composition and quantified CO2 in the range of 0-20 kPa. A standard curve was generated with high fidelity (R2 = 0.998) along with an analytical resolution of 0.5 kPa (3.7 mm Hg). Additionally, the sensor is fully autoclavable for applications requiring sterility and has a long working lifetime. This flexible, simple-to-manufacture sensor has a myriad of potential applications and represents a new, straightforward means for optical carbon dioxide measurement. DA - 2022/9/27/ PY - 2022/9/27/ DO - 10.3389/fchem.2022.983523 VL - 10 SP - SN - 2296-2646 KW - CO2 KW - in-line sensor KW - flexible sensor KW - process analytical technology KW - cell culture KW - biomanufacturing ER - TY - JOUR TI - Purification of Adeno-Associated Virus (AAV) Serotype 2 from Spodoptera frugiperda (Sf9) Lysate by Chromatographic Nonwoven Membranes AU - Fan, Jinxin AU - Barbieri, Eduardo AU - Shastry, Shriarjun AU - Menegatti, Stefano AU - Boi, Cristiana AU - Carbonell, Ruben G. T2 - MEMBRANES AB - 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. DA - 2022/10// PY - 2022/10// DO - 10.3390/membranes12100944 VL - 12 IS - 10 SP - SN - 2077-0375 KW - adeno-associated virus (AAV) KW - viral vector purification KW - nonwovens KW - membrane adsorber KW - anion exchange KW - cation exchange KW - membrane chromatography ER - TY - JOUR TI - Development of peptide ligands for the purification of a-1 antitrypsin from cell culture fluids AU - Chu, Wenning AU - Prodromou, Raphael AU - Moore, Brandyn AU - Elhanafi, Driss AU - Kilgore, Ryan AU - Shastry, Shriarjun AU - Menegatti, Stefano T2 - JOURNAL OF CHROMATOGRAPHY A AB - 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. DA - 2022/8/30/ PY - 2022/8/30/ DO - 10.1016/j.chroma.2022.463363 VL - 1679 SP - SN - 1873-3778 KW - a-1 antitrypsin KW - Peptide ligands KW - Affinity chromatography KW - Protein purification KW - CHO cell culture harvest ER - TY - JOUR TI - Mobile On Demand COVID-19 Vaccine Production Units for Developing Countries AU - Fahr, Steffen AU - Pena-Benavides, Samantha Ayde AU - Thiel, Lukas AU - Sengoba, Carl AU - Karacasulu, Kaan AU - Ihling, Nina AU - Sosa-Hernandez, Juan Eduardo AU - Gilleskie, Gary AU - Woodley, John M. AU - Parra-Saldivar, Roberto AU - Mansouri, Seyed Soheil AU - Roh, Kosan T2 - INDUSTRIAL & ENGINEERING CHEMISTRY RESEARCH AB - Two years into the COVID-19 pandemic and more than one year after the approval of the first vaccine, bottlenecks in production and supply chain infrastructure continue to delay vaccination campaigns in the Global South. Mobile on Demand (MOD) vaccine manufacture may help quickly ramp up production capacity while bypassing infrastructure bottlenecks. Such decentralized small-scale factories can help tip the scales in the battle against COVID-19 and future pandemics. In this work, we designed two MOD vaccine manufacturing units based on a protein antigen expressed in yeast and in vitro transcription of mRNA. Each unit consists of three shipping containers and can produce on the order of 10,000 vaccine doses daily for competitive prices and in close proximity of their end users. Abandoning economies of scale may lead to a moderate increase in production costs that may be outweighed by reduced closed-vial dose wastage and an earlier protection of vulnerable populations. DA - 2022/8/19/ PY - 2022/8/19/ DO - 10.1021/acs.iecr.2c01217 VL - 61 IS - 35 SP - SN - 0888-5885 UR - http://dx.doi.org/10.1021/acs.iecr.2c01217 ER - TY - JOUR TI - Towards continuous mAb purification: Clearance of host cell proteins from CHO cell culture harvests via "flow-through affinity chromatography" using peptide-based adsorbents AU - Sripada, Sobhana Alekhya AU - Chu, Wenning AU - Williams, Taufika Islam AU - Teten, Matthew A. AU - Mosley, Brian J. AU - Carbonell, Ruben G. AU - Lenhoff, Abraham M. AU - Cramer, Steven M. AU - Bill, Jerome AU - Yigzaw, Yinges AU - Roush, David J. AU - Menegatti, Stefano T2 - BIOTECHNOLOGY AND BIOENGINEERING AB - The 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. DA - 2022/4/22/ PY - 2022/4/22/ DO - 10.1002/bit.28096 SP - SN - 1097-0290 KW - CHO KW - flow-through chromatography KW - host cell proteins KW - monoclonal antibodies KW - peptide-based adsorbents ER - TY - JOUR TI - Epigenome-wide association study of bronchopulmonary dysplasia in preterm infants: results from the discovery-BPD program AU - Wang, Xuting AU - Cho, Hye-Youn AU - Campbell, Michelle R. AU - Panduri, Vijayalakshmi AU - Coviello, Silvina AU - Caballero, Mauricio T. AU - Sambandan, Deepa AU - Kleeberger, Steven R. AU - Polack, Fernando P. AU - Ofman, Gaston AU - Bell, Douglas A. T2 - CLINICAL EPIGENETICS AB - Bronchopulmonary dysplasia (BPD) is a lung disease in premature infants caused by therapeutic oxygen supplemental and characterized by impaired pulmonary development which persists into later life. While advances in neonatal care have improved survival rates of premature infants, cases of BPD have been increasing with limited therapeutic options for prevention and treatment. This study was designed to explore the relationship between gestational age (GA), birth weight, and estimated blood cell-type composition in premature infants and to elucidate early epigenetic biomarkers associated with BPD.Cord blood DNA from preterm neonates that went on to develop BPD (n = 14) or not (non-BPD, n = 93) was applied to Illumina 450 K methylation arrays. Blood cell-type compositions were estimated using DNA methylation profiles. Multivariable robust regression analysis elucidated CpGs associated with BPD risk. cDNA microarray analysis of cord blood RNA identified differentially expressed genes in neonates who later developed BPD.The development of BPD and the need for oxygen supplementation were strongly associated with GA (BPD, p < 1.0E-04; O2 supplementation, p < 1.0E-09) and birth weight (BPD, p < 1.0E-02; O2 supplementation, p < 1.0E-07). The estimated nucleated red blood cell (NRBC) percent was negatively associated with birth weight and GA, positively associated with hypomethylation of the tobacco smoke exposure biomarker cg05575921, and high-NRBC blood samples displayed a hypomethylation profile. Epigenome-wide association study (EWAS) identified 38 (Bonferroni) and 275 (false discovery rate 1%) differentially methylated CpGs associated with BPD. BPD-associated CpGs in cord blood were enriched for lung maturation and hematopoiesis pathways. Stochastic epigenetic mutation burden at birth was significantly elevated among those who developed BPD (adjusted p = 0.02). Transcriptome changes in cord blood cells reflected cell cycle, development, and pulmonary disorder events in BPD.While results must be interpreted with caution because of the small size of this study, NRBC content strongly impacted DNA methylation profiles in preterm cord blood and EWAS analysis revealed potential insights into biological pathways involved in BPD pathogenesis. DA - 2022/12// PY - 2022/12// DO - 10.1186/s13148-022-01272-0 VL - 14 IS - 1 SP - SN - 1868-7083 KW - Preterm infant KW - Cord blood KW - DNA methylation KW - Epigenome-wide association study KW - Gestational age KW - Stochastic epimutation KW - Nucleated red blood cell KW - cDNA microarray KW - Bronchopulmonary dysplasia KW - Lung ER - TY - JOUR TI - Using Proteomic Approaches to Unravel the Response of Ctenocephalides felis felis to Blood Feeding and Infection With Bartonella henselae AU - Andre, Marcos Rogerio AU - Neupane, Pradeep AU - Lappin, Michael AU - Herrin, Brian AU - Smith, Vicki AU - Williams, Taufika Islam AU - Collins, Leonard AU - Bai, Hongxia AU - Jorge, Gabriel Lemes AU - Balbuena, Tiago Santana AU - Bradley, Julie AU - Maggi, Ricardo G. AU - Breitschwerdt, Edward B. T2 - FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY AB - Among the Ctenocephalides felis felis-borne pathogens, Bartonella henselae, the main aetiological agent of cat scratch disease (CSD), is of increasing comparative biomedical importance. Despite the importance of B. henselae as an emergent pathogen, prevention of the diseases caused by this agent in cats, dogs and humans mostly relies on the use of ectoparasiticides. A vaccine targeting both flea fitness and pathogen competence is an attractive choice requiring the identification of flea proteins/metabolites with a dual effect. Even though recent developments in vector and pathogen -omics have advanced the understanding of the genetic factors and molecular pathways involved at the tick-pathogen interface, leading to discovery of candidate protective antigens, only a few studies have focused on the interaction between fleas and flea-borne pathogens. Taking into account the period of time needed for B. henselae replication in flea digestive tract, the present study investigated flea-differentially abundant proteins (FDAP) in unfed fleas, fleas fed on uninfected cats, and fleas fed on B. henselae-infected cats at 24 hours and 9 days after the beginning of blood feeding. Proteomics approaches were designed and implemented to interrogate differentially expressed proteins, so as to gain a better understanding of proteomic changes associated with the initial B. henselae transmission period (24 hour timepoint) and a subsequent time point 9 days after blood ingestion and flea infection. As a result, serine proteases, ribosomal proteins, proteasome subunit α-type, juvenile hormone epoxide hydrolase 1, vitellogenin C, allantoinase, phosphoenolpyruvate carboxykinase, succinic semialdehyde dehydrogenase, glycinamide ribotide transformylase, secreted salivary acid phosphatase had high abundance in response of C. felis blood feeding and/or infection by B. henselae. In contrast, high abundance of serpin-1, arginine kinase, ribosomal proteins, peritrophin-like protein, and FS-H/FSI antigen family member 3 was strongly associated with unfed cat fleas. Findings from this study provide insights into proteomic response of cat fleas to B. henselae infected and uninfected blood meal, as well as C. felis response to invading B. henselae over an infection time course, thus helping understand the complex interactions between cat fleas and B. henselae at protein levels. DA - 2022/1/28/ PY - 2022/1/28/ DO - 10.3389/fcimb.2022.828082 VL - 12 SP - SN - 2235-2988 KW - bartonellosis KW - cat flea KW - cat scratch disease KW - flea-pathogen interface KW - proteome ER - TY - JOUR TI - A direct comparison between membrane adsorber and packed column chromatography performance (vol 1612, 460629, 2020) AU - Boi, Cristiana AU - Malavasi, Andrea AU - Carbonell, Ruben G. AU - Gilleskie, Gary T2 - JOURNAL OF CHROMATOGRAPHY A DA - 2022/3/15/ PY - 2022/3/15/ DO - 10.1016/j.chroma.2022.462852 VL - 1666 SP - SN - 1873-3778 ER - TY - JOUR TI - A concept for continuous virus manufacture using a moving bed bioreactor: Growth of MDCK cells to confluence on paper as a model support AU - Duffy, Colleen M. AU - Overton, Laurie AU - Flickinger, Michael C. T2 - CHEMICAL ENGINEERING AND PROCESSING-PROCESS INTENSIFICATION AB - A moving-bed bioreactor (MBB) could intensify growth of adherent mammalian cells for viral vaccines. A continuously fed sterile paper passing through a thin liquid layer as a flexible cell substrate is a new concept for bioprocess intensification (BPI). Paper could enable cell expansion with reduced footprint and reduced media consumption as the first stage of a 2-stage growth + infection continuous process. This study focused only on cell growth (stage 1). We report a simple 32 mm paper disc method to evaluate growth of adherent Madin Darby canine kidney cells (MDCK CCL-34) adapted to 5% FBS to confluence on unmodified papers to screen substrates. Bibulous paper was found to be the best substrate for proliferation of MDCK. An MBB process was simulated using paper discs to test growth to confluence (stage 1). Growth was characterized using staining, image analysis; confocal microscopy. Cells grew on the surface of bibulous paper to a confluence of >90% in 192 h. Extending this concept by stabilizing MDCK on paper (end of stage 1) by engineering cells to survive freezing or lyoprotection would enable live cells to be shipped as modules to multiple manufacturing sites for infection resulting in rapid, reproducible viral vaccine manufacture. DA - 2022/1// PY - 2022/1// DO - 10.1016/j.cep.2021.108667 VL - 170 SP - SN - 1873-3204 KW - Cell culture on paper KW - Moving bed bioreactor KW - Continuous virus manufacture KW - Intensification of vaccine manufacture KW - MDCK cell growth on paper ER -