Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture

doi: 10.3389/fbioe.2023.1193454

Figure Lengend Snippet: Description of large-scale CVA21 cell culture in microcarrier single-use bioreactors and purification by glutathione affinity chromatography. Chromatography elution infectivity yield from clarified bulk and residual host cell DNA and bovine serum albumin concentrations shown. *Extrapolated HC-DNA concentration indicated if below the lower limit of quantitation, except when indistinguishable from no template controls.

Article Snippet: At the laboratory scale, MRC-5 cells were cultured in 3 L glass bioreactors with a BioFlo 320 controller (Cat#: 1379962911, Eppendorf, Hamburg, Germany) on 1 g/L Cytodex 1 microcarriers (Cat#: 17548702, Cytiva, Marlborough, MA) or in iCELLis Nano fixed-bed bioreactors (Pall Corporation, Port Washington, NY) with a 10 cm bed height with WMEM and 10% BCS controlled at a target temperature of 37°C and pH of 7.2 as described previously ( ).

Techniques: Cell Culture, Purification, Affinity Chromatography, Chromatography, Infection, Concentration Assay, Quantitation Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture

doi: 10.3389/fbioe.2023.1193454

Figure Lengend Snippet: Optimization of microcarrier bioreactor temperature during CVA21 infection. (A) Relative infectivity sampled across 4–5 days post-infection controlled at 34°C (blue diamond), 35.5°C (green square), 37°C (yellow triangle), and 38.5°C (red circle). Error bars represent one standard deviation of an average of two replicate conditions. (B) Average peak relative infectivity, defined as the highest sample infectivity across the 4–5-day bioreactor infection phase, as a function of infection temperature across three experiments. Error bars represent one standard deviation of an average at each temperature condition. See for a description of experimental replicates.

Article Snippet: At the laboratory scale, MRC-5 cells were cultured in 3 L glass bioreactors with a BioFlo 320 controller (Cat#: 1379962911, Eppendorf, Hamburg, Germany) on 1 g/L Cytodex 1 microcarriers (Cat#: 17548702, Cytiva, Marlborough, MA) or in iCELLis Nano fixed-bed bioreactors (Pall Corporation, Port Washington, NY) with a 10 cm bed height with WMEM and 10% BCS controlled at a target temperature of 37°C and pH of 7.2 as described previously ( ).

Techniques: Infection, Standard Deviation

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture

doi: 10.3389/fbioe.2023.1193454

Figure Lengend Snippet: Impact of cell culture controlled at 37°C and 34°C during infection on downstream GSH affinity chromatography performance. (A) SDS-PAGE of CB (100x pre-dilution) and GSH elution (no pre-dilution) harvested from bioreactors controlled at 37°C (lane 1–2) and 34°C (lane 3–4) from experiment 1. (B) GSH elution sample relative ratio of the 34°C–37°C material from experiment 1 (green, horizontal bar) and experiment 2 (blue, dotted) for infectious particles by relative infectivity assay and total genomes by RT-qPCR. (C) GSH elution sample % empty capsid content by RP-UPLC for the 34°C and 37°C materials from experiment 1 (green, horizontal bar) and experiment 2 (blue, dotted).

Article Snippet: At the laboratory scale, MRC-5 cells were cultured in 3 L glass bioreactors with a BioFlo 320 controller (Cat#: 1379962911, Eppendorf, Hamburg, Germany) on 1 g/L Cytodex 1 microcarriers (Cat#: 17548702, Cytiva, Marlborough, MA) or in iCELLis Nano fixed-bed bioreactors (Pall Corporation, Port Washington, NY) with a 10 cm bed height with WMEM and 10% BCS controlled at a target temperature of 37°C and pH of 7.2 as described previously ( ).

Techniques: Cell Culture, Infection, Affinity Chromatography, SDS Page, Quantitative RT-PCR

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture

doi: 10.3389/fbioe.2023.1193454

Figure Lengend Snippet: Characterization of GSH chromatography elution purified from large-scale microcarrier bioreactors controlled at 37°C (yellow, horizontal bar) and 34°C (blue, dotted) during infection. Data represent average values from batches shown in , and error bars represent one standard deviation. (A) Relative comparison of batch productivity at GSH elution. (B) Comparison of viral genomes by RT-qPCR and viral particles by VP1 CE Western assays at GSH elution. (C) Comparison of % empty capsids by RP-UPLC in the GSH elution.

Article Snippet: At the laboratory scale, MRC-5 cells were cultured in 3 L glass bioreactors with a BioFlo 320 controller (Cat#: 1379962911, Eppendorf, Hamburg, Germany) on 1 g/L Cytodex 1 microcarriers (Cat#: 17548702, Cytiva, Marlborough, MA) or in iCELLis Nano fixed-bed bioreactors (Pall Corporation, Port Washington, NY) with a 10 cm bed height with WMEM and 10% BCS controlled at a target temperature of 37°C and pH of 7.2 as described previously ( ).

Techniques: Chromatography, Purification, Infection, Standard Deviation, Comparison, Quantitative RT-PCR, Western Blot