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: Unless otherwise indicated, laboratory-scale GSH affinity chromatography was performed with GSTPrep FF 16/10 columns packed with GSH Sepharose 4 FF resin (Cat#: 28936550, Cytiva) run at a 6 min residence time using an AKTA Pure 150 M FPLC system (Cytiva) with UNICORN system control software (Cytiva).

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: Investigation of the GSH affinity resin supplier, residence time, and binding capacity. (A) Evaluation of GSH elution infectivity yield at different residence times using 1 mL prepacked columns packed with Glutathione Sepharose 4 FF (blue square), Glutathione Sepharose 4B (green circle), and Pierce Glutathione (yellow diamond) with 37°C infection temperature CB. (B) Impact of residence time on infectivity yield (circle, solid line) and relative empty capsid content (diamond, dashed line) using 20 mL Glutathione Sepharose 4 FF columns with 37°C infection temperature CB (orange) and 34°C infection temperature CB (purple). Empty capsid content relative to the 6 min residence time 37°C infection temperature CB condition is shown. (C) GSH affinity chromatography breakthrough experiments using 20 mL Glutathione Sepharose 4 FF columns loaded at a 6 min residence time with 34°C infection temperature CB (red square) and concentrated and purified full capsid material (blue diamond). Purified material was diluted to the same particle concentration in CB prior to loading.

Article Snippet: Unless otherwise indicated, laboratory-scale GSH affinity chromatography was performed with GSTPrep FF 16/10 columns packed with GSH Sepharose 4 FF resin (Cat#: 28936550, Cytiva) run at a 6 min residence time using an AKTA Pure 150 M FPLC system (Cytiva) with UNICORN system control software (Cytiva).

Techniques: Binding Assay, Infection, Affinity Chromatography, Purification, Concentration 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 GSH chromatography wash buffer NaCl concentration and pH conditions with 34°C infection temperature CB. (A) Total viral particle distribution by anti-VP1 CE Western blot in a NaCl wash gradient from 150–1,000 mM NaCl (dotted black line) at pH 7 (green square), pH 8 (yellow circle), and pH 9 (red diamond). (B) BSA impurity clearance by BSA ELISA across GSH chromatography steps operated at pH 7 (green square), pH 8 (yellow circle), and pH 9 (red diamond).

Article Snippet: Unless otherwise indicated, laboratory-scale GSH affinity chromatography was performed with GSTPrep FF 16/10 columns packed with GSH Sepharose 4 FF resin (Cat#: 28936550, Cytiva) run at a 6 min residence time using an AKTA Pure 150 M FPLC system (Cytiva) with UNICORN system control software (Cytiva).

Techniques: Chromatography, Concentration Assay, Infection, Western Blot, Enzyme-linked Immunosorbent 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 GSH chromatography elution buffer GSH and NaCl concentration at pH 8. (A) Analysis of GSH elution gradient fractions from 100–600 µM GSH for 37°C infection temperature harvests: microcarrier CB produced with serum at infection, total viral particle distribution by anti-VP1 CE Western blot (green circle), and empty:full capsid ratio by anti-VP4 CE Western blot (purple circle); iCELLis CB produced without serum at infection, total viral particle distribution (blue diamond), and empty:full capsid ratio (yellow diamond). (B) Analysis of GSH elution gradient fractions from 0–125 mM NaCl: 37°C infection temperature material total viral particle distribution (green circle) and % empty capsid (purple circle); 34°C infection temperature material total viral particle distribution by anti-VP1 CE Western blot (blue diamond) and % empty capsid by RP-UPLC (yellow diamond).

Article Snippet: Unless otherwise indicated, laboratory-scale GSH affinity chromatography was performed with GSTPrep FF 16/10 columns packed with GSH Sepharose 4 FF resin (Cat#: 28936550, Cytiva) run at a 6 min residence time using an AKTA Pure 150 M FPLC system (Cytiva) with UNICORN system control software (Cytiva).

Techniques: Chromatography, Concentration Assay, Infection, Produced, Western Blot

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: Unless otherwise indicated, laboratory-scale GSH affinity chromatography was performed with GSTPrep FF 16/10 columns packed with GSH Sepharose 4 FF resin (Cat#: 28936550, Cytiva) run at a 6 min residence time using an AKTA Pure 150 M FPLC system (Cytiva) with UNICORN system control software (Cytiva).

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

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: Microcarrier cell culture infected with CVA21 at 37°C produced empty capsids (empty capsid A) that were separated from full, infectious virions across GSH affinity chromatography. However, cell culture infected at 34°C generated empty capsids (empty capsid B) that bound and co-purified with infectious virions such that a polishing chromatography step using CEX was implemented to clear the residual empty capsids from the GSH elution. CVA21 virus particles were characterized by several assays including high-throughput viral infectivity, SDS-PAGE, CE Western, RP-UPLC, and qPCR.

Article Snippet: Unless otherwise indicated, laboratory-scale GSH affinity chromatography was performed with GSTPrep FF 16/10 columns packed with GSH Sepharose 4 FF resin (Cat#: 28936550, Cytiva) run at a 6 min residence time using an AKTA Pure 150 M FPLC system (Cytiva) with UNICORN system control software (Cytiva).

Techniques: Cell Culture, Infection, Produced, Affinity Chromatography, Generated, Purification, Chromatography, Virus, High Throughput Screening Assay, SDS Page, Western Blot