Application of cation exchange chromatography in bind and elute and flowthrough mode for the purification of enteroviruses

Konstantinidis, Spyridon; Poplyk, Murphy R.; Swartz, Andrew R.; Rustandi, Richard R.; Thompson, Rachel; Wang, Sheng‐Ching

doi:10.1016/j.chroma.2022.463259

Cited by 2 publications

(13 citation statements)

References 25 publications

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“…At lower NaCl elution concentrations, full capsids may be preferentially displaced by free GSH in the elution buffer prior to empty capsids, but further development would be necessary to increase resolution. As an alternative, polishing chromatography using CEX was established as an effective means of empty capsid separation with highly infectious particle yield (Supplementary Figure S7) (Konstantinidis et al, 2022). Purification of the hightiter 34 °C infection temperature harvests with GSH affinity combined with polishing chromatography was selected as the ideal strategy to increase productivity while maintaining high full capsid purity (Scheme 1).…”

Section: Discussionmentioning

confidence: 99%

“…The AEX column was stripped using AEX Equil buffer with 300 mM and 1 M NaCl. The AEX FT sample was adjusted to pH 4 with a sodium citrate buffer containing a final concentration of 400 mM NaCl and loaded to a cation exchange (CEX) 5-mL POROS 50 HS GoPure column (Cat#: A36637, Thermo Fisher Scientific) to clear residual empty capsids present in the GSH elution from cell culture harvests produced at 34 °C during infection as described previously (Konstantinidis et al, 2022). Full capsids were eluted from the CEX column with an 800 mM NaCl buffer.…”

Section: Ion-exchange Polishing Chromatography Of the Gsh Elutionmentioning

confidence: 99%

“…Despite the aforementioned efforts to reduce empty capsids and serum proteins in the GSH elution, polishing chromatography was investigated to separate the residual impurities. Anion exchange (AEX) and cation exchange (CEX) chromatography methods were established, as described in a previous work (Konstantinidis et al, 2022). AEX of the the GSH elution was evaluated by direct loading to a POROS 50 HQ packed column, where CVA21 virions flowed through at 75 mM NaCl at pH 8 and residual serum proteins bound (Supplementary Figure S7C).…”

Section: Elution Buffer Conditions and Demonstration Of Polishing Chr...mentioning

confidence: 99%

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

Swartz

Shieh

Gulasarian

et al. 2023

Front. Bioeng. Biotechnol.

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Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two–three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250 L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5 L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.

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Section: Discussionmentioning

confidence: 99%

Section: Ion-exchange Polishing Chromatography Of the Gsh Elutionmentioning

confidence: 99%

Section: Elution Buffer Conditions and Demonstration Of Polishing Chr...mentioning

confidence: 99%

See 1 more Smart Citation

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

Swartz

Shieh

Gulasarian

et al. 2023

Front. Bioeng. Biotechnol.

Self Cite

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“…Recently, a chimeric SARS-CoV-2 vaccine candidate, VSVΔG-SARS-CoV-2, was developed by replacing the live vesicular stomatitis virus glycoprotein gene with a coding sequence for the SARS-CoV-2 spike glycoprotein (Espeseth et al, 2022). VSVΔG-SARS-CoV-2, or V590, was produced via adherent Vero cell culture (Konstantinidis, Reinhart, et al, 2022).…”

Section: Introductionmentioning

confidence: 99%

“…For example, its regeneration requires the use of solvents (Konstantinidis, Reinhart, et al, 2022), it represents a unique technology and, given its suitability to LVV purification, procuring it for commercial scale manufacturing may face supply chain limitations. Finally, its adsorption mechanism is characterized by irreversible binding of solutes diffusing into the pores of the resin (Carta et al, 2022;Mi et al, 2021Mi et al, , 2023.…”

Section: Introductionmentioning

confidence: 99%

Purification processes of live virus vaccine candidates expressed in adherent Vero cell lines via multimodal chromatography in flowthrough mode

Konstantinidis

Poplyk

et al. 2023

Biotech & Bioengineering

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Live virus vaccine (LVV) purification, employing chromatography, can be challenged by low binding capacities and elution yields. Alternatively, processes relying solely on enzymatic digestion steps and size‐based membrane separations can be limited by suboptimal reduction of process related impurities and poorly scalable unit operations. Here, we demonstrate that the combination of flowthrough mode chromatography and an ultrafiltration/diafiltration (UF/DF) unit operation delivers a purification process for two different LVV candidates, V590 and Measles, expressed in adherent Vero cells. For V590, chromatography with mixed mode cation exchange resins returned final product yields of ∼50% and logarithmic reduction values (LRVs) of 1.7–>3.4 and 2.5–3.0 for host cell DNA (hcDNA) and host cell proteins (HCPs), respectively. For Measles, chromatography with mixed mode anion exchange resins returned final product yields of ∼50% and LRVs of 1.6 and 2.2 for hcDNA and HCPs, respectively. For both V590 and Measles processing, the employed resins cleared a key HCP, fibronectin, which could foul the UF/DF unit operation, and thusly enabling it to further reduce HCPs and to formulate the final LVV products. This integrated purification process utilizes the complementary action of the two unit operations and its applicability across LVVs supports its consideration for their processing.

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Application of cation exchange chromatography in bind and elute and flowthrough mode for the purification of enteroviruses

Cited by 2 publications

References 25 publications

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

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

Purification processes of live virus vaccine candidates expressed in adherent Vero cell lines via multimodal chromatography in flowthrough mode

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