Quantitation of Coxsackievirus A21 Viral Proteins in Mixtures of Empty and Full Capsids Using Capillary Western

Gillespie, Paul F.; Rustandi, Richard R.; Swartz, Andrew R.; Liu, Shang; Raffaele, Jessica; Prout, Ashley; Cunningham, Nicholas; Dawod, Mohamed; Deng, James Z.; Wang, Shiyi; Olson, Jessica; Shieh, Yvonne; Loughney, John W.

doi:10.1089/hum.2022.147

Cited by 3 publications

(1 citation statement)

References 23 publications

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“…Capillary electrophoresis (CE) Western blot was performed using a Protein Simple Jess system (Protein Simple, Santa Clara, CA) and a 12-230 kDa 25-capillary Jess separation kit (SM-W004) based on a published procedure (Gillespie et al, 2022). Samples were prepared following the manufacturer's instructions using the EZ Standard Pack (PS-ST01EZ) and anti-rabbit detection module (DM-001).…”

Section: Quantitative Capillary Electrophoresis Western Blotmentioning

confidence: 99%

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: Quantitative Capillary Electrophoresis Western Blotmentioning

confidence: 99%

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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Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western

Shieh,

Swartz,

Rustandi

2024

Electrophoresis

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Therapeutic messenger RNA (mRNA) has been demonstrated as a scalable and versatile vaccine platform for the rapid development and manufacture of new vaccine candidates. mRNA is synthesized enzymatically through in vitro transcription (IVT) using bacteriophage T7 RNA polymerase (T7 RNAP), a 99 kDa protein with high binding affinity for the promoter sequence and a low error rate. Post‐IVT, mRNA is purified to remove impurities, but if T7 RNAP is insufficiently cleared, undesirable clinical side effects may result. Therefore, it is important to quantitate T7 RNAP concentrations in IVT and process intermediates to understand clearance during downstream purification. A high‐throughput T7 RNAP assay was developed using Simple Western (SW), a capillary immunoassay technology, to quantitate concentrations as low as 5.3 ng/mL with good precision and accuracy. Compared to existing T7 RNAP immunoassays or total protein assays such as bicinchoninic acid assays or Bradford, the SW T7 RNAP assay is specific to T7 RNAP, requires <10 µL of sample volume, and consists of minimal sample handling and hands‐on time. This work highlights the development and optimization of a highly sensitive and robust T7 RNAP quantitation assay using the SW platform.

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Recent applications of quantitative mass spectrometry in biopharmaceutical process development and manufacturing

2023

Journal of Pharmaceutical and Biomedical Analysis

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Quantitation of Coxsackievirus A21 Viral Proteins in Mixtures of Empty and Full Capsids Using Capillary Western

Cited by 3 publications

References 23 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

Detection of residual T7 RNA polymerase used in mRNA in vitro transcription by Simple Western

Recent applications of quantitative mass spectrometry in biopharmaceutical process development and manufacturing

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