Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography

Marichal-Gallardo, Pavel; Börner, Kathleen; Pieler, Michael; Sonntag‐Buck, Vera; Obr, Martin; Bejarano, David Alejandro; Wolff, Michael W.; Kräusslich, Hans‐Georg; Reichl, Udo; Grimm, Dirk

doi:10.1089/hum.2019.284

Cited by 32 publications

(30 citation statements)

References 44 publications

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“…Membrane‐based SXC was used successfully as a capture step with virtually full product recovery for both the total IAV content (aHA assay) and the HA antigen content (SRID assay) as previously reported for a similar process (Marichal‐Gallardo et al, 2017). Compared to other chromatography techniques for purification of IAV and other viruses, SXC appears to be comparable or better in terms of product recovery and ease of use, as also discussed previously (Marichal‐Gallardo et al, 2017, 2021). The ability to load and recover the product at physiological salt concentration and pH value minimizes the risk of losing biological activity compared with other techniques.…”

Section: Discussionsupporting

confidence: 59%

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells

Bissinger

Marichal-Gallardo

et al. 2021

Biotech & Bioengineering

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Seasonal influenza epidemics occur both in northern and southern hemispheres every year. Despite the differences in influenza virus surface antigens and virulence of seasonal subtypes, manufacturers are well‐adapted to respond to this periodical vaccine demand. Due to decades of influenza virus research, the development of new influenza vaccines is relatively straight forward. In similarity with the ongoing coronavirus disease 2019 pandemic, vaccine manufacturing is a major bottleneck for a rapid supply of the billions of doses required worldwide. In particular, egg‐based vaccine production would be difficult to schedule and shortages of other egg‐based vaccines with high demands also have to be anticipated. Cell culture‐based production systems enable the manufacturing of large amounts of vaccines within a short time frame and expand significantly our options to respond to pandemics and emerging viral diseases. In this study, we present an integrated process for the production of inactivated influenza A virus vaccines based on a Madin–Darby Canine Kidney (MDCK) suspension cell line cultivated in a chemically defined medium. Very high titers of 3.6 log10(HAU/100 µl) were achieved using fast‐growing MDCK cells at concentrations up to 9.5 × 106 cells/ml infected with influenza A/PR/8/34 H1N1 virus in 1 L stirred tank bioreactors. A combination of membrane‐based steric‐exclusion chromatography followed by pseudo‐affinity chromatography with a sulfated cellulose membrane adsorber enabled full recovery for the virus capture step and up to 80% recovery for the virus polishing step. Purified virus particles showed a homogenous size distribution with a mean diameter of 80 nm. Based on a monovalent dose of 15 µg hemagglutinin (single‐radial immunodiffusion assay), the level of total protein and host cell DNA was 58 µg and 10 ng, respectively. Furthermore, all process steps can be fully scaled up to industrial quantities for commercial manufacturing of either seasonal or pandemic influenza virus vaccines. Fast production of up to 300 vaccine doses per liter within 4–5 days makes this process competitive not only to other cell‐based processes but to egg‐based processes as well.

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Section: Discussionsupporting

confidence: 59%

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells

Bissinger

Marichal-Gallardo

et al. 2021

Biotech & Bioengineering

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“…The cell factor (described in Section 2.2-2) used to convert the permittivity signal to an online viable cell concentration was equal to 0.57, 0.65, and 0.44 for run 1, run 2, and the perfusion control run, respectively adenovirus (Fernandes et al, 2013;Moleirinho et al, 2020), 41% for MVA (Léon et al 2016), 52% for influenza virus , and 20%-60% for AAV production (Moleirinho et al, 2020;Terova et al, 2018) were reported. Successful application of membrane-based SXC for influenza virus, yellow fever virus, AAV, baculovirus, hepatitis C virus, and Orf virus purifications have been reported (Lothert, Offersgaard, et al, 2020;Lothert, Pagallies, et al, 2020;Lothert, Sprick, et al, 2020;Marichal-Gallardo et al, 2021;Marichal-Gallardo, 2019). This suggests that the integrated process established here may also be transferrable to other virus manufacturing processes (Bissinger et al, 2021).…”

Section: Discussionmentioning

confidence: 99%

A high cell density perfusion process for Modified Vaccinia virus Ankara production: Process integration with inline DNA digestion and cost analysis

Gränicher

Babakhani

Göbel

et al. 2021

Biotech & Bioengineering

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By integrating continuous cell cultures with continuous purification methods, process yields and product quality attributes have been improved over the last 10 years for recombinant protein production. However, for the production of viral vectors such as Modified Vaccinia virus Ankara (MVA), no such studies have been reported although there is an increasing need to meet the requirements for a rising number of clinical trials against infectious or neoplastic diseases. Here, we present for the first time a scalable suspension cell (AGE1.CR.pIX cells) culture-based perfusion process in bioreactors integrating continuous virus harvesting through an acoustic settler with semi-continuous chromatographic purification. This allowed obtaining purified MVA particles with a space-time yield more than 600% higher for the integrated perfusion process (1.05 × 10 11 TCID 50 /L bioreactor /day) compared to the integrated batch process. Without further optimization, purification by membrane-based steric exclusion chromatography resulted in an overall product recovery of 50.5%. To decrease the level of host cell DNA before chromatography, a novel inline continuous DNA digestion step was integrated into the process train. A detailed cost analysis comparing integrated production in batch versus production in perfusion mode showed that the cost per dose for MVA was reduced by nearly one-third using this intensified small-scale process.semi-continuous production, steric exclusion chromatography, suspension cell culture in bioreactor, upstream and downstream processing, viral vector | INTRODUCTIONTo date, the implementation of an integrated perfusion process is an option to decrease manufacturing costs and to potentially increase the quality of a product (Bielser et al., 2018;Walther et al., 2015Walther et al., , 2019Xu & Chen, 2016). A considerable amount of research has been conducted on the integrated continuous production of recombinant pro-

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“…The IAV DIPs DI244 and OP7 were produced in a cell culture-based process using a 500 mL laboratory scale stirred tank bioreactor, followed by purification and concentration by membrane-based steric exclusion chromatography [ 44 , 45 ], as described previously [ 29 , 30 ]. Production titers of 3.3 and 3.67 log hemagglutination (HA) units/100µL (quantified by the HA assay [ 46 ]) and 5.6 × 10 8 and 1.12 × 10 11 DI vRNAs/mL (quantified by real-time RT-qPCR [ 29 , 42 , 47 ]) were achieved for DI244 and OP7, respectively.…”

Section: Methodsmentioning

confidence: 99%

Antiviral Activity of Influenza A Virus Defective Interfering Particles against SARS-CoV-2 Replication In Vitro through Stimulation of Innate Immunity

Rand

Kupke

Shkarlet

et al. 2021

Cells

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) emerged in late 2019 and resulted in a devastating pandemic. Although the first approved vaccines were already administered by the end of 2020, worldwide vaccine availability is still limited. Moreover, immune escape variants of the virus are emerging against which the current vaccines may confer only limited protection. Further, existing antivirals and treatment options against COVID-19 show only limited efficacy. Influenza A virus (IAV) defective interfering particles (DIPs) were previously proposed not only for antiviral treatment of the influenza disease but also for pan-specific treatment of interferon (IFN)-sensitive respiratory virus infections. To investigate the applicability of IAV DIPs as an antiviral for the treatment of COVID-19, we conducted in vitro co-infection experiments with cell culture-derived DIPs and the IFN-sensitive SARS-CoV-2 in human lung cells. We show that treatment with IAV DIPs leads to complete abrogation of SARS-CoV-2 replication. Moreover, this inhibitory effect was dependent on janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. Further, our results suggest boosting of IFN-induced antiviral activity by IAV DIPs as a major contributor in suppressing SARS-CoV-2 replication. Thus, we propose IAV DIPs as an effective antiviral agent for treatment of COVID-19, and potentially also for suppressing the replication of new variants of SARS-CoV-2.

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Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography

Cited by 32 publications

References 44 publications

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells

Towards integrated production of an influenza A vaccine candidate with MDCK suspension cells

A high cell density perfusion process for Modified Vaccinia virus Ankara production: Process integration with inline DNA digestion and cost analysis

Antiviral Activity of Influenza A Virus Defective Interfering Particles against SARS-CoV-2 Replication In Vitro through Stimulation of Innate Immunity

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