Upstream and Downstream Processes for Viral Nanoplexes as Vaccines

Lothert, Keven; Dekevic, Gregor; Loewe, Daniel; Salzig, Denise; Czermak, Peter; Wolff, Michael W.

doi:10.1007/978-1-0716-0795-4_12

Cited by 4 publications

(2 citation statements)

References 84 publications

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“…Prior to all chromatographic procedures, the cell culture-derived KHV was clarified by consecutive centrifugation, as described above. The SXC runs were performed either on an Äkta Prime Plus or an Äkta Pure 25 (Cytiva) according to a standard procedure (Lothert et al, 2021), described here in short. Each single-use column consisted of 10 membrane discs of regenerated cellulose with a nominal pore size of either 1 μm (GE Healthcare Life Sciences) or 3-5 μm (kindly provided by Sartorius Stedim).…”

Section: Sxc Purification Proceduresmentioning

confidence: 99%

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography

Eilts,

Jordan,

Harsy

et al. 2023

Journal of Fish Diseases

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Koi herpesvirus (KHV) is the causative agent of a koi herpesvirus disease (KHVD) inducing high mortality rates in common carp and koi (Cyprinus carpio). No widespread effective vaccination strategy has been implemented yet, which is partly due to side effects of the immunized fish. In this study, we present an evaluation of the purification of infectious KHV from host cell protein and DNA, using the steric exclusion chromatography. The method is related to conventional polyethylene glycol (PEG) precipitation implemented in a chromatographic set‐up and has been applied for infectious virus particle purification with high recoveries and impurity removal. Here, we achieved a yield of up to 55% of infectious KHV by using 12% PEG (molecular weight of 6 kDa) at pH 7.0. The recoveries were higher when using chromatographic cellulose membranes with 3–5 μm pores in diameter instead of 1 μm. The losses were assumed to originate from dense KHV precipitates retained on the membranes. Additionally, the use of >0.6 M NaCl was shown to inactivate infectious KHV. In summary, we propose a first step towards a purification procedure for infectious KHV with a possible implementation in fish vaccine manufacturing.

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Section: Sxc Purification Proceduresmentioning

confidence: 99%

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography

Eilts,

Jordan,

Harsy

et al. 2023

Journal of Fish Diseases

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“…To use YF-VLP as a passive immunization agent, a fast and efficient production must be established. There are many ways to produce VLPs using different expression systems, e.g., mammalian cells [ 11 ], insect cells [ 12 ], microbial cells [ 13 ], or plants [ 14 ]. Mammalian cells offer the decisive advantage of being able to produce important correct post-translational modification patterns, such as glycosylation.…”

Section: Introductionmentioning

confidence: 99%

A Bioreactor-Based Yellow Fever Virus-like Particle Production Process with Integrated Process Analytical Technology Based on Transient Transfection

Dekevic,

Tertel,

Tasto

et al. 2023

Viruses

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Yellow Fever (YF) is a severe disease that, while preventable through vaccination, lacks rapid intervention options for those already infected. There is an urgent need for passive immunization techniques using YF-virus-like particles (YF-VLPs). To address this, we successfully established a bioreactor-based production process for YF-VLPs, leveraging transient transfection and integrating Process Analytical Technology. A cornerstone of this approach was the optimization of plasmid DNA (pDNA) production to a yield of 11 mg/L using design of experiments. Glucose, NaCl, yeast extract, and a phosphate buffer showed significant influence on specific pDNA yield. The preliminary work for VLP-production in bioreactor showed adjustments to the HEK cell density, the polyplex formation duration, and medium exchanges effectively elevated transfection efficiencies. The additive Pluronic F-68 was neutral in its effects, and anti-clumping agents (ACA) adversely affected the transfection process. Finally, we established the stirred-tank bioreactor process with integrated dielectric spectroscopy, which gave real-time insight in relevant process steps, e.g., cell growth, polyplex uptake, and harvest time. We confirmed the presence and integrity of YF-VLP via Western blot, imaging flow cytometry measurement, and transmission electron microscopy. The YF-VLP production process can serve as a platform to produce VLPs as passive immunizing agents against other neglected tropical diseases.

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Affinity chromatography for virus-like particle manufacturing: Challenges, solutions, and perspectives

Ma,

Tian,

Shi

et al. 2024

Journal of Chromatography A

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Upstream and Downstream Processes for Viral Nanoplexes as Vaccines

Cited by 4 publications

References 84 publications

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography

Purification and concentration of infectious koi herpesvirus using steric exclusion chromatography

A Bioreactor-Based Yellow Fever Virus-like Particle Production Process with Integrated Process Analytical Technology Based on Transient Transfection

Affinity chromatography for virus-like particle manufacturing: Challenges, solutions, and perspectives

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