Selection of chromatographic methods for the purification of cell culture-derived Orf virus for its application as a vaccine or viral vector

Lothert, Keven; Pagallies, Felix; Feger, Thomas; Amann, Ralf; Wolff, Michael W.

doi:10.1016/j.jbiotec.2020.07.023

Cited by 23 publications

(34 citation statements)

References 64 publications

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“…For comparative reasons, the CP were chosen to be spherical and of an average size (190 nm) of viruses, which were successfully purified by the SXC in previous studies [ 15 , 17 , 19 ]. In order to characterize the CP aggregation behavior, the pH-and PEG 8000 -dependent size and charge distributions were assessed.…”

Section: Resultsmentioning

confidence: 99%

“…All SXC experiments were conducted on an Äkta Pure 25 (Cytiva, Marlborough, MA, USA) with online UV (260 nm), pre-column pressure, and dynamic light scattering (DLS) (Nano DLS Particle Size Analyzer, Brookhaven Instruments, Holtsville, NY, USA) monitoring, controlled via the Unicorn 7.1 software (Cytiva, Marlborough, MA, USA). The execution of the SXC runs was adapted from Lothert et al [ 17 ] with minor adjustments. In short, the single-use adsorbent was a stack of ten layers of regenerated cellulose membranes (Whatman, Maidstone, UK), 13 mm in diameter and 1 µm nominal pore size, inserted into a stainless-steel filter holder (Pall Life Sciences, Port Washington, NY, USA).…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, pressure limits are of utmost importance for the stationary phase as well as for the LC system. In the past, convective media, such as monoliths [ 1 , 2 , 13 , 14 ] and membranes [ 15 , 16 , 17 , 18 , 19 ], were applied to counteract diffusion limitations and clogging caused by the virus particles’ large size. As a secondary effect, the high viscosity of the PEG solutions was easier to handle with these stationary phases.…”

Section: Introductionmentioning

confidence: 99%

“…As a secondary effect, the high viscosity of the PEG solutions was easier to handle with these stationary phases. Last, in-line mixing, representing the blending in the mixer and tubes of the LC immediately before loading onto the stationary phase, was proposed to reduce a possible pressure increase [ 1 , 18 ], but several authors worked on a laboratory scale with off-line mixing without describing such limitations [ 10 , 15 , 16 , 17 , 18 ].…”

Section: Introductionmentioning

confidence: 99%

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The Suitability of Latex Particles to Evaluate Critical Process Parameters in Steric Exclusion Chromatography

et al. 2022

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The steric exclusion chromatography (SXC) is a rather new method for the purification of large biomolecules and biological nanoparticles based on the principles of precipitation. The mutual steric exclusion of a nonionic organic polymer, i.e., polyethylene glycol (PEG), induces target precipitation and leads to their retention on the chromatographic stationary phase. In this work, we investigated the application of latex particles in the SXC by altering the particle’s surface charge as well as the PEG concentration and correlated both with their aggregation behavior. The parameters of interest were offline precipitation kinetics, the product recovery and yield, and the chromatographic column blockage. Sulfated and hydroxylated polystyrene particles were first characterized concerning their aggregation behavior and charge in the presence of PEG and different pH conditions. Subsequently, the SXC performance was evaluated based on the preliminary tests. The studies showed (1) that the SXC process with latex particles was limited by aggregation and pore blockage, while (2) not the aggregate size itself, but rather the aggregation kinetics dominated the recoveries, and (3) functionalized polystyrene particles were only suitable to a limited extent to represent biological nanoparticles of comparable size and charge.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The Suitability of Latex Particles to Evaluate Critical Process Parameters in Steric Exclusion Chromatography

et al. 2022

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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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Protein adsorption on core‐shell resins for flow‐through purifications: Effect of protein molecular size, shape, and salt concentration

Wang²,

Winters³

et al. 2022

Biotechnology Progress

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This work addresses the functional properties of the core‐shell resins Capto Core 400 and 700 for a broad range of proteins spanning 66.5 to 660 kDa in molecular mass, including bovine serum albumin (BSA) in monomer and dimer form, fibronectin, thyroglobulin, and BSA conjugates with 10 and 30 kDa poly(ethylene glycol) chains. Negatively charged latex nanoparticles (NPs) with nominal diameters of 20, 40, and 100 nm are also studied as surrogates for bioparticles. Protein binding and its trends with respect to salt concentration depend on the protein size and are different for the two agarose‐based multimodal resins. For the smaller proteins, the amount of protein bound over practical time scales is limited by the resin surface area and is larger for Capto Core 400 compared with Capto Core 700. For the larger proteins, diffusion is severely restricted in Capto Core 400, resulting in lower binding capacities than those observed for Capto Core 700 despite the larger surface area. Adding 500 mM NaCl reduces the local bound protein concentration and diffusional hindrance resulting in higher binding capacities for the large proteins in Capto Core 400 compared with low ionic strength conditions. The NPs are essentially completely excluded from the Capto Core 400 pores. However, 20 and 40 nm NPs bind significantly to Capto Core 700, further hindering protein diffusion. A model is provided to predict the dynamic binding capacities as a function of residence time.

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Selection of chromatographic methods for the purification of cell culture-derived Orf virus for its application as a vaccine or viral vector

Cited by 23 publications

References 64 publications

The Suitability of Latex Particles to Evaluate Critical Process Parameters in Steric Exclusion Chromatography

The Suitability of Latex Particles to Evaluate Critical Process Parameters in Steric Exclusion Chromatography

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

Protein adsorption on core‐shell resins for flow‐through purifications: Effect of protein molecular size, shape, and salt concentration

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