A scalable, integrated downstream process for production of a recombinant measles virus-vectored vaccine

Steppert, Petra; Mosor, Magdalena; Stanek, Larissa; Burgstaller, Daniel; Palmberger, Dieter; Preinsperger, Shirin; Aguilar, Patrícia Pereira; Müllner, Matthias; Csar, Patrick; Jungbauer, Alois

doi:10.1016/j.vaccine.2022.01.004

Cited by 12 publications

(25 citation statements)

References 20 publications

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“…Various applications of Capto Core 700 have been reported to purify viruses [13][14][15][16][17][18][19] and VLPs [20][21][22] by removing host cell proteins and host-cell DNA impurities in a flow-through mode. 8,9,13,14,19,23,24 In addition, these resins may be suitable for purifying extracellular vesicles such as exosomes, 20,25,26 large proteins, 27 human mesenchymal stem cells, 28 as well as novel vaccine molecules, [29][30][31] all in a flow-through mode.…”

mentioning

confidence: 99%

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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mentioning

confidence: 99%

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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“…Despite efforts are done to reduce DNA from enveloped virus preparations using different unit operations such as filtration, ultracentrifugation, or chromatographic purification, 1,3–5 endonuclease treatment is an almost mandatory step in manufacturing of viral vaccines 6 . Nevertheless, DNA in the form of chromatin is often neglected 1 .…”

Section: Introductionmentioning

confidence: 99%

“…A common problem during enveloped virus purification is the removal of host cell DNA 6 . Especially the presence of DNA in form of chromatin is often overlooked in vaccine production and one of the reasons for high host cell DNA in virus preparations even after several purification steps 1 .…”

Section: Introductionmentioning

confidence: 99%

“…A very commonly used endonuclease for DNA digestion in biopharmaceuticals production is the Benzonase® from Merck KGaA (Germany). It is very often used after the clarification step to reduce the double stranded DNA (dsDNA) amount before further processing 1,3,6,11,14 . High salt concentrations commonly found in clarified harvest material are reducing and inhibiting the enzyme activity of commonly used endonucleases like Benzonase®.…”

Section: Introductionmentioning

confidence: 99%

“…This resin material is specifically used for the purification of viruses and bigger particles as they cannot enter the pores 15,16 . At the inside of the pores, positively and hydrophobically active ligands can bind the process related impurities like host cell proteins and DNA 6,17,18 …”

Section: Introductionmentioning

confidence: 99%

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Removal of chromatin by salt‐tolerant endonucleases for production of recombinant measles virus

Mayer

Frank

Preinsperger

et al. 2023

Biotechnology Progress

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Host cell DNA is a critical impurity in downstream processing of enveloped viruses. Especially, DNA in the form of chromatin is often neglected. Endonuclease treatment is an almost mandatory step in manufacturing of viral vaccines. In order to find the optimal performer, four different endonucleases, two of them salt tolerant, were evaluated in downstream processing of recombinant measles virus. Endonuclease treatment was performed under optimal temperature conditions after clarification and before the purification by flow‐through chromatography with a core shell chromatography medium: Capto™ Core 700. Virus infectivity was measured by TCID50. DNA and histone presence in process and purified samples was determined using PicoGreen™ assay and Western blot analysis using an anti‐histone antibody, respectively. All tested endonucleases allowed the reduction of DNA content improving product purity. The salt‐tolerant endonucleases SAN and M‐SAN were more efficient in the removal of chromatin compared with the non‐salt‐tolerant endonucleases Benzonase® and DENARASE®. Removal of chromatin using M‐SAN was also possible without the addition of extra salt to the cell culture supernatant. The combination of the endonuclease treatment, using salt‐tolerant endonucleases with flow‐through chromatography, using core–shell particles, resulted in high purity and purification efficiency. This strategy has all features for a platform downstream process of recombinant measles virus and beyond.

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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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A scalable, integrated downstream process for production of a recombinant measles virus-vectored vaccine

Cited by 12 publications

References 20 publications

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

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

Removal of chromatin by salt‐tolerant endonucleases for production of recombinant measles virus

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

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