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dc.contributor.advisorWolff, Michael W.
dc.contributor.advisorSpengler, Bernhard
dc.contributor.advisorCzermak, Peter
dc.contributor.authorEilts, Friederike
dc.date.accessioned2023-12-21T11:12:24Z
dc.date.available2023-12-21T11:12:24Z
dc.date.issued2023-12-04
dc.identifier.urihttps://jlupub.ub.uni-giessen.de//handle/jlupub/18820
dc.identifier.urihttp://dx.doi.org/10.22029/jlupub-18185
dc.description.abstractThe COVID-19 (coronavirus disease 2019) pandemic has shown the importance of ready-at-hand- tools to bring pharmaceutical products to market. This includes the availability of plat-forms to target new diseases as well as their production process. Such a platform could be available with the Orf virus (ORFV), a promising viral vector with applications as gene thera-peutic, antiviral, oncolytic, immunomodulatory, and vaccination agent. However, production processes for the ORFV are not established yet. A recent publication on a lab-scale purifica-tion process of the ORFV suggested the use of the steric exclusion chromatography (SXC) with high infectious virus yields and impurity removal. This rather new method is closely related to the conventional polyethylene glycol (PEG) precipitation, however, including a po-rous chromatographic media in the process. By preferential and steric exclusion mechanisms, the precipitates accrete inside the stationary phase and are retained. Several critical process parameters of the method are known and easily controlled, i.e., the PEG composition, the buffer composition as well as the pH and the ionic strength, the mixing technique prior to column loading, the flow rate, and the stationary phase. Nevertheless, high variability be-tween different runs is frequently observed. In this work, three unexplored parameters for ORFV processing with SXC were identified: the pore size of the stationary phase, the incuba-tion time and mixing strategy of the PEG / ORFV solution, and the addition of salts. Here, small pore sizes and long incubation times induced filtration effects, which cause pressure surges. The pressure is often a constraint in SXC application due to the high viscosity of the PEG. Therefore, a reduction of the PEG concentration while maintaining high yields is desira-ble. This could be achieved by adding different chaotropic ions to the PEG/ORFV mixture as proven for Mg2+. Furthermore, characterization of degrading and stabilizing conditions on the infectivity of the new platform virus was rarely published. In this work, this gap was addressed by an extensive study of mechanical, chemical, and thermal stress conditions on ORFV infec-tivity. In summary, the ORFV is extremely robust against degradation under conditions rou-tinely applied for pharmaceutical virus production. Nevertheless, care should be taken with heat and the implementation of ultrasonication in the production process. Generally, the ORFV should be stored under refrigerated conditions (4 °C), although the virus showed no substantial loss of infectivity if stored at 37 °C for two days or frozen. With regards to ion typically applied in purification processes, only ammonium salts should be avoided. Further stabilization is expected by the addition of proteins, sugars, and some amino acids. Based on these findings, a formulation buffer for ORFV storage with 1 % recombinant human serum albumin and 5 % sucrose was proposed. In the case of extended storage under heated condi-tions, albumin can be substituted with arginine. In conclusion, this comprehensive study of preferable process and storage conditions and critical process parameters of the SXC for the ORFV can inform future scale-up options to successfully implement SXC in pharmaceutical ORFV production processes with high infectious titer recoveries.de_DE
dc.description.sponsorshipBundesministerium für Bildung und Forschung (BMBF); ROR-ID:04pz7b180de_DE
dc.description.sponsorshipBundesländerde_DE
dc.description.sponsorshipEuropäische Kommission; ROR-ID:00k4n6c32de_DE
dc.description.sponsorshipSonstige Drittmittelgeber/-innende_DE
dc.language.isoende_DE
dc.relation.hasparthttps://doi.org/10.1016/j.virusres.2023.199213de_DE
dc.relation.hasparthttps://doi.org/10.1016/j.vaccine.2023.06.047de_DE
dc.relation.hasparthttps://doi.org/10.3390/membranes12050488de_DE
dc.relation.hasparthttps://doi.org/10.3390/membranes12111070de_DE
dc.relation.hasparthttps://doi.org/10.1016/j.jviromet.2022.114614de_DE
dc.rightsIn Copyright*
dc.rights.urihttp://rightsstatements.org/page/InC/1.0/*
dc.subjectDownstream Processingde_DE
dc.subjectSteric Exclusion Chromatographyde_DE
dc.subjectOrf virusde_DE
dc.subjectParapoxvirus ovisde_DE
dc.subjectViral vectorde_DE
dc.subjectFormulationde_DE
dc.subject.ddcddc:570de_DE
dc.subject.ddcddc:620de_DE
dc.titleIdentification of critical process parameters for the downstream processing of cell culture-derived Orf virus particlesde_DE
dc.typedoctoralThesisde_DE
dcterms.dateAccepted2023-11-29
local.affiliationPZI Promotionszentrum für Ingenieurwissenschaftende_DE
local.projectEXIST-Forschungstransfer grant (03EFKBW171); European Regional Development Fund as part of the Union’s response to the COVID-19 pandemic (IGJ-ERDF-Program Hesse - React EU 20008790)de_DE
thesis.levelthesis.doctoralde_DE


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