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Herpes simplex virus type 1 (HSV-1) has several therapeutic indications including cancer, dermatological diseases and neurological disorders. For the clinical studies, high 106–108 plaque forming unit per mL (PFU mL-1) formulated virus doses are required. Supplying this amount of virus is challenging since the upstream process titer is influenced by numerous, non-linearly dependent parameters as well as by the genetic nature of the virus. The aim of this work was to characterize and mathematically model the wild-type HSV-1 production process as a basis for producing high quantity and quality virus. Three HSV-1 permissive cell lines (Vero, suspension HEK-293 and BHK-21) were considered for the production. However, due to the superior Vero cell productivity, the process development focused on Vero cells growing on Cytodex 1 microcarrier in a stirred tank bioreactor. Results showed that temperature (33 and 37 °C), pH (6.5–7.4) and medium exchange before time of infection (TOI) are not only factors for improving the virus titer but also the virus quality (ratio of PFU to virus genome copies). For the first time, the HSV-1 production—from cell inoculation until harvest—was described with a mathematical model. This model was used to maximize the virus titer by varying the TOI (2–3.5 days) and multiplicity of infection (MOI) (0.0001–0.1), which was possible without compromising the virus quality. This work revealed that ammonium accumulation in Vero cell cultures inhibits HSV-1 propagation, even more severely in higher cell density cultures. Using pyruvate or GlutaMAX supplementation instead of L-glutamine reduced the ammonium accumulation and increased the virus titer 2.9-fold in high cell density cultures (3.5 x 106 cells mL-1) in shaking flask, respectively. Alternatively, applying a daily medium exchange after TOI in glutamine cultures also led to comparable titers to pyruvate and GlutaMAX cultures. Overall, increasing the cell density from 1.0 x 106 cells mL-1 to 3.7 x 106 cells mL-1 for the TOI increased the titer 1.9-fold and space-time yield 1.6-fold in pyruvate bioreactor cultures. Genetically modified HSV-1 vectors were produced by applying the wild-type HSV-1-based process. While the insertion sites and transgenes (for targeting tumor cells or immune checkpoint inhibitor) into the virus genome decreased the virus titer 2.7–4.5 fold, the production of these viruses was still economically feasible. Yet attenuating the virus (by deletion of UL39 and mutation of UL37 gene) resulted an order of magnitude lower titer than the wild type virus and will require a complementing Vero cell line to achieve economic production titers., Das Herpes-simplex-Virus Typ 1 (HSV-1) hat mehrere therapeutische Indikationen, darunter Krebs, Hautkrankheiten und neurologische Störungen. Für klinische Studien werden hoch formulierte Virusdosen im Bereich von 106 bis 108 Plaque formenden Einheiten per mL (PFU mL-1) benötigt. Die Bereitstellung dieser Virusmenge stellt eine Herausforderung dar, da der Titer des Upstream-Prozesses von zahlreichen, nicht linear abhängigen Parametern, sowie von der genetischen Beschaffenheit des Virus beeinflusst wird. Ziel dieser Arbeit war es, den Wildtyp-HSV-1-Produktionsprozess zu charakterisieren und mathematisch zu modellieren, um eine Grundlage für die Produktion von Viren in hoher Quantität und Qualität zu schaffen. Drei HSV-1-permissive Zelllinien (Vero, HEK-293 und BHK-21) wurden für die Produktion in Betracht gezogen. Aufgrund der überlegenen Produktivität der Vero-Zellen konzentrierte sich die Prozessentwicklung auf Vero-Zellen, die auf Cytodex 1 in einem Rührkessel Bioreaktor gewachsen sind. Die Ergebnisse zeigten, dass sowohl die Wahl der Temperatur (33 und 37 °C) und des pH-Werts (6,5-7,4) als auch der Austausch des Mediums vor dem Infektionszeitpunkt (TOI) den Virustiter und die Virusqualität (Verhältnis von PFU zu Virusgenomkopien) beeinflussen. Zum ersten Mal wurde die HSV-1-Produktion—von der Zellinokulation bis zur Ernte—eines mathematischen Modells beschrieben. Dieses Modell wurde verwendet, um den Virustiter durch Variation der TOI (2-3,5 Tage) und der Multiplizität der Infektion (MOI) (0,0001-0,1) zu maximieren, was ohne Beeinträchtigung der Virusqualität möglich war. Diese Arbeit enthüllte, dass die Anreicherung von Ammonium in Vero-Zellkulturen die Vermehrung von HSV-1 hemmt und dieser Effekt mit erhöhter Zelldichte zunehmend ist. Durch Verwendung von Pyruvat oder GlutaMAX anstelle von L-Glutamin konnte diese Ammoniumakkumulation verringert werden, was zu einem 2,9-fach erhöhten Virustiter in Kulturen mit hoher Zelldichte (3,5 x 106 Zellen mL-1) im Schüttelkolben führte. Alternativ dazu führte ein täglicher Mediumwechsel nach TOI zu vergleichbaren Erträgen mit Glutamin wie unter Verwendung von Pyruvat und GlutaMAX. Insgesamt verbesserte die Erhöhung der Zelldichte von 1,0 x 106 auf 3,7 x 106 Zellen mL-1 den Titer um das 1,9-fache und die Raumzeitausbeute um das 1,6-fache in Pyruvat-Bioreaktorkulturen. Zusätzlich wurden gentechnisch veränderte HSV-1-Vektoren durch Anwendung des Wildtyp-HSV-1-basierten Verfahrens hergestellt. Obwohl die Insertionsstellen und Transgene (für Zielsteuerung auf Tumorzellen oder den Immun-Checkpoint-Inhibitor) den Virustiter um das 2,7- bis 4,5-fache verringerten, war die Herstellung dieser Viren dennoch wirtschaftlich umsetzbar. Eine Abschwächung des Virus (durch Deletion von UL39 und Mutation des UL37-Gens) reduzierte den Titer jedoch um eine Größenordnung gegenüber dem Wildtyp-Virus und erfordert eine komplementäre Vero-Zelllinie, um wirtschaftliche Produktionserträge zu erzielen.

Biotechnological processes have become more and more popular for a wide range of productsin recent years, but are frequently constrained by substrate and product inhibition. This process limitation can be alleviated by process intensification, in particular by in-situ separation of the inhibiting products via extraction. This study applies reactive extraction of the polyamide monomer hexane-1,6-diamine from a cell-containing aqueous biotechnological production medium. For this purpose, a cascaded option tree method was developed and applied to determine the most promising solvents and reactive extractants for the separation of the selected diamine. After a preliminary identification of suitable solvent classes, equilibrium experiments were conducted to determine partition coefficients and separation efficiencies. In these experiments D2EHPA and isostearic acid were identified as promising reactive extractants and kerosene as well as oleylalcohol were identified as suitable solvents. As a next step, the coalescence behavior was investigated in a standardized settling cell. D2EHPA diluted in oleyl alcohol was selected as promising option for realization in an extraction column. First, the simulation of the extraction column, based on single-drop experiments measurements, was successfully validated in pilot-scale experiments. Second, the extraction column was used as bioreactor with integrated aeration and product extraction, applying a four-phase system, investigating the effect on drop distributions and holdup of the dispersed phases. Interestingly, aeration increased the holdup and decreased the drop size of the organic extractive phase, thereby potentially improving the separation efficiency of reactive extraction. Therefore, the feasibility of the in-situ reactive extraction of hexane-1,6-diamine from a cell containing biotechnological production media in an extraction column operating with four phases was proven.

Dissertation, Rheinisch-Westfälische Technische Hochschule, 2018; Aachen 1 Online-Ressource (XIV, 192 Seiten) : Illustrationen, Diagramme (2018). = Dissertation, Rheinisch-Westfälische Technische Hochschule, 2018, Biotechnological development in shake flask necessitates vital engineering parameters e.g. volumetric power input, mixing time, gas-liquid mass transfer coefficient, hydromechanical stress and effective shear rate. This work provides ample experimental data for future validations representing shake flask fluid flow hydrodynamics i.e. in-phase (IP) and out-of-phase (OP) (phase phenomenon). Phase phenomenon was earlier expressed based on power input and that can now be expressed via liquid distribution. Optical fluorescence technique measurement producing 7644 data curves and 546 three-dimensional (3D) liquid distribution plots (hydrophilic), resulted from leading edge of bulk liquid (LB) and tail of bulk liquid (TB) analysis from 15-40 mL filling volume (VL), 150-450 rpm shaking frequency (n), 0-120 g/L of polyvinylpyrrolidone in fluorescent (fluorescent-PVP) solutions at 25 mm shaking diameter. The toroidal shapes of LB and TB are clearly asymmetrical and the measured TB differed by the elongation of the liquid particularly towards the torus part of the shake flask. Four new evaluation criteria proposed from 2184 evaluated cases, shed some light on how we closely relate 3D liquid distribution to phase phenomenon. The evaluation criteria on measured data are film thickness of the rotating bulk liquid, slope relative percentage of LB curves, circular angle of LB positions and 3D bulk liquid distribution’s peak shape change ($\geq$270$^\circ$ from 3D plot). A new compensated critical phase number (Phcrit) from theoretical to measured IP and OP has been revised from 1.26 to 0.91. Thus, phase change of liquid flow in 250-mL shake flask at 25 mm shaking diameter occurs at a little higher viscosity level of the fluorescent-PVP solutions than expected by the theoretical (calculated) phase number values. Additional to this, a non-viscosity model which serves as a simulated data is compared to measured liquid distribution investigated, illustrating range from similar to varying maximum liquid height (Hmax). Effect of hydrophobicity to liquid distribution angular position strengthen the fact to further understand the film and bulk liquid parameters. In conclusion, the 3D liquid distribution data collected at varying filling volume and shaking frequency, comprising of LB and TB values relative to the direction of the centrifugal acceleration are essential for validating future numerical solutions using CFD to predict vital engineering parameters in shake flask., Published by Aachen