Dynamic oncolytic measles virus production

Oncolytic viruses can be effective weapons against cancer with few treatment options. For example the tissue culture–adapted Edmonston strains of measles virus (MV) have altered its receptor specificity and became selectively oncolytic with attenuated pathogenicity. Russel et al. showed in 2014 full remission in an advanced stage multiple myeloma patient after systemic application of genetically modified MV. In this clinical trial, the patient was treated by intravenous infusion of 1011 TCID50 (50% tissue culture infectious dose) - of an engineered MV encoding human sodium iodide symporter. Appropriate medical treatment with oncolytic viruses calls for high concentrations and absolute product purity. The main challenge in the field of viral bioprocess design is the low product stability. Although oncolytic MV production is feasible in a stirred tank bioreactor, the poor knowledge about the virus release and inactivation process hampers oncolytic MV production in industrial scale. As published by Weiss et al. already a simple transfer of the MV production process from a static cultivation system (e.g. T-Flask) into a dynamic system (e.g. STR) can dramatically reduce MV yield. As static systems are only suitable for small-scale processes, the process transfer into scalable dynamic systems is a bottleneck for a broad application of MV as cancer drugs. Beside their limited scalability, T-flasks or cell factories only allows the MV production in a batch mode. Through this process mode, the MV particles can be harvest only once and at an assumed time of harvest (TOH). In consideration of the short MV half-life of one and two hours at 37°C and 32°C respectively, the TOH is a critical point in bioprocessing. But measles viruses are known to be sensitive against the production temperature. Therefore an infection cycle adapted virus harvest with related synchronal purification is required. Virus membrane filtration represents a beneficial trade-off. Membrane-based filtration like cross flow filtration can process potentially large volumes and yielding high host cell concentration in the bioreactor. On the other hand, membrane based filtration processes are very unspecific. To remain the advantages of membrane filtration and increase the selectivity of the purification, membrane chromatography is a true alternative. Application of adsorptive membranes based on the electric interaction between charged components of the liquid phase including viruses and ionic groups immobilized on the solid membrane matrix, ion exchange membrane chromatography (IEMC) is a potentially simple and efficient method for MV concentration and purification. In present work, a continuous bioprocess concept for oncolytic MV production and purification for the use in cancer therapy will be presented

A rapid method for an offline glycerol determination during microbial fermentation

Background: The purpose of this work was to find a rapid method for glycerol detection during microbial fermentations. The method requirements were, first, to avoid sample pretreatment, and second, to measure glycerol precisely especially out of fermentation broth. Results: This was achieved by combining two reaction principles — the Malaprade reaction and the Hantzsch reaction. In the Malaprade reaction, glycerol is converted into formaldehyde. This forms a dye in the Hantzsch reaction after which adsorption is than detected. The subsequent assay was investigated with two different fermentation media, a chemically undefined and a chemically defined media, used for Pichia pastoris fermentation. In both media, as well as in real fermentation samples, glycerol content could be reproducibly detected with the method. Moreover, measurements were more precise than using a standard glycerol detection kit. Conclusions: With this rapid assay, glycerol could be detected easily in microbial fermentation broth. It is reliable over a wide concentration range including advantages such as an easy assay set-up, a short assay time and no sample pretreatment.Citation: Kuhn J, H Müller, D Salzig, P Czermak:A rapid method for an offline glycerol determination during microbial fermentation, Electronic Journal of Biotechnology 18 (2015) 3, 252-255 http://dx.doi.org/10.1016/j.ejbt.2015.01.00

Bioprocess Development for Human Mesenchymal Stem Cell Therapy Products

Mesenchymal stem cells (MSCs) are advanced therapy medicinal products used in cell therapy applications. Several MSC products have already advanced to phase III clinical testing and market approval. The manufacturing of MSCs must comply with good manufacturing practice (GMP) from phase I in Europe and phase II in the US, but there are several unique challenges when cells are the therapeutic product. Any GMP-compliant process for the production of MSCs must include the expansion of cells in vitro to achieve a sufficient therapeutic quantity while maintaining high cell quality and potency. The process must also allow the efficient harvest of anchorage-dependent cells and account for the influence of shear stress and other factors, especially during scale-up. Bioreactors are necessary to produce clinical batches of MSCs, and bioprocess development must therefore consider this specialized environment. For the last 10 years, we have investigated bioprocess development as a means to produce high-quality MSCs. More recently, we have also used bioreactors for the cocultivation of stem cells with other adult cells and for the production of MSC-derived extracellular vesicles. This review discusses the state of the art in bioprocess development for the GMP-compliant manufacture of human MSCs as products for stem cell therapy

Manufacturing of Human Umbilical Cord Mesenchymal Stromal Cells on Microcarriers in a Dynamic System for Clinical Use

Citation: Florian Petry, J. Robert Smith, Jasmin Leber, Denise Salzig, Peter Czermak, and Mark L. Weiss, “Manufacturing of Human Umbilical Cord Mesenchymal Stromal Cells on Microcarriers in a Dynamic System for Clinical Use,” Stem Cells International, vol. 2016, Article ID 4834616, 12 pages, 2016. doi:10.1155/2016/4834616The great properties of human mesenchymal stromal cells (hMSCs) make these cells an important tool in regenerative medicine. Because of the limitations of hMSCs derived from the bone marrow during isolation and expansion, hMSCs derived from the umbilical cord stroma are a great alternative to overcome these issues. For a large expansion of these cells, we performed a process transfer from static culture to a dynamic system. For this reason, a microcarrier selection out of five microcarrier types was made to achieve a suitable growth surface for the cells. The growth characteristics and metabolite consumption and production were used to compare the cells growth in 12-well plate and spinner flask. The goal to determine relevant process parameters to transfer the expansion process into a stirred tank bioreactor was achieved

Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium

Citation: Denise Salzig, Jasmin Leber, Katharina Merkewitz, Michaela C. Lange, Natascha Köster, and Peter Czermak, “Attachment, Growth, and Detachment of Human Mesenchymal Stem Cells in a Chemically Defined Medium,” Stem Cells International, vol. 2016, Article ID 5246584, 10 pages, 2016. doi:10.1155/2016/5246584The manufacture of human mesenchymal stem cells (hMSCs) for clinical applications requires an appropriate growth surface and an optimized, preferably chemically defined medium (CDM) for expansion. We investigated a new protein/peptide-free CDM that supports the adhesion, growth, and detachment of an immortalized hMSC line (hMSC-TERT) as well as primary cells derived from bone marrow (bm-hMSCs) and adipose tissue (ad-hMSCs). We observed the rapid attachment and spreading of hMSC-TERT cells and ad-hMSCs in CDM concomitant with the expression of integrin and actin fibers. Cell spreading was promoted by coating the growth surface with collagen type IV and fibronectin. The growth of hMSC-TERT cells was similar in CDM and serum-containing medium whereas the lag phase of bm-hMSCs was prolonged in CDM. FGF-2 or surface coating with collagen type IV promoted the growth of bm-hMSCs, but laminin had no effect. All three cell types retained their trilineage differentiation capability in CDM and were detached by several enzymes (but not collagenase in the case of hMSC-TERT cells). The medium and coating did not affect detachment efficiency but influenced cell survival after detachment. CDM combined with cell-specific surface coatings and/or FGF-2 supplements is therefore as effective as serum-containing medium for the manufacture of different hMSC types

A tailor-made purification strategy for oncolytic measles viruses using membrane-based processes

Cancer patients can benefit from the Measles virus, since in the early 70s a relation between cancer remission and an infection with Measles was first mentioned (Bluming, Ziegler 1971). Further studies confirmed this oncolytic activity and therefore, the Measles virus became highly interesting for the application in cancer treatment. However, for the widespread application as a therapeutic agent several bottlenecks have to be overcome in context of quantity and quality. For one therapeutic dose of oncolytic Measles viruses (OMV) at least 1011 infectious particles are needed (one vaccination contains ~103 TCID50) (Russell et al. 2014). Besides that, the impurities, such as host cell proteins (HCP) and host cell DNA (hcDNA), must be reduced to appropriate limits set by regulatory authorities. The full recovery of OMV infectivity must also addressed. This underlines the need of a tailor-made downstream processing. After we established a high titer production process, achieving OMV titers of 1011 TCID50 mL-1 (Grein et al. 2017), we are now focused on the downstream processing of OMV. For this purpose we characterized the OMV regarding process parameters used in DSP, such as stability towards ionic strength, osmolality, agglomeration and shear stress. Based on this, a clarification step was conducted, followed by the further purification with tangential flow filtration (TFF). By using polyether sulfone flat sheet membranes in concentration mode, we were able to recover the infectious virus and lowered the impurities by ~70% for hcDNA and ~80% for protein content. In the next purification step, we applied a discontinuous diafiltration and could deplete the impurities by ~95% in total. These results showed that TFF is an appropriate tool for the purification and formulation of OMV. References Bluming, Avrum Z.; Ziegler, John L. (1971): Regression of Burkitt\u27s Lymphoma in association with Measles infection. In The Lancet 298 (7715), pp. 105–106. DOI: 10.1016/S0140-6736(71)92086-1. Grein, Tanja A.; Schwebel, Felix; Kress, Marco; Loewe, Daniel; Dieken, Hauke; Salzig, Denise et al. (2017): Screening different host cell lines for the dynamic production of measles virus. In Biotechnology progress. DOI: 10.1002/btpr.2432. Russell, Stephen J.; Federspiel, Mark J.; Peng, Kah-Whye; Tong, Caili; Dingli, David; Morice, William G. et al. (2014): Remission of disseminated cancer after systemic oncolytic virotherapy. In Mayo Clinic proceedings 89 (7), pp. 926–933. DOI: 10.1016/j.mayocp.2014.04.003

A rapid method for an offline glycerol determination during microbial fermentation

Background: The purpose of this work was to find a rapid method for glycerol detection during microbial fermentations. The method requirements were, first, to avoid sample pretreatment, and second, to measure glycerol precisely especially out of fermentation broth. Results: This was achieved by combining two reaction principles \u2014 the Malaprade reaction and the Hantzsch reaction. In the Malaprade reaction, glycerol is converted into formaldehyde. This forms a dye in the Hantzsch reaction after which adsorption is than detected. The subsequent assay was investigated with two different fermentation media, a chemically undefined and a chemically defined media, used for Pichia pastoris fermentation. In both media, as well as in real fermentation samples, glycerol content could be reproducibly detected with the method. Moreover, measurements were more precise than using a standard glycerol detection kit. Conclusions: With this rapid assay, glycerol could be detected easily in microbial fermentation broth. It is reliable over a wide concentration range including advantages such as an easy assay set-up, a short assay time and no sample pretreatment

Aeration and Shear Stress Are Critical Process Parameters for the Production of Oncolytic Measles Virus

Oncolytic Measles virus is a promising candidate for cancer treatment, but clinical studies have shown that extremely high doses (up to 1011 TCID50 per dose) are required to effect a cure. Very high titers of the virus must therefore be achieved during production to ensure an adequate supply. We have previously shown that Measles virus can be produced in Vero cells growing on a Cytodex 1 microcarrier in serum-containing medium using a stirred-tank reactor (STR). However, process optimization and further process transfer or scale up requires the identification of critical process parameters, particularly because the use of STRs increases the risk of cell damage and lower product yields due to shear stress. Using a small-scale STR (0.5 L working volume) we found that Measles virus titers are sensitive to agitator-dependent shear, with shear stress ≥0.25 N m−2 reducing the titer by more than four orders of magnitude. This effect was observed in both serum-containing and serum-free medium. At this scale, virus of titers up to 1010 TCID50 mL−1 could be achieved with an average shear stress of 0.1 N m−2. We also found that the aeration method affected the virus titer. Aeration was necessary to ensure a sufficient oxygen supply to the Vero cells, and CO2 was also needed to regulate the pH of the sodium bicarbonate buffer system. Continuous gassing with air and CO2 reduced the virus titer by four orders of magnitude compared to head-space aeration. The manufacture of oncolytic Measles virus in a STR can therefore be defined as a shear-sensitive process, but high titers can nevertheless be achieved by keeping shear stress levels below 0.25 N m−2 and by avoiding extensive gassing of the medium

Three-Dimensional Bioreactor Technologies for the Cocultivation of Human Mesenchymal Stem/Stromal Cells and Beta Cells

Diabetes is a prominent health problem caused by the failure of pancreatic beta cells. One therapeutic approach is the transplantation of functional beta cells, but it is difficult to generate sufficient beta cells in vitro and to ensure these cells remain viable at the transplantation site. Beta cells suffer from hypoxia, undergo apoptosis, or are attacked by the host immune system. Human mesenchymal stem/stromal cells (hMSCs) can improve the functionality and survival of beta cells in vivo and in vitro due to direct cell contact or the secretion of trophic factors. Current cocultivation concepts with beta cells are simple and cannot exploit the favorable properties of hMSCs. Beta cells need a three-dimensional (3D) environment to function correctly, and the cocultivation setup is therefore more complex. This review discusses 3D cultivation forms (aggregates, capsules, and carriers) for hMSCs and beta cells and strategies for large-scale cultivation. We have determined process parameters that must be balanced and considered for the cocultivation of hMSCs and beta cells, and we present several bioreactor setups that are suitable for such an innovative cocultivation approach. Bioprocess engineering of the cocultivation processes is necessary to achieve successful beta cell therapy

A Combined Ultrafiltration/Diafiltration Process for the Purification of Oncolytic Measles Virus

Measles virus (MV) is an important representative of a new class of cancer therapeutics known as oncolytic viruses. However, process intensification for the downstream purification of this fragile product is challenging. We previously found that a mid-range molecular weight cut-off (300 kDa) is optimal for the concentration of MV. Here, we tested continuous and discontinuous diafiltration for the purification of MV prepared in two different media to determine the influence of high and low protein loads. We found that a concentration step before diafiltration improved process economy and MV yield when using either serum-containing or serum-free medium. We also found that discontinuous diafiltration conferred a slight benefit in terms of the permeate flow, reflecting the repetitive dilution steps and the ability to break down parts of the fouling layer on the membrane. In summary, the combined ultrafiltration/diafiltration process is suitable for the purification of MV, resulting in the recovery of ~50% infectious virus particles with a total concentration factor of 8 when using 5 diavolumes of buffer