Key parameters of measles virus production for oncolytic virotherapy

Attenuated measles virus has revealed selective tumor cell killing and is currently tested in clinical trials for the therapy of cancer patients. The amount of infectious particles per dose needed for oncolytic therapy can be more than a million times higher compared to vaccination. This requires highly effective production processes which guarantee the measles virus quantities needed for its use in cancer therapy. Referring to measles virus production itself, several factors are influencing process parameters and subsequent virus yields. These factors are medium optimization, feeding of nutrients, an optimal multiplicity of infection, localization of produced virus particles, temperature sensitivity and time of harvest. This review summarizes the available data concerning measles virus production in cell culture and factors influencing the virus yield

Bioreactor Expansion of Human Mesenchymal Stem Cells According to GMP Requirements

In cell therapy, the use of autologous and allogenic human mesenchymal stem cells is rising. Accordingly, the supply of cells for clinical applications in highest quality is required. As hMSCs are considered as an advanced therapy medicinal products (ATMP), they underlie the requirements of GMP and PAT according to the authorities (FDA and EMA). The production process of these cells must therefore be documented according to GMP, which is usually performed via a GMP protocol based on standard operating procedures. This chapter provides an example of such a GMP protocol for hMSC, here a genetically modified allogenic cell line, based on a production process in a microcarrier-based stirred tank reactor including process monitoring according to PAT and final product quality assurance

Influence of process conditions on measles virus stability

Recombinant measles viruses are currently tested in clinical trials as oncolytic agent to be applied in cancer therapy. Contrary to their use as vaccine where 10³ infectious virus particles per dose are needed, for cancer therapy 10[superscript 9] virus particles should be provided per dose. This leads to other challenges for the production process when compared to vaccine production. This study presents measles virus stability with regard to conditions during production and storage of the virus. Relevant process parameters such as temperature (4-37°C), pH (pH 4-11), conductivity (1.5 to 137.5 mS cmˉ¹) and oxygen partial pressure were analyzed. The infectivity of measles virus particles decreased highly at 37 and 32°C, while at 22 and 4°C it remained stable for several hours or even days, respectively. The thermal inactivation reactions followed first order kinetics and the thermodynamic parameters enthalpy and entropy were estimated. Towards changes in pH measles virus particles were very sensitive, while no inactivation could be observed with varying conductivity. Measles virus incubation at an oxygen partial pressure of 100% did not lead to any loss of infectivity. The results show which parameters should be considered and controlled strongly in the production process to further raise measles virus yields for the high amount needed in cancer therapy approaches

Dielectric Spectroscopy and Optical Density Measurement for the Online Monitoring and Control of Recombinant Protein Production in Stably Transformed Drosophila melanogaster S2 Cells

The production of recombinant proteins in bioreactors requires real-time process monitoring and control to increase process efficiency and to meet the requirements for a comprehensive audit trail. The combination of optical near-infrared turbidity sensors and dielectric spectroscopy provides diverse system information because different measurement principles are exploited. We used this combination of techniques to monitor and control the growth and protein production of stably transformed Drosophila melanogaster S2 cells expressing antimicrobial proteins. The in situ monitoring system was suitable in batch, fed-batch and perfusion modes, and was particularly useful for the online determination of cell concentration, specific growth rate (µ) and cell viability. These data were used to pinpoint the optimal timing of the key transitional events (induction and harvest) during batch and fed-batch cultivation, achieving a total protein yield of ~25 mg at the 1-L scale. During cultivation in perfusion mode, the OD880 signal was used to control the bleed line in order to maintain a constant cell concentration of 5 × 107 cells/mL, thus establishing a turbidostat/permittistat culture. With this setup, a five-fold increase in productivity was achieved and 130 mg of protein was recovered after 2 days of induced perfusion. Our results demonstrate that both sensors are suitable for advanced monitoring and integration into online control strategies

Non-invasive raman spectroscopy and quantitative real-time PCR distinguish among undifferentiated human mesenchymal stem cells and redifferentiated nucleus pulposus cells and chondrocytes In Vitro

Background: The most common cause of lower back pain is the pathological degeneration of the nucleus pulposus (NP). Promising NP regeneration strategies involving human mesenchymal stem cells (hMSCs) would require specific markers to confirm successful differentiation into the NP lineage and to distinguish the articular cartilage (AC). Objective: We sought specific NP mRNA markers that are upregulated in native NP cells but not in dedifferentiated NP cells, undifferentiated hMSCs or chondrocytes. We also considered the suitability of non-invasive Raman spectroscopy to distinguish among these classes of cells. Method: We used quantitative real-time PCR and Raman spectroscopy to analyse undifferentiated hMSCs in monolayers and embedded in hydrogels, and compared the results with dedifferentiated and redifferentiated human NP and AC cells. Results: The redifferentiation of NP cells induced the expression of annexin A3 (ANXA3), collagen type II (COL2) and proteoglycan mRNAs , whereas the redifferentiation of AC cells only induced proteoglycan expression. Redifferentiated NP cells expressed higher levels of ANXA3, COL2, paired box 1 (PAX1) and OCT4 mRNA than redifferentiated AC cells. Redifferentiated NP cells and undifferentiated hMSC-TERT cells expressed similar amount of OCT4 mRNA, indicating that only ANXA3, COL2 and PAX1 are promising markers for redifferentiated NP cells. Raman spectra clearly differed among the three cell types and highlighted their differentiation status. Conclusion: We recommend ANXA3, COL2 and PAX1 as markers to determine the success of hMSC-based differentiation to regenerate NP cells. Raman spectroscopy can be used to determine cell type and differentiation status especially in the context of clinical trials

Comparison of cell-based versus cell-free mammalian systems for the production of a recombinant human bone morphogenic growth factor

The human bone morphogenetic protein-2 (hBMP2) is a glycoprotein, which induces de novo bone formation. Here, recombinant production in stably transfected Chinese Hamster Ovary (CHO) cells is compared to transient expression in Human Embryo Kidney (HEK) cells and cell-free synthesis in CHO cell lysates containing microsomal structures as sites of post-translational processing. In case of the stably transfected cells, growth rates and viabilities were similar to those of the parent cells, while entry into the death phase of the culture was delayed. The maximum achievable rhBMP2 concentration in these cultures was 153 pg/mL. Up to 280 ng/mL could be produced in the transient expression system. In both cases the rhBMP-2 was found to interact with the producer cells, which presumably contributed to the low yields. In the cell-free system, hBMP2 yields could be increased to almost 40 μg/mL, reached within three hours. The cell-free system thus approached productivities for the active (renatured) protein previously only recorded for bacterial hosts, while assuring comprehensive post-translational processing

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