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

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

Cytotoxic Evaluation of e-Liquid Aerosol using Different Lung-Derived Cell Models

The in vitro toxicological evaluation of e-liquid aerosol is an important aspect of consumer protection, but the cell model is of great significance. Due to its water solubility, e-liquid aerosol is deposited in the conducting zone of the respiratory tract. Therefore, primary normal human bronchial epithelial (NHBE) cells are more suitable for e-liquid aerosol testing than the widely used alveolar cell line A549. Due to their prolonged lifespan, immortalized cell lines derived from primary NHBE cells, exhibiting a comparable in vitro differentiation, might be an alternative for acute toxicity testing. In our study, A549 cells freshly isolated NHBE cells and the immortalized cell line CL-1548 were exposed at the air-liquid interface to e-liquid aerosol and cigarette mainstream smoke in a CULTEX® RFS compact module. The cell viability was analyzed 24 h post-exposure. In comparison with primary NHBE cells, the CL-1548 cell line showed lower sensitivity to e-liquid aerosol but significantly higher sensitivity compared to A549 cells. Therefore, the immortalized cell line CL-1548 is recommended as a tool for the routine testing of e-liquid aerosol and is preferable to A549 cells

Response to Polosa et al. Comments on Scheffler et al. Cytotoxic Evaluation of E-Liquid Aerosol Using Different Lung Derived Cell Models. Int. J. Environ. Res. Public Health, 2015, 12, 12466-12474

Referring to the comments of Polosa and colleagues [1] on our latest in vitro e-liquid aerosol testing publication, we would like to give our statements about some points. [...

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

Evaluation of E-Cigarette Liquid Vapor and Mainstream Cigarette Smoke after Direct Exposure of Primary Human Bronchial Epithelial Cells

E-cigarettes are emerging products, often described as “reduced-risk” nicotine products or alternatives to combustible cigarettes. Many smokers switch to e-cigarettes to quit or significantly reduce smoking. However, no regulations for e-cigarettes are currently into force, so that the quality and safety of e-liquids is not necessarily guaranteed. We exposed primary human bronchial epithelial cells of two different donors to vapor of e-cigarette liquid with or without nicotine, vapor of the carrier substances propylene glycol and glycerol as well as to mainstream smoke of K3R4F research cigarettes. The exposure was done in a CULTEX® RFS compact  module, allowing the exposure of the cells at the air-liquid interface. 24 h post-exposure, cell viability and oxidative stress levels in the cells were analyzed. We found toxicological effects of e-cigarette vapor and the pure carrier substances, whereas the nicotine concentration did not have an effect on the cell viability. The viability of mainstream smoke cigarette exposed cells was 4.5–8 times lower and the oxidative stress levels 4.5–5 times higher than those of e-cigarette vapor exposed cells, depending on the donor. Our experimental setup delivered reproducible data and thus provides the opportunity for routine testing of e-cigarette liquids to ensure safety and quality for the user

High-resolution mass spectrometric analysis of the secretome from mouse lung endothelial progenitor cells.

Recently, we isolated and characterized resident endothelial progenitor cells from the lungs of adult mice. These cells have a high proliferation potential, are not transformed and can differentiate into blood- and lymph-vascular endothelial cells under in vitro and in vivo conditions. Here we studied the secretome of these cells by nanoflow liquid chromatographic mass spectrometry (LC-MS). For analysis, 3-day conditioned serum-free media were used. We found 133 proteins belonging to the categories of membrane-bound or secreted proteins. Thereby, several of the membrane-bound proteins also existed as released variants. Thirty-five proteins from this group are well known as endothelial cell- or angiogenesis-related proteins. The MS analysis of the secretome was supplemented and confirmed by fluorescence activated cell sorting analyses, ELISA measurements and immunocytological studies of selected proteins. The secretome data presented in this study provides a platform for the in-depth analysis of endothelial progenitor cells and characterizes potential cellular markers and signaling components in hem- and lymphangiogenesis