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.

Opportunities to debottleneck the downstream processing of the oncolytic measles virus.

Oncolytic viruses (including measles virus) offer an alternative approach to reduce the high mortality rate of late-stage cancer. Several measles virus strains infect and lyse cancer cells efficiently, but the broad application of this therapeutic concept is hindered by the large number of infectious particles required (108-1012 TCID50 per dose). The manufacturing process must, therefore, achieve high titers of oncolytic measles virus (OMV) during upstream production and ensure that the virus product is not damaged during purification by applying appropriate downstream processing (DSP) unit operations. DSP is currently a production bottleneck because there are no specific platforms for OMV. Infectious OMV must be recovered as intact, enveloped particles, and host cell proteins and DNA must be reduced to acceptable levels to meet regulatory guidelines that were developed for virus-based vaccines and gene therapy vectors. Handling such high viral titers and process volumes is technologically challenging and expensive. This review considers the state of the art in OMV purification and looks at promising DSP technologies. We discuss here the purification of other enveloped viruses where such technologies could also be applied to OMV. The development of DSP technologies tailored for enveloped viruses is necessary to produce sufficient titers for virotherapy, which could offer hope to millions of patients suffering from incurable cancer.

Tangential Flow Filtration for the Concentration of Oncolytic Measles Virus: The Influence of Filter Properties and the Cell Culture Medium.

The therapeutic use of oncolytic measles virus (MV) for cancer treatment requires >108 infectious MV particles per dose in a highly pure form. The concentration/purification of viruses is typically achieved by tangential flow filtration (TFF) but the efficiency of this process for the preparation of MV has not been tested in detail. We therefore investigated the influence of membrane material, feed composition, and pore size or molecular weight cut-off (MWCO) on the recovery of MV by TFF in concentration mode. We achieved the recovery of infectious MV particles using membranes with a MWCO ≤ 300 kDa regardless of the membrane material and whether or not serum was present in the feed. However, serum proteins in the medium affected membrane flux and promoted fouling. The severity of fouling was dependent on the membrane material, with the cellulose-based membrane showing the lowest susceptibility. We found that impurities such as proteins and host cell DNA were best depleted using membranes with a MWCO ≥ 300 kDa. We conclude that TFF in concentration mode is a robust unit operation to concentrate infectious MV particles while depleting impurities such as non-infectious MV particles, proteins, and host cell DNA.

Forced Degradation Studies to Identify Critical Process Parameters for the Purification of Infectious Measles Virus.

Oncolytic measles virus (MV) is a promising treatment for cancer but titers of up to 1011 infectious particles per dose are needed for therapeutic efficacy, which requires an efficient, robust, and scalable production process. MV is highly sensitive to process conditions, and a substantial fraction of the virus is lost during current purification processes. We therefore conducted forced degradation studies under thermal, pH, chemical, and mechanical stress to determine critical process parameters. We found that MV remained stable following up to five freeze-thaw cycles, but was inactivated during short-term incubation (< 2 h) at temperatures exceeding 35 °C. The infectivity of MV declined at pH < 7, but was not influenced by different buffer systems or the ionic strength/osmolality, except high concentrations of CaCl2 and MgSO4. We observed low shear sensitivity (dependent on the flow rate) caused by the use of a peristaltic pump. For tangential flow filtration, the highest recovery of MV was at a shear rate of ~5700 s-1. Our results confirm that the application of forced degradation studies is important to identify critical process parameters for MV purification. This will be helpful during the early stages of process development, ensuring the recovery of high titers of active MV particles after purification.

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.

High titer oncolytic measles virus production process by integration of dielectric spectroscopy as online monitoring system.

Oncolytic viruses offer new hope to millions of patients with incurable cancer. One promising class of oncolytic viruses is Measles virus, but its broad administration to cancer patients is currently hampered by the inability to produce the large amounts of virus needed for treatment (1010 -1012 virus particles per dose). Measles virus is unstable, leading to very low virus titers during production. The time of infection and time of harvest are therefore critical parameters in a Measles virus production process, and their optimization requires an accurate online monitoring system. We integrated a probe based on dielectric spectroscopy (DS) into a stirred tank reactor to characterize the Measles virus production process in adherent growing Vero cells. We found that DS could be used to monitor cell adhesion on the microcarrier and that the optimal virus harvest time correlated with the global maximum permittivity signal. In 16 independent bioreactor runs, the maximum Measles virus titer was achieved approximately 40 hr after the permittivity maximum. Compared to an uncontrolled Measles virus production process, the integration of DS increased the maximum virus concentration by more than three orders of magnitude. This was sufficient to achieve an active Measles virus concentration of > 1010 TCID50 ml-1 .

Screening different host cell lines for the dynamic production of measles virus.

Measles virus (MV) has a natural affinity for cancer cells and oncolytic MV preparations have therefore been investigated in several clinical trials as a potential treatment for cancer. The main bottleneck in the administration of oncolytic MV to cancer patients is the production process, because very large doses of virus particles are required for each treatment. Here, we investigated the productivity of different host cells and found that a high infection efficiency did not necessarily result in high virus yields because virus release is also dependent on the host cell. As well as producing large numbers of active MV particles, host cells must perform well in dynamic cultivation systems. In screening experiments, the highest productivity was achieved by Vero and BJAB cells, but only the Vero cells maintained their high virus productivity when transferred to a stirred tank reactor. We used dielectric spectroscopy as an online monitoring system to control the infection and harvest times, which are known to be critical process parameters. The precise control of these parameters allowed us to achieve higher virus titers with Vero cells in a stirred tank reactor than in a static cultivation system based on T-flasks, with maximum titers of up to 1011 TCID50 ml-1 . © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:989-997, 2017.

Virus separation using membranes.

Industrial manufacturing of cell culture-derived viruses or virus-like particles for gene therapy or vaccine production are complex multistep processes. In addition to the bioreactor, such processes require a multitude of downstream unit operations for product separation, concentration, or purification. Similarly, before a biopharmaceutical product can enter the market, removal or inactivation of potential viral contamination has to be demonstrated. Given the complexity of biological solutions and the high standards on composition and purity of biopharmaceuticals, downstream processing is the bottleneck in many biotechnological production trains. Membrane-based filtration can be an economically attractive and efficient technology for virus separation. Viral clearance, for instance, of up to seven orders of magnitude has been reported for state of the art polymeric membranes under best conditions.This chapter summarizes the fundamentals of virus ultrafiltration, diafiltration, or purification with adsorptive membranes. In lieu of an impractical universally applicable protocol for virus filtration, application of these principles is demonstrated with two examples. The chapter provides detailed methods for production, concentration, purification, and removal of a rod-shaped baculovirus (Autographa californica M nucleopolyhedrovirus, about 40 × 300 nm in size, a potential vector for gene therapy, and an industrially important protein expression system) or a spherical parvovirus (minute virus of mice, 22-26 nm in size, a model virus for virus clearance validation studies).

Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatography.

Significant progress in the application of viral vectors for gene delivery into mammalian cells and the use of viruses as biopesticides requires downstream processing that can satisfy application-specific demands on performance. In the present work the stability and ion exchange membrane chromatography of a recombinant of Autographa californica M nucleopolyhedrovirus is studied. To adjust the degree of purification the effect of ionic conductivity or pH on the viral infectivity was assessed (0.77-78.00mS/cm, pH 3-8). Infectivity decreased rapidly by several orders of magnitude at below 5mS/cm (i.e., 0.49MPa osmotic pressure change) or at below pH 5.5 (rationalized with particle aggregation). The virus was concentrated and purified via adsorption (0.2-1.1×10(16)pfu/m(3) chromatographic bed volume, 0.6-1.1×10(12)pfu/m(2) membrane area facing the incident fluid flow) and elution at pH 6.1 and 6.35mS/cm from three strong anion exchange membranes. Virus recovery and concentration in accord with the volume reduction were obtained using a polyether sulfone-based membrane with quaternary ammonium ligands. The level of host cell protein (down to below the detection limit) and suspended DNA (below 93pg DNA per 10(6)pfu) are reported for each membrane employed, for the purpose of comparability, under equal adsorption or elution conditions respectively.

Alternatives to dimethylsulfoxide for serum-free cryopreservation of human mesenchymal stem cells.

Human mesenchymal stem cells (hMSCs) have some favorable characteristics like high plasticity, multilineage differentiation potential, and comparably easy handling in vitro, making them of interest for many clinical and therapeutic approaches including cell therapy. For routine applications, these cells have to be stored over a certain period of time without loss of cell vitality and function. An easy way to preserve cells is to store them at temperatures between -80 degrees C and -196 degrees C (liquid nitrogen). To prevent cells from the damage caused by the cryopreservation process and to achieve high cell recovery and vitality, cryoprotectants are used. Typically dimethylsulfoxide, often in combination with serum, is used as a cryoprotectant. However, for clinical approaches, the use of dimethylsulfoxide and serum in patients is problematic for several reasons. Therefore, the cryopreservation of human mesenchymal stem cells for cell therapeutic applications without dimethylsulfoxide and serum demands investigation. In this work, non-toxic alternatives to dimethylsulfoxide such as glycerol or the compatible solutes, proline and ectoin, were analyzed in a serum-free cryomedium with respect to their cryoprotective properties. Different concentrations of the cryoprotectants (1-10% (w/v) ectoin or proline, respectively, or 5-20% (v/v) glycerol) and certain incubation times (0-60 minutes) were investigated with regard to post-thaw cell vitality and cell growth. Our results showed that, in general, cryopreservation with ectoin led to high post-thaw cell survival of up to 72% whereas after cryopreservation with glycerol and proline, the hMSC cells were completely dead (glycerol) or had only poor cell survival (proline, 22%). Moreover, the morphology of the hMSC cells changed to a large and flat phenotype after cryopreservation with proline. These results indicate that glycerol and proline are not suitable for cryopreservation of hMSC. In contrast, ectoin has the potential to replace dimethylsulfoxide as a cryoprotectant in a serum-free cryomedium.