LOEWE Zentrum für
& Bioressourcen

Seminar announcement 12th November 2014

Denise Salzig

Bioprocess development for special bioresources

As the LOEWE center ZIB stands not only for insects and insect-derived molecules, I decided to speak about the other bioresources. From the therapeutic view interesting bioresources are therapeutic cells and viruses. From the view of a bioprocess engineer, which we are at IBPT, cells and viruses form a special class of products which led to completely different process requirement than e.g. for protein production. As at IBPT we intensely investigated stem cell expansion processes and viral production processes, I would like to present the major insights of our work.

Cell therapy has evolved into a distinct healthcare sector observable by an increasing number of clinical trials and FDA-approved commercial products. As stem cells are a complex and viable product, their manufacturing has to meet several requirements. Production in a Good Manufacturing Practice (GMP) environment and compliance with Process Analytical Technology (PAT) over the whole expansion process is challenging. One important cell source for clinical applications are hMSCs (human mesenchymal stem cells). To enable their use as advanced therapy medicinal products (ATMP), high quantities of undifferentiated cells with high viability are required. During several projects expansion concepts for hMSCs were developed at IBPT. For cell therapy processes where e.g. hMSCs form the product, it is indispensable to know process characteristics exactly. Unfortunately, bioengineering investigations were often performed under non-realistic conditions using neither the culture medium/carrier nor the parameters (e.g. low agitation, aeration) relevant for hMSC expansion. Therefore at IBPT, we investigated bioengineering characteristics (power input, shear stress, kLa, mixing time) under conditions realistic for hMSC expansion. During hMSC manufacturing, the cell harvest step is of great importance. In contrast to manufacturing of other therapeutics (e.g. antibodies), the hMSCs must stay viable and unaffected. As hMSCs grow strictly adherent to a surface, cell harvest is ultimately required. In consideration of cGMP guidelines, cell harvest is a challenging assignment. As required by the authorities, detachment enzymes of non-animal origin are needed. At IBPT hMSC harvest from bioreactor systems is investigated.

The potential anticancer activity of the lytic measles virus has been proven in several studies. Beside the growing interest and the numerous investigations in cancer virotherapy only little is known about the production of the infectious oncolytic measles virus particles. Most reports on measles virus production deal with vaccine production which has a different production set-up when compared to oncolytic virus production. These studies do not meet current state of (oncolytic) virus production under cGMP which includes PAT and avoidance of raw materials of mammalian origin. Furthermore, the application of oncolytic measles virus requires up to 1,000,000-times higher doses than used for vaccination. This is a huge challenge for measles virus manufacturing. So the subject of the presented project is to scale up the production of MV in Vero cells in order to achieve an optimized and standardized fermentation process by providing more MV particles for the use in cancer therapy. The cells have been adapted to a commercially available serum-free medium. The growth surface was provided by micro carriers for the cultivation of adherent cells in stirred systems. Similar maximum titers were reached for cell-associated and extracellular MV. Repeated harvesting of the extracellular MV showed no negative influence on MV yields, but raised cumulative virus titers. In addition research on the temperature stability of the MV was carried out and it has been shown that the MV in the supernatant under culture conditions was very unstable due to temperature inactivation. To meet commercial and regulatory requirements, this process must be high yielding, scalable and reproducible. These requirements are met by establishing a cell culture process employing stirred system and serum-free cell culture medium. For the control of the several online methods are used. The description of MV production kinetics is used for a successful optimization of the process.

Dr.-Ing.  Denise Salzig, Technische hochschule Mittelhessen and LOEWE ZIB

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