Dirk Nehring

Perfusion cultures and modelling of oxygen uptake with three-dimensional chondrocyte pellets

Chondrocyte pellets were cultivated in a perfused flow chamber and supplied with medium by a constant flow rate from a conditioning vessel. In this conditioning vessel the medium was aerated and used medium was exchanged semi-continuously. The higher amount of DNA and glycosaminoglycane (GAG) in these pellets compared to control cultures under stationary conditions showed a positive effect of the reactor system, compared to standard culture conditions. A diffusion reaction model was applied to calculate the oxygen uptake of the cell pellet and to describe the oxygen profile within the pellet. The model included diffusion within the cell pellet and oxygen uptake of the cells. Calculated data were compared to experimental data obtained by tissue engineered chondrocyte cell pellets. Model calculations agreed rather well with experimental data.

Experimental Study of a Ceramic Microsparging Aeration System in a Pilot‐Scale Animal Cell Culture

The oxygen supply of cell cultures with the aid of free gas bubbles is an efficient process strategy in pharmaceutical production. If the cell‐damaging impact of gas bubbles is reduced, direct aeration becomes a practical solution with scale‐up potential and comparatively high oxygen transfer rates. In this paper a microsparging aeration system made of porous ceramic was compared with bubble‐free membrane aeration. The sparging system was used for the long‐term cultivation of mammalian cells in 2‐to 100‐L scale bioreactors and produced bubble sizes of 100–500 μm in diameter. Using a scale of 2.5 and 30 L, a cell density of 2.6 × 106 cells/mL was attained. When a 100‐L scale was used, a density of 1.1 × 106 cells/mL was achieved, whereas a comparable membrane‐aerated system showed a cell density of 2.2 × 106 cells/mL. At relatively low agitation rates of less than 70 rpm in the sparged bioreactors, a homogeneous and constant oxygen concentration was kept in the medium. As a result of the different foam‐forming tendency caused by the lower gas flow of the ceramic sparger compared to that of the standard aeration systems, we were able to develop an appropriate process control strategy. Furthermore, oxygen transfer measurements for the common stainless steel sparger and the ceramic sparger showed a 3‐fold higher oxygen transfer coefficient for the ceramic sparger.

Fixed Bed Reactors for the Cultivation of Mammalian Cells: Design, Performance and Scale-Up

Fixed-bed reactors have gained growing attention for the cultivation of mammalian cells. They allow for a low shear stress cultivation of adherent and non-adherent cells due to the immobilization of cells within macroporous carriers. Their potential has been demonstrated for many cell culture purposes. Some of the recent developments are presented in this review, including improved antibody production by hybridoma cells, high performance cultivation of a hepatoblas- toma cell line and cultivation of cells for the production of retroviral vectors. Furthermore, criteria for the selection of process strategies and scale-up concepts are addressed.

Production of Retroviral Pseudotype Vectors in Fixed Bed Reactors for Use in Gene Therapy

To achieve an efficient large scale manufacturing of viral vectors for gene therapy, traditional concepts in engineering design must be augmented by consideration of the biological properties of the viruses and the cells by which they are produced. Fixed bed bioreactors represent an interesting alternative in the vector production because of the high volumetric productivity and the possibility to work in perfusion. Based on specific examples, production of retroviral pseudotype vector, derived from the murine leukaemia virus carrying the HIV-1 envelop protein MLV (HIV-1, donated by Prof. Chichutek, PEI, Langen), the relevant aspects for process design (reactor configuration, mode of operation etc.) are discussed. Especially methods for increase of vector titre by means of integrated cross flow filtration steps will be addressed. Extended mathematical models for growth kinetic and product formation were used to optimise the process parameters to achieve high vector titres. The results of our studies emphasise that not only optimisation of packaging cell lines but also optimisation of the cultivation and purification process is needed in order to achieve appropriate concentrations of retroviral particles for gene therapy application.