(2010) ATROSAB, a humanized antagonistic anti-tumor necrosis factor receptor one-specific antibody, mAbs, 2:6, 639-647,

Scalability is a major demand for high-yield, stable bioprocess systems in animal cell culture-based biopharmaceutical production. Increased yields can be achieved through high-density cell culture, such as in the combination of microcarrier and fluidized bed bioreactor technology. To minimize inocula volume in industrial applications of fluidized bed fermentation systems, it is crucial to increase the bed volume in the reactor during the fermentation process. We tested scale-up strategy for the production of recombinant human arylsulfatase B (ASB) enzyme used in enzyme replacement therapy in patients afflicted with mucopolysaccharidosis type VI (MPS VI). This enzyme was derived from Chinese hamster ovary (CHO) cells cultivated as adherent cell culture on Cytoline macroporous microcarriers (Amersham Biosciences, Uppsala, Sweden) using a Cytopilot Mini fluidized bed bioreactor (FBR; Amersham Biosciences, Vogelbusch, Austria). Both 1:2 expansion (herein referred to as the addition of fresh, not-yet-colonized microcarriers) and 1:6 expansion of the carrier bed were performed successfully; the cells restarted to proliferate for colonizing these newly added carriers; and the stability of the culture was not negatively affected.

The success of culturing CHO cells solely depends on functionality of the used media. Cell culture technology is more than 50 years old, and the knowledge of cell requirements increased steadily. In the beginning, animal-sourced components were the key to growth. Nowadays state-of-the-art media do not contain any animal or naturally sourced components. The compositions are based on scientific awareness of the needs of the cells. The result is high lot-to-lot consistency and high performance.In this book section, a method for the development of a synthetic, animal component-free medium is described. The composition is based on public available formulations and information based on the work of many scientists printed in numerous papers and manuscripts. The method shall help beginners to design their own medium, although some knowledge of biochemistry and animal cells is still required.

Due to the inherent risks of animal-derived raw materials, the biopharmaceutical industry has an increasing demand for serum-free and protein-free media for industrial cell culture bioprocesses. The absence of serum often changes the characteristics of mammalian cells, especially growth, productivity, and adherence properties. This study is mainly focused on the influence of media additives on cell adherence characteristics. An array of different carboxymethyl dextrans and different ferric citrate concentrations was tested with a number of CHO clones, using standard cell culture Roux-flasks and Cytoline 1 macroporous microcarriers. A prototype mixing system with controlled shear force input was developed as a screening system for adherence characteristics. The results of this evaluation revealed a negatively correlated dose-dependent influence on adhesion for ferric citrate. It was also found that certain carboxymethyl dextrans are capable of increasing the adherence on Roux-flasks and microcarriers.

In this publication different detachment factors were tested for enhancing carrier to carrier transfer for scale-up of macroporous microcarrier based bioprocesses. Two Chinese hamster ovary cell lines, CHO-K1 and a genetically engineered CHO-K1 derived cell line (CHO-MPS), producing recombinant human Arylsulfatase B, were examined. The cells were grown on Cytoline 1microcarriers (Amersham Biosciences, Uppsala, Sweden) in protein-free and chemically defined medium respectively. Fully colonised microcarriers were used at passage ratios of approximately 1:10 for carrier to carrier transfer experiments. To accelerate the colonisation of the non-colonised, freshly added microcarriers the detachment reagents trypsin, papain, Accutasetrade mark (PAA, Linz, Austria), heparin and dextransulphate were used. Both cell lines showed good results with trypsin, Accutase and dextransulphate (Amersham Biosciences, Uppsala, Sweden), while papain failed to enhance carrier to carrier transfer in comparison to the non-treated reference. The maximum growth rate of cells on microcarriers with 2% dextransulphate in the medium was 0.25 +/- 0.02d(-1) and 0.27 +/- 0.03d(-1) for the CHO-MPS and CHO-K1, respectively. TheCHO-K1 grew best after detachment with trypsin (mu = 0.36 +/- 0.03d(-1)). This indicates, that one of the key parameters for carrier to carrier transfer is the uniform distribution of cells on the individual carriers during the initial phase. When this distribution can be improved, growth rate increases, resulting in a faster and more stable process.