Roland Wagner

Improved ion-pair high-performance liquid chromatographic method for the quantification of a wide variety of nucleotides and sugar—nucleotides in animal cells

An improved method including extraction procedures is presented for the analysis of nucleotides in suspension-cultivated animal cells. Quantification was performed by ion-pair high-performance liquid chromatography after perchloric acid extraction. It was found that the amount of perchloric acid taken for extraction influenced the yield and that cell washing procedures caused deterioration of the analysis results for triphosphates. More than thirty nucleotides and sugar-nucleotides were separated within 25 min using a Supelcosil reversed-phase column (3 microns) with tetrabutylammonium hydrogensulphate as pairing agent and methanol-pH gradient elution. Cultivated hybridoma cells showed variations in intracellular nucleotide concentrations as well as relative amounts during different growth phases, which could reflect the physiological state of a cell culture.

Production of recombinant human interleukin-2 with BHK cells in a hollow fibre and a stirred tank reactor with protein-free medium

For the production of recombinant human interleukin-2 (IL-2) two different culture processes, a 1-2 liter homogeneous stirred bubble-free aerated system and a dense cell hollow fibre bioreactor were compared. Cultivations were carried out with serum- or protein-free medium formulations. In the stirred culture 0.75 mg IL-2 were produced with 1 l of perfused medium at a maximum cell number of 3 X 10(10). The product yield in the hollow fibre module was only 0.23 mg l-1 at a maximum cell number of 6 X 10(10). In contrast to results with hybridoma or EBV-transformed cell lines, in which hollow fibre bioreactors showed comparable efficiency to perfused stirred tank reactors, the tissue-like cell density is disadvantageous as adherent cells tend to stick together leaving insufficient intercellular space for removal of product.

CONDITIONS FOR THE PRODUCTION OF RECOMBINANT IL-2 IN STIRRED SUSPENSION CULTURE USING A PROTEIN FREE MEDIUM

ABSTRACT A suspension culture in a homogeneous stirred bubble free aerated system with continuous medium exchange was established for the cell line BHK 21 pSVIL2. High cell densities of up to 3·107 ml–1 were reached. The production of IL-2, which did not exceed 1 mg d–1 in previous microcarrier processes based on 1-l-bioreactors, was increased to 4 mg d–1. Using a cell specific supplementation of amino acids IL-2 production increased to 6 mg d–1 at high cell densities for 4 weeks of cultivation. Compared with other previous serum-free medium formulations cultivation with the protein-free medium had the lowest costs and also lowered the burden on downstream processing. Data are shown concerning the further optimization of the production process going from a microcarrier to a suspension culture.

ACTIVATION OF A SPECIFIC PROTEOLYTIC ACTIVITY IN SUSPENSION CULTURES OF RECOMBINANT ADHERENT CELLS

ABSTRACT During comparison of microcarrier and suspension cultures with recombinant BHK cells a specific proteolytic activity appeared only when the cells grew in suspension. The protease was detected by its specificity in cleaving the recombinant product into smaller peptides. Approximately one-third of the total amount of the product was inactivated as estimated by Western-blot analysis. No proteolytic activity was recognized when using an anchorage dependent state of growth which was investigated by several fermentations with different microcarrier types. Key words : proteases, suspension culture, microcarrier culture, recombinant proteins, interleukin 2

INTRACELLULAR CONCENTRATION OF ATP AND OTHER NUCLEOTIDES DURING CONTINUOUS CULTIVATION OF HYBRIDOMA CELLS

Abstract A murine hybridoma cell line, which produces a monoclonal murine IgG 2a antibody, was cultivated in a stirred reactor equipped with a bubble-free aeration and a continuous perfusion system. During growth of up to 3.1·10 7 viable cells per ml, intracellular amounts of ATP, ADP, AMP, NAD, GTP, UTP and CTP were measured by ion-pair HPLC technique after perchloric acid extraction of sedimented cells. We found very stable values for adenylate energy charge, whereas the ratio of trinucleotides of the purine pool to these of the pyrimidine pool changed rapidly during different growth phases. Keywords: Adenylate energy charge; nucleotide triphosphates; purine-pyrimidine ratio; growth cycle; ion-pair HPLC

CHARACTERIZATION OF PROTEASE ACTIVITY IN SERUM-FREE CULTURE SUPERNATANTS OF HYBRIDOMAS AND RECOMBINANT MAMMALIAN CELLS

ABSTRACT [3H]-labelled Casein was used for a quantitative determination of protease activity in cell culture supernatants during long-term cultivation of BHK and hybridoma cells. They produced recombinant human interleukin 2 an IgG2a-antibody respectively in serum- or protein free medium. Protease activities were characterized by inhibitor studies and specific p-NA derivates. Only 20% of the total protease activity in hybridoma cells and up to 50% in BHK cells is based om serine type proteases.

COMPARISON OF THE PRODUCTION EFFICIENCY OF MAMMALIAN CELLS GROWN IN A FLUIDIZED BED AND IN A STIRRED TANK BIOREACTOR

Abstract A perfused fluidized bed bioreactor packed with porous glass spheres and a stirred tank perfusion bioreactor equipped with a double membrane stirrer for bubble-free aeration and medium perfusion were used for the cultivation of a murine hybridoma cell line which produces a monoclonal murine IgG 2a antibody (MAb). The production efficiency of both systems was compared. A productivity per unit reactor volume of 159.7 mg/d/l and 42.8 mg/d/l was obtained with the fluidized bed bioreactor and the stirred tank bioreactor, respectively. The reported results show an approximately 4-fold increase in the MAb production rate attained with the perfused fluidized bed bioreactor based on porous glass spheres. Keywords: Fluidized bed bioreactor; stirred tank bioreactor;production efficiency; monoclonal antibody.

THE PRODUCTION OF RECOMBINANT HUMAN INTERLEUKIN-2 IN A FLUIDIZED BED BIOREACTOR

ABSTRACT A perfused fluidized bed bioreactor packed with SIRAN porous glass spheres was used for the cultivation of an anchorage dependent recombinant BHK cell line producing human interleukin-2, constitutively. Cultivations were carried out with different medium formulations over 80 days. Maximal cell densities and product yield were maintained even when protein-free medium was perfused, as a result less than 10% cell wash out. Final cell densities of the order of 3.8·10 8 ml -1 intrasphere volume were attained while the interleukin-2 production rate was 0.45 mg d -1 . The production rate showed a 1.9-fold decrease compared with an homogeneous stirred bubble-free aerated system. This result was in contrast to that achieved with hybridoma cell lines, where better performances with respect to productivity were obtained with the fluidized bed bioreactor. Key words : Interleukin-2, protein-free medium, porous glass fluidized bed bioreactor, double membrane stirrer bioreactor.