ABSTRACT
A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran(®)-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.
ABSTRACT
A high-performance liquid chromatographic method has been developed to determine cefpodoxime levels in chinchilla plasma and middle ear fluid (MEF) to be used in studying otitis media. Cefpodoxime and the internal standard, cefuroxime, were separated on an ODS column (250 x 2.1 mm I.D., 5 microns Hypersil), using a mobile phase of 25 mM acetate buffer (pH 4.3)/15 mM triethylamine-acetonitrile (92.5:7.5, v/v). Following elution of cefpodoxime and the internal standard, at 3.5 and 5.9 min respectively, the acetonitrile concentration was increased to 1:1 (v/v) in a step function to elute endogenous compounds retained on the column. Sample preparation involved protein precipitation with acetonitrile. This fast, efficient protein precipitation procedure together with UV detection allows a quantitation limit of 50 ng/ml with a 50-microliters sample size. Recoveries (mean +/- S.D., n = 3) at 0.1 microgram/ml in MEF were 90.3 +/- 2.9% and 88.6 +/- 1.2% for cefpodoxime and cefuroxime respectively. Recoveries (mean +/- S.D., n = 3) at 0.1 microgram/ml in plasma were 72.1 +/- 7.3% and 81.1 +/- 1.1% for cefpodoxime and cefuroxime respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering cefpodoxime proxetil.
