TY  - THES
AB  - Background: The IgG molecule basically contains an asparagine-linked bi-antennary complex-type oligosaccharide on the C H 2 domain of each heavy chain, which is necessary for effector functions of the antibody. R24, a mouse IgG3 monoclonal antibody (mab) against ganglioside GD3, can block tumor growth as reported in a series of clinical trials on patients with metastatic melanoma. The purpose of this study was to explore (i) the specific rates of antibody production and (ii) the oligosaccharide profiles of mab R24 produced in bioreactor batches using three CO2/HCO3 and a HEPES buffered system.
Methods: Mab R24 (ATCC, HB8445) was produced in CO2/HCO3 (pH 6.9, 7.2, and 7.4) and HEPES (pH 7.2) buffered serum-free medium in a bubble-free aerated 2L-bioreactor using the repeated batch modus. The specific mab production rate qR24 (µg/10^6 cells x d) was determined during logarithmic growth and the IgG3 mab was purified to homogeneity (as judged by SDS/PAGE under reducing conditions) by protein A chromatography. Sialic acids (Neu5Ac und Neu5Gc) were identified as their fluorescent DMB derivatives by RP18 column chromatography. Oligosaccharides were released by hydrazinolysis and PNGaseF digest and their structures were determined by HPAEC-PAD, MALDI-TOF und ESI-QTOF mass spectrometry.
Results: Specific production rates and glycosylation pattern of mab R24 varied significantly over the extracellular pH range of 6.9 to 7.4. Maximum product yields were obtained at pH 6.9 (42.9 µg/10^6 cells x d), whereas lowest yields were obtained in pH 7.4 adjusted batches (10.8 µg/10^6 cells x d). HPAEC-PAD profiles revealed agalacto-biantennary chains as the major oligosaccharide in all preparations (40-50 percent). Considerable different amounts of fully galactosylated carbohydrate chains were detected in pH 7.4 (26 percent), pH 7.2 and pH 6.9 (both 17 percent) and HEPES (14 percent) derived material. A 3:1 molar ratio of Neu5Ac to Neu5Gc was detected in HEPES derived mab, whereas all the other preparations revealed equimolar ratios of Neu5Ac and Neu5Gc. Thus, different culture pH leads to differences in specific production rates and glycosylation pattern of murine R24 IgG3 monoclonal antibody.
DA  - 2001
KW  - ,  , Glycoprotein , Oligosaccharides , Sialic acids , R24
LA  - ger
PY  - 2001
TI  - Auswirkungen der Kulturbedingungen auf die Produktion, Glykosylierung und Funktion des monoklonalen Maus-Antikörpers R 24
UR  - https://nbn-resolving.org/urn:nbn:de:hbz:361-4619
Y2  - 2024-11-21T16:20:23
ER  -