Effects of buffering conditions and culture pH on production rates and glycosylation of clinical phase I anti‐melanoma mouse IgG3 monoclonal antibody R24

Publisher Website
Google Scholar
Abstract
R24, a mouse IgG3 monoclonal antibody (MAb) against ganglioside GD3 (Neu5Acα8Neu5Acα3Gal β4Glcβ1Cer), can block tumor growth as reported in a series of clinical trials in patients with metastatic melanoma. The IgG molecule basically contains an asparagine-linked biantennary complex type oligosaccharide on the CH2 domain of each heavy chain, which is necessary for its in vivo effector function. The purpose of this study was to investigate the biotechnological production and particularly the glycosylation of this clinically important MAb in CO2/HCO3− (pH 7.4, 7.2, and 6.9) and HEPES buffered serum-free medium. Growth, metabolism, and IgG production of hybridoma cells (ATCC HB-8445) were analyzed on a 2-L bioreactor scale using fed-batch mode. Specific growth rates (μ) and MAb production rates (qIgG) varied significantly with maximum product yields at pH 6.9 (qIgG = 42.9 μg 10−6 cells d−1, μ = 0.30 d−1) and lowest yields in pH 7.4 adjusted batches (qIgG = 10.8 μg 10−6 cells d−1, μ = 0.40 d−1). N-glycans were structurally characterized by high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), and electrospray-ionization quadrupole time-of-flight (ESI-QTOF) mass spectrometry (MS). The highest relative amounts of agalacto and monogalacto biantennary complex type oligosaccharides were detected in the pH 7.2 (46% and 38%, respectively) and pH 6.9 (44% and 40%, respectively) cultivations and the uppermost quantities of digalacto (fully galactosylated) structures in the pH 7.4 (32%) and the HEPES (26%) buffered fermentation. In the experiments with HEPES buffering, antibodies with a molar Neu5Ac/Neu5Gc ratio of 3.067 were obtained. The fermentations at pH 7.2 and 6.9 resulted in almost equal molar Neu5Ac/Neu5Gc ratios of 1.008 and 0.985, respectively, while the alkaline shift caused a moderate overexpression of Neu5Ac deduced from the Neu5Ac/Neu5Gc quotient of 1.411. Different culture buffering gave rise to altered glycosylation pattern of the MAb R24. Consequently, a detailed molecular characterization of MAb glycosylation is generally recommended as a part of the development of MAbs for targeted in vivo immunotherapy to assure biochemical consistency of product lots and oligosaccharide-dependent biological activity. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 83: 321–334, 2003.
Funding Information
  • the DFG Graduate Program “Fundamentals in Cellular Biotechnical Processes”
  • the DFG Special Collaborative Program 549 “Macromolecular Processing and Signalling in the Extracellular Matrix”
  • HbfG Grant for the ESI-QTOF mass spectrometer (Micromass, Manchester, UK) at the University of Münster