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

Abstract

R24, a mouse IgG3 monoclonal antibody (MAb) against ganglioside GD3 (Neu5Acalpha8Neu5Acalpha3Gal beta4Glcbeta1Cer), 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 C(H)2 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 CO(2)/HCO(3) (-) (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 (mu) and MAb production rates (q(IgG)) varied significantly with maximum product yields at pH 6.9 (q(IgG) = 42.9 microg 10(-6) cells d(-1), mu = 0.30 d(-1)) and lowest yields in pH 7.4 adjusted batches (q(IgG) = 10.8 microg 10(-6) cells d(-1), mu = 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.

Obtención de anticuerpos monoclonales de ratón contra proteasa de cisteína 5 recombinante de Entamoeba histolytica

“…Para evitar la multiplicación de estos híbridos no secretores de anticuerpos, los cuales por ser metabólicamente más eficientes podría dominar el cultivo, inmediatamente después de evaluar los sobrenadantes de las colonias se procedió al proceso de clonación y reclonación. Este proceso además fue de gran importancia debido a que la población de hibridomas obtenida después de la fusión fue heterogénea con relación a los anticuerpos secretados por ella y se precisó seleccionar los híbridos productores del anticuerpo específico de interés, asegurando que las inmunoglobulinas secretadas por el cultivo fueran idénticas y así cerciorar la estabilidad y el carácter monoclonal del mismo lo que permitió al final de esta etapa seleccionar un único clon secretor (3G8C3) del anticuerpo específico contra EhCP5r (17,18,(30)(31)(32).…”

Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature?

“…In this context, an inverse relationship between cell proliferation rate and cell specific recombinant protein production has been reported in a variety of circumstances; the use of specific metabolites or nutrient limitations (Altamirano et al, 2001;Carvalhal et al, 2003), reduction in culture pH (Miller et al, 1988;Müthing et al, 2003), reduced culture temperature (Fox et al, 2004;Kaufmann et al, 1999;Yoon et al, 2003b), or hyperosmotic pressure (Kim and Lee, 2002b;Lee and Lee, 2000). However, as each of the above changes in the physico-chemical environment of cells may affect multiple cellular processes (metabolic or other) simultaneously, it is extremely difficult to evaluate the basis of the relationship between qP and m in every case.…”

Application of Quality by Design Paradigm to the Manufacture of Protein Therapeutics

“…pH causes GnTI, ManII, GalT and SiaT to mislocalize in Golgi [156] Viability, growth phase and temperature EPO/CHO:  branching at 32°C<T<37°C  temperature decreases UDPGlcNAc and UDP-GalNAc concentration [157] Serum and lipid supplements IFN /CHO:  branching with lipid supplementation…”