Glycosylation of an immunoglobulin produced from a murine hybridoma cell line: The effect of culture mode and the anti‐apoptotic gene, bcl‐2

Abstract

The impact of bcl-2 over-expression on the glycosylation pattern of an antibody produced by a bcl-2 transfected hybridoma cell line (TB/C3.bcl-2) was investigated in suspension batch, continuous and high cell density culture (Flat hollow fibre, Tecnomouse system). In all culture modes bcl-2 over-expression resulted in higher cell viability. Analysis of the glycans from the IgG of batch cultures showed that >95% of the structures were neutral core fucosylated asialo biantennary oligosaccharides with variable terminal galactosylation (G0f, G1f and G2f) consistent with previous analysis of glycans from the conserved site at Asn-297 of the IgG protein. The galactosylation index (GI) was determined as an indicator of the glycan profile (=(G2 + 0.5* G1)/(G0 + G1 + G2)). GI values in control cultures were comparable to bcl-2 cultures during exponential growth (0.53) but declined toward the end of the culture when there was a loss in cell viability. Low dilution rates in chemostat culture were associated with reduced galactosylation of the IgG glycans in both cell lines. However, at the higher dilution rates the GI for IgG was consistently higher in the TB/C3.bcl-2 cultures. In the hollow fibre bioreactor the galactosylation of the IgG glycans was considerably lower than in suspension batch or continuous cultures with GI values averaging 0.38. Similar low galactosylation values have been found previously for high density cell cultures and these are consistent with the low values obtained when the dissolved oxygen level is maintained at a low value (10%) in controlled suspension cultures of hybridomas.

Rapid whole monoclonal antibody analysis by mass spectrometry: An Ultra scale‐down study of the effect of harvesting by centrifugation on the post‐translational modification profile

“…The characterization of the post-translational modifications that occur on therapeutic proteins produced in mammalian cells has previously focused on the glycosylation profile because of their impact on efficacy (Hossler et al, 2009;Jefferis, 2009;Kawasaki et al, 2009), and to this end have usually involved digestion of the protein (Bailey et al, 2005;Bongers et al, 2000;Boyiadzis and Foon, 2008;Damen et al, 2009;Fernandez et al, 2001;Hooker et al, 1995;Korecka et al, 2004;Rousseaux et al, 1989;Schahs et al, 2007;Stigter et al, 2007) or cleavage of the glycan moiety (Fernandez et al, 2001;Jefferis, 2005;Kamoda et al, 2004;Majid et al, 2007;Mechref et al, 2005;Qian et al, 2007;Robinson et al, 1994;Yu et al, 2005) from the protein backbone or a combination of the two. To enable the simultaneous and rapid analysis of different PTMs, and to allow the investigation as to how processing conditions can affect this, LC-MS analysis of intact monoclonal antibodies was used in conjunction with USD bioprocessing mimics.…”

Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose

“…Differences in galactosylation have been observed when varying the production system (ascites, hollow fiber, stirred flask, etc.) or the medium type (serum, serum free, chemically defined) (Cabrera et al, 2004;Kumpel et al, 1994;Lund et al, 1993;Majid et al, 2007;Patel et al, 1992;Serrato et al, 2007). However, these factors were undesirable to evaluate in our platform.…”

A simultaneous assessment metric for MAb quantity and glycan quality

“…Similarly, various metrics do exist that assess the chemical reactions that occur during the glycosylation process. One method frequently employed to assess the progression of glycosylation is a series of independent glycosylation indices (Aghamohseni et al 2014;Ohadi et al 2013;Liu et al 2014;Majid et al 2007). Two common forms that measure the galactosylation and sialylation processes include the galactosylation index (GI) and sialylation index (SI):…”