Fucose content of monoclonal antibodies can be controlled by culture medium osmolality for high antibody-dependent cellular cytotoxicity

Abstract

Antibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of oligosaccharides bound to monoclonal antibodies (MAbs). As MAbs with a low fucose content exhibit high ADCC activity, it is important to control the defucosylation levels (deFuc%) of MAbs and to analyze the factors that affect deFuc%. In this study, we observed that the deFuc% was inversely related to culture medium osmolality for MAbs produced in the rat hybridoma cell line YB2/0, with r (2) values as high as 0.92. Moreover, deFuc% exhibited the same correlation irrespective of the type of compound used for regulating osmolality (NaCl, KCl, fucose, fructose, creatine, or mannitol) at a culture scale ranging from 1 to 400 L. We succeeded in controlling MAb deFuc% by maintaining a constant medium osmolality in both perfusion and fed-batch cultures. In agreement with these observations, reverse transcription PCR analyses revealed decreased transcription of genes involved in glycolysis, GDP-fucose supply, and fucose transfer under hypoosmotic conditions.

Fig. 1

Dependence of MAb defucosylation levels (deFuc%) produced by YB2/0 cells on initial medium osmolality. The medium was diluted with distilled water to attain the indicated osmolality at the beginning of the culture. Fed-batch method was used in a 5-L bioreactor. Independent experiments under similar conditions exhibited similar results in 1-L bioreactors

Fig. 2

Dependence of defucosylation levels (deFuc%) of IgG1 MAbs produced from NS0 (a) and SP2/0 (b) cells on initial medium osmolality. Two independent experiments under similar conditions exhibited similar results

Fig. 3

Defucosylation levels (deFuc%) of MAbs produced by YB2/0 cells cultured under various osmolalities. Cells were cultured by a perfusion method that enabled arbitrary changes to be made to the medium osmolality. aOpen circles observed medium osmolality, closed diamonds osmolality of feeding medium, closed squares observed deFuc%. b Reactor cell viability (open triangles). c Reactor viable cell density (closed circles). Reproducibility is ensured because several conditions were adopted, although reproducibility is just one aspect of perfusion culture

Fig. 4

Relationship between defucosylation levels (deFuc%) of MAbs produced and medium osmolalities attained by supplementing with different compounds: closed squares NaCl, closed triangles KCl, closed circles fucose, open squares mannitol, open triangles fructose, open circles creatine

Fig. 5

Relationship between defucosylation levels (deFuc%) and initial medium osmolalities analyzed under four culture scale conditions. MAbs were produced under 78 different physical conditions: four reactor sizes (1, 5, 30, and 400 L) each with different blades and aspect ratios (L/D), as well as different osmolalities

Fig. 6

RT-PCR analysis of cultured YB2/0 cells in 80-L pre-culture and 400-L main fed-batch culture in bioreactors. FUT8 and GMD transcript amounts relative to β-actin, calculated from the fluorescence intensity of each band measured by FluoroImager SI, are shown below the RT-PCR panels. Two independent experiments under similar conditions exhibited similar results

Fig. 7

Defucosylation levels (deFuc%) of MAbs produced with different methods to regulate medium osmolality. Open symbols hyperosmotic, cross symbols hypoosmotic, closed symbols constant osmotic conditions. Cells were cultured in a 5-L fed-batch bioreactor with custom-made medium containing low NaCl. Osmolality was adjusted by supplementing with NaCl. a glucose concentration in the medium, b medium osmolality, c deFuc% MAbs harvested on day 11, and d viable cell density. Independent experiments under similar conditions exhibited similar initial results in 1-L bioreactors