The impact of microcarrier culture optimization on the glycosylation profile of a monoclonal antibody

Abstract

Microcarriers are widely used for the large-scale culture of attachment-dependent cells with increased cell densities and, ultimately, higher product yield. In these processes, the specific culture conditions can affect the quality of the product, which is closely related to its glycosylation pattern. Furthermore, the lack of studies in the area reinforces the need to better understand the effects of microcarrier culture in product glycosylation. Consequently, in this work, the glycosylation profile of a monoclonal antibody (mAb) produced by adherent CHO-K1 cells grown in Cytodex 3 was evaluated under different conditions, and compared to that obtained of typical adherent cultures. It was found that microcarrier cultures result in a glycosylation profile with different characteristics from T-flask cultures, with a general increase in galactosylation and decrease in fucosylation levels, both with a potentially positive impact on mAb activity. Sialylation also varied but without a general tendency. This study then showed that the specific culture conditions used in microcarrier culture influence the mAb glycan profile, and each functional element (galactose, core fucose, sialic acid) is independently affected by these conditions. In particular, great reductions of fucosylation (from 79 to 55%) were obtained when using half volume at inoculation, and notable decreases in sialylation (from 23 to 2%) and glycoform heterogeneity (from 20 to 11 glycoforms) were observed for shake flask culture, potentially associated with the improved cell densities achieved in these culture vessels.

Rapid Analysis of Cell Surface N-Glycosylation from Living Cells Using Mass Spectrometry

“…Furthermore, earlier studies revealed that L-selectin and human protein C expressed in HEK 293 cells contained the GalNAc motif as well 24 25 , suggesting that the HEK 293 cell line is able to produce GalNAc epitopes on glycoproteins. 17 The core-fucosylation and traces of the antigenic Neu5Gc found in this study in the N-glycome of CHO-K1 cells are in accordance with the literature published on the N-glycome of recombinant glycoproteins expressed in CHO-K1 cells 26,27 , namely anti-HER2 monoclonal antibodies 28 , human plasminogen activator 29 , human alpha-1-antitrypsin (A1AT) 30 and human erythropoietin 31 .…”

Chondrogenic differentiation of human adipose-derived stem cells using microcarrier and bioreactor combination technique

“…Microcarriers are widely used for the large-scale culture of attachment-dependent cells (12,13), as they possess an increased surface area for cell adhesion, and thus, numerous seed cells can be harvested in a short time period (14). The microcarrier dynamic culture is the combination of using microcarriers with a bioreactor, and has been widely used in the field of tissue engineering.…”

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…A review reported that commercially available Mabs showed that GI from CHO-derived Mabs is lower than that in murine cell derived Mabs (Raju and Jordan, 2012). There are several reports of non-commercialized Mabs from CHO cells with high GI values around 4.5 (Costa et al, 2013), although the intrinsic difference between cell lines remains unclear. For Mab production, no evidence showed a relationship between extracellular galactosidase and external galactose content (Gramer et al, 2011;Quellhorst et al, 1999).…”