Galactose supplementation enhance sialylation of recombinant Fc-fusion protein in CHO cell: an insight into the role of galactosylation in sialylation

Abstract

Sialic acid levels of therapeutic glycoprotein play an important role in plasma half-life. An undesirable decrease of sialic acid content was observed when we increased Fc-fusion protein productivity fourfold in a GS-CHO cell line by bioprocess optimization. We investigated the potential mechanism for the sialic acid content reduction. We found that limited nucleotide sugar precursor and the extracellular sialidase were not responsible for the reduction of the sialic acid content after titer improvement. Oligosaccharide analysis revealed that the lack of protein galactosylation was the potential cause for the reduction of sialic acid content. Thus we validated this notion by evaluated galactose supplementation in 2 L bioreactors. Cell culture performance was not impacted by addition of up to 40 mM galactose except for the glucose consumption rate. Addition of 20 mM galactose to the bioreactor resulted in the increase of 44 % for total sialic acid content and 20.3 % for sialylated glycans. These data were further validated when the process was run on 200 L scaled bioreactor. These data together show that the galactosylation plays an apparent role in sialylation in our current system.

Supplementing glycosylation: A review of applying nucleotide-sugar precursors to growth medium to affect therapeutic recombinant protein glycoform distributions

“…This strategy has been reliably reported across several different research groups, utilizing different cell platforms, expressing different proteins, to increase galactosylation (Figure 4). Even beyond galactosylated glycoforms, supplementation of Gal has also been demonstrated to relieve bottlenecks towards improving the sialylation of Fc-fusion glycoproteins in GS-CHO cultures (Liu et al, 2015). Increased galactosylation is generally accepted to be of therapeutic benefit, with lack of galactosylation associated endogenously with several disease states (discussed in Section 4).…”

“…A N U S C R I P T (Gu and Wang, 1998). Interestingly, researchers that do not observe increases in sialylation typically reported a deficit of galactosylation that could be improved with galactose feeding (Hills et al, 2001;Kildegaard et al, 2015;Liu et al, 2015). Liu and colleagues (2015) supplementing galactose improved galactosylation in cultures, which subsequently also lead to increased sialylation by 14%.…”

Tailoring recombinant protein quality by rational media design

“…This finding was further confirmed in GS-CHO cell line expressing the human tumor necrosis factor receptor linked to the Fc portion of human IgG1. The addition of 20 mM of galactose in 2-L bench scale and 200-L pilot scale fed-batch cultures led to 20.3% increase of sialylated glycans 140 .…”

Process and metabolic engineering perspectives of lactate production in mammalian cell cultures

“…Substitution of glucose by galactose, for example, produced significantly less lactate but decreased specific cell growth rate and peak viable cell density [19]. To enhance cell biomass, a biphasic culture strategy (alternating the consumption of glucose and galactose) has been proposed in CHO cell batch and fed-batch cultures [20,21]. Using this strategy, high cell densities have been achieved with significantly decreased (up 66%) lactate accumulation and improved galactosylation in fed-batch cultures [20][21][22].…”

“…To enhance cell biomass, a biphasic culture strategy (alternating the consumption of glucose and galactose) has been proposed in CHO cell batch and fed-batch cultures [20,21]. Using this strategy, high cell densities have been achieved with significantly decreased (up 66%) lactate accumulation and improved galactosylation in fed-batch cultures [20][21][22]. Aternatives to glutamine (glutamate, pyruvate, TCA cycle intermediaries and dipeptides) have also been developed.…”