Production of α2,6-sialylated IgG1 in CHO cells

Abstract

The presence of a2,6-sialic acids on the Fc N-glycan provides anti-inflammatory properties to the IgGs through a mechanism that remains unclear. Fc-sialylated IgGs are rare in humans as well as in industrial host cell lines such as Chinese hamster ovary (CHO) cells. Facilitated access to well-characterized a2,6-sialylated IgGs would help elucidate the mechanism of this intriguing IgG's effector function. This study presents a method for the efficient Fc glycan a2,6-sialylation of a wild-type and a F243A IgG1 mutant by transient co-expression with the human a2,6-sialyltransferase 1 (ST6) and b1,4-galactosyltransferase 1 (GT) in CHO cells. Overexpression of ST6 alone only had a moderate effect on the glycoprofiles, whereas GT alone greatly enhanced Fc-galactosylation, but not sialylation. Overexpression of both GT and ST6 was necessary to obtain a glycoprofile dominated by a2,6-sialylated glycans in both antibodies. The wild-type was composed of the G2FS(6)1 glycan (38%) with remaining unsialylated glycans, while the mutant glycoprofile was essentially composed of G2FS(6)1 (25%), G2FS(3,6)2 (16%) and G2FS(6,6)2 (37%). The a2,6-linked sialic acids represented over 85% of all sialic acids in both antibodies. We discuss how the limited sialylation level in the wild-type IgG1 expressed alone or with GT results from the glycan interaction with Fc's amino acid residues or from intrinsic galactosyl-and sialyl-transferases substrate specificities.

Preparation of legionaminic acid analogs of sialo-glycoconjugates by means of mammalian sialyltransferases

“…The codon-optimized (human codon bias) gene encoding the human ST6Gal1 protein (NP15907) intralumenal domain (aa 27-406) with a human VEGFa (P15692) signal peptide linked at its N-terminus and a C-terminal GHHHHHHHHHHG tag at its C-terminus was chemically synthesized by GenScript (Piscataway, NJ) and cloned into the pTT5 mammalian expression vector [12,13]. The secreted ST6Gal1 enzyme was expressed in CHO-EBNA1 (CHO-3E7) cells according to previously published protocols [14,15]. The clarified culture medium supernatant was harvested at 8 days post-transfection and the secreted ST6Gal1 was purified by immobilized metal-affinity chromatography (IMAC).…”

Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids

“…They found that 100 μM Ac 4 ManNAz was sufficient to produce the azidomAb as detected by fluorescence and not mass spectrometry [39]. By contrast, in the present study, CHO cells were transfected with double mutant TZMm2, GT, and human α-2, 6-sialyltransferase 1 which has previously demonstrated high yields of sialylation [42]. Each cell batch (A1-A6, B1-B6) was incubated with increasing amounts of Ac 4 ManNAz to determine the metabolic expression of SiaNAz.…”

“…This approach was further compared to the in vitro enzymatic addition of SiaNAz in terminal position using cytidine monophosphate (CMP)-SiaNAz and soluble β-galactosamide α-2,6-sialyltransferase 1. The highlight of this report is therefore to evaluate how much SiaNAz can be metabolically incorporated onto mAbs produced under conditions already known to express high sialylation levels with ManNAc [41,42]. In parallel, a study was conducted on the production of lightly sialylated EG2-hFc mAb [44,45] in CHO cells with increasing concentrations of Ac 4 ManNAz.…”

“…This mAb has been engineered with two important amino acid substitutions (F241A and F243A). These substitutions have previously been shown to improve terminal galactosylation and sialylation [41][42][43]. This approach was further compared to the in vitro enzymatic addition of SiaNAz in terminal position using cytidine monophosphate (CMP)-SiaNAz and soluble β-galactosamide α-2,6-sialyltransferase 1.…”

“…Lastly, this mAb was purified on a protein A resin and stored in H 2 O prior to digestion and analysis. Over the course of this production, aliquots of the sample were analyzed for the presence of sialylation using SNA and ECL biotinylated lectins following the manufacturers' protocols [51] which has previously been discussed [42].…”