TY - JOUR
AU - Rosenlöcher, Julia
AB - Abstract N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient's bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4 and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties. asialoglycoprotein receptor, coagulation factor VII, N-acetylgalactosamine, mannose receptor, sialylation Introduction Between 2015 and 2018, around 80% of marketed biopharmaceuticals derived from mammalian cells (Walsh, 2018). These products often require mammalian posttranslational modifications (PTMs) for adequate protein folding, stability, biological activity and bioavailability as well as for reduced immunogenicity (Walsh et al., 1990; Leyte et al., 1991; Galili, 2005; Arnold et al., 2007; Padler-Karavani et al., 2008; Seested et al., 2010; Mi et al., 2014). Today, most biopharmaceuticals are produced in Chinese hamster ovary (CHO), baby hamster kidney (BHK) or the murine myeloma cell lines NS0 and Sp2/0 (Estes and Melville, 2014). Although PTMs from these rodent sources are often sufficiently human-like for clinical use, they can differ from those found on plasma or human cell line-derived proteins (Berkner, 1993; Grancha et al., 2011; Kannicht et al., 2013) and confer adverse properties such as immunogenicity (Tangvoranuntakul et al., 2003; Padler-Karavani et al., 2008; Ghaderi et al., 2010; Berg et al., 2014) and enhanced clearance (Ghaderi et al., 2010) linked to nonhuman N-glycan structures including galactose-α1,3-galactose (α-Gal) and N-glycolylneuraminic acid (Neu5Gc) (Déglon et al., 2003; Chung et al., 2008; Diaz et al., 2009; Bosques et al., 2010). Moreover, CHO and BHK cells also lack machinery for producing certain human N-glycan structures like bisecting N-acetylglucosamine (bis-GlcNAc) or α2,6-linked sialic acids (Grabenhorst et al., 1999; Xu et al., 2011). Certain N-glycan structures are efficient ligands for abundant clearance receptors of the liver, and they can thus impair the pharmacokinetics of a therapeutic protein. For example, terminal galactose (Gal) and especially terminal N-acetylgalactosamine (GalNAc) trigger rapid clearance via the asialoglycoprotein receptor (ASGP-R; ASGR1 and ASGR2) (Baenziger and Fiete, 1980; Baenziger and Maynard, 1980) whereas terminal sialylation reduces ASGP-R binding (Seested et al., 2010; Mi et al., 2014). Similarly, terminal mannose, fucose, N-acetylglucosamine (GlcNAc) (Shepherd et al., 1981; Largent et al., 1984; Dong et al., 1999; Lee et al., 2002) and sulfated GalNAc (Leteux et al., 2000; Lee et al., 2002) all bind the mannose receptor (MR; MRC1), leading to rapid clearance of the protein (Lee et al., 2002). Every N-glycan structure results from an interplay of enzymes—mainly glycosyltransferases and glycosidases. Typically, antennary GlcNAc gets capped by Gal, followed by sialic acid. However, each of the two enzymes beta-1,4-N-acetylgalactosaminyltransferase 3 (B4GALNT3) or beta-1,4-N-acetylgalactosaminyltransferase 4 (B4GALNT4) can instead cap GlcNAc with GalNAc if a peptide recognition motif is present on the protein (Miller et al., 2008; Fiete et al., 2012a, 2012b). GalNAc may then get capped by an α2,6-linked sialic acid via ST6 beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1) (Stockell Hartree and Renwick, 1992; Dell et al., 1995), or may be sulfated by either N-acetylgalactosamine-specific carbohydrate sulfotransferase 8 (CHST8) or 9 (CHST9) (Hiraoka et al., 2001). Additionally, the subterminal GlcNAc of either Galβ4GlcNAc or GalNAcβ4GlcNAc may get fucosylated by α1,3/4-fucosyltransferases (Dell et al., 1995; Vries et al., 1995). The human embryonic kidney 293 (HEK 293) cell line is an efficient and widely used protein expression platform that is easy to transfect both transiently and stably; that grows and produces in serum-free suspension culture; and that is the source of several market-approved biopharmaceuticals (Chapple et al., 2006; Butler and Spearman, 2014; Swiech et al., 2015; Dumont et al., 2016). The FreeStyle 293-F cell line (HEK 293-F) is a clonal isolate adapted to high-density growth in animal-origin-free suspension culture and is therefore generally well-suited for large-scale biopharmaceutical production. However, GalNAc or sulfated GalNAc was found on several HEK 293 cell-derived glycoproteins, i.e. on coagulation factor VII (FVII) (Böhm et al., 2015), low-density lipoprotein receptor homolog SorLA/LR11 (Fiete et al., 2007), protein C (Yan et al., 1993), L-selectin (Wedepohl et al., 2010) and tissue factor pathway inhibitor (Smith et al., 1992). In addition, compared to BHK cell, CHO cell, or human plasma origin, sialylation was also lower in HEK 293 cell-derived FVII (Böhm et al., 2015), alpha(1)-proteinase inhibitor (Goh and Ng, 2018), erythropoietin (Croset et al., 2012), protein C (Yan et al., 1993) and cystatin F (Croset et al., 2012). This currently limits the use of HEK 293 cells in biopharmaceutical production, since these structures may adversely affect the pharmacokinetic properties of the glycoprotein. To reduce N-glycan GalNAc and increase sialylation in the HEK 293-F cell line, we first knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 and then knocked-in either sialyltransferase ST6GAL1 or ST3 beta-galactoside alpha-2,3-sialyltransferase 6 (ST3GAL6) by CRISPR/Cas9. We chose FVII to assess the effects on N-glycosylation because FVII from unmodified HEK 293 cells carries ample terminal GalNAc and little sialylation (Böhm et al., 2015). However, since FVII has a very short in vivo half-life of only ~2.5 h (Lindley et al., 1994; Kubisz and Stasko, 2004), which limits clinical use in various hematologic conditions, we here expressed the therapeutically interesting FVII-albumin fusion protein (FVII-alb) whose half-life is substantially prolonged (Weimer et al., 2008; Golor et al., 2013; Herzog et al., 2014). We found that double knock-out of B4GALNT3 and B4GALNT4 eliminated both GalNAc and sulfated GalNAc, reduced antenna fucosylation, increased antennarity as well as bis-GlcNAc and improved sialylation. Additional knock-in of ST6GAL1 or ST3GAL6 further increased sialylation and in the case of ST6GAL1 to a level reported for plasma-derived FVII and beyond those reported for CHO, BHK or HEK 293 cell-derived FVII (Böhm et al., 2015). Both knock-ins also further reduced fucosylation. Glyco-engineered FVII-alb showed reduced in vitro ASGP-R as well as MR binding and improved in vivo pharmacokinetics. Results N-glycosylation analysis of glyco-engineered FVII-alb We expressed FVII-alb in eight HEK 293-F cell lines: Wild-type (wt) HEK 293-F; knock-out (KO) of B4GALNT3 (NT3-KO), B4GALNT4 (NT4-KO) and B4GALNT3 as well as B4GALNT4 (NT3/4-KO); knock-in (KI) of ST6GAL1 or ST3GAL6 in the wild-type and NT3/4-KO cells (wt + ST6GAL1-KI; wt + ST3GAL6-KI; NT3/4-KO + ST6GAL1-KI; NT3/4-KO + ST3GAL6-KI). Furthermore, we expressed FVII without the albumin fusion in wild-type cells. FVII and NovoSeven®, an activated FVII produced in BHK cells, were analyzed for comparison. To characterize N-glycosylation of the FVII-alb and FVII variants, we separated and identified the different N-glycan species by LC–MS and analyzed composition and content of monosaccharides and sialic acids orthogonally by ultra high performance liquid chromatography (UHPLC) (Exemplary HILIC chromatograms with N-glycan peak annotation see Supplementary Figure S1, Supplementary Tables S1 and S2). These approaches—each with relative standard deviations of less than 5% (Supplementary Figures S2 and S3)—concurringly revealed that the NT3/4-KO completely eliminated GalNAc on FVII-alb (Figure 1A). While both NT3-KO and NT4-KO individually reduced the level of GalNAc (NT3-KO by 74%; NT4-KO by 12%), neither alone sufficed for complete elimination. Interestingly, all GalNAc declines were accompanied by proportional increases in Gal (Figure 1B), which suggests that engineered cells replaced GalNAc with Gal. Knock-in of ST6GAL1 or ST3GAL6 did not affect GalNAc or Gal levels. Fig. 1 Open in new tabDownload slide N-linked GalNAc (A) and galactose (B) units per molecule of FVII and FVII-alb. For monosaccharide analysis, all groups N = 3 technical replicates, except NovoSeven® N = 2. Fig. 1 Open in new tabDownload slide N-linked GalNAc (A) and galactose (B) units per molecule of FVII and FVII-alb. For monosaccharide analysis, all groups N = 3 technical replicates, except NovoSeven® N = 2. Along with Gal levels, the NT3-KO and NT3/4-KO also increased sialylation ~ 2.5-fold whereas the NT4-KO had no such effect (Figure 2A). Additional knock-in of ST3GAL6 or ST6GAL1 on the double KO background further increased sialylation to ~ 3.3 or ~ 4.5 fold above wild-type, respectively—i.e. to levels similar or beyond those for BHK cell-derived NovoSeven®. Of note, the ~3.8 mol sialic acid per mol FVII-alb from the NT3/4-KO + ST6GAL1-KI cell line suggest that sialylation is almost complete because FVII-alb contains two N-glycosylation sites—Asparagine 145 and 322 of FVII—which predominantly carry biantennary N-glycans. Both knock-ins also increased sialylation on the wild-type background but there considerably less so. In general, ST6GAL1 was more efficient than ST3GAL6. In wt HEK 293-F and NT4-KO cells, 35% of sialic acids on biantennary N-glycans were α2,3-linked (Figure 2B). Simultaneous KO of B4GALNT3 and B4GALNT4 or KI of ST3GAL6 yielded 48% α2,3-linked sialic acids and the combined KO/KI yielded 63%. In contrast, knock-in of ST6GAL1 in wild-type and NT3/4-KO cells lead to almost exclusive α2,6-linked sialic acids. All sialic acids found on HEK 293-F cell-derived proteins were N-acetylneuraminic acid (Neu5Ac) while 2.7% nonhuman Neu5Gc was detected on NovoSeven®. Fig. 2 Open in new tabDownload slide (A) Sialic acid units per molecule FVII and FVII-alb. For sialic acid analysis, all groups N = 3 technical replicates. (B) Percentage of α2,3 and α2,6-linked sialic acids on biantennary N-glycans in the N-glycan profiling. Fig. 2 Open in new tabDownload slide (A) Sialic acid units per molecule FVII and FVII-alb. For sialic acid analysis, all groups N = 3 technical replicates. (B) Percentage of α2,3 and α2,6-linked sialic acids on biantennary N-glycans in the N-glycan profiling. We also found that 21% of GalNAc on FVII-alb from wt HEK 293-F cells was either sialylated (12%) or sulfated (9%) and that neither the NT3-KO nor ST3GAL6-KI changed this considerably (Figure 3A). However, the NT4-KO and ST6GAL1-KI halved sulfation to 5% and 4%, and the KI additionally increased GalNAc sialylation to 65%. All GalNAc on NovoSeven® was unmodified. Fig. 3 Open in new tabDownload slide (A) Percentage of GalNAc modified with α2,6-linked sialic acid or 4-linked sulfate in the N-glycan profiling. (B) N-linked fucose units per molecule of FVII and FVII-alb in the N-glycan profiling. Fucose units located on the N-glycan core and the antenna are indicated. Fig. 3 Open in new tabDownload slide (A) Percentage of GalNAc modified with α2,6-linked sialic acid or 4-linked sulfate in the N-glycan profiling. (B) N-linked fucose units per molecule of FVII and FVII-alb in the N-glycan profiling. Fucose units located on the N-glycan core and the antenna are indicated. We next examined antenna fucosylation because it can lead to clearance by members of the MR family. As substantially all N-glycans were core fucosylated (Supplementary Tables S1 and S2), we considered all fucose beyond the 2 mol/mol threshold as antennary. As shown in Figure 3B, both FVII and FVII-alb from wt HEK 293-F cells carried on average nearly two antennary fucoses. This decreased to well below one antennary fucose per FVII-alb molecule from NT3-KO, NT3/4-KO and ST6GAL1-KI cells. The combined NT3/4-KO + ST6GAL1-KI synergistically almost fully abrogated antenna fucosylation. The ST3GAL6-KI also profoundly reduced antennary fucose of FVII-alb from NT3/4-KO but not wild-type cells. Some of our glyco-engineering also increased triantennarity and the incorporation of bis-GlcNAc, both of which are almost absent in FVII and FVII-alb from wt HEK 293-F cells. B4GALNT3 appeared to play a key role as the NT3-KO raised bis-GlcNAc to 26% and triantennarity to 10% (Figure 4). The additional knock-out of B4GALNT4 raised both levels to 38% and 16% although the NT4-KO alone did not have any notable effect. And a further increase yet—to 56% bis-GlcNAc and 18% triantennarity—resulted from the knock-in of ST3GAL6 (but not ST6GAL1) on the NT3/4-KO background. Fig. 4 Open in new tabDownload slide Percentage of bis-GlcNAc containing (A) and triantennary (B) N-glycans in the N-glycan profiling. Fig. 4 Open in new tabDownload slide Percentage of bis-GlcNAc containing (A) and triantennary (B) N-glycans in the N-glycan profiling. Overall, our analyses suggested that FVII and FVII-alb from wt HEK293-F cells carry substantially identical N-glycans, and that our glyco-engineering may considerably reduce binding to clearance receptors—to the ASGP-R by replacing GalNAc with Gal, increasing sialylation and changing sialic acid linkage-type distribution and to the MR by reducing antenna fucosylation and GalNAc sulfation. Binding of glyco-engineered FVII-alb to ASGP-R and MR We assessed ASGP-R and MR binding by surface plasmon resonance (SPR) using methods with relative standard deviations of less than 10% (Supplementary Figure S4). In accordance with its much higher GalNAc and antennary fucose levels, FVII from wt HEK 293-F cells bound both clearance receptors much more readily than NovoSeven® (Figure 5). Strikingly, however, the albumin fusion itself dramatically reduced FVII binding in both cases, and our glyco-engineering of FVII-alb reduced it further. Fig. 5 Open in new tabDownload slide Binding of FVII and FVII-alb to ASGP-R (A) and MR (B) relative to NovoSeven®. All groups N = 3 technical replicates measured on three independent immobilizations. The nonglycosylated human serum albumin negative control did not bind either receptor. Fig. 5 Open in new tabDownload slide Binding of FVII and FVII-alb to ASGP-R (A) and MR (B) relative to NovoSeven®. All groups N = 3 technical replicates measured on three independent immobilizations. The nonglycosylated human serum albumin negative control did not bind either receptor. Binding to the ASGP-R dropped through both ST6GAL1-KI and NT3-KO, and although the NT3/4-KO did not reduce binding further, its combination with a ST6GAL1-KI yielded synergy effects that almost completely abrogated binding (Figure 5A). We found the same near-complete loss of ASGP-R binding with the ST3GAL6-KI on the NT3/4-KO background although the ST3GAL6-KI alone had no notable effect. MR binding was less affected but decreased slightly after both sialyltransferase knock-ins and after NT3-KO as well as NT3/4-KOs. But there was no considerable synergy effect from any KO/KI combination (Figure 5B). It should be noted that FVII-alb from the NT4-KO cell line was purified on an ÄKTA instead of a Tecan system, which was used for all other proteins. The different purification did not considerably affect N-glycan profiling results of FVII-alb from wt HEK 293-F cells (Supplementary Figure S5A). However, we attributed the increase in ASGP-R and MR binding of FVII-alb from the NT4-KO compared to the wt HEK 293-F cell line—in the absence of any meaningful difference in N-glycosylation—to this difference. When purified by the same method, FVII-alb from both wt HEK 293-F and NT4-KO cells bound ASGP-R and MR identically (Supplementary Figure S5B). To determine whether reduced clearance receptor binding in vitro results in lower clearance in vivo, we next assessed the pharmacokinetics of the FVII-alb variants in rat. Pharmacokinetics of glyco-engineered FVII-alb We intravenously injected rats with 270 μg/kg NovoSeven® or activated FVII (FVIIa) from wt HEK 293-F cells, or with 650 μg/kg activated FVII-alb (FVIIa-alb) from wt HEK 293-F, NT3-KO, or NT3/4-KO cells (all doses equimolar). Compared to NovoSeven®, plasma recovery of FVIIa from wt HEK 293-F cells was extremely poor at only 10% 5 min after injection (Figure 6A and B). While albumin fusion itself rescued recovery, the NT3-KO and NT3/4-KO substantially improved FVIIa-alb recovery further. Overall, this corroborated our in vitro ASGP-R and MR binding data, although albumin fusion alone had a smaller effect in vivo. Fig. 6 Open in new tabDownload slide (A) FVIIa concentrations over time in rat plasma (four animals sampled per time point; logarithmic scale). For better comparability, only the molecular weight-adjusted FVIIa fraction of FVIIa-alb was plotted. Recovery (B), terminal half-life (C) and AUC (D) of FVIIa and FVIIa-alb variants were calculated from the plasma concentration curves using noncompartmental analysis. Fig. 6 Open in new tabDownload slide (A) FVIIa concentrations over time in rat plasma (four animals sampled per time point; logarithmic scale). For better comparability, only the molecular weight-adjusted FVIIa fraction of FVIIa-alb was plotted. Recovery (B), terminal half-life (C) and AUC (D) of FVIIa and FVIIa-alb variants were calculated from the plasma concentration curves using noncompartmental analysis. Albumin fusion also extended FVIIa terminal half-life compared to NovoSeven®, specifically ~2.2-fold for FVIIa-alb from wt HEK 293-F cells. In addition, the increase was ~4-fold for both glyco-engineered FVIIa-alb variants which further demonstrates reduced clearance even over a course of several hours (Figure 6C). Poor recovery of FVIIa from wt HEK 293-F cells precluded meaningful half-life analysis for this protein. Recovery and half-life together affect plasma levels over time as represented by the area under the curve (AUC). While AUC increased ~1.8-fold by albumin fusion alone (FVIIa-alb from wt HEK 293-F cells compared to NovoSeven®), additional KO of NT3 and NT3/4 increased AUC ~4.9-fold and ~5.4-fold, respectively (Figure 6D). Taken together, these data confirmed that our glyco-engineering improves FVII-alb pharmacokinetics. Discussion N-glycosylation analysis of glyco-engineered FVII-alb We improved the utility of the HEK 293-F cell line as an expression system for therapeutic proteins by engineering its N-glycosylation machinery at the genomic level; specifically by combining GalNAc transferase knock-outs with a sialyltransferase knock-in. Complete elimination of GalNAc on N-glycans required a double knock-out of B4GALNT3 and B4GALNT4, while each knock-out alone merely reduced the level of GalNAc. Hence, both enzymes apparently contribute to the high-GalNAc phenotype of HEK 293 cell-derived glycoproteins, a notion well in line with earlier reports by others (Sato et al., 2003; Gotoh et al., 2004). GalNAc reduction also increased sialylation—and GalNAc elimination increased it further—most likely because α2,3-sialyltransferases cannot add sialic acids to GalNAc but can add them to the Gal that replaced GalNAc in the NT3/4-KO cells (Harduin-Lepers et al., 2001; Mi et al., 2014). Levels of both bis-GlcNAc and triantennary glycans also increased after B4GALNT-KO. While both structures were almost absent on FVII-alb from wild-type cells, they surged to 38% and 16%, respectively, in the NT3/4-KO. Interestingly, we found neither any bis-GlcNAc nor triantennarity on N-glycan structures that contained GalNAc, regardless of genetic background. This suggests that the presence of GalNAc has an impact on the occurrence of bisection and antennarity, potentially due to low N-acetyl-glucosaminyltransferase III, IV and V (MGAT3-MGAT5) activity toward N-glycans containing GalNAc. To our knowledge, this relationship has not been described before, but the finding integrates well with earlier studies which invariably found GalNAc in a background of predominantly nonbisected, biantennary N-glycans (Green et al., 1985; Baenziger and Green, 1988; Bergweff et al., 1992; Siciliano et al., 1993; Dell et al., 1995). We also observed reduced antenna fucosylation after KO of B4GALNT3 and B4GALNT3/4 and KI of ST6GAL1 and ST3GAL6. In the KO cell lines, this might be explained by the increased levels of bis-GlcNAc, which were shown to reduce the addition of terminal glycan structures (Koyota et al., 2001; Lu et al., 2016; Nakano et al., 2019). Furthermore, more of the Galβ4GlcNAc antennae were sialylated, switching the enzymatic flux away from fucosyltransferases acting on nonsialylated antennae and toward fucosyltransferases acting on α2,3-sialylated antennae. Narimatsu et al. (2019) showed that FUT4, FUT10 and FUT11 were the most abundant α1,3- or α1,4-fucosyltransferases in HEK 293 cells, all of which prefer nonsialylated over α2,3-sialylated antennae (Kumar et al., 1991; Sherwood et al., 2002; Toivonen et al., 2002; Mollicone et al., 2009). The potentially low availability of fucosyltransferases acting on α2,3-sialylated N-glycans could explain the reduced antenna fucosylation after B4GALNT 3/4-KO and ST3GAL6-KI. The mutual exclusivity of antenna fucosylation and α2,6-sialylation (Paulson et al., 1978; Dell et al., 1995), together with elevated sialyltransferase levels, which seemingly outcompeted the fucosyltransferases, may explain the reduction of antenna fucosylation in the ST6GAL1-KI cell lines. We found our N-glycan profiling results for NovoSeven® (Supplementary Table S3) to be consistent with reports by Montacir et al. (2018). Likewise, the N-glycans we found on FVII from wt HEK 293-F cells matched those reported by Böhm et al. (2015), i.e. they were antenna-fucosylated, high-GalNAc, biantennary structures with little sialylation (Supplementary Table S4). However, our assignment of certain N-glycans differs from that of Böhm et al. This results from our interpretation of a 486 Da fragment as a sulfated GalNAcβ4GlcNAc disaccharide (predicted mass 486.1155 Da), whereas Böhm et al. interpreted an almost identical mass (486.1584 Da) as three hexoses and assigned hybrid and high-mannose structures (Supplementary Table S5). We assignment sulfated instead of high-mannose glycans based on MS–MS fragmentation (Supplementary Figure S6), negative charges found in negative ion mode MALDI-TOF-MS (data not shown), higher mass precision (Supplementary Table S5), better matching HILIC elution times (Supplementary Table S6) and abundance of the structures in question in the wild-type and knock-out cell lines (Supplementary Table S7). However, the differences in Böhm et al.’s and our analysis could also originate from the suspension adaption of the HEK 293-F cell line and the resulting differences in medium and culture conditions (Jenkins et al., 1995; Kunkel et al., 1998; Chee Furng Wong et al., 2005; Lefloch et al., 2006; Pacis et al., 2011; Costa et al., 2013; Fan et al., 2015). High sialylation is generally desirable on circulating therapeutic proteins because it usually improves pharmacokinetic properties by reducing clearance rates (Seested et al., 2010; Mi et al., 2016), although some exceptions apply—e.g. sialylation seems not to affect the clearance of certain IgGs (Huang et al., 2006; Millward et al., 2008) or coagulation factor VIII (Pipe et al., 2016; Swystun et al., 2019); and it seems to impair the antibody-dependent cellular cytotoxicity of IgGs (Scallon et al., 2007). To improve the pharmacokinetics of HEK 293-F cell-derived FVII-alb, we thus additionally sought to improve the fairly poor sialylation of HEK 293 cells (Croset et al., 2012; Böhm et al., 2015; Goh and Ng, 2018; Canis et al., 2018) by knock-in of ST6GAL1 or ST3GAL6. On a wild-type HEK 293-F background, the ST6GAL1-KI led to more sialylation than the ST3GAL6-KI because it sialylates both GalNAc and Gal (Stockell Hartree and Renwick, 1992) while ST3GAL6 only sialylates Gal (Harduin-Lepers et al., 2001; Mi et al., 2014)—a substrate barely available in the high-GalNAc environment of this genetic background. Since GalNAc cannot be sialylated and sulfated at the same time (Mi et al., 2008), the ST6GAL1-KI also decreased GalNAc sulfation by removing sulfotransferase substrate. On an NT3/4-KO background, however, where GalNAc is essentially absent and fully replaced by Gal, both KIs increased sialylation—but again ST6GAL1 led to more sialylation. In fact, on the double-KO background, the ST6GAL1-KI yielded near-complete sialylation—a level beyond that of BHK, CHO or HEK cell-derived FVII and on par with pdFVII (Böhm et al., 2015). Therefore, sialylation appears limited by both GalNAc level and sialic acid transfer rate in HEK 293-F cells. In addition to the near-complete sialylation, HEK 293-F cell-derived FVII-alb—in contrast to NovoSeven®—contained no nonhuman, potentially immunogenic Neu5Gc (Tangvoranuntakul et al., 2003; Padler-Karavani et al., 2008; Ghaderi et al., 2010). But why did ST6GAL1 sialylate more effectively than ST3GAL6 in the NT3/4-KO background? We suspect that this relates to a higher level of bis-GlcNAc in the NT3/4-KO. After all, a deliberate increase of bis-GlcNAc via overexpression of MGAT3 did impair α2,3-sialylation by ST3GAL6 but not α2,6-sialylation by ST6GAL1 (Lu et al., 2016; Nakano et al., 2019). In all our samples with meaningful amounts of bisected N-glycans, these were two to three times less likely to carry sialic acids than the nonbisected N-glycans. Currently, the mechanism behind the ST3GAL6-mediated increase of bis-GlcNAc remains unclear, as does the reason for increased triantennarity. Binding of glyco-engineered FVII-albumin to ASGP-R and MR Our various changes to FVII-alb N-glycosylation reduced binding to clearance receptors in vitro. This was consistent with the findings from others. Specifically, ASGP-R binding was diminished when terminal GalNAc was either sialylated (wt + ST6GAL1-KI), replaced by Gal (NT3-KO; NT3/4-KO) or replaced by sialylated Gal (NT3-KO; NT3/4-KO; NT3/4-KO + ST6GAL1; NT3/4-KO + ST3GAL6)—all in agreement with reports by Baenziger and Fiete (1980), Baenziger and Maynard (1980), Seestedt et al. (2010) and Mi et al. (2014). And similarly, MR binding was reduced when levels of sulfated GalNAc (wt + ST3Gal6-KI, wt + ST6GAL1-KI) and antenna fucosylation (NT3-KO, NT3/4-KO; wt + ST6GAL1-KI; NT3/4-KO + ST6GAL1; NT3/4-KO + ST3GAL6) went down—in agreement with reports by Shepherd et al. (1981), Largent et al. (1984), Pontow et al. (1993), Fiete et al. (1998), Dong et al. (1999), Liu et al. (2000) and Lee et al. (2002). Yet ASGP-R binding did not only depend on the absolute sialylation level as evidenced by the fact that it was very similar for the wt + ST6GAL1-KI and the NT3/4-KO + ST3GAL6-KI cell lines (see Figure 2A)—and yet residual ASGP-R binding of FVII-alb remained higher in the former. We did not investigate the underlying mechanism in detail, but we noted that N-glycosylation differs in three conceivably relevant aspects between those two cell lines. First, only the wt + ST6GAL1-KI causes sialylated GalNAc, which retains some ASGP-R affinity (Park et al., 2003, 2005). Second, it also maintains residual levels of nonsialylated GalNAc, which binds the ASGP-R with greater avidity than the nonsialylated Gal of the NT3/4-KO + ST3GAL6-KI (Baenziger and Maynard, 1980). Third, both cell lines attach sialic acids to Gal in different linkage forms—i.e. mainly α2,6-linked sialic acids in the wt + ST6Gal1-KI and α2,3-linked sialic acids in the NT3/4-KO + ST3GAL6-KI cell lines. That Gal capped with α2,3-linked sialic acid does not bind to the ASGP-R while it does when capped with α2,6-linked sialic acid was indeed shown by Park et al. (2005). Other studies showed faster, ASGP-R-dependent in vivo clearance of proteins capped with α2,6 instead of α2,3-linked sialic acids (André et al., 1997; André et al., 2004; Unverzagt et al., 2002; Steirer et al., 2009; Chung et al., 2020). These differences may account both individually and collectively for the different ASGP-R affinities. Although linkage type and sialylation level also differed between the ST6GAL1 and ST3GAL6 KIs on the NT3/4-KO background, FVII-alb from either cell line did not bind the ASGP-R. But this need not contradict the notion that linkage can affect ASGP-R binding; it may simply reflect that N-glycans with α2,3-linked sialic acids bind the ASGP-R less readily than N-glycans with α2,6-linked sialic acids and that the difference in linkage is balanced by the overall higher α2,6 sialylation, which leads to similar ASGP-R binding. Furthermore, it may reflect that FVII-alb from both the NT3/4-KO + ST3GAL6-KI and from the NT3/4-KO + ST6GAL1-KI cell lines do not bind the ASGP-R anymore, which renders the advantage of α2,3 over α2,6-linkage undetectable. Pharmacokinetics of glyco-engineered FVII-albumin It should be noted that the ASGP-R and MR binding studies were performed using recombinant human receptors, while the PK experiment was performed in rats. However, since most glycan receptors are widely conserved across mammalian species, they are generally considered suitable models for the human situation (Taylor and Drickamer, 2019). The MR and the ASGR1 subunit of the ASGP-R are highly conserved between mammals (Spiess and Lodish, 1985; Harris et al., 1992; Su et al., 2005) and the orthologs share similar ligand specificities (Park and Baenziger, 2004; Martinez-Pomares et al., 2005). Recovery rates 5 min after intravenous injection of our wt HEK 293-F cell-derived FVIIa and FVIIa-alb were substantially lower than those reported for similar proteins of CHO cell origin (Weimer et al., 2008; Zollner et al., 2014). This reflects the absence of GalNAc and the higher level of sialylation of the CHO cell-derived protein (Böhm et al., 2015). When we shifted FVII-alb N-glycosylation to a more CHO cell-like profile—via expression in the NT3/4-KO cell line—recovery increased to a level similar to those reported for CHO. The terminal half-life of FVIIa-alb from wt HEK 293-F cells (365 min) substantially increased through both NT3-KO (668 min) and NT3/4-KO (654 min). These improved values exceeded previously reported values for FVIIa-alb products in rat models around two-fold—and we saw a similar difference with NovoSeven® (Weimer et al., 2008; Appa et al., 2010; Seested et al., 2011; Zollner et al., 2014). However, our analyses included postinjection time points up to 18 (NovoSeven®) and 66 h (FVIIa-alb) while all other cited studies terminated between 4 and 24 h. If analyzed for those same intervals, we find similar values for both NovoSeven® and our engineered proteins (Supplementary Table S8). Our B4GALNT3 and B4GALNT3/4 knock-outs improved both FVIIa-alb recovery and terminal half-life, which together increased AUC. While not included in our pharmacokinetic study, we suspect that our NT3/4-KO + ST6GAL1-KI cell line may yield even further improvements given the even higher sialylation and lower clearance receptor binding of the FVII-alb product derived from it. Impact of albumin fusion on receptor binding and pharmacokinetics As albumin is not glycosylated itself (Sheffield et al., 2000), N-glycosylation of FVII and FVII-alb from wt HEK 293-F cells were nearly identical. Therefore, it cannot explain the significant loss of ASGP-R and MR binding after albumin fusion. This is more probably a result of steric hindrance. In fact, the C-type lectin-like domains of the ASGP-R (Baenziger and Fiete, 1980; Lee et al., 1983) and MR (Weis et al., 1992; Taylor and Drickamer, 1993), and the cysteine-rich domain of the MR (Roseman and Baenziger, 2001) all show the highest affinities toward multivalent oligosaccharides with more than two interaction partners. Since most N-glycans on FVII are biantennary, ASGP-R and MR binding may require simultaneous access to both FVII N-glycans—an interaction the albumin fusion may impair. In accordance with this, Zollner et al. (2014) and Weimer et al. (2008) already saw the higher recovery of FVIIa-alb over FVIIa, but because glycosylation was not analyzed in these studies, its role remained previously unknown. Conclusion Double knock-out of B4GALNT3 and B4GALNT4 in HEK 293-F cells eliminated GalNAc from FVII-alb N-glycans and increased sialylation. This glyco-engineering reduced FVII-alb clearance receptor binding and improved pharmacokinetics. The double-KO cell line seems therefore well-suited for the production of glycoproteins susceptible to high levels of GalNAc or sulfated GalNAc, including lutropin (Baenziger and Green, 1988), thyroid-stimulating hormone (Baenziger and Green, 1988), chorionic gonadotropin (Birken et al., 1996), prolactin-like protein A (Manzella et al., 1997), glycodelin (Dell et al., 1995), carbonic anhydrase 6 (Hooper et al., 1995), urokinase-type plasminogen activator (Bergweff et al., 1992), pro-opiomelanocortin (Siciliano et al., 1993) and other proteins with a peptide recognition motif for B4GALNT3 and B4GALNT4 (Miller et al., 2008; Fiete et al., 2012a, 2012b). Additional knock-in of ST6GAL1 further improved sialylation beyond levels reported for CHO and BHK cells (Böhm et al., 2015). This further reduced FVII-alb clearance receptor binding, and in combination with the absence of unfavorable, nonhuman N-glycans, should render this cell line a preferred host for the production of therapeutic glycoproteins with improved pharmacokinetics. Materials and methods Materials Unless otherwise stated, all chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany) or Carl Roth (Karlsruhe, Germany). Solvents were of liquid chromatography mass spectrometry (LC–MS) grade quality. NovoSeven® was acquired from Novo Nordisk A/S (Bagsværd, Denmark). Cell lines and cell culture All cells were maintained in shaking incubators at 37°C, humid atmosphere and 8% CO2 in a serum-free proprietary culture medium. FreeStyle 293-F cells were acquired from Thermo Fisher Scientific (Dreieich, Germany). B4GALNT3 and B4GALNT4 knock-out cell pools and clones were generated by Transposagen Biopharmaceuticals (Lexington, USA) using CRISPR/Cas9, by transfecting a plasmid encoding for Cas9 and the respective guide RNAs (B4GALNT3: 5’-GAGAGAACTGGCCAAGGCTC-3′; B4GALNT4: 5’-GTTCTTCCACTTGGGGGACA-3′). Knock-out clones were selected based on the absence of wild-type B4GALNT3 and/or B4GALNT4 DNA sequences and small in-frame mutations. ST6GAL1 and ST3GAL6 overexpressing cell pools were generated by CRISPR/Cas9 knock-in of the plasmid AAVS1-SA-hygro-EF1α-MCS (BioCat, Heidelberg, Germany), encoding for human ST6GAL1 or ST3GAL6, into the AAVS1 integration site of HEK 293-F cells variants, using Lipofectamine™ CRISPRMAX™ Transfection Reagent (Thermo Fisher Scientific, Dreieich, Germany), a published guide RNA (5’-GGGGCCACUAGGGACAGGAU-3′, Mali et al. 2013) and Cas9 protein (Synthego, Menlo Park, USA). Stably overexpressing cell pools were selected using hygromycin B (Thermo Fisher Scientific, Dreieich, Germany). A modified version of the UCOE control vector (Merck, Darmstadt, Germany), encoding for human FVII or FVII fused to human albumin via a rigid linker consisting of six proline-alanine-proline-alanine-proline repeats, was transfected into the different HEK 293-F cell lines using FuGENE® HD transfection reagent (Promega, Mannheim, Germany), followed by the selection of stably expressing cells using puromycin (Sigma-Aldrich, Taufkirchen, Germany). For the production of FVII and FVII-alb, FVII and FVII-alb expressing cell pools were cultured in batch mode in a baffled shake flask for five days before supernatants were collected by centrifugation. Purification and quantification FVII and FVII-alb were captured from culture supernatants by affinity chromatography using the VIISelect chromatography resin (GE Healthcare, Freiburg, Germany) on a Tecan EVO 200 system configured for protein purification (Tecan Group, Crailsheim, Germany), eluted in 0.05 M glycine pH 3 and rebuffered into the water using PD-10 Sephadex G-25 M desalting columns (GE Healthcare, Freiburg, Germany). FVII-alb from the NT4-KO cell line was purified on an ÄKTA pure system (GE Healthcare, Freiburg, Germany), using the same chromatography method but rebuffering first in 0.025 M sodium acetate and subsequently into water. FVII and FVII-alb were quantified by enzyme-linked immunosorbent assay (ELISA) using the ZYMUTEST Faktor VII Antigen kit (Coachrom, Maria Enzersdorf, Austria) according to the kit protocol. Calibration curves were generated using the Human Coagulation Factor VII Concentrate BRP batch 2 (Sigma Aldrich, Taufkirchen, Germany) and an in-house FVII-alb standard. Monosaccharide analysis Monosaccharides were released from 75 μg freeze-dried protein with 200 μL 4 M triflouracetic acid at 100°C for 3 h, freeze-dried, labeled by reductive amination with 2-aminobenzoic acid (2-AA) using the LudgerTag 2-AA Monosaccharide Release and Labeling kit (Ludger, Oxfordshire, United Kingdom) according to the kit protocol but using 10 μL of sodium acetate, 2-AA dye and cyanoborohydride mixture, respectively. From the labeled samples, 2 μL were dissolved in 178 μL Solvent A (0.2% (v/v) butylamine, 0.5% (v/v) orthophosphoric acid, 1% (v/v) tetrahydrofuran in ddH2O) and 1 μL were separated by UHPLC on an UltiMate™ 3000 system (Thermo Scientific, Waltham, USA) equipped with a fluorescence detector FLD-3100 using an XBridge BEH C18 XP column (Waters Corporation, Milford, USA). Fluorescence signals were detected at 425 nm, peaks were integrated automatically according to predefined parameters using the Chromeleon™ 7.2 chromatography software (Thermo Scientific, Waltham, USA) and compared to standard curves ranging from 0.16 to 10 nmol of the respective monosaccharides. To compensate for small differences in the amount of analyzed protein, monosaccharides levels were calculated relative to mannose (2 N-glycans and 3 mannoses per glycan). NovoSeven® was injected twice and all other samples were injected three times and means, as well as standard deviations, were calculated. Reproducibility was assessed by analyzing three biologic FVII-alb replicates produced individually in the wt HEK 293-F cell line. Sialic acid analysis Sialic acid analysis was performed using the Signal™ DMB Labeling kit (ProZyme, Hayward, USA) according to the kit protocol with minor modifications. Sialic acids were released from 40 μg freeze-dried protein with 100 μL acetic acid at 80°C for 2 h, again freeze-died, dissolved in 5 μL water and labeled by reductive amination with 20 μL 1,2-diamino-4,5-methylenedioxybenzene (DMB) dissolved in sodium dithionite, acetic acid and β-mercaptoethanol. Labeled samples were freeze-dried, dissolved in 525 μL water and 1.5 μL were used for subsequent UHPLC analysis. UHPLC analysis was performed on an UltiMate™ 3000 system (Thermo Scientific, Waltham, USA) equipped with a fluorescence detector FLD-3100 using a XBridge BEH C18 XP column (Waters Corporation, Milford, USA). Fluorescence signals were detected at 448 nm, peaks were integrated automatically according to predefined parameters using the Chromeleon™ 7.2 chromatography software (Thermo Scientific, Waltham, USA) and compared to Neu5Ac and Neu5Gc standard curves ranging from 0.27 to 17.14 pmol per injection. Each sample was injected 3 times and means, as well as standard deviations, were calculated. Reproducibility was assessed by analyzing three FVII-alb replicates produced individually in the wt HEK 293-F cell line. Preparation of released N-glycans Proteins were denatured for 3 min at 90°C in 1% RapiGest SF and 4 mM tris(2-carboxyethyl)phospine (TCEP, Thermo Fisher Scientific, Dreieich, Germany) according to the adapted deglycosylation workflow for complex and challenging glycoprotein samples. N-glycans were released from 15 μg protein by Rapid PNGase F, labeled with RapiFluor-MS and purified on a hydrophilic interaction liquid chromatography (HILIC) μElution Plate with the GlycoWorks RapiFluor-MS N-glycan kit (Waters, Milford, USA). Glycans eluted from the HILIC μElution Plate were lyophilized, reconstituted in 4.5 μL H2O and diluted with 15.5 μL GlycoWorks Sample Diluent-DMF/ACN. The RapiFlour-MS Intact mAB Standard (Waters, Milford, USA) was used as control. N-glycan profiling and quantification A sample volume of 3 μL was separated via an ACQUITY UPLC Glycan BEH Amide column (Waters, Milford, USA) with HILIC retention mechanism using a liquid chromatography system with coupled mass spectrometry (LC–MS), according to the GlycoWorks RapiFluor-MS N-glycan kit (Waters, Milford, USA) instructions. LC–MS experiments were performed on an ACQUITY H-Class Bio equipped with fluorescence detector, and coupled to a Xevo G2-XS Q-TOF, piloted by UNIFI 1.9.4 software (Waters, Milford, USA). Data acquisition was performed in MSE mode by electrospray ionization with a measuring range of 100–2000 m/z, a capillary voltage of 2.75 kV, a sample cone of 80 V, a source temperature of 120°C, a desolvation temperature of 500°C and a high energy ramp of 20–30 eV. Peak areas of the fluorescence chromatograms were used for relative quantification. Only peaks making up more than 0.5% of the total fluorescence signal were considered for quantification. For peaks containing multiple structures, the intensity of the mass signal (charge variants 2H+ and/or 3H+) was used to determine the relative proportions of each structure of the peak fluorescence. From the relative abundance of the detected N-glycans we calculated the average amount of each monosaccharide per FVII molecule, based on the assumption that both N-glycosylation sites of FVII are occupied, which in turn is based on multiple reports where only fully N-glycosylated FVII was detected (Fenaille et al., 2008; Böhm et al., 2015; Montacir et al., 2018) and most of the secreted FVII had two N-glycans due to retaining of FVII without both N-glycans in the endoplasmatic reticulum (Bolt et al., 2005). Peak identification was achieved by dextran ladder calibration (expressed in glucose units (GU); based on fluorescence signal), Sialidase A (ProZyme, Hayward, USA) and Sialidase S (ProZyme, Hayward, USA) digestion, MS–MS fragmentation with peak annotation in GlycoWorkbench (Ceroni et al., 2008) and independent Q-TOF mass spectrometry confirmation. Reproducibility of the complete N-glycan profiling workflow was assessed by analyzing three biologic FVII-alb replicates produced individually in the wt HEK 293-F cell line. ASGP-R and MR binding by SPR Binding studies were performed at 25°C using a Biacore T200 instrument (GE Healthcare, Freiburg, Germany) equipped with CM5 sensor chips (GE Healthcare, Freiburg, Germany) and equilibrated with running buffer (ASGP-R: 20 mM HEPES, 150 mM NaCl, 5 mM CaCl2, 0.005% Tween20, pH 7.4; MR: 10 mM HEPES, 150 mM NaCl, 1 mM MgCl2, 5 mM CaCl2, 0.005% Tween 20, pH 7.4). Recombinant human ASGR1 (R&D Systems, Minneapolis, USA)—the ASGP-R subunit containing the C-type lectin domain—and recombinant human MR (R&D Systems, Minneapolis, USA) were immobilized using the Amine Coupling kit (GE Healthcare, Freiburg, Germany) according to the manufacturer’s instructions. The immobilization levels of the receptors were between 2500 and 4000 RU for ASGP-R and between 6300 and 9500 RU for MR. Flow cell 1 was immobilized only with a running buffer for 2 min and was used as a reference to correct for systematic noise and instrument drift. SPR measurements were performed with a flow of 30 μL/min and 600 nM of the analytes. FVII, FVII-alb and human serum albumin (Pan Biotech, Aidenbach, Germany) were injected for 180 s followed by a 200 s dissociation phase with running buffer. The surface was regenerated with regeneration buffer (50 mM HEPES, 100 mM EDTA pH 7.4) for 5–15 s. The measurements also included zero-concentration cycles of analyte every fourth cycle for blank subtraction. All samples were measured in triplicates and each receptor was immobilized three times at different Fc positions. Biacore T200 Evaluation Software 3.0 (GE Healthcare, Freiburg, Germany) was used to analyze the SPR data. After double reference subtraction and molecular weight adjustment, the report points for binding (5 s before sample injection stop) were used as readout and were normalized to NovoSeven®. Reproducibility was assessed by analyzing three biologic FVII-alb replicates produced individually in the wt HEK 293-F cell line. Pharmacokinetic study in rats NovoSeven® from BHK, FVII produced in wt HEK-F cells and FVII-alb produced in wt HEK-F, NT3-KO and NT3/4-KO cells were tested in a pharmacokinetic study. For comparability to NovoSeven®, FVII and FVII-alb were activated by incubation of 0.86 mg/mL FVII and 1.5 mg/mL FVII-alb variants in 45 mM glycine, 90 μM ZnCl2, 90 mM Tris, 10 mM CaCl2 at pH 8.0 and 4°C for approximately 4 days. Activation was monitored by reducing SDS-PAGE. Equimolar amounts of activated FVII (270 μg/kg of FVIIa and 650 μg/kg of FVIIa-Alb constructs) were injected into the tail vein of 16 male rats (CD® (Sprague Dawley) IGS Rat; Charles River, Sulzfeld, Germany) per construct with 276 g mean body weight at the start of the study. Blood was sampled from the retrobulbar venous plexus under isoflurane anesthesia 0 min, 5 min, 15 min, 1 h, 3 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48 h, 54 h, 60 h, 66 h and 72 h after injection (four animals per time point), immediately cooled, centrifuged and sodium citrate stabilized plasma was stored at −80°C until ELISA measurement. Pharmacokinetic parameters (AUC and terminal half-life) were determined using noncompartmental analysis in the SAS software version 9.4 M5 (SAS Institute, Cary, USA) and recovery 5 min after administration was calculated with Excel 2013 (Microsoft, Redmond, USA), assuming a plasma volume of 11.3 mL per rat (Probst et al., 2006). All animal activities were approved by the Animal Protection Authority in Hamburg (Germany) “Behörde für Gesundheit und Verbraucherschutz, Amt für Verbraucherschutz, Lebensmittelsicherheit und Veterinärwesen” and performed in accordance with the “Guide on the Care and Use of Laboratory Animals”. Acknowledgements The authors thank Julia Kaim, Janine Wöhlk, Ulrike Noller, Thomas Orlik, Marius Lechner, Melina Bitsch and Ronny Schmidt for supporting the production, purification and quantification of FVII and FVII-alb samples; Laurenz Trawnicek for support with the pharmacokinetic analysis; and Tilo Schwientek, Barbara Solecka-Witulska and Tobias Stuwe for critically reviewing the manuscript. Funding declaration This work was founded by Octapharma Biopharmaceuticals GmbH and supported by the Cooperative Research Training Group: Tissue Analytics for Stem Cell based Diagnostics and Therapy (TASCDT), funded by the Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg. R.U., R.P.W., M.K., A.B., P.R., A.S., M.P., M.K., G.K., C.K. and J.R. are employees of Octapharma Biopharmaceuticals GmbH, Heidelberg, Germany, a fully owned subsidiary of Octapharma AG. Octapharma AG owns a patent application for the multiallelic knock-out of B4GALNT3 and B4GALNT4 (WO2017220527A1). This patent application was initially filed by the Glycotope GmbH. Abbreviations 2-AA, 2-aminobenzoic acid; ACN, Acetonitrile; AUC; BHK, Baby hamster kidney; bis-GlcNAc, Bisecting N-acetylglucosamine; CHO, Chinese hamster ovary; CRISPR/Cas9, Clustered regularly interspaced short palindromic repeats/CRISPR associated 9; DMB, 1,2-diamino-4,5-methylenedioxybenzene; DMF, N,N-Dimethylformamide; ELISA, Enzyme-linked immunosorbent assay; FVII, Coagulation factor VII; FVIIa, Activated coagulation factor VII; FVIIa-alb, Activated coagulation factor VII albumin fusion protein; FVII-alb, Coagulation factor VII albumin fusion protein; GalNAc, N-acetylgalactosamine; GU, Glucose units; HEK 293, Human embryonic kidney 293; HEK 293-F, FreeStyle 293-F; HILIC, Hydrophilic interaction liquid chromatography; KI, Knock-in; KO, Knock-out; LC–MS, Liquid chromatography with coupled mass spectrometry; MALDI-TOF-MS, Matrix assisted laser desorption ionization—time of flight mass spectrometry; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; NT3/4-KO, B4GALNT3 and B4GALNT4 knock-out; NT3/4-KO + ST3GAL6-KI, NT3 + 4 knock-out and ST3Gal6 knock-in; NT3/4-KO + ST6GAL1-KI, NT3 + 4 knock-out and ST6Gal1 knock-in; NT3-KO, B4GALNT3 knock-out; NT4-KO, B4GALNT4 knock-out; PTMs, Posttranslational modifications; SPR, Surface plasmon resonance; TCEP, tris(2-carboxyethyl)phosphine; UHPLC, Ultra high performance liquid chromatography; wt, Wild-type; wt + ST3GAL6-KI, ST3Gal6 knock-in; wt + ST6GAL1-KI, ST6Gal1 knock-in; α-Gal, Galactose-α-1,3-galactose. References André S , Unverzagt C, Kojima S, Dong X, Fink C, Kayser K, Gabius HJ. 1997 . Neoglycoproteins with the synthetic complex biantennary nonasaccharide or its alpha 2,3/alpha 2,6-sialylated derivatives: Their preparation, assessment of their ligand properties for purified lectins, for tumor cells in vitro, and in tissue sections, and their biodistribution in tumor-bearing mice . Bioconjug Chem . 8 : 845 – 855 . Google Scholar Crossref Search ADS PubMed WorldCat André S , Unverzagt C, Kojima S, Frank M, Seifert J, Fink C, Kayser K, von der Lieth C-W, Gabius H-J. 2004 . Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico, in vitro and in vivo . Eur J Biochem . 271 : 118 – 134 . Google Scholar Crossref Search ADS PubMed WorldCat Appa RS , Theill C, Hansen L, Møss J, Behrens C, Nicolaisen EM, Klausen NK, Christensen MS. 2010 . Investigating clearance mechanisms for recombinant activated factor VII in a perfused liver model . Thromb Haemost . 104 : 243 – 251 . Google Scholar Crossref Search ADS PubMed WorldCat Arnold JN , Wormald MR, Sim RB, Rudd PM, Dwek RA. 2007 . The impact of glycosylation on the biological function and structure of human immunoglobulins . Annu Rev Immunol . 25 : 21 – 50 . Google Scholar Crossref Search ADS PubMed WorldCat Baenziger JU , Fiete D. 1980 . Galactose and N-acetylgalactosamine-specific endocytosis of glycopeptides by isolated rat hepatocytes . Cell . 22 : 611 – 620 . Google Scholar Crossref Search ADS PubMed WorldCat Baenziger JU , Green ED. 1988 . Pituitary glycoprotein hormone oligosaccharides: Structure, synthesis and function of the asparagine-linked oligosaccharides on lutropin, follitropin and thyrotropin . Biochim Biophys Acta . 947 : 287 – 306 . Google Scholar Crossref Search ADS PubMed WorldCat Baenziger JU , Maynard Y. 1980 . Human hepatic lectin. Physiochemical properties and specificity . J Biol Chem . 255 : 4607 – 4613 . Google Scholar Crossref Search ADS PubMed WorldCat Berg EA , Platts-Mills TAE, Commins SP. 2014 . Drug allergens and food--the cetuximab and galactose-alpha-1,3-galactose story . Ann Allergy Asthma Immunol . 112 : 97 – 101 . Google Scholar Crossref Search ADS PubMed WorldCat Bergweff AA , Thomas-Oates JE, van Oostrum J, Kamerling JP, Vliegenthart JFG. 1992 . Human urokinase contains Ga1NA c β(1-4)[Fucα(1-3)]G1cNA c β(1-2) as a novel terminal element in N -linked carbohydrate chains . FEBS Let . 314 : 389 – 394 . Google Scholar Crossref Search ADS WorldCat Berkner KL . 1993 . Expression of recombinant vitamin K-dependent proteins in mammalian cells: Factors IX and VII . Methods Enzymol . 222 : 450 – 477 . Google Scholar Crossref Search ADS PubMed WorldCat Birken S , Maydelman Y, Gawinowicz MA, Pound A, Liu Y, Hartree AS. 1996 . Isolation and characterization of human pituitary chorionic gonadotropin . Endocrinology . 137 : 1402 – 1411 . Google Scholar Crossref Search ADS PubMed WorldCat Böhm E , Seyfried BK, Dockal M, Graninger M, Hasslacher M, Neurath M, Konetschny C, Matthiessen P, Mitterer A, Scheiflinger F. 2015 . Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells . BMC Biotechnol . 15 :87. Google Scholar OpenURL Placeholder Text WorldCat Bolt G , Kristensen C, Steenstrup TD. 2005 . Posttranslational N-glycosylation takes place during the normal processing of human coagulation factor VII . Glycobiology . 15 : 541 – 547 . Google Scholar Crossref Search ADS PubMed WorldCat Bosques CJ , Collins BE, JW3 M, Sarvaiya H, Murphy JL, Dellorusso G, Bulik DA, Hsu I-H, Washburn N, Sipsey SF, et al. 2010 . Chinese hamster ovary cells can produce galactose-alpha-1,3-galactose antigens on proteins . Nat Biotechnol . 28 : 1153 – 1156 . Google Scholar Crossref Search ADS PubMed WorldCat Butler M , Spearman M. 2014 . The choice of mammalian cell host and possibilities for glycosylation engineering . Curr Opin Biotechnol . 30 : 107 – 112 . Google Scholar Crossref Search ADS PubMed WorldCat Canis K , Anzengruber J, Garenaux E, Feichtinger M, Benamara K, Scheiflinger F, Savoy L-A, Reipert BM, Malisauskas M. 2018 . In-depth comparison of N-glycosylation of human plasma-derived factor VIII and different recombinant products: From structure to clinical implications . J Thromb Haemost . 16 : 1592 – 1603 . Google Scholar Crossref Search ADS WorldCat Ceroni A , Maass K, Geyer H, Geyer R, Dell A, Haslam SM. 2008 . GlycoWorkbench: A tool for the computer-assisted annotation of mass spectra of glycans . J Proteome Res . 7 : 1650 – 1659 . Google Scholar Crossref Search ADS PubMed WorldCat Chapple SDJ , Crofts AM, Shadbolt SP, McCafferty J, Dyson MR. 2006 . Multiplexed expression and screening for recombinant protein production in mammalian cells . BMC Biotechnol . 6 : 49 . Google Scholar Crossref Search ADS PubMed WorldCat Chee Furng Wong D , Tin Kam Wong K, Tang Goh L, Kiat Heng C, Gek Sim Yap M. 2005 . Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures . Biotechnol Bioeng . 89 : 164 – 177 . Google Scholar Crossref Search ADS PubMed WorldCat Chung CH , Mirakhur B, Chan E, Le Q-T, Berlin J, Morse M, Murphy BA, Satinover SM, Hosen J, Mauro D, et al. 2008 . Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose . N Engl J Med . 358 : 1109 – 1117 . Google Scholar Crossref Search ADS PubMed WorldCat Costa AR , Withers J, Rodrigues ME, McLoughlin N, Henriques M, Oliveira R, Rudd PM, Azeredo J. 2013 . The impact of cell adaptation to serum-free conditions on the glycosylation profile of a monoclonal antibody produced by Chinese hamster ovary cells . N Biotechnol . 30 : 563 – 572 . Google Scholar Crossref Search ADS PubMed WorldCat Croset A , Delafosse L, Gaudry J-P, Arod C, Glez L, Losberger C, Begue D, Krstanovic A, Robert F, Vilbois F, et al. 2012 . Differences in the glycosylation of recombinant proteins expressed in HEK and CHO cells . J Biotechnol . 161 : 336 – 348 . Google Scholar Crossref Search ADS PubMed WorldCat Déglon N , Aubert V, Spertini F, Winkel L, Aebischer P. 2003 . Presence of gal-alpha1,3Gal epitope on xenogeneic lines: Implications for cellular gene therapy based on the encapsulation technology . Xenotransplantation . 10 : 204 – 213 . Google Scholar Crossref Search ADS PubMed WorldCat Dell A , Morris HR, Easton RL, Panico M, Patankar M, Oehninger S, Koistinen R, Koistinen H, Seppala M, Clark GF. 1995 . Structural analysis of the oligosaccharides derived from Glycodelin, a human glycoprotein with potent immunosuppressive and contraceptive activities . J Biol Chem . 270 : 24116 – 24126 . Google Scholar Crossref Search ADS PubMed WorldCat Diaz SL , Padler-Karavani V, Ghaderi D, Hurtado-Ziola N, Yu H, Chen X, Brinkman-Van der Linden ECM, Varki A, Varki NM. 2009 . Sensitive and specific detection of the non-human sialic acid N-glycolylneuraminic acid in human tissues and biotherapeutic products . PLoS One . 4 :e4241. Google Scholar OpenURL Placeholder Text WorldCat Dong X , Storkus WJ, Salter RD. 1999 . Binding and uptake of agalactosyl IgG by mannose receptor on macrophages and dendritic cells . J Immunol . 163 : 5427 – 5434 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Dumont J , Euwart D, Mei B, Estes S, Kshirsagar R. 2016 . Human cell lines for biopharmaceutical manufacturing. History, status, and future perspectives . Crit Rev Biotechnol . 36 : 1110 – 1122 . Google Scholar Crossref Search ADS PubMed WorldCat Estes S , Melville M. 2014 . Mammalian cell line developments in speed and efficiency . Adv Biochem Eng Biotechnol . 139 : 11 – 33 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Fan Y , Jimenez Del Val I, Müller C, Wagtberg Sen J, Rasmussen SK, Kontoravdi C, Weilguny D, Andersen MR. 2015 . Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation . Biotechnol Bioeng . 112 : 521 – 535 . Google Scholar Crossref Search ADS PubMed WorldCat Fenaille F , Groseil C, Ramon C, Riandé S, Siret L, Chtourou S, Bihoreau N. 2008 . Mass spectrometric characterization of N- and O-glycans of plasma-derived coagulation factor VII . Glycoconj J . 25 : 827 – 842 . Google Scholar Crossref Search ADS PubMed WorldCat Fiete D , Beranek M, Baenziger JU. 2012 . Molecular basis for protein-specific transfer of N-acetylgalactosamine to N-linked glycans by the glycosyltransferases β1,4-N-acetylgalactosaminyl transferase 3 (β4GalNAc-T3) and β4GalNAc-T4 . J Biol Chem . 287 : 29194 – 29203 . Google Scholar Crossref Search ADS PubMed WorldCat Fiete D , Mi Y, Oats EL, Beranek MC, Baenziger JU. 2007 . N-linked oligosaccharides on the low density lipoprotein receptor homolog SorLA/LR11 are modified with terminal GalNAc-4-SO4 in kidney and brain . J Biol Chem . 282 : 1873 – 1881 . Google Scholar Crossref Search ADS PubMed WorldCat Fiete DJ , Beranek MC, Baenziger JU. 1998 . A cysteine-rich domain of the "mannose" receptor mediates GalNAc-4-SO4 binding . Proc Natl Acad Sci USA . 95 : 2089 – 2093 . Google Scholar Crossref Search ADS PubMed WorldCat Galili U . 2005 . The alpha-gal epitope and the anti-gal antibody in xenotransplantation and in cancer immunotherapy . Immunol Cell Biol . 83 : 674 – 686 . Google Scholar Crossref Search ADS PubMed WorldCat Ghaderi D , Taylor RE, Padler-Karavani V, Diaz S, Varki A. 2010 . Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins . Nat Biotechnol . 28 : 863 – 867 . Google Scholar Crossref Search ADS PubMed WorldCat Goh JB , Ng SK. 2018 . Impact of host cell line choice on glycan profile . Crit Rev Biotechnol . 38 : 851 – 867 . Google Scholar Crossref Search ADS PubMed WorldCat Golor G , Bensen-Kennedy D, Haffner S, Easton R, Jung K, Moises T, Lawo J-P, Joch C, Veldman A. 2013 . Safety and pharmacokinetics of a recombinant fusion protein linking coagulation factor VIIa with albumin in healthy volunteers . J Thromb Haemost . 11 : 1977 – 1985 . Google Scholar Crossref Search ADS PubMed WorldCat Gotoh M , Sato T, Kiyohara K, Kameyama A, Kikuchi N, Kwon Y-D, Ishizuka Y, Iwai T, Nakanishi H, Narimatsu H. 2004 . Molecular cloning and characterization of β1,4- N -acetylgalactosaminyltransferases IV synthesizing N , N ′-diacetyllactosediamine 1 . FEBS Lett . 562 : 134 – 140 . Google Scholar Crossref Search ADS PubMed WorldCat Grabenhorst E , Schlenke P, Pohl S, Nimtz M, Conradt HS. 1999 . Genetic engineering of recombinant glycoproteins and the glycosylation pathway in mammalian host cells . Glycoconj J . 16 : 81 – 97 . Google Scholar Crossref Search ADS PubMed WorldCat Grancha S , Navajas R, Maranon C, Paradela A, Albar JP, Jorquera JI. 2011 . Incomplete tyrosine 1680 sulphation in recombinant FVIII concentrates . Haemophilia . 17 : 709 – 710 . Google Scholar Crossref Search ADS PubMed WorldCat Green ED , van Halbeek H, Boime I, Baenziger JU. 1985 . Structural elucidation of the disulfated oligosaccharide from bovine lutropin . J Biol Chem . 260 : 15623 – 15630 . Google Scholar Crossref Search ADS PubMed WorldCat Harduin-Lepers A , Vallejo-Ruiz V, Krzewinski-Recchi M-A, Samyn-Petit B, Julien S, Delannoy P. 2001 . The human sialyltransferase family . Biochimie . 83 : 727 – 737 . Google Scholar Crossref Search ADS PubMed WorldCat Herzog E , Harris S, McEwen A, Henson C, Pragst I, Dickneite G, Schulte S, Zollner S. 2014 . Recombinant fusion protein linking factor VIIa with albumin (rVIIa-FP). Tissue distribution in rats . Thromb Res . 134 : 495 – 502 . Google Scholar Crossref Search ADS PubMed WorldCat Hiraoka N , Misra A, Belot F, Hindsgaul O, Fukuda M. 2001 . Molecular cloning and expression of two distinct human N-acetylgalactosamine 4-O-sulfotransferases that transfer sulfate to GalNAc beta 1→4GlcNAc beta 1→R in both N- and O-glycans . Glycobiology . 11 : 495 – 504 . Google Scholar Crossref Search ADS PubMed WorldCat Hooper LV , Beranek MC, Manzella SM, Baenziger JU. 1995 . Differential expression of GalNAc-4-sulfotransferase and GalNAc-transferase results in distinct glycoforms of carbonic anhydrase VI in parotid and submaxillary glands . J Biol Chem . 270 : 5985 – 5993 . Google Scholar Crossref Search ADS PubMed WorldCat Huang L , Biolsi S, Bales KR, Kuchibhotla U. 2006 . Impact of variable domain glycosylation on antibody clearance: An LC/MS characterization . Anal Biochem . 349 : 197 – 207 . Google Scholar Crossref Search ADS PubMed WorldCat Ikehara Y , Sato T, Niwa T, Nakamura S, Gotoh M, Ikehara SK, Kiyohara K, Aoki C, Iwai T, Nakanishi H, et al. 2006 . Apical Golgi localization of N,N'-diacetyllactosediamine synthase, beta4GalNAc-T3, is responsible for LacdiNAc expression on gastric mucosa . Glycobiology . 16 : 777 – 785 . Google Scholar Crossref Search ADS PubMed WorldCat Jenkins N , Castro P, Menon S, Ison A, Bull A. 1995 . Effect of lipid supplements on the production and glycosylation of recombinant interferon-γ expressed in CHO cells. In: Buckland BC, Aunins JG, Bibila TA, editors. Cell Culture Engineering 4. Improvements of Human Health . Dordrecht : Springer . p. 209 – 215 . Google Scholar Google Preview OpenURL Placeholder Text WorldCat COPAC Kannicht C , Ramstrom M, Kohla G, Tiemeyer M, Casademunt E, Walter O, Sandberg H. 2013 . Characterisation of the post-translational modifications of a novel, human cell line-derived recombinant human factor VIII . Thromb Res . 131 : 78 – 88 . Google Scholar Crossref Search ADS PubMed WorldCat Koyota S , Ikeda Y, Miyagawa S, Ihara H, Koma M, Honke K, Shirakura R, Taniguchi N. 2001 . Down-regulation of the alpha-gal epitope expression in N-glycans of swine endothelial cells by transfection with the N-acetylglucosaminyltransferase III gene. Modulation of the biosynthesis of terminal structures by a bisecting GlcNAc . J Biol Chem . 276 : 32867 – 32874 . Google Scholar Crossref Search ADS PubMed WorldCat Kubisz P , Stasko J. 2004 . Recombinant activated factor VII in patients at high risk of bleeding . Hematology . 9 : 317 – 332 . Google Scholar Crossref Search ADS PubMed WorldCat Kunkel JP , Jan DCH, Jamieson JC, Butler M. 1998 . Dissolved oxygen concentration in serum-free continuous culture affects N-linked glycosylation of a monoclonal antibody . J Biotechnol . 62 : 55 – 71 . Google Scholar Crossref Search ADS PubMed WorldCat Largent BL , Walton KM, Hoppe CA, Lee YC, Schnaar RL. 1984 . Carbohydrate-specific adhesion of alveolar macrophages to mannose-derivatized surfaces . J Biol Chem . 259 : 1764 – 1769 . Google Scholar Crossref Search ADS PubMed WorldCat Lee SJ , Evers S, Roeder D, Parlow AF, Risteli J, Risteli L, Lee YC, Feizi T, Langen H, Nussenzweig MC. 2002 . Mannose receptor-mediated regulation of serum glycoprotein homeostasis . Science . 295 : 1898 – 1901 . Google Scholar Crossref Search ADS PubMed WorldCat Lee YC , Townsend RR, Hardy MR, Lönngren J, Arnarp J, Haraldsson M, Lönn H. 1983 . Binding of synthetic oligosaccharides to the hepatic gal/GalNAc lectin. Dependence on fine structural features . J Biol Chem . 258 : 199 – 202 . Google Scholar Crossref Search ADS PubMed WorldCat Lefloch F , Tessier B, Chenuet S, Guillaume J-M, Cans P, Goergen J-L, Marc A. 2006 . Related effects of cell adaptation to serum-free conditions on murine EPO production and glycosylation by CHO cells . Cytotechnology . 52 : 39 – 53 . Google Scholar Crossref Search ADS PubMed WorldCat Leteux C , Chai W, Loveless RW, Yuen CT, Uhlin-Hansen L, Combarnous Y, Jankovic M, Maric SC, Misulovin Z, Nussenzweig MC, et al. 2000 . The cysteine-rich domain of the macrophage mannose receptor is a multispecific lectin that recognizes chondroitin sulfates a and B and sulfated oligosaccharides of blood group Lewis(a) and Lewis(x) types in addition to the sulfated N-glycans of lutropin . J Exp Med . 191 : 1117 – 1126 . Google Scholar Crossref Search ADS PubMed WorldCat Leyte A , van Schijndel HB, Niehrs C, Huttner WB, Verbeet MP, Mertens K, van Mourik JA. 1991 . Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor . J Biol Chem . 266 : 740 – 746 . Google Scholar Crossref Search ADS PubMed WorldCat Lindley CM , Sawyer WT, Macik BG, Lusher J, Harrison JF, Baird-Cox K, Birch K, Glazer S, Roberts HR. 1994 . Pharmacokinetics and pharmacodynamics of recombinant factor VIIa . Clin Pharmacol Ther . 55 : 638 – 648 . Google Scholar Crossref Search ADS PubMed WorldCat Liu Y , Chirino AJ, Misulovin Z, Leteux C, Feizi T, Nussenzweig MC, Bjorkman PJ. 2000 . Crystal structure of the cysteine-rich domain of mannose receptor complexed with a sulfated carbohydrate ligand . J Exp Med . 191 : 1105 – 1116 . Google Scholar Crossref Search ADS PubMed WorldCat Lu J , Isaji T, Im S, Fukuda T, Kameyama A, Gu J. 2016 . Expression of N-Acetylglucosaminyltransferase III suppresses α2,3-sialylation, and its distinctive functions in cell migration are attributed to α2,6-sialylation levels . J Biol Chem . 291 : 5708 – 5720 . Google Scholar Crossref Search ADS PubMed WorldCat Mali P , Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. 2013 . RNA-guided human genome engineering via Cas9 . Science . 339 : 823 – 826 . Google Scholar Crossref Search ADS PubMed WorldCat Manzella SM , Dharmesh SM, Cohick CB, Soares MJ, Baenziger JU. 1997 . Developmental regulation of a pregnancy-specific oligosaccharide structure, NeuAcalpha2,6GalNAcbeta1,4GlcNAc, on select members of the rat placental prolactin family . J Biol Chem . 272 : 4775 – 4782 . Google Scholar Crossref Search ADS PubMed WorldCat Mi Y , Coonce M, Fiete D, Steirer L, Dveksler G, Townsend RR, Baenziger JU. 2016 . Functional consequences of mannose and Asialoglycoprotein receptor ablation . J Biol Chem . 291 : 18700 – 18717 . Google Scholar Crossref Search ADS PubMed WorldCat Mi Y , Fiete D, Baenziger JU. 2008 . Ablation of GalNAc-4-sulfotransferase-1 enhances reproduction by altering the carbohydrate structures of luteinizing hormone in mice . J Clin Invest . 118 : 1815 – 1824 . Google Scholar Crossref Search ADS PubMed WorldCat Mi Y , Lin A, Fiete D, Steirer L, Baenziger JU. 2014 . Modulation of mannose and asialoglycoprotein receptor expression determines glycoprotein hormone half-life at critical points in the reproductive cycle . J Biol Chem . 289 : 12157 – 12167 . Google Scholar Crossref Search ADS PubMed WorldCat Millward TA , Heitzmann M, Bill K, Längle U, Schumacher P, Forrer K. 2008 . Effect of constant and variable domain glycosylation on pharmacokinetics of therapeutic antibodies in mice . Biologicals . 36 : 41 – 47 . Google Scholar Crossref Search ADS PubMed WorldCat Montacir O , Montacir H, Eravci M, Springer A, Hinderlich S, Mahboudi F, Saadati A, Parr MK. 2018 . Bioengineering of rFVIIa biopharmaceutical and structure characterization for biosimilarity assessment . Bioengineering (Basel) . 5 : 7 . Google Scholar Crossref Search ADS WorldCat Nakano M , Mishra SK, Tokoro Y, Sato K, Nakajima K, Yamaguchi Y, Taniguchi N, Kizuka Y. 2019 . Bisecting GlcNAc is a general suppressor of terminal modification of N-glycan . Mol Cell Proteomics . 18 : 2044 – 2057 . Google Scholar Crossref Search ADS PubMed WorldCat Pacis E , Yu M, Autsen J, Bayer R, Li F. 2011 . Effects of cell culture conditions on antibody N-linked glycosylation—what affects high mannose 5 glycoform . Biotechnol Bioeng . 108 : 2348 – 2358 . Google Scholar Crossref Search ADS PubMed WorldCat Padler-Karavani V , Yu H, Cao H, Chokhawala H, Karp F, Varki N, Chen X, Varki A. 2008 . Diversity in specificity, abundance, and composition of anti-Neu5Gc antibodies in normal humans. Potential implications for disease . Glycobiology . 18 : 818 – 830 . Google Scholar Crossref Search ADS PubMed WorldCat Park EI , Manzella SM, Baenziger JU. 2003 . Rapid clearance of sialylated glycoproteins by the asialoglycoprotein receptor . J Biol Chem . 278 : 4597 – 4602 . Google Scholar Crossref Search ADS PubMed WorldCat Park EI , Mi Y, Unverzagt C, Gabius H-J, Baenziger JU. 2005 . The asialoglycoprotein receptor clears glycoconjugates terminating with sialic acid alpha 2,6GalNAc . Proc Natl Acad Sci USA . 102 : 17125 – 17129 . Google Scholar Crossref Search ADS PubMed WorldCat Pipe SW , Montgomery RR, Pratt KP, Lenting PJ, Lillicrap D. 2016 . Life in the shadow of a dominant partner: The FVIII-VWF association and its clinical implications for hemophilia a . Blood . 128 : 2007 – 2016 . Google Scholar Crossref Search ADS PubMed WorldCat Pontow SE , Kery V, Stahl PD. 1993 . Mannose receptor . Int Rev Cytol . 137 ( Part B ): 221 – 244 . Google Scholar Crossref Search ADS WorldCat Probst RJ , Lim JM, Bird DN, Pole GL, Sato AK, Claybaugh JR. 2006 . Gender differences in the blood volume of conscious Sprague-Dawley rats . J Am Assoc Lab Anim Sci . 45 : 49 – 52 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Rabouille C , Hui N, Hunte F, Kieckbusch R, Berger EG, Warren G, Nilsson T. 1995 . Mapping the distribution of Golgi enzymes involved in the construction of complex oligosaccharides . J Cell Sci . 108 : 1617 – 1627 . Google Scholar PubMed OpenURL Placeholder Text WorldCat Roseman DS , Baenziger JU. 2001 . The mannose/N-acetylgalactosamine-4-SO4 receptor displays greater specificity for multivalent than monovalent ligands . J Biol Chem . 276 : 17052 – 17057 . Google Scholar Crossref Search ADS PubMed WorldCat Roth J , Berger EG. 1982 . Immunocytochemical localization of galactosyltransferase in HeLa cells: Codistribution with thiamine pyrophosphatase in trans-Golgi cisternae . J Cell Biol . 93 : 223 – 229 . Google Scholar Crossref Search ADS PubMed WorldCat Sato T , Gotoh M, Kiyohara K, Kameyama A, Kubota T, Kikuchi N, Ishizuka Y, Iwasaki H, Togayachi A, Kudo T, et al. 2003 . Molecular cloning and characterization of a novel human beta 1,4-N-acetylgalactosaminyltransferase, beta 4GalNAc-T3, responsible for the synthesis of N,N'-diacetyllactosediamine, galNAc beta 1-4GlcNAc . J Biol Chem . 278 : 47534 – 47544 . Google Scholar Crossref Search ADS PubMed WorldCat Scallon BJ , Tam SH, McCarthy SG, Cai AN, Raju TS. 2007 . Higher levels of sialylated Fc glycans in immunoglobulin G molecules can adversely impact functionality . Mol Immunol . 44 : 1524 – 1534 . Google Scholar Crossref Search ADS PubMed WorldCat Seested T , Appa RS, Christensen EI, Ioannou YA, Krogh TN, Karpf DM, Nielsen HM. 2011 . In vivo clearance and metabolism of recombinant activated factor VII (rFVIIa) and its complexes with plasma protease inhibitors in the liver . Thromb Res . 127 : 356 – 362 . Google Scholar Crossref Search ADS PubMed WorldCat Seested T , Nielsen HM, Christensen EI, Appa RS. 2010 . The unsialylated subpopulation of recombinant activated factor VII binds to the asialo-glycoprotein receptor (ASGPR) on primary rat hepatocytes . Thromb Haemost . 104 : 1166 – 1173 . Google Scholar Crossref Search ADS PubMed WorldCat Sewell R , Bäckström M, Dalziel M, Gschmeissner S, Karlsson H, Noll T, Gätgens J, Clausen H, Hansson GC, Burchell J, et al. 2006 . The ST6GalNAc-I sialyltransferase localizes throughout the Golgi and is responsible for the synthesis of the tumor-associated sialyl-Tn O-glycan in human breast cancer . J Biol Chem . 281 : 3586 – 3594 . Google Scholar Crossref Search ADS PubMed WorldCat Sheffield WP , Marques JA, Bhakta V, Smith IJ. 2000 . Modulation of clearance of recombinant serum albumin by either glycosylation or truncation . Thromb Res . 99 : 613 – 621 . Google Scholar Crossref Search ADS PubMed WorldCat Shepherd VL , Lee YC, Schlesinger PH, Stahl PD. 1981 . L-Fucose-terminated glycoconjugates are recognized by pinocytosis receptors on macrophages . Proc Natl Acad Sci USA . 78 : 1019 – 1022 . Google Scholar Crossref Search ADS PubMed WorldCat Siciliano RA , Morris HR, McDowell RA, Azadi P, Rogers ME, Bennett HP, Dell A. 1993 . The Lewis x epitope is a major non-reducing structure in the sulphated N-glycans attached to Asn-65 of bovine pro-opiomelanocortin . Glycobiology . 3 : 225 – 239 . Google Scholar Crossref Search ADS PubMed WorldCat Smith PL , Skelton TP, Fiete D, Dharmesh SM, Beranek MC, MacPhail L, Broze GJ, Baenziger JU. 1992 . The asparagine-linked oligosaccharides on tissue factor pathway inhibitor terminate with SO4-4GalNAc beta 1, 4GlcNAc beta 1,2 Mana alpha . J Biol Chem . 267 : 19140 – 19146 . Google Scholar Crossref Search ADS PubMed WorldCat Stockell Hartree A , Renwick AGC. 1992 . Molecular structures of glycoprotein hormones and functions of their carbohydrate components . Biochem J . 287 : 665 – 679 . Google Scholar Crossref Search ADS PubMed WorldCat Swiech K , Freitas MC de , Covas DT, Picanco-Castro V. 2015 . Recombinant glycoprotein production in human cell lines . Methods Mol Biol 1258 : 223 – 240 . Google Scholar Crossref Search ADS PubMed WorldCat Swystun LL , Ogiwara K, Rawley O, Brown C, Georgescu I, Hopman W, Labarque V, Male C, Thom K, Blanchette VS, et al. 2019 . Genetic determinants of VWF clearance and FVIII binding modify FVIII pharmacokinetics in pediatric hemophilia a patients . Blood . 134 : 880 – 891 . Google Scholar Crossref Search ADS PubMed WorldCat Tangvoranuntakul P , Gagneux P, Diaz S, Bardor M, Varki N, Varki A, Muchmore E. 2003 . Human uptake and incorporation of an immunogenic nonhuman dietary sialic acid . Proc Natl Acad Sci USA . 100 : 12045 – 12050 . Google Scholar Crossref Search ADS PubMed WorldCat Taylor ME , Drickamer K. 1993 . Structural requirements for high affinity binding of complex ligands by the macrophage mannose receptor . J Biol Chem . 268 : 399 – 404 . Google Scholar Crossref Search ADS PubMed WorldCat Unverzagt C , André S, Seifert J, Kojima S, Fink C, Srikrishna G, Freeze H, Kayser K, Gabius H-J. 2002 . Structure−activity profiles of complex Biantennary Glycans with Core Fucosylation and with/without additional α2,3/α2,6 sialylation. Synthesis of Neoglycoproteins and their properties in lectin assays, cell binding, and organ uptake † . J Med Chem . 45 : 478 – 491 . Google Scholar Crossref Search ADS PubMed WorldCat de VT , Srnka CA, Palcic MM, Swiedler SJ, van den Eijnden DH, Macher BA. 1995 . Acceptor specificity of different length constructs of human recombinant alpha 1,3/4-fucosyltransferases. Replacement of the stem region and the transmembrane domain of fucosyltransferase V by protein a results in an enzyme with GDP-fucose hydrolyzing activity . J Biol Chem . 270 : 8712 – 8722 . Google Scholar Crossref Search ADS PubMed WorldCat Walsh G . 2018 . Biopharmaceutical benchmarks 2018 . Nat Biotechnol . 36 : 1136 – 1145 . Google Scholar Crossref Search ADS PubMed WorldCat Walsh MT , Watzlawick H, Putnam FW, Schmid K, Brossmer R. 1990 . Effect of the carbohydrate moiety on the secondary structure of beta 2-glycoprotein. I. Implications for the biosynthesis and folding of glycoproteins . Biochemistry . 29 : 6250 – 6257 . Google Scholar Crossref Search ADS PubMed WorldCat Wang J-R , Gao W-N, Grimm R, Jiang S, Liang Y, Ye H, Li Z-G, Yau L-F, Huang H, Liu J, et al. 2017 . A method to identify trace sulfated IgG N-glycans as biomarkers for rheumatoid arthritis . Nat Commun . 8 :631. Google Scholar OpenURL Placeholder Text WorldCat Wedepohl S , Kaup M, Riese SB, Berger M, Dernedde J, Tauber R, Blanchard V. 2010 . N-glycan analysis of recombinant L-selectin reveals sulfated GalNAc and GalNAc-GalNAc motifs . J Proteome Res . 9 : 3403 – 3411 . Google Scholar Crossref Search ADS PubMed WorldCat Weimer T , Wormsbächer W, Kronthaler U, Lang W, Liebing U, Schulte S. 2008 . Prolonged in-vivo half-life of factor VIIa by fusion to albumin . Thromb Haemost . 99 : 659 – 667 . Google Scholar Crossref Search ADS PubMed WorldCat Weis WI , Drickamer K, Hendrickson WA. 1992 . Structure of a C-type mannose-binding protein complexed with an oligosaccharide . Nature . 360 : 127 – 134 . Google Scholar Crossref Search ADS PubMed WorldCat Xu X , Nagarajan H, Lewis NE, Pan S, Cai Z, Liu X, Chen W, Xie M, Wang W, Hammond S, et al. 2011 . The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line . Nat Biotechnol . 29 : 735 – 741 . Google Scholar Crossref Search ADS PubMed WorldCat Yan SB , Chao YB, van Halbeek H. 1993 . Novel Asn-linked oligosaccharides terminating in GalNAc beta (1→4)Fuc alpha (1→3)GlcNAc beta (1→.) are present in recombinant human protein C expressed in human kidney 293 cells . Glycobiology . 3 : 597 – 608 . Google Scholar Crossref Search ADS PubMed WorldCat Zollner S , Schuermann D, Raquet E, Mueller-Cohrs J, Weimer T, Pragst I, Dickneite G, Schulte S. 2014 . Pharmacological characteristics of a novel, recombinant fusion protein linking coagulation factor VIIa with albumin (rVIIa-FP) . J Thromb Haemost . 12 : 220 – 228 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)
TI - Glyco-engineered HEK 293-F cell lines for the production of therapeutic glycoproteins with human N-glycosylation and improved pharmacokinetics
JF - Glycobiology
DO - 10.1093/glycob/cwaa119
DA - 2021-01-05
UR - https://www.deepdyve.com/lp/oxford-university-press/glyco-engineered-hek-293-f-cell-lines-for-the-production-of-fDAf5f3PMX
SP - 1
EP - 1
VL - Advance Article
IS - 
DP - DeepDyve
ER -