Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells

Abstract

BACKGROUND &

Methods: Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS &

Discussion: The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures.

Conclusions: From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

Fig. 1

SDS-PAGE analysis of rFVII in cell culture supernatants and after purification. a Western blot after SDS-PAGE stained for human FVII: lane 1: molecular mass marker. pdFVII (lane 2), pdFVIIa (lane 3), commercially available rFVIIa (lane 4), supernatants from CHOrFVII clones (lanes 5–7) and from HEK293rFVII clones (lanes 8–10). 50 ng rFVII based on antigen were loaded per lane and resolved under reducing conditions by SDS-PAGE. Human FVII was detected by dual-antibody staining after blotting on a nitrocellulose membrane. Molecular mass marker sizes are shown on the left. b Silver-stained SDS-PAGE: 220 ng per lane each purified HEK293rFVII (lane 1), commercially available rFVIIa (lane 2), were resolved by SDS-PAGE and protein bands were made visible by silver staining. M: molecular mass standard, molecular mass marker sizes are shown on the left.. The FVII whole molecule, the heavy (HC) and light chain (LC) of rFVIIa are marked with arrows

Fig. 2

Proposed N-glycan structures on N145 and N322 for pdFVII, HEK293rFVII, BHKrFVII, and CHOrFVII. Legend: GlcNAc, Man, Gal, Fuc, GalNAc, sialic acid (NANA). Relative abundances of N-glycan structures at each N-glycosylation site detected by LC-MS are shown. Mean values four clones of HEK293rFVII, two clones of CHOrFVII, and values for one BHKrFVII clone and one pdFVII preparation are shown. Only structures detected at equal or higher 2 % are shown. The data for CHOrFVII, HEK293rFVII and BHKrFVII of each clone and of pdFVII can be found in Additional file 1: Table S1 in the supplementary file

Fig. 3

a and b Mass spectra of the N-glycosylated peptides containing the light and heavy chain glycosylation sites (N145 and N322) of pdFVII and rFVII isolated from CHO, HEK293 and BHK. a Mass spectra of the light chain peptides (N145). Relative intensities of glycan structures were calculated based on the masses of the 3-fold positively charged peptides. b Mass spectra of the heavy chain peptides (N322) based on the 4-fold positively charged peptides. Both mass spectra of all samples were recorded within the same system setup. Only intensities of manually identified peaks were used for the calculation of relative amounts of N-glycan species. Major identified oligosaccharide structures are shown for each FVII isolate. Theoretical and calculated molecular masses, and relative abundances of glycan structures can also be found in the Additional file 1: Tables S4A-H

Fig. 4

Oligosaccharide maps of BHK-, CHO-, and HEK293-derived rFVII. N-linked oligosaccharides obtained after PNGase F digest were separated according to their charge by HPAEC with PAD. Positions of neutral and bi-antennary/mono-sialylated to tetra-antennary/tetra-sialylated structures are marked

Fig. 5

Peptide maps of CHO-, HEK293-, BHK-derived rFVII, and pdFVII. Peptides were separated by HPLC on a C18 column after reduction, alkylation and tryptic digest. Shown are chromatograms with retention times of respective peptides detected at 214 nm of one representative preparation of each FVII isolate, all four recorded within one sequence

Fig. 6

a and b Mass spectra of γ-carboxylated peptides of CHO-, HEK293-, BHK-derived rFVII, and pfVII. Shown are mass spectra of peptides T1-5 (a) with peaks corresponding to peptides containing 8, 9, and 10 Gla residues indicated, and T1-6 (b), which mostly contained 10 Gla residues. All spectra were recorded within one system setup. Corresponding data can also be found in Additional file 1: Tables S1A-D in the supplementary file

Fig. 7

Mass spectra of the O-glycosylated peptide T7 detected from CHO-, HEK293-, BHK-derived rFVII, and pdFVII. Shown are masses of the T7 peptide corresponding to the peptides containing O-glycans on S52 and S60. The major forms on CHOrFVII and HEK293rFVII were GlcXyl2 on S52 and Fuc on S60, whereas BHKrFVII contained mostly one Glc on S52 and Fuc on S60, and pdFVII mostly GlcXyl or GlcXyl2 on S52 and Fuc on S60 and. Symbols: Fucose; Glucose; Xylose. In Additional file 1: Tables S3A-D, raw data can be found