Stable high volumetric production of glycosylated human recombinant IFNalpha2b in HEK293 cells

Abstract

Background: Mammalian cells are becoming the prevailing expression system for the production of recombinant proteins because of their capacity for proper protein folding, assembly, and post-translational modifications. These systems currently allow high volumetric production of monoclonal recombinant antibodies in the range of grams per litre. However their use for large-scale expression of cytokines typically results in much lower volumetric productivity.

Results: We have engineered a HEK293 cell clone for high level production of human recombinant glycosylated IFNalpha2b and developed a rapid and efficient method for its purification. This clone steadily produces more than 200 mg (up to 333 mg) of human recombinant IFNalpha2b per liter of serum-free culture, which can be purified by a single-step cation-exchange chromatography following media acidification and clarification. This rapid procedure yields 98% pure IFNalpha2b with a recovery greater than 70%. Purified IFNalpha2b migrates on SDS-PAGE as two species, a major 21 kDa band and a minor 19 kDa band. N-terminal sequences of both forms are identical and correspond to the expected mature protein. Purified IFNalpha2b elutes at neutral pH as a single peak with an apparent molecular weight of 44,000 Da as determined by size-exclusion chromatography. The presence of intramolecular and absence of intermolecular disulfide bridges is evidenced by the fact that non-reduced IFNalpha2b has a greater electrophoretic mobility than the reduced form. Treatment of purified IFNalpha2b with neuraminidase followed by O-glycosidase both increases electrophoretic mobility, indicating the presence of sialylated O-linked glycan. A detailed analysis of glycosylation by mass spectroscopy identifies disialylated and monosialylated forms as the major constituents of purified IFNalpha2b. Electron transfer dissociation (ETD) shows that the glycans are linked to the expected threonine at position 106. Other minor glycosylated forms and non-sialylated species are also detected, similar to IFNalpha2b produced naturally by lymphocytes. Further, the HEK293-produced IFNalpha2b is biologically active as shown with reporter gene and antiviral assays.

Conclusion: These results show that the HEK293 cell line is an efficient and valuable host for the production of biologically active and glycosylated human IFNalpha2b.

Figure 1

Expression plasmid encoding human IFNα2b cDNA. A) The pYD7-IFNα2b expression plasmid has been used to generate the D9 clone. (Amp) ampicillin, (Blast) blasticidin, (CMV) cytomegalovirus promoter, (enh MLP) adenovirus major late promoter, (IFNα2b) human codon-optimized sequence for human IFNα2b gene, (pA) polyadenylation sequence, (pMB1ori) bacterial origin of replication, (Puro) puromycin, (OriP) Epstein-Barr virus origin of replication, (SV40pA) simian virus 40 polyadenylation sequence, (TPL) adenovirus tripartite leader.B) Amino acid sequence of human IFNα2b. Signal peptide is underlined. The two intramolecular disulfide bridges are C₁-C₉₈ and C₂₉-C₁₃₈. The glycan-linked threonine (Thr₁₀₆) is underscored.

Figure 2

Kinetics of cell growth and IFNα2b production from D9 clone in fed-batch culture. D9 cells were seeded at a cell density of 0,25 × 10⁶ cells per mL, fed with 0,1% TN1 the next day and sampled every day. A) Coomassie-stained SDS-PAGE analysis of the culture medium (20 μL) collected daily. B) Cell counts and viability were measured at the indicated times.

Figure 3

Purification of IFNα2b by cation-exchange chromatography. A) A typical chromatographic profile of a purification of HEK293-produced IFNα2b from a 400 mL fed-batch culture is illustrated. Solid line shows the 280 nM absorbance profile. Dotted line shows pH variations. IFNα2b elutes in a single peak between 1000 and 1200 mL. B) Coomassie-stained SDS-PAGE analysis of 20 μL samples collected at different steps of production and purification of IFNα2b. 1- crude harvest. 2- precipitate (equivalent to 200 μL of harvest volume). 3- clarified harvest. 4- flow through SO3^- column. 5- wash SO3^- column. 6- elution peak SO3^- column. 7- desalted IFNα2b in PBS.

Figure 4

Purified IFNα2b is not aggregated and forms dimers at neutral pH independent of intermolecular cystine formation. Following a desalting step in neutral PBS, purified IFNα2b was analysed for dimer formation. A) Twenty mg of purified IFNα2b were analysed on a Superdex 75 HR16/60 column equilibrated with PBS at pH 7,0. The arrows and numbers above indicate the elution volumes of molecular weight standards eluted in the same conditions. Purified IFNα2b elutes in the same volume as ovalbumin, a 44 kDa globular protein. B) Coomassie-stained SDS-PAGE analysis of samples (20 μL) of each of the 10 fractions (4 mL) collected between elution volumes 40–80 mL. C) Coomassie-stained SDS-PAGE analysis of reduced and non-reduced IFNα2b from HEK293 cells.

Figure 5

HEK293-produced human IFNα2b is sialylated and O-glycosylated. IFNα2b was deglycosylated as described in material and methods. 1- 10 μg of purified HEK-produced IFNα2b. 2- 10 μg of purified HEK-produced IFNα2b digested with neuraminidase. 3- 10 μg of purified HEK-produced IFNα2b digested with O-glycosidase. 4- 10 μg of purified E. coli-produced IFNα2b.

Figure 6

ESI-MS analysis of the intact IFNα2b glycoprotein. A) ESI mass spectrum exhibiting the glycoform profiles associated with each charge state of the protein and B) the glycoprotein molecule weight profile reconstructed from the mass spectrum in panel A. The most intense peak at 20,213 Da appears to be composed of the mature peptide chain plus a single core type-1 disialylated glycan (Hex₁HexNAc₁SA₂).

Figure 7

CID and ETD analysis of the tryptic glycopeptides from IFNα2b. A) CID-MS/MS spectrum of the triply protonated ion at m/z 1426.8 corresponding to the disialylated glycopeptide of T84-112. The spectrum is dominated by the sequential neutral loss of the glycan components from the doubly protonated glycopeptide ion. The principal b and y fragment ions arising from fragmentation of the peptide backbone are indicated in the spectrum as are the compositions the glycan oxonium ions observed m/z 494.9, 657.0 and 948.0, respectively. The sequence of the peptide is provided in the inset. B) CID-MS/MS spectrum of the triply protonated ion at m/z 1340.8 corresponding to the monosialylated glycopeptide of T84-112. Note that the neutral loss corresponding to a second sialic acid is missing from this spectrum as is the corresponding oxonium ion at m/z 948.0. C) ETD MS/MS spectrum of the triply protonated, monosialylated T84-112 glycopeptide at m/z 1340.8. The higher m/z region of the ETD spectrum contained the most informative fragment ions and is presented here. The c ion series indicated in the spectrum clearly identified the site of O-linkage as Threonine 106 of the mature protein.

Figure 8

HEK293-produced human IFNα2b is biologically active. The biological activity of HEK293-produced human IFNα2b was assayed with a gene reporter assay and compared to E. coli-produced human recombinant IFNα2b as described in material and methods. The activity of the secreted alkaline phosphatase is plotted against the concentration of IFNα2b produced in the two hosts. Each point represents the average ± SEM of 3 experiments performed in triplicate.