Cell Line for Producing Recombinant Glycoproteins with Di-Antennary N-Glycans, Methods Using the Same, and Recombinant Glycoproteins

Abstract

The present invention relates to a genetically modified cell line with reduced expression of GnTIVa/b and/or GnTV, a method for the production of glycoproteins having N-glycans with a reduced number of tri- and/or tetra-antennary N-glcyans using said cell line, and respective glycoproteins.

Claims

1. A cell line that is genetically modified to reduce the expression of at least one of GnTIVa/b and GnTV.

2. The cell line according to claim 1, wherein the expression of all of GnTIVa/b and GnTV is reduced.

3. The cell line according to claim 2, wherein the expression of GnTIVa/b and/or GnTV is completely abolished.

4. The cell line according to claim 3, wherein the cell line is further genetically modified to overexpress -galactoside -2,6-sialyltransferase 1 (ST6Gal1) and/or -2,3-sialyltransferase 4 (ST3Gal4).

5. The cell line according to claim 4, wherein the cell line is an insect cell line, an avian cell line, or a mammalian cell line, preferably a human cell line.

6. The cell line according to claim 5, wherein the cell line is derived from a cell line, selected from the group consisting of Muscovy Duck cells (AGE.CR), African green monkey kidney epithelial cells (Vero), Madin Darby canine kidney cells (MDCK), baby hamster kidney cells (BHK), Chinese hamster ovary (CHO) cells, human hepatocarcinoma cell lines (HepG2, Huh7), human embryonic kidney 293 (HEK293) cells, human neuronal precursor cells (AGE1.HN, NC5T11), human embryonic retinoblast cells (Per.C6), myeloma cell lines (HMCLs, MM.1, U266, RPMI8226), CIVIL tumor cell lines (NM, NM-F9), hybrid HEK293 and lymphoma cell (HKB11), and human amniocytes-derived CAP cells.

7. The cell line according to claim 5, wherein the cell line is derived from human primary amniocytes comprising at least one nucleic acid encoding the gene products of the adenoviral E1 and pIX regions.

8. A recombinant glycoprotein having N-glycans with a significantly increased proportion of di-antennary N-glycans when compared to the same recombinant glycoprotein expressed in a cell line with non-reduced expression of GnTIVa/b and/or GnTV.

9. The recombinant glycoprotein according to claim 8, wherein the proportion of tetra-antennary N-glycans is significantly reduced when compared to the same recombinant glycoprotein expressed in a cell line with non-reduced expression of GnTIVa/b and/or GnTV.

10. The recombinant glycoprotein according to claim 9, wherein at least 80% of the antennae of the N-glycans are terminated by 2,6-linked sialic acid.

11. The recombinant glycoprotein according to claim 10, wherein the glycoprotein is selected from the group consisting of al-antitrypsin (AAT), hepatocyte growth factor (HGF), Factor VII (FVII), Factor VIII (FVIII), Factor IX (FIX), von-Willebrand-Factor (vWF), and C1 esterase inhibitor (C1-inhibitor; C1 Inh).

12. The recombinant glycoprotein according to claim 10, wherein the glycoprotein is a mammalian glycoprotein.

13. The recombinant glycoprotein according to claim 8, wherein the glycoprotein has been expressed in a cell line according to claim 1.

14. A method for the expression of a recombinant glycoprotein of interest in a cell, said glycoprotein having N-glycans with a significantly increased proportion of di-antennary N-glycans when compared to the same recombinant glycoprotein expressed in a cell line with non-reduced expression of GnTIVa/b and/or GnTV, comprising the steps of: (a) providing a cell line according to claim 1; (b) expressing the glycoprotein of interest in said cell line; and (c) isolating the glycoprotein from the cell or the cell culture supernatant.

15. The method according to claim 14, wherein the recombinant glycoprotein is a recombinant glycoprotein according to claim 8.

16. The recombinant glycoprotein according to claim 12, wherein the mammalian glycoprotein is a human glycoprotein.

Description

[0052] The figures show:

[0053] FIG. 1:

[0054] Biosynthesis of tri- and tetra-antennary branched N-glycans. Tri- and tetra-antennary complex N-glycans are generated by the action of GnTIV and GnTV. These branches can be elongated further with e.g. galactose, N-acetylgalactosamine, sialic acid, and fucose.

[0055] FIG. 2:

[0056] Branch specificity of ST6Gal1 activity. ST6Gal1 catalyzes sialylation of the primary branches of tri- and tetra-antennary N-glycans, whereas sialylation of the additional branches of tri- and tetra-antennary N-glycans is catalyzed by ST3Gal4.

[0057] FIG. 3:

[0058] DSA and PHA-L lectin immunoblot analysis of N-glycans from human AAT purified from human serum (Prolastin) in comparison to rhAAT expressed in CAP wild-type cells.

[0059] FIG. 4:

[0060] DSA lectin immunoblot of recombinant hAAT expressed in wildtype CAP cells (control) or CAP cells harboring a GnTIVb knock-out in the presence or absence of sialyltransferases. CAP wild-type cells or CAP cells harboring a GnTIVb knock-out (without (A) or with additional overexpression of sialyltransferases ST3Gal4 (B) or ST6Gal1 (C)) were modified to stably express rhAAT. rhAAT was purified from cell culture supernatants and was subject to lectin blot analysis. Datura stramonium (DSA) lectin detects core 1.fwdarw.4 branched N-glycans synthesized by GnTIVb. Therefore, a diminished signal in the DSA blot means a decreased amount of N-glycans with 1.fwdarw.4 branches, indicating a decreased or absent GnTIVb enzyme activity, resulting in N-glycan structures without a 1.fwdarw.4 branch and therefore with only di- or tri-antennary structures. As loading control, the same samples were subjected to western blot analysis using a hAAT specific antibody.

[0061] FIG. 5:

[0062] PHA-L lectin immunoblot of recombinant hAAT expressed in wildtype CAP cells or CAP cells harboring a GnTV knock-out in the presence or absence of sialyltransferases. CAP wild-type cells or CAP cells harboring a GnTV knock-out (with or without additional overexpression of ST6Gal1) were modified in order to stably express rhAAT. rhAAT was purified from cell culture supernatants and was subject to lectin blot analysis. Phytohemagglutinin-L (PHA-L) lectin detects core 1.fwdarw.6 branched N-glycans synthesized by GnTV. Therefore, a diminished signal in the PHA-L blot is indicating a decreased or absent GnTV enzyme activity, resulting in N-glycan structures without a 1.fwdarw.6 branch and therefore with only di- or tri-antennary structures. Binding of PHA-L lectin to the 1.fwdarw.6 branched N-glycan is inhibited by sialic acid, therefore signal is decreased in the samples purified from cells additionally expressing ST6Gal1. Nevertheless, a diminished signal in the GnTV knock-out sample in comparison to the CAP wild-type sample can be observed. As loading control the same samples were subject to western blot analysis using an hAAT specific antibody.

[0063] FIG. 6:

[0064] Summary of MALDI-TOF mass spectrum analysis of rhAAT N-glycans of protein expressed in CAP wild-type or in CAP GnTIVb with or without additional overexpression of ST3Gal4 or ST6GAL1. Displayed are the sums of relative amounts of the hybrid, di-, tri- and tetra-antennary N-glycan structures of CAP GnTIVb normalized to CAP wt. Glycan fragments generated by MALDI-TOF or negligible signal were not used for this analysis. Knock-out of GnTIVb results in a 3- to 15-fold decrease in the total amounts of tetra-antennary N-glycans. Residual amounts of tetra-antennary N-glycans could be due to the enzymatic activity of GnTIVa.

[0065] FIG. 7:

[0066] DSA and PHA-L lectin immunoblot of recombinant hAAT expressed in wildtype CAP cells or CAP cells harboring a GnTV or GnTIVb/GnTV knock-out. CAP wild-type cells or CAP single cell clones harboring a GnTV, or GnTIVb/GnTV double knock-out were modified in order to stably express rhAAT. rhAAT was purified from cell culture supernatants and was subject to lectin blot analysis. DSA lectin detects core 1.fwdarw.4 branched N-glycans synthesized by GnTIVb. Therefore, a diminished signal in the DSA blot means a decreased amount of N-glycans with 1.fwdarw.4 branches. PHA-L lectin detects core 1.fwdarw.6 branched N-glycans synthesized by GnTV. Therefore, a diminished signal in the PHA-L blot means a decreased amount of N-glycans with 1.fwdarw.6 branches. AAT purified from CAP single cell clones with the GnTIVb/GnTV double knock-out display only a minor signal in both lectin blot analysis in comparison to the wild type control indicating that almost all N-glycans are di-antennary.

[0067] FIG. 8:

[0068] Comparison of rhAAT isoform patterns by IEF analysis. As the backbones of the different rhAAT are identical, changes in the IEF are most likely due to changes in the sialic acid content. rhAAT was expressed in wild-type, GnTIVb, GnTV, or GnTIVb/GnTV CAP cells with additional expression of ST6Gal. AAT purified from knock-out cells shows a less acidic pl, indicating a decrease in the total amount of sialic acids per molecule. In addition, this indicates a decrease of potential NeuAc binding sites due to a shift from tri- and tetra-antennary structures to di-antennary N-glycan structures.

[0069] FIG. 9:

[0070] ECL lectin immunoblot of recombinant hAAT expressed in wildtype CAP cells or CAP cells harboring a GnTIVb/GnTV knock-out. Erythrina crista-galli (ECL) lectin detects 1-4 linked terminal galactose on N-linked glycans. Due to the branch specificity of ST6Gal1 (Gal1.fwdarw.4GlcNAc1.fwdarw.2Man1.fwdarw.3 and Gal1.fwdarw.4GlcNAc1.fwdarw.2Man1.fwdarw.6), overexpression of ST6Gal1 results in complete sialylation of di-antennary N-glycans present on rhAAT expressed in GnTIVb/GnTV cells, whereas rhAAT expressed in wild-type CAP cells with additional overexpression of ST6Gal1 still harbors non-sialylated N-glycan branches. Neuraminidase catalyzes the hydrolysis of N-acetyl-neuraminic acid residues from oligosaccharide, thus neuraminidase treated samples serve as positive control. As loading control, the same samples were subjected to western blot analysis using an hAAT specific antibody.

[0071] FIG. 10:

[0072] DSA and PHA-L lectin immunoblot of recombinant rhC1 Inh expressed in wildtype CAP cells or CAP cells harboring a GnTIVb/GnTV double knock-out in comparison to plasma derived C1 Inhibitor (Berinert). CAP wild-type cells or CAP-ST6Gal1 single cell clones harboring a GnTIVb/GnTV double knock-out were modified in order to stably express rhC1 Inh. rhC1 Inh was purified from cell culture supernatants and was subjected to lectin blot analysis. Lectin Datura stramonium (DSA) lectin detects core 1.fwdarw.4 branched N-glycans synthesized by GnTIVb. Therefore, a diminished signal in the DSA blot proves a reduced amount of N-glycans with 1.fwdarw.4 branches. Phytohemagglutinin-L (PHA-L) lectin detects core 1.fwdarw.6 branched N-glycans synthesized by GnTV. Therefore, a diminished signal in the PHA-L blot proves a decreased amount of N-glycans with 1.fwdarw.6 branches. Plasma derived C1 Inh. as well as C1 Inh purified from CAP single cell clones with the GnTIVb/GnTV double knock-out display only a minor signal in both lectin blot analysis in comparison to the wild type control indicating that almost all N-glycans are di-antennary. The corresponding densitometrical analysis was normalized on the input using the C1-Inhibitor specific western blot as well as on the signal intensity of the CAP wild type.

[0073] The present invention will be further illustrated by the following examples without being limited thereto.

EXAMPLES

[0074] Material and Methods:

[0075] Cell Lines

[0076] Cell lines used in the present application are indicated in Table 1 below.

TABLE-US-00001 TABLE 1 Stable cell lines used in the present invention. Rec. Overexpression Expression of Protein of Cell Line GnTV/GnTIVb Expression Sialyltransferases CAP wild-type rhAAT CAP GnTIVb rhAAT CAP -GnTV rhAAT CAP GnTIVb/GnTV rhAAT CAP wild-type rhAAT ST6Gal1 CAP GnTIVb rhAAT ST6Gal1 CAP -GnTV rhAAT ST6Gal1 CAP GnTIVb/GnTV rhAAT ST6Gal1 CAP wild-type rhAAT ST3Gal4 CAP GnTIVb rhAAT ST3Gal4 CAP wild-type rhC1 lnh CAP GnTIVb/GnTV rhC1 lnh ST6Gal1

[0077] Cell Culture and Fermentation

[0078] The permanent human amniocyte cell line CAP 1D5 and all their derivates with diminished expression of GnTIVa/b and/or GnTV were cultured in suspension, either in chemically defined, animal component free CAP-CDM medium (CEVEC Pharmaceuticals, Germany) supplemented with 6 mM stable glutamine (biochrom, Germany), or in serum free PEM media (Life Technologies) supplemented with 4 mM stable glutamine (biochrom, Germany).

[0079] All cell lines were cultivated at 37 C. in shaker flasks (Corning, #431143, 125 mL (25 mL wv) or #431252, 3000 mL (1000 mL wv)) at 5% CO.sub.2, and 185 rpm. During fermentation CAP cells were fed at d3, d5, and d7 with 10% CAP-CDM feed solution (CEVEC Pharmaceuticals, Germany) and 4 mM stable glutamine (biochrom, Germany).

[0080] Cloning

[0081] For the generation of the CAP cell lines stably expressing ST3Gal4 and/or ST6Gal1, the cells were nucleofected with the corresponding nuclei acid constructs.

[0082] For designing the ST3Gal4 cDNA, sequence information of the precursor protein and mature protein was based of the database entry UniProt Q11206. For cloning, a ClaI restriction site and a Kozak sequence were added 5 of the start codon of the human ST3Gal4 cDNA and an EcoRV restriction site was added 3 of the stop codon to be inserted between the ClaI and EcoRV restriction sites in the pStbI-Neo-CMV-MCS() vector resulting in the expression plasmid pStbI-Neo-CMV-ST3Gal4. This vector contains a CMV promoter driving the expression of the gene of interest, followed by an SV40 intron for improved, splicing-mediated mRNA transport and a multiple cloning site for the insertion of the gene of interest. The selection marker is driven by the human ubiquitin (UbC) promoter. cDNA synthesis was performed at GeneArt (Germany, Life Technologies).

[0083] For designing the ST6Gal1 cDNA, sequence information of the precursor protein and mature protein was based of the database entry UniProt P15907. For cloning, a ClaI restriction site and a Kozak sequence were added 5 of the start codon of the human ST6Gal1 cDNA and a EcoRV restriction site was added 3 of the stop codon to be inserted between the ClaI and EcoRV restriction sites in the pStbI-Neo-CMV-MCS() vector resulting in the expression plasmid pStbI-Neo-CMV-ST6Gal1. cDNA synthesis was performed at GeneArt (Germany, Life Technologies).

[0084] Nucleofection and Pool Generation

[0085] Nucleofection was performed using a Nucleofector (LONZA) with the appropriate Nucleofector Kit (KitV) according to the manufacturer's protocol. Briefly, during exponential growth phase of the culture 110.sup.7 cells were harvested via centrifugation (150 g for 5 min) and resuspended in 100 l complete nucleofector solution and mixed with a total of 5 g plasmid. Nucleofection was performed using the X001 program (CAP cells). After the pulse, cells were recovered in 12 ml complete cell culture media in a 125 ml shaking flask. The cells were cultured as before at 37 C., 5% CO.sub.2, and 185 rpm. 72 to 96 h post-nucleofection cells were selected with 200 g/ml neomycin in order to generate stable pools.

[0086] Purification of AAT from Cell Culture Supernatants

[0087] Following harvest by tangential flow filtration and sterile filtration, the rhAAT containing cell culture supernatant was concentrated and diafiltrated against buffer for the first chromatography step. The diafiltrated solution was loaded on an AAT affinity chromatography column. Subsequently, rhAAT protein was eluted in the linear gradient with increasing MgCl.sub.2 concentrations. The pooled fractions were concentrated and the buffer was exchanged against formulation buffer using desalting columns.

[0088] Purification of C1 Inh from Cell Culture Supernatants

[0089] Following harvest by tangential flow filtration and sterile filtration, C1 Inh was purified from the cell culture supernatant by anion exchange chromatography column followed by a phenyl hydrophobic interaction chromatography column, which was run in negative mode. The flow-through of this chromatography step was collected, concentrated and the buffer was exchanged against formulation buffer using desalting columns.

[0090] Lectin Immunoblotting

[0091] Lectins are proteins that bind specific carbohydrate structures. Biotin-coupled lectins can therefore be used to analyze N-linked glycans. DSA lectin detects core 1.fwdarw.4 branched N-glycans synthesized by GnTIVb. PHA-L lectin detects core 1.fwdarw.6 branched N-glycans synthesized by GnTV. Purified protein or cell culture supernatants with or without diminished expression of GnTIVa/b and/or GnTV and with or without co-expression of ST3Gal4 and/or ST6Gal1 were separated as described above and blotted onto Amersham Hybond ECL nitrocellulose membrane (GE healthcare). The membrane was blocked for 1 h at RT with PBSTB (phosphate-buffered saline, pH=7.4, supplemented with 0.1% Tween 20 and 1% BSA). Afterwards, the membrane was incubated with the lectin diluted 1:2000 in PBSTB. After washing the membrane with PBST (phosphate-buffered saline, pH=7.4, supplemented with 0.1% Tween 20), the membrane was stained with streptavidin-coupled horseradish peroxidase for 1 h at RT (1:1000 diluted in PBSTB). The HRP signal was amplified using anti-streptavidin IgG and anti IgG-HRP. The proteins were detected using the Pierce ECL WB Substrate Kit via a chemiluminescence detector (INTAS).

[0092] IEF Analysis

[0093] Isoelectric focusing (IEF) was performed in order to analyze the isoelectric point (pi) of rhAAT purified from CAP cells expressing rhAAT with or without diminished expression of GnTIVa/b and/or GnTV and with or without co-expression of ST3Gal4 and/or ST6Gal1. The degree of sialylation correlates with a given proteins acidity and, therefore, with its pl. IEF analysis was done according to the manufacturers protocol (Invitrogen). Briefly, 5 g of purified protein were loaded on pH3-7 gels and subjected to electrophoresis (1 h 100 V, 1 h 200 V, 30 min 500 V). Proteins were stained with SimplyBlue SafeStain according to the manufacturers protocol (Invitrogen).

Example 1

[0094] Cells of the human amniocyte cell line CAP 1D5 were stably transfected with a plasmid coding for rhAAT. After pool generation cells were subject to fed-batch in order to generate sufficient amounts of recombinant AAT.

[0095] To determine the degree of core 1,6- and or core 1,4-branched N-glycans of AAT expressed in wild-type CAP cells in comparison to plasma-derived AAT (Prolastin), DSA and PHA-L lectin blots were performed. PHA-L reacts specifically with core 61,6-branched products synthesized by GnTV and DSA detects core 1.fwdarw.4 branched N-glycans synthesized by GnTIVb. Therefore, the increased signal in blots shown in FIG. 3 indicates a higher degree of tri- and/or tetra-antennary N-glycans in the AAT derived from wild-type CAP cells in comparison to the plasma derived AAT.

[0096] Lectin blot analysis (FIG. 3) reveals that as in other mammalian cell lines, expression of recombinant glycoproteins results in N-glycan structures with elevated amount of tri- and tetra-antennary structures compared to the corresponding plasma-derived human protein.

Example 2

[0097] Cells of the human amniocyte cell line CAP 1D5 with either wild type expression or diminished expression of GnTIVb were stably transfected with a vector encoding rhAAT. After pool generation, cell lines were subject to fed-batch in order to make sufficient amounts of recombinant AAT. rhAAT was purified from cell culture supernatants and was subject to lectin blot analysis. DSA lectin detects core 1.fwdarw.4 branched N-glycans synthesized by GnTIVb. The reduction in DSA-signal shown in FIG. 4A is due to a reduced or absent GnTIVb enzyme activity, resulting in N-glycan structures without a 1.fwdarw.4 branch and therefore without tetra-antennary structures. As loading control same samples were subject to western blot analysis using a hAAT specific antibody (FIG. 4).

[0098] These results were confirmed by MALDI-TOF analysis of N-glycans from AAT derived in CAP wildtype cells and CAP GnTIVb (FIG. 6), as knock-out of GnTIVb results in a 3- to 4-fold decrease in the total amounts of tetra-antennary N-glycans. Residual amount of tetra-antennary N-glycan structures are probably due to residual GnTIVb activity in the modified CAP cells or due to GnTIVa enzyme activity.

Example 3

[0099] Cells of the human amniocyte cell line CAP 1D5 with either wild type expression or diminished expression of GnTV were stably transfected. After pool generation cell lines were subject to fed-batch in order to make sufficient amounts of recombinant AAT.

[0100] Purified recombinant AAT was then analyzed to determine the degree of core 1,6-branched N-glycans in the AAT expressed in GnTV vs. wild-type CAP cells. In order to do so, a PHA-L (phytohemagglutinin-L) lectin blot was performed. PHA-L reacts specifically with core 131,6-branched glycostructures synthesized by GnTV.

[0101] As shown in FIG. 5 (lane 2 and 4) and FIG. 7B a diminished signal in the PHA-L blot was detected, indicating a decreased or absent GnTV enzyme activity, decreased amount of 1,6-branched products, and, therefore, reduced amount of tri- and/or tetra-antennary N-glycans.

Example 4

[0102] In order to investigate if an absent enzyme activity of GnTIVb and GnTV results in only di-antennary N-glycans, the following experiment was performed. CAP cells with either wild type expression or reduced expression of GnTIVb and GnTV (GnTIVb/GnTV cells) were nucleofected with a vector coding for rhAAT. After pool generation cell lines were subject to fed-batch in order to generate sufficient amounts of recombinant AAT. Subsequently, AAT was purified from the cell culture supernatant.

[0103] Next, the N-glycan structures from rhAAT expressed in wild-type or GnTIVb/GnTV CAP cells were analyzed by DSA and PHA-L lectin blots (FIG. 7).

[0104] Interestingly, in the DSA as well as in the PHA-L lectin blots only a background signal was detectable for the rhAAT expressed in GnTIVb/GnTV CAP, strongly suggesting that rhAAT expressed in these cells contains no or only minimal amounts of tri or tetra-antennary N-glycan structures.

Example 5

[0105] CAP wild-type cells expressing rhAAT or cells with reduced expression of GnTIVb and/or GnTV expressing rhAAT were further transfected with an additional vector encoding for ST6Gal1 or ST3Gal4. After pool generation, generated cell lines were subject to fed-batch in order to generate sufficient amounts of recombinant AAT. Subsequently, AAT was purified from the cell culture supernatant of the following cell lines: CAP-AAT, CAP-AAT+ST6Gal1, CAP-AAT+ST3Gal4, GnTIVb-AAT, GnTIVb-AAT+ST6Gal1, GnTIVb-AAT+ST3Gal4, GnTV-AAT, GnTV-AAT+ST6Gal1, AAGnTIVb/V-AAT, and AAGnTIVbN-AAT+ST6Gal1.

[0106] FIGS. 4 and 7 show that additional expression of sialyltransferases like ST3Gal4 or ST6Gal1 has no influence on the diminished amount of 1,6-branched N-glycans caused by the diminished expression of GnTIVb. In addition, FIGS. 5 and 7 show that additional expression of sialyltransferases has also no influence on the reduced amount of 1,4-branched N-glycans caused by the diminished expression of GnTV. Of note, binding of PHA-L lectin to the 1.fwdarw.6 branched N-glycan is inhibited by sialic acid, so that signal is decreased in the samples purified from cells additionally expressing ST6Gal1. Nevertheless, a diminished signal of the GnTV knock-out in comparison to the CAP wild-type cells can be observed.

[0107] As the backbones of the different rhAAT are identical, changes in the IEF indicate changes in the sialic acid content. rhAAT expressed in GnTIVb, GnTV, or GnTIVb/GnTV CAP cells with additional expression of ST6Gal1 results in a modified rhAAT which slightly shifts towards a less acidic pl, in comparison to the rhAAT expressed in parental CAP cells overexpressing ST6Gal1. This indicates a decrease in the total amount of sialic acids per molecule. The observation is consistent with a decrease of potential NeuAc binding sites due to a shift from tri- and tetra-antennary structures to di-antennary N-glycan structures (FIG. 8). This rational is supported by the ECL blot shown in FIG. 9.

Example 6

[0108] To explore if, like hAAT (see example 1), also rhC1 Inh from mammalian expression systems displays an increased amount of tri- and tetra-antennary N-glycan structures which is not seen in the serum derived counterpart, the degree of core 1,6- and or core 1,4-branched N-glycans of C1 Inh expressed in wild-type CAP cells in comparison to plasma-derived C1 Inh (Berinert) was determined by DSA (Datura stramonium) and PHA-L (Phytohemagglutinin-L) blots. PHA-L reacts specifically with core 61,6-branched antennae synthesized by GnTV and DSA detects core 1.fwdarw.4 branched N-glycans synthesized by GnTIVb. Therefore, the signal ratio in blots shown in FIG. 10 (lane1, CAP wild type, and lane 2, Berinert) indicates a significantly higher degree of tri- and/or tetra-antennary N-glycans in the C1 Inh derived from wild-type CAP cells in comparison to the plasma derived C1 Inh.

[0109] Therefore, the lectin blot analysis in FIG. 10 reveals that, like for rhAAT, expression of the recombinant glycoprotein C1 Inh in unmodified CAP cells results in N-glycan structures with elevated amounts of tri- and tetra-antennary structures compared to the corresponding plasma-derived human protein (Berinert).

[0110] In order to investigate if an absent enzyme activity of GnTIVb and GnTV results in only di-antennary N-glycans, not only in rhAAT but also in the complex glycoprotein C1 Inh, non-glycoengineered CAP cells with wild type expression of GnTIVb and GnTV and CAP-ST6Gal1 single cell clones with reduced expression of GnTIVb and GnTV (GnTIVb/GnTV cells) were nucleofected with a vector coding for rhC1 Inh. After pool generation, cell lines were subjected to fed-batch in order to generate sufficient amounts of recombinant C1 Inh. Subsequently, C1 Inh was purified from the cell culture supernatant. Next, the N-glycan structures from rhC1 Inh expressed in wild-type or GnTIVb/GnTV CAP-ST6Gal1 cells were analyzed by DSA and PHA-L lectin blots (FIG. 10).

[0111] Interestingly, in the DSA as well as in the PHA-L lectin blots, only a background signal was detectable for the rhC1 Inh expressed in GnTIVb/GnTV CAP-ST6Gal1 cells, strongly suggesting that rhC1 Inh expressed in these cells contains no or only minimal amounts of tri or tetra-antennary N-glycan structures.

[0112] The present invention discloses the following amino acid sequences.

TABLE-US-00002 HumanGnTV SEQIDNO:1 MALFTPWKLSSQKLGFFLVTFGFIWGMMLLHFTIQQRTQPESSSMLREQI LDLSKRYIKALAEENRNVVDGPYAGVMTAYDLKKTLAVLLDNILQRIGKL ESKVDNLVVNGTGTNSTNSTTAVPSLVALEKINVADIINGAQEKCVLPPM SDGYPHCEGKIKWMKDMWRSDPCYADYGVDGTCSFFIYLSEVENWCPHLP WRAKNPYEEADHNSLAEIRTDFNILYSMMKKHEEFRWMRLRIRRMADAWI QAIKSLAEKQNLEKRKRKKVLVHLGLLTKESGFKIAETAFSGGPLGELVQ WSDLITSLYLLGHDIRISASLAELKEIMKKVVGNRSGCPTVGDRIVELIY IDIVGLAQFKKTLGPSWVHYQCMLRVLDSFGTEPEFNHANYAQSKGHKTP WGKWNLNPQQFYTMFPHTPDNSFLGFVVEQHLNSSDIHHINEIKRQNQSL VYGKVDSFWKNKKIYLDIIHTYMEVHATVYGSSTKNIPSYVKNHGILSGR DLQFLLRETKLFVGLGFPYEGPAPLEAIANGCAFLNPKFNPPKSSKNTDF FIGKPTLRELTSQHPYAEVFIGRPHVWTVDLNNQEEVEDAVKAILNQKIE PYMPYEFTCEGMLQRINAFIEKQDFCHGQVMWPPLSALQVKLAEPGQSCK QVCQESQLICEPSFFQHLNKDKDMLKYKVTCQSSELAKDILVPSFDPKNK HCVFQGDLLLFSCAGAHPRHQRVCPCRDFIKGQVALCKDCL HumanGnTIVa SEQIDNO:2 MRLRNGTVATALAFITSFLTLSWYTTWQNGKEKLIAYQREFLALKERLRI AEHRISQRSSELNTIVQQFKRVGAETNGSKDALNKFSDNTLKLLKELTSK KSLQVPSIYYHLPHLLKNEGSLQPAVQIGNGRTGVSIVMGIPTVKREVKS YLIETLHSLIDNLYPEEKLDCVIVVFIGETDIDYVHGVVANLEKEFSKEI SSGLVEVISPPESYYPDLTNLKETFGDSKERVRWRTKQNLDYCFLMMYAQ EKGIYYIQLEDDIIVKQNYFNTIKNFALQLSSEEWMILEFSQLGFIGKMF QAPDLTLIVEFIFMFYKEKPIDWLLDHILWVKVCNPEKDAKHCDRQKANL RIRFRPSLFQHVGLHSSLSGKIQKLTDKDYMKPLLLKIHVNPPAEVSTSL KVYQGHTLEKTYMGEDFFWAITPIAGDYILFKFDKPVNVESYLFHSGNQE HPGDILLNTTVEVLPFKSEGLEISKETKDKRLEDGYFRIGKFENGVAEGM VDPSLNPISAFRLSVIQNSAVWAILNEIHIKKATN HumanGnTIVb SEQIDNO:3 MRLRNGTFLTLLLFCLCAFLSLSWYAALSGQKGDVVDVYQREFLALRDRL HAAEQESLKRSKELNLVLDEIKRAVSERQALRDGDGNRTWGRLTEDPRLK PWNGSHRHVLHLPTVFHHLPHLLAKESSLQPAVRVGQGRTGVSVVMGIPS VRREVHSYLTDTLHSLISELSPQEKEDSVIVVLIAETDSQYTSAVTENIK ALFPTEIHSGLLEVISPSPHFYPDFSRLRESFGDPKERVRWRTKQNLDYC FLMMYAQSKGIYYVQLEDDIVAKPNYLSTMKNFALQQPSEDWMILEFSQL GFIGKMFKSLDLSLIVEFILMFYRDKPIDWLLDHILWVKVCNPEKDAKHC DRQKANLRIRFKPSLFQHVGTHSSLAGKIQKLKDKDFGKQALRKEHVNPP AEVSTSLKTYQHFTLEKAYLREDFFWAFTPAAGDFIRFRFFQPLRLERFF FRSGNIEHPEDKLFNTSVEVLPFDNPQSDKEALQEGRTATLRYPRSPDGY LQIGSFYKGVAEGEVDPAFGPLEALRLSIQTDSPVWVILSEIFLKKAD