Highly active glycoproteins-process conditions and an efficient method for their production
The present invention relates to highly active glycoproteins with specific sialylation degrees, a pharmaceutical composition for use in diagnosis or therapy comprising the glycoproteins, a method for the determination of highly active glycoproteins and of the conditions for their production, a method for producing the highly active glycoproteins, and a method for differential sialylation of secretory glycoproteins. The invention also relates to the use of the recombinantly expressed highly active glycoproteins for biological purposes and for prophylactic and/or therapeutic treatment or diagnosis of diseases, particularly bone marrow transplantation, neutropenia, cytopenia, AML and myelodysplastic syndromes, cancer, HIV and/or diseases of hematopoietic systems.
1. A process for the production of a highly active sialylation form of a target glycoprotein, said highly active sialylation form of said target glycoprotein having a sialylation degree that is lower than that of a reference glycoprotein, yet said highly active sialylation form of said target glycoprotein also having an activity that is higher than that of said reference glycoprotein, wherein said sialylation degree also corresponds to a determined concentration of at least one sialic acid precursor additive, said process comprising:
(a) providing a human expression cell line which harbors at least one defect in the sugar nucleotide biosynthetic pathway of sialic acids, said at least one defect being a defect in at least one enzyme selected from the group consisting of UDP-GlcNAc-2-epimerase, N-acetylmannosamine kinase, N-acetylglucosamine kinase, Neu5Ac-9-P-synthetase, Neu5Ac-9-P-phosphatase, CMP-Neu5Ac synthetase and CMP-sialic acid transporter, and said human expression cell line being transfected with a nucleic acid encoding the target glycoprotein;
(b) determining a concentration of said at least one sialic acid precursor additive that is able to reconstitute said defect by a process comprising:
(i) expressing a plurality of different sialylation forms of said target glycoprotein by differential sialylation using different concentrations of said at least one sialic acid precursor additive;
(ii) determining the activity of each of the different sialylation forms in comparison to the reference glycoprotein; and
(iii) selecting said highly active sialylation form of said target glycoprotein from among the different sialylation forms expressed, and determining the concentration of the at least one sialic acid precursor additive that correlates with the expression of said highly active sialylation form of said target glycoprotein; and
(c) expressing the highly active sialylation form of said target glycoprotein in said human expression cell line in a medium supplemented with the at least one sialic acid precursor additive at the concentration determined in step (b)(iii).
2. The process of claim 1 , wherein the defect in the biosynthetic pathway of sialic acids is a loss-of-function of a protein involved in the sugar transportation, and wherein the protein involved in the sugar transportation is a CMP-sialic acid transporter.
3. The process of claim 1 , wherein the addition of said sialic acid precursor additive results in glycoproteins with natural sialic acid modifications.
4. The process of claim 1 , wherein the defect in the biosynthetic pathway of sialic acids results in a decreased or absent enzymatic activity of UDP-N-acetylglucosamine-2-epimerase.
5. The process of claim 1 , wherein the glycoprotein is secreted by the cells of the expression cell line.
6. The process of claim 1 , wherein the expression cell line is selected from the group consisting of NM-F9 (deposited under the accession number DSM ACC2606), and NM-D4 (deposited under the accession number DSM ACC2605).
7. The process of claim 1 , wherein the glycoprotein is selected from the group consisting of Glycophorin A, erythropoietin (EPO), granulocyte colony stimulating factor (G-CSF), granulocytemacrophage colony stimulating factor (GM-CSF), follicle stimulating hormone (FSH), human chorionic gonadotrophin (hCG), luteinizing hormone (LH), an interferon, an interleukin, an antibody, or one or more sialylated fragments of said glycoproteins.
8. The process of claim 1 , wherein at least one sialic acid precursor additive is selected from the group consisting of ManNAc, acetylated ManNAc, peracetylated ManNAc and fetuin.
9. The process of claim 1 , wherein the glycoprotein is GM-CSF, the expression cell line is NM-F9 (deposited under the accession number DSM ACC2606), the sialic acid precursor additive is ManNAc, and the ManNac concentration determined in step b) and employed in step c) is 90 mM.
10. A process for the production of a highly active glycoprotein GM-CSF, comprising:
(a) providing a human expression cell line which is NM-F9 (deposited under the accession number DSM ACC2606), and transfecting said cell line with a nucleic acid encoding the glycoprotein GM-CSF;
(b) expressing said GM-CSF glycoprotein in said NM-F9 cell line in a medium supplemented with ManNAc at a concentration of 90 mM.