Production of recombinant lubricin
Disclosed are new recombinant isoforms of human-like lubricin or PRG4 glycoprotein having outstanding lubrication properties and a novel glycosylation pattern, and methods for their manufacture at high levels enabling commercial production.
1. A method of manufacture of a recombinant lubricin glycoprotein comprising the steps of:
culturing, in a medium, Chinese hamster ovary (CHO) cells transfected with and which express the human PRG4 gene and post translationally glycosylate the expression product, wherein said culturing is for a time and under culture conditions sufficient to produce a lubricin glycoprotein comprising at least 30% by weight glycosidic residues and at a concentration in the medium of at least 0.4 g lubricin glycoprotein/liter, wherein at least 90% of said glycosidic residues are core 1 glycosidic residues; and
purifying the lubricin glycoprotein from said medium.
2. The method of claim 1 , wherein the CHO cells are CHO-M cells comprising a nucleic acid encoding the human PRG4 gene.
3. The method of claim 1 , wherein the CHO cells are transfected with a first vector comprising a nucleic acid encoding a chromatin element and a second vector comprising a nucleic acid encoding the human PRG4 gene.
4. The method of claim 3 , wherein the chromatin element is a boundary element, matrix attachment region, locus control region or a universal chromatin opening element.
5. The method of claim 4 , wherein the chromatin element is a matrix attachment region.
6. The method of claim 1 , wherein the culturing is for a time and under culture conditions sufficient to produce said lubricin glycoprotein at a concentration in the medium of at least 0.5 g/liter.
7. The method of claim 1 , wherein the culturing is for a time and under culture conditions sufficient to produce said lubricin glycoprotein at a concentration in the medium of at least 0.8 g/liter.
8. The method of claim 1 , wherein at least 95% by weight of glycosylation of the lubricin glycoprotein is core 1 glycosylation.
9. The method of claim 1 , wherein at least 99% by weight of glycosylation of the lubricin glycoprotein is core 1 glycosylation.
10. The method of claim 1 , wherein the glycosidic residues are enriched in sulfated saccharide side chains as compared with native human lubricin.
11. The method of claim 1 , wherein the lubricin glycoprotein comprises a monomeric lubricin species co-purified from said culture medium and in admixture with a multimeric lubricin species.
12. The method of claim 1 , wherein the lubricin glycoprotein comprises a dimeric lubricin species.
13. The method of claim 1 , wherein the lubricin glycoprotein has a molecular weight of at least 1200 kDa and comprises at least five disulfide-bonded or non-covalently associated individually glycosylated amino acid chains.
14. The method of claim 1 , wherein the culturing is in at least 10, 50, or 100 liters of medium.
15. The method of claim 1 , wherein the lubricin glycoprotein comprises at least 35% by weight glycosidic residues.
16. The method of claim 1 , wherein the recombinant lubricin glycoprotein comprises the amino acid sequence of SEQ ID NO:1.