IP Library › Granted Patent US 12,435,115
Granted Patent B2
US 12,435,115 · App. 17/975,572 · Granted Oct 7, 2025

Production of recombinant lubricin

Inventors: Tannin A. Schmidt (Avon, CT); Gregory D. Jay (Norfolk, MA)
Assignee: Lubris, LLC
C07K14/4725A61K9/0014A61K9/0019A61K9/0048A61K9/008A61K31/728A61K38/1709A61K47/36C12P21/005
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,435,115
App. No.
17/975,572
Granted
Oct 7, 2025
Kind
B2
Abstract

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.

Claims (20)

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 produces a lubricin glycoprotein comprising at least 30% by weight glycosidic residues, 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 1 , wherein the glycosidic residues are enriched in sulfated saccharide side chains as compared with native human lubricin.

5. The method of claim 1 , wherein the lubricin glycoprotein produces a static coefficient of friction no greater than 150% of the static coefficient of friction of purified native bovine lubricin as measured in a cartilage on cartilage friction test.

6. The method of claim 1 , wherein the lubricin glycoprotein purified from said culture medium comprises monomeric, dimeric and/or multimeric lubricin species.

7. The method of claim 1 , wherein the human PRG4 gene comprises the nucleic acid sequence of SEQ ID NO:2.

8. 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.

9. The method of claim 8 , wherein the chromatin element is a matrix attachment region.

10. The method of claim 1 , wherein at least 95% by weight of glycosylation of the lubricin glycoprotein is core 1 glycosylation.

11. The method of claim 1 , wherein at least 99% by weight of glycosylation of the lubricin glycoprotein is core 1 glycosylation.

12. 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.

13. The method of claim 1 , wherein the lubricin glycoprotein comprises a dimeric lubricin species.

14. 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.

15. The method of claim 1 , wherein the culturing is in at least 10, 50, or 100 liters of medium.

16. The method of claim 1 , wherein the lubricin glycoprotein comprises at least 35% by weight glycosidic residues.

17. The method of claim 1 , wherein the lubricin glycoprotein comprises the amino acid sequence of residues 25-1404 of SEQ ID NO:1.

18. The method of claim 17 , wherein the culturing produces said lubricin glycoprotein at a concentration in the medium of at least 0.5 g/liter.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded May 29, 2025
From: SCHMIDT, TANNIN; JAY, GREGORY D.
To: LUBRIS LLC
Reel/Frame 071249/0932 →
Continuity (5)
Continuation 16904978 · Jun 18, 2020
Continuation 16183497 · Nov 7, 2018
Continuation 15030825
Provisional Application 61894366 · Oct 22, 2013
Related Publication 20230234999A1 · Jul 27, 2023
References Cited (109)
US 6743774B1 · Jay · 2004 [cited by applicant]
US 6960562B2 · Jay · 2005 [cited by applicant]
US 7001881B1 · Jay · 2006 [cited by applicant]
US 7129062B2 · Mermod et al. · 2006 [cited by applicant]
US 7415381B2 · Jay · 2008 [cited by applicant]
US 7618941B2 · Jay · 2009 [cited by applicant]
US 7642236B2 · Flannery et al. · 2010 [cited by applicant]
US 7893029B2 · Flannery et al. · 2011 [cited by applicant]
US 8026346B2 · Jay · 2011 [cited by applicant]
US 8252917B2 · Mermod et al. · 2012 [cited by applicant]
US 8506944B2 · Sullivan et al. · 2013 [cited by applicant]
US 8551467B2 · Sullivan et al. · 2013 [cited by applicant]
US 8563028B2 · Sullivan et al. · 2013 [cited by applicant]
US 8680057B2 · Jay · 2014 [cited by applicant]
US 8795723B2 · Gervais et al. · 2014 [cited by applicant]
US 8945604B2 · Sullivan et al. · 2015 [cited by applicant]
US 8980840B2 · Truitt, III et al. · 2015 [cited by applicant]
US 9107885B2 · Sullivan et al. · 2015 [cited by applicant]
US 9138457B2 · Sullivan et al. · 2015 [cited by applicant]
US 9248161B2 · Sullivan et al. · 2016 [cited by applicant]
US 9393285B2 · Sullivan et al. · 2016 [cited by applicant]
US 9421241B2 · Sullivan et al. · 2016 [cited by applicant]
US 9585936B2 · Sullivan et al. · 2017 [cited by applicant]
US 9730865B2 · Sullivan et al. · 2017 [cited by applicant]
US 9730978B2 · Sullivan et al. · 2017 [cited by applicant]
US 9982027B2 · Schmidt · 2018 [cited by applicant]
US 10125180B2 · Schmidt et al. · 2018 [cited by applicant]
US 10383796B2 · Truitt et al. · 2019 [cited by applicant]
US 10500250B2 · Jay et al. · 2019 [cited by applicant]
US 10500251B2 · Jay et al. · 2019 [cited by applicant]
US 10723773B2 · Schmidt et al. · 2020 [cited by applicant]
US 10960047B2 · Jay et al. · 2021 [cited by applicant]
US 10967048B2 · Jay et al. · 2021 [cited by applicant]
US 11213566B2 · Truitt, III et al. · 2022 [cited by applicant]
US 20030087342A1 · Mermod et al. · 2003 [cited by applicant]
US 20040072741A1 · Jay · 2004 [cited by applicant]
US 20040229804A1 · Jay · 2004 [cited by applicant]
US 20060240037A1 · Fey et al. · 2006 [cited by applicant]
US 20070111327A1 · Jay · 2007 [cited by applicant]
US 20070249557A1 · Jay · 2007 [cited by applicant]
US 20080139458A1 · Jay et al. · 2008 [cited by applicant]
US 20080287369A1 · Jay · 2008 [cited by applicant]
US 20090068247A1 · Jay · 2009 [cited by applicant]
US 20090104148A1 · Jay et al. · 2009 [cited by applicant]
US 20090155200A1 · Jay · 2009 [cited by applicant]
US 20100098749A1 · Barenholz et al. · 2010 [cited by applicant]
US 20100204087A1 · Jay · 2010 [cited by applicant]
US 20110059902A1 · Sullivan et al. · 2011 [cited by applicant]
US 20110061117A1 · Mermod et al. · 2011 [cited by applicant]
US 20110142908A1 · Sullivan et al. · 2011 [cited by applicant]
US 20120052077A1 · Truitt, III et al. · 2012 [cited by applicant]
US 20120134925A1 · Sullivan et al. · 2012 [cited by applicant]
US 20120231449A1 · Mermod et al. · 2012 [cited by applicant]
US 20120321611A1 · Sullivan et al. · 2012 [cited by applicant]
US 20120321693A1 · Sullivan et al. · 2012 [cited by applicant]
US 20130039865A1 · Truitt, III et al. · 2013 [cited by applicant]
US 20130116186A1 · Jay · 2013 [cited by applicant]
US 20130143264A1 · Nicolas et al. · 2013 [cited by applicant]
US 20130196930A1 · Flannery et al. · 2013 [cited by applicant]
US 20130315973A1 · Jay · 2013 [cited by applicant]
US 20140179611A1 · Jay · 2014 [cited by applicant]
US 20160235809A1 · Sullivan et al. · 2016 [cited by applicant]
US 20160250286A1 · Schmidt · 2016 [cited by applicant]
US 20170312335A1 · Truitt, III et al. · 2017 [cited by applicant]
US 20180140546A1 · Sullivan et al. · 2018 [cited by applicant]
US 20180161393A1 · Jay et al. · 2018 [cited by applicant]
WO WO1998008934A1 · 1998 [cited by applicant]
WO WO2005102363A2 · 2005 [cited by applicant]
WO WO2006012492A2 · 2006 [cited by applicant]
WO WO20080143816A1 · 2008 [cited by applicant]
WO WO2015061488A1 · 2015 [cited by applicant]
WO WO2015081121A1 · 2015 [cited by applicant]
WO WO2016123123A1 · 2016 [cited by applicant]
WO WO2016187414A1 · 2016 [cited by applicant]
“Selexis Lubris Partnership Advances Difficult-to-Express Protein Towards Clinic,” Selexis Archived Press Release, Jun. 27, 2012 (Jun. 27, 2012), pp. 1-2. Retrieved from the Internet: www.selexis.com/#!12-selexis-lubris… [cited by applicant]
Abubacker S et al., (2016), ‘Full-Length Recombinant Human Proteoglycan 4 Interacts with Hyaluronan to Provide Cartilage Boundary Lubrication,’ Ann Biomed Eng, 44(4):1128-37. [cited by applicant]
Ali et al., (2014) “The O-glycomap of Lubricin, a Novel Mucin Responsible for Joint Lubrication, Identified by Site-specific Glycopeptide Analysis”, Molecular & Cellular Proteomics 13: 10.1074/mcp.M114.040865, 3396-3409… [cited by applicant]
Appendix 1B: Symbol Nomenclature for Glycans (SNFG) in Essentials of Glycobiology, 3rd Ed. Varki et al., eds., 2015-2017, Cold Springs Harbor Laboratory Press: Cold Spring Harbor, NY, (1 page). [cited by applicant]
Brockhausen et al., Chapter 10: O-GalNAc Glycans in “Essentials of Glycobiology, 3rd Ed.” Varki et al., eds., 2015-2017, Cold Springs Harbor Laboratory Press: Cold Spring Harbor, NY, (20 pages). [cited by applicant]
Brockhausen I et al., ‘Chapter 9. O-GalNAc Glycans,’ Essentials of Glycobiology, (2nd Ed, 2009), A Varki et al., (Eds), Cold Spring Harbor Laboratory Press, Cold Spring, NY (Pub), pp. 115-127 (retrieved from the interne… [cited by applicant]
Croset et al., (2012), ‘Differences in the Glycosylation of Recombinant Proteins Expressed in HEK and CHO Cells,’ Journal of Biotechnology 161, 336-348. [cited by applicant]
Dorosz S et al., (2003), ‘Cartilage Boundary Lubricating Ability of Full-Length Human Recombinant PRG4—Alone and in Combination with Hyaluronan,’ 59th Annual Meeting of the Orthopaedic Research Society, San Antonio, TX,… [cited by applicant]
Dorosz S et al., (2003), ‘Cartilage Boundary Lubricating Ability of Full-Length Human Recombinant PRG4—Alone and in Combination with Hyaluronan,’ 59th Annual Meeting of the Orthopaedic Research Society, San Antonio, TX,… [cited by applicant]
Estrella RP et al., (2010), ‘The Glycosylation of Human Synovial Lubricin: Implications for its Role in Inflammation,’ Biochem J, 429(2):359-67. [cited by applicant]
Flannery et al., “Prevention of Cartilage Degeneration in a Rat Model of Osteoarthritis by Intraarticular Treatment with Recombinant Lubricin”, Arthritis & Rheumatism, American College of Rheumatology, Mar. 3, 2009, vol… [cited by applicant]
Girod PA et al., (2007), ‘Genome-wide Prediction of Matrix Attachment Regions that Increase Gene Expression in Mammalian Cells,’ Nat Methods, 4(9):747-53. [cited by applicant]
Gleghorn JP et al., (2009), ‘Boundary Mode Lubrication of Articular Cartilage by Recombinant Human Lubricin,’ J Orthop Res, 27(6):771-7. [cited by applicant]
Harraghy N et al., (2008), ‘Sustained Transgene Expression Using MAR Elements,’ Curr Gene Ther, 8(5):353-66. [cited by applicant]
Hart CM and Laemmli UK, (1998), ‘Facilitation of Chromatin Dynamics by SARs,’ Curr Opin Genet Dev, 8(5):519-25. [cited by applicant]
Ikegawa S et al., (2000), ‘Isolation Characterization and Mapping of the Mouse and Human PRG4 (Proteoglycan 4) Genes,’ Cytogenet Cell Genet, 90(3-4):291-7. [cited by applicant]
International Search Report (Form ISA/201) for International Application No. PCT/US2014/061827 mailed Feb. 5, 2015 (4 pages). [cited by applicant]
Jay GD et al, Homology of lubricin and superficial zone protein (SZP): products of megakaryocyte stimulating factor (MSF) gene expression by human synovial fibroblasts and articular chondrocytes localized to chromosome … [cited by applicant]
Jay GD et al., (2001), ‘Boundary Lubrication by Lubricin is Mediated by O-Linked β(1-3)Gal-GalNAc Oligosaccharides,’ Glycoconj J, 18(10):807-15. [cited by applicant]
Jay GD et al., (2007), ‘Association Between Friction and Wear in Diarthrodial Joints Lacking Lubricin,’ Arthritis Rheum, 56(11):3662-9. [cited by applicant]
Jay GD et al., (2010), ‘Prevention of Cartilage Degeneration and Restoration of Chondroprotection by Lubricin Tribosupplementation in the Rat Following Anterior Cruciate Ligament Transection,’ Arthritis Rheum, 62(8):238… [cited by applicant]
Jay GD, (2004), ‘Lubricin and Surfacing of Articular Joints,’ Curr Opin Orthop, 15(5):355-9. [cited by applicant]
Jenuwein T et al., (1997), ‘Extension of Chromatin Accessibility by Nuclear Matrix Attachment Regions,’ Nature, 385(6613):269-72. [cited by applicant]
Jones Arc et al., (2002), ‘Degradation of PRG4/SZP by Matrix Proteases,’ 49th Annual Meeting of the Orthopaedic Reserach Study, Paper #0133, 1 page. [cited by applicant]
Jones Arc et al., (2007), ‘Binding and Localization of Recombinant Lubricin to Articular Cartilage Surfaces,’ J Orthop Res, 25(3):283-92. [cited by applicant]
Kooyman J et al., (2010), ‘Cartilage Boundary Lubricating Properties of Native Proteoglycan 4 Purified from Normal Bovin Synovial Fluid,’ 56th Annual Meeting of the Orthopaedic Research Society, New Orleans, LA, Mar. 6-… [cited by applicant]
Könitzer, (2015), “A global RNA-seq-driven analysis of CHO host and production cell lines reveals distinct differential expression patterns of genes contributing to recombinant antibody glycosylation”, Biotechnol. J., 1… [cited by applicant]
Le Fourn et al., (2014), “CHO Cell engineering to prevent polypeptide aggregation and improve therapeutic protein secretion,” Metabolic Engineering 21:91-102. [cited by applicant]
Lee DW et al., (2013), ‘Stick-Slip Friction and Wear of Articular Joints,’ Proc Natl Acad Sci USA, 110(7):E567-74. [cited by applicant]
Ludwig TE et al., (2012), ‘Diminished Cartilage-Lubricating Ability of Human Osteoarthritic Synovial Fluid Deficient in Proteoglycan 4 Restoration Through Proteoglycan 4 Supplementation,’ Arthritis Rheum, 64(12):3963-71. [cited by applicant]
Schmidt TA et al., (2007), ‘Boundary Lubrication of Articular Cartilage Role of Synovial Fluid Constituents,’ Arthritis Rheum, 56(3):882-91. [cited by applicant]
Schmidt TA et al., (2009), ‘Disulfide-Bonded Multimers of Proteoglycan 4 PRG-4 are Present in Normal Synovial Fluids,’ Biochim Biophys Acta, 1790(5):375-84. [cited by applicant]
Supplementary European Search Report for European Patent No. EP14856188, Dated Apr. 6, 2017, 3 pages. [cited by applicant]
Written Opinion of the International Searching Authority (Form ISA/237) for International Application No. PCT/US2014/061827 mailed Feb. 5, 2015 (7 pages). [cited by applicant]
Yang et al., (2014), “The GalNAc-type OGlycoproteome of CHO Cells Characterized by the Simple Cell Strategy”, Molecular & Cellular Proteomics, 13:3224-3225. [cited by applicant]