title: Scalable Production of a Multifunctional Protein (TSG-6) That Aggregates with Itself and the CHO Cells That Synthesize It.
abstract: TNF-α stimulated gene/protein 6 (TNFAIP6/TSG-6) is a multifunctional protein that has a number of potential therapeutic applications. Experiments and clinical trials with TSG-6, however, have been limited by the technical difficulties of producing the recombinant protein. We prepared stable clones of CHO cells that expressed recombinant human TSG-6 (rhTSG-6) as a secreted glycoprotein. Paradoxically, both cell number and protein production decreased dramatically when the clones were expanded. The decreases occurred because the protein aggregated the synthesizing CHO cells by binding to the brush border of hyaluronan that is found around many cultured cells. In addition, the rhTSG-6 readily self-aggregated. To address these problems, we added to the medium an inhibitor of hyaluronan synthesis and heparin to compete with the binding of TSG-6 to hyaluronan. Also, we optimized the composition of the culture medium, and transferred the CHO cells from a spinner culture system to a bioreactor that controlled pH and thereby decreased pH-dependent binding properties of the protein. With these and other improvements in the culture conditions, we obtained 57.0 mg ± 9.16 S.D. of rhTSG-6 in 5 or 6 liter of medium. The rhTSG-6 accounted for 18.0% ± 3.76 S.D. of the total protein in the medium. We then purified the protein with a Ni-chelate column that bound the His tag engineered into the C-terminus of the protein followed by an anion exchange column. The yield of the purified monomeric rhTSG-6 was 4.1 mg to 5.6 mg per liter of culture medium. After intravenous injection into mice, the protein had a longer plasma half-life than commercially available rhTSG-6 isolated from a mammalian cell lysate, apparently because it was recovered as a secreted glycoprotein. The bioactivity of the rhTSG-6 in suppressing inflammation was demonstrated in a murine model.
authors.affiliation: Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.
authors.forename: Dong-Ki
authors.initials: DK
authors.lastname: Kim
authors.affiliation: Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.
authors.forename: Hosoon
authors.initials: H
authors.lastname: Choi
authors.affiliation: Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.
authors.forename: Hidetaka
authors.initials: H
authors.lastname: Nishida
authors.affiliation: Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.
authors.forename: Joo Youn
authors.initials: JY
authors.lastname: Oh
authors.affiliation: Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.
authors.forename: Carl
authors.initials: C
authors.lastname: Gregory
authors.affiliation: Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.
authors.forename: Ryang Hwa
authors.initials: RH
authors.lastname: Lee
authors.affiliation: Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.
authors.forename: Ji Min
authors.initials: JM
authors.lastname: Yu
authors.affiliation: Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.
authors.forename: Jun
authors.initials: J
authors.lastname: Watanabe
authors.affiliation: Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.
authors.forename: Su Yeon
authors.initials: SY
authors.lastname: An
authors.affiliation: Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Bartosh
authors.affiliation: Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine at Scott and White, Temple, Texas, United States of America.
authors.forename: Darwin J
authors.initials: DJ
authors.lastname: Prockop
mesh_terms: D053687:Adult Stem Cells / Q000166:cytology* / Q000378:metabolism; D000818:Animals; D015815:Cell Adhesion Molecules / Q000008:administration & dosage / Q000737:chemistry* / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D006207:Half-Life; D006801:Humans; D006820:Hyaluronic Acid / Q000378:metabolism; D007249:Inflammation / Q000139:chemically induced / Q000378:metabolism / Q000517:prevention & control*; D008070:Lipopolysaccharides / Q000633:toxicity; D051379:Mice; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000737:chemistry* / Q000378:metabolism
chemical_list: D015815:Cell Adhesion Molecules; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; C072273:TNFAIP6 protein, human; D006820:Hyaluronic Acid