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.
Highlights
TNF-α stimulated gene/protein 6 (TNFAIP6/TSG-6) is a multifunctional endogenous protein that is expressed by a variety of cells in response to stimulation by pro-inflammatory cytokines [1,2,3,4,5]
We developed conditions to overcome two major challenges in developing scalable production of the protein: the tendency of recombinant human TSG-6 (rhTSG-6) to aggregate the cells that synthesize it by cross-linking the brush border of hyaluronan that surrounds many cultured cells, and its tendency to self-aggregate in an apparently irreversible manner
A frozen vial of about 106 passage 1 hMSCs was thawed, and plated at 200 to 500 cells/cm2 in 150 mm diameter plates with 30 ml of complete culture medium (CCM) that consisted of αminimal essential medium (α-MEM; Invitrogen, Carlsbad, CA), 17% fetal bovine serum (FBS; lot-selected for rapid growth of hMSCs; Atlanta Biologicals, Inc, Norcross, GA), 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine (Invitrogen)
Summary
TNF-α stimulated gene/protein 6 (TNFAIP6/TSG-6) is a multifunctional endogenous protein that is expressed by a variety of cells in response to stimulation by pro-inflammatory cytokines [1,2,3,4,5]. TSG-6 binds to a large number of components of the extracellular matrix including hyaluronan, heparin, heparan sulfate, thrombospondins-1 and -2, fibronectin, and pentraxin 3 [6,7,8,9,10]. These interactions primarily act to stabilize or remodel the extracellular matrix. One of the more complex interactions is that the protein catalytically transfers the heavy chains of inter-α-trypsin inhibitor onto hyaluronan [11] It thereby helps stabilize the extracellular matrix, e.g. during cumulus expansion prior to ovulation [12,13,14,15]. TSG-6 thereby reduces the large, second phase of inflammation that is frequently an excessive and deleterious response to sterile injuries [25]
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