Table 1.
Composition of optimized chemically defined protein free medium (OCDPF medium).
Fig 1.
CHO cell line stably transfected to synthesize rhTSG-6 forms aggregates resulting in a decrease of protein production.
Cells were incubated in CD CHO basal media and in spinner culture. (A i) Diagram of expression plasmid of TSG-6 under control of human elongation factor promoter 1α and insertion of sequences for Myc and His tags at the C-terminus. (A ii) Expression of rhTSG-6 (antibody clone A38.1.20) and the His tag by immunocytochemistry. Scale bar = 10 μm. (A iii) Expression by Western blotting. (B) Number of control CHO cells (CHO-S) and rhTSG-6 synthesizing cells (rhTSG-6/CHO-S). Data are presented as mean ± S.D. (C) rhTSG-6 content and pH of medium from cultures of rhTSG-6/CHO-S cells. Data are presented as mean ± S.D. (D) Western blots with antibodies to the His-tag of medium from cultures of rhTSG-6/CHO-S cells. Monomeric rhTSG-6 was detected after day 3 but not after day 4, apparently because of aggregation. Abbreviations: IN, input sample; FT, flow through. (E) Aggregates of rhTSG-6/CHO-S cells after 4 days of culture. Scale bars = 500 μm and 250 μm (insert). (F) Immunolabeling with rhTSG-6 antibody (clone A38.1.20), HABP (biotin-conjugated HA binding proteins), and DAPI staining of aggregates. Scale bars = 100 μm and 50 μm (insert).
Fig 2.
Addition of heparin to medium improved yield of rhTSG-6 in spinner cultures.
(A) Effect on number of rhTSG-6/CHO-S cells. (B) Effect on yield of rhTSG-6. (C) Effect on yields of protein aggregates/complexes (H-rhTSG-6) and monomeric rhTSG-6 (L-rhTSG-6). Western blots with antibodies to hTSG-6 on medium from day 4 cultures of rhTSG-6/CHO-S cells. (E) Effects on pH of medium. Values are mean ± S.D. of 3 replicates of each sample in (A), (B), and (D).
Fig 3.
Synthesis of rhTSG-6 by culture of rhTSG-6/CHO-S cells in OCDPF medium and in a bioreactor that controlled pH.
(A) Expansion of cells and oxygen saturation. (B) Yield of rhTSG-6 and pH of medium. (C) Photomicrographs demonstrating that rhTSG-6/CHO-S cells are largely single-cell suspensions. Scale bars = 100 μm and 50 μm (insert) (D) Yield of monomeric rhTSG-6. Western blot of medium with antibodies to hTSG-6. Values in (A) and (B) are means of 3 replicates.
Fig 4.
Purification of rhTSG-6 from 5 or 6 liters cultured media of the bioreactor.
(A) Schematic for the purification steps. (B) Assay by SDS-PAGE of rhTSG-6 eluted from the Q-Sepharose FF column. Symbols: IN, input sample; CB, Coomassie Blue staining of gel. (C) Endotoxin content of conditioned culture medium, eluate from the His-tag column and Q-Sepharose FF column. Values are mean ± S.D. of 3 replicates. (D) Deglycosylation of the purified rhTSG-6. Arrows indicate deglycosylated forms rhTSG-6.
Table 2.
Synthesis and Purification of rhTSG-6 in Bioreactor.
Fig 5.
In vivo half-life of rhTSG-6 proteins in plasma of mice.
(A) rhTSG-6 (50 μg) was injected through the tail vein and the blood was collected at times indicated. After isolation of the plasma, levels of rhTSG-6 proteins were determined by ELISA. Values are mean and SEM from 3 mice. Distribution (t1/2α) and elimination (t1/2β) were calculated by non-linear least squares regression using GraphPad Prism program. Myeloma-derived rhTSG-6 (R&D Systems): t1/2α = 0.15 h, t1/2β = 0.20 h, CHO cell-derived rhTSG-6: t1/2α = 0.08 h, t1/2β = 0.47 h. The results showed a tendency for the CHO cell-derived rhTSG-6 to have a longer half-life. (B) The difference plasma levels at 24 h after infusion of 50 μg rhTSG-6 of the two proteins. Comparison of the two groups was made by Student’s t test with Welch’s correction. Data are presented as mean ± S.E.M. from 6 mice.
Fig 6.
Purified rhTSG-6 suppressed LPS-induced inflammation in mice.
(A) Schematic for the experiment. (B, C) rhTSG-6 suppressed LPS-induced levels of mRNA for IL-6 and IFNγ in spleen. Statistical calculation was performed by a one-way ANOVA followed by the Tukey multiple comparison post hoc test. Data are presented as mean ± S.E.M. Symbol: RQ, relative quantity.