9)

9). the early-phase procedure, had been attained by prolonging seed lifestyle age group and getting rid of BGP so. This technique was successfully scaled up in 500-L bioreactors also. Furthermore, we confirmed that higher concentrations of reactive oxygen species were present in the high-iron Chinese hamster ovary cell cultures compared to that in the low-iron cultures, suggesting a possible mechanism for the drug substance coloration caused by high-iron media. Finally, hypotheses for the mechanisms Flurbiprofen of titer improvement by both high-iron and long-term culture are discussed. 0.0001), (1-b) final viability ( 0.01), (1-c) final titer ( 0.0001), (1-d) specific productivity (qP) ( 0.0001), (1-e) specific consumption rate of glutamic acid (qGlu) ( 0.0001), (1-f) specific consumption rate of glutamine (qGln) (P 0.001), and (1-g) specific production rate of ammonium (qNH4) ( 0.0001). Impact of seed passages on CHO cell culture performance During process development, we found that the longer seed passages improved titer in low-iron media, which was unexpected. Thus, the effect Flurbiprofen of different seed passages was studied in detail using chemically defined media containing 10?M iron (Fig.?2). Seeds between passages 7C11 from the vial thaw of master cell bank (MCB) vials are usually sufficient to expand the seed culture for clinical good-manufacturing-practices (GMP) manufacturing. Therefore, seed passages between 5 and 13 (i.e., 2 additional passages at both Flurbiprofen the low and high ends) were used for this experiment. Peak VCD (Fig.?2-a) and final titer (Fig.?2-c) almost linearly increased when the seed passage increased from 5 to 13, but the final viability (Fig.?2-b) and qp (Fig.?2-d) had no significant difference between the different seed passages. The nutrient consumption rates of qGlu (Fig.?2-e) and qGln (Fig.?2-f) increased, while the toxic production rate of qNH4 (Fig.?2-f) was reduced with the increase of passage numbers. Open in a separate window Figure 2. Flurbiprofen Impact of seed passage numbers on CHO cell cultures using low-iron media in fed-batch production 250-mL shake flasks for 12?days (n = 3): One-way analysis of (2-a) peak VCD ( 0.0001), (2-b) final viability (P = 0.188), AXIN2 (2-c) final titer ( 0.0001), (2-d) qP (P = 0.307), (2-e) qGlu ( 0.0001), (2-f) qGln ( 0.001), and (2-g) qNH4 ( 0.0001). Because the protein titer continued to increase during the manufacturing seed passage range between 5 Flurbiprofen and 13 (Fig.?2-c), we hypothesized that the trend would continue and that passages longer than passage 13 (P13) would produce even higher titer. Therefore, longer term seed passages up to 33 were examined. Protein titer almost linearly increased from P8 to P18, and then maintained at a similar high level from P18 to P33 (Fig.?3-b). However, qp values decreased when the passages were beyond P23 (Fig.?3-c), which was mainly due to the fact that peak VCD increased with increasing passages from P23 to P33 (Fig.?3-a), but titer remained at the same level (Fig.?3-b). Open in a separate window Figure 3. One-way analysis of (3-a) Peak VCD ( 0.0001), (3-b) Day14 titer (normalized) ( 0.0001) and (3-c) qP (P = 0.0197) in fed-batch production 125-mL shake flasks containing low-iron media after 7C8 passages of master cell bank (MCB) and different development cell banks (DCBs) (n = 4). Passage 8: P8 seed from MCB vial thaw; Passage 12: P7 seed from the P5-DCB made from 5th passage of MCB; Passage 15: P7 seed from the P8-DCB; Passage 18: P8 seed from the P10-DCB; Passage 20: P8 seed from the P12-DCB; Passage 23: P8 seed from the P15-DCB; Passage 28: P8 seed from the P20-DCB; Passage 33: P8 seed from the P25-DCB. Drug substance color evaluation at 7-L bioreactor scale Based on the aforementioned results from shake flasks, it is clear that the upstream titer can be improved after longer term seed culture in low-iron media. We then sought to evaluate whether long-term seed culture would cause drug substance coloration, as the drug substance coloration was a major.