Improving titer while maintaining quality of final formulated drug substance via optimization of CHO cell culture conditions in low-iron chemically defined media

Abstract

During biopharmaceutical process development, it is important to improve titer to reduce drug manufacturing costs and to deliver comparable quality attributes of therapeutic proteins, which helps to ensure patient safety and efficacy. We previously reported that relative high-iron concentrations in media increased titer, but caused unacceptable coloration of a fusion protein during early-phase process development. Ultimately, the fusion protein with acceptable color was manufactured using low-iron media, but the titer decreased significantly in the low-iron process. Here, long-term passaging in low-iron media is shown to significantly improve titer while maintaining acceptable coloration during late-phase process development. However, the long-term passaging also caused a change in the protein charge variant profile by significantly increasing basic variants. Thus, we systematically studied the effect of media components, seed culture conditions, and downstream processing on productivity and quality attributes. We found that removing β-glycerol phosphate (BGP) from basal media reduced basic variants without affecting titer. Our goals for late-phase process development, improving titer and matching quality attributes to the early-phase process, were thus achieved by prolonging seed culture age and removing BGP. This process was also successfully scaled up in 500-L bioreactors. In addition, we demonstrated 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 of titer improvement by both high-iron and long-term culture are discussed.

Keywords: CHO long-term culture; basic variants; iron; manufacturing process development; protein drug substance color; titer, quality; β-glycerol phosphate.

Figure 1.

Impact of iron concentrations in basal production media on CHO cell cultures at passage 9 in fed-batch production 250-mL shake flasks for 12 days (n = 3): One-way analysis of (1-a) peak viable cell density (VCD) (P < 0.0001), (1-b) final viability (P < 0.01), (1-c) final titer (P < 0.0001), (1-d) specific productivity (q_P) (P < 0.0001), (1-e) specific consumption rate of glutamic acid (q_Glu) (P < 0.0001), (1-f) specific consumption rate of glutamine (q_Gln) (P< 0.001), and (1-g) specific production rate of ammonium (q_NH4) (P < 0.0001).

Figure 2.

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 (P < 0.0001), (2-b) final viability (P = 0.188), (2-c) final titer (P < 0.0001), (2-d) q_P (P = 0.307), (2-e) q_Glu (P < 0.0001), (2-f) q_Gln (P < 0.001), and (2-g) q_NH4 (P < 0.0001).

Figure 3.

One-way analysis of (3-a) Peak VCD (P < 0.0001), (3-b) Day14 titer (normalized) (P < 0.0001) and (3-c) q_P (P = 0.0197) in fed-batch production 125-mL shake flasks containing low-iron media after 7–8 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.

Figure 4.

Comparison of final drug substance color made from CHO cell cultures grown in the basal media containing different iron concentrations with different seed passages at different bioreactor scales.

Figure 5.

Scale-up comparison of (5-a) viable cell density and (5-b) titer for different processes: Tox process contained 110 µM iron and 0.9 g/L BGP inoculated with P9 seeds (n = 2 at 500-L scale); Process A contained 10 µM iron and 0.9 g/L BGP inoculated with P9 seeds (n = 5 at 600-L scale); prototype Process B contained 20 µM iron and 0.9 g/L BGP inoculated with P20 seeds (n = 2 with one each at 50-L and 500-L scale); Process B contained 20 µM iron and without BGP inoculated with P20 seeds (n = 2 at 500-L scale).

Figure 6.

One-way analysis of (6-a) acidic species (P < 0.0001), (6-b) main species (P < 0.0001), and (6-c) basic species (P = 0.798) during downstream purification steps (n = 7). PAVIB: Protein A viral inactivation bulk pool; AEX pool: anion exchange chromatography pool; CEX pool: Cation exchange chromatography pool.

Figure 7.

Impact of different upstream process parameters (Table 3) in fed-batch production 250-mL shake flasks (n = 3): (7-a) on basic variants on day10 (D10), day12 (D12) and day14 (D14); (7-b) on the prediction profiles.

Figure 8.

Impact of different iron concentrations and seed passages on reactive oxygen species (ROS) residuals on day 3 (n = 4 for 12 g/L galactose conditions only), day 6 (n = 8 for all DOE conditions) and day 10 (n = 8) in the fed-batch production 250-mL shake flasks from the DOE design shown in Table 3.

Figure 9.

Cell line creation and Master Cell Bank (MCB) Genealogy: RCB: Research Cell Bank; DCB: Development Cell Bank; all seed cultures were split every three days per passage.