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Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

Du Z, Treiber D, McCarter JD, Fomina-Yadlin D, Saleem RA, McCoy RE, Zhang Y, Tharmalingam T, Leith M, Follstad BD, Dell B, Grisim B, Zupke C, Heath C, Morris AE, Reddy P - Biotechnol. Bioeng. (2014)

Bottom Line: Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted.In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature.Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures.

View Article: PubMed Central - PubMed

Affiliation: Cell Sciences and Technology, Amgen Inc., 1201 Amgen Court West, Seattle, Washington. zhimeidu@gmail.com.

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Selective CDK4/6 inhibition by small molecule compound. (A) Schematic diagram of the cell cycle and function of a selective CDK4/6 specific inhibitor. E2F, E2 transcription factor; DP, E2F dimerization partner; R, restriction checkpoint. (B) Whole cell extracts with or without CCI treatment were assayed for protein levels of phospho-Rb (p-Rb), phospho-ERK (p-ERK), phospho-S6 (p-S6) by Western blot. Rab11 was used as a loading control.
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fig01: Selective CDK4/6 inhibition by small molecule compound. (A) Schematic diagram of the cell cycle and function of a selective CDK4/6 specific inhibitor. E2F, E2 transcription factor; DP, E2F dimerization partner; R, restriction checkpoint. (B) Whole cell extracts with or without CCI treatment were assayed for protein levels of phospho-Rb (p-Rb), phospho-ERK (p-ERK), phospho-S6 (p-S6) by Western blot. Rab11 was used as a loading control.

Mentions: To induce a complete and selective cell cycle G0/G1 arrest without potentiating other cellular pathways, we used a small molecule cell cycle inhibitor (CCI) to selectively inhibit the kinase activity of CDK4/6 (Fig. 1A). While potently inhibiting CDK4/6, the compound was highly selective showing little activity when screened at 1 μM compound concentration against the vast majority of >440 other kinases, including CDK2, CDK3, CDK5, and multiple cyclin-dependent kinase-like (CDKL) kinases, confirming and expanding previous reports (Barvian et al., 2000; Ekholm and Reed, 2000; Fry et al., 2001, 2004; Toogood, 2001) (data not shown). In addition, the inhibitory effect of CCI on different selected kinases, including CDK4/6, was tested over a large concentration range (0.001–10 µM) of CCI in the kinase assays to obtain IC50 values. The results demonstrate the potency and selectivity of CCI against CDK4/6 compared to inhibition against various other kinases (Table 1995). The specific inhibitory effect on Rb phosphorylation was further confirmed from by Western blotting (Fig. 1B). Both ERK and S6 kinase signaling networks are involved in cell proliferation and protein expression. As shown in Figure 1B, phosphorylation of ERK and S6 kinases were not affected, suggesting that the CCI has no effect on these two signaling pathways. To test its effect on recombinant CHO cell growth, different dosages of the compound were added to the cultures of two different mAb-expressing cell lines at day 0 with seeding density at 0.5 × 105 c/mL, while VCD and viability were measured daily for 5 days. Compared to the control, in which cell growth reached 3–5 million cells per mL, cell growth after CCI treatment was significantly inhibited, and the maximum effect on growth arrest was attained between 5–10µM at 24 h of treatment (Fig. 2A and B). Despite the growth arrest, no significant cytotoxicity was observed at these concentrations (5–10 µM) of the compound as deduced from ending viabilities compared to the control (Fig. 2C and D) for both cell lines. Similar results were observed with an additional 10 different Amgen recombinant cell lines with different productivity, growth rate and product quality profiles using the same dosages of CCI, suggesting a broad treatment effect (data not shown).


Use of a small molecule cell cycle inhibitor to control cell growth and improve specific productivity and product quality of recombinant proteins in CHO cell cultures.

Du Z, Treiber D, McCarter JD, Fomina-Yadlin D, Saleem RA, McCoy RE, Zhang Y, Tharmalingam T, Leith M, Follstad BD, Dell B, Grisim B, Zupke C, Heath C, Morris AE, Reddy P - Biotechnol. Bioeng. (2014)

Selective CDK4/6 inhibition by small molecule compound. (A) Schematic diagram of the cell cycle and function of a selective CDK4/6 specific inhibitor. E2F, E2 transcription factor; DP, E2F dimerization partner; R, restriction checkpoint. (B) Whole cell extracts with or without CCI treatment were assayed for protein levels of phospho-Rb (p-Rb), phospho-ERK (p-ERK), phospho-S6 (p-S6) by Western blot. Rab11 was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4282109&req=5

fig01: Selective CDK4/6 inhibition by small molecule compound. (A) Schematic diagram of the cell cycle and function of a selective CDK4/6 specific inhibitor. E2F, E2 transcription factor; DP, E2F dimerization partner; R, restriction checkpoint. (B) Whole cell extracts with or without CCI treatment were assayed for protein levels of phospho-Rb (p-Rb), phospho-ERK (p-ERK), phospho-S6 (p-S6) by Western blot. Rab11 was used as a loading control.
Mentions: To induce a complete and selective cell cycle G0/G1 arrest without potentiating other cellular pathways, we used a small molecule cell cycle inhibitor (CCI) to selectively inhibit the kinase activity of CDK4/6 (Fig. 1A). While potently inhibiting CDK4/6, the compound was highly selective showing little activity when screened at 1 μM compound concentration against the vast majority of >440 other kinases, including CDK2, CDK3, CDK5, and multiple cyclin-dependent kinase-like (CDKL) kinases, confirming and expanding previous reports (Barvian et al., 2000; Ekholm and Reed, 2000; Fry et al., 2001, 2004; Toogood, 2001) (data not shown). In addition, the inhibitory effect of CCI on different selected kinases, including CDK4/6, was tested over a large concentration range (0.001–10 µM) of CCI in the kinase assays to obtain IC50 values. The results demonstrate the potency and selectivity of CCI against CDK4/6 compared to inhibition against various other kinases (Table 1995). The specific inhibitory effect on Rb phosphorylation was further confirmed from by Western blotting (Fig. 1B). Both ERK and S6 kinase signaling networks are involved in cell proliferation and protein expression. As shown in Figure 1B, phosphorylation of ERK and S6 kinases were not affected, suggesting that the CCI has no effect on these two signaling pathways. To test its effect on recombinant CHO cell growth, different dosages of the compound were added to the cultures of two different mAb-expressing cell lines at day 0 with seeding density at 0.5 × 105 c/mL, while VCD and viability were measured daily for 5 days. Compared to the control, in which cell growth reached 3–5 million cells per mL, cell growth after CCI treatment was significantly inhibited, and the maximum effect on growth arrest was attained between 5–10µM at 24 h of treatment (Fig. 2A and B). Despite the growth arrest, no significant cytotoxicity was observed at these concentrations (5–10 µM) of the compound as deduced from ending viabilities compared to the control (Fig. 2C and D) for both cell lines. Similar results were observed with an additional 10 different Amgen recombinant cell lines with different productivity, growth rate and product quality profiles using the same dosages of CCI, suggesting a broad treatment effect (data not shown).

Bottom Line: Additionally, the product quality attributes of the mAb, with regard to high molecular weight (HMW) and glycan profile, are not negatively impacted.In fact, high mannose is decreased after treatment, which is in contrast to other established growth control methods such as reducing culture temperature.Overall, our observations showed that cell cycle arrest by directly targeting CDK4/6 using selective inhibitor compound can be utilized consistently and rapidly to optimize process parameters, such as cell growth, qP, and glycosylation profile in recombinant antibody production cultures.

View Article: PubMed Central - PubMed

Affiliation: Cell Sciences and Technology, Amgen Inc., 1201 Amgen Court West, Seattle, Washington. zhimeidu@gmail.com.

Show MeSH
Related in: MedlinePlus