Valeric acid induces cell cycle arrest at G1 phase in CHO cell cultures and improves recombinant antibody productivity

Abstract

To find a more effective chemical reagent for improved monoclonal antibody (mAb) production, eight chemical reagents (curcumin, quercein, DL-sulforaphane, thymidine, valeric acid, phenyl butyrate, valproic acid, and lithium chloride) known to induce cell cycle arrest were examined individually as chemical additives to recombinant CHO (rCHO) cell cultures producing mAb. Among these chemical additives, valeric acid showed the best production performance. Valeric acid decreased specific growth rate (μ), but increased culture longevity and specific mAb productivity (qmAb ) in a dose-dependent manner. The beneficial effect of valeric acid on culture longevity and qmAb outweighed its detrimental effect on μ, resulting in 2.9-fold increase in the maximum mAb concentration when 1.5 mM valeric acid was added to the cultures. Furthermore, valeric acid did not negatively affect the mAb quality attributes with regard to aggregation, charge variation, and galactosylation. Unexpectedly, galactosylation of the mAb increased by the 1.5 mM valeric acid addition. Taken together, the results obtained here demonstrate that valeric acid is an effective chemical reagent to increase mAb production in rCHO cells.

Proteomic Analysis of Host Cell Protein Dynamics in the Culture Supernatants of Antibody-Producing CHO Cells

“…The rCHO cell line producing rituximab (SM-0.025) was established by transfection of a vector containing glutamine synthetase (GS) and antibody genes into GS knockout CHO-K1 cells using Lipofectamine 2000 (Invitrogen, Carsbad, CA), following the manufacturer’s protocols, and subsequent selection using 25 μM methionine sulfoximine (MSX, Sigma-Aldrich, St. Louis, MO)47. SM-0.025 cells were adapted to grow in a serum-free suspension culture in 125 mL Erlenmeyer flasks (Corning, Corning, NY) containing 50 mL of culture medium and incubated in a climo-shaking incubator (ISF1-X, Adolf Kuhner AG, Birsfelden, Switzerland).…”

Comparative proteomic analysis of three Chinese hamster ovary (CHO) host cells

“…Meanwhile, gene regulation and culture condition optimization has also been used to induce G1 phase arrest during cell cycle to improve cellular metabolism [38, 39]. Different from those previous studies, the comparative proteomics in our study has identified some cytoskeletal proteins (i.e.…”

High throughput screening identifies novel, cell cycle‐arresting small molecule enhancers of transient protein expression

“…Thus, we investigated whether the compounds influenced cell cycle progression. Several studies have shown that an increase of cells in G1 phase correlates with higher recombinant protein levels in stable CHO cell lines . For transient gene expression, Tait et al, showed that luciferase activity and hence luciferase expression increased in CHO cells upon treatment with cell cycle arresting agents (nocodazole: G2/M arrest; hydroxyurea: G1 arrest) .…”

“…Several studies have shown that an increase of cells in G1 phase correlates with higher recombinant protein levels in stable CHO cell lines. 12,17,20 For transient gene expression, Tait et al, showed that luciferase activity and hence luciferase expression increased in CHO cells upon treatment with cell cycle arresting agents (nocodazole: G2/M arrest; hydroxyurea: G1 arrest). 36 Furthermore, coexpression of the cell cycle regulators p18, p21, and p27, which induce G1 arrest, 37 was found to improve antibody expression in HEK293E and CHO cells, respectively.…”

“…Various SMs have been reported to enhance protein production in hybridoma or CHO stable cell lines, mostly by suppressing growth and increasing specific productivity. 12 Examples include the mTOR inhibitor rapamycin, 13,14 chemical chaperones; 4-phenylbutyric acid and glycerol, 15,16 cell cycle inhibitors; palbociclib, valeric/pentanoic acid and LiCl, [17][18][19][20] and histone deacetylase (HDAC) inhibitors; sodium butyrate and valproic acid. 12,[21][22][23][24][25] A screen of 192 SMs in a stable CHO cell line expressing a recombinant monoclonal antibody identified 4 compounds that robustly increased titers by 40%.…”