TY - JOUR
T1 - Butyrated ManNAc analog improves protein expression in Chinese hamster ovary cells
AU - Yin, Bojiao
AU - Wang, Qiong
AU - Chung, Cheng Yu
AU - Ren, Xiaozhi
AU - Bhattacharya, Rahul
AU - Yarema, Kevin J.
AU - Betenbaugh, Michael J.
N1 - Funding Information:
This research was supported by the Maryland Innovation Initiative
Funding Information:
Maryland Innovation Initiative, Grant number: 2015-MII-1944; National Science Foundation, Grant numbers: CBET-1264802, CBET-1512265
Funding Information:
This research was supported by the Maryland Innovation Initiative (grant no. 2015-MII-1944) and the National Science Foundation (grants no. CBET-1264802 and CBET-1512265).
Publisher Copyright:
© 2018 Wiley Periodicals, Inc.
PY - 2018/6
Y1 - 2018/6
N2 - The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4-O-Bu3ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4-O-Bu3ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ∼20% decrease in maximal viable cell density and ∼30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4-O-Bu3ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4-O-Bu3ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up-regulated more in sodium butyrate treated wild-type, erythropoietin, or IgG expressing CHO-K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4-O-Bu3ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4-O-Bu3ManNAc-treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4-O-Bu3ManNAc-treatment. These findings demonstrate that 1,3,4-O-Bu3ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4-O-Bu3ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.
AB - The chemical additive sodium butyrate (NaBu) has been applied in cell culture media as a direct and convenient method to increase the protein expression in Chinese hamster ovary (CHO) and other mammalian cells. In this study, we examined an alternative chemical additive, 1,3,4-O-Bu3ManNAc, for its effect on recombinant protein production in CHO. Supplementation with 1,3,4-O-Bu3ManNAc for two stable CHO cell lines, expressing human erythropoietin or IgG, enhanced protein expression for both products with negligible impact on cell growth, viability, glucose utilization, and lactate accumulation. In contrast, sodium butyrate treatment resulted in a ∼20% decrease in maximal viable cell density and ∼30% decrease in cell viability at the end of cell cultures compared to untreated or 1,3,4-O-Bu3ManNAc treated CHO cell lines for both products. While NaBu treatment enhanced product yields more than the 1,3,4-O-Bu3ManNAc treatment, the NaBu treated cells also exhibited higher levels of caspase 3 positive cells using microscopy analysis. Furthermore, the mRNA levels of four cell apoptosis genes (Cul2, BAK, BAX, and BCL2L11) were up-regulated more in sodium butyrate treated wild-type, erythropoietin, or IgG expressing CHO-K1 cell lines while most of the mRNA levels of apoptosis genes in 1,3,4-O-Bu3ManNAc treated cell lines remained equal or increased only slightly compared to the levels in untreated CHO cell lines. Finally, lectin blot analysis revealed that the 1,3,4-O-Bu3ManNAc-treated cells displayed higher relative sialylation levels on recombinant EPO, consistent with the effect of the ManNAc component of this additive, compared to control while NaBu treatment led to lower sialylation levels than control, or 1,3,4-O-Bu3ManNAc-treatment. These findings demonstrate that 1,3,4-O-Bu3ManNAc has fewer negative effects on cell cytotoxicity and apoptosis, perhaps as a result of a more deliberate uptake and release of the butyrate compounds, while simultaneously increasing the expression of multiple recombinant proteins, and improving the glycosylation characteristics when applied at comparable molarity levels to NaBu. Thus, 1,3,4-O-Bu3ManNAc represents a highly promising media additive alternative in cell culture for improving protein yields without sacrificing cell mass and product quality in future bioproduction processes.
KW - CHO cells
KW - ManNAc analog
KW - antibody recombinant protein expression
KW - sialylation
KW - sodium butyrate
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U2 - 10.1002/bit.26560
DO - 10.1002/bit.26560
M3 - Article
C2 - 29427449
AN - SCOPUS:85043695680
VL - 115
SP - 1531
EP - 1541
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
SN - 0006-3592
IS - 6
ER -