Insights into the loss of protein sialylation in an fc-fusion protein-producing CHO cell bioprocess
- 229 Downloads
Sialylation affects circulating half-life, charge distribution, and other biochemical properties of therapeutic glycoproteins. Loss of protein sialylation during glycoprotein-producing bioprocesses could lead to a low final protein sialylation level and bring negative effects on subsequent clinical efficacy. In this work, an Fc-fusion protein-producing Chinese hamster ovary cell fed-batch culture process was studied and insights into the loss of protein sialylation during the Fc-fusion protein production phase (days 5 to 13) were presented. The results showed that the decreased total sialic acid content was 13.84 μg/mg during the production phase, which accounted for 24% of the total sialic acid content on day 5. The lost sialic acids were predominantly from α 2-3 sialylation on N- and O-glycans. Through cell-free incubation and kinetics studies, it was found that the decreased sialic acid content caused by extracellular sialic acid degradation and incomplete glycan biosynthesis were 7.79 μg/mg and 6.05 μg/mg, respectively. The two processes had a nearly equal contribution to the loss of final product sialylation. Detailed characterizations revealed that decreases in sialic acid content were due either to extracellular sialic acid degradation via hydrolysis of α 2-3 sialic acids probably by released cytosolic sialidase or to a lack of galactosylated glycan availability for sialylation during late-stage glycosylation. Our work provides a better understanding of losses in protein sialylation during glycoprotein manufacturing.
KeywordsChinese hamster ovary cells Fc-fusion protein Sialylation Extracellular degradation Intracellular biosynthesis
Compliance with ethical standards
This work was supported by the Major Programs of Development Foundation of Shanghai Zhangjiang National Independent Innovation Demonstration Zone (No. ZJ2015-ZD-002).
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- Bongers J, Devincentis J, Fu J, Huang P, Kirkley DH, Leister K, Liu P, Ludwig R, Rumney K, Tao L, Wu W, Russell RJ (2011) Characterization of glycosylation sites for a recombinant IgG1 monoclonal antibody and a CTLA4-Ig fusion protein by liquid chromatography–mass spectrometry peptide mapping. J Chromatogr A 1218(45):8140–8149. https://doi.org/10.1016/j.chroma.2011.08.089 CrossRefPubMedGoogle Scholar
- Ha TK, Lee GM (2014) Effect of glutamine substitution by TCA cycle intermediates on the production and sialylation of fc-fusion protein in Chinese hamster ovary cell culture. J Biotechnol 180(0):23-29 doi: https://doi.org/10.1016/j.jbiotec.2014.04.002
- Harazono A, Kobayashi T, Kawasaki N, Itoh S, Tada M, Hashii N, Ishii A, Arato T, Yanagihara S, Yagi Y, Koga A, Tsuda Y, Kimura M, Sakita M, Kitamura S, Yamaguchi H, Mimura H, Murata Y, Hamazume Y, Sato T, Natsuka S, Kakehi K, Kinoshita M, Watanabe S, Yamaguchi T (2011) A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals. Biologicals 39(3):171–180. https://doi.org/10.1016/j.biologicals.2011.04.002 CrossRefPubMedGoogle Scholar
- Hassinen A, Pujol FM, Kokkonen N, Pieters C, Kihlström M, Korhonen K, Kellokumpu S (2011) Functional organization of Golgi N- and O-glycosylation pathways involves pH-dependent complex formation that is impaired in cancer cells. J Biol Chem 286(44):38329–38340. https://doi.org/10.1074/jbc.M111.277681 CrossRefPubMedPubMedCentralGoogle Scholar
- Houel S, Hilliard M, Yu YQ, McLoughlin N, Martin SM, Rudd PM, Williams JP, Chen W (2013) N- and O-glycosylation analysis of etanercept using liquid chromatography and quadrupole time-of-flight mass spectrometry equipped with electron-transfer dissociation functionality. Anal Chem 86(1):576–584. https://doi.org/10.1021/ac402726h CrossRefPubMedGoogle Scholar
- Liu L, Gomathinayagam S, Hamuro L, Prueksaritanont T, Wang W, Stadheim TA, Hamilton SR (2013) The impact of glycosylation on the pharmacokinetics of a TNFR2: fc fusion protein expressed in glycoengineered Pichia pastoris. Pharm Res 30(3):803–812. https://doi.org/10.1007/s11095-012-0921-3 CrossRefPubMedGoogle Scholar
- Liu J, Wang J, Fan L, Chen X, Hu D, Deng X, Fai Poon H, Wang H, Liu X, Tan W-S (2015b) Galactose supplementation enhance sialylation of recombinant fc-fusion protein in CHO cell: an insight into the role of galactosylation in sialylation. World J Microbiol Biotechnol 31(7):1147–1156. https://doi.org/10.1007/s11274-015-1864-8 CrossRefPubMedGoogle Scholar
- Qian Y, Lewis AM, Sidnam SM, Bergeron A, Abu-Absi NR, Vaidyanathan N, Deresienski A, Qian N-X, Borys MC, Li ZJ (2017) LongR3 enhances fc-fusion protein N-linked glycosylation while improving protein productivity in an industrial CHO cell line. Process Biochem 53:201–209. https://doi.org/10.1016/j.procbio.2016.11.018 CrossRefGoogle Scholar
- Xu X, Nagarajan H, Lewis NE, Pan S, Cai Z, Liu X, Chen W, Xie M, Wang W, Hammond S, Andersen MR, Neff N, Passarelli B, Koh W, Fan HC, Wang J, Gui Y, Lee KH, Betenbaugh MJ, Quake SR, Famili I, Palsson BO, Wang J (2011) The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line. Nat Biotechnol 29(8):735–741. https://doi.org/10.1038/nbt.1932 CrossRefPubMedPubMedCentralGoogle Scholar
- Yang Z, Wang S, Halim A, Schulz MA, Frodin M, Rahman SH, Vester-Christensen MB, Behrens C, Kristensen C, Vakhrushev SY, Bennett EP, Wandall HH, Clausen H (2015) Engineered CHO cells for production of diverse, homogeneous glycoproteins. Nat Biotechnol 33(8):842–844. https://doi.org/10.1038/nbt.3280 CrossRefPubMedGoogle Scholar
- Yin B, Gao Y, Chung C-y, Yang S, Blake E, Stuczynski MC, Tang J, Kildegaard HF, Andersen MR, Zhang H, Betenbaugh MJ (2015) Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation. Biotechnol Bioeng 112(11):2343–2351. https://doi.org/10.1002/bit.25650 CrossRefPubMedGoogle Scholar