Synthesis of Erythropoietin

Since approval by the FDA in 1989, erythropoietin (EPO) has been used extensively for the treatment of anemia – especially in those with chronic kidney disease, undergoing chemotherapy, or have acquired immune deficiency syndrome (AIDS).¹ EPO is a highly glycosylated glycoprotein containing three N-linked and one O-linked glycosylation sites. These glycans constitute 40% of the weight of the glycoprotein and are important for activity.² It has been found that the glycans play pivotal roles in the solubility, stability, and activity of EPO.³ Therapeutic EPO is obtained by recombinant expression in mammalian cell lines such as Human Embryonic Kidney (HEK) or Chinese Hamster Ovary (CHO) cells. Although this approach can produce large quantities of glycoprotein, it leads to heterogeneous mixtures of glycoforms (same protein with different glycan attachments). Glycosylation occurs in a non-template mediated driven manner during post-translational modification in the ER and Golgi. Due to the mixture of glycoforms present, it has been challenging to perform structure-activity relationship (SAR) studies to establish the optimal glycan structures for biological activity. This has led to a desire to develop homogenous EPO glycoforms and two methods have been pursued based on chemical and chemoenzymatic synthesis. 

The chemical synthesis of EPO, initially developed by Danishesky and coworkers, leverages the power of solid-phase peptide synthesis (SPPS), native chemical ligation, and attachment of the N-/O-glycans in a highly regioselective format.⁴ Through this approach, it was possible to obtain a homogenous glycoform of EPO with similar bioactivity to commercial EPO. Chemoenzymatic synthesis of EPO, developed by Wang and coworkers, utilizes template mediated driven of protein synthesis in cells to produce the polypeptide backbone followed by using endoglycosidases to cleave off and reattach homogenous glycans of their choosing.⁵ The strategies, while powerful, have neither yet to surpass the therapeutic efficacy of recombinant EPO produced in mammalian cell lines. However, as the field is rapidly evolving, we may soon see a homogenously produced EPO with greater in vivo efficacy than commercial recombinant EPO.

References

1. D. Goldsmith, Clin. J. Am. Soc. Nephrol. 2010, 5, 929–935.

2. M. Takeuchi, S. Takasaki, H. Miyazaki, T. Kato, S. Hoshi, N. Kochibe, A. Kobata, J. Biol. Chem. 1988, 263, 3657–3663.

3. M. S. Dordal, F. F. Wang, E. Goldwasser, Endocrinology 1985, 116, 2293–2299.

4. Wang, P.; Dong, S.; Shieh, J.-H. .; Peguero, E.; Hendrickson, R.; Moore, M. A. S.; Danishefsky, S. J. Erythropoietin Derived by Chemical Synthesis. Science 2013, 342 (6164), 1357–1360. https://doi.org/10.1126/science.1245095.

5. Yang, Q.; An, Y.; Zhu, S.; Zhang, R.; Loke, C. M.; Cipollo, J. F.; Wang, L.-X. Glycan Remodeling of Human Erythropoietin (EPO) through Combined Mammalian Cell Engineering and Chemoenzymatic Transglycosylation. ACS Chemical Biology 2017, 12 (6), 1665–1673. https://doi.org/10.1021/acschembio.7b00282.