Table 1. Key synthetic EPO protein glycoforms and folding from literature.

Synthetic EPO	Glycan/position				Net charge a	Folding protocol b	Folding yield	Ref
	24	38	83	126
‘click’ EPO1–165	GalNAc-‘click’	Gal-‘click’	Glc-‘click’	GalNAcα1-O-(CH2)3-‘click’	0	1	N/A c	This work
SEP-PEGylated EPO1–166	Anionic tetraantennary polymer	Asn	Asn	Anionic tetraantennary polymer	–8	1	25–40%	18
Lys24,38,83 EPO1–165	N/A-Lys d	N/A-Lys d	Lys d	Ser	+3	1	26%	11
Consensus EPO1–166	Biantennary dodecasaccharide e	Biantennary dodecasaccharide e	Biantennary dodecasaccharide e	Sia-Man-Sia(Man)	–8	1	37%	17
Glycotruncated consensus EPO1–166	(GluNAc)2	(GluNAc)2	(GluNAc)2	Sia-Man-Sia(Man)	–2	1 f	N/A g	17
Disialyl83 EPO1–166 h	Asn	Asn	Biantennary dodecasaccharide i	Ser	–2	2	65% j	45
Disialyl24,38,83 EPO1–166 k	Biantennary dodecasaccharide i	Biantennary dodecasaccharide i	Biantennary dodecasaccharide i	Ser	–6	2	68% j , l	20
Disialyl38,83 EPO1–166 k	Asn	Biantennary dodecasaccharide i	Biantennary dodecasaccharide i	Ser	–4	2	58% j , l	20
Disialyl24,83 EPO1–166 k	Biantennary dodecasaccharide i	Asn	Biantennary dodecasaccharide i	Ser	–4	2	43% j , l	20
Disialyl24,38 EPO1–166 k	Biantennary dodecasaccharide i	Biantennary dodecasaccharide i	Asn	Ser	–4	2	49% j , l	20
Disialyl83 EPO1–166 k	Asn	Asn	Biantennary dodecasaccharide i	Ser	–2	2	37% j , l	20

^aNet charge introduced at positions 24, 38, 83 and 126 only.

^bFolding protocol 1 = the polypeptide was firstly denatured in a degassed solution of GnHCl (6 M, 5.8 mL) and 100 mM tris (pH = 8.5). The resulting ‘click’ EPO stock solution (0.1 mg mL^–1) was then dialyzed against an initial degassed first folding solution containing guanidine (3 M), tris (100 mM), l-cysteine (4 mM) and l-cystine (0.5 mM) adjusted to pH 8.5. After 18 h, the first folding solution was replaced by a second folding solution of guanidine (1 M) and tris (100 mM) adjusted to pH 8.0, and the dialysis allowed to proceed for a further 21 h. The EPO solution was then transferred into a third folding solution of tris (10 mM) adjusted to pH 7.0 and dialyzed for 26 h. After the last dialysis, the ‘click’ EPO solution was concentrated to approx. 2 mL and purification by RP-HPLC was attempted; folding protocol 2 = the folding procedure was altered to begin with 8 M GnHCl at pH 7.5, the subsequent steps follow protocol 1. In some cases, minor variance in the times or temperature at each step are made by some authors.

^cYield could not be determined as the correctly folded product could not be isolated from the partially folded adduct.

^dResidue mutated from native sequence.

^eBiantennary dodecasacharide = (Sia-Gal-GluNAc-Man)₂-Man-GluNAc-GluNAc(Fuc).

^fNo data specified, presumably folding attempted as per consensus glycoform.

^gNo yield reported but peptide reported to aggregate significantly compared to consensus form.

^hSequence mutated E²¹ → A and Q⁷⁸ →A.

ⁱBiantennary dodecasaccharide = (NeuNAc-Gal-GlcNAc-Man)₂-Man-GlcNAc-GlcNAc.

^jApproximation based on peak area.

^kSequence mutated Q⁷⁸→A.

^lFolding yields were improved by 18–25% when performed with 0.01 mg mL^–1 peptide concentration, due to minimizing formation of misfolded oligomers.