Comparative study of idursulfase beta and idursulfase in vitro and in vivo
Chihwa Kim1, Jinwook Seo2, Yokyung Chung2, Hyi-jeong Ji3, Jaehyun Lee2, Jongmun Sohn2, Byoungju Lee2, Euicheol Jo1.
1Protein Engineering team, MOGAM Institute for Biomedical Research, Yongin, Republic of Korea
2Researchand DevelopmentResearch Center, Green Cross Corporation, Yongin, Republic of Korea
3Corporate Development, Green Cross Corporation, Yongin, Republic of Korea
Correspondence and reprint requests: Chihwa Kim, Ph.D.
Protein Engineering Team, MOGAM Institute for Biomedical Research
107, 30 beon-gil, Ihyeon-ro,Giheung-gu, Yongin, 446-799, Republic of Korea
Tel: +82-31-260-9836
E-mail: ,
Supplementary figure 1.M6PR expression in normal and patient fibroblasts.Normal and patient fibroblasts were harvested and treated with anti-M6PR antibody (anti-IGF2R antibody) or isotype antibody, respectively, for 30 min on ice. After washout, Cells were read on FACSCalibur (BD bioscience, MD, USA) and the resulting data were analyzed by Flowjo program (Flowjo, Ashland, OR, USA).
Supplementary figure 2.Intracellular IDS protein activity in patient fibroblasts after cellular uptake of two enzymes. Cell lysates (A; KH38, B; GM00615) were obtained after treating two enzymes for 24 hours and assayed for the specific activity of the IDS enzymesusingthe 4-MU method. Specific activities were presented as nmol per min per microgram. All experiments were performedthree times.Thedata are presented as averages and the error bar representsstandard deviation.
Supplementary figure 3.Radiographs of the mouse bones.The two enzymeswere intravenously administered at1mg/kg, once a week for 6 months. The quantitative data of this micro-CT analysis were presented infigure 3C and 3D.
Supplementary figure 4. Compararison of the immunogenicity of two enzymesfollowingADA analysis conducted withdifferent antigens. Each enzyme (10 μg/ml) solution wascoated as the antigen for detection of anti-drug antibodies,. All plasma samples were subjected to antibody titration, and the data are presentedas OD value of 1:1000 dilution samples. Idursulfase as the coating antigenshowed higher antibody titraion than idursulfase beta as the antigen.
Supplementary figure 5.Evaluation of tolerant IDS mice. IDS KO and the tolerant IDS-/-C84Ttg mice received IDS proteins i.v. for 6 months and their blood were collectedevery four weeks. All plasma samples were diluted, 1;10~1:10,000. Antibody formation in the tolerant micefoundsubstantiallyweaker than in the IDS KO mice.
Supplement table 1. Two therapeutic drugs for MPS II patients
Contents / Idursulfase beta / IdursulfaseAmino acids / 525 / 525
Molecular weight (kDa) / 76 / 76
Product cell line / CHO-DG44 / HT-1080
Cultivation / Serum-free / With serum
M6P contents (mol/mol) 17 / 2.38±0.07 / 2.42 ±0.28
Formylglycine (%) 17 / 79.40±0.92 / 68.12±2.22
Kuptake (nM),
normal cell (p<0.05) 17 / 5.09±0.96 / 6.50±1.28
Supplementary table 2. Sialic acid content analysis
SA / Idursulfase beta / IdursulfaseMolar ratio
SA/protein (mol/mol) / Neu5Ac / 18.94 ± 0.28 / 20.08 ± 0.06
Protein content for calculation of molar ratio wasdetermined by BCA method
Supplementary table 3. Monosaccharide composition analysis
Enzymes / monosaccharide / MonosaccharideAmount (pmol/10μL) / Relative
STDEV(%) / Molar ratio
monosaccharide/protein (pmol/pmol)
Idursulfase beta / Fucose / 52.97 / 2.09 / 2.77
GalN / 0.57 / 9.57 / 0.03
GlcN / 541.49 / 0.27 / 28.35
Galactose / 311.16 / 0.95 / 16.29
Glucose / 9.35 / 68.69 / -
Mannose / 394.73 / 1.81 / 20.67
Idursulfase / Fucose / 77.18 / 1.19 / 4.46
GalN / 2.43 / 1.53 / 0.14
GlcN / 512.14 / 1.12 / 29.60
Galactose / 281.32 / 1.44 / 16.26
Glucose / 8.89 / 49.46 / -
Mannose / 362.14 / 2.17 / 20.93
Protein content for calculation of molar ratio wasdetermined by amino acidanalysis
Supplementarytable4.Glycan structure proposals forthe most abundant peaks.
Composition / Structure proposal / TheoreticalMass (Da)
Hex5HexNAc2 / / 1377.5
Hex3HexNAc4 / / 1459.5
Hex3HexNAc4dHex1 / / 1605.6
Hex4HexNAc4 / / 1621.6
Hex5HexNAc3dHex1 / / 1726.6
Hex6HexNAc3 / / 1742.6
Hex4HexNAc4dHex1 / / 1767.6
Supplementarytable4. Continued
Composition / Structure proposal / TheoreticalMass (Da)
Hex5HexNAc4 / / 1783.7
Hex4HexNAc4 / / 1824.7
Hex9HexNAc2 / / 1883.7
Hex9HexNAc2 / / 1905.4
Hex5HexNAc4dHex1 / / 1929.7
Hex4HexNAc5dHex1 / / 1970.7
Supplementarytable4. Continued
Composition / Structure proposal / TheoreticalMass (Da)
Hex5HexNAc5dHex1 / / 2132.8
Hex6HexNAc5 / / 2148.8
Hex6HexNAc5dHex1 / / 2294.75
Hex7HexNAc6dHex1 / / 2660