images.nature.com · Web viewAll plasma samples were subjected to antibody titration, and the data...
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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
2Research and Development Research 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: [email protected],
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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).
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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 enzymes using the
4-MU method. Specific activities were presented as nmol per min per microgram. All
experiments were performed three times. The data are presented as averages and the error bar
represents standard deviation.
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Supplementary figure 3. Radiographs of the mouse bones. The two enzymes were
intravenously administered at 1 mg/kg, once a week for 6 months. The quantitative data of
this micro-CT analysis were presented in figure 3C and 3D.
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Supplementary figure 4. Compararison of the immunogenicity of two enzymes following
ADA analysis conducted with different antigens. Each enzyme (10 μg/ml) solution was
coated as the antigen for detection of anti-drug antibodies,. All plasma samples were
subjected to antibody titration, and the data are presented as OD value of 1:1000 dilution
samples. Idursulfase as the coating antigen showed higher antibody titraion than idursulfase
beta as the antigen.
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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 collected every four weeks.
All plasma samples were diluted, 1;10~1:10,000. Antibody formation in the tolerant mice
found substantially weaker than in the IDS KO mice.
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Supplement table 1. Two therapeutic drugs for MPS II patients
Contents Idursulfase beta Idursulfase
Amino 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
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Supplementary table 2. Sialic acid content analysis
SA Idursulfase beta Idursulfase
Molar ratioSA/protein (mol/mol)
Neu5Ac 18.94 ± 0.28 20.08 ± 0.06
Protein content for calculation of molar ratio was determined by BCA method
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Supplementary table 3. Monosaccharide composition analysis
Enzymes monosaccharideMonosaccharide
Amount (pmol/10 μL)
RelativeSTDEV(%)
Molar ratiomonosaccharide/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 was determined by amino acid analysis
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Supplementary table 4. Glycan structure proposals for the 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
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Supplementary table 4. Continued
Composition Structure proposal TheoreticalMass (Da)
Hex5HexNAc4 1783.7
Hex4HexNAc4 1824.7
Hex9HexNAc2 1883.7
Hex9HexNAc2 1905.4
Hex5HexNAc4dHex1 1929.7
Hex4HexNAc5dHex1 1970.7
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Supplementary table 4. Continued
Composition Structure proposal TheoreticalMass (Da)
Hex5HexNAc5dHex1 2132.8
Hex6HexNAc5 2148.8
Hex6HexNAc5dHex1 2294.75
Hex7HexNAc6dHex1 2660