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: chkim@mogam.re.kr,

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 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.

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.

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.

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.

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

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

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

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

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

Supplementary table 4. Continued

Composition Structure proposal TheoreticalMass (Da)

Hex5HexNAc5dHex1 2132.8

Hex6HexNAc5 2148.8

Hex6HexNAc5dHex1 2294.75

Hex7HexNAc6dHex1 2660