G-CSFPEG

G-CSF ()G-CSF G-CSF G-CSF

PEG()

PEGrhG-CSFrhG-CSF

mPEG-PALG-CSF =mPEG-PALNmPEG-PALPEG

Figure 1. SDS-PAGE analysis of the orthogonal samples stained with Coomassie Blue. Lane 1-9: nine samples in the orthogonal tests corresponding to the test number. The Optimal Conditions:

Time48 hours

pH5.0

Temperature20

Molar ratio PEG/rhG-CSF10:1

Figure 2. Much mono-PEGylated rhG-CSF was obtained under the optimal conditions (48 h, pH5.0, molar ratio of mPEG to rhG-CSF 10: 1; 20 ), more than under the conditions of Test 5 or 7 in the orthogonal tests. A B

Chart12

100

52.7

66.5

72.9

Control Lane 5 Lane 7 Optimal

Mono-PEGylated rhG-CSF( % of control)

Sheet1

10052.766.572.9

rhG-CSF57optimal

Sheet1

Control Lane 5 Lane 7 Optimal

Mono-PEGylated rhG-CSF( % of control)

Sheet2

Sheet3

Figure 3. The profile of cation exchange fast protein liquid chromatogram (CTA FPLC) of the PEGylated rhG-CSF. The average molecular weights of mPEG-PAL used are (A) 20 kDa and (B) 5 kDa, respectively.

PEG(5 kD)-G-CSFFigure 4. The FPLC profile of PEG(5 kD)-G-CSF after purification.

PEG(20 kD)-G-CSFFigure 5. The FPLC profile of PEG(20 kD)-G-CSF after purification.

Mono-PEG-G-CSFLANE A: MarkerLANE B: Mono-PEG(5 kDa)-G-CSFLANE C: Mono-PEG(20 kDa)-G-CSFLANE D: G-CSF

Figure 6. SDS-PAGE analysis of the purified mono-PEG-G-CSF stained with Coomassie brilliant Blue.

Mono-PEG-G-CSFFigure 7. PEGylation Retained the Activity of rhG-CSF in vitro. (Although The relative activities of PEG(5kD)-G-CSF and PEG(20kD)-G-CSF was down-regulated to about 67% and 40%, respectively, of that of unmodified rhG-CSF.)

Mono-PEG-G-CSFFigure 8. PEGylation increased the thermal stability of rhG-CSF. Left: pH 7.0 in 37 ; Right: pH 7.0, at 60

Chart1

100100100000000

99.303299153798.942029758798.62327044930.610.610.220.220.510.51

98.605764672698.623270449398.620.150.150.230.230.320.32

99.152274921498.695555005797.69555500570.880.880.350.350.180.18

97.65880618998.0196.94202975870.510.510.560.560.50.5

96.993632892597.265494741295.95395947980.210.210.840.840.470.47

96.605764672697.942029758794.150.850.850.650.651.221.22

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity remaining(%)

GABE

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:22

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.58232.62710.13060.12740.12780.12620.12600.1238......

B......2.53912.58972.41522.41542.36662.35882.33191.2827......

C......2.41502.43312.42692.39162.37752.34252.32832.2829......

D......2.02012.13202.31592.24582.11321.95531.90211.7159......

E......1.85581.86512.32092.24192.14512.01051.89421.7068......

F......1.70591.74532.09021.88121.91261.75211.70691.7217......

G......1.69561.70342.04841.80531.71521.70551.71051.6956......

H......1.70541.71230.13200.13370.13480.13160.13260.1412......

1.10471.08231.1271

1.06441.03911.08970.91520.91540.86660.85880.8319-0.2173

0.924050.91500.93310.92690.89160.87750.84250.82830.7829

0.576050.52010.63200.81590.74580.61320.45530.40210.2159

0.360450.35580.36510.82090.74190.64510.51050.39420.2068

0.22560.20590.24530.59020.38120.41260.25210.20690.2217

0.19950.19560.20340.54840.30530.21520.20550.21050.1956

0.208850.20540.2123

01234567

0100.000100.000100.000

196.34596.00996.68282.84682.86478.44777.74175.306

283.66584.54282.78883.90580.71079.43376.26574.98070.870

352.06448.05556.07373.85767.51255.50841.21536.39919.544

432.63432.87432.39374.31067.15958.39646.21235.68418.720

520.39419.02421.76453.42634.50737.35022.82118.72920.069

618.05918.07318.04649.64227.63619.48018.60219.05517.706

718.90718.97818.836

E

Time(min)0510204080160

83.66551.53431.07228.41520.71218.89218.887

23.14.24.354.955.25.25

IU/mL)2511.66291239425.44094102064.90365061193.2352028872.72047051032.6278012977

10046.6521.7619.6112.9410.8810.51

ApH2.2

Timehr012481632

83.66583.375577079881.786910473478.93998370677.002806191775.142572644270.870

22.052.12.152.182.22.26

IU/mL)2524.148408223123.325824788422.531261565322.067574907321.763764082420.8771979857

10096.5993.3090.1388.2787.0683.51

B(pH11.0)

Timehr012481632

83.66574.083461573367.335023083256.95211369643.7132253136.041459219719.1318910111

22.212.42.753.283.755.1

IU/mL)2521.613430782718.946457081414.865088937510.29488771587.43254446882.9157280986

10086.4575.7959.4641.1829.7311.66

GABE

0

0

0

0

0

0

0

0

GTS

00

00

00

00

00

00

00

TABE

0

0

0

0

0

0

0

TTS

0

0

0

0

0

0

0

FABE

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:31

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.9

123456789101112

A......2.73892.75440.14260.13520.13290.13780.13790.2667......

B......2.68042.69242.45282.46412.45822.39852.44232.4519......

C......2.46762.45992.44822.45362.44922.46732.50192.4615......

D......2.17352.20372.38132.27152.14641.89621.75711.7597......

E......1.93841.96522.40872.29092.15622.12391.80531.7723......

F......1.81471.89592.46732.45962.42962.40492.38122.3507......

G......1.75481.75932.45152.43102.42042.39252.37132.3753......

H......1.76841.74590.13560.13540.13450.13250.13610.1385......

1.246651.23891.2544

1.18641.18041.19240.95280.96410.95820.89850.94230.9519

0.963750.96760.95990.94820.95360.94920.96731.00190.9615

0.68860.67350.70370.88130.77150.64640.39620.25710.2597

0.45180.43840.46520.90870.79090.65620.62390.30530.2723

0.35530.31470.39590.96730.95960.92960.90490.88120.8507

0.257050.25480.25930.95150.93100.92040.89250.87130.8753

0.257150.26840.2459

100100100

95.16795.27895.05776.90777.81977.34372.52476.05976.834

77.30778.10276.52376.53676.97276.61678.07780.87077.609

55.23654.36356.09971.13662.27352.17531.98020.75220.962

36.24135.38637.08573.34763.83952.96650.35924.64321.979

28.50025.40231.56178.07777.45675.03473.04171.12868.666

20.61920.56720.67176.80275.14774.29272.04070.32970.651

20.62721.66419.603

01234567

T1(37

Timehr0248163264

78.10276.72177.39576.98075.30178.46577.222

22.12.082.092.112.052.08

IU/mL)2523.325824788423.651441168123.488068730423.164701547324.148408223123.6514411681

10093.3094.6193.9592.6696.5994.61

T260

Timehr00.5124816

78.10272.241504560563.055936718152.570828961241.16958592322.69755428221.470659456

22.22.52.93.65.255.5

IU/mL)2521.763764082417.677669529713.39716828178.24692444232.62780129772.2097086912

10087.055056329670.710678118753.588673126832.987697769310.51120519078.8388347648

S(

Timehr012481632

78.10277.439664218376.301557833674.663007506772.540156590570.728065219169.6585680846

22.052.082.142.22.252.32

IU/mL)2524.148408223123.651441168122.687978882921.763764082421.022410381320.0267469397

10096.5994.6190.7587.0684.0980.11

FABE

000

000

000

000

000

000

000

000

FTS

0

0

0

0

0

0

0

0

E

0

0

0

0

0

0

0

T1

0

0

0

0

0

0

0

S

0

0

0

0

0

0

0

A

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:27

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.55272.63540.13440.13260.13240.13190.13210.1312......

B......2.40572.55092.29032.28122.28012.28092.27052.2597......

C......2.27042.29822.28902.28392.27892.26322.26192.2581......

D......1.99791.91912.28132.26772.26712.08092.06052.0097......

E......1.83931.76682.28512.27862.25392.21322.11191.9981......

F......1.71821.65032.28572.28152.25052.11891.95871.7852......

G......1.70101.60642.28932.27912.23872.10581.97501.7514......

H......1.74321.58990.13180.13370.13230.13160.13410.1375......

1.144051.10271.1854

1.02830.95571.10090.84030.83120.83010.83090.82050.8097

0.83430.82040.84820.83900.83390.82890.81320.81190.8081

0.50850.54790.46910.83130.81770.81710.63090.61050.5597

0.353050.38930.31680.83510.82860.80390.76320.66190.5481

0.234250.26820.20030.83570.83150.80050.66890.50870.3352

0.20370.25100.15640.83930.82910.78870.65580.52500.3014

0.216550.29320.1399

0100100100

189.77086.66992.87270.88770.12070.02770.09469.21768.306

272.97774.39971.55470.77870.34869.92668.60168.49268.171

344.63049.68739.57370.12868.98168.93053.22351.50247.216

431.01535.30426.72570.44969.90067.81764.38355.83846.238

520.61024.32216.89770.49970.14567.53056.42842.91428.277

617.97822.76213.19470.80369.94366.53555.32344.28925.426

719.19626.58911.802

E

Time(min)0510204080160

72.97770.65170.04467.03255.87643.60126.852

22.022.052.152.53.13.5

IU/mL)2524.6624.1522.5317.6811.668.84

10098.6296.5990.1370.7146.6535.36

ApH2.2

Timehr012481632

72.97770.83370.23469.97669.34868.85468.239

22.022.042.062.12.132.14

IU/mL)2524.65624.31623.98223.32622.84622.688

10098.62397.26595.92693.30391.38390.752

B(pH11.0)

Timehr012481632

72.97770.28969.44168.37458.80353.67046.727

22.042.072.132.42.622.8

IU/mL)2524.31623.81622.84618.94616.26714.359

10097.26595.26491.38375.78665.06757.435

A

0

0

0

0

0

0

0

0

B

0

0

0

0

0

0

0

T2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:29

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.77322.68730.14160.14580.14360.13980.13760.1346......

B......2.57042.58932.49922.51702.51272.50132.50962.5134......

C......2.53062.49292.51192.52612.51332.49712.51842.4918......

D......2.25432.24072.48142.42932.35842.23112.11502.1074......

E......2.00961.96892.47912.43062.39132.27172.16541.9801......

F......1.83581.84762.53102.49022.49452.48922.47352.4635......

G......1.80311.74662.49752.49582.48682.47912.46972.4532......

H......1.79521.75760.14190.13580.13560.13300.13580.1381......

1.150251.19321.1073

0.999850.99041.00930.91920.93700.93270.92130.92960.9334

0.931750.95060.91290.93190.94610.93330.91710.93840.9118

0.66750.67430.66070.90140.84930.77840.65110.53500.5274

0.409250.42960.38890.89910.85060.81130.69170.58540.4001

0.26170.25580.26760.95100.91020.91450.90920.89350.8835

0.194850.22310.16660.91750.91580.90680.89910.88970.8732

0.19640.21520.1776

100100100

87.07783.00491.15079.91381.46181.08780.09680.81781.148

81.05679.66882.44481.01782.25281.13979.73081.58279.270

58.09056.51259.66878.36673.83667.67256.60546.51245.851

35.56336.00435.12178.16673.94970.53260.13550.89334.784

22.80321.43824.16782.67879.13179.50479.04477.67976.809

16.87218.69815.04679.76579.61778.83578.16677.34875.914

17.03718.03616.039

01234567

T1(37

Timehr0248163264

81.05680.46581.85681.11379.91381.20080.209

22.021.971.992.031.982

IU/mL)2524.65625.52525.17424.48625.34925.000

10098.623102.101100.69697.942101.396100.000

T260

Timehr00.5124816

81.05678.26559443673.892632036569.102369050258.369919582748.702455987840.3173223212

22.092.252.52.953.33.75

IU/mL)2523.48821.02217.67812.94110.1537.433

10093.95284.09070.71151.76340.61329.730

S(

Timehr012481632

81.05681.221473592779.374049119879.169745707578.604651162877.513584003576.3616605086

21.992.072.082.092.12.11

IU/mL)2525.17423.81623.65123.48823.32623.165

100100.69695.26494.60693.95293.30392.659

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:25

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.69012.70120.12910.12450.12190.12230.12170.1226......

B......2.59222.61932.50222.49172.47472.46442.45472.4510......

C......2.49532.51092.49842.49262.48492.47722.46992.4655......

D......2.27222.31982.48032.44162.41012.31682.17682.0643......

E......2.03302.00402.46242.46412.40252.30612.19792.0856......

F......1.87681.89692.48122.46132.37642.21252.13101.9979......

G......1.85241.87222.49012.47382.36572.24922.12742.0239......

H......1.84151.89670.51610.62610.21320.39200.21730.2197......

1.015651.01011.0212

0.925750.91220.93930.82220.81170.79470.78440.77470.7710

0.82310.81530.83090.81840.81260.80490.79720.78990.7855

0.6160.59220.63980.80030.76160.73010.63680.49680.3843

0.33850.35300.32400.78240.78410.72250.62610.51790.4056

0.206850.19680.21690.80120.78130.69640.53250.45100.3179

0.18230.17240.19220.81010.79380.68570.56920.44740.3439

0.18910.16150.2167

0100100100

191.14490.30891.98080.95379.91978.24577.23176.27675.912

281.04080.71581.36580.57980.00879.25078.49277.77377.340

360.64058.62862.65278.79774.98671.88562.69948.91437.838

433.33734.94731.72777.03477.20271.13761.64550.99239.935

520.36119.48321.24078.88576.92668.56752.42944.40531.300

617.94417.06818.82179.76278.15767.51356.04344.05133.860

718.60415.98921.220

E

Time(min)0510204080160

81.04079.32477.54168.04054.23644.22832.580

22.052.152.63.23.54.1

IU/mL)2524.1522.5316.4910.888.845.83

10096.5990.1365.9843.5335.3623.33

ApH2.2

Timehr012481632

81.04080.76679.96478.74877.86177.02576.626

22.012.042.092.142.182.2

IU/mL)2524.8324.3223.4922.6922.0721.76

10099.3197.2793.9590.7588.2787.06

B(pH11.0)

Timehr012481632

81.04077.91676.09471.51162.17249.95338.886

22.142.22.42.753.33.75

IU/mL)2522.6921.7618.9514.8710.157.43

10090.7587.0675.7959.4640.6129.73

000

000

000

000

000

000

000

000

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:12

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.7

123456789101112

A......2.71042.72500.12750.12900.13030.12840.12400.1195......

B......2.66942.65642.51822.50982.52352.50282.51152.5164......

C......2.52262.51642.50742.51962.51692.51442.49772.5111......

D......2.24462.23022.47692.40812.33482.17172.09571.9932......

E......2.01292.09372.49312.42222.35682.20642.11251.9778......

F......1.89741.91202.49842.51282.47472.45122.44562.4495......

G......1.82871.87082.49622.47832.46772.46522.45842.4342......

H......1.85491.86450.13310.13450.13300.12800.13240.1346......

1.06771.06041.0750

1.01291.01941.00640.86820.85980.87350.85280.86150.8664

0.86950.87260.86640.85740.86960.86690.86440.84770.8611

0.58740.59460.58020.82690.75810.68480.52170.44570.3432

0.40330.36290.44370.84310.77220.70680.55640.46250.3278

0.25470.24740.26200.84840.86280.82470.80120.79560.7995

0.199750.17870.22080.84620.82830.81770.81520.80840.7842

0.20970.20490.2145

0100100100

194.87696.13493.61981.31580.52881.81179.87380.68781.146

281.44382.29080.59580.30381.44681.19380.95979.39580.650

355.02356.07353.97277.44771.00364.13848.86241.74432.144

437.74934.22341.27478.96472.32466.19852.11243.31730.702

523.85123.33124.37279.46180.80977.24175.04074.51574.881

618.69616.85220.54079.25477.57876.58576.35175.71473.448

719.63819.32319.953

T1(37

Timehr0248163264

81.44380.80980.98781.50280.41680.04180.898

22.032.021.9922.042.03

IU/mL)2524.4924.6625.1725.0024.3224.49

10097.9498.62100.70100.0097.2797.94

T260

Timehr00.5124816

81.44378.205488433171.663388592365.168118385350.487028191442.530673410131.4226842746

22.092.32.63.243.74.3

IU/mL)2523.4920.3116.4910.587.695.08

10093.9581.2365.9842.3430.7820.31

S(

Timehr012481632

81.44379.357497424479.193593706176.912990540475.695420061875.114732602874.1640910368

22.042.072.122.172.222.25

IU/mL)2524.31623.81623.00522.22121.46421.022

10097.26595.26492.01988.88485.85784.090

000

000

000

000

000

000

000

000

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

E

Time(min)0510204080160

T10098.6296.5990.1370.7146.6538.36

F10096.5990.1365.9843.5332.3625.33

G10046.6521.7616.2312.9410.8810.51

T00.090.350.561.591.243.85

F00.150.961.121.562.783.25

G01.252.893.120.950.510.29

000000000

0000.090.090.150.151.251.25

0000.350.350.960.962.892.89

0000.560.561.121.123.123.12

0001.591.591.561.560.950.95

0001.241.242.782.780.510.51

0003.853.853.253.250.290.29

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (min)

Relative Activity Remaining (%)

T1(37

Timehr0248163264

T10099.3098.6199.1597.6696.9996.61

F10098.9498.6298.7098.0197.2797.94

G10098.6298.6297.7096.9495.9594.15

T00.610.150.880.510.210.85

F00.220.230.350.560.840.65

G00.510.320.180.50.471.22

000000

0.610.610.220.220.510.51

0.150.150.230.230.320.32

0.880.880.350.350.180.18

0.510.510.560.560.50.5

0.210.210.840.840.470.47

0.850.850.650.651.221.22

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity remaining(%)

S(

Timehr012481632

T10098.2096.9695.3193.9593.2092.66

F10097.2795.2692.0288.8885.8684.09

G10096.5994.6190.7587.0683.0980.11

T01.520.510.610.230.150.47

F00.050.840.910.620.970.59

G00.890.490.560.611.020.52

000000000

0001.521.520.050.050.890.89

0000.510.510.840.840.490.49

0000.610.610.910.910.560.56

0000.230.230.620.620.610.61

0000.150.150.970.971.021.02

0000.470.470.590.590.520.52

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

ApH2.2

Timehr012481632

T10098.62397.26595.42693.30391.38390.752

F10097.8196.2793.9590.7588.2787.06

G10096.5993.9090.1386.2784.0683.51

T00.410.390.481.050.461.28

F00.380.850.911.091.271.21

G00.280.911.260.612.020.52

000000000

0000.410.410.380.380.280.28

0000.390.390.850.850.910.91

0000.480.480.910.911.261.26

0001.051.051.091.090.610.61

0000.460.461.271.272.022.02

0001.281.281.211.210.520.52

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

B(pH11.0)

Timehr012481632

T10096.2792.5885.3775.7965.0757.43

F10092.7587.0675.7959.4643.6135.73

G10086.4575.7959.4639.1822.7311.66

T00.681.123.951.521.322.58

F01.253.301.581.692.093.69

G01.582.542.690.855.151.08

000000000

0000.680.681.251.251.581.58

0001.121.123.33.32.542.54

0003.953.951.581.582.692.69

0001.521.521.691.690.850.85

0001.321.322.092.095.155.15

0002.582.583.693.691.081.08

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

T260

Timehr00.5124816

T10092.9585.0970.7151.7640.6132.73

F10090.9581.2362.9842.3430.7822.31

G10086.0971.7150.5927.5810.518.84

T00.563.122.151.591.624.52

F00.150.961.121.561.261.15

G02.151.951.024.121.121.58

000000

0.560.560.150.152.152.15

3.123.120.960.961.951.95

2.152.151.121.121.021.02

1.591.591.561.564.124.12

1.621.621.261.261.121.12

4.524.521.151.151.581.58

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

Chart2

100100100000000

92.952274921490.952274921486.090.560.560.150.152.152.15

85.089641525481.225239635671.71067811873.123.120.960.961.951.95

70.710678118762.975395538650.58867312682.152.151.121.121.021.02

51.763246192142.337265618127.581.591.591.561.564.124.12

40.612619817830.778610333610.51120519071.621.621.261.261.121.12

32.730177875122.30630990898.83883476484.524.521.151.151.581.58

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

GABE

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:22

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.58232.62710.13060.12740.12780.12620.12600.1238......

B......2.53912.58972.41522.41542.36662.35882.33191.2827......

C......2.41502.43312.42692.39162.37752.34252.32832.2829......

D......2.02012.13202.31592.24582.11321.95531.90211.7159......

E......1.85581.86512.32092.24192.14512.01051.89421.7068......

F......1.70591.74532.09021.88121.91261.75211.70691.7217......

G......1.69561.70342.04841.80531.71521.70551.71051.6956......

H......1.70541.71230.13200.13370.13480.13160.13260.1412......

1.10471.08231.1271

1.06441.03911.08970.91520.91540.86660.85880.8319-0.2173

0.924050.91500.93310.92690.89160.87750.84250.82830.7829

0.576050.52010.63200.81590.74580.61320.45530.40210.2159

0.360450.35580.36510.82090.74190.64510.51050.39420.2068

0.22560.20590.24530.59020.38120.41260.25210.20690.2217

0.19950.19560.20340.54840.30530.21520.20550.21050.1956

0.208850.20540.2123

01234567

0100.000100.000100.000

196.34596.00996.68282.84682.86478.44777.74175.306

283.66584.54282.78883.90580.71079.43376.26574.98070.870

352.06448.05556.07373.85767.51255.50841.21536.39919.544

432.63432.87432.39374.31067.15958.39646.21235.68418.720

520.39419.02421.76453.42634.50737.35022.82118.72920.069

618.05918.07318.04649.64227.63619.48018.60219.05517.706

718.90718.97818.836

E

Time(min)0510204080160

83.66551.53431.07228.41520.71218.89218.887

23.14.24.354.955.25.25

IU/mL)2511.66291239425.44094102064.90365061193.2352028872.72047051032.6278012977

10046.6521.7619.6112.9410.8810.51

ApH2.2

Timehr012481632

83.66583.375577079881.786910473478.93998370677.002806191775.142572644270.870

22.052.12.152.182.22.26

IU/mL)2524.148408223123.325824788422.531261565322.067574907321.763764082420.8771979857

10096.5993.3090.1388.2787.0683.51

B(pH11.0)

Timehr012481632

83.66574.083461573367.335023083256.95211369643.7132253136.041459219719.1318910111

22.212.42.753.283.755.1

IU/mL)2521.613430782718.946457081414.865088937510.29488771587.43254446882.9157280986

10086.4575.7959.4641.1829.7311.66

GABE

0

0

0

0

0

0

0

0

GTS

00

00

00

00

00

00

00

TABE

0

0

0

0

0

0

0

TTS

0

0

0

0

0

0

0

FABE

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:31

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.9

123456789101112

A......2.73892.75440.14260.13520.13290.13780.13790.2667......

B......2.68042.69242.45282.46412.45822.39852.44232.4519......

C......2.46762.45992.44822.45362.44922.46732.50192.4615......

D......2.17352.20372.38132.27152.14641.89621.75711.7597......

E......1.93841.96522.40872.29092.15622.12391.80531.7723......

F......1.81471.89592.46732.45962.42962.40492.38122.3507......

G......1.75481.75932.45152.43102.42042.39252.37132.3753......

H......1.76841.74590.13560.13540.13450.13250.13610.1385......

1.246651.23891.2544

1.18641.18041.19240.95280.96410.95820.89850.94230.9519

0.963750.96760.95990.94820.95360.94920.96731.00190.9615

0.68860.67350.70370.88130.77150.64640.39620.25710.2597

0.45180.43840.46520.90870.79090.65620.62390.30530.2723

0.35530.31470.39590.96730.95960.92960.90490.88120.8507

0.257050.25480.25930.95150.93100.92040.89250.87130.8753

0.257150.26840.2459

100100100

95.16795.27895.05776.90777.81977.34372.52476.05976.834

77.30778.10276.52376.53676.97276.61678.07780.87077.609

55.23654.36356.09971.13662.27352.17531.98020.75220.962

36.24135.38637.08573.34763.83952.96650.35924.64321.979

28.50025.40231.56178.07777.45675.03473.04171.12868.666

20.61920.56720.67176.80275.14774.29272.04070.32970.651

20.62721.66419.603

01234567

T1(37

Timehr0248163264

78.10276.72177.39576.98075.30178.46577.222

22.12.082.092.112.052.08

IU/mL)2523.325824788423.651441168123.488068730423.164701547324.148408223123.6514411681

10093.3094.6193.9592.6696.5994.61

T260

Timehr00.5124816

78.10272.241504560563.055936718152.570828961241.16958592322.69755428221.470659456

22.22.52.93.65.255.5

IU/mL)2521.763764082417.677669529713.39716828178.24692444232.62780129772.2097086912

10087.055056329670.710678118753.588673126832.987697769310.51120519078.8388347648

S(

Timehr012481632

78.10277.439664218376.301557833674.663007506772.540156590570.728065219169.6585680846

22.052.082.142.22.252.32

IU/mL)2524.148408223123.651441168122.687978882921.763764082421.022410381320.0267469397

10096.5994.6190.7587.0684.0980.11

FABE

000

000

000

000

000

000

000

000

FTS

0

0

0

0

0

0

0

0

E

0

0

0

0

0

0

0

T1

0

0

0

0

0

0

0

S

0

0

0

0

0

0

0

A

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:27

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.55272.63540.13440.13260.13240.13190.13210.1312......

B......2.40572.55092.29032.28122.28012.28092.27052.2597......

C......2.27042.29822.28902.28392.27892.26322.26192.2581......

D......1.99791.91912.28132.26772.26712.08092.06052.0097......

E......1.83931.76682.28512.27862.25392.21322.11191.9981......

F......1.71821.65032.28572.28152.25052.11891.95871.7852......

G......1.70101.60642.28932.27912.23872.10581.97501.7514......

H......1.74321.58990.13180.13370.13230.13160.13410.1375......

1.144051.10271.1854

1.02830.95571.10090.84030.83120.83010.83090.82050.8097

0.83430.82040.84820.83900.83390.82890.81320.81190.8081

0.50850.54790.46910.83130.81770.81710.63090.61050.5597

0.353050.38930.31680.83510.82860.80390.76320.66190.5481

0.234250.26820.20030.83570.83150.80050.66890.50870.3352

0.20370.25100.15640.83930.82910.78870.65580.52500.3014

0.216550.29320.1399

0100100100

189.77086.66992.87270.88770.12070.02770.09469.21768.306

272.97774.39971.55470.77870.34869.92668.60168.49268.171

344.63049.68739.57370.12868.98168.93053.22351.50247.216

431.01535.30426.72570.44969.90067.81764.38355.83846.238

520.61024.32216.89770.49970.14567.53056.42842.91428.277

617.97822.76213.19470.80369.94366.53555.32344.28925.426

719.19626.58911.802

E

Time(min)0510204080160

72.97770.65170.04467.03255.87643.60126.852

22.022.052.152.53.13.5

IU/mL)2524.6624.1522.5317.6811.668.84

10098.6296.5990.1370.7146.6535.36

ApH2.2

Timehr012481632

72.97770.83370.23469.97669.34868.85468.239

22.022.042.062.12.132.14

IU/mL)2524.65624.31623.98223.32622.84622.688

10098.62397.26595.92693.30391.38390.752

B(pH11.0)

Timehr012481632

72.97770.28969.44168.37458.80353.67046.727

22.042.072.132.42.622.8

IU/mL)2524.31623.81622.84618.94616.26714.359

10097.26595.26491.38375.78665.06757.435

A

0

0

0

0

0

0

0

0

B

0

0

0

0

0

0

0

T2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:29

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.77322.68730.14160.14580.14360.13980.13760.1346......

B......2.57042.58932.49922.51702.51272.50132.50962.5134......

C......2.53062.49292.51192.52612.51332.49712.51842.4918......

D......2.25432.24072.48142.42932.35842.23112.11502.1074......

E......2.00961.96892.47912.43062.39132.27172.16541.9801......

F......1.83581.84762.53102.49022.49452.48922.47352.4635......

G......1.80311.74662.49752.49582.48682.47912.46972.4532......

H......1.79521.75760.14190.13580.13560.13300.13580.1381......

1.150251.19321.1073

0.999850.99041.00930.91920.93700.93270.92130.92960.9334

0.931750.95060.91290.93190.94610.93330.91710.93840.9118

0.66750.67430.66070.90140.84930.77840.65110.53500.5274

0.409250.42960.38890.89910.85060.81130.69170.58540.4001

0.26170.25580.26760.95100.91020.91450.90920.89350.8835

0.194850.22310.16660.91750.91580.90680.89910.88970.8732

0.19640.21520.1776

100100100

87.07783.00491.15079.91381.46181.08780.09680.81781.148

81.05679.66882.44481.01782.25281.13979.73081.58279.270

58.09056.51259.66878.36673.83667.67256.60546.51245.851

35.56336.00435.12178.16673.94970.53260.13550.89334.784

22.80321.43824.16782.67879.13179.50479.04477.67976.809

16.87218.69815.04679.76579.61778.83578.16677.34875.914

17.03718.03616.039

01234567

T1(37

Timehr0248163264

81.05680.46581.85681.11379.91381.20080.209

22.021.971.992.031.982

IU/mL)2524.65625.52525.17424.48625.34925.000

10098.623102.101100.69697.942101.396100.000

T260

Timehr00.5124816

81.05678.26559443673.892632036569.102369050258.369919582748.702455987840.3173223212

22.092.252.52.953.33.75

IU/mL)2523.48821.02217.67812.94110.1537.433

10093.95284.09070.71151.76340.61329.730

S(

Timehr012481632

81.05681.221473592779.374049119879.169745707578.604651162877.513584003576.3616605086

21.992.072.082.092.12.11

IU/mL)2525.17423.81623.65123.48823.32623.165

100100.69695.26494.60693.95293.30392.659

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:25

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.69012.70120.12910.12450.12190.12230.12170.1226......

B......2.59222.61932.50222.49172.47472.46442.45472.4510......

C......2.49532.51092.49842.49262.48492.47722.46992.4655......

D......2.27222.31982.48032.44162.41012.31682.17682.0643......

E......2.03302.00402.46242.46412.40252.30612.19792.0856......

F......1.87681.89692.48122.46132.37642.21252.13101.9979......

G......1.85241.87222.49012.47382.36572.24922.12742.0239......

H......1.84151.89670.51610.62610.21320.39200.21730.2197......

1.015651.01011.0212

0.925750.91220.93930.82220.81170.79470.78440.77470.7710

0.82310.81530.83090.81840.81260.80490.79720.78990.7855

0.6160.59220.63980.80030.76160.73010.63680.49680.3843

0.33850.35300.32400.78240.78410.72250.62610.51790.4056

0.206850.19680.21690.80120.78130.69640.53250.45100.3179

0.18230.17240.19220.81010.79380.68570.56920.44740.3439

0.18910.16150.2167

0100100100

191.14490.30891.98080.95379.91978.24577.23176.27675.912

281.04080.71581.36580.57980.00879.25078.49277.77377.340

360.64058.62862.65278.79774.98671.88562.69948.91437.838

433.33734.94731.72777.03477.20271.13761.64550.99239.935

520.36119.48321.24078.88576.92668.56752.42944.40531.300

617.94417.06818.82179.76278.15767.51356.04344.05133.860

718.60415.98921.220

E

Time(min)0510204080160

81.04079.32477.54168.04054.23644.22832.580

22.052.152.63.23.54.1

IU/mL)2524.1522.5316.4910.888.845.83

10096.5990.1365.9843.5335.3623.33

ApH2.2

Timehr012481632

81.04080.76679.96478.74877.86177.02576.626

22.012.042.092.142.182.2

IU/mL)2524.8324.3223.4922.6922.0721.76

10099.3197.2793.9590.7588.2787.06

B(pH11.0)

Timehr012481632

81.04077.91676.09471.51162.17249.95338.886

22.142.22.42.753.33.75

IU/mL)2522.6921.7618.9514.8710.157.43

10090.7587.0675.7959.4640.6129.73

000

000

000

000

000

000

000

000

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:12

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.7

123456789101112

A......2.71042.72500.12750.12900.13030.12840.12400.1195......

B......2.66942.65642.51822.50982.52352.50282.51152.5164......

C......2.52262.51642.50742.51962.51692.51442.49772.5111......

D......2.24462.23022.47692.40812.33482.17172.09571.9932......

E......2.01292.09372.49312.42222.35682.20642.11251.9778......

F......1.89741.91202.49842.51282.47472.45122.44562.4495......

G......1.82871.87082.49622.47832.46772.46522.45842.4342......

H......1.85491.86450.13310.13450.13300.12800.13240.1346......

1.06771.06041.0750

1.01291.01941.00640.86820.85980.87350.85280.86150.8664

0.86950.87260.86640.85740.86960.86690.86440.84770.8611

0.58740.59460.58020.82690.75810.68480.52170.44570.3432

0.40330.36290.44370.84310.77220.70680.55640.46250.3278

0.25470.24740.26200.84840.86280.82470.80120.79560.7995

0.199750.17870.22080.84620.82830.81770.81520.80840.7842

0.20970.20490.2145

0100100100

194.87696.13493.61981.31580.52881.81179.87380.68781.146

281.44382.29080.59580.30381.44681.19380.95979.39580.650

355.02356.07353.97277.44771.00364.13848.86241.74432.144

437.74934.22341.27478.96472.32466.19852.11243.31730.702

523.85123.33124.37279.46180.80977.24175.04074.51574.881

618.69616.85220.54079.25477.57876.58576.35175.71473.448

719.63819.32319.953

T1(37

Timehr0248163264

81.44380.80980.98781.50280.41680.04180.898

22.032.021.9922.042.03

IU/mL)2524.4924.6625.1725.0024.3224.49

10097.9498.62100.70100.0097.2797.94

T260

Timehr00.5124816

81.44378.205488433171.663388592365.168118385350.487028191442.530673410131.4226842746

22.092.32.63.243.74.3

IU/mL)2523.4920.3116.4910.587.695.08

10093.9581.2365.9842.3430.7820.31

S(

Timehr012481632

81.44379.357497424479.193593706176.912990540475.695420061875.114732602874.1640910368

22.042.072.122.172.222.25

IU/mL)2524.31623.81623.00522.22121.46421.022

10097.26595.26492.01988.88485.85784.090

000

000

000

000

000

000

000

000

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

E

Time(min)0510204080160

T10098.6296.5990.1370.7146.6538.36

F10096.5990.1365.9843.5332.3625.33

G10046.6521.7616.2312.9410.8810.51

T00.090.350.561.591.243.85

F00.150.961.121.562.783.25

G01.252.893.120.950.510.29

000000000

0000.090.090.150.151.251.25

0000.350.350.960.962.892.89

0000.560.561.121.123.123.12

0001.591.591.561.560.950.95

0001.241.242.782.780.510.51

0003.853.853.253.250.290.29

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (min)

Relative Activity Remaining (%)

T1(37

Timehr0248163264

T10099.3098.6199.1597.6696.9996.61

F10098.9498.6298.7098.0197.2797.94

G10098.6298.6297.7096.9495.9594.15

T00.610.150.880.510.210.85

F00.220.230.350.560.840.65

G00.510.320.180.50.471.22

000000

0.610.610.220.220.510.51

0.150.150.230.230.320.32

0.880.880.350.350.180.18

0.510.510.560.560.50.5

0.210.210.840.840.470.47

0.850.850.650.651.221.22

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity remaining(%)

S(

Timehr012481632

T10098.2096.9695.3193.9593.2092.66

F10097.2795.2692.0288.8885.8684.09

G10096.5994.6190.7587.0683.0980.11

T01.520.510.610.230.150.47

F00.050.840.910.620.970.59

G00.890.490.560.611.020.52

000000000

0001.521.520.050.050.890.89

0000.510.510.840.840.490.49

0000.610.610.910.910.560.56

0000.230.230.620.620.610.61

0000.150.150.970.971.021.02

0000.470.470.590.590.520.52

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

ApH2.2

Timehr012481632

T10098.62397.26595.42693.30391.38390.752

F10097.8196.2793.9590.7588.2787.06

G10096.5993.9090.1386.2784.0683.51

T00.410.390.481.050.461.28

F00.380.850.911.091.271.21

G00.280.911.260.612.020.52

000000000

0000.410.410.380.380.280.28

0000.390.390.850.850.910.91

0000.480.480.910.911.261.26

0001.051.051.091.090.610.61

0000.460.461.271.272.022.02

0001.281.281.211.210.520.52

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

B(pH11.0)

Timehr012481632

T10096.2792.5885.3775.7965.0757.43

F10092.7587.0675.7959.4643.6135.73

G10086.4575.7959.4639.1822.7311.66

T00.681.123.951.521.322.58

F01.253.301.581.692.093.69

G01.582.542.690.855.151.08

000000000

0000.680.681.251.251.581.58

0001.121.123.33.32.542.54

0003.953.951.581.582.692.69

0001.521.521.691.690.850.85

0001.321.322.092.095.155.15

0002.582.583.693.691.081.08

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

T260

Timehr00.5124816

T10092.9585.0970.7151.7640.6132.73

F10090.9581.2362.9842.3430.7822.31

G10086.0971.7150.5927.5810.518.84

T00.563.122.151.591.624.52

F00.150.961.121.561.261.15

G02.151.951.024.121.121.58

000000

0.560.560.150.152.152.15

3.123.120.960.961.951.95

2.152.151.121.121.021.02

1.591.591.561.564.124.12

1.621.621.261.261.121.12

4.524.521.151.151.581.58

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

Mono-PEG-G-CSF pHFigure 9. PEGylated rhG-CSF had distinctly higher acid-base stability than unmodified rhG-CSF. Left: pH 2.2; Right: pH 11.0.

Chart3

100100100000000

98.623270449397.8196.59363289250.410.410.380.380.280.28

97.265494741296.265494741293.90329915370.390.390.850.850.910.91

95.426411932593.952274921490.12504626110.480.480.910.911.261.26

93.303299153790.751915531786.27029962911.051.051.091.090.610.61

91.383145022988.270299629184.05505632960.460.461.271.272.022.02

90.751915531787.055056329683.50879194281.281.281.211.210.520.52

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

GABE

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:22

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.58232.62710.13060.12740.12780.12620.12600.1238......

B......2.53912.58972.41522.41542.36662.35882.33191.2827......

C......2.41502.43312.42692.39162.37752.34252.32832.2829......

D......2.02012.13202.31592.24582.11321.95531.90211.7159......

E......1.85581.86512.32092.24192.14512.01051.89421.7068......

F......1.70591.74532.09021.88121.91261.75211.70691.7217......

G......1.69561.70342.04841.80531.71521.70551.71051.6956......

H......1.70541.71230.13200.13370.13480.13160.13260.1412......

1.10471.08231.1271

1.06441.03911.08970.91520.91540.86660.85880.8319-0.2173

0.924050.91500.93310.92690.89160.87750.84250.82830.7829

0.576050.52010.63200.81590.74580.61320.45530.40210.2159

0.360450.35580.36510.82090.74190.64510.51050.39420.2068

0.22560.20590.24530.59020.38120.41260.25210.20690.2217

0.19950.19560.20340.54840.30530.21520.20550.21050.1956

0.208850.20540.2123

01234567

0100.000100.000100.000

196.34596.00996.68282.84682.86478.44777.74175.306

283.66584.54282.78883.90580.71079.43376.26574.98070.870

352.06448.05556.07373.85767.51255.50841.21536.39919.544

432.63432.87432.39374.31067.15958.39646.21235.68418.720

520.39419.02421.76453.42634.50737.35022.82118.72920.069

618.05918.07318.04649.64227.63619.48018.60219.05517.706

718.90718.97818.836

E

Time(min)0510204080160

83.66551.53431.07228.41520.71218.89218.887

23.14.24.354.955.25.25

IU/mL)2511.66291239425.44094102064.90365061193.2352028872.72047051032.6278012977

10046.6521.7619.6112.9410.8810.51

ApH2.2

Timehr012481632

83.66583.375577079881.786910473478.93998370677.002806191775.142572644270.870

22.052.12.152.182.22.26

IU/mL)2524.148408223123.325824788422.531261565322.067574907321.763764082420.8771979857

10096.5993.3090.1388.2787.0683.51

B(pH11.0)

Timehr012481632

83.66574.083461573367.335023083256.95211369643.7132253136.041459219719.1318910111

22.212.42.753.283.755.1

IU/mL)2521.613430782718.946457081414.865088937510.29488771587.43254446882.9157280986

10086.4575.7959.4641.1829.7311.66

GABE

0

0

0

0

0

0

0

0

GTS

00

00

00

00

00

00

00

TABE

0

0

0

0

0

0

0

TTS

0

0

0

0

0

0

0

FABE

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:31

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.9

123456789101112

A......2.73892.75440.14260.13520.13290.13780.13790.2667......

B......2.68042.69242.45282.46412.45822.39852.44232.4519......

C......2.46762.45992.44822.45362.44922.46732.50192.4615......

D......2.17352.20372.38132.27152.14641.89621.75711.7597......

E......1.93841.96522.40872.29092.15622.12391.80531.7723......

F......1.81471.89592.46732.45962.42962.40492.38122.3507......

G......1.75481.75932.45152.43102.42042.39252.37132.3753......

H......1.76841.74590.13560.13540.13450.13250.13610.1385......

1.246651.23891.2544

1.18641.18041.19240.95280.96410.95820.89850.94230.9519

0.963750.96760.95990.94820.95360.94920.96731.00190.9615

0.68860.67350.70370.88130.77150.64640.39620.25710.2597

0.45180.43840.46520.90870.79090.65620.62390.30530.2723

0.35530.31470.39590.96730.95960.92960.90490.88120.8507

0.257050.25480.25930.95150.93100.92040.89250.87130.8753

0.257150.26840.2459

100100100

95.16795.27895.05776.90777.81977.34372.52476.05976.834

77.30778.10276.52376.53676.97276.61678.07780.87077.609

55.23654.36356.09971.13662.27352.17531.98020.75220.962

36.24135.38637.08573.34763.83952.96650.35924.64321.979

28.50025.40231.56178.07777.45675.03473.04171.12868.666

20.61920.56720.67176.80275.14774.29272.04070.32970.651

20.62721.66419.603

01234567

T1(37

Timehr0248163264

78.10276.72177.39576.98075.30178.46577.222

22.12.082.092.112.052.08

IU/mL)2523.325824788423.651441168123.488068730423.164701547324.148408223123.6514411681

10093.3094.6193.9592.6696.5994.61

T260

Timehr00.5124816

78.10272.241504560563.055936718152.570828961241.16958592322.69755428221.470659456

22.22.52.93.65.255.5

IU/mL)2521.763764082417.677669529713.39716828178.24692444232.62780129772.2097086912

10087.055056329670.710678118753.588673126832.987697769310.51120519078.8388347648

S(

Timehr012481632

78.10277.439664218376.301557833674.663007506772.540156590570.728065219169.6585680846

22.052.082.142.22.252.32

IU/mL)2524.148408223123.651441168122.687978882921.763764082421.022410381320.0267469397

10096.5994.6190.7587.0684.0980.11

FABE

000

000

000

000

000

000

000

000

FTS

0

0

0

0

0

0

0

0

E

0

0

0

0

0

0

0

T1

0

0

0

0

0

0

0

S

0

0

0

0

0

0

0

A

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:27

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.55272.63540.13440.13260.13240.13190.13210.1312......

B......2.40572.55092.29032.28122.28012.28092.27052.2597......

C......2.27042.29822.28902.28392.27892.26322.26192.2581......

D......1.99791.91912.28132.26772.26712.08092.06052.0097......

E......1.83931.76682.28512.27862.25392.21322.11191.9981......

F......1.71821.65032.28572.28152.25052.11891.95871.7852......

G......1.70101.60642.28932.27912.23872.10581.97501.7514......

H......1.74321.58990.13180.13370.13230.13160.13410.1375......

1.144051.10271.1854

1.02830.95571.10090.84030.83120.83010.83090.82050.8097

0.83430.82040.84820.83900.83390.82890.81320.81190.8081

0.50850.54790.46910.83130.81770.81710.63090.61050.5597

0.353050.38930.31680.83510.82860.80390.76320.66190.5481

0.234250.26820.20030.83570.83150.80050.66890.50870.3352

0.20370.25100.15640.83930.82910.78870.65580.52500.3014

0.216550.29320.1399

0100100100

189.77086.66992.87270.88770.12070.02770.09469.21768.306

272.97774.39971.55470.77870.34869.92668.60168.49268.171

344.63049.68739.57370.12868.98168.93053.22351.50247.216

431.01535.30426.72570.44969.90067.81764.38355.83846.238

520.61024.32216.89770.49970.14567.53056.42842.91428.277

617.97822.76213.19470.80369.94366.53555.32344.28925.426

719.19626.58911.802

E

Time(min)0510204080160

72.97770.65170.04467.03255.87643.60126.852

22.022.052.152.53.13.5

IU/mL)2524.6624.1522.5317.6811.668.84

10098.6296.5990.1370.7146.6535.36

ApH2.2

Timehr012481632

72.97770.83370.23469.97669.34868.85468.239

22.022.042.062.12.132.14

IU/mL)2524.65624.31623.98223.32622.84622.688

10098.62397.26595.92693.30391.38390.752

B(pH11.0)

Timehr012481632

72.97770.28969.44168.37458.80353.67046.727

22.042.072.132.42.622.8

IU/mL)2524.31623.81622.84618.94616.26714.359

10097.26595.26491.38375.78665.06757.435

A

0

0

0

0

0

0

0

0

B

0

0

0

0

0

0

0

T2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:29

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.77322.68730.14160.14580.14360.13980.13760.1346......

B......2.57042.58932.49922.51702.51272.50132.50962.5134......

C......2.53062.49292.51192.52612.51332.49712.51842.4918......

D......2.25432.24072.48142.42932.35842.23112.11502.1074......

E......2.00961.96892.47912.43062.39132.27172.16541.9801......

F......1.83581.84762.53102.49022.49452.48922.47352.4635......

G......1.80311.74662.49752.49582.48682.47912.46972.4532......

H......1.79521.75760.14190.13580.13560.13300.13580.1381......

1.150251.19321.1073

0.999850.99041.00930.91920.93700.93270.92130.92960.9334

0.931750.95060.91290.93190.94610.93330.91710.93840.9118

0.66750.67430.66070.90140.84930.77840.65110.53500.5274

0.409250.42960.38890.89910.85060.81130.69170.58540.4001

0.26170.25580.26760.95100.91020.91450.90920.89350.8835

0.194850.22310.16660.91750.91580.90680.89910.88970.8732

0.19640.21520.1776

100100100

87.07783.00491.15079.91381.46181.08780.09680.81781.148

81.05679.66882.44481.01782.25281.13979.73081.58279.270

58.09056.51259.66878.36673.83667.67256.60546.51245.851

35.56336.00435.12178.16673.94970.53260.13550.89334.784

22.80321.43824.16782.67879.13179.50479.04477.67976.809

16.87218.69815.04679.76579.61778.83578.16677.34875.914

17.03718.03616.039

01234567

T1(37

Timehr0248163264

81.05680.46581.85681.11379.91381.20080.209

22.021.971.992.031.982

IU/mL)2524.65625.52525.17424.48625.34925.000

10098.623102.101100.69697.942101.396100.000

T260

Timehr00.5124816

81.05678.26559443673.892632036569.102369050258.369919582748.702455987840.3173223212

22.092.252.52.953.33.75

IU/mL)2523.48821.02217.67812.94110.1537.433

10093.95284.09070.71151.76340.61329.730

S(

Timehr012481632

81.05681.221473592779.374049119879.169745707578.604651162877.513584003576.3616605086

21.992.072.082.092.12.11

IU/mL)2525.17423.81623.65123.48823.32623.165

100100.69695.26494.60693.95293.30392.659

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:25

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.69012.70120.12910.12450.12190.12230.12170.1226......

B......2.59222.61932.50222.49172.47472.46442.45472.4510......

C......2.49532.51092.49842.49262.48492.47722.46992.4655......

D......2.27222.31982.48032.44162.41012.31682.17682.0643......

E......2.03302.00402.46242.46412.40252.30612.19792.0856......

F......1.87681.89692.48122.46132.37642.21252.13101.9979......

G......1.85241.87222.49012.47382.36572.24922.12742.0239......

H......1.84151.89670.51610.62610.21320.39200.21730.2197......

1.015651.01011.0212

0.925750.91220.93930.82220.81170.79470.78440.77470.7710

0.82310.81530.83090.81840.81260.80490.79720.78990.7855

0.6160.59220.63980.80030.76160.73010.63680.49680.3843

0.33850.35300.32400.78240.78410.72250.62610.51790.4056

0.206850.19680.21690.80120.78130.69640.53250.45100.3179

0.18230.17240.19220.81010.79380.68570.56920.44740.3439

0.18910.16150.2167

0100100100

191.14490.30891.98080.95379.91978.24577.23176.27675.912

281.04080.71581.36580.57980.00879.25078.49277.77377.340

360.64058.62862.65278.79774.98671.88562.69948.91437.838

433.33734.94731.72777.03477.20271.13761.64550.99239.935

520.36119.48321.24078.88576.92668.56752.42944.40531.300

617.94417.06818.82179.76278.15767.51356.04344.05133.860

718.60415.98921.220

E

Time(min)0510204080160

81.04079.32477.54168.04054.23644.22832.580

22.052.152.63.23.54.1

IU/mL)2524.1522.5316.4910.888.845.83

10096.5990.1365.9843.5335.3623.33

ApH2.2

Timehr012481632

81.04080.76679.96478.74877.86177.02576.626

22.012.042.092.142.182.2

IU/mL)2524.8324.3223.4922.6922.0721.76

10099.3197.2793.9590.7588.2787.06

B(pH11.0)

Timehr012481632

81.04077.91676.09471.51162.17249.95338.886

22.142.22.42.753.33.75

IU/mL)2522.6921.7618.9514.8710.157.43

10090.7587.0675.7959.4640.6129.73

000

000

000

000

000

000

000

000

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:12

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.7

123456789101112

A......2.71042.72500.12750.12900.13030.12840.12400.1195......

B......2.66942.65642.51822.50982.52352.50282.51152.5164......

C......2.52262.51642.50742.51962.51692.51442.49772.5111......

D......2.24462.23022.47692.40812.33482.17172.09571.9932......

E......2.01292.09372.49312.42222.35682.20642.11251.9778......

F......1.89741.91202.49842.51282.47472.45122.44562.4495......

G......1.82871.87082.49622.47832.46772.46522.45842.4342......

H......1.85491.86450.13310.13450.13300.12800.13240.1346......

1.06771.06041.0750

1.01291.01941.00640.86820.85980.87350.85280.86150.8664

0.86950.87260.86640.85740.86960.86690.86440.84770.8611

0.58740.59460.58020.82690.75810.68480.52170.44570.3432

0.40330.36290.44370.84310.77220.70680.55640.46250.3278

0.25470.24740.26200.84840.86280.82470.80120.79560.7995

0.199750.17870.22080.84620.82830.81770.81520.80840.7842

0.20970.20490.2145

0100100100

194.87696.13493.61981.31580.52881.81179.87380.68781.146

281.44382.29080.59580.30381.44681.19380.95979.39580.650

355.02356.07353.97277.44771.00364.13848.86241.74432.144

437.74934.22341.27478.96472.32466.19852.11243.31730.702

523.85123.33124.37279.46180.80977.24175.04074.51574.881

618.69616.85220.54079.25477.57876.58576.35175.71473.448

719.63819.32319.953

T1(37

Timehr0248163264

81.44380.80980.98781.50280.41680.04180.898

22.032.021.9922.042.03

IU/mL)2524.4924.6625.1725.0024.3224.49

10097.9498.62100.70100.0097.2797.94

T260

Timehr00.5124816

81.44378.205488433171.663388592365.168118385350.487028191442.530673410131.4226842746

22.092.32.63.243.74.3

IU/mL)2523.4920.3116.4910.587.695.08

10093.9581.2365.9842.3430.7820.31

S(

Timehr012481632

81.44379.357497424479.193593706176.912990540475.695420061875.114732602874.1640910368

22.042.072.122.172.222.25

IU/mL)2524.31623.81623.00522.22121.46421.022

10097.26595.26492.01988.88485.85784.090

000

000

000

000

000

000

000

000

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

E

Time(min)0510204080160

T10098.6296.5990.1370.7146.6538.36

F10096.5990.1365.9843.5332.3625.33

G10046.6521.7616.2312.9410.8810.51

T00.090.350.561.591.243.85

F00.150.961.121.562.783.25

G01.252.893.120.950.510.29

000000000

0000.090.090.150.151.251.25

0000.350.350.960.962.892.89

0000.560.561.121.123.123.12

0001.591.591.561.560.950.95

0001.241.242.782.780.510.51

0003.853.853.253.250.290.29

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (min)

Relative Activity Remaining (%)

T1(37

Timehr0248163264

T10099.3098.6199.1597.6696.9996.61

F10098.9498.6298.7098.0197.2797.94

G10098.6298.6297.7096.9495.9594.15

T00.610.150.880.510.210.85

F00.220.230.350.560.840.65

G00.510.320.180.50.471.22

000000

0.610.610.220.220.510.51

0.150.150.230.230.320.32

0.880.880.350.350.180.18

0.510.510.560.560.50.5

0.210.210.840.840.470.47

0.850.850.650.651.221.22

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity remaining(%)

S(

Timehr012481632

T10098.2096.9695.3193.9593.2092.66

F10097.2795.2692.0288.8885.8684.09

G10096.5994.6190.7587.0683.0980.11

T01.520.510.610.230.150.47

F00.050.840.910.620.970.59

G00.890.490.560.611.020.52

000000000

0001.521.520.050.050.890.89

0000.510.510.840.840.490.49

0000.610.610.910.910.560.56

0000.230.230.620.620.610.61

0000.150.150.970.971.021.02

0000.470.470.590.590.520.52

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

ApH2.2

Timehr012481632

T10098.62397.26595.42693.30391.38390.752

F10097.8196.2793.9590.7588.2787.06

G10096.5993.9090.1386.2784.0683.51

T00.410.390.481.050.461.28

F00.380.850.911.091.271.21

G00.280.911.260.612.020.52

000000

0.410.410.380.380.280.28

0.390.390.850.850.910.91

0.480.480.910.911.261.26

1.051.051.091.090.610.61

0.460.461.271.272.022.02

1.281.281.211.210.520.52

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

B(pH11.0)

Timehr012481632

T10096.2792.5885.3775.7965.0757.43

F10092.7587.0675.7959.4643.6135.73

G10086.4575.7959.4639.1822.7311.66

T00.681.123.951.521.322.58

F01.253.301.581.692.093.69

G01.582.542.690.855.151.08

000000000

0000.680.681.251.251.581.58

0001.121.123.33.32.542.54

0003.953.951.581.582.692.69

0001.521.521.691.690.850.85

0001.321.322.092.095.155.15

0002.582.583.693.691.081.08

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

T260

Timehr00.5124816

T10092.9585.0970.7151.7640.6132.73

F10090.9581.2362.9842.3430.7822.31

G10086.0971.7150.5927.5810.518.84

T00.563.122.151.591.624.52

F00.150.961.121.561.261.15

G02.151.951.024.121.121.58

000000

0.560.560.150.152.152.15

3.123.120.960.961.951.95

2.152.151.121.121.021.02

1.591.591.561.564.124.12

1.621.621.261.261.121.12

4.524.521.151.151.581.58

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

Chart4

100100100000000

96.265494741292.751915531786.45372313080.680.681.251.251.581.58

92.5887.055056329675.78582832551.121.123.33.32.542.54

85.3775.785828325559.46035575013.953.951.581.582.692.69

75.785828325559.460355750139.17955086341.521.521.691.690.850.85

65.067092772143.612619817822.73017787511.321.322.092.095.155.15

57.434917749935.730177875111.66291239422.582.583.693.691.081.08

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

GABE

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:22

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.58232.62710.13060.12740.12780.12620.12600.1238......

B......2.53912.58972.41522.41542.36662.35882.33191.2827......

C......2.41502.43312.42692.39162.37752.34252.32832.2829......

D......2.02012.13202.31592.24582.11321.95531.90211.7159......

E......1.85581.86512.32092.24192.14512.01051.89421.7068......

F......1.70591.74532.09021.88121.91261.75211.70691.7217......

G......1.69561.70342.04841.80531.71521.70551.71051.6956......

H......1.70541.71230.13200.13370.13480.13160.13260.1412......

1.10471.08231.1271

1.06441.03911.08970.91520.91540.86660.85880.8319-0.2173

0.924050.91500.93310.92690.89160.87750.84250.82830.7829

0.576050.52010.63200.81590.74580.61320.45530.40210.2159

0.360450.35580.36510.82090.74190.64510.51050.39420.2068

0.22560.20590.24530.59020.38120.41260.25210.20690.2217

0.19950.19560.20340.54840.30530.21520.20550.21050.1956

0.208850.20540.2123

01234567

0100.000100.000100.000

196.34596.00996.68282.84682.86478.44777.74175.306

283.66584.54282.78883.90580.71079.43376.26574.98070.870

352.06448.05556.07373.85767.51255.50841.21536.39919.544

432.63432.87432.39374.31067.15958.39646.21235.68418.720

520.39419.02421.76453.42634.50737.35022.82118.72920.069

618.05918.07318.04649.64227.63619.48018.60219.05517.706

718.90718.97818.836

E

Time(min)0510204080160

83.66551.53431.07228.41520.71218.89218.887

23.14.24.354.955.25.25

IU/mL)2511.66291239425.44094102064.90365061193.2352028872.72047051032.6278012977

10046.6521.7619.6112.9410.8810.51

ApH2.2

Timehr012481632

83.66583.375577079881.786910473478.93998370677.002806191775.142572644270.870

22.052.12.152.182.22.26

IU/mL)2524.148408223123.325824788422.531261565322.067574907321.763764082420.8771979857

10096.5993.3090.1388.2787.0683.51

B(pH11.0)

Timehr012481632

83.66574.083461573367.335023083256.95211369643.7132253136.041459219719.1318910111

22.212.42.753.283.755.1

IU/mL)2521.613430782718.946457081414.865088937510.29488771587.43254446882.9157280986

10086.4575.7959.4641.1829.7311.66

GABE

0

0

0

0

0

0

0

0

GTS

00

00

00

00

00

00

00

TABE

0

0

0

0

0

0

0

TTS

0

0

0

0

0

0

0

FABE

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:31

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.9

123456789101112

A......2.73892.75440.14260.13520.13290.13780.13790.2667......

B......2.68042.69242.45282.46412.45822.39852.44232.4519......

C......2.46762.45992.44822.45362.44922.46732.50192.4615......

D......2.17352.20372.38132.27152.14641.89621.75711.7597......

E......1.93841.96522.40872.29092.15622.12391.80531.7723......

F......1.81471.89592.46732.45962.42962.40492.38122.3507......

G......1.75481.75932.45152.43102.42042.39252.37132.3753......

H......1.76841.74590.13560.13540.13450.13250.13610.1385......

1.246651.23891.2544

1.18641.18041.19240.95280.96410.95820.89850.94230.9519

0.963750.96760.95990.94820.95360.94920.96731.00190.9615

0.68860.67350.70370.88130.77150.64640.39620.25710.2597

0.45180.43840.46520.90870.79090.65620.62390.30530.2723

0.35530.31470.39590.96730.95960.92960.90490.88120.8507

0.257050.25480.25930.95150.93100.92040.89250.87130.8753

0.257150.26840.2459

100100100

95.16795.27895.05776.90777.81977.34372.52476.05976.834

77.30778.10276.52376.53676.97276.61678.07780.87077.609

55.23654.36356.09971.13662.27352.17531.98020.75220.962

36.24135.38637.08573.34763.83952.96650.35924.64321.979

28.50025.40231.56178.07777.45675.03473.04171.12868.666

20.61920.56720.67176.80275.14774.29272.04070.32970.651

20.62721.66419.603

01234567

T1(37

Timehr0248163264

78.10276.72177.39576.98075.30178.46577.222

22.12.082.092.112.052.08

IU/mL)2523.325824788423.651441168123.488068730423.164701547324.148408223123.6514411681

10093.3094.6193.9592.6696.5994.61

T260

Timehr00.5124816

78.10272.241504560563.055936718152.570828961241.16958592322.69755428221.470659456

22.22.52.93.65.255.5

IU/mL)2521.763764082417.677669529713.39716828178.24692444232.62780129772.2097086912

10087.055056329670.710678118753.588673126832.987697769310.51120519078.8388347648

S(

Timehr012481632

78.10277.439664218376.301557833674.663007506772.540156590570.728065219169.6585680846

22.052.082.142.22.252.32

IU/mL)2524.148408223123.651441168122.687978882921.763764082421.022410381320.0267469397

10096.5994.6190.7587.0684.0980.11

FABE

000

000

000

000

000

000

000

000

FTS

0

0

0

0

0

0

0

0

E

0

0

0

0

0

0

0

T1

0

0

0

0

0

0

0

S

0

0

0

0

0

0

0

A

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:27

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.55272.63540.13440.13260.13240.13190.13210.1312......

B......2.40572.55092.29032.28122.28012.28092.27052.2597......

C......2.27042.29822.28902.28392.27892.26322.26192.2581......

D......1.99791.91912.28132.26772.26712.08092.06052.0097......

E......1.83931.76682.28512.27862.25392.21322.11191.9981......

F......1.71821.65032.28572.28152.25052.11891.95871.7852......

G......1.70101.60642.28932.27912.23872.10581.97501.7514......

H......1.74321.58990.13180.13370.13230.13160.13410.1375......

1.144051.10271.1854

1.02830.95571.10090.84030.83120.83010.83090.82050.8097

0.83430.82040.84820.83900.83390.82890.81320.81190.8081

0.50850.54790.46910.83130.81770.81710.63090.61050.5597

0.353050.38930.31680.83510.82860.80390.76320.66190.5481

0.234250.26820.20030.83570.83150.80050.66890.50870.3352

0.20370.25100.15640.83930.82910.78870.65580.52500.3014

0.216550.29320.1399

0100100100

189.77086.66992.87270.88770.12070.02770.09469.21768.306

272.97774.39971.55470.77870.34869.92668.60168.49268.171

344.63049.68739.57370.12868.98168.93053.22351.50247.216

431.01535.30426.72570.44969.90067.81764.38355.83846.238

520.61024.32216.89770.49970.14567.53056.42842.91428.277

617.97822.76213.19470.80369.94366.53555.32344.28925.426

719.19626.58911.802

E

Time(min)0510204080160

72.97770.65170.04467.03255.87643.60126.852

22.022.052.152.53.13.5

IU/mL)2524.6624.1522.5317.6811.668.84

10098.6296.5990.1370.7146.6535.36

ApH2.2

Timehr012481632

72.97770.83370.23469.97669.34868.85468.239

22.022.042.062.12.132.14

IU/mL)2524.65624.31623.98223.32622.84622.688

10098.62397.26595.92693.30391.38390.752

B(pH11.0)

Timehr012481632

72.97770.28969.44168.37458.80353.67046.727

22.042.072.132.42.622.8

IU/mL)2524.31623.81622.84618.94616.26714.359

10097.26595.26491.38375.78665.06757.435

A

0

0

0

0

0

0

0

0

B

0

0

0

0

0

0

0

T2

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:29

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.77322.68730.14160.14580.14360.13980.13760.1346......

B......2.57042.58932.49922.51702.51272.50132.50962.5134......

C......2.53062.49292.51192.52612.51332.49712.51842.4918......

D......2.25432.24072.48142.42932.35842.23112.11502.1074......

E......2.00961.96892.47912.43062.39132.27172.16541.9801......

F......1.83581.84762.53102.49022.49452.48922.47352.4635......

G......1.80311.74662.49752.49582.48682.47912.46972.4532......

H......1.79521.75760.14190.13580.13560.13300.13580.1381......

1.150251.19321.1073

0.999850.99041.00930.91920.93700.93270.92130.92960.9334

0.931750.95060.91290.93190.94610.93330.91710.93840.9118

0.66750.67430.66070.90140.84930.77840.65110.53500.5274

0.409250.42960.38890.89910.85060.81130.69170.58540.4001

0.26170.25580.26760.95100.91020.91450.90920.89350.8835

0.194850.22310.16660.91750.91580.90680.89910.88970.8732

0.19640.21520.1776

100100100

87.07783.00491.15079.91381.46181.08780.09680.81781.148

81.05679.66882.44481.01782.25281.13979.73081.58279.270

58.09056.51259.66878.36673.83667.67256.60546.51245.851

35.56336.00435.12178.16673.94970.53260.13550.89334.784

22.80321.43824.16782.67879.13179.50479.04477.67976.809

16.87218.69815.04679.76579.61778.83578.16677.34875.914

17.03718.03616.039

01234567

T1(37

Timehr0248163264

81.05680.46581.85681.11379.91381.20080.209

22.021.971.992.031.982

IU/mL)2524.65625.52525.17424.48625.34925.000

10098.623102.101100.69697.942101.396100.000

T260

Timehr00.5124816

81.05678.26559443673.892632036569.102369050258.369919582748.702455987840.3173223212

22.092.252.52.953.33.75

IU/mL)2523.48821.02217.67812.94110.1537.433

10093.95284.09070.71151.76340.61329.730

S(

Timehr012481632

81.05681.221473592779.374049119879.169745707578.604651162877.513584003576.3616605086

21.992.072.082.092.12.11

IU/mL)2525.17423.81623.65123.48823.32623.165

100100.69695.26494.60693.95293.30392.659

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:25

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.69012.70120.12910.12450.12190.12230.12170.1226......

B......2.59222.61932.50222.49172.47472.46442.45472.4510......

C......2.49532.51092.49842.49262.48492.47722.46992.4655......

D......2.27222.31982.48032.44162.41012.31682.17682.0643......

E......2.03302.00402.46242.46412.40252.30612.19792.0856......

F......1.87681.89692.48122.46132.37642.21252.13101.9979......

G......1.85241.87222.49012.47382.36572.24922.12742.0239......

H......1.84151.89670.51610.62610.21320.39200.21730.2197......

1.015651.01011.0212

0.925750.91220.93930.82220.81170.79470.78440.77470.7710

0.82310.81530.83090.81840.81260.80490.79720.78990.7855

0.6160.59220.63980.80030.76160.73010.63680.49680.3843

0.33850.35300.32400.78240.78410.72250.62610.51790.4056

0.206850.19680.21690.80120.78130.69640.53250.45100.3179

0.18230.17240.19220.81010.79380.68570.56920.44740.3439

0.18910.16150.2167

0100100100

191.14490.30891.98080.95379.91978.24577.23176.27675.912

281.04080.71581.36580.57980.00879.25078.49277.77377.340

360.64058.62862.65278.79774.98671.88562.69948.91437.838

433.33734.94731.72777.03477.20271.13761.64550.99239.935

520.36119.48321.24078.88576.92668.56752.42944.40531.300

617.94417.06818.82179.76278.15767.51356.04344.05133.860

718.60415.98921.220

E

Time(min)0510204080160

81.04079.32477.54168.04054.23644.22832.580

22.052.152.63.23.54.1

IU/mL)2524.1522.5316.4910.888.845.83

10096.5990.1365.9843.5335.3623.33

ApH2.2

Timehr012481632

81.04080.76679.96478.74877.86177.02576.626

22.012.042.092.142.182.2

IU/mL)2524.8324.3223.4922.6922.0721.76

10099.3197.2793.9590.7588.2787.06

B(pH11.0)

Timehr012481632

81.04077.91676.09471.51162.17249.95338.886

22.142.22.42.753.33.75

IU/mL)2522.6921.7618.9514.8710.157.43

10090.7587.0675.7959.4640.6129.73

000

000

000

000

000

000

000

000

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:12

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.7

123456789101112

A......2.71042.72500.12750.12900.13030.12840.12400.1195......

B......2.66942.65642.51822.50982.52352.50282.51152.5164......

C......2.52262.51642.50742.51962.51692.51442.49772.5111......

D......2.24462.23022.47692.40812.33482.17172.09571.9932......

E......2.01292.09372.49312.42222.35682.20642.11251.9778......

F......1.89741.91202.49842.51282.47472.45122.44562.4495......

G......1.82871.87082.49622.47832.46772.46522.45842.4342......

H......1.85491.86450.13310.13450.13300.12800.13240.1346......

1.06771.06041.0750

1.01291.01941.00640.86820.85980.87350.85280.86150.8664

0.86950.87260.86640.85740.86960.86690.86440.84770.8611

0.58740.59460.58020.82690.75810.68480.52170.44570.3432

0.40330.36290.44370.84310.77220.70680.55640.46250.3278

0.25470.24740.26200.84840.86280.82470.80120.79560.7995

0.199750.17870.22080.84620.82830.81770.81520.80840.7842

0.20970.20490.2145

0100100100

194.87696.13493.61981.31580.52881.81179.87380.68781.146

281.44382.29080.59580.30381.44681.19380.95979.39580.650

355.02356.07353.97277.44771.00364.13848.86241.74432.144

437.74934.22341.27478.96472.32466.19852.11243.31730.702

523.85123.33124.37279.46180.80977.24175.04074.51574.881

618.69616.85220.54079.25477.57876.58576.35175.71473.448

719.63819.32319.953

T1(37

Timehr0248163264

81.44380.80980.98781.50280.41680.04180.898

22.032.021.9922.042.03

IU/mL)2524.4924.6625.1725.0024.3224.49

10097.9498.62100.70100.0097.2797.94

T260

Timehr00.5124816

81.44378.205488433171.663388592365.168118385350.487028191442.530673410131.4226842746

22.092.32.63.243.74.3

IU/mL)2523.4920.3116.4910.587.695.08

10093.9581.2365.9842.3430.7820.31

S(

Timehr012481632

81.44379.357497424479.193593706176.912990540475.695420061875.114732602874.1640910368

22.042.072.122.172.222.25

IU/mL)2524.31623.81623.00522.22121.46421.022

10097.26595.26492.01988.88485.85784.090

000

000

000

000

000

000

000

000

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

E

Time(min)0510204080160

T10098.6296.5990.1370.7146.6538.36

F10096.5990.1365.9843.5332.3625.33

G10046.6521.7616.2312.9410.8810.51

T00.090.350.561.591.243.85

F00.150.961.121.562.783.25

G01.252.893.120.950.510.29

000000000

0000.090.090.150.151.251.25

0000.350.350.960.962.892.89

0000.560.561.121.123.123.12

0001.591.591.561.560.950.95

0001.241.242.782.780.510.51

0003.853.853.253.250.290.29

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (min)

Relative Activity Remaining (%)

T1(37

Timehr0248163264

T10099.3098.6199.1597.6696.9996.61

F10098.9498.6298.7098.0197.2797.94

G10098.6298.6297.7096.9495.9594.15

T00.610.150.880.510.210.85

F00.220.230.350.560.840.65

G00.510.320.180.50.471.22

000000

0.610.610.220.220.510.51

0.150.150.230.230.320.32

0.880.880.350.350.180.18

0.510.510.560.560.50.5

0.210.210.840.840.470.47

0.850.850.650.651.221.22

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity remaining(%)

S(

Timehr012481632

T10098.2096.9695.3193.9593.2092.66

F10097.2795.2692.0288.8885.8684.09

G10096.5994.6190.7587.0683.0980.11

T01.520.510.610.230.150.47

F00.050.840.910.620.970.59

G00.890.490.560.611.020.52

000000000

0001.521.520.050.050.890.89

0000.510.510.840.840.490.49

0000.610.610.910.910.560.56

0000.230.230.620.620.610.61

0000.150.150.970.971.021.02

0000.470.470.590.590.520.52

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

ApH2.2

Timehr012481632

T10098.62397.26595.42693.30391.38390.752

F10097.8196.2793.9590.7588.2787.06

G10096.5993.9090.1386.2784.0683.51

T00.410.390.481.050.461.28

F00.380.850.911.091.271.21

G00.280.911.260.612.020.52

000000

0.410.410.380.380.280.28

0.390.390.850.850.910.91

0.480.480.910.911.261.26

1.051.051.091.090.610.61

0.460.461.271.272.022.02

1.281.281.211.210.520.52

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

B(pH11.0)

Timehr012481632

T10096.2792.5885.3775.7965.0757.43

F10092.7587.0675.7959.4643.6135.73

G10086.4575.7959.4639.1822.7311.66

T00.681.123.951.521.322.58

F01.253.301.581.692.093.69

G01.582.542.690.855.151.08

000000

0.680.681.251.251.581.58

1.121.123.33.32.542.54

3.953.951.581.582.692.69

1.521.521.691.690.850.85

1.321.322.092.095.155.15

2.582.583.693.691.081.08

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

T260

Timehr00.5124816

T10092.9585.0970.7151.7640.6132.73

F10090.9581.2362.9842.3430.7822.31

G10086.0971.7150.5927.5810.518.84

T00.563.122.151.591.624.52

F00.150.961.121.561.261.15

G02.151.951.024.121.121.58

000000

0.560.560.150.152.152.15

3.123.120.960.961.951.95

2.152.151.121.121.021.02

1.591.591.561.564.124.12

1.621.621.261.261.121.12

4.524.521.151.151.581.58

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

Mono-PEG-G-CSFFigure 10. PEGylation significantly enhanced proteolytic stability of rhG-CSF. Left: human serum; Right: trypsin.

Chart5

100100100000000

98.195555005797.265494741296.59363289251.521.520.050.050.890.89

96.963799804495.263799804494.60576467260.510.510.840.840.490.49

95.305764672692.018765062590.75191553170.610.610.910.910.560.56

93.952274921488.884268116787.05505632960.230.230.620.620.610.61

93.203299153785.856543643883.08964152540.150.150.970.971.021.02

92.65880618984.089641525480.1069877590.470.470.590.590.520.52

PEG(20kD)-G-CSF

PEG(5kD)-G-CSF

G-CSF

Time (h)

Relative Activity Remaining (%)

GABE

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:22

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.8

123456789101112

A......2.58232.62710.13060.12740.12780.12620.12600.1238......

B......2.53912.58972.41522.41542.36662.35882.33191.2827......

C......2.41502.43312.42692.39162.37752.34252.32832.2829......

D......2.02012.13202.31592.24582.11321.95531.90211.7159......

E......1.85581.86512.32092.24192.14512.01051.89421.7068......

F......1.70591.74532.09021.88121.91261.75211.70691.7217......

G......1.69561.70342.04841.80531.71521.70551.71051.6956......

H......1.70541.71230.13200.13370.13480.13160.13260.1412......

1.10471.08231.1271

1.06441.03911.08970.91520.91540.86660.85880.8319-0.2173

0.924050.91500.93310.92690.89160.87750.84250.82830.7829

0.576050.52010.63200.81590.74580.61320.45530.40210.2159

0.360450.35580.36510.82090.74190.64510.51050.39420.2068

0.22560.20590.24530.59020.38120.41260.25210.20690.2217

0.19950.19560.20340.54840.30530.21520.20550.21050.1956

0.208850.20540.2123

01234567

0100.000100.000100.000

196.34596.00996.68282.84682.86478.44777.74175.306

283.66584.54282.78883.90580.71079.43376.26574.98070.870

352.06448.05556.07373.85767.51255.50841.21536.39919.544

432.63432.87432.39374.31067.15958.39646.21235.68418.720

520.39419.02421.76453.42634.50737.35022.82118.72920.069

618.05918.07318.04649.64227.63619.48018.60219.05517.706

718.90718.97818.836

E

Time(min)0510204080160

83.66551.53431.07228.41520.71218.89218.887

23.14.24.354.955.25.25

IU/mL)2511.66291239425.44094102064.90365061193.2352028872.72047051032.6278012977

10046.6521.7619.6112.9410.8810.51

ApH2.2

Timehr012481632

83.66583.375577079881.786910473478.93998370677.002806191775.142572644270.870

22.052.12.152.182.22.26

IU/mL)2524.148408223123.325824788422.531261565322.067574907321.763764082420.8771979857

10096.5993.3090.1388.2787.0683.51

B(pH11.0)

Timehr012481632

83.66574.083461573367.335023083256.95211369643.7132253136.041459219719.1318910111

22.212.42.753.283.755.1

IU/mL)2521.613430782718.946457081414.865088937510.29488771587.43254446882.9157280986

10086.4575.7959.4641.1829.7311.66

GABE

0

0

0

0

0

0

0

0

GTS

00

00

00

00

00

00

00

TABE

0

0

0

0

0

0

0

TTS

0

0

0

0

0

0

0

FABE

SAFIRE; Serial number: 12901300318; Firmware: V 2.20 08/02 Safire; XFLUOR4 Version: V 4.50

Date:8/10/08

Time:19:31

Measurement mode:Absorbance

Measurement wavelength:570nm

Number of flashes:10

Plate definition file:COS96ft.pdf

Part of the plate:A3 - H10

Time between move and read:100ms

RawdataTemperature: 28.9

123456789101112

A......2.73892.75440.14260.13520.13290.13780.13790.2667......

B......2.68042.69242.45282.46412.45822.39852.44232.4519......

C......2.46762.45992.44822.45362.44922.46732.50192.4615......

D......2.17352.20372.38132.27152.14641.89621.75711.7597......

E......1.93841.96522.40872.29092.15622.12391.80531.7723......

F......1.81471.89592.46732.45962.42962.40492.38122.3507......

G......1.75481.75932.45152.43102.42042.39252.37132.3753......