TWI356065B - Method of producing hydroxyalkyl starch derivative - Google Patents

Description

1356065 A7 B7 V. INSTRUCTIONS (73) The compound (Μ) is a polypeptide. Further, the present invention relates to the use of the above-described base-based powdered-killing derivative for the preparation of an agent for treating anemia or hematopoietic insufficiency or a related disease thereof. According to a preferred embodiment, the invention relates to a pharmaceutical composition as described above, wherein the polypeptide is an antithrombin (ΑΤ), preferably ΑΤ III (Levy JH, Weisinger A, Ziomek CA, Echelard Y, recombinant antithrombin: role in manufacturing and cardiovascular disease, speech on thrombosis and hemostasis 27, 4 (2001) 405-416; Edmunds T, Van Patten Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed SM , Pollock J, Hanson E, Bernasconi R, Higgins E, Manavalan P, Ziomek C, Meade H, McPherson J, Cole ES, Human antithrombin prepared by gene transfer: against human blood stasis Comparison of structure and function of thrombin, Blood 91, 12 (1998) 4661-4671; Minnema MC, Chang ACK, Jansen PM, Lubbers YTP, Pratt BM, Whittaker BG, Taylor FB, Hack CE, Friedman B, recombinant human resistance Thrombin III improves survival and reduces inflammation in response to a fatal attack by E. coli, Blood 95, 4 (2000) 1117-1123; Van Patten SM, Hanson EH, Bernasconi R, Zhang K, Manavaln P, Cole ES, McPherson JM, Edmunds T, Methionine Oxidation of residues in anticoagulase, Journal of Biochemistry 274, 15 (1999) 10268-10276). · -75- This paper size applies to the national standard (CNS) A4 specification (210 X297 mm) 1356065 A7 B7 V. Inventive Note (74) According to other preferred embodiments, the present invention relates to a polypeptide as G CSF or A pharmaceutical composition of IFN-β. According to a particularly preferred embodiment, the invention relates to a pharmaceutical composition as described above, wherein the polypeptide is erythropoietin. According to another embodiment, the present invention relates to a pharmaceutical composition as described above, wherein the functional group γ is _SH and the compound (L) is a compound of the formula Ζι_L'-X, wherein the functional group Z! It is selected from the group consisting of the following base circles Η^Ν—Η〆h2n -〇, R'/01

N 2 H

^MnG HN o=s-=ro N-H \ 2N Η h2n

HN

HN

N 2 H h^n

G Ministry of Economic Affairs Intellectual Property Bureau employees consumption cooperatives printed in which G is Ο or S, and if it occurs twice, it is independently 〇 or s, and R' is a methyl group, and wherein the functional group X is selected from the following groups Group of groups

0 -76- This paper size iSffi towel standard (CNS) A4 specification (210 x 297 public Chu) ' ----

1356065

Wherein Hal is Cl, Βγ τ, .7Γ. η ^ or 1 and wherein L' is an organic chain or L' in which a bridged acetonitrile and X are absent. According to a preferred embodiment, the invention relates to a medical group as described above. Wherein the functional group γ is selected from the group consisting of an acid group, a hydrazino group, a hemyl acid, and a condensate group, and the compound (6) is a compound of the formula ζ丨·L, _x, wherein the functional group Z1 is selected Group h2n—h2nj Η, Ν* , 0, R./〇V Η 4 composed of the following groups

Η2Ν^Νγ- GΗ Η Η,ΝΎΝ\ G Η2Ν, 〇 NI-S— II ο

Η2Ν, Νγα G Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the employee consumption cooperative, where G is 0 or S, and if it appears twice, it is independently 〇 or s, and R' is methyl, and the functional group X is Groups consisting of the following groups: η2ν, Ν, Η, Ν· 'Η; Η2Ν, γ G Η ΗΗ, ΝγΝ' -77- ΗΑ Μt\~fr οη/丫G, 'recognition of the scale applicable to China ^^ 1356065 A7 B7 V. INSTRUCTIONS (76) where G is Ο or S, and if it occurs twice, it is independently Ο or S, and R' is methyl, and wherein L' is a bridging 丨The organic chain with X or L' does not exist. According to another embodiment, the present invention relates to a pharmaceutical composition as described above, wherein the functional group Y is -SH, the compound (D) is a compound of the formula Zr D'-W, and the compound (L) is A compound of the formula Z2-L'-X wherein the functional group Z1 is selected from the group consisting of the following groups

N 2 H

N 2 H

HN

N 2 H ο R, ο

NH Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing

N 2 H h2n

HN

HN

rG

Onsno I NH / N 2 H

HN

N 2 H HN 〆

G 8 7

This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1356065 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (77) where G is Ο or S, and if two Further, the system is independently Ο or S, and R' is a methyl group, wherein the functional group X is selected from the group consisting of the following groups

Wherein Hal is a, Br or I, wherein the functional group W or the functional group Z2 is -SH, and the functional group 2 or the functional group W is selected from the group consisting of the following groups

Wherein Hal is Cl, Br or I, or wherein the functional group W or the functional group Z2 is selected from the group consisting of the above-mentioned active ester or, optionally, a carboxyl group of the active ester, and the functional group Z2 or the functional group W is selected from Group of the following groups -79- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

1356065 A7. B7 V. INSTRUCTIONS (78) h2n——h2n η2ν γ" H2rr0\ r, /〇, n*

H, N,

G

G

H h'm'- O Η2Ν, Νγα where G is O or S ' and if it occurs twice, it is independently 〇 or s, and R' is f-group, and wherein D is a bridge Ζι and w The organic chain or D' does not exist, and L' is an organic chain or L that bridges the heart and the ruthenium, and does not exist. According to another embodiment, the present invention relates to a pharmaceutical composition as described above, wherein the functional group γ is selected from the group consisting of an aldehyde group, a ketone group, a hemiacetal group and an acetal group, and the compound (D) Is a compound of the formula ZrD'-W, and the compound (L) is a compound of the formula Z2-L, -X wherein the functional group Z i is selected from the group consisting of the following groups

N 2 H

〆 H2N

HN 〆 h2n ο R' ο

NH Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing

N 2 H

N 2 H

\ HN YGTG HN HN OMSNO NH / h2n

〆 H2N

HN

G

This paper scale applies to China National Standard (CNS) A4 specification (210x297 public) 1356065 A7 V. Inventive Note (79) where G is Ο or S, and if it appears twice, it is independently 〇 or s and R' Is a group of groups wherein the functional group X is selected from the group consisting of the following groups h2n-, 0,

FT h2n

r HN

HN G \_ Y HN \ N 2 H onsno 1 NH / N 2 Η

N 2 H

HN

G wherein G is deuterium or S, and if present twice, is independently 〇 or s, and R' is fluorenyl 'the functional group W or the functional group Z2 is -SH, and the functional group 2 or functional group W is selected from the group consisting of the following groups

智慧 Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, printed in which Hal is Cl, Br or I, or a group in which the functional group W or the functional group Z2 is selected from the above-mentioned active esters or, as the case may be, converted into the carboxyl groups of the active esters, and The functional group Z2 or the functional group W is selected from the group consisting of the following groups -81- 'The paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

1356065 A7 B7 V. INSTRUCTIONS (80) Ν 2 Η Ν 2 Η ΗΝ Ν 2 Η ο R, ο

NH Ν- 2 Η Ν 2 Η \ ΗΝ YGYG ΗΝ ΗΝ N 2 Η Ν 2 Η

NH

HN

OHSHO

G Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed G is Ο or S, and if it appears twice, it is independently Ο or S, and R' is methyl, and D' is a bridge Z1 and The organic chain of W or D' does not exist, and L' is a bridge Z, and the organic chain of X or L' does not exist. According to a particularly preferred embodiment, the invention relates to a pharmaceutical composition as described above, wherein the hydroxyalkyl starch is reacted with a compound of the formula in an aqueous medium.

Moreover, the reaction product is reacted with erythropoietin. According to a highly preferred embodiment, the invention relates to the above pharmaceutical composition, wherein the erythropoietin is oxidized by sodium iodate prior to the reaction. -82- This paper scale is applicable to China National Standard (CNS) A4 specification (210x297 mm) Ministry of Economic Affairs Intellectual Property Office Staff Consumption Cooperative Printing 1356065 A7 B7 V. Invention Description (81) According to another preferred embodiment, The invention relates to a pharmaceutical composition as described above, wherein the erythropoietin is partially de-acidified before the reaction and is then oxidized with sodium chlorate. According to another preferred embodiment of the present invention, a pharmaceutical composition comprising a hydroxyalkyl starch derivative prepared by using sulfur-EPO which has been completely reduced according to Example 6 is excluded. The above pharmaceutical compositions are particularly suitable for the treatment of anemia or hematopoietic dysfunction or its related diseases. The term "therapeutically effective amount" as used herein refers to an amount which provides a defined condition and the therapeutic effect of the ingested method. It is preferred to take the erythropoietin isoform recombination via the parenteral route for the particular path chosen for the condition to be treated. It is preferred to take the erythropoietin isomeric recombinant in a manner that is part of the plastic formulation, wherein the dosage form comprises a suitable carrier (such as human serum albumin), a suitable diluent (such as a buffered saline solution), and/or is suitable for use. Agent. The required dose will be an amount sufficient to increase the blood draw ratio of the patient and will vary with the severity of the condition to be treated, the method of administration employed, and the like. Preferably, the therapeutic use of the pharmaceutical composition of the present invention is such that the increase in hemoglobin value in the blood exceeds 6.8 millimoles per liter. For this, the pharmaceutical composition can have a weekly hemoglobin value of 毫 6 millimoles. / liter and 1 6 mA / -83- This paper scale applies to China National Standard (CNS) A4 specification (21〇x297 public Chu)

1356065 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing B7 V. Invention description (82) * : - ... · *V: . Shame.- ;;; If the hemoglobin value exceeds 8.7 milligrams per liter, it is best to discontinue treatment until the hemoglobin value is less than 8.1 millimoles per exposure. Preferably, the compositions of the present invention are used in a dosage form suitable for subcutaneous or intravenous or parenteral 'Z-primary injection. Suitable excipients and carriers for this are, for example, sodium dihydrogen sulphate, disodium hydrogen sulfate, sodium chloride, polysorbate 80, HAS and water for injection. The composition can be administered three times a week, preferably twice a week, more preferably once a week, and the best line is once every two weeks. Preferably, the pharmaceutical composition is administered in an amount of 0.01·10 μg/kg of the patient's body weight, preferably 0.1 to 5 μg/kg, 0.1 to 1 μg/kg or 0·2·0.9 μg/kg. 0.3-0.7 μg/kg and the best system. 4-0·6 μg/kg volume. In general, it is preferred to take 10 micrograms to 200 micrograms per dose, preferably 15 micrograms to 100 micrograms. The invention further relates to a HAS-polypeptide useful in a method of treatment of a human or animal body in accordance with the present invention. The invention further relates to the use of a HAS-EPO conjugate of the invention for the preparation of a medicament for the treatment of anemia or hematopoietic dysfunction or a related disease thereof. If the compound (L) used in accordance with the present invention contains one or more palmar centers, the -84- paper scale applies to the Chinese National Standard (CNS) A4 specification (210 x 297 mm).

1356065 A7 B7 V. INSTRUCTIONS (83) With respect to each palm center, the compound (II) may exist in the R configuration or in the S configuration or in the racemic compound form. If the compound Φ) used in the present invention comprises one or more palm centers, the compound (D) may exist in the R configuration or in the S configuration or in the racemic compound form with respect to each palm center. The invention is further illustrated by the following examples, tables and figures, which are not intended to limit the scope of the invention in any way. EXAMPLES OF EXAMPLES Example 1: Formation of Hydroxyethyl Starch by Reductive Amination of Unoxidized Reduction End Example 1.1: Reaction of Hydroxyethyl Starch with 1,3-Diamino-2-Hydroxypropane Ministry of Economic Affairs Intellectual Property Office Staff Consumption Cooperative printing Η2Ν^γ^ΝΗ2

OH a) In a solution of 200 mg of hydroxyethyl starch (HES18/0.4 (MW=18,000D, DS=0.4)) dissolved in 5 ml of water, add 0.83 mmol-85- This paper scale applies to Chinese national standards ( CNS) A4 size (210 x 297 mm) 1356065 A7 B7 V. INSTRUCTION DESCRIPTION (84) 1,3-Diamino-2-ridinopropane (Sigma Aldrich, Taufkirchen, D) and 50 mg of sodium cyanoborohydride NaCNBH3. The resulting mixture was incubated at 80 ° C for 17 hours. The reaction mixture was added to a 1:1 cold mixture (vol/vol) of 160 ml of acetone and ethanol. The precipitate was collected by centrifugation and dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, D) and lyophilized. b) culture of a mixture of 0.83 mmol of 1,3-diamino-2-hydroxypropane and 50 mg of sodium cyanoborohydride NaCNBH3 in 200 mg of hydroxyethyl starch is feasible and can be used at 25 ° C Let it go for 3 days. Example 1.; 2: Reaction of hydroxyethyl starch with 1,2-dihydroxy-3-aminopropane

H2N, Y~〇H OH Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed a) in 200 mg of hydroxyethyl starch (HES18/0.4 (MW=18,000D, DS=0.4)) dissolved in 5 ml of water, 0.83 mmol of 1,2-diaminopropylpropane (Sigma Aldrich, Taufkirchen, D) and 50 mg of sodium cyanoborohydride NaCNBH3 were added. The resulting mixture was incubated at 80 ° C for 17 hours, and the reaction mixture was added to a 1:1 cold mixture (volume/volume) of 160 ml of acetone and ethanol. Collected by centrifugation -86- The paper size is applicable to the national specifications of the country (21〇χ 297 public Chu V " 1356065 A7 B7 V. Invention description (85) Shen Dian and water for 4 days (SnakeSkin dialysis tube, 3.5 KD Removed, Perbio Science Deutschland GmbH, Bonn, D) and lyophilized b) from 0.83 mmol of 1,2-diamino-2-aminopropane and 5 mg of sodium cyanoborohydride NaCNBH: $200 The cultivation of a mixture of milligrams of ethyl starch solution is feasible and can be carried out at 25 ° C for 3 days. The reaction of 1,2-di-propyl-2-aminopropanone with HES is confirmed indirectly by quantitative measurement of formic acid, such as G. Avigad Anal. Biochem· 134 (1983) 449-504 As described, the 1,2-diol in the reaction product is formed by oxidative decomposition of periodate. Example 1.3: Reaction of hydroxyethyl starch with 1,4-diaminobutane 痤 a) in 200 mg of hydroxyethyl starch (HES 18/0.4 (MW = 18,000 D, DS = 0.4)) dissolved in 5 ml To the solution of water, 0.83 mmol of 1,4-diaminobutylbutane (Sigma Aldrich, Taufkirchen, D) and 50 mg of sodium cyanoborohydride NaCNBH3 were added. The resulting mixture was incubated at 80 ° C for 17 hours. The reaction mixture was added to a 1:1 cold mixture (vol/vol) of 160 ml of acetone and ethanol. Collecting sediment by centrifugation-87- This paper scale is applicable to China National Standard (CNS) A4 specification (210x297 mm) 1356065 A7 B7 V. Invention description (86) and dialysis by water for 4 days (SnakeSkin dialysis tube, 3.5 KD removal) , Perbio Science Deutschland GmbH, Bonn, D) and cold; b) culture of a mixture of 0.83 mmol of 1,4-diaminobutane and 50 mg of sodium cyanoborohydride NaCNBH3 in 200 mg of hydroxyethyl starch is feasible and can be carried out at 25 ° C. day. Example 1.4: Reaction of hydroxyethyl starch with 1-hydrothio- 2-aminoethane The Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, printed a) at 200 mg of hydroxyethyl starch (HES18/0.4 (MW=18,000D) , DS = 0.4)) dissolved in 5 ml of water, adding 0.83 mmol of 1-arylthio-2-aminoethane (Sigma Aldrich, Taufkirchen, D) and 50 mg of sodium cyanoborohydride NaCNBH3» The mixture was incubated at 80 ° C for 17 hours. The reaction mixture was added to a cold mixture (volume/volume) of 160 ml of acetone and ethanol. The precipitate was collected by centrifugation and dialyzed against water (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, D) and lyophilized and dried. b) Adding 2.0 mg of 1-hydrothio-2-aminoethane and 50 mg of sodium cyanoborohydride NaCNBH3 to 200 mg of hydroxyethyl starch solution -88- This paper scale applies to China National Standard (CNS) A4 Specification (210x297 mm) 1356065 A7 B7 V. INSTRUCTIONS ο The culture of the formed mixture is feasible and can be carried out at 25 ° C for 3 days. Example 2: Formation of hydroxyethyl powder by coupling with unoxidized reducing end Derivative Example 2.1: Reaction of Hydroxyethyl Starch with Carbonium H2N\ Human^NH, 2 NN 2 Η Η 0.96 g of HES18/0.4 (MW = 18,000 D, DS = 0.4) was dissolved in 8 ml of aqueous 0.1 M acetic acid Sodium buffer (pH 5.2) was added with 8 mmol of mash (Sigma Aldrich, Taufkirchen, D). After stirring at 25 ° C for an hour, the reaction mixture was added to 160 ml of a 1 * 1 cold mixture (vol/vol) of acetone and ethanol. The precipitated product was collected by centrifugation and dissolved in 40 ml of water and dialyzed against water for 3 days (SnakeSkin dialysis tube, 3.5 KD removed, Perbio Science Deutschland GmbH, Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Cooperatives)

Bonn, D) and lyophilized. , the reaction of diterpenoids Example 2.2: hydroxyethyl starch and

-89- 0

H2N This paper scale applies to China National Standard (CNS) A4 specification (210x297 public Chu) 1356065 A7 B7 V. Description of invention (88) Dissolve 0.96 g of HES18/0.4 (MW=18,000D, DS=0_4) in 8 ml of water 0.1 M sodium acetate buffer (pH 5.2) was added with 8 millimolar adipic acid eucalyptus (Lancaster Synthesis, Frankfurt/Main, D). After 18 hours at 25 C, the reaction mixture was added to a 1:1 cold mixture (vol/vol) of 160 ml of acetone and ethanol. The precipitated product was collected by centrifugation, dissolved in 40 ml of water and dialyzed against water for 3 days (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, D) and cooled to dryness. Example 2.3: Reaction of hydroxyethyl starch with 1,4-phenylene-bis-3-aminothiourea

Printed by the Ministry of Economic Affairs' Intellectual Property Office Staff Consumer Cooperative, 0.96 g of HES18/0.4 (MW=18,000D, DS=0.4) was dissolved in 8 ml of aqueous 0.1 M sodium acetate buffer (pH 5.2) and 8 mmol was added. 4-phenylene bis-3-aminophosphine (Lancaster Synthesis, Frankfurt/Main, D). After stirring at 25 ° C for 18 hours, 8 ml of water was added to the reaction mixture and the suspension was centrifuged at 4,500 rpm for 15 minutes. The supernatant was decanted and then added to a 1:1 cold mixture (vol/vol) of 160 ml of ketone and ethanol. Collect the precipitated product by centrifugation, dissolve it in 40 ml of water and centrifuge at 4,500 rpm. 15-90- This paper scale is applicable to China National Standard (CNS) A4 specification (210x297 mm). 1356065 A7 B7 V. Description of invention (89) minute. The supernatant was dialyzed against water for 3 days (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, D) and lyophilized β Example 2.4: Hydroxyethyl starch and 0-[2-(2-amine oxygen) The reaction of keto-ethoxy)-ethyl]-hydroxylamine 0-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine can be as described in Boturyn et al., Tetrahedron 53 (1997). It is synthesized from commercially available materials in 2 steps as described in the page 5485-5492. Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, 0.96 g of HES18/0.4 (MW=18,000D, DS=0.4) dissolved in 8 ml of aqueous 0.1 M sodium acetate buffer (pH 5.2) and added 8 mM 〇- [2-(2-Aminooxy-ethoxy)-ethyl]-hydroxylamine. After stirring at 25 ° C for 18 hours, the reaction mixture was added to a 1 : 1 cold mixture (vol/vol) of 160 ml of copper and ethanol. The precipitated product was collected by centrifugation, dissolved in 40 ml of water and dialyzed against water for 3 days (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, D) and lyophilized. Example 2.4(a): Reaction of hydroxyethyl starch with 0-[2-(2.aminooxy-ethoxy)-ethyl]-hydroxylamine-91- This paper scale applies to Chinese National Standard (CNS) A4 Specification (210x297 mm) 1356065 A7 B7 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed V. Description of the Invention (90) The oxidized HES is prepared as described in DE 196 28 705 A1. 200 mg of oxidized HES18/0.4 (MW = 18000 D, DS = 0.4) was heated in vacuo at 80 °C for 17 hours and dissolved in 2 ml of dry DMSO (Fluka, Sigma-Aldrich Chemie GmbH, Taufkirchen, D). In this solution, 2 mmol of 〇-[2-(2-aminooxy-ethoxy)-ethyl]-hydroxylamine was added. After 5 days of incubation at 65 ° C, the reaction mixture was added to 20 ml of ice-cold 2-propanol at -20. (The culture was carried out for 1 hour. The precipitated product was collected by centrifugation at 4 ° C, washed with 42 ml of ice-cold 2-propanol, dissolved in 10 ml of water and dialyzed against water for 27 hours (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Sciences Deutschland GmbH, Bonn, D) and lyophilized. Example 3: Formation of hydroxyethyl starch derivatives by reaction with oxidized reducing ends Example 3.1: Hydroxyethyl starch and carbon oxime Reaction H2N, human Γ, ίί. ΝΚ 0.12 mM Οχο-HES 10/0.4 (MW=10,000 D, DS=(K4 'produced according to DE 196 28 705 A1) is dissolved in 3 ml absolute dimethyl In sulfoxide (DMSO) and 15 mM carbon-92-dropped under nitrogen, this paper scale is applicable to Chinese national standard ((^3) 八4 specification (21〇χ297 mm) 1356065 A7 B7 V. Invention description (91) Stir-fry (Sigma Aldrich, Taufkirchen, D) was dissolved in a mixture of 15 ml of DMSO. After stirring at 65 ° C for 88 hours, the reaction mixture was added to 160 ml of a 1:1 cold mixture of acetone and ethanol (volume / volume / Volume). The sediment was collected by centrifugation and dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5 KD) Removed, Perbio Science Deutschland GmbH, Bonn, D) and lyophilized. Example 3.2: Reaction of hydroxyethyl starch with 1,4 phenylene-3-aminothiourea Η2Ν

Ji will dissolve 0.12 millimoles Oxo-HES 10/0.4 (MW=10,000D, Printed by the Ministry of Economic Affairs, Intellectual Property Office, Consumer Cooperatives, DS=0.4, prepared according to DE 196 28 705 A1) in 3 ml absolute dimethyl To a mixture of 15 mM 1,4-phenyl-bis-3-carbazone (Lancaster Synthesis, Frankfurt/Main, D) dissolved in 15 ml of DMSO was added dropwise in sulfoxide (DMSO) under nitrogen. . After stirring at 65 ° C for 88 hours, the reaction mixture was added to a 1:1 cold mixture (vol/vol) of 6 ml of acetone and ethanol. The precipitate was collected by centrifugation and dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5 KD removed,

Perbio Science Deutschland GmbH, Bonn, D) and cold-image drying 0 -93- This paper scale applies to China National Standard (CNS) A4 specification (210x297 public Chu) 1356065 A7 B7 V. Invention description (9〇 Example 3.3: Hydroxy B Reaction of the base starch with hydrazine H2N-NH2 1.44 g (0.12 mmol) of Oxo-HES 10/0.4 (MW = 10, OOOD, DS = 0.4, prepared according to DE 196 28 705 A1) dissolved in 3 ml Absolute dimercaptosulfoxide (DMSO) was added dropwise to a mixture of 0.47 ml (15 mmol) and dissolved in 15 ml of DMSO under nitrogen. After stirring at 40 ° C for 19 hours, the reaction mixture was added to 160. 1:1 mixture of ethanol and acetone in 1:1 (vol/vol). The precipitated product was collected by centrifugation, dissolved in 4 mL of water and dialyzed against 0.5% (v/v) aqueous solution of triethylamine for 2 days. It was dialyzed against water for 2 days (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, Germany) and lyophilized. Example 3.4: Reaction of hydroxyethyl starch with hydroxylamine to produce v〇, NH2 ^ - economy Ministry of Intellectual Property Bureau employee consumption cooperative printed 0-[2-(2-aminooxy) The oxy)-ethyl]-hydroxylamine can be synthesized from commercially available materials in two steps as described by Boturyn et al. (Boturyn, Boudali, Constant, Defrancq, Lhomme, 1997, Tetrahedron, 53, 5485) -94-ben Paper scale applicable to China National Standard (CNS) A4 specification (210 x 297 mm) 1356065 A7 B7 V. Description of invention (93) 1.44 g (0.12 mmol) Ox〇-HES 10/0.4 (MW=10,000 D, DS =0.4, prepared according to DE 196 28 705 A1) dissolved in 3 ml of absolute dimethyl hydrazine (DMSO) and 2.04 g (15 mmol) 0-[2-(2-) was added dropwise under nitrogen. Aminooxyethoxylated ethyl-hydroxy]-hydroxylamine was dissolved in a mixture of 15 ml of DMSO. After stirring at 65 ° C for 48 hours, the reaction mixture was added to a mixture of 160 ml of ethanol and acetone: / volume). The precipitated product was collected by centrifugation, dissolved in 40 ml of water and dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, D) and cold rolled and dried. . Example 3.5 • • Hydroxyethyl starch reacts with diammonium adipate Η 2Ν,

The Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, printed 1.74 g (15 mmol) of lanthanum adipic acid (Lancaster Synthesis, Frankfurt/Main, D) dissolved in 20 ml of absolute dimethyl hydrazine at 65 ° C. 1.40 g (0.12 mmol) of Oxo-HES 10/0.4 dissolved in 3 ml absolute DMSO (MW = 10,000 D, DS = 0, 4, according to DE 196 28 705 A1) was added dropwise in (DMSO) under nitrogen. Made by). After 68 hours of scrambling at 60 ° C, the reaction mixture was added to 200 ml of water. The solution containing the reaction product was dialyzed against 0.5% (v/v) aqueous solution of triethylamine for 2 days and dialyzed against water for 2 days (SnakeSkin dialysis tube, 3.5 KD shift-95- paper scale applicable to China National Standard (CNS) M specification (210X297 mm) 1356065 A7 B7 V. Description of invention (94), except Perbio Science Deutschland GmbH, Bonn, Germany) and freeze-dried. Example 3.6: Reaction of hydroxyethyl starch with 1,4-diaminobutane 1.44 g (0.12 mmol) 〇x〇-HES 10/0.4 (MW = 10,000 D, DS = 0.4, according to DE 196 28 705 A1 was dissolved in 3 ml of dry dimethyl sulfoxide (DMSO) and 1.51 ml (15 mmol) of 1,4-diaminobutyrate (Sigma Aldrich, Taufkirchen, D) Dissolved in a mixture of 15 ml of DMSO. After stirring at 40 t for 19 hours, the reaction mixture was added to 160 ml of a 1:1 mixture (vol/vol) of ethanol and acetone. The precipitated amine-HESlOICD/tM was collected by centrifugation and dissolved in 40 ml of water. Dialysis with water for 4 days (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, Germany) and freeze-drying 0 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Print Example 4: Oxidation of erythrocyte auxin Oxidized erythropoietin was prepared as described in Example 8. As the oxidized erythrocyte auxin, EPO-GT-1-A (EPO-GT-1 which was not acid-hydrolyzed but oxidized by mild periodate) as described in Example 8.11 (c) was used. 96. This paper scale applies to national standard (cns) A4 specification (210x297 public) 1356065 A7 B7 V. Description of invention (95) Example 5: Coupling of hydroxyethyl starch derivative with oxidized erythrocyte auxin of Example 4. Example 5.1: Reaction of oxidized erythropoietin with the reaction product of Example 2.1 The oxidized EPO (1.055 μg/μl) dissolved in 20 mM PBS buffer was adjusted to pH with 5 Μ sodium acetate buffer (pH 5.2). 5.3. In 19 μl of EPO solution, 18 μl of the HES derivative solution prepared according to Example 2.1 (MW 18 kD; 18.7 μg/ml in 0.1 乙酸 sodium acetate buffer (pH 5.2)) was added, and the mixture was Incubate at 25 ° C for 16 hours. After lyophilization, the crude product was analyzed by SDS-Page in NuPAGE 10% Bis_Tris gel/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions provided by Invitrogen. The gel was stained overnight with Roti-Blue Coomassie stain (Roth, Karlsruhe, D). The results of the experiment printed by the Intellectual Property Office of the Intellectual Property Bureau of the Ministry of Economic Affairs are shown in Figure 1. Migration to a higher molecular weight by the protein band indicates successful coupling. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 5.2: Reaction of oxidized erythrocyte auxin with the reaction product of Example 2.3 -97- This paper scale applies _ National Standard (CNS) A4 specification (210x297 mm) 1356065 A7 B7 V. Description of invention (96) to 5 Μ Sodium acetate buffer (pH 5.2) was adjusted to pH 5.3 with oxidized EPO (1.055 μg/μl) dissolved in 20 mM PBS buffer. In 19 μl of EPO solution, 18 μl of a HES derivative solution (MW 18 kD; 18.7 μg/ml in 0.1 乙酸 sodium acetate buffer (pH 5.2)) prepared according to Example 2.3 was added, and the mixture was Incubate at 25 ° C for 16 hours. After the cold edge was dried, the crude product was analyzed by SDS-Page using NuPAGE 10% Bis-Tris gel/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions provided by invitrogen. * Example 5.3: Reaction of oxidized erythrocyte auxin with the reaction product of Example 2.4 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative printed oxidized EPO dissolved in 20 mM PBS buffer with 5 Μ sodium acetate buffer (pH 5.2) (1. 〇 55 μg/μl) was adjusted to pH 5.3. In 19 μl of EPO solution, 18 μl of the HES derivative solution (MW 18 kD; 18.7 μg/ml in 〇.1 乙酸 sodium acetate buffer (pH 5.2)) prepared according to Example 2.4 was added, and the mixture was At 25. (: cultured for 16 hours. After cold-dried, as described in the instruction manual provided by invitrogen, using NuPAGE 10% Bis-Tris gel/MOPS buffer solution (invitrogen, Carlsbad, CA, USA) by SDS_Page Crude product. The gel was dyed overnight with Roti-Blue Coomassie stain (R〇th, Karlsruhe, D) -98- This paper scale applies to Chinese National Standard (CNS) A4 size (210x297 mm) 1356065 A7 B7 BRIEF DESCRIPTION OF THE INVENTION (97) The experimental results are shown in Figure 2. The migration of the protein band to a higher molecular weight indicates successful coupling. The increased bandwidth is attributed to the molecular weight distribution of the HES derivative used and to the protein. Number of HES derivatives. Example S.4 • Reaction of oxidized erythrocyte auxin with the reaction product of Example 3.1 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperative Printed with 5 Μ sodium acetate buffer (pH 5.2) will dissolve in 20 The oxidized EPO (1. 〇 55 μg/μl) in mM PBS buffer was adjusted to pH 5.3. In 19 μl of EPO solution, 18 μl of HES derivative solution prepared according to Example 3.1 (MW 10kD) was added. ; 18.7 micro G/ml is dissolved in 〇.1 乙酸 sodium acetate buffer (pH 5.2), and the mixture is incubated at 25 ° C for 16 hours. After lyophilization, as described in the instructions provided by Invitrogen, by SDS- Page The crude product was analyzed using NuPAGE 10% Bis-Tris gel/MOPS buffer (Invitrogen, Carlsbad, CA, USA). The gel was stained overnight with Roti-Blue Coomassie stain (Roth, Karlsruhe, D). This is shown in Figure 3. The migration of the protein band to a higher molecular weight indicates successful coupling. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. - This paper size applies to China National Standard (CNS) A4 specification (210x297 mm) 1356065 A7 B7 V. Description of invention (98) Example 5.5: Reaction of oxidized erythrocyte auxin with the reaction product of Example 3.2 buffered with 5 Μ sodium acetate The solution (pH 5.2) was adjusted to pH 5.3 with oxidized EPO (1.055 μg/μl) dissolved in 20 mM PBS buffer. In 19 μl of EPO solution, 18 μl of HES prepared according to Example 3.2 was added. Derivative solution (MW 10 kD; 18.7 μg/ml was dissolved in 0.1 乙酸 sodium acetate buffer (pH 5.2)), and the mixture was incubated at 25 ° C for 16 hours. After lyophilization, the crude product was analyzed by SDS_Page using NuPAGE 10% Bis-Tris gel/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions provided by Invitrogen. The gel was stained overnight with Roti-Blue Coomassie stain (Roth, Karlsruhe, D). The results of the experiment printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives, are shown in Figure 3. "The migration of the protein band to a higher molecular weight indicates that the coupling is successful. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein.

Example 6: Formation of sulfur-EPO by reduction of erythrocyte auxin 241.5 μg Dissolved in 500 μl of 0.1 M sodium borate buffer, 5 mM EDTA, 10 mM DTT (Lancaster, Morcambe, UK) -100-paper The scale applies to the Chinese National Standard (CNS) A4 specification (210x297 mm) 1356065 A7 B7 V. The invention (") erythrocyte auxin (EPO-GT-1, see Example 8) is incubated at 37 ° C for 1 hour. DTT was removed by centrifugation at 13,000 rpm using a VIVASPIN 0.5 ml concentrator, l〇KD MWCO (VIVASCIENCE, Hannover, D), followed by 3 washes with borate buffer and phosphate buffer (0.1 M, 9.15 M NaCl, Wash 50 nl MEDTA, pH 7.2) twice. The gel was stained overnight with Roti-Blue Coomassie stain (Roth, Karlsruhe, D). Example 7: Coupling of a hydroxyethyl ash powder derivative with thio-erythroglobin using a crosslinking compound In the following examples, Ν-(α-maleimide ethoxylated) amber ia was used. An amine ester (AMAS) is used as a crosslinking compound.

Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed Example 7.1: The reaction of thio-erythroglobin with the reaction product of Example 2.1 and the cross-linking compound was prepared according to Example 2.1 and dissolved in 200 μl of 0.1 Μ sodium phosphate buffer. Add 50 μmol of HES derivative to liquid (0.1 Μ, 9.15 Μ NaCl, 50 mM EDTA, pH 7.2), add ι〇 microliter 2.5 μMule AMAS (Sigma -101- This paper scale applies to Chinese National Standard Specification (210 χ 297 gong) PCT) 1356065 A7 B7 V. Description of invention (100)

Aldrich, Taufkirchen, D) A solution in DMSO. The clear solution was incubated at 25 for 80 minutes and at 40 ° C for 20 minutes. The remaining AMAS was removed by centrifugation at 3,000 rpm using a VIVASPIN 0.5 ml concentrator, 5KD MWC(R) (VIVASCIENCE, Hannover, D), and washed 4 times with phosphate buffer for 30 minutes. To the remaining solution, 15 μg of the thiopurine obtained according to Example 5 (1 μg/μl in phosphate buffer) was added, and the mixture was incubated at 25 ° C for 16 hours. After cold drying, the crude product was analyzed by SDS-Page using NuPAGE 10% Bis-Tris gel/MOPS buffer (Invitrogen, Carlsbad, Indole, 118) as described in the instructions provided by Invitrogen. The gel was stained overnight by the ruler ^-:61116 (1:0011138816 staining agent (Roth, Karlsruhe, D). The results of the printing experiment of the Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative are shown in Figure 4. Migration by protein band The higher molecular weight indicates successful coupling. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 7.2: Thio-erythrocytoin with Example 2.2 and cross-linking The reaction product of the reaction product of the compound was prepared according to Example 2.2 and dissolved in 200 μl of 〇·1 Μ Sodium Phosphate-102- This paper scale is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 1356065 Ministry of Economy Intellectual Property Bureau Staff Consumer Cooperative Printed A7 B7 V. Inventive Note (101) Add 50 μl of 2.5 micromolar AMAS (Sigma) to 50 nmol HES derivative of flush (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) Aldrich, Taufkirchen, D) A solution dissolved in DMSO. The clear solution was incubated at 25 ° C for 80 minutes and at 40 ° C for 20 minutes. Using VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, D) The remaining AMAS was removed by centrifugation at 13,000 rpm and washed in phosphate buffer for 4 times for 30 minutes. In the remaining solution, 15 μg of thioguanidine prepared according to Example 5 (1 μg/μl dissolved) was added. In phosphate buffer), and the mixture was incubated at 25 ° C for 16 hours. After drying in cold east, using NuPAGE 10% Bis-Tris gel by SDS-Page as described in the instructions provided by Invitrogen The crude product was analyzed by MOPS buffer (Invitrogen, Carlsbad, CA, USA). The gel was stained overnight with Roti-Blue Coomassie stain (Roth, Karlsruhe, D). The results of the experiment are shown in Figure 5. Migration by protein band The higher molecular weight indicates successful coupling. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 7.3: Thio-erythroglobin and Example 2.3 and Reaction of the reaction product of the compound -103- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210x297 mm)

1356065 A7 B7 V. INSTRUCTION DESCRIPTION (102) In 50 nmol of HES derivative prepared according to Example 2.3 and dissolved in 200 μl of 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7_2), A solution of 10 microliters of 2.5 micromolar AMAS (Sigma Aldrich, Taufkirchen, D) dissolved in DMSO was added. The clear solution was incubated at 25 ° C for 80 minutes and at 40 ° C for 20 minutes. The remaining AM AS was removed by centrifugation at 13,000 rpm using a VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, D), and washed 4 times with sulphate buffer for 30 minutes. To the remaining solution, 15 μg of the sulfur-based EPO prepared according to Example 5 (1 μg/μl in phosphate buffer) was added, and the mixture was incubated at 25 ° C for 16 hours. After cold-blown drying, the crude product was analyzed by SDS-Page using NuPAGE 10% Bis-Tris gel/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions provided by Invitrogen. The gel was stained overnight with Roti-Blue Coomassie stain (Roth, Karlsruhe, D). The results of the experiment printed by the Intellectual Property Office of the Intellectual Property Bureau of the Ministry of Economic Affairs are shown in Figure 5. Migration to a higher molecular weight by the protein band indicates successful coupling. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. -104- This paper scale applies to China National Standard (CNS) A4 specification (210x297 mm) 1356065 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Description of invention (1〇3) Example 7.4: Sulfur-red blood cell growth The reaction of the product with the reaction product of Example 2.4 and the crosslinking compound was carried out in 50 nmol HES prepared according to Example 2.4 and dissolved in 200 μl of 0.1 M sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2). To a solution of 10 microliters of 2.5 micromolar AMAS (Sigma Aldrich, Taufkirchen, D) dissolved in DMSO was added. The clear solution was incubated at 25 ° C for 80 minutes and at 40 ° C for 20 minutes. The remaining AMAS was removed by centrifugation at 13,000 rpm using a VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, D), and washed 4 times with phosphate buffer for 30 minutes. To the remaining solution, 15 μg of thioguanidine prepared according to Example 5 (1 μg/μl in phosphate buffer) was added, and the mixture was incubated at 25 ° C for 16 hours. After cold drying, the crude product was analyzed by SDS-Page using NuPAGE 10% Bis-Tris gel/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions provided by Invit^ogen. The gel was stained overnight with Roti-Blue Coomassie stain (Roth, Karlsruhe, D). The experimental results are shown in Figure 4. Migration to a higher molecular weight by the protein band indicates successful coupling. The increased bandwidth is attributed to the molecular weight distribution of the HES derivative used and the HES derivative of this protein. The paper size is applicable to the National Standard (CNS) A4 specification (210x297 mm).

1356065 A7 B7 V. INSTRUCTIONS (l〇4) Number of organisms Example 7.5: Reaction of thio-erythroglobin with the reaction product of Example 1.1 and a cross-linking compound. (: 50 nmol HES prepared in the next 17 hours and under the culture conditions of 3 days at 25 ° C and dissolved in 200 μl of sodium citrate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2) To the derivative, 10 μl of a 2.5 micromolar AMAS (Sigma Aldrich, Taufkirchen, D) solution in DMSO was added. The clear solution was incubated at 25 ° C for 80 minutes and at 40 ° C for 20 minutes. AMAS can be removed by centrifugation at 13,000 rpm with a VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, D), and washed 4 times with phosphate buffer for 30 minutes. Printed by the Intellectual Property Intelligence Bureau of the Ministry of Economic Affairs. To the remaining solution, 15 μg of thioguanidine prepared according to Example 5 (1 μg/μl dissolved in phosphate buffer) was added, and the mixture was incubated at 25 ° C for 16 hours. After lyophilization, as in Invitrogen The crude product was analyzed by SDS-Page using NuPAGE 10% Bis-Tris gel/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instruction manual. Roti-Blue Coomassie stain (Roth, Karlsruhe) , D) dye the gel overnight. Shown in Figure 5. Migration by protein band to higher score -106- This paper scale applies to China National Standard (CNS) A4 specification (210x297 mm) 1356065 A7 B7 V. Description of invention (105) Sub-indicator for occasional cooperation Success. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 7.6: Reaction of thio-erythroglobin with the reaction product of Example 1.3 and a cross-linking compound Prepared according to Example 1.3 at 80. (: squat hour and at 25: (under 3 days of culture conditions) and dissolved in 200 μl of o.im sodium phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2)-^ 50nmol HES derivative, 10 μl of a solution of 2.5 micromolar AMAS (Sigma Aldrich, Taufkirchen, D) dissolved in DMSO was added. The clear solution was incubated at 25 ° C for 80 minutes and at 40 Incubate for 20 minutes at ° C. The remaining AMAS can be removed by centrifugation at 13,000 rpm with a VIVASPIN 0.5 ml concentrator, 5 KD MWCO (VIVASCIENCE, Hannover, D), and washed 4 times with phosphate buffer for 30 minutes. The Intellectual Property Office of the Intellectual Property Office of the Ministry of Economic Affairs printed the remaining solution, adding 15 μg of the thiopurine (1 μg/μl dissolved in phosphate buffer) prepared according to Example 5, and the mixture was cultured at 25 C. 16 hours. After cooling, the crude product was analyzed by SDS-Page using NuPAGE 10% Bis-Tris gel/MOPS buffer (invitrogen, Carlsbad, CA, USA) as described in the instruction manual provided by invitr〇gen. The gel was stained overnight with R〇ti_BiUe Coomassie stain (Roth, Karlsruhe, D). -107- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 public) 1356065 A7 B7 V. Description of invention (1〇6) The experimental results are shown in Figure 5. Migration to a higher molecular weight by the protein band indicates successful coupling. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 7.7: Reaction of the reaction product of thio-erythroglobin with Example 3.1 and a crosslinking compound was prepared according to Example 3.1 and dissolved in 200 μl of phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH). 7.2) 50 nmol HES derivative, 10 μl of a solution of 2.5 micromolar AMAS (Sigma Aldrich, Taufkirchen, D) dissolved in DMSO was added, and the clear solution was incubated at 25 ° C for 80 minutes and at 40 ° C Cultivate for 20 minutes. The AMAS can be removed by centrifugation at 13,000 rpm with a VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany) and washed 4 times with phosphate buffer for 30 minutes. Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative Printed In the remaining solution, 15 μg of thiol hydrazine (1 μg/μl dissolved in phosphate buffer) was added, and the mixture was incubated at 25 ° C for 16 hours. After lyophilization, the crude product was analyzed by SDS-Page using NuPAGE 10% Bis-Tris gel/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions provided by Invitrogen. Roti-Blue Coomassie stain (Roth, Karlsruhe, D) -108- This paper size applies to the Chinese National Standard (CNS) A4 specification (210 x 297 mm) 1356065 A7 B7 V. Description of invention (107) Gel overnight dyeing. The experimental results are shown in Figure 6. Migration to a higher molecular weight by the protein band indicates successful coupling. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 7.8: Reaction of thio-erythroglobin with the reaction product of Example 3.2 and the cross-linked compound. Prepared according to Example 3.2 and dissolved in 200 μl of phosphate buffer.

To 50 nmol of HES derivative (0.1 M, 9_15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 micromolar AMAS (Sigma Aldrich, Taufkirchen, D) dissolved in DMSO was added, and the clear solution was at 25* Incubate for 80 minutes at t and incubate for 2 min at 40 °C. The AMAS can be removed by centrifugation at 13,000 rpm with a VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany) and washed with phosphate buffer. 4 times for 30 minutes. Ministry of Economic Affairs, Intellectual Property Bureau, employee consumption cooperation, printing

I In the remaining solution, 15 μg of thio-indole (1 μg/μl in phosphate buffer) was added, and the mixture was incubated at 25 ° C for 16 hours. After cooling and drying, the crude product-109- was analyzed by SDS-Page using NuPAGE 10% Bis-Tris gel/MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instruction manual provided by Invitrogen. This paper size applies to the Chinese National Standard (CNS) A4 specification (210x297 mm) 1356065 A7 B7 V. Description of the invention (ι〇0. The gel is dyed overnight with Roti-Blue Coomassie stain (Roth, Karlsruhe, D). The experimental results are shown in Figure 6. The migration of the protein band to a higher molecular weight indicates successful coupling. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 7.9: Reaction of the reaction product of thio-erythroglobin with Example 3.3 and a cross-linking compound. Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative printed in accordance with Example 3.3 and dissolved in 200 μl of phosphate buffer (0.1 To 50 nmol of HES derivative of M, 9.15 M NaCl, 50 mM EDTA, pH 7.2), 10 μl of a 2.5 μm-mole AMAS (Sigma Aldrich, Taufkirchen, D) solution in DMSO was added and the clarification was carried out. Incubate for 80 minutes at 25 ° C and incubate for 20 minutes at 40 ° C. The AMAS can be removed by centrifugation at 13,000 rpm with a VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany) and buffered with phosphate. The solution was washed 4 times for 30 minutes. In the remaining solution, 15 μg of thiol hydrazine (1 μg/μl in phosphate buffer) was added, and the mixture was incubated at 25 ° C for 16 hours. Afterwards, as described in the instruction manual provided by Invitrogen, the SDS-Page uses the NuPAGE 10% Bis-Tris gel-110- paper scale for the Chinese National Standard (CNS) A4 specification (210 X 297 mm). 1356065 A7 B7 V. INSTRUCTIONS (l〇9) /MOPS Buffer (Invitrogen, Carlsbad, CA, USA) Analysis of crude product. Gelatin stained overnight with Roti-Blue Coomassie stain (R〇th, Karlsruhe, D) The experimental results are shown in Figure 6. The migration of the protein band to a higher molecular weight indicates successful coupling. The increased bandwidth is attributed to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. . Example 7.10: Reaction of the reaction product of thio-erythroglobin with Example 3.4 and a cross-linking compound Ministry of Economics Intellectual Property Office Staff Consumer Cooperative printed in accordance with Example 3.4 and dissolved in 200 μl of phosphate buffer (0.1 To 50 nmol of HES derivative of 9.1, 9.15 Μ NaCl, 50 mM EDTA, pH 7.2), 10 μl of a solution of 2.5 micromolar AMAS (Sigma Aldrich, Taufkirchen, D) dissolved in DMSO was added, and the clarified solution was at 25 ° C. The culture was carried out for 80 minutes and cultured at 40 ° C for 20 minutes. The AMAS can be removed by centrifugation at 13,000 rpm with a VIVASPIN 0.5 ml concentrator, 5KD MWCO (VIVASCIENCE, Hannover, Germany) and washed 4 times with phosphate buffer for 30 minutes. To the remaining solution, 15 μg of thio-indole (1 μg/μl in phosphate buffer) was added, and the mixture was incubated at 25 ° C for 16 hours. After freezing and drying, as described in the instruction manual provided by Invitrogen-111- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1356065 A7 B7 5. The invention description (uo) SDS-Page The crude product was analyzed using NuPAGE 10% Bis-Tris gel/MOPS buffer (Invitrogen, Carlsbad, CA, USA). The gel was stained overnight with Roti-Blue Coomassie stain (Roth, Karlsruhe, D). The experimental results are shown in Figure 6. Migration to a higher molecular weight by the protein band indicates successful coupling. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 7.11: Reaction of the reaction product of thio-erythroglobin with Example 3.5 and a crosslinking compound was prepared according to Example 3.5 and dissolved in 200 μl of phosphate buffer (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH). 7.2) 50 nmol HES derivative, add 10 μl of 2.5 micromolar AMAS (Sigma Aldrich, Taufkirchen, D) solution dissolved in DMSO' and the clear solution was incubated at 25t for 80 minutes and cultured at 4 (TC 20) Minutes. The Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, printed the AMAS can be removed by centrifugation at 13,000 rpm with VIVASPIN 0.5 ml concentrator ' 5KD MWCO (VIVASCIENCE, Hannover, D) and washed 4 times with phosphate buffer for up to 30 In the remaining solution, add 15 μg of thio-anthracene (1 μg/μl in phosphate buffer), and the mixture is incubated at 25 ° C for 16 hours. • 112- This paper scale applies to Chinese national standards. (CNS) A4 size (210x297 mm) 1356065 A7 B7 V. INSTRUCTIONS (111) After lyophilization, use NuPAGE 10% Bis-Tris gel by SDS-Page as described in the instruction manual provided by Invitrogen MOPS Buffer (I Nvitrogen, Carlsbad, CA, USA) Analysis of the crude product. The gel was stained overnight with Roti-Blue Coomassie stain (R〇th, Karlsruhe, D). The results are shown in Figure 6. Migration by protein band to High molecular weight indicates successful cooperation. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of HES derivatives attached to the protein. Example 7.12: Thio-erythroblastin with Example 3.6 and cross-linking The reaction product of the reaction of the compound was prepared according to Example 3.6 and dissolved in 50 μl of HES printed by 200 μl of Phosphate Buffer Economy Department Intellectual Property Office Staff Consumer Cooperative (0.1 M, 9.15 M NaCl, 50 mM EDTA, pH 7.2). To the derivative, 10 μl of a 2.5 μm molar AMAS (Sigma Aldrich, Taufkirchen, D) solution in DMSO was added and the clear solution was incubated at 25 ° C for 80 minutes and at 4 (TC for 20 minutes).

The AMAS can be removed by centrifugation at 13 〇〇〇 rpm with a VIVASPIN 0.5 ml concentrator ' 5KD MWCO (VIVASCIENCE, Hannover, Germany) and washed 4 times with phosphate buffer for 30 minutes. 15 micrograms of thio-anthracene (1 μg/μl ▲ at -113- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 public « ) 1356065 A7 B7 V. Description of invention (II2 ) Phosphate buffer In solution, and the mixture was incubated at 25 ° C for 16 hours. After lyophilization, the crude product was analyzed by SDS-Page using NuPAGE 10% Bis-Tris gel / MOPS buffer (Invitrogen, Carlsbad, CA, USA) as described in the instructions provided by Invitrogen. The gel was stained overnight with Roti-Blue Coomassie stain (Roth, Karlsruhe, D). The experimental results are shown in Figure 6. The higher molecular weight of the protein band migration I indicates that the coupling is successful. The increased bandwidth is due to the molecular weight distribution of the HES derivative used and the number of organisms attached to the protein. Example 8: Preparation of HES-EPO conjugates Abstract: Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperatives, HES-EPO conjugates, HES derivatives (average mw 丨8, 〇〇〇^ deafness; hydroxyethyl substitution degree) 0.4() was coupled to the oxidized citric acid residue of a partially (mild phytate) oxidized on the oligo-bond of recombinant human Ep. The modification introduced based on the structural analysis of hydrocarbons does not affect the structural integrity of the heart oligosaccharide chain, as the MALDI/TOF-MS of the glycan treated with mild acid treatment and H^s is unrecognizable and unmodified. Ερ(: The intact neutral acetylcholine type chain of the neutral group seen in the product. The results indicated that the EPO formulation was modified without prior shifting -114-

1356065 A7 B7 V. INSTRUCTIONS (U3) In addition to some tannic acid, each EPO molecule binds at least 3 modified HES-residues. EPO variants lacking about 50% of the prolonged acid residues of the previous protein showed a similar high molecular weight mobility (60-110 KDa, relative to the BRP EPO standard of 4 KDa) in SDS-PAGE. The HES modified by HES is stable at room temperature and pH 3-10 under standard ion exchange chromatography conditions. Based on the protein determination value of the UV absorption value of the RP-HPLC EPO protein assay method obtained from the European Pharmacopoeia and the BRP EPO standard preparation background, the red blood cells are normally smaller than the international BRP EPO reference standard. EPO-biological analysis of the murine system indicated that HES-modified EPO had a specific activity (IU/mg) of 2.5-3.0 fold higher in this assay. Example 8.1: Materials and methods (a) Released by N-glycosidase digestion N. Linked to the Department of Intellectual Property, Intellectual Property Office, Staff Consumer Cooperative, printed samples and 25 units (according to the manufacturer's specifications, Roche Diagnostics, Germany) The recombinant PNGase F was cultured overnight at 37 °C. Complete digestion can be monitored by specific mobility shifts of proteins in SDS-PAGE. The released N-Galco can be isolated from the polypeptide by the addition of 3 volumes of cold 100% ethanol and incubation at -20 °C for at least 2 hours (Schroeter S et al., 1999). Apply Chinese National Standard (CNS) A4 size (210 X 297 mm) at 13000 rpm -115- paper scale at 4 ° C 1356065 A7 B7 V. Inventive Note (II4) Remove the precipitate by centrifugation for 10 minutes Protein. The sphere was then washed twice with 500 microliters of ice-cold 75% ethanol. The oligosaccharides in the concentrate were dried in a vacuum centrifuge (Speed Vac concentrator, Savant Instruments, USA). The glycan sample was desalted using a Hypercarb tube (25 mg or 1 mg of HyperCarb) as follows: The column was cleaned with 3 x 5 〇〇 microliters of 80% acetonitrile (vol/vol) dissolved in 0.1% TFA. Then, wash it with 3 X 500 μl of water. The sample is diluted with water to a final volume of 300 microliters to 600 microliters before being loaded into the tube, and the tube is thoroughly washed with water. The oligosaccharide was dissolved in 1.2 ml (25 mg tube; as for 100 mg tube, 1.8 ml) 25% water-soluble acetonitrile, wherein the water contained 0.1% trifluoroacetic acid (vol/vol) to 2M NH4OH The dissolved oligosaccharides were neutralized and dried in a Speed Vac concentrator. In some instances, the digested sugar released by N-cholinease was desalted by adsorption of a digestion mixture of a sample of <100 micrograms of total (sugar) protein onto a 100 mg HyperCarb tube. (b) Analysis of the use of a Bruker ULTRAFLEX time-of-flight (T〇F/T) by the Matrix-assisted Laser Desorption Ionization/Time-of-Flight Mass Spectrometer (MALDI/TOF-MS) for the Intellectual Property Office of the Oligosaccharide Ministry of Commerce 〇F) Apparatus: Analysis of natural deanated oligosaccharides using 2,5-dihydroxybenzoic acid as a UV absorbing material in positive ion mode and negative ion mode, both of which utilize reflectors. For Ms-MS analysis, the selected parent ion is subjected to laser enthalpy dissociation (LID) and the resulting fragment ion is separated by the second TOF phase (LIFT) of the instrument by 1 microliter and the approximate concentration is 1-116-ben. The paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210 x 297 mm). 1356065 A7 B7 V. INSTRUCTION DESCRIPTION (The sample solution of ιι〇lOpmol/μL is mixed with each substrate in equal amounts. The mixture is spotted in a stainless steel The target was dried at room temperature prior to analysis. Example 8.2 Preparation and characterization of recombinant human EPO (EPO-GT-1) as described (Muelle PP et al, 1999, Doraer AJ et al., 1984) by recombination The body CHO cells express EPO and are characterized according to the method described in the European Pharmacopoeia (Repression Drugs 4, Monography 01/2002: 1316: Concentrated Red Blood Soluble Solution). The final product has a tannic acid content of 12 nMol (+/- 1.5 nMol)/nMol protein. As described (Nimtz et al., 1999, Grabenhorst, 1999), the structure of N-linked oligosaccharides was determined by HPAEC-PAD and by MALDI/TOF-MS. Containing di-, tri-, and tetradecanoate oligosaccharides (2-12%, 15-28%, and 60-80%, respectively) The sulphation and pentoxide chain are present in small amounts. The overall glycosylation profile of the EPO formulation is similar to the international BRP EPO standard formulation. The Department of Economic Intelligence's Intellectual Property Office employee consumption cooperative prints the isoelectric focusing pattern of the recombinant EPO. Similar to the international BRP reference EPO standard formulation showing the corresponding isomeric recombination. 25% EPO protein has no purine-glycosylation at Ser126 of the polypeptide chain. Example 8.3 Preparation of partially de-ethanated EPO type in 20 mM sodium phosphate EPO GT-1 protein-117-^ paper scale in buffer (pH 7.0) is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 1356065 A7 B7 V. Invention description (ι 〇 (2·84) (mg/ml) heated to 8〇〇c, then add 100 μl of IN H2S04 per ml of EP0 solution; continue to culture for 5 minutes, 10 minutes and 60 minutes respectively, resulting in different degrees of acidification of EPO preparations with 0-4 Quantitative measurement of citric acid oligosaccharides can be performed after the release of oligosaccharides by the polypeptide N-glycosidase and the separation of the N-linked strands can be desalted using Hypercarb guanidine (25 HHyperSep Hypercarb; ThermoHypersil-Keystone, UK). Way "Using 1N NaOH to neutralize the EPO preparation and freezing it under liquid A, and storing it at _2 ° °c until further use. Example 8.4 Acidification EPO type periodate oxidation Ministry of Economics Intellectual Property Bureau Printed by the Employees' Cooperative. In a solution of 10 mg of untreated or mildly acid-treated EPO dissolved in 3.5 ml of 20 mM sodium phosphate buffer (pH 7.0), 1.5 ml of 0.1 M sodium acetate buffer (pH 5.5) was added, and The mixture was cooled to 〇 ° C in an ice bath; 500 μl of 10 mM sodium periodate was added and the reaction mixture was kept in the dark for 60 minutes. Then 10 microliters of glycerol was added and the incubation was continued for another 10 minutes in the dark. Desalting in a laboratory centrifuge equipped with a fixed angle rotor at 3000 rpm using a VIVASPIN concentrator (1〇, 〇〇〇MWCO, PES Vivascience AG, Hannover, Germany) according to the manufacturer's recommendations to separate the partially oxidized reagents EP0 type. After freezing in liquid nitrogen, the EPO formulation was stored at -20 ° C in a final volume of 4 ml. -118- The paper size is applicable to China National Standard (CNS) A4 specification (210X 297 mm) 1356065 A7 B7 V. INSTRUCTIONS (117) Order several 10 micrograms of partially oxidized EPO preparation for N-glycosidase and The oligosaccharides were separated using a Hypercarb tube. Oligosaccharides were deacylated by aminic acid treatment and analyzed by HPAEC-PAD, and as described (Nimtz et al., 1990 and 1993), the residence time is similar to the residence time of these standard oligosaccharides. Example 8.S Reduction of EPO disulfide 5 mg EPO-GT-1 with dithiothreitol 5 ml 0.1 M Tris/HCl buffer (pH in the presence of 37 ° C and 30 mM dithiothreitol (dtT) The culture was incubated for 60 minutes in 8.1; the removal of DTT was accomplished in 4 buffer exchange cycles at 4 °C using a Vivaspin concentrator. The final reduced EPO formulation was frozen in liquid nitrogen and stored in 50 mM sodium acetate buffer (pH 5 5) at -20 °C. Example 8.6 EPO Protein Determination The Ministry of Economic Affairs, Intellectual Property Office, Employees, Consumer Cooperatives, and the quantitative determination of EPO protein are based on the European Pharmacopoeia (European Pharmacopoeia 4 Monography 01/2002: 1316: erythropoietin concentrated solution) in a color path of 1 cm. The tube was carried out by measuring the uv absorption of 280 nm. Moreover, EPO can be quantified by RP-C4 column (Vydac protein C4, catalog number 214TP5410, Grace Vydac, CA, USA) by RP-HPLC method; and phylogenetic system using erythropoietin BRP 1 reference standard School-119- This paper scale applies to China National Standard (CNS) A4 specification (210 X297 mm) 1356065 A7 B7 V. Illustrative (118) Pharmacopoeia, Conseil de l'Europe BP 907-F67029, Strasbourg Cedex 1).

Example 8.7 Galactose Oxidase Oxidation De-ethanated EPO 4·485 mg EPO that has been completely decapitated in 16 μl of hydrogen peroxide decomposing enzyme (6214 units/200 ml) and 80 μl of galactose oxidase (2250 units) /ml, cultured in 20 mM sodium phosphate buffer (pH 6.8) in the presence of DaciWww i/ewi/ro/i/es (Sigma-Aldrich, Steinheim, Germany); overnight culture at 37 ° C; Twenty microliters of galactose oxidase was added to each of the 4 hours after the start of the culture and after 8 hours. Example 8.8 Preparation of EPO Samples for Bioanalysis Purification of EPO Samples Printed by the EPO of the EPO Protein Formulation Efficient by Peroxidation of Periodate or Galactose Oxidase by Active HES (Intellectual Property Bureau) Removal of the HES derivative) was carried out at room temperature. The EP0 culture mixture (nearly 5 mg of EPO protein) was diluted 1:10 with buffer A (20 mM N-morpholine propane sulfonic acid [MOPS/NaOH] dissolved in double distilled water, pH 8.0). Coated on a column containing 3 ml of Q-Sepharose HP (Pharmacopoeia code 17-1014-03, lot number 220211), wherein the column was subjected to a flow rate of 0.5 ml/min to 10 column volumes (CV) Buffer A Daping-120- This paper scale is applicable to country t. National Standard (CNS) A4 specification (210x297 mm) 1356065 A7 B7 V. Invention description (ll9) Balance. The column was washed with 6-8 CV of buffer A (flow rate = 0.8 ml/min) and buffer B (20 mM of wheylin ethanesulfonic acid [MES/NaOH] dissolved in double distilled water, 0.5 M NaCl, ρΗ6·5) was eluted at a flow rate of 0.5 liter/min. The ruthenium was detected by UV absorption at 280 nm and dissolved in about 6 ml. The column was regenerated using 3 CV of buffer C (20 mM MES, 1.5 Μ NC1, which was adjusted to a secondary distilled water of pH 6.5) and again equilibrated with 10 CV of buffer A (flow rate = 0.7 ml/min). The EPO eluate obtained from the Q-Sepharose step was buffer exchanged with a Vivaspin concentrator and phosphate buffered saline (PBS) in 3 centrifugation cycles per sample; samples were taken in PBS Adjusted to 2 ml and stored at -20 ° C. Only a 25% HES-modified partial de-acidification was obtained from the Q-Sepharose gel solution and then the EPO plastic was oxidized by mild periodate. Because under the conditions used, the basic EPO type could not be bonded to the Q_Sepharose gel and could be found in the flow through together with the unreacted HES derivative. Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed Example 8.9 High pH Anion Exchange Chromatography with Pulse Current Detector (HPAEC_PAD) Utilizing CarboPac PA1 Tube with High pH Anion Exchange (HP AE) Chromatography Column (0.4x25 cm) combined with a pulse current detector-121- This paper scale is applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 1356065 A7 B7 V. Invention description (12〇) (Into 0 01〇1^81〇1^System 〇1^, 118 入)) Analyzed purified natural and de-decanolated oligosaccharides (SChriiter et al., 1999; Nimtz et al., 1" 9). The potential (E) and pulse width (T) of the detector are: El: +50 millivolts, T1: 480 milliseconds; E2: +500 millivolts, T2: 120 milliseconds; E3: -500 millivolts, T3: 60 Milliseconds, and the output range is 500-1500nA. The oligosaccharide was then injected onto a CarboPacPAl column equilibrated with 1% solvent A. For the deuterated oligosaccharide, the solvent B was added by linear gradient (0-20%) in 40 minutes, followed by linear addition of solvent b by 20%-100% for 5 minutes (flow rate: 1 ML/min). Solvent A is 0.2M dissolved in NaOH of secondary distilled water, solvent B is composed of 〇.6M NaOAc dissolved in solvent A. For natural oligosaccharides, the column is 100% solvent C (0.1M dissolved in secondary The NaOH of distilled water is equilibrated, and the solvent D is added by linear gradient (0-35%) within 48 minutes, followed by linear addition of solvent D by 35%-1〇〇% for 1 minute (flow rate) : 1 ml / min). Solvent D consisted of 0.6 M NaOAc dissolved in solvent C. Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed Example 8.10 Analysis of Monosaccharide Composition of N-Glycan, HES Modified N-Glycan and EPO Protein by GC-MS in Methanol Decomposition, N-Re-Ethylation and Analysis of the corresponding methyl glycoside of monosaccharides by GC/MS after trimethylsulfonation [Chaplin, MF· (1982) A fast and sensitive method for the analysis of hydrocarbons, 扪oc/zew. /23, 336- 341]. These analyses are based on the 30-meter DB5-122- paper scale applicable to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1356065 Α7 ______Β7 V. Description of invention (121) Capillary column operating in positive ion ΕΙ mode The Finnigan GCQ ion fat mass spectrometer (Fmnigan MAT, San J〇se, CA) was performed. Temperature program. Isothermally at 80 ° C for 2 minutes, then 1 〇. 〇/min is increased to 300t » Monosaccharide is confirmed by its residence time and characteristic fragment pattern. Uncorrected Tongzi wave peak integration results are used for qualitative analysis. The monosaccharide system which produces more than one peak due to the anomerization and/or the presence of the oxime-type compound and the pyran type compound can be measured by adding all the main peaks. 0.5 microgram of inositol was used as an internal standard compound. Example 8.11 Example of Result 8.11 (a) Characterization of N-glycans of epo_gt-1 by mild acid treatment (partial deanoflation) Printed by the Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative as shown in Figure 7, by which N_ The glycoside enzyme was cultured to release the N•linked oligosaccharide and thereafter, the SDS-PAGE analysis of the EP〇_G1M preparation which had been treated with mild acid for $, 1 or 60 minutes. The N-linked oligosaccharide was subjected to HPAEC-PAD oligosaccharide map identification (Fig. 8). Untreated contains 90% of N-linked oligosaccharides with 3 or 4 citric acid residues, whereas after 5 minutes of incubation in the presence of mild acid, <4% by weight of hydrocarbon chains have 3 or 4 涎Acid residue. The HpAEc pAD that has been denitatized with N-glycan is detected by untreated EPO-GT4 and is still being acid treated -123- This paper scale applies to China National Standard (CNS) A4 specification (21〇χ297 Gong Chu)~~1 ' *--- 1356065 A7 B7 V. INSTRUCTIONS (l22) The proportion of stable neutral oligosaccharides in the preparation of 5, 10 or 60 minutes. MALDI/TOF-MS, which has been de-acidified, shows that <90% of the proximal algae is still present after mild acid treatment of the protein. Example 8.11(b) Characterization of EPO-GT-1 treated with phytate Figure 10 shows the comparison of SDS- with mildly periodate treated EPO type which has been acid treated or untreated for 5 and 10 minutes in advance. PAGE mobility. The conditions for the periodate oxidizing citric acid did not change the SDS-PAGE pattern of the EPO formulation (compared to Figure 7). Oxidation of tannic acid caused oligosaccharides to migrate to earlier dissolution times in HPAEC-PAD analysis (compare Figures 8 and 11) Example 8.11 (c) Characterization of HES-modified EPO derivatives (aa) to follow Example 2.4 The HES derivative X modified by hydroxylamine was used to carry out the HES modification of EPO-GT-1-A. The Ministry of Economy, Intellectual Property Bureau, and the Consumer Cooperatives printed 400 micrograms of HES derivatives modified with hydroxylamine. X is added in 20 μl of 0.5^1 00 ( (buffer & 115.5) 20 μg £0 0 1'·1 (oxidized EPO by mild periodate, which is oxidized in mild periodate) The reaction was carried out in liquid nitrogen before 30 minutes, 2, 4 and 17 hours, respectively, to stop the reaction. Next, sample-124- paper size is applicable to China National Standard (CNS) A4 specification (210 x 297 mm). 1356065 A7 B7 V. Description of invention (123) ----- Store at -20 °C until further Analyze it. SDS-PAGE sample buffer was added and the sample was heated to 9 〇 >> and coated on SDS-gel. As shown in Figure 12, increasing the incubation time resulted in an increase in the shift toward higher protein molecular weight. After incubation for 17 hours in the presence of a few aminoamine-modified HES derivative X, a diffusion-prone Coomassie-stained protein band migration was detected based on the position of the fractionated standard (see the left part of Figure 12). To the £ field between 60 and i1KDa. After treatment with N-saccharide 4 enzyme, 'most of the protein moves toward the position of the de-N-glycosylated EPO (see Figure 12. 'Right gel; arrow a indicates the migration position of N-glycosidase, arrow B indicates - The migration site of the glycosylated epo), it is presumed that the diffusion protein band seen in the region between the 28 KDa and the 36 KDa molecular weight standard represents the EPO type which has been modified by HES and the 0-glycosylation position of the molecule. In view of the specificity of N-glycosidase, the result of this is derived from the fact that HES modification occurs in the oxime (bb) HES-EPO conjugate of the EPO protein glycoside oxidized by iodate. Characterization of the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, printed as described above, synthesis of HES-EPO conjugate I (EPO-GT-1 derived from the oxidation of mild periodate, ie from EPO-GT-1 -A), II (EPO-GT-1 derived from 5-minute acid hydrolysis and mild periodate oxidation), III (EPO derived from 1 minute acid hydrolysis and mild peracid salt oxidation) GT-1)»includes a controlled culture (K) containing -125- This paper scale applies to the Chinese National Standard (CNS) A4 specification (21〇X 297 public) 1356065 A7 B7 V. Description of invention (I24) An unmodified EPO-GT-1 was added to the same amount of unmodified hes under the same buffer conditions. The culture mixtures were subjected to further purification to carry out biochemical analysis of the HES-EPO derivatives. The culture HES-EPO conjugates I, II and III and the control culture K were subjected to a Q-far lipid gel purification step to remove the ion exchange column flow as described in "Materials and Methods" (Example 8.8). Excess unreacted HES-reagent expected in the reaction. Due to the high acidity of the basic EPO type contained in the sample and in in the prior acid treatment, we expect that the flow through contains a large amount of modified 源自» derived from these cultures, as shown in Figure 13, almost all samples I The EPO material is retained in the Q-Sepharose column, which is only approximately 20-30°/. Samples II and III can be recovered in the fractions containing high salt concentrations. When compared to the control EPO, all of the proteinaceous material derived from the culture containing the HES derivative X in the flow through and the high salt-containing lysate fraction had a significantly higher molecular weight in SDS-PAGE. Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperatives For more detailed characterization, HES-modified EPO samples A and K (see Figure 11) are compared to iodate-oxidized EPO-GT-1-A. The sample was subjected to N-glycosidase treatment and released N-glycans as depicted in Figures 14a and 14b, while two low molecular weights were generated at the sites of the standard EPO formulation with glycosylated and unglycosylated EPO types. band. As for sample a, another band was detected to migrate to the 28 KDa mw standard position, and it is suggested that the HES modification is at the 〇-glycan of this EPO variant (refer to Example 8.11(c)(aa)). This belt -126- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1356065 A7 B7 V. Invention Description (I25) (and severely modified by HES The N-glycosylated Epo of the 咼mw type, see Figures 14a and 14b) disappears after the sample is gently hydrolyzed, and the HES is modified with the iodate-oxidized ruthenium residue of erythropoietin. See you in agreement. Numerous N-glycosidase culture mixtures are hydrolyzed using conditions that completely remove the fatty acid residues of the oligodomain (and the extended acid-linked HES derivative); after neutralization, the mixture is then adsorbed onto a small Hypercarb column to Desalting. The column was thoroughly rinsed with water' followed by dissolution of the neutral oligosaccharide bonded to 40% acetonitrile dissolved in water containing 0.1 〇/〇 trifluoroacetic acid. The resulting oligosaccharide was subjected to MALDI/TOF-MS. For the odor of the conjugate type, the spectra of the deanated oligosaccharide fractions of sample a, ΕΡΟ·GT-bA and sample K show the same mass below: m/z=1810Da (biantenna), 2175=three antennae, 2540 = four antennae, 2906 = four antennae plus one N-acetyl galactosamine repeat and 3271 = four antennas plus two N-acetyl thiolactam repeats; detected corresponding to the lack of fucose (- 146) and a small signal of galactose (-162), which can be attributed to the acid hydrolysis conditions used to remove the acid (see MALDI of Figures 17 '18 and 19). In a parallel experiment, the N-glycosidase digestion mixture was adsorbed onto a liter of RP-C18 tube E (without prior acid hydrolysis of oligosaccharides) and dissolved at 5 °/ of water containing 〇 1% TFA. The acetonitrile was dissolved; under these conditions, the Ep〇 protein was completely retained on the RP-material. The oligosaccharide was washed out of the column with 5% acetonitrile dissolved in water containing 1% TFA. The wheat-N-glycosylated EP0 protein was dissolved in 70% acetonitrile dissolved in water containing 〇 1〇/〇TFA. Neutralized and obtained from N-Sugar -127- This paper scale applies Chinese National Standard (CNS) A4 specification (210 x 297 mm)

1356065 A7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing _______ ________ B7 V. Invention Description (126) The oligode of the enzyme treatment of sample A, EPO-GT-1-A and sample K RP-C18 steps, Desalting is then carried out using the Hypercarb tube described above. The mild acid treatment was carried out under quantitative conditions (see Figure 15) and κ (see Figure 16), and the isolated vinegar was subjected to HPAEC-PAD pattern identification. The UPAEC-PAD profile obtained from the natural substance of the HES modified sample shows only negligible oligosaccharide signals, while the oligo vinegar derived from EPO GTd-A appears to be treated with a mild periodate. The same polymerase profile shown in the sample of EPO-GT-1). The solute profile obtained from the oligosaccharide controlling the EPO sample (K) produced the expected pattern (compared to the profile of Figure 8). For comparison, natural oligosaccharide profiles containing international BRP-Ep® standards were used for comparison and as a reference standard. After mild acid hydrolysis, all oligosaccharide preparations showed a qualitative property as expected for the hydrocarbon chain of the type of di-, tri- and tetra-antenna conjugates used as starting materials in the current study as described in the section on preparation of Ερ〇. And the natural oligosaccharide structure dissolution profile of the same composition is quantified (see Figure 16). This result demonstrates that the HES modification of the ruthenium sample forms a covalent linkage of the HES derivative, and the HES derivative can be dissociated from the Ep 〇 protein by the N-glycosidase and is acid-stable because it utilizes known deacidification of hydrocarbons. The mild acid treatment conditions of the compound can be removed from the N-glycan (see Figure 14a+b) ° (cc) HES-EPO and HES-EPO N-glycans by GC-MS

-128-

1356065 A7 B7 V. Inventive Note (127) Analysis of Monosaccharide Composition To further confirm that the EES modification of EPO occurs in the N-glycan of the molecule, the EPO sample is digested with N-glycosidase and the EPO protein is adsorbed in the RP- The C18 tube was placed on the crucible, and the oligosaccharide material was washed out as described above. As shown in Table 2, glucose and hydroxyethylated glucose derivatives were detected only in the EPO-modified EPO protein of the cysteine residue and the oligosaccharide fraction of the EPO sample A2. Example 8.11 (d) In-vivo analysis The bioactive Ministry of Economics of the EES-modified EPO is printed in a normal red blood cell system, and the EP0_bio-tiller is performed according to the procedure described in the European Pharmacopoeia; The laboratory for EPO analysis utilizes the international BRP EPO reference standard formulation. For the HES-modified EP〇A2 formulation, the average value of the specific activity of EPO of 294,600 units/mg protein was measured as compared with the international BRP EPO reference standard formulation contained in the sample for activity analysis. A nearly three times higher specific activity. The results of the research are summarized in Table 3. References for Examples 1 to 8: -129- 1356065 Printed by the Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative A7 B7 V. Description of Invention (l28)

Nimtz M, Noll G, Paques EP, Conradt HS Hydrocarbon structure of human tissue plasminogen activator expressed as recombinant Chinese hamster ovary cells. FEBS Lett. 1990 Oct. 1 ; 271 (1-2) : 14-8

Dorner AJ, Wasley LC, Kaufman RJ. Increased synthesis of secreted proteins induces the expression of glucose-regulated proteins in butyrate-treated Chinese hamster ovary cells J. Biol Chem. 1989 Dec 5 ; 264 (34) : 20602-7

Mueller PP, Schlenke P, Nimtz M, Conradt HS, Hauser H Quality of recombinant glycoprotein in BHK-21 cells under proliferative control Biotechnol Bioeng. 1999 Dec 5 ; 65(5) : 529-36

Nimtz M, Martin W, Wray V, Kloppel KD, Augustin J, Conradt HS Structure of human erythropoietin citrate oligosaccharide expressed as recombinant BHK-21 cells

Eur J Biochem. 1993 Apr. 1 ; 213(1) : 39-56

Hermentin P, Witzel R, Vliegenthart JF, Kamerling JP, Nimtz M, Conradt HS Method for the identification of N-glycans by high pH anion exchange chromatography with pulse current detector • 130- This paper scale applies China National Standard (CNS) A4 specification (210 X 297 mm)

1356065 A7

Anal Biochem. 1992 June ; 203(2) : 281-9

Schroter S, Derr P, Conradt HS, Nimtz M, Hale g

Kirchhoff C. Male specific modification of human CD52. J Biol Chem. 1999 Oct. 15 ; 274(42) : 29862-73 Example 9 Recombinant EPO 镅诰 A) Production of recombinant EP0 in chyme animal cells in CH0 cells was made as follows: Human EPO cDNA The plastid vector is selected into a eukaryotic expression vector (pCR3 and pCREPO as described hereinafter). Site-directed mutagenesis using the standard procedure (Grabenhorst, Nimtz, Costa et al., 1999, Human Synthetic Lewis oligosaccharide and citrate Lewis oligosaccharide motifs printed on human αΐ 3/ in the Intellectual Property Office of the Ministry of Bioeconomics. 4 In vivo specificity of fucosyltransferase III-VII versus conjugate type Ν-glycans - simultaneous performance studies of ΒΗΚ-21 cells together with human beta microproteins, J. Biol. Chem" 273(47) , 30985-30994).

Cys* is a stable expression of human EPO or its amino acid variants (eg -131- This paper size applies to National Standard (CNS) A4 specification (210x297 mm) 1356065 A7 B7 V. Description of invention (uo) 29-&gt The CHO cell line of Ser/Ala or Cys-33-> Ser/Ala, Ser-126-»Ala, etc.) was produced by calcium phosphate precipitation and selected as described (Grabenhorst et al.) with G418-sulfate. Three days after transfection, cells were transplanted 1:5 and housed in DMEM containing i〇% fbs and 1.5 g/L G418 sulfate. Using this selection procedure, typically 100-500 strains survive and additionally the culture medium is selected there for a period of 2-3 weeks of propagation. The degree of EPO expression in the cell culture supernatant of the fusion growth monolayer was then analyzed by Western blot analysis and by IEF/Western dot analysis. EPO was prepared from Stabilized Strain in a stirred flask or in a 21 perfusion reactor. EPOs of different glycoforms with different NeuAc contents (eg, 2-8, 4-10, 8-12 NeuAc residues) were separated according to published experimental procedures using a combination of various chromatographic analysis procedures as described below. come out. Literature: Ministry of Economic Affairs, Intellectual Property Bureau, employee consumption cooperative, printing

Grabenhorst, Conradt, 1999, cytoplasmic, transmembrane and cognac regions of glycosyltransferases specify their in vivo functional low localization and stability in high-kilstein, J. Biol Chem., 274(51), 36107 -16; Grabenhorst, Schlenke, Pohl, Nimtz, Conradt, 1999, Genetic engineering of recombinant cold proteins and glycosylation pathways in mammalian host cells, Glycoconj J., 16(2), 81-97 i Mueller, Schlenke, Nimtz, Conradt, -132- This paper scale applies to China National Standard (CNS) A4 specification (210 x 297 mm) 1356065 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (Ul)

Hauser, 1999, Quality of recombinant glycoprotein products in proliferating controlled βηΚ-21 cells, Biotechnology and Bioengineering, 65(5), 529_536; Schlenke, Grabenhorst, Nimtz, Conradt, 1999, with human type extension The structure and characteristics of the acid-transformed transfected BHK-21, cell assay, 30(1-3), 17-25. B) Manufacture in insect cells As described in the literature, cells were infected with a recombinant baculovirus vector containing human EPO cDNA under the control of a polyhedrin promoter, and then prepared from insect cell strains SF9 and SF21. Infect cells grown in serum-free medium at a cell density of 2x10 or 2xl〇7 cells/ml and measure the EPO titer in the cell culture supernatant daily by blue agarose gel chromatography, Ion exchange chromatography on a Q_ agarose gel and finally RP-HPLC on the C4 phase to purify EP0. The purity of the product can be viewed by SDS-PAGE and N-terminal sequencing. Detailed hydrocarbon structure analysis (N- and 〇-glycosylation) was performed according to published procedures. References: -133- This paper scale applies to the China Standard (CNS) A4 specification (210x297 mm) "-

1356065 A7 B7 V. Description of invention (132)

Grabenhorst, Hofer, Nimtz, Jager, Conradt, 1993, Biosynthesis and secretion of human interleukin 2 glycoprotein variants from Sf21 cells infected with baculovirus. Characterization of Peptides and Post-translational Modification, Eur J Biochem., 215(1), 189-97; Quelle, Caslake, Burkert,

Wojchowski, 1989, High performance and purification of recombinant human erythropoietin produced by baculovirus vector, Blood, 74(2), 652· 7 » Example 10

Reactive HES Derivatives 1. SH-Reactive HES

1.1 EMCH reacts with OX0-HES12KD to form SH-reactive HES12KD B

0.M4 g (0.012 mmol) 〇xo-HES12KD (Fresenius German patent DE 196 28 705 A1) was dissolved in 0.3 ml of absolute dimethyl sulfoxide (DMSO) -134 by the Intellectual Property Office of the Ministry of Economic Affairs. - This paper size applies to China National Ladder (CNS) A4 specification (210x297 mm) 1356065 A7 B7 V. Inventive Note (l33 and 34 mg (0.15 mmol) EMCH is added dropwise under nitrogen (Perbio Science, Deutschland GmbH , Bonn, Germany) dissolved in a mixture of 1.5 ml of DMSO. After stirring at 60 ° C for 19 hours, the reaction mixture was added to a mixture of 16 ml of ethanol and propylene. The precipitate was collected by centrifugation and dissolved again. It was re-precipitated in 3 ml of DMSO as described above. SH·reactive-HES12KD B was obtained by centrifugation and dried in vacuo. The coupling reaction with thio-EPO is described in Examples 1.1, 2.2. : In this reaction, all crosslinkers which can exhibit a sulfhydryl functional group and a maleimide functional group separated by a spacer can be used. Other examples of such molecules are shown in Table 1; A", which can be made by Pe in Bonn, Germany. Also available from rbio Science Deutschland GmbH. In addition, another class of crosslinkers which exhibit an active disulfide functional group to replace the maleimide functional group can also be used. 1.2 HES glycosylamine alizarin acetamide derivative economics wisdom Printed by the Property Bureau Staff Consumer Cooperatives a) Formation of choline amines ^Manger, Wong, Rademacher, Dwek, 1992, Biochemistry, 31, 10733-10740;

Manger, Rademacher, Dwek, 1992, Biochemistry, 31, 10724-10732) 135- This paper scale applies to National Standards (CNS) A4 (210x297 mm) 1356065 A7 B7 V. Description of invention (l34) 1 mg HES12KD The sample was dissolved in 3 ml of saturated ammonium bicarbonate. Then, while incubating at 30 ° C for 120 hours, additional solid ammonium hydrogencarbonate was added to maintain the saturation of the solution. The amine-HES12KDC was desalted by direct freeze drying of the reaction mixture.

b) Deuterated glycosylamine C with chloroacetic anhydride A 1 mg sample of the amine-HES12KD C was dissolved in 1 ml of 1 M sodium bicarbonate and cooled on ice. Solid chloroacetic anhydride (~5 mg) crystals were added and the reaction mixture was allowed to warm to room temperature. The pH is monitored and if the pH drops to 7.0, additional base is added. After 2 hours at room temperature, a second base and anhydride were added. After 6 hours, the product chloroacetamide-HESD1 (X = C1) was desalted by a mixture of bed Amberlite MB-3 (H) (OH) ion exchange resin. c) Brewing of sulphonylamine 2 with bromoacetic acid (25/^^, Αϋ, 7W/, Withers, 1993, Carbohydr. Res., 250, 195) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed such as Thomas3 (3Thomas , 1977, A/eMoi/ey V”/g, 362, 362) Prepare the preparation of bromoacetic anhydride. Dissolve 1 mg of the amino-HES12KD C sample in 0.1 ml dry DMF and cool on ice, then add 5 Mg of bromoacetic acid anhydride. The reaction mixture was slowly warmed to room temperature and the solution was stirred for 3 hours. The reaction mixture was added to 1 ml of -20 ° C ethanol and acetone 1:1 mixed -136 - This paper scale applies to Chinese national standards (CNSA4 specification (210x297 mm) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1356065 A7 B7 V. Invention description (135) In the compound, the precipitate was collected by centrifugation, and then dissolved in 0.1 ml of DMF and as Reprecipitated as described above. Obtaining bromoacetamide-HES D2 (X=Br) by centrifugation and drying it in a vacuum. The coupling reaction with thio-EP〇 is as in Example 11, 1.2 households/1 The sentence is synthesized as described for D2. The corresponding iodine-derivative D3 (X=I) is replaced by N-City-produced imino iodoacetate. Bromoacetic anhydride

And all the steps are carried out in the dark in the β substitution method: other active forms of arginine can be used to deuterate amine groups, such as - bromide or - vapor-ester, such as Ν-hydroxy amber imidate, An ester having a substituted phenol (p-nitrophenol, pentafluoropan, trichloroethylene, etc.) Further, all cross-linking agents having an amine-based reactive group and a halogenated ethylidene functional group separated by a spacer can be used. An example of this is SBAp. This and other molecules are available from Perbi〇 Science Deutschland, Bonn, Germany.

Purchased by GmbH. It's on the table! The middle is marked with "D,". For use as a connection • 137- This paper scale applies to the national standard (CNS) A4 specification (10) café; ^------^

1356065 A7 B7 V. INSTRUCTIONS (136) Amine-HES and thio-EPO, without the crosslinker of the isolated halogenated acetamide-HES derivative, see the notes of Examples 11, 1.2. 1.3 Amino-HESE1 Haloacetamide Derivatives a) 1,4-Diaminobutane is reacted with Oxo-HES12KD to form an amine group-HES12KD E4 (4S. Frie, Diplomarbeit, Fachhochschule Hamburg, 1998) 1.44 g (0.12 mmol) Oxo-HES12KD was dissolved in 3 ml of dry dimethyl hydrazine (DMSO) and 1.51 ml (15 mmol) of 1,4-diaminobutane was dissolved in 15 ml under nitrogen. In a mixture of DMSO. After stirring at 40 ° C for 19 hours, the reaction mixture was added to a mixture of 160 ml of a 1: 1 mixture of ethanol and acetone. The precipitated amine-HES12KD E was collected by centrifugation, dissolved in 40 ml of water and dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, Germany) and dried by cold shower. Of

Printed by the Consumer Intellectual Property Office of the Ministry of Economic Affairs, b) The description of chloracetamide-HES12KD D1 as described above, -138- The paper size applies to the Chinese National Standard (CNS) A4 specification (210x297 mm) 1356065 A7 B7 , Invention Description (137) Preparation of chloroacetamide-HES12KD F1. c) Preparation of bromoacetamide-HES12KD F2 (X = Br) as described in 1-3 for bromoacetamide-HES12KD D2. The coupling reaction with thio-EPO is described in Examples 11, 1.2. d) The corresponding iodine-derivative F3 (X = I) is not separated before it reacts with thio-EPO. The experimental system is described in Examples 11, 1.1. Alternative method: See 1.2.

2. CHO-Reactive HES 2.1 醯肼-HES

J Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed a) Reaction of Ox-HES 12KD 1.44 g (0.12 mmol) of Oxo-HES12KD was dissolved in 3 ml of absolute dimethyl sulfoxide (DMSO) and nitrogen Add 0.47 milli-139- drop to the paper. Applicable to China National Standard (CNS) A4 specification (210 X 297 mm) 1356065 A7 B7 V. Description of invention (138) L (15 mmol) 醯肼 dissolved in 15 In a mixture of milliliters of DMSO" at 40. (After stirring for 19 hours, the reaction mixture was added to 160 ml of a 1:1 mixture of ethanol and acetone. The precipitated product J was collected by centrifugation, and dissolved in 40 ml of water at 0.5% (vol/vol) Aqueous solution of ethylamine was dialyzed for 2 days and dialyzed against water for 2 days (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, Germany) and cooled and dried with a coupled reaction system with deuterated Glyco-EPO Described in Example 12, 2.2.

Printed by the Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, b) Reaction of diammonium adipate with Oxo-HES12KD 1.74 g (15 mmol) of diammonium adipate dissolved in 20 ml absolute at 65 °C Methyl hydrazine (DMSO) and 1.44 g (0.12 mmol) of Oxo-HES12KD dissolved in 3 ml absolute DMSO were added dropwise under nitrogen. After stirring at 60 ° C for 68 hours, the reaction mixture was added to 200 ml of water. The solution containing L was dialyzed against an aqueous solution of 0.5% (v/v) triethylamine for 2 days and dialyzed against water for 2 days (SnakeSkin dialysis tubing, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, Germany) and cold-dried It. The coupling reaction with oxidized Glyco-EPO is described in Example 12, 2.2 - 140- This paper scale applies to China National Standard (CNS > A4 specification (210 X 297 mm) 1356065 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative Printed in V. Invention (139). Alternative method: In addition, two derivatives in which the sulfhydryl group is separated by any spacer may be used. 3. Other amino groups-HES12KD derivatives I and H1 D or F aminolysis The effect was carried out by dissolving 1 mg of each dentate acetamide sample in 0.1 ml of saturated ammonium carbonate. Then, while incubating at 30 ° C for 120 hours, additional solid ammonium carbonate was added to maintain the saturation of the solution. The mixture was added to 1 ml of a 1:1 mixture of ethanol and acetone at -20 ° C. The precipitate was collected by centrifugation, and then decomposed in 0.05 ml of water and precipitated again as described. The product amine was obtained by centrifugation. The base-HES oxime or I is dried in vacuo. The coupling reaction with oxidized Glyco-EPO is described in Examples 12, 4.1.

K -141- This paper size is applicable to China National Standard (CNS) A4 specification (210x297 mm) 1356065 A7 B7 V. Invention description (M〇)

4. HES12KDK modified by hydroxylamine group Printing of 0-[2-(2-aminooxy-ethoxy)ethyl]-hydroxylamine by the Intellectual Property Office of the Ministry of Economic Affairs, as described by Boturyn et al. 5 (5 Boturyn, Boudali, Constant, Defrancq, Lhomme, 1997, Tetrahedron, 53, 5485) was synthesized in 2 steps from commercially available materials. 1.44 g (0.12 mmol) of Oxo-HES12KD was dissolved in 3 ml of absolute dimercaptopurine (DMSO) and 2.04 g (15 mmol) of 0-[2-(2-amine oxygen) was added dropwise under nitrogen. The base-ethoxy)-ethyl]-hydroxylamine was dissolved in a mixture of 15 ml of DMSO. After stirring at 65 ° C for 48 hours, the reaction mixture was added to 160 ml of a 1:1 mixture of ethanol and acetone. The precipitated product K was collected by centrifugation, dissolved in 40 ml of water and dialyzed against water for 4 days (SnakeSkin dialysis tube, 3.5 KD removal, Perbio Science Deutschland GmbH, Bonn, Germany) and dried by cold drying. The coupling reaction with oxidized Glyco-EPO is described in Examples 12, 3.1. Alternative method: -142- This paper is suitable for China National Standard (CNS) A4 specification (210x297 mm) 1356065

V. INSTRUCTIONS (141) Further, a derivative in which two hydroxylamine groups are separated by any spacer can be used.

5. Sulfur-HES12KD

5.1 Addition to Oxo-HES12KD

NH

, SH Μ Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1.44 g (0.12 mmol) Oxo-HES12KD dissolved in 3 mM absolute dimethyl sulfoxide (DMSO) and added 1.16 g under nitrogen (15 Millolamine was dissolved in a mixture of 15 ml of DMSO. After 24 hours at 4 ° C, the reaction mixture was added to a mixture of 160 ml of ethanol and a 1:1 mixture. The precipitated product was collected by centrifugation, dissolved in 40 liters of water and dialyzed against 0.5% (v/v) aqueous solution of tris-amine in water for 2 days and dialyzed against water for 2 days (SnakeSkin dialysis tube, 3.5 KD) Removed, Perbio Science Deutschland GmbH, Bonn, Germany) and cool and dry. The coupling reaction with oxidized Glyco-EPO is described in Example 12, 2.1 t. Alternative method: • 143· This paper size applies to the National Standard for Sleepy (CNS) A4 specification (210 X 297 mm) 1356065 A7 B7 V. Description of invention (156) Reagents in the sample The conjugate of the protein conjugate was obtained by lyophilization after overnight dialysis. Note on procedures: Knee adducts are somewhat unstable at extreme pH. For applications that may include low pH treatment, we can reduce the domains to hydrazine by treatment with 30 mM sodium hydride in PBS, buffer. This extra step is not required for most applications. 2.2 Example Experimental Procedure 8 Directly coupled oxidized Glyco-EPO to 醯肼·ΗΕ 812κ〇 [中. 4 Order Printed by the Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative A. The oxidized Glyc〇_Ep〇 solution obtained from 6.1.1: 5 mg/ml of Glyco-EPO dissolved in acetate buffer. B. Brewing-HES12KDL or j:i〇mg/ml is dissolved in acetate buffer. 〇. Acetate buffer: 〇.1] ^ sodium acetate solution, 1) 115.5 D. Gel filtration column: for example, X 45 cm). E. Coomassie® Protein Analysis Reagent (perbi〇Science -158- This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 public) 1356065 A7 U B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention Description (157)

Deutschland GmbH, Bonn, Germany) F. PBS, phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4. Method 1 ml of hydrazine-HES12KD L solution and 1 ml of oxidized Glyco-EPO solution were combined and the reaction mixture was reacted with stirring for 16 hours at room temperature. The reaction mixture was applied to Sephadex® G-200 (1.5 X 45 cm) equilibrated with PBS and 1 ml of the fraction was collected. The Coomassie protein analysis reagent was used to monitor the protein content in the fraction, and all the fractions containing the protein conjugate were concentrated and dried overnight by water to obtain a coupling by cold drying. The results of the occasional cooperation are shown in Fig. 24. The observed change in molecular weight demonstrates that the coupling is successful. This viscous material is caused by the heterogeneity of HES. Figure 25 demonstrates that the HES is coupled to the hydrocarbon moiety of the hydrocarbon side chain. Note on the procedure: The ruthenium adduct is somewhat unstable at extreme pH. For applications that may involve low pH treatment, we can reduce hydrazine to hydrazine by treatment with 30 mM sodium cyanoborohydride in PBS buffer. For most applications, this extra step is not required. -159- This paper size applies to China National Standard (CNS) A4 specification (210x297 mm)

Ministry of Economic Affairs Intellectual Property Bureau J1·Consumer Cooperative Printed 1356065 Α7 Β7 V. Invention Description (158) 3. Bias with several kipo-derivatives 8 (8R〇se, 1994, J/w. CAew. «Sbc., 116, 30) 3.1 Example Experimental Procedure 9 Glyco-EPO oxidized to hydroxylamine-HES12KD K. Materials A. Oxidized Glyco-EPO solution obtained from 6.1.1: 5 mg/ml Glyco-EPO was dissolved in acetate buffer. B. Hydroxylamine-HES12KDK: 10 mg/ml is dissolved in acetate buffer. C. Acetate buffer: 〇.1 Μ sodium acetate solution, pH 5.5 D. Gel filtration column: for example, Sephadex® G-200 (1.5 x 45 cm). E. Coomassie® Protein Assay Reagent (Perbio Science Deutschland GmbH, Bonn, Germany) F. PBS, phosphate buffered saline: 10 mM sodium phosphate, 150 mM NaCl, pH 7.4. Method Combined with 1 ml of hydroxylamine-HES12KD K solution and 1 ml of oxygen 1 -160- paper scale applicable to China National Standard (CNS) A4 specification (210 X 297 mm)

1356065 A7 B7 V. INSTRUCTIONS (1S9) Glyco-EPO was mixed and the reaction mixture was allowed to react at room temperature for 16 hours. The reaction mixture was applied to SePhade X® G-200 (1.5 x 45 cm) equilibrated with pBs and a 1 ml fraction was collected. The protein content in the fraction was monitored using the Coomassie Protein Tiller reagent'. All the libraries containing the protein conjugate were concentrated and dialyzed against water overnight, and the conjugate was obtained by freeze drying. The obtained conjugate is shown in Fig. 24. The change in molecular weight observed in the gel coat 2 proved that the coupling was successful. This viscous material is caused by the heterogeneity of HES. Figure 25 demonstrates that the HES is coupled to the hydrocarbon moiety of the hydrocarbon side chain.

Ministry of Economic Affairs, Intellectual Property Office, Employees, Consuming, Shanghai-level XM13, EPO N-glycan-characterized recombinant EPO, or partially de-storylated EP0 (produced by finite mild acid hydrolysis) The galactose oxidase was incubated with galactose oxidase at 37 ° C for 3 〇 4 hours in the presence of hydrogen peroxide decomposing enzyme in 〇 5 M sodium phosphate buffer pH 7.0. By removing 50 micrograms of the EPO moiety, followed by treatment of the protein with the polypeptide N-glucanase, before and after removal of the acid, as described (Grabenhorst et al, 1999 Nimtz et al, 1993/1994; Schlenke et al, 1999) The released N-linked oligosaccharide (method of detecting de-glycosylated polypeptide by SDS-PAGE-161- This paper scale applies to national standard (CNS) A4 specification (210x297 mm) ) 1356065 Ministry of Economic Affairs Intellectual Property Bureau staff consumption cooperation Du printing A7 B7 V. Invention Description (160) Monitoring) HPAEC-PAD map identification. The measurement of the amount of oxidized galactose residues in each Ep oligosaccharide can be carried out by typical changes observed by HPAEC-PAD and can also be confirmed by MALDI/TOD MS of the oligo-sense mixture. Example 14 Characterization of HAOs that have been modified with HAS Separation of EPO-modified EPO-type and unreacted EPO or HAS-precursor molecules can be performed by gel filtration using, for example, Ultrogel AcA 44/54 or similar gel filtration media. achieve. Alternatively, EPO is separated by immunoaffinity on a 4 ml column to remove unreacted HAS, followed by gel filtration (eg, using a matrix that can separate globular proteins with a relative molecular weight between 20 kDa and 200 kDa), The column contains a monoclonal antibody coupled to Affigel (Bi〇Ra(i). The HAS modified by HPS was analyzed by SDS-PAGE (using 12.5 or 10% acrylamide gel) to coat the gel with Coomassie Brilliant Blue. After staining, it is identified by its higher molecular weight detection than untreated EPO. The higher molecular weight of the HAO-modified HPO polypeptide can also be analyzed by Western blotting using multiple antibodies against recombinant human EPO. Knowledge-162- This paper size applies to China National Standard (CNS) A4 specification (210x297 mm)

1356065 A7 V. INSTRUCTIONS (W-type EPO-type N-Polymer modification can be successfully removed from EPO protein by polymorphic N-polyacetase (recombinant N•glycosidase obtained from Roch, Germany, its use) 25 units/mg EPO protein was obtained at 37 ° C for 16 hours); SDS-PAGE analysis produced EPO. The protein typically changed to nearly 20 KDa. The mobility position of the unmodified Ep〇 treated by N-glycosidase. And the modification of EPO 〇-glycan by galactose oxidase in Serl26 can be detected by the sdS- of the deglycosylated product compared to the untreated Ν Ν 糖 glycosylated hydrazine. The PAGE mobility can be proved. If necessary, the modified ΕΡΟ can be classified by SDS-PAGE on the C8-phase by RP-HPLC. The HAS 0 glycan modification can also be eliminated by β_ The glycan was detected and analyzed in the western blot of anti-recombinant human ΕΡ0. The epo was analyzed by the _〇_glycosylation type. Example 15 Ministry of Economic Affairs Intellectual Property Bureau Employees Consumption Cooperatives And modified ΕΡΟ type ΕΡ 0 type such as the European Pharmacopoeia (2000, Erythrop〇iet Ini Solutio concentrate, 1316, 780-785) is quantified by UV measurement and compared to the international BRP reference EP0 standard. Or, by Rp_ • 163 - this paper scale applies the Chinese National Standard (CNS) A4 specification ( 210x297 mm) 1356065 A7 B7 V. INSTRUCTIONS (162) HPLC analysis using RP-C4-column and 254 μm absorption to determine the concentration of EPO'. The calibration system uses 20, 40, 80 and 120 micrograms of BRP standard EPO reference. Formulation. Example 16

MffES modified recombinant human EPO in vivo. The purified EES-modified EPO was tested using the erythropoietin bioactivity assay as described by Krystal [Krystal, 1984, Exp. Heamatol., 11, 649-660]. Anemia was induced by treatment with phenylhydrazine hydrochloride in NMRI mice and spleen cells were collected and used as described in [Fibi et al., 1991, Blood, 77, 1203 and subsequent pages]. EPO dilutions were grown at 3 x 105 cells/twist in a 96 well microtiter plate. After 24 hours at 37 ° C in a humid atmosphere (5% CO 2 ), the cells were labeled with 3H-thymidine per well for 4 hours. The incorporated radioactivity is determined by liquid phase flash counting. International reference EPO standards (BRP-standards) are used for comparison. Alternatively, EPO biological activity can be measured by in vivo analysis of EPO-sensitive cell line TF-1 (Kitamura et al, J. cell Phys., 140, 323-334). The exponentially growing cells were washed to be free of growth factors and additionally cultured for 48 hours in the presence of a series of EPO dilutions. As stated by Mosmann-164- This paper scale applies to the S National Standard (CNS) A4 specification (210 31297 mm) ^-----The Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1356065 Α7 Β7 V. Invention Description (163) [Mosman, 1983, J. Imnrnno, Methods, 65, 55-63] Analysis of cell proliferation using a 减少 reduction assay. Example 17 In vivo Activity Assay for EPO and HAS Modified Ep〇 Type In vivo activity assays were measured in erythrocyte normal mice 4 days after receiving a predetermined dose of EPO or modified EP〇 in animals. The way red blood cells increase is carried out. The analysis was performed using the BRp EP0 standard, which was calibrated against the WHO EPO standard in the erythrocytosis mouse assay. The β Ep 〇 sample was diluted with saline buffered saline, which contained 1 mg/ml bovine serum albumin (Sigma). The Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, printed each animal via a subcutaneous sputum. 5 ml of EPO test solution dissolved in Dulbecco's buffered saline (corresponding to 1〇〇, 8〇, 4〇 or 2〇IU/ml brp standard) EPO EPO protein equivalent) infection. Blood samples were collected 4 days after infection and reticular red blood cells were stained with acridine orange: Quantitative measurement of reticular red blood cells was performed by flow cytometry in a total of 30,000 blood cells within 5 hours after blood collection (see Ph. Eur , 2000, Erythropoietini Solutio concentrate, 1316, 780-785 and European Pharmacopoeia (1996/2000, appendix 2002)) 〇 165- This paper scale applies to the Chinese National Standard (CNS) A4 specification (210 x 297 public Chu) 1356065 A7 B7 , invention description (164) ^ specific amount of unmodified or modified sputum intravenous injection of rabbits. ▲ samples were taken at specific times and serum was prepared. The amount of serum erythropoietin was measured by /# in vivo analysis or by EPQ·specific commercial ELISA. Example 19 蚤 蚤 蚤 立 在 在 在 In the mice: each animal received 300 IU EPO / kg subcutaneously. After 7 days of treatment, the blood volume ratio of each animal was measured. A significant increase in blood volume ratio was observed in all animals treated with modified EPO, which is the expected result given the relatively short half-life of untreated EPO. The average change in the blood volume of the modified epo treated population is significantly different from that of the untreated EPO and control populations. Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperatives In rabbits, rabbits were treated with a single dose of unmodified or HAS modified EPO, where the dose corresponds to 200 or up to 800 ng/kg body weight. Blood samples were measured at 2, 6, 16, 24, and 48 hours using a commercially available EPO-specific ELISA to determine the plasma concentration. The mean plasma EP0 concentration was measured and as described: (Zettlmissl et al., 1989, J. Biol. 264, -166- This paper scale applies to Chinese national standards ((3) Magic 8 4 specifications (210 x 297 mm) 1356065 Ministry of Economy Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (165) 21153-21159) The average initial half-life (α-phase) and the terminal phase half-life (β-phase) were calculated from the ELISA values.

Sytkowski, Lunn, Risinger and Davis, 1999, contain erythropoietin fusion proteins of the same repeat domain that exhibit higher biological properties, J. Biol. Chem., 274, 24773-24778. Example 20 Evaluation of in vivo biological activity of HES-modified recombinant human IL-2 The modified IL2 system was recovered by gel filtration on Ultrogel AcA 54. The corresponding fractions were sterile filtered and assayed for IL2 biological activity using IL2-dependent murine CTLL-2 cell lines [Gillis, Ferm, On and Smith, 1978, J. Immunol., 120, 2027-2032]. The active system is related to the international reference IL2 standard preparation. -167- This paper size applies to Yin Guo National Standard (CNS) A4 specification (210x297 mm)

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. 5 \ ο « : Lord. ••SO·. ‘ V • ο m D | 介. \ 转80 ί Type a Ph 〇Chemical name mealμ!Γ δ 1 inch set S 1 town t key to ¢- έ f S S 3 S .1 3 ^ 1 ^ If ^ ει. Ν-Amber y Base-(4-vinyl wall sulfhydryl) benzoate sulfonate-SMPB sulfonate-LC-SMPT SVSB Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed 1356065 A7 B7 V. Invention Description (l66) Table 2 Monosaccharide composition analysis of glycans obtained from HES modified EPO and control samples **Single I. Glycan obtained from A2 II. Glycan obtained from EPO-GT-1A III. Obtained from K2 Glycan III. Glycan obtained from A2 IV. Glycan obtained from EPO-GT-1A V. Glycan obtained from K2 VI. EPO protein modified by cysteine* Fucose 1,935 3,924 2,602 2,246 4,461 2,601 2,181 Mannose 6,028 11,020 9,198 6,379 11,668 6,117 6,260 Galactose 8,886 19,935 14,427 10,570 16,911 11,555 10,386 Glucose 17,968 — 21,193 Traces 33,021 GlcNAc 7,839 21,310 14,440 11,360 15,953 10,503 10,498 GlcHel 5,583 — — 5,926 — — 14,857 GlcHe2 1,380 — — 1,552 — — 3,775 NeuNAc 5,461 822 4,5 04 3,895 4,871 13,562 13,003 Inositol 1,230 2,310 1,620 2,050 1,320 1,134 1,087 *This equivalent of the Cys-HEs modified EPO protein line was analyzed for composition: EPO protein was chromatographed as described above in QQ-Sepharose The hose was separated from the HES culture mixture and desalted by centrifugation using a Vivaspin 5 separation device. ** The monosaccharide assay was performed by a single GC operation of trimethylsulfonylalkyl glucoside; -168- This paper size applies to China National Standard (CNS) A4 specification (210 X 297 mm)

1356065 A7 B7 V. INSTRUCTIONS (l67) The electronic integral value of the peak has not been corrected for the loss during the derivative process and the recovery of each compound. Table 3 Sample No. Sample Description The specific activity of the EPO sample calculated (based on A280nm and RP· HPLC) 850247 1. HES modified EPO A2 344,000 U/mg 850248 2. EPO-GT-1-A 82,268 U/ MG850249 3. Control EPO K2 121,410 U/mg 850250 4.BRPEPO standard 86,702 U/mg 850251 1. Dilute 309,129 U/mg 850252 in 4 volumes PBS 2. Dilute 94,500 U/mg 850253 in 4 volumes PBS 3. Dilute 114,100 U/mg 850254 in 4 volumes of PBS 4. Dilute 81,200 U/mg 850255 in 4 volumes of PBS 5. Diluted 230,720 U/mg in 4 volumes of PBS

-I I > 4. Set. Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed Schematic Description Circle 1 Figure 1 shows the SDSPage analysis of the HES-EPO conjugate prepared according to Example 5.1. Adhesive A: Roti®-Mark PRESTAINED (Carl -169- This paper scale applies to China National Standard (CNS) A4 specification (210x297 mm) 1356065 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Description of the invention (168)

Roth GmbH+Co, Karlsruhe, D); molecular weight of the protein label (expressed in kD) from top to bottom: 245, 123, 77, 42, 30, 25.4 and 17. Colloid B: Crude product after coupling according to Example 5.1" Colloid C: EPO starting material. Figure 2 Figure 2 shows the SDSPage analysis of the HES-EPO conjugate prepared according to Example 5.3. Colloid A: The crude product after coupling according to Example 5.3. Rubber Road B: EPO starting material. Colloid C: protein label R〇ti®-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D); molecular weight of the protein label (expressed in kD) from top to bottom: 245, 123, 77, 42, 30, 25.4 and 17. Figure 3 Figure 3 shows the SDSPage analysis of the HES-EPO conjugate prepared according to Examples 5.4 and 5.5. Glue A: Protein label R〇ti®-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D); molecular weight of protein label (in kD) from top to bottom: -170- This paper scale applies to Chinese national standards (CNS) A4 specification (210 X 297 mm)

1356065 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (丨69) 245, 123, 77, 42, 30, 25.4 and 17. Colloid B: The crude product after coupling according to Example 5.4. Colloid C: The crude product after coupling according to Example 5.5. Glue D: EPO starting material. Figure 4 Figure 4 shows the SDSPage analysis of the HES-EPO conjugate prepared according to Examples 7.1 and 7.4. Gelatin Α: Protein label Roti®-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D); from top to bottom , molecular weight of protein label (expressed in kD): 245, 123, 77, 42, 30, 25.4 and 17. Colloid B: The crude product after coupling according to Example 7.4. Colloid C: The crude product after coupling according to Example 7.1. Glue D: EPO starting material. Figure 5 Figure 5 shows the SDSPage analysis of the HES-EPO conjugate prepared according to Examples 7.2, 7.3, 7.5 and 7.6. Colloid A: Protein label R〇ti®-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D); molecular weight of protein label (in kD) from top to bottom: -171- This paper scale applies to Chinese national standards (CNS) A4 size (210x297 mm)

A7 Ministry of Economic Affairs Intellectual Property Bureau staff consumption cooperation; 41 printing 1356065 __B7_ V. Invention description (l7〇) 245, 123, 77, 42, 30, 25.4 and 17. The gum lane B·· the crude product after coupling according to Example 7.6 with the example 1.3 b) as the substrate. Colloid C: The crude product after coupling according to Example 7.5 using the substrate of Example 1.1 b). Colloid D: The crude product after coupling according to Example 7.6, using Example 1.3 a). Colloid E: Crude product after coupling with Example 1.1 a), according to Example 7.5. Colloid F: The crude product after coupling according to Example 7.2. Glue G: The crude product after coupling according to Example 7.3. Glue K: EPO starting material. Figure 6 Figure 6 shows the SDSPage analysis of HES-EPO conjugates prepared according to Examples 7.7, 7.8, 7.9, 7.10, 7.11 and 7.12. Colloid A: Protein label Roti®-Mark PRESTAINED (Carl Roth GmbH+Co, Karlsruhe, D); molecular weight of the protein label (expressed in kD) from top to bottom: 245, 123, 77, 42, 30, 25.4 and 17 ° Colloid B: Crude product after coupling according to Example 7.11. Colloid C: The crude product after coupling according to Example 7.10. Colloid D: The crude product after coupling according to Example 7.7. -172- This paper size is applicable to China National Standard (CNS) A4 specification (210x297 mm)

1356065 A7 B7 V. Inventive Note (m) Crude product after coupling according to Example 7.8. Crude product EPO starting material after coupling according to Example 7.12. Crude product after coupling according to Example 7.9 < Rubber Road E Plastic Road F G Glue κ Figure 7 SDS_pAGE analyte of Ep〇GT1, which has been incubated for 5 minutes with mild acid, gelatin 2, mild acid treatment for 1 min = gel lane 3; mild acid treatment for 60 min = wins 4 Untreated, 〇 = gel lane ^ shows removal of N· poly _ after ' Ε 〇 迁移 mobility shift (+pNGASE). Round 8 from untreated EPQ & freely cultured for 5 minutes, 10 minutes under mild acid hydrolysis conditions And the oligosaccharide HPAEC-PAD pattern separated by Ep〇 for 60 minutes. Roman numeral ^ indicates the dissolution position of 1 and the acidified biantenna structure; 11 = tri-extension three-antenna structure (two substances) ' 111 = tetradecyanate four Tentacle structure + 2 N_ ethyl lactosamine amine economics intellectual property bureau employee consumption cooperative printing base, IV = tetrazoic acid tetra-antennary structure + 1 N-ethyl-lactylamine repeating group 'V-four-extension acidification Four antennae structure + no N_ ethyl basal milk _ repeating group. oligosaccharide structure without or with 1-4 acid extensions The lytic zone is represented by a chain. After the gull is decapitated, the N_linked oligosaccharide HpAEC_pAD; the display team B 173-X 297 mm) This paper scale applies to the Chinese National Standard (CNS) A4 1356065 A7 B7 Ministry of Economics intellectual property Bureau employee consumption cooperative printing 5, invention description (l72) lysine position of the basal neuraminic acid; number 1-9 indicates the dissolution of the standard oligosaccharide. position; 1 = double antennae; 2 = three antennae (2-4 heterogeneous 3) three antennae (2-6 isomers); 4 = four antennae; 5 = three antennae plus 1 repeat; 6 = four antennae plus 1 repeat; 7 = three antennas plus 2 repeats Base; 8 = four antennae plus 2 repeats and 9 = four antennas plus 3 repeats. Circle 10 has been subjected to mild treatment of peroxyacid oxidation of decanoate residues and SDS-PAGE analysis of untreated EPO. 1 = oxidation by iodate but not acid treatment; 2 = oxidation by iodate and acid treatment for 5 minutes; 3 = oxidation by iodate and acid treatment for 10 minutes; 4 = oxidation by iodate but not acid Treatment; 5 = BRP EPO standard without iodate and without acid treatment. Figure 11 HPAEC-PAD pattern of natural oligosaccharides isolated from untreated EPO and EPO separated by epiudate treatment for 5 minutes and 10 minutes, followed by periodate treatment. The solvation zone having no or 1-4 decanoic acid oligosaccharide structures is indicated by brackets 1-5. Figure 12 SDS-PAG analysis during the HES-modified EPO-GT-1-A time: Several 20 micrograms of EPO-GT-1-A reacted with hydroxylamine-modified HES derivative X for 30 minutes, 2 4 and 17 hours. Rubber Road -174- This paper size is applicable to China National Standard (CNS) A4 specification (210 X 297 mm)

1356065 A7 B7

V. Description of invention (I73) Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1=30 minutes reaction time, rubber lane 2=2 hours reaction time; rubber lane 3=4 hours reaction time, rubber lane 4=17 hour reaction time ; Glue 5 = EPO-GT-1-A without HES modification. The left panel shows that the mobility of EPO-GT-1-a in the presence of a modified amine-modified HES derivative X varies with increasing incubation time (flow rate: 1 ml/min): gel lane 1 = 3 〇 minutes reaction time; gel lane 2 = 2 hours reaction time; gel lane 3 = 4 hours reaction time; gel lane 4 = 17 hours reaction time; rubber lane 5 = HES-modified EPO-GT-1-A. The graph on the right shows the analysis of the same sample after treatment with ν·curonase. Figure 13 sds_pag analysis of the Q-Sepharose fraction of the HES-EPO conjugate. Each 1% flow fraction and 1% fractions dissolved at high salt concentration were concentrated in a Speed Vac concentrator and loaded onto the gel in sample buffer. The EPO protein was stained with Coomassie Blue. A = sample I; sample II, C = sample III; K = control EPO-GT-1; Al, B1, C1 and Κ1 indicate flow-through fractions; Α2, Β2, C2 and Κ2 indicate fractions dissolved at high salt concentration . Figure 14a SDS-PAG analysis of HES modified EPO sample A2 (see Figure 13), control EPO sample K2 and EPO-GT-1-A, wherein the EPO formulations were digested in the presence of N-glycosidase to remove N-linked oligosaccharides. All EPO samples show low mobility or low molecular weight containing 〇-glycan -175- This paper size applies _ National Standard (CNS) A4 (210x297 mm)

1356065 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 B7 invention description (m), state changes. After de-I-glycosylation, the ubiquinone-glycosylated protein band of the EPO sample was reduced by Ms and the diffusion protein band was detected around 30 kDa, presumably indicating that HES modification occurred in On the tannic acid of the 0-glycan residue (see the arrow with the asterisk. Head).闺14b HES-modified EP0 sample A2 (see Figure 13), control EP0 sample K2 and EPO-GT-1-A after mild hydrolysis analysis, where the samples were untreated or already in N-glycosidase Digestion in the presence of N-linked oligosaccharides (see circle 14a). The high molecular weight form of A2 before treatment with N-glycosidase and A after treatment (see brackets with or without arrows) disappeared after the sample was processed. The BRf EP0 standard used by the company is mild acid treatment

囷1S HPAEC-PAD analysis of N-linked oligosaccharide substances released from HES modified samples a, EPO-GT-1-A, and controlled EP0 samples cultured without unmodified HES (K). Roman numeral Ι-V indicates the dissolution position of I = diterpene acidated biantennary structure; 11 = triterpene acidated three antennal structure (two isomers); 111 = tetradecanoate four antenna system + 2 N-acetyl groups Lactosamine repeating group; IV = tetradecanoate tetra-antennary structure + 1 N-ethyl decyl lactosamine repeating group; V = tetradecanoate tetra-antennary structure + no N-acetyl galactosamine repeating group; As indicated in the description of 11, the brackets indicate that the double-acidification _, three-176- paper scale applies the Chinese National Standard (CNS) A4 specification (210x297 mm)'

1356065 A7 B7 V. INSTRUCTIONS (I75) The dissolution zone of the decanoate- and tetradecanoate N-glycans. Figure 16 HPAEC-PAD analysis of N-linked oligosaccharide material released from HES-modified sample A, EPO-GT-1-A, and control EPO sample (K) without unmodified HES. Shows the residence time of the standard oligosaccharide mixture: numbers 1-9 indicate the location of the standard oligosaccharide: 1 = biantenna; 2 = triantennary (2-4 isomers); 3 = triantennary (2-6 different) Structure); 4 = four antennae; 5 = three antennae plus 1 repeat; 6 = four antennae plus 1 repeat; 7 = three antennas plus 2 repeats; 8 = four antennas plus 2 repeats and 9 = four antennas plus 3 repeats. Figures 17 to 23 Printed by the Intellectual Property Office of the Ministry of Economic Affairs, Consumers' Cooperatives, Figures 17 to 23 represent the separation of the enzymes from the HES-modified EPO and the controlled EPO, which have been released by the enzyme and chemically deacidified. MALDI/TOF mass spectrum. The main signal lines at w/zl809.7, 2174.8, 2539.9, 2905.0, and 3270.1 correspond to a two- to four-antennary coupling type Ν-glycan structure having no or one or two fluorenyl-ethyl galactosamine repeat groups. It is accompanied by a weak signal due to the loss of fucose or galactose, where the loss of fucose or galactose is due to deacidification of the sample for acid hydrolysis conditions for MS analysis. Figure 17 MALDI/TOF Spectrogram: Desiccated Acid-177 by HES-Modified EPO A2 This paper scale applies to China National Standard (CNS) A4 specification (210 x 297 mm) Ministry of Economic Affairs Intellectual Property Office staff consumption Cooperatives printed 1356065 A7 B7 V. Description of invention (176) oligosaccharides. Figure 18 MALDI/TOF spectrum: EP0GT-1-A has been de-acidified. Figure 19 MALDI/TOF spectrum: decarboxylated oligosaccharides of EP0K2. Figure 20 MALDI/TOF spectrum: EPO-GT-1 has been de-acidified. Figure 21 MALDI/TOF spectrograms · Demylated oligosaccharides of EPO GT-1 which have been subjected to acid hydrolysis for 5 minutes. Figure 22 MALDI/TOF spectrum: Deionated oligosaccharides of EPO GT-1 which have been subjected to acid hydrolysis for 10 minutes. Figure 23 MALDI/TOF spectrum: Deionated oligosaccharides of EPO GT-1 that have been acid hydrolyzed for 60 minutes. Figure 24-178- This paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm)

1356065 A7 B7 V. INSTRUCTIONS (m) Figure 24 shows SDSPage analysis of two HES-EPO conjugates mw: labeled gel lane 1: HES-EPO prepared according to example experimental procedure 8: EPO coupling to 醯肼_ HES 12KD L Colloid 2: HES-EPO prepared according to the experimental procedure 9: EPO coupling to hydroxylamine-HES 12KD KC: controlled (uncoupled EPO); upper band representing EPO dimer Figure 25 Figure 25 The HES-coupled to the hydrocarbon moiety of the hydrocarbon side chain is demonstrated by a HAS-modified EPO plastic that exhibits digestion with a polypeptide N-glycosidase.

Gel lane 1: HES-EPO gel lane 2 prepared according to the experimental procedure 8 after digestion with N-glycosidase: prepared by N-glycosidase digestion according to the experimental procedure 9

HES-EPO Ministry of Economic Affairs Intellectual Property Bureau Bayer Consumer Cooperative Printed Plastic Road 3: BRPEPO Standard Rubber Road 4: BRPEPO Standard Mw after N-Glycosidase Digestion: Label (Bio-Rad SDS-PAGE Standard Low) Scope Catalog No. 161-0305, Bio-Rad Laboratories, Hercules, CA, USA) -179- This paper size applies to the Chinese National Standard (CNS) A4 specification (210 x 297 mm)

Claims (1)

1356065 Patent application scope A8 B8 C8 D8 Patent application No. 93123642 ROC Patent Application. No. 93123642 I. After the application: seeking for profit _> replacement "Tai-PfW Amended Claims in Chinese - EnclTnn (Republic of China, August, 100 (Submitted on August 22, 2011) A method for producing a hydroxyalkyl starch derivative containing an additional compound (M) having a structure HAS according to formula (1), Η 0) Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperative, Printed, USA, its 1R3 system is independently hydrogen or a straight chain or branched chain alkyl group, and the method includes sucking (a) a base-based base powder in it a non-oxidized reducing end which is reacted with a compound (L) of the formula 1 wherein L' is a bridge & an organic chain with or without L, and wherein the z-line and the base group are non-oxygen a hydroxyalkyl starch derivative obtained by reacting a hydroxyalkyl starch of the formula (I) at a non-oxidative reducing end thereof with a compound (D) of the formula Z^D'-W, a compound of the formula Z2-L, _x (]1) wherein the functional group Z2 is reacted with a functional group W contained in the hydroxyalkyl starch, wherein D' is an organic chain bridging the B and w or wherein D is absent ', and the &<>> reacts with the non-oxidized reducing end of the hydroxyalkyl powder, and wherein L, is an organic chain of bridging enthalpy and X or the lack of l, -180 of this paper scale applies to Chinese national standards ( CNS) A4 specification (210x297 mm) 1356065 Α8 Β8 C8 D8 VI. Patent application scope (i) where the official The group W or the functional group Z2 is -SH, and the functional group Z2 or the functional group W is selected from the group consisting of QN-a_s. ο ο where Hal is α, Br or I, or (ii) wherein the functional group W or The functional group Z2 is selected from the group consisting of an active ester or a carboxyl group which may optionally be converted into an active ester, and the functional group Z2 or the functional group W is selected from the group consisting of HN-H2N Η〆R〆Ν·Η Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, Printed Η G Η2Ν,丫G Ν, Η2Νν ?{ Ν一S— Η II Ο 丫G where G is Ο or S, and if it appears twice, it is independently 0 or S, and R' is methyl, -181 This paper scale is applicable to China National Standard (CNS) A4 specification (210x297 mm) 1356065 A8 B8 C8 D8 VI. Patent application scope to obtain a hydroxyalkyl starch containing functional group X a derivative, wherein ZHf is selected from the group consisting of h2n* H2N \ R, 〆0, ίί H2N 丫G r5 & H2NTf .N. Η2Ν-Νγ°' G ” where G is Ο or S ' and if present Twice, the system is independently 〇 or S, and R' is a methyl group, (b) the method additionally Including reacting a functional group X with a functional group of the other compound (Μ), wherein the functional group is selected from the group consisting of an aldehyde group, a ketone group, a hemiacetal group, and an acetal group, and a functional group thereof X is selected from the group consisting of η2ν- Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, η2ν··κ, FT H, T h2n τ'. -τ where G is 〇 or s, and if it occurs twice, Independently -182 This paper scale applies to the Chinese National Standard (CNS) A4 specification (2ΐ〇χ297 public) 1356065 A8 B8 C8 D8 r The patent application scope is 0 or S, and R' is a methyl group, or the functional group thereof Y is a thiol group and the functional group X is selected from the group consisting of 0 N- as_s Ha, ^ printed by the Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative ο where Hal is α, Br or I, and wherein the additional compound ) is a polypeptide. 2. The method of claim 1, wherein R, R2 and R3 are independently hydrogen or 2-hydroxyethyl. 3. The method of claim 1 or 2 wherein the hydroxyalkyl starch is hydroxyethyl starch. 4. The method of claim 1 or 2, wherein the hydroxyalkyl starch is reacted with the compound (L) via a functional group Z] with a reducing end of the hydroxyalkyl starch, and the resulting reaction product is passed through The reaction of the functional group X contained in the compound (L) with the functional group Y contained in the additional compound (M) is reacted with the compound (M). 5. The method of claim 1 or 2, wherein the hydroxyalkylate-183 paper scale is applicable to the Chinese National Standard (CNS) A4 specification (210x297 mm) 1356065 A8 B8 C8 D8 6. Patent application range The reaction with the reducing end of the hydroxyalkyl starch is carried out via the functional group Z with the compound (L). The compound (L) is reacted with the hydroxyalkyl group before the functional group X contained in the compound (L). The reaction of the functional group γ contained in the additional compound (M) is reacted with the compound (M). 6. The method of claim 1 or 2, wherein the hydroxyalkyl starch is reacted with the compound (D) via a reaction of the functional group Z! contained in the compound (D) with the reducing end of the hydroxyalkyl starch. Obtaining a first hydroxyalkylated starch derivative, and wherein the first hydroxyalkyl starch derivative is reacted with a compound (L) via a functional group Z2 contained in the compound (L) and a functional group W contained in the compound (D) The reaction to obtain a second hydroxyalkyl starch derivative 'and wherein the second hydroxyalkyl starch derivative is reacted via the functional group X contained in the compound (L) with the functional group γ contained in the additional compound (M) Reacts with the compound (M). The method of claim 1 or 2, wherein the hydroxyalkyl starch is reduced by the functional group Ζι contained in the compound (D) and the hydroxyalkyl starch. The reaction at the end is reacted with the compound (D) to obtain a first mercaptoalkyl starch derivative, and the first hydroxyalkyl starch derivative is a functional group contained in the compound (D) and a functional group contained in the compound (L). The reaction of the group Z2 is carried out in the reaction with the compound (L), wherein the compound (1) is contained in the functional group X contained in the compound (L) and the additional compound (M) before being reacted with the first hydroxyalkyl starch derivative. The reaction of the functional group γ is reacted with the compound (M). -184 This paper scale applies to China National Standard (CNS) A4 specification (21〇χ297 mm) 1356065 A8 B8 C8 D8 Six patent application scope 8. The method of claim 1 or 2, wherein the polypeptide is red blood cell generation Prime. A hydroxyalkyl starch derivative comprising an additional compound (M) which can be obtained by a process for producing a hydroxyalkyl starch having a structure according to formula (I). The Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives, prints that R1, R_2, and R>3 are independently hydrogen or a straight or branched bond, and the method includes (a) a hydroxyalkyl starch of formula (I). Reacting with a compound (L) of the formula ZrL'-X at its non-oxidized reducing end, wherein L' is a bridged Z, an organic chain with X or a lack of L' therein, and wherein the Z! and hydroxyalkyl starch a non-oxidative reduction end reaction, or a hydroxyalkyl starch obtainable by reacting a hydroxyalkyl starch of the formula (I) with a compound of the formula Z^D'-W (D) at its non-oxidized reducing end Derivatives, compound (L) anti-185 with formula Z2-L'-X This paper scale is applicable to China National Standard (CNS) A4 specification (210x297 mm) 1356065 A8 B8 C8 D8 VI. The functional group B is reacted with a functional group W contained in the hydroxyalkyl starch, wherein D' is an organic chain bridging acetamethylene and W or a D' is absent therein, and wherein the Zt system and the hydroxyalkyl group are a non-oxidative reduction end reaction, and wherein L' is an organic chain of bridges 22 and hydrazine or L' is absent, (i) wherein the functional group W or the functional group Z2 is -SH, and the functional group Z2 or the functional group W is selected from the group consisting of Hal wherein a is a, Br or I, or (Π) The functional group W or the functional group Z2 is selected from the group consisting of an active ester or a carboxyl group which may be converted into an active ester, and the functional group Z2 or the functional group W is selected from the group of economic ministry of the Ministry of Economics Employee Consumption Cooperative Printed -186 This paper scale applies to China National Standard (CNS) A4 specification (210 X 297 mm) 1356065 A8 B8 C8 D8 VI. Patent application scope HN- Μ, Η〆FT .0. ΗA, / H2N 〆丫G Η Η^ν ?1 〇G υ G G where G is 〇 or S, and if it occurs twice, it is independently 〇 or S, and R is a methyl group to obtain a functional group X a hydroxyalkyl starch derivative, wherein the Ζι is selected from the group consisting of the following: Ministry of Economic Affairs, Intellectual Property Office, employee consumption cooperative, printed Wherein G is 0 or S, and if present twice, is independently 〇 or S, and R' is fluorenyl, (b) the method additionally comprises a functional group of functional group X and the additional compound (M) Y reaction, - wherein the functional group Y is selected from the group consisting of aldehyde group, ketone group, hemiacetal-187 paper size applicable to China National Standard (CNS) A4 specification (210x297 public) 1356065 A8 B8 C8 D8 VI. Patent application scope a group consisting of a base and an acetal group, and wherein the functional group X is selected from the group consisting of 2Ν Ν 2 Η ΗΝ η2ν ο \ R ο Ν 2 Η ^mng ΗΝ onsMO NH \ N 2 Η ΗΝ YG ΗΝ \ Ν 2 Η Ν Η2 ΗΝ G Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed Ha丨fei α_ Wherein G is 0 or S, and if present twice, is independently 0 or S, and R' is methyl, or wherein the functional group Y is a thiol group and the functional group X is selected from the group consisting of 0 N 〇 wherein Hal is a, Br or I, and wherein the additional compound (Μ) is a polypeptide. 10. The hydroxyalkyl starch derivative of claim 9, wherein Ri, R2 and R3 are independently wind or 2-ylethyl. -188 This paper size is applicable to China National Standard (CNS) A4 specification (210x297 mm) 1356065 A8 B8 C8 D8 VI. Patent application scope 11. For the hydroxyalkyl starch derivative of claim 9 or 10, The hydroxyalkyl starch is hydroxyethyl starch. 12. A hydroxyalkyl starch derivative as claimed in claim 9 or 1 wherein the hydroxyalkyl starch is via the functional group! The reaction with the reducing end of the hydroxyalkyl starch is reacted with the compound (L), and the resulting reaction product is reacted via the functional group X contained in the compound (L) with the functional group γ contained in the additional compound (M). Compound (M) is reacted. 13. The hydroxyalkyl starch derivative as claimed in claim 9 or 1 wherein the hydroxyalkyl starch is reacted with the compound (L) via a reaction of a functional group with a reducing end of the hydroxyalkyl starch, wherein The compound (L) is reacted with the compound (M) via the reaction of the functional group X contained in the compound with the functional group Y contained in the additional compound (M) before the reaction with the hydroxyalkyl starch. Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumers' Cooperatives 14. For the hydroxyalkyl starch derivative of the ninth or first aspect of the patent application, wherein the hydroxyalkyl starch is via the functional group Z contained in the compound (〇) The reaction of the reducing end of the base group powder is reacted with the compound (D) to obtain a first hydroxyalkyl starch derivative, and the first transalkylidene starch derivative is a functional group contained in the compound (L). The reaction of Z2 with the functional group W contained in the compound (D) is reacted with the compound (L) to obtain a second hydroxyalkyl starch derivative, and wherein the second -189 paper size is applicable to the Chinese National Standard (CNS) A4 specification. (Ex. X 297 public) 1356065 Six patent application scope 8 8 8 8 AB c D base alkyl starch derivative via functional group χ contained in compound (L) and functional group γ contained in the other compound (Μ) The reaction with the compound (Μ). The hydroxyalkyl starch derivative according to claim 9 or claim 1, wherein the hydroxyalkyl starch is reacted with the functional group Z contained in the compound (D) and the reducing end of the hydroxyalkyl starch. Reacting with the compound (D) to obtain a first hydroxyalkyl starch derivative, and the first hydroxyalkyl starch derivative is via the functional group W contained in the compound (D) and the functional group Z2 contained in the compound (L) The reaction with the compound (L) wherein the compound (L) is subjected to the functional group X contained in the compound (L) and the functional group contained in the additional compound (M) before the reaction with the first hydroxyalkyl starch derivative The reaction of γ is reacted with the compound (M). 16. The hydroxyalkyl starch derivative according to claim 9 or 10, wherein the polypeptide is erythropoietin. The Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, printed a pharmaceutical composition comprising a therapeutically effective amount of a hydroxyl group comprising an additional compound (M) obtainable by a process for producing a hydroxyalkyl temple powder derivative. Alkyl starch derivative, the hydroxyalkyl starch has a structure according to formula (I) -190 This paper scale is applicable to China National Standard (CNS) A4 specification (210x297 mm) 1356065 A8 B8 C8 D8 VI. Patent application scope The Ministry of Education, Ministry of Education, Intellectual Property Office, and the Consumer Cooperatives, in which R!, R2 and R3 are independently hydrogen or a straight chain or a branched chain, the method comprising (a) a hydroxyalkyl starch of formula (I) Reacting with a compound (L) of the formula ZrL'-X at its non-oxidized reducing end, wherein L' is an organic chain bridging acetamidine with X or lacking L' therein, and wherein Z is a transalkyl group a non-oxidative reducing end reaction of starch, or a hydroxyalkylated starch obtained by reacting a hydroxyalkyl starch of the formula (I) with a compound of the formula ZrD'-W (D) at its non-oxidized reducing end And reacting with a compound (L) of the formula Z2-L'-X, wherein the functional group 2 is reacted with a functional group W contained in the hydroxyalkyl starch, wherein D' is a bridge B1 and W An organic chain or a lack of D' therein, and wherein the Z! system reacts with a non-oxidative reducing end of the hydroxyalkyl starch, and wherein L' is an organic chain bridging Z2 and X or wherein L' is absent, (i) wherein The functional group W or the functional group Z2 is -SH, and the functional group Z2 or the functional group W is selected from the group consisting of -191 The scale applies to the Chinese National Standard (CNS) A4 specification (210x297 mm) (3) 6065 ', the application scope of the patent A8 B8 C8 D8 P N- η^Ύ >c° ο where Hal is Q, Br or I, or (ii) The functional group W or the functional group Z2 is selected from the group consisting of an active ester or a carboxyl group which may be converted into an active ester, and the functional group Z2 or the functional group W is selected from the group consisting of H2N-h2n Η〆Ό R R R R R R R R R R R R R R R R R R Is Ο or S, and R' is a methyl group to obtain a hydroxyalkyl starch derivative containing a functional group X, wherein the acetamidine is selected from the group consisting of: 192. This paper scale applies to the Chinese National Standard (CNS) A4. Specifications (210x297 mm) 1356065 A8 B8 C8 η η2ν"ν\ Η2Ν,0\ r,/〇,n Η Η <丫G Η . w Η2Ν,Νγ^\ G where G is 〇 or s, and if it appears twice, it is independently 〇 or S, and R' is a fluorenyl group, (b) the method additionally comprises reacting a functional group hydrazine with a functional group Y of the additional compound (M), wherein the functional group γ is selected from the group consisting of a thiol group, a ketone group, and a semi-shrinkage a group consisting of a base and an acetal group, and wherein the functional group X is selected from the group consisting of H2N-HjN Η〆R·〆0, Η, NT r h2n, H2N* NSh printed by the Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative No HV3, G where G is 〇 or S, and if it occurs twice, it is independently 〇 or S, and R' is sulfhydryl, or -193 This paper scale applies to China National Standard (CNS) A4 specification (210x297 1356065 Α8 Β8 C8 D8 The scope of the patent application wherein the functional group Y is a thiol group and the functional group X is selected from the group consisting of 0 Printed by the Ministry of Economic Affairs, Intellectual Property Office, Staff Consumer Cooperative, where Hal is a, Br or I, and wherein the additional compound (Μ) is a polypeptide. 18. The pharmaceutical composition of claim 17, wherein the polypeptide is antithrombin type III (AT III). 19. The pharmaceutical composition of claim 17, wherein the polypeptide is erythropoietin. 20. The pharmaceutical composition according to claim 17, wherein the hydroxyalkyl starch is reacted in an aqueous medium with a compound (L) according to the following formula and the reaction product is reacted with a compound (Μ), wherein Μ) is erythropoietin. -194 This paper size is applicable to China National Standard (CNS) A4 specification (210x297 mm) 1356065 A8 B8 C8 D8 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing VI. Patent application scope 21. For example, the patent application scope 20 medicine A composition wherein the erythropoietin is oxidized by sodium iodate prior to the reaction. 22. The pharmaceutical composition of claim 20, wherein the erythropoietin is partially de-acidified prior to the reaction and then oxidized by sodium permanganate. -195 This paper size applies to China National Standard (CNS) A4 specification (210x297 mm)