Process for preparation of physiologically active polypeptide complex

Abstract

Disclosed is a method for the preparation of a complex in which a physiologically active polypeptide is covalently bonded to an immunoglobulin constant region via a non-peptidyl linker. The method is characterized by the employment of a reducing agent, by which conventional problems of low production yield and modification of the polypeptide can be overcome. The physiologically active polypeptide-non-peptidyl polymer-immunoglobulin constant region complex can be produced with high purity and yield as well as at low cost. Thus, the method is industrially useful. Moreover, by exhibiting a prolonged action profile, the physiologically active polypeptide-non-peptidyl polymer-immunoglobulin constant region complex can be effectively used for developing long-acting formulations of physiologically active polypeptides which have improved drug compliance.

Claims

1. A method for preparing a complex of insulin - polyethylene glycol-immunoglobulin Fc region, comprising: (1) reacting a polyethylene glycol having two aldehydes as functional groups with an insulin in the presence of a reducing agent at a concentration of from 2 to 20 mM and an alcohol; and (2) reacting the reaction mixture of step (1) with an immunoglobulin Fc region in the presence of a reducing agent at a concentration of 5 to 40 mM and an alcohol, wherein the reaction in step (1) is carried out for 1 to 16 hrs at a temperature of 4° C. to room temperature at pH 6.0, the reaction in step (2) is carried out for 13 to 43 hrs at a pH of 8.2, wherein the reducing agent in step (1) and the reducing agent in step (2) are sodium cyanoborohydride (SCB), and wherein the alcohol in step (1) is 45% (v/v) isopropanol and the alcohol in step (2) is 10% (v/v) ethanol.

2. The method of claim 1, further comprising separating a conjugate of the insulin-polyethylene glycol from the reaction mixture after step (1).

3. The method of claim 1, wherein the reducing agents used in steps (1) and (2) function to reduce a reversible imine double bond produced from bonding between the aldehyde group of the polyethylene glycol and an amine group of the insulin or the immunoglobulin Fc region to form a covalent bond.

4. The method of claim 1, wherein the polyethylene glycol is covalently bonded to each of the insulin and the immunoglobulin Fc region through the two aldehyde functional groups thereof.

5. The method of claim 1, wherein the functional groups of the polyethylene glycol are bonded to each of an amine group of the insulin and the immunoglobulin Fc region, wherein the amine group is present at an N-terminus or on a side chain of Lys residue.

6. The method of claim 1, wherein molecular weight of the polyethylene glycol ranges from 1 to 100 kDa.

7. The method of claim 1, wherein the immunoglobulin Fc region is aglycosylated.

8. The method of claim 1, wherein the immunoglobulin Fc region consists of one to four domains selected from the group consisting of C.sub.H1, C.sub.H2, C.sub.H3 and C.sub.H4 domains.

9. The method of claim 1, wherein the immunoglobulin Fc region further comprises a hinge region.

10. The method of claim 1, wherein the immunoglobulin Fc region is selected from the group consisting of constant regions derived from IgG, IgA, IgD, IgE, IgM, or combinations or hybrids thereof.

11. The method of claim 1, wherein the immunoglobulin Fc region is selected from the group consisting of constant regions of IgG1, IgG2, IgG3, IgG4, a combination thereof, and a hybrid thereof.

12. The method of claim 1, wherein the immunoglobulin Fc region is an IgG4 Fc region.

13. The method of claim 12, wherein the immunoglobulin Fc region is an aglycosylated human IgG4 Fc region.

14. The method of claim 1, wherein the step (2) is carried out in the presence of the reducing agent at a concentration of 20 mM.

15. A method for preparing a complex of insulin-polyethylene glycol-immunoglobulin Fc region, comprising: (1) reacting a polyethylene glycol having two aldehydes as functional groups with an insulin in the presence of a reducing agent at a concentration of from 2 to 20 mM and an alcohol; (2) isolating a complex of the polyethylene glycol and the insulin from reaction mixture of (1); (3) reacting the isolated complex of step (2) with the immunoglobulin Fc region in the presence of a reducing agent at a concentration of 5 to 40 mM and an alcohol, wherein the reducing agent in step (1) and the reducing agent in step (3) are sodium cyanoborohydride (SCB); wherein the step (1) is carried out for 1 to 16 hrs at a temperature of 4° C. to room temperature at a pH of 6.0, and the reaction in step (3) is carried out at a pH of 8.2 for 13-43 hrs at a temperature of 25° C., and wherein the alcohol in step (1) is 45% (v/v) isopropanol and the alcohol in step (3) is 10% (v/v) ethanol.

Description

MODE FOR THE INVENTION

(1) A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention.

Example 1

PEGylation of Insulin Using Sodium Cyanoborohydride as Reducing Agent and Purification of Mono-PEGylated Insulin

(2) Insulin powder was dissolved in 10 mM HCl, and PEGylated at the N-terminus of the beta chain with 3.4K propion-ALD2 PEG (PEG with two propionaldehyde groups, IDB, Korea). In this regard, 5 mg/ml insulin was reacted with PEG at a molar ratio of 1:2 at 4° C. to room temperature for 2 hrs. The reaction was performed in 50 mM sodium citrate buffer at pH 6.0 in 45% isopropanol in the presence of 2-20 mM sodium cyanoborohydride as a reducing agent. The reaction mixture was loaded onto an SP-HP (GE Healthcare) column, followed by eluting with a buffer containing sodium citrate (pH 3.0) and 45% EtOH, and using a concentration gradient of KCl to purify mono-PEGylated insulin.

(3) PEGylation yields of insulin according to conditions of the reducing agent sodium cyaborohydride during the preparation of a complex comprising the insulin and the immunoglobulin Fc region are summarized in Table 1, below.

Example 2

Changes in Production Yields of a Complex of Mono-PEGylated Insulin-Immunoglobulin Fc Region According to Conditions of Sodium Cyanoborohydride as Reducing Agent Used in PEGylation

(4) To examine the production yield of the insulin-PEG-immunoglobulin Fc region complex, the mono-PEGylated insulin prepared in Example 1 was reacted with at a molar ratio of 1:1 with an immunoglobulin Fc at 25° C. for 13 hrs, with the total protein concentration set to be 20 mg/ml. This coupling reaction was carried out in 100 mM HEPES buffer containing 22 mM potassium phosphate and 10% ethanol at pH 8.2, in the presence of 20 mM sodium cyanoborohydride as a reducing agent.

(5) The reaction mixture was loaded onto a Source 15Q (GE Healthcare) column, followed by eluting with Tris-HCl (pH 7.5) buffer and using a concentration gradient of NaCl to separate and purify unreacted insulin, unreacted immunoglobulin Fc region, an insulin-PEG-immunoglobulin Fc region complex, and an immunoglobulin Fc region coupled with two or more mono-PEGylated insulin (insulin-PEG) moieties. Production yields of the insulin-PEG-immunoglobulin Fc region complex were determined UV absorbance at 280 nm after purification by chromatography.

(6) In Table 1, yields of the coupling reaction with an immunoglobulin Fc region are summarized according to conditions of sodium cyanoborohydride used as a reducing agent in the PEGylation of insulin.

(7) TABLE-US-00002 TABLE 1 Conc. of Sodium cyanoboro- RxnTi Rxn PEGylation Coupling Total hydride me Temp. yield (%) yield (%) yield (%) 2 mM 2 hr 4° C. 23.3 31.7 7.39 4 mM 2 hr 4° C. 37.6 31 11.66 4 mM 2 hr RT 39.2 28.8 11.29 8 mM 2 hr RT 40.4 27.1 10.95 8 mM 4 hr 4° C. 40.4 27 10.9 20 mM  2 hr 4° C. 42.2 26.8 11.3

Example 3

PEGylation of Insulin Using Borane Pyridine Complex as Reducing Agent and Purification of Mono-PEGylated Insulin

(8) Insulin powder was dissolved in 10 mM HCl, and PEGylated at the N-terminus of the beta chain with 3.4K propion-ALD2 PEG (PEG with two propionaldehyde groups, IDB, Korea). In this regard, 5 mg/ml insulin was reacted with PEG at a molar ratio of 1:2 at 4° C. for 2 hrs. The reaction was performed in 50 mM sodium citrate buffer at pH 6.0 in 45% isopropanol in the presence of 3-20 mM borane pyridine complex as a reducing agent. The reaction mixture was loaded onto an SP-HP (GE Healthcare) column, followed by eluting with a buffer containing sodium citrate (pH 3.0) and 45% EtOH, and using a concentration gradient of KCl to purify mono-PEGylated insulin.

(9) PEGylation yields of insulin according to conditions of the reducing agent borane pyridine complex during the preparation of a complex comprising the insulin and the immunoglobulin Fc region are summarized in Table 2, below.

Example 4

Changes in Production Yields of a Complex of Mono-PEGylated Insulin-Immunoglobulin Fc Region According to Conditions of Borane Pyridine Complex as Reducing Agent Used in PEGylation

(10) To examine the production yield of the insulin-PEG-immunoglobulin Fc region complex, the mono-PEGylated insulin prepared in Example 3 was reacted with at a molar ratio of 1:1 with an immunoglobulin Fc at 25° C. for 13 hrs, with the total protein concentration set to be 20 mg/ml. This coupling reaction was carried out in 100 mM HEPES buffer containing 22 mM potassium phosphate and 10% ethanol at pH 8.2, in the presence of 20 mM sodium cyanoborohydride as a reducing agent.

(11) The reaction mixture was loaded onto a Source 15Q (GE Healthcare) column, followed by eluting with Tris-HCl (pH 7.5) buffer and using a concentration gradient of NaCl to separate and purify unreacted insulin, unreacted immunoglobulin Fc region, an insulin-PEG-immunoglobulin Fc region complex, and an immunoglobulin Fc region coupled with two or more mono-PEGylated insulin (insulin-PEG) moieties. Production yields of the insulin-PEG-immunoglobulin Fc region complex were determined UV absorbance at 280 nm after purification by chromatography.

(12) In Table 2, yields of the coupling reaction with an immunoglobulin Fc region are summarized according to conditions of borane pyridine complex used as a reducing agent in the PEGylation of insulin.

(13) TABLE-US-00003 TABLE 2 Conc. of Borane pyridine PEGylation yield Coupling Total complex (%) yield (%) yield (%)  3 mM 25.4 35.1 8.92 10 mM 47.6 34.8 16.6 20 mM 50.8 34.2 17.4

Example 5

Yields of Coupling Reaction and Formation of Immunoglobulin Fc Aberrant According to Concentration of Sodium Cyanoborohydride and Reaction Time

(14) To examine the formation of immunoglobulin Fc aberrant according to the concentrations of the reducing agent, and reaction times in the coupling reaction, the mono-PEGylated insulin was reacted at a molar ratio of 1:1 with an immunoglobulin Fc at 25° C. for 13-43 hrs, with the total protein concentration set to be 20 mg/ml. This coupling reaction was carried out in 100 mM HEPES buffer containing 22 mM potassium phosphate and 10% ethanol, pH 8.2, in the presence of 5-40 mM sodium cyanoborohydride.

(15) The reaction mixture was loaded onto a Source 15Q (GE Healthcare) column, followed by eluting with Tris-HCl (pH 7.5) buffer and using a concentration gradient of NaCl to separate and purify unreacted insulin, unreacted immunoglobulin Fc region, an insulin-PEG-immunoglobulin Fc region complex, and an immunoglobulin Fc region coupled with two or more mono-PEGylated insulin (insulin-PEG) moieties. Production yields of the insulin-PEG-immunoglobulin Fc region complex were determined UV absorbance at 280 nm after purification by chromatography.

(16) In Table 3, yields of the coupling reaction to prepare a complex comprising the insulin and the immunoglobulin Fc region are summarized according to concentrations of sodium cyanoborohydride used as a reducing agent, and reaction times in the coupling reaction.

(17) TABLE-US-00004 TABLE 3 Rxn Time 5 mM SCB 20 mM SCB 40 mM SCB 13 hrs 35.2% 37.5% 37.5% 18 hrs 36.1% 37.7% 37.7% 37 hrs 36.7% 37.3% 37.5% 43 hrs 36.8% 37.2% 36.8%

(18) The formation of immunoglobulin Fc aberrants according to the concentrations of the reducing agent in the coupling reaction was monitored by LC on a Propac SAX-10 (DIONEX) column eluting with Tris-HCl (pH 8.0) buffer and using a concentration gradient of NaCl.

(19) In Table 4, production yields of immunoglobulin Fc aberrants are given according to concentrations of sodium cyanoborohydride and reaction times in the coupling reaction to prepare a complex comprising the insulin and the immunoglobulin Fc region.

(20) TABLE-US-00005 TABLE 4 Rxn Time 5 mM SCB 20 mM SCB 40 mM SCB 13 hrs 4.6% 7.0% 7.8% 18 hrs 6.2% 9.0% 9.8% 37 hrs 11.7% 14.5% 15.3% 43 hrs 12.7% 15.5% 16.8%

Example 6

PEGylation of Immunoglobulin Fc Using Sodium Cyanoborohydride as Reducing Agent and Purification of Mono-PEGylated Immunoglobulin Fc

(21) The N-terminus of immunoglobulin Fc was PEGylated with 5K propion-ALD2 PEG (PEG with three propionaldehyde groups, NOF, Japan). In this regard, 10 mg/ml immunoglobulin Fc was reacted with PEG at a molar ratio of 1:2 at 4° C. to room temperature for 4.5 hrs. The reaction was performed in 100 mM potassium phosphate buffer at pH 6.0 in the presence of 20 mM sodium cyanoborohydride as a reducing agent. The reaction mixture was loaded onto a Source 15Q column, followed by eluting with Tris-HCl (pH 7.5) buffer and using a concentration gradient of NaCl to purify mono-PEGylated immunoglobulin Fc.

Example 7

Preparation of a Complex of Mono-PEGylated Immunoglobulin Fc Region-Insulin Using Sodium Cyanoborohydride as Reducing Agent

(22) To prepare an insulin-PEG-immunoglobulin Fc region complex, the mono-PEGylated immunoglobulin Fc prepared in Example 6 was reacted with at a molar ratio of 1:4 with an insulin at 4° C. for 13 hrs, with the total protein concentration set to be 20 mg/ml. This coupling reaction was carried out in 100 mM potassium phosphate buffer at pH 6.0 in the presence of 20 mM sodium cyanoborohydride as a reducing agent.

(23) The reaction mixture was loaded onto a Source 15Q (GE Healthcare) column for the primary purification. And the secondary purification was additionally performed with a Source 15ISO (GE Healthcare) column to obtain an insulin-PEG-immunoglobulin Fc region complex.