KR101974305B1 - Method for preparation of biological active polypeptide conjugate - Google Patents

Abstract

The present invention relates to a physiologically active polypeptide and a non-peptide polymer linked by a covalent bond and a non-peptide polymer conjugate using an organic solvent in order to improve persistence and stability of the physiologically active polypeptide in vivo And a method for producing a physiologically active polypeptide conjugate having a carrier bound thereto. The physiologically active polypeptide and the non-peptide polymer conjugate can be produced with high purity and yield by the production method of the present invention, and the physiologically active polypeptide-conjugate prepared using the same can reduce the manufacturing cost, And the half-life of blood is remarkably increased, which can be usefully used for the development of sustained-release formulations of physiologically active polypeptides that can increase patient compliance with medicines.

Description

Method for preparation of biologically active polypeptide conjugate}

The present invention is a physiologically active polypeptide and a non-peptide polymer linker in which a physiologically active polypeptide and a non-peptide polymer are covalently linked using an organic solvent in order to improve the in vivo persistence and stability of the physiologically active polypeptide. , And to a method of preparing a physiologically active polypeptide conjugate in which a carrier is bound to the linker.

In general, because bioactive polypeptides have low stability and are easily denatured and are easily degraded by proteolytic enzymes in the blood and easily removed through the kidneys or liver, the concentration in the blood of protein drugs containing bioactive polypeptides as pharmacological components and Protein drugs need to be administered frequently to patients to maintain potency. However, in the case of protein drugs, which are mostly administered to patients in the form of injections, frequent injections in order to maintain the blood level of the active polypeptide cause tremendous pain to the patient. In order to solve this problem, efforts have been made to increase the blood stability of protein drugs and to maximize drug efficacy by maintaining high blood drug concentrations for a long time. Such long-acting preparations of protein drugs should increase the stability of the protein drugs, and at the same time, the potency of the drug itself should be maintained sufficiently high and should not induce an immune response in the patient.

As a method for stabilizing proteins and suppressing contact with proteolytic enzymes and kidney loss, conventionally, a polymer with high solubility, such as polyethylene glycol (hereinafter abbreviated as “PEG”), is chemically applied to the protein drug surface. The method of adding has been used. PEG is known to be effective in stabilizing proteins by increasing solubility by non-specifically binding to a specific site or various sites of the target protein, preventing hydrolysis of the protein, and not causing any special side effects (Sada et al., J. Fermentation Bioengineering 71: 137-139, 1991). However, the stability of the protein may be increased by binding of PEG, but the titer of the bioactive protein is significantly lowered, and as the molecular weight of PEG increases, the reactivity with the protein decreases, resulting in a decrease in yield. Accordingly, the present inventors have provided a conjugate in which a physiologically active polypeptide and a carrier are linked to a non-peptide polymer. However, there is still an increasing demand for a method of manufacturing the conjugate with high yield and high purity, and there is a disadvantage in that the method of first binding a non-peptide polymer to a carrier, which is a conventional method, is expensive.

On the other hand, insulin is a peptide secreted from beta cells of the human pancreas and plays a very important role in regulating blood sugar in the body. When the insulin is not secreted properly or the secreted insulin does not function properly in the body, blood sugar in the body cannot be controlled and rises, and this condition is called diabetes. The case mentioned above is called type 2 diabetes, and the case where blood sugar rises due to the inability to secrete insulin from the pancreas is called type 1 diabetes.

In the case of type 2 diabetes, an oral hypoglycemic agent based on a chemical substance is used for treatment, and some patients are treated with insulin. On the other hand, in the case of type 1 diabetes, the administration of insulin is essentially required.

Insulin therapy, which is currently widely used, is a method of administering insulin through injections before and after meals. However, this insulin therapy causes a lot of pain and discomfort to patients because it must be used continuously three times a day. Various attempts have been made to overcome this problem, and as one of them, there has been an attempt to deliver the peptide drug into the body by inhalation through the oral cavity or nasal cavity by increasing the biomembrane permeability of the peptide drug. However, this method has significantly lower efficiency of delivery of the peptide to the body compared to injections, and thus, there are still many difficulties in maintaining the activity of the peptide drug in the body under the required conditions.

On the other hand, there was a method of delaying absorption after subcutaneous administration of an excessive amount of the drug, and through this, there was a method of maintaining a continuous blood concentration by administering once a day. Some of them have been approved as medicines (Lantus, Sanofi-aventis) and are currently being administered to patients. In addition, a study was conducted to strengthen the binding of insulin polymer by modifying fatty acids in insulin and to increase the duration by binding with albumin in the administration site and blood, and it was approved as a drug (Levemir, NovoNordisk). However, this method has a side effect of causing pain at the administration site, and it is still a burden to the patient due to the administration interval through injection once a day.

On the other hand, after the peptide drug is absorbed into the body, efforts have been made to increase the stability in the blood of the peptide drug and to maximize the drug efficacy by maintaining a high blood drug concentration for a long time. Such long-acting formulations of the peptide drug improve the stability of the peptide drug. At the same time, the potency of the drug itself should be kept high enough and should not induce an immune response in the patient. As a long-acting formulation of such a peptide drug, a method of chemically adding a highly soluble polymer material such as polyethylene glycol (PEG) to the surface of the peptide has been used.

PEG is effective in inhibiting loss by kidney and preventing hydrolysis by increasing the molecular weight of the peptide by non-specific binding to a specific site or various sites of the target peptide, and does not cause any special side effects. For example, WO2006/076471 binds to NPR-A and activates the production of cGMP to reduce blood pressure in the arteries, and thus B-type natriuretic peptide is used as a treatment for congestive heart failure. peptide, BNP) to sustain physiological activity, and US 6,924,264 describes a method of increasing the duration in vivo by binding PEG to the lysine residue of exendin-4. However, this method can increase the PEG molecular weight to extend the in vivo duration of the peptide drug, while increasing the molecular weight significantly lowers the potency of the peptide drug, and also lowers the reactivity with the peptide, resulting in a decrease in yield. .

WO 02/46227 describes a fusion protein of GLP-1 and exendin-4 or its analogs using recombinant gene technology and human serum albumin or immunoglobulin fragments (Fc), and US 6,756,480 describes parathyroid hormone (PTH) And the analogue and the Fc fusion protein are described. While this method can overcome the problem of low pegylation yield and non-specificity, the effect of increasing the blood half-life is not as drastic as expected, and in some cases, the potency is low. In order to maximize the effect of increasing the blood half-life, various kinds of peptide linkers are sometimes used, but there is a possibility that an immune response may be induced. In addition, when a peptide having a disulfide bond such as BNP is used, the probability of misfolding is high, making it difficult to apply, and when there are amino acid residues that do not exist in nature, it is impossible to produce it in the form of genetic recombination. There is a problem.

Under this background, the present inventors have made diligent efforts to find a method that can simultaneously increase the blood half-life of a physiologically active polypeptide including insulin and maintain its activity in vivo. As a result, the physiologically active polypeptide and the physiologically active polypeptide in a reaction solution containing an organic solvent If the non-peptide polymer is first linked, the manufacturing cost is reduced, and it is confirmed that the bioactive polypeptide and the non-peptide polymer linker can be produced with high yield and purity. In this case, the present invention was completed by confirming that the in vivo activity was maintained high and showed a significantly improved effect of increasing blood half-life than the known inframe fusion method.

One object of the present invention is to provide a method for preparing a physiologically active polypeptide and a non-peptide polymer linker in order to prolong the half-life in blood while maintaining the in vivo activity of the physiologically active polypeptide.

Another object of the present invention is to provide a method for preparing a physiologically active polypeptide conjugate in which a carrier is covalently linked to the physiologically active polypeptide and a non-peptide polymer conjugate.

As an aspect for achieving the above object, the present invention provides a step of (1) reacting a physiologically active polypeptide and a non-peptide polymer to be linked in a reaction solution containing an organic solvent; And (2) separating and purifying a physiologically active polypeptide conjugate to which a non-peptide polymer is covalently bonded from the reaction mixture of step (1). Provides a way.

In the present invention, the organic solvent means a liquid organic compound capable of dissolving solids, gases, and liquids, and for the purposes of the present invention, a non-peptide polymer is covalently bonded to the N-terminus of a physiologically active polypeptide in high yield and purity. In order to be able to, it may be included in the reaction solution.

As the organic solvent, all organic solvents commonly used in the art may be used, but are not limited thereto, but preferably all primary, secondary, and tertiary alcohols may be used, and alcohols having 1 to 10 carbon atoms may be used. It may, more preferably, the alcohol may be isopropanol, ethanol or methanol. As the organic solvent, a suitable organic solvent may be freely selected according to the type of physiologically active polypeptide.

The organic solvent is included in the reaction solution for linking between the physiologically active polypeptide and the non-peptide polymer, but is not limited thereto, but may preferably be included in an amount of 10 to 60% based on the total amount of the reaction solution, and more preferably 30 To 55%, even more preferably 45 to 55%. In addition, the pH of the reaction solution is not limited thereto, but may be preferably 4.5 to 7.0, more preferably 5.5 to 6.5. At this time, in the reaction solution having a weakly acidic pH, the physiologically active polypeptide generally has a low solubility, but when the organic solvent of the present invention is used, the solubility problem is improved to facilitate the reaction. There is this.

In one embodiment of the present invention, in order to selectively bind PEG to the N-terminus of the beta chain of insulin, isopropanol as an organic solvent was included in the reaction solution, and the PEGylation reaction of insulin was performed at various pHs, and the organic solvent was reacted. When included in the solution, it was confirmed that various kinds of impurities can be efficiently reduced to prepare a physiologically active polypeptide and a non-peptide polymer linker with high purity and yield (Table 1). High purity physiologically active polypeptide conjugates can be prepared. In an embodiment of the present invention, the content of isopropanol and the pH of the reaction solution may change the content of impurities and the ratio of mono-PEGylated insulin, especially when isopropanol is 45 to 55% based on the total amount of the reaction solution at pH 6.0. , It was confirmed that the content of mono-PEGylated insulin is the maximum (Table 1).

In the present invention, the term "bioactive polypeptide" is a generic term for a polypeptide having a certain physiological action in a living body, has a common point of having a polypeptide structure, and has various physiological activities. The physiological activity regulates genetic expression and physiological function to correct an abnormal condition due to a lack or excessive secretion of a substance involved in function regulation in the living body, and may include general protein therapeutics. .

In the present invention, the physiologically active polypeptide includes without limitation, as long as it has physiological activity in vivo, examples of which include insulin, luteinizing hormone (LTH), follicle stimulating hormone (FSH), blood coagulation factor 8 (Factor VIII), There are blood coagulation factor VII, adiponectin, antibody, antibody fragment scFv, Fab, Fab', F(ab')2 or relaxin, preferably insulin. have. The physiologically active polypeptide may have two or more N-terminals (amino termini).

In the present invention, the insulin is secreted by the pancreas when blood sugar in the body is high, absorbs sugar from liver, muscle, and adipose tissue, stores it as glycogen, and controls blood sugar by inhibiting it from being used as an energy source by decomposing fat. Magnetism is a peptide. Such peptides include insulin agonists, precursors, derivatives, fragments, variants, and the like, preferably natural insulin, fast-acting insulin, long-acting insulin, and the like. .

Natural insulin is a hormone secreted by the pancreas, and generally plays a role in regulating blood sugar in the body by promoting the absorption of glucose in cells and inhibiting the breakdown of fat. Insulin is processed in the form of a precursor of proinsulin that does not have a glycemic control function, and becomes insulin with a glycemic control function. The insulin amino acid sequence is as follows.

Alpha chain:

Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr-Gln-Leu-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO: 1)

Beta Chain:

Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe- Tyr-Thr-Pro-Lys-Thr (SEQ ID NO: 2)

Insulin agonist refers to a substance that exhibits the same biological activity as insulin by binding to a receptor of insulin in vivo regardless of the structure of insulin.

Insulin derivatives show homology in amino acid sequence at least 80% or more compared to natural insulin, and some groups of amino residues are chemically substituted (e.g., alpha-mthylatio, alpha-hydroxylation), elimination (e.g., deamination) or It can be in the form of modified (eg, N-methylation, glycosylation, fatty acid), and refers to a peptide that has the function of regulating blood sugar in the body.

Insulin fragment refers to a form in which one or more amino acids are added or deleted at the amino terminal or carboxy terminal of insulin, and the added amino acid may be an amino acid that does not exist in nature (eg, D-type amino acid), and such insulin fragment Retains the function of regulating blood sugar in the body.

Insulin variant refers to a peptide having at least one amino acid sequence different from that of insulin, and having a function of regulating blood sugar in the body.

The preparation methods used in each of the insulin agonists, derivatives, fragments, and variants can be used independently or in combination. For example, peptides having one or more different amino acid sequences and having a function of regulating blood sugar in the body that are deaminated at the N-terminal amino acid residue are also included.

As a specific aspect, the insulin used in the present invention may be produced through a recombinant method, and may also be produced by a method synthesized through a solid phase synthesis method.

In addition, the insulin used in the present invention may be characterized in that a non-peptidyl polymer is bound to the N-terminus (amino terminal) of the beta chain. In the case of insulin, when the alpha chain is modified, the activity decreases and the safety is poor. Therefore, in the present invention, by linking a non-peptide polymer to the N-terminus of the beta chain of insulin, the safety is improved while maintaining the activity of insulin.

In the present invention, the term "coagulation factor" refers to a blood coagulation-related protein that acts to protect the body through blood coagulation in case of bleeding due to a wound, and blood coagulation involves 12 factors included in these coagulation factors. It is a series of reactions that occur. Among them, factor 8 of blood coagulation is also called anti-hemophilia factor, and it is a factor that causes hemophilia in case of genetic deficiency, and factor 7 of blood coagulation is also called Proconvertin, and is a blood coagulant that can activate and cause blood coagulation. It is an argument that can be used as

In the present invention, the term "adiponectin" refers to a protein secreted from adipocytes and is a substance involved in fat and sugar metabolism, reducing the risk of heart disease and diabetes in adulthood, and allowing the muscles to convert fat into energy. It is a protein known to suppress appetite. In addition, the term "Relaxin" refers to a hormone that is secreted from the ovarian corpus luteum to relax pubic bonds and promote childbirth, and is a hormone that plays a role in gently relaxing bones and joints of the whole body.

In the present invention, "non-peptide polymer" refers to a biocompatible polymer in which two or more repeat units are bonded, and the repeat units are linked to each other through an arbitrary covalent bond, not a peptide bond.

Non-peptide polymers usable in the present invention include polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, PLA ( It may be selected from the group consisting of biodegradable polymers such as polylactic acid, polylactic acid) and PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof, preferably polyethylene It is glycol. Derivatives thereof already known in the art and derivatives that can be easily prepared at the technical level in the art are also included in the scope of the present invention.

The disadvantage of the peptide linker used in the fusion protein produced by the existing inframe fusion method is that it is easily cleaved by the protease in vivo, so that the effect of increasing the blood half-life of the active drug by the carrier cannot be obtained as expected. will be. However, in the present invention, a polymer that is resistant to protease can be used to maintain the blood half-life of the peptide, similar to the carrier. Therefore, the non-peptidyl polymer that can be used in the present invention can be used without limitation as long as it has the same role as the above, that is, a polymer that is resistant to proteases in vivo. It is preferred that the non-peptidyl polymer has a molecular weight in the range of 1 to 100 kDa, preferably in the range of 1 to 20 kDa. In addition, the non-peptide polymer of the present invention combined with the physiologically active polypeptide may be a combination of different types of polymers as well as one type of polymer.

The non-peptidyl polymer used in the present invention may have a reactive group at both ends or three ends capable of being bonded to a physiologically active polypeptide and a carrier.

The reactive group of the non-peptidyl polymer is not limited thereto, but preferably a reactive aldehyde group, propion aldehyde group, butyl aldehyde group, maleimide group, orthopyridyl disulfide, thiol, or succinimide derivative Can be In the above, as the succinimide derivative, succinimidyl propionate, hydroxy succinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate may be used. Particularly, in the case of having a reactive aldehyde group at the end of the non-peptide polymer, it is effective in minimizing a non-specific reaction and binding to a physiologically active polypeptide and a carrier at the end of the non-peptide polymer, respectively. The final product resulting from reductive alkylation by aldehyde bonds is much more stable than those linked by amide bonds. The aldehyde reactor selectively reacts to the amino terminus at low pH and can form covalent bonds with lysine residues at high pH, for example, pH 9.0.

Both terminal or three terminal reactive groups of the non-peptidyl polymer may be the same or different from each other. For example, it may have a maleimide group at one end and an aldehyde group, a propion aldehyde group, or a butyl aldehyde group at the other end. In the case of using poly(ethylene glycol) having hydroxy reactive groups at both ends as a non-peptidyl polymer, the hydroxy groups may be activated by a known chemical reaction to the various reactive groups, or poly(ethylene glycol) having a commercially available modified reactive group Ethylene glycol) can be used to prepare the protein conjugate of the present invention.

On the other hand, in the step (2) of the present invention, the process of separating and purifying a linker containing a physiologically active polypeptide having a non-peptide polymer covalently bonded to the N-terminus can be used without limitation. And, preferably, ion exchange chromatography can be used.

In another aspect, the present invention comprises the steps of: (1) preparing a physiologically active polypeptide and a non-peptide polymer linker by the above method; And (2) a carrier selected from the group consisting of an immunoglobulin Fc region, an antibody, albumin, and transferrin is covalently linked to the non-peptidyl polymer of the linker, so that the ends of the non-peptide polymer are physiologically active polypeptides and carriers, respectively. It provides a method for producing a physiologically active polypeptide conjugate, comprising the step of generating a peptide conjugate conjugated with.

In the present invention, "carrier" refers to a substance that is bound together with a drug, and is generally a substance that is bound to a drug to increase or decrease or eliminate the physiological activity of the drug. For the purposes of the present invention, the drug bound to the carrier is preferably a physiologically active polypeptide, and may be bound by a non-peptide polymer, while minimizing the decrease in the in vivo activity of the bound physiologically active polypeptide. It is intended to increase the in vivo stability of. The carrier is not limited thereto, but may preferably be an immunoglobulin Fc region, an antibody, albumin, or transferrin.

Since the immunoglobulin Fc region is a biodegradable polypeptide that is metabolized in vivo, it is safe to be used as a drug carrier. In addition, since the immunoglobulin Fc region has a relatively low molecular weight compared to the entire immunoglobulin molecule, it is advantageous in terms of preparation, purification, and yield of conjugates, as well as the homogeneity of substances by removing the Fab portion that exhibits high heterogeneity because the amino acid sequence is different for each antibody. This can be expected to significantly increase and decrease the likelihood of inducing antigenicity in the blood.

In the present invention, "immunoglobulin Fc region" refers to the heavy and light chain variable regions of immunoglobulins, heavy chain constant region 2 (CH2) and heavy chain constant region 3 ( It means a CH3) moiety, and also includes a hinge moiety in the heavy chain constant region. In addition, as long as the immunoglobulin Fc region of the present invention has substantially the same or improved effect with the native type, some or all of the heavy chain constant region 1 (CH1) and/or light chain constant region except for the heavy and light chain variable regions of the immunoglobulin. It may be an extended Fc region comprising 1 (CL1). In addition, it may be a region from which some considerably long amino acid sequences corresponding to CH2 and/or CH3 have been removed. That is, the immunoglobulin Fc region of the present invention includes (1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, (2) a CH1 domain and a CH2 domain, (3) a CH1 domain and a CH3 domain, (4) a CH2 domain and a CH3 domain. Domain, (5) a combination of one or two or more domains and an immunoglobulin hinge region (or part of the hinge region), (6) a heavy chain constant region, and may be a dimer of each domain and a light chain constant region.

In addition, the immunoglobulin Fc region of the present invention includes a native amino acid sequence as well as a sequence derivative thereof. An amino acid sequence derivative means that one or more amino acid residues in a natural amino acid sequence have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, may be used as a suitable site for modification. In addition, various kinds of derivatives are possible, such as a site capable of forming a disulfide bond is removed, several amino acids at the N-terminus in a native Fc are removed, or a methionine residue may be added to the N-terminus of a native Fc. Do. In addition, in order to eliminate the effector function, the complement binding site, for example, the C1q binding site may be removed, or the ADCC site may be removed. Techniques for preparing a sequence derivative of such an immunoglobulin Fc region are disclosed in International Patent Publication No. 97/34631, International Patent Publication No. 96/32478, and the like.

Amino acid exchanges in proteins and peptides that do not totally alter the activity of the molecule are known in the art (H. Neuroth, R.L. Hill, The Proteins, Academic Press, New York, 197 9). The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly.

In some cases, it is modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amylation, etc. may be modified.

The Fc derivative described above is a derivative that exhibits the same biological activity as the Fc region of the present invention, but has increased structural stability against heat and pH of the Fc region.

In addition, these Fc regions may be obtained from natural types isolated in vivo from animals such as humans and cattle, goats, pigs, mice, rabbits, hamsters, rats, guinea pigs, etc., or obtained from transformed animal cells or microorganisms. It may be recombinant or a derivative thereof. Here, the method of obtaining from the natural form can be obtained by separating the entire immunoglobulin from a human or animal living body and then treating a protease. When papain is treated, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab)2. This can be separated from Fc or pF'c using size-exclusion chromatography or the like.

Preferably, it is a recombinant immunoglobulin Fc region obtained by obtaining a human-derived Fc region from a microorganism.

In addition, the immunoglobulin Fc region may be a natural type sugar chain, an increased sugar chain compared to the natural type, a reduced sugar chain compared to the natural type, or a form in which sugar chains are removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms may be used for the increase or decrease of such immunoglobulin Fc sugar chains. Here, the immunoglobulin Fc region from which the sugar chain has been removed from the Fc significantly decreases the binding power of complement (c1q), and the antibody-dependent cytotoxicity or complement-dependent cytotoxicity is reduced or eliminated, so it does not cause an unnecessary immune response in vivo. Does not. In this respect, a form more suitable for the original purpose as a drug carrier will be referred to as an immunoglobulin Fc region in which sugar chains have been removed or non-glycosylated.

In the present invention, "deglycosylation" refers to an Fc region in which sugar is removed by an enzyme, and "aglycosylation" refers to an Fc region that is not glycosylated by producing in a prokaryotic animal, preferably E. coli. .

In addition, the immunoglobulin Fc region may be an Fc region derived from IgG, IgA, IgD, IgE, or IgM, or a combination thereof or a hybrid thereof. It is preferably derived from IgG or IgM, which is the most abundant in human blood, and most preferably derived from IgG known to enhance the half-life of the ligand binding protein.

Meanwhile, in the present invention, "combination" means that when forming a dimer or multimer, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin forms a bond with a single-chain polypeptide of a different origin. That is, it is possible to prepare a dimer or multimer from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragment.

In the present invention, "hybrid" is a term meaning that sequences corresponding to immunoglobulin Fc fragments of two or more different origins exist in the single-chain immunoglobulin Fc region. In the case of the present invention, various types of hybrid are possible. That is, from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc, a hybrid of a domain consisting of 1 to 4 domains is possible, and may include a hinge.

Meanwhile, IgG can also be divided into subclasses of IgG1, IgG2, IgG3 and IgG4, and in the present invention, a combination or hybridization thereof is possible. It is preferably the IgG2 and IgG4 subclasses, and most preferably the Fc region of IgG4, which has little effector functions such as complement dependent cytotoxicity (CDC).

That is, the most preferable immunoglobulin Fc region for a drug carrier of the present invention is a non-glycosylated Fc region derived from human IgG4. The human-derived Fc region is more preferable than the non-human-derived Fc region, which can cause an undesirable immune response such as generating a new antibody against it by acting as an antigen in a human body.

The physiologically active polypeptide conjugate prepared by the production method of the present invention can be prepared with high purity and yield, and its persistence and stability in vivo are improved, and thus medication compliance can be significantly improved during treatment with a physiologically active polypeptide. In one embodiment of the present invention, as a result of confirming the in vivo disappearance half-life using the insulin-PEG-immunoglobulin Fc conjugate prepared by the production method of the present invention, 15.73 hours for intravenous administration and 16.98 hours for subcutaneous administration, about 0.5 hours. It was confirmed that the persistence was 30 times longer than that of phosphorus natural insulin (FIG. 6), and as a result of conducting an in vivo efficacy test, it was confirmed that the blood sugar-lowering effect lasted for about 4 days (FIG. 7).

In the present invention, it is also possible to provide a physiologically active polypeptide long-acting preparation comprising the physiologically active polypeptide conjugate.

In the present invention, "administration" means introducing a predetermined substance to a patient by any suitable method, and the route of administration of the conjugate may be administered through any general route as long as the drug can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc. may be used, but are not limited thereto. However, when administered orally, since the peptide is digested, the oral composition is preferably coated with an active agent or formulated to be protected from degradation in the stomach. Preferably, it can be administered in the form of an injection. In addition, long-acting formulations can be administered by any device capable of moving the active substance to the target cells.

The long-acting formulation including the conjugate of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc. for oral administration, and buffers, preservatives, painlessness, etc. for injections. An agent, a solubilizing agent, an isotonic agent, a stabilizer, and the like can be mixed and used. In the case of topical administration, a base agent, an excipient, a lubricant, a preservative, and the like can be used. The formulation of the long-acting formulation of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, when administered orally, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, and in the case of injections, it can be prepared in the form of unit dosage ampoules or multiple dosage forms. In addition, it can be formulated into solutions, suspensions, tablets, pills, capsules, sustained-release preparations, and the like.

Meanwhile, examples of suitable carriers, excipients and diluents for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used. In addition, fillers, anti-aggregating agents, lubricants, wetting agents, flavoring agents, preservatives, and the like may additionally be included.

The long-acting formulation of the present invention is determined according to the type of drug as the active ingredient, along with various related factors such as the disease to be treated, the route of administration, the age, sex and weight of the patient, and the severity of the disease. Since the long-acting formulation of the present invention has excellent in vivo persistence and potency, the number and frequency of administration of the pharmaceutical formulation of the present invention can be remarkably reduced. In addition, since the long-acting formulation maintains very high persistence and stability in vivo of the physiologically active polypeptide, it is effective in treating diseases using the same.

Through the production method of the present invention, a bioactive polypeptide and a non-peptide polymer conjugate can be prepared with high purity and yield, and the bioactive polypeptide conjugate prepared by using the same can reduce the manufacturing cost and increase in vivo activity. It is maintained relatively high, and the half-life in the blood is significantly increased, and thus it can be usefully used in the development of a long-acting formulation of a physiologically active polypeptide that can increase patient compliance.

1 shows the results of analysis using SEC HPLC to confirm that the insulin-PEG linker is modified in the beta chain. A: insulin reducing conditions, B: PEG-insulin reducing conditions.
FIG. 2 shows the results of analysis using peptide mapping to confirm that the insulin-PEG conjugate was modified by 95% or more in phenylalanine (B1F) No. 1 of the beta chain. A: insulin peptide mapping result, B: PEG-insulin peptide mapping result, C: PEG-insulin sequence and predicted cleavage site during peptide mapping (red line).
3 shows the results of RP-HPLC and SE-HPLC analysis for each buffer condition of the insulin-PEG conjugate. Solid arrows indicate mono-PEGylated insulin peaks, and dotted arrows indicate bridge-shaped PEGylated impurity peaks. The conditions of each buffer solution are as follows.
A: (a) 100mM Potassium phosphate (pH6.0) buffer; (b) 100mM Potassium phosphate (pH6.0), 30% isopropanol buffer; (c) 100mM Potassium phosphate (pH6.0), 45% isopropanol buffer; (d) 100mM Potassium phosphate (pH6.0), 55% isopropanol buffer
B: (a) 50mM Sodium Citrate (pH6.0), 45% isopropanol buffer; (b) 50mM Sodium Citrate (pH6.0), 55% isopropanol buffer;
C: (a) 50mM Sodium acetate (pH4.0) buffer; (b) 50mM Sodium acetate (pH4.0), 10% isopropanol buffer; (c) 50mM Sodium acetate (pH4.0), 20% isopropanol buffer; (d) 50mM Sodium acetate (pH4.0), 30% isopropanol buffer; (e) 50mM Sodium acetate (pH4.0), 40% isopropanol buffer; (f) 50mM Sodium acetate (pH4.0), 45% isopropanol buffer; (g) 50mM Sodium acetate (pH4.0), 50% isopropanol buffer.
Figure 4 shows the RP HPLC analysis results (A) and SE HPLC analysis results (B) of the insulin-PEG-immunoglobulin Fc conjugate.
5 shows the results of SDS PAGE analysis of the insulin-PEG-immunoglobulin Fc conjugate. M: size marker, 1: insulin-PEG-immunoglobulin Fc conjugate reduction conditions, 2: insulin-PEG-immunoglobulin Fc conjugate non-reduction conditions.
6 shows the results of a pharmacokinetic confirmation test for confirming the persistence in vivo of the insulin-PEG-immunoglobulin Fc conjugate. iv: intravenous administration, sc: subcutaneous administration.
7 shows the results of in vivo efficacy tests of insulin-PEG-immunoglobulin Fc conjugates.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are for illustrative purposes only, and the scope of the present invention is not limited thereto.

Example 1: of insulin Pegylation ( PEGylation ) Reaction and mono Pegylated Insulin tablets

After dissolving the insulin powder in 10 mM HCl, in order to PEGylate 3.4K propion-ALD2 PEG (PEG with two propionaldehyde groups, IDB, Korea) at the N-terminus of the insulin beta chain, insulin: PEG The molar ratio of 1: 2 to 4, and the insulin concentration of 3 to 5 mg/ml were reacted at 4 to 8° C. for about 2 hours. At this time, the reaction was carried out in 50mM Sodium Citrate pH6.0, 45% isopropanol, and 4-20mM NaCNBH ₃ The reaction was carried out by adding a reducing agent. The reaction solution was purified using a buffer containing Sodium Citrate (pH3.0), 45% EtOH, and an SP-HP (GE Healthcare) column using a KCl concentration gradient.

The prepared mono-PEGylated insulin was confirmed to be 98% or more PEGylated to phenylalanine #1 (B1F) of the beta chain using SE-HPLC and Peptide mapping analysis (FIGS. 1 and 2).

Example 2: of the reaction solution pH and Organic solvent Mono according to concentration PEG -Changes in the ratio of the yield of insulin and the content of impurities

When an organic solvent such as isopropanol is used in the reaction solution during the reaction between insulin and PEG, in order to compare the reaction yield of mono PEG-insulin and whether impurities are generated during preparation, the molar ratio of insulin and 3.4K Propion-ALD2 PEG is 1: 2, the insulin concentration was 3 mg/mL, and the reaction was carried out at 4°C for 4 hours. At this time, the reaction solution was 50mM Sodium Citrate pH 6.0, 45% isopropanol buffer solution; 50mM Sodium Citrate pH 6.0, 55% isopropanol buffer; 100mM Potassium phosphate pH6.0 buffer; 100 mM Potassium phosphate pH 6.0, 30% isopropanol buffer; 100 mM Potassium phosphate pH 6.0, 45% isopropanol buffer; 100 mM Potassium phosphate pH 6.0, 55% isopropanol buffer; 50mM Sodium acetate pH4.0 buffer; 50mM Sodium acetate pH4.0, 10% isopropanol buffer; 50mM Sodium acetate pH4.0, 20% isopropanol buffer; 50mM Sodium acetate pH4.0, 30% isopropanol buffer; 50mM Sodium acetate pH4.0, 40% isopropanol buffer; Sodium acetate pH4.0, 45% isopropanol buffer; And 50mM Sodium acetate pH4.0, 50% isopropanol buffer was used, and 20 mM NaCNBH ₃ as a reducing agent was added thereto. After each reaction solution was purified in the same manner as in Example 1, the purification profile is shown in FIG. 3. When PEG is reacted in the form of a bridge between the alpha chain and the beta chain of insulin at the same time, the coupling reaction between mono-PEGylated insulin and immunoglobulin Fc becomes impossible, so the ratio of these impurities is shown in Table 1. I got it. As shown in Table 1, the ratio of mono-PEGylated insulin varies depending on the content of isopropanol contained in the reaction solution and the pH condition, and when the content of isopropanol is about 45-55% at pH 6.0, mono-PEGylated insulin It was confirmed that the content of was the maximum. Under low pH conditions, the content of bridge-type PEGylated impurities increased as the content of isopropanol increased, resulting in a decrease in the yield of mono-PEGylated insulin.

Buffer condition Organic solvent Bridge type PEGylation impurity content Mono PEG-insulin content 100mM Potassium phosphate (pH6.0) - 1.9% 12.0% 30% isopropanol 8.1% 32.0% 45% isopropanol 10.5% 44.0% 55% isopropanol 6.4% 21.0% 50mM Sodium Citrate (pH6.0) 45% isopropanol 9.9% 45.0% 55% isopropanol 12.3% 43.0% 50mM Sodium acetate (pH4.0), 0.2M NaCl - 4.7% 18.0% 10% isopropanol 6.9% 18.0% 20% isopropanol 19.3% 27.0% 30% isopropanol 25.7% 31.0% 40% isopropanol 28.9% 32.0% 45% isopropanol 31.0% 34.0% 50% isopropanol 33.9% 34.0%

Example 3: mono Pegylated Insulin and immunoglobulins Fc Conjugate manufacturing

In order to prepare the insulin-PEG-immunoglobulin Fc conjugate, the molar ratio of mono-PEGylated insulin and immunoglobulin Fc obtained using the method of Example 1 was 1:1, and the total protein concentration was 20. It was reacted at 25° C. for 15 to 17 hours at -50 mg/ml. At this time, the reaction solution was 100mM HEPES, 22 mM Potassium phosphate, 10% ethanol, pH 8.2, and 20mM NaCNBH ₃ as a reducing agent was added.

After the reaction was terminated, the reaction solution was subjected to primary purification using a Source 15Q (GE Healthcare) column to remove residual immunoglobulin Fc and mono-PEGylated insulin. At this time, it was eluted using a Tris-HCl (pH7.5) buffer and a NaCl concentration gradient.

Thereafter, Source 15ISO (GE Healthcare) was used as a secondary column to remove the remaining immunoglobulin Fc and multi-PEGylated insulin conjugate, thereby obtaining an insulin-PEG-immunoglobulin Fc conjugate. At this time, it was eluted using a concentration gradient of Ammonium Sulfate containing Tris-HCl (pH7.5).

The eluted insulin-PEG-immunoglobulin Fc conjugate was analyzed using RP-HPLC, SE-HPLC and SDS PAGE, and it was confirmed that it was prepared with a high purity of 98% or more (FIGS. 4 and 5).

Example 4: Measurement of half-life of long-acting insulin conjugate in vivo

In order to confirm the persistence in vivo of the insulin-PEG-immunoglobulin Fc conjugate prepared in Example 3 in vivo, the pharmacokinetics was measured through intravenous administration and subcutaneous administration using a normal male rat (Normal SD rat). Confirmed. After a single intravenous and subcutaneous administration of the long-acting insulin conjugate to normal male mice at 0.1 mg/kg (insulin standard), the change in blood concentration over time was measured using an insulin ELISA kit. The pharmacokinetic parameters were calculated. The half-life of the long-acting insulin conjugate in vivo was 15.73 hours when administered intravenously and 16.98 hours when administered subcutaneously, which was 30 times longer than natural insulin, which is about 0.5 hours, and bioavailability when administered subcutaneously is 54%. It was confirmed that it was about (Fig. 6).

Example 5: of insulin conjugate in vivo Efficacy test

In order to compare the in vivo efficacy of the insulin conjugate, a blood glucose lowering effect comparison test was conducted in mice with diabetes induced by Streptozotocin. Diabetes was induced after intraperitoneal administration of streptozotocin dissolved in 10 mM citric acid buffer (pH4.5) at 60 mg/kg to normal rats fasted for 16 hours. Subsequently, a single subcutaneous administration of the insulin conjugate at a dose of 0.5 mg/kg to rats with elevated blood sugar levels of 500 mg/dL and then comparing the blood glucose-lowering effect showed that the blood glucose drop of the insulin conjugate lasted about 4 days after administration, and the blood glucose level increased on the fifth day It was confirmed that it rises to the vehicle level (FIG. 7).

<110> Hanmi Science Co., Ltd. <120> Method for preparation of biological active polypeptide conjugate <130> PA110844-KR-D1-D1 <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 21 <212> PRT <213> Homo sapiens <400> 1 Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu 1 5 10 15 Glu Asn Tyr Cys Asn 20 <210> 2 <211> 30 <212> PRT <213> Homo sapiens <400> 2 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr 20 25 30

Claims (22)

(1) A process for producing insulin and a non-peptide polymer conjugate characterized by reacting insulin and a non-peptide polymer in a reaction solution containing an organic solvent so that a carrier can be additionally linked to the non-peptide polymer step; And
(2) separating and purifying the insulin and the non-peptide polymer conjugate from the reaction mixture of step (1)
Wherein the insulin has two or more N-termini,
The non-peptide polymer has a double-ended or three-terminal reactor,
Wherein the reaction solution comprises sodium citrate and an organic solvent in an amount of 45 to 55% by volume based on the total amount of the reaction solution, the pH of the reaction solution is 4.5 to 6.5,
A method for producing an insulin and a non-peptide polymer conjugate capable of further linking a carrier to a non-peptide polymer,
Wherein the organic solvent is an alcohol having 1 to 10 carbon atoms.
delete The process according to claim 1, wherein the alcohol is selected from the group consisting of isopropanol, ethanol and methanol.
The process according to claim 1, wherein the alcohol is isopropanol.
delete The method of claim 1, wherein the insulin is selected from the group consisting of substitution, addition, deletion, modification, or a combination of one or more amino acids in a native insulin, an insulin variant, Insulin derivative, or a fragment thereof.
The method according to claim 1, wherein the non-peptide polymer is bound to the N-terminus of the beta chain of the insulin.
7. The method according to claim 6, wherein the non-peptide polymer is bound to the amine group or thiol group of the insulin or insulin derivative.
The method of claim 1, wherein the non-peptide polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, Wherein the polymer is selected from the group consisting of a polymer, a lipid polymer, chitin, hyaluronic acid, and combinations thereof.
The composition of claim 1, wherein the non-peptide polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polyvinyl ethyl ether, &Lt; / RTI &gt;
The non-peptide polymer of claim 1, wherein the non-peptide polymer has a reactor selected from the group consisting of aldehyde groups, propionaldehyde groups, butylaldehyde groups, maleimide groups, orthopyridyl disulfide, thiol and succinimide derivatives Gt;
12. The method according to claim 11, wherein the succinimide derivative is succinimidyl propionate, succinimidyl carboxymethyl, hydroxy succinimidyl or succinimidyl carbonate.
The method according to claim 1, wherein the non-peptide polymer has reactors of aldehyde groups at both terminals.
The process according to claim 1, wherein the separation and purification are carried out by ion exchange chromatography.
delete delete (1) preparing an insulin and a non-peptide polymer conjugate by the method according to any one of claims 1, 3, 4, and 6 to 14; And
(2) covalently linking a non-peptide polymer of the linkage with a carrier selected from the group consisting of immunoglobulin Fc region, antibody, albumin and transferrin so that the ends of the non-peptide polymer are bound to insulin and the carrier respectively Peptide conjugate. &Lt; RTI ID = 0.0 &gt;
Insulin conjugate.
18. The method according to claim 17, wherein the immunoglobulin Fc region consists of one to four selected from the group consisting of CH1, CH2, CH3 and CH4 domains.
18. The method of claim 17, wherein the immunoglobulin Fc region further comprises a hinge region.
18. The method according to claim 17, wherein said immunoglobulin Fc region is an Fc region derived from IgG, IgA, IgD, IgE or IgM.
18. The method of claim 17, wherein the immunoglobulin Fc region is an IgG4 Fc region.
18. The method of claim 17, wherein the immunoglobulin Fc region is a human unchromosomal IgG4 Fc region.

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