CROSS REFERENCE TO RELATED APPLICATIONS

This application is a National Stage of International Application No. PCT/KR2016/010762 filed Sep. 26, 2016, claiming priority based on Korean Patent Application No. 10-2015-0135874, filed Sep. 24, 2015.

TECHNICAL FIELD

The present invention relates to a protein complex prepared by linking an immunoglobulin Fc region to a physiologically active polypeptide via a non-peptidyl polymer, in which the non-peptidyl polymer is specifically linked to a N-terminus of the immunoglobulin Fc region, a preparation method thereof, and a pharmaceutical composition including the same for improving in vivo duration and stability of the physiologically active polypeptide. Additionally, the present invention relates to a population of protein complexes which include the protein complexes prepared by the above method.

BACKGROUND ART

In general, polypeptides are easy to denature owing to their low stability, and are decomposed by proteolytic enzymes in blood to be readily removed through the kidney or liver. Therefore, in order to maintain the blood concentration and titer of a protein drug including a polypeptide as a pharmacologically active ingredient, it is necessary to frequently administer the protein drug to patients. However, in the case of protein drugs administered to patients primarily in the form of an injectable formulation, frequent injections to maintain the blood concentration of active polypeptides may cause excessive suffering in patients. To solve such problems, there has been constant effort to maximize pharmacological efficacy by increasing the blood stability of the protein drug and maintaining its blood concentration for a longer time. Such long-acting formulations of protein drugs are required to increase the stability of protein drugs and at the same time to maintain the potency of the drugs themselves at a sufficiently high level, as well as to cause no immune reaction in patients.

In the prior art, for stabilizing proteins and inhibiting contact with proteolytic enzymes and loss through the kidney, a method for chemically adding polymers having a high solubility such as polyethylene glycol (hereinafter, referred to as “PEG”) to the surface of protein drugs has been used. It has been known that PEG is effective in stabilizing proteins and preventing the hydrolysis of proteins by non-specifically binding to a specific site or various sites of the target protein to increase the solubility thereof without causing any adverse side effects (Sada et al.,J. Fermentation Bioengineering71: 137-139, 1991). However, binding of PEG may increase stability of the protein but remarkably reduce the titer of the physiologically active protein. As a molecular weight of PEG is increased, its reactivity with the protein is also reduced, leading to low yield. Another problem which may be generated by binding of PEG to proteins is linkage isomerization of a conjugate, which is caused by competition of many possible linkage sites.

This is an important issue in the preparation of conjugate drugs using PEG, and there is a demand for homogeneous drugs prepared under conditions eliciting maximal efficacy and effect. If a large amount of related compounds generated by binding of PEG to a binding site which affects efficacy of the drug is included, an undesirable reduction in the efficacy may be caused.

DISCLOSURE

Technical Problem

The present inventors have studied a method capable of linking a physiologically active polypeptide conjugate at a site showing the highest efficacy by a process suitable for mass-production of the conjugate, and as a result, they have developed a method of preparing a high-purity conjugate with the highest efficacy by specifying a binding site of an immunoglobulin Fc fragment which is included as a carrier, thereby completing the present invention.

Technical Solution

An object of the present invention is to provide a protein complex prepared by linking a physiologically active polypeptide and an immunoglobulin Fc fragment via a non-peptidyl polymer, in which the non-peptidyl polymer is site-specifically linked to a N-terminus of the immunoglobulin Fc fragment.

Another object of the present invention is to provide a method of preparing the protein complex.

Still another object of the present invention is to provide a pharmaceutical composition for improving in vivo duration and stability of the physiologically active polypeptide, the composition including the protein complex as an active ingredient.

Advantageous Effects

The present invention relates to a protein complex with improved in vivo duration, which is prepared using a specific site during preparation of the protein complex. Particularly, the present invention relates to a protein complex in which a physiologically active polypeptide, a non-peptidyl polymer and a N-terminus of an immunoglobulin Fc region are linked to each other by a covalent bond, thereby improving in vivo duration of the physiologically active polypeptide. The protein complex prepared by the present invention may be applied to preparation of long-acting formulations of various physiologically active polypeptide drugs.

