KR20160120239A - Stabilized Formulations of Interferon beta Mutant - Google Patents

Abstract

The present invention relates to a stabilized pharmaceutical agent of human interferon beta variant (R27T) including R27T, acetic acid buffer, arginine, mannitol, poloxamer 188, and methionine. Since the stabilized R27T pharmaceutical agent according to the present invention includes acetic acid buffer, arginine, mannitol, poloxamer 188, and methionine, formation of R27T protein aggregate is inhibited and thermodynamical and structural stability is improved, thereby allowing long-term storage. Thus, the stabilized R27T pharmaceutical agent according to the present invention is expected to be usefully applied for prevention, alleviation and treatment of multiple sclerosis, cancer, autoimmune disease, viral infection, HIV infection, hepatitis type C, rheumarthritis, and so forth.

Description

Stabilized Formulations of Interferon beta Mutant < RTI ID = 0.0 >

The present invention relates to stabilized pharmaceutical preparations of R27T comprising human interferon beta variant (R27T), acetic acid buffer, arginine, mannitol, Poloxamer 188, and methionine.

Interferons (IFNs) are a type of cytokine that exhibits anti-viral activity, inhibits cell proliferation, and has a function to regulate the natural immune response. IFN is classified into IFN-α, IFN-β and IFN-γ according to the cell origin (leukocyte, fibroblast, T cell), and interferon-beta (IFN- ), Which is 22 kD in size and 18 kD when the sugar chain is removed.

Studies on the clinical application of IFN-β have been actively under way, and in particular, they are in the spotlight as a symptom relief, relief, or remedy for multiple sclerosis. In addition, antiviral activity, cell growth inhibition, Toxicity-enhancing activity, immunoregulatory activity, induction or inhibition of differentiation of target cells, activation of macrophages, increase of cytokine production, increase of cytotoxic T cell effect, increase of natural killing cell, etc. Have been reported to be effective in the treatment of cancer, autoimmune disorders and virus infections, diseases related to HIV, hepatitis C, and rheumatoid arthritis.

Human IFN-? Is also a kind of glycoprotein. Since the sugar chain portion connected to the protein plays an important role in the activity of the protein, the activity of the glycoprotein may be increased by adding the sugar chain. In other words, protein glycosylation is known to affect many biochemical properties such as stability, solubility, intracellular trafficking activity, pharmacokinetics and antigenicity.

Accordingly, there has been reported an example in which a human IFN-beta mutant having an increased or improved activity or function by introducing a sugar chain into a human native IFN-beta protein (Korean Patent Registration No. 10-0781666). R27T is a recombinant human IFN-? Mutant (hereinafter referred to as rhINF-?) Designed by substituting threonine (Thr) for arginine (Arg) at the 27th position for additional glycation at the 25th position of IFN- ), Showing increased stability, decreased protein aggregation tendency and increased half-life compared to wild-type INF-β1a (Rebif). That is, R27T is a biobetter version of rhINF-β produced by additional saccharification via position-directed mutagenesis.

On the other hand, one of the major challenges in the development of protein drugs is to produce products with long shelf life of more than 24 months by giving sufficient chemical, physical and biological stability. Achieving stability, however, is still a difficult problem due to the complexity of protein structures with various levels of intrinsic susceptibility, macromolecules, and secondary, tertiary and quaternary structures in the proteolytic pathway.

As the first recombinant peptide hormone, insulin was approved for the first time in 1982 and successfully produced for more than 30 years, success stories of numerous recombinant protein / peptide drugs have been reported. However, in the process of development of biopharmaceuticals, especially in formulations, there are still challenges due to several factors such as protein aggregation, physicochemical instability, low half life, low solubility, pharmacokinetic properties.

In particular, protein aggregation is one of the major problems that occur easily in almost all biopharmaceutical processes during storage, as the therapeutic proteins are structurally / thermodynamically unstable in solution. Therapeutic proteins are susceptible to structural changes due to a variety of factors during purification, processing and storage, and such problems can be exacerbated when proteins are exposed to high temperatures, high / low pH, shear strain and surface adsorption . In addition, protein-based biopharmaceuticals have the potential for physical degradation such as unfolding, coagulation, and insoluble granulation due to non-native aggregation, thus avoiding protein aggregation or physical denaturation and maximizing stability Optimization of formulation systems is required, including the development of stable pH ranges, suitable buffer systems, and excipients.

Conventionally, various biophysical analysis methods such as Dynamic Light Scattering (DLS), Differential Scanning Calorimetry (DSC), Circular Dichroism (CD), Size Exclusion Chromatography (SEC), and ATR- Efforts have been made to rapidly and efficiently produce protein preparations such as factors and antibodies and were used mainly to observe the diversity of protein structure under various solution conditions such as solution pH, buffer concentration, buffer concentration, protein concentration and excipient. However, since many data can be difficult to interpret due to their diversity and complexity, new data analysis methods and stabilization systems are needed to find consistency between the results and to show a meaningful association to define the physical state of the target protein.

Accordingly, the inventors of the present invention have conducted intensive studies to increase the usefulness of the interferon beta mutant R27T protein as a stabilized pharmaceutical preparation by inhibiting the formation of aggregates and improving the thermodynamic and structural stability. As a result, Thereby completing the invention.

Accordingly, it is an object of the present invention to provide a stabilized pharmaceutical preparation of human interferon beta mutant (R27T) of novel composition.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to achieve the object of the present invention, the present invention provides a human interferon beta mutant (R27T), an acetic acid buffer solution at a concentration of 5 to 100 mM, arginine at a concentration of 10 to 150 mM, mannitol (R27T) comprising Mannitol at a concentration of 0.1 to 10 mg / mL, Poloxamer 188 at a concentration of 0.1 to 10 mg / mL, and methionine at a concentration of 0.5 to 5 mM.

In one embodiment of the present invention, the human interferon beta mutant (R27T) is characterized in that arginine, which is the 27th amino acid of human interferon beta, is substituted with threonine to include an N-linked sugar chain in the asparagine residue which is the 25th amino acid .

In another embodiment of the present invention, the concentration of the acetic acid buffer solution is 10 to 30 mM.

In another embodiment of the present invention, the acetic acid buffer solution has a pH of 3.6 to 4.4.

In another embodiment of the present invention, the concentration of the arginine is 50 to 100 mM.

In still another embodiment of the present invention, the concentration of the mannitol is 150 to 250 mM.

In another embodiment of the present invention, the concentration of the poloxamer 188 is 0.1 to 1 mg / mL.

In another embodiment of the present invention, the concentration of the methionine is 0.5 to 2 mM.

In another embodiment of the present invention, the stabilized pharmaceutical preparation is characterized in that the pH is in the range of 3.6 to 4.4.

In another embodiment of the present invention, the stabilized pharmaceutical preparation is a pharmaceutical composition for preventing or treating a disease selected from the group consisting of multiple sclerosis, cancer, autoimmune disease, viral infection disease, HIV infection disease, hepatitis C and rheumatoid arthritis .

In another embodiment of the present invention, the stabilized pharmaceutical preparation is for oral or parenteral administration.

In another embodiment of the present invention, the stabilized pharmaceutical preparation is characterized by being a liquid or lyophilized formulation.

The stabilized R27T pharmaceutical preparation according to the present invention inhibits the formation of aggregates of R27T protein by including acetic acid buffer, arginine, mannitol, poloxamer 188, and methionine, and improves thermodynamic and structural stability to enable long-term storage, It is expected that the compounds of the present invention can be usefully used for the prevention, improvement and treatment of diseases such as scleroderma, cancer, autoimmune diseases, viral infection diseases, HIV infection diseases, hepatitis C, and rheumatoid arthritis.

1 shows the gene and protein sequence of the human interferon beta mutant (R27T).
2 shows the result of evaluating the thermodynamic stability according to the concentration of R27T (0.8 mg / mL; 0.5 mg / mL; 0.3 mg / mL; 0.05 mg / mL) in a preparation using a differential scanning calorimeter (DSC).
Figures 3a and 3b are the results of evaluating the physicochemical stability of R27T (0.8 mg / ml) at various pH ranges using DLS-zeta potential (Figure 3a) and DSC-transition temperature (Figure 3b).
Fig. 4 shows the result of evaluating the optimal buffer for R27T by measuring T _m by performing DSC after adding four acidic buffers (phosphoric acid, acetic acid, citric acid, and histidine).
Figure 5 shows the results of evaluating optimal pH conditions in four acidic buffers (phosphoric acid, acetic acid, citric acid, histidine) using a multi-objective robust design method (RD).
FIG. 6 shows the results of the measurement of the amount of residual monomer in the protein solution by performing size exclusion chromatography (SEC) after storing the R27T preparation containing 20 mM acetic acid buffer at various pH at 37 DEG C for 11 days, This is the result of evaluating the pH effect on the storage stability of the preparation.
FIG. 7 shows the results of evaluating the pH effect on the structural stability of the formulation containing 20 mM acetic acid buffer by measuring the T _m by performing DSC analysis on the R27T preparation containing 20 mM acetic acid buffer at pH 3.4 to 4.4.
Figures 8a and 8b, as one subjected to DSC analysis for each R27T formulation that contains 20 mM acetate buffer, and other kinds of excipients pH 4.2 measured T _m, mixture addition of a vehicle (Fig. 8a) and a single (Fig. 8 (b)) of the R27T formulation to evaluate the structural stability of the R27T formulation. (Formulation: Formulation containing Arginine HCl and Polysorbate 20) (Reference: Conventional Rebif preparation; Formulation: Mannitol, Poloxamer 188, Methionine, and Benzyl alcohol)
FIG. 9 shows the results of SEC analysis of each R27T preparation containing 20 mM acetic acid buffer at pH 4.2 and other excipients at 40 DEG C for 9 days to measure the residual monomer content of the protein solution, Storage stability of the R27T preparation according to the addition.
Figure 10 shows that R27T stabilizing agents added at different concentrations (10, 20, and 50 mM) and pH (3.8, 4.2) of acetic acid buffer at 4 ° C or 25 ° C, After 2 weeks of storage, SEC was performed to measure the residual monomer and aggregate amount of the protein solution to evaluate the concentration and pH effect of the acetic acid buffer solution on the storage stability.
Figure 11 shows the effect of increasing the concentration of the R27T preparation (F1 to F8) in a low concentration (100 占 퐂 / ml) or a high concentration (640 占 퐂 / ml) in 20 mM acetic acid buffer at pH 3.8, arginine, poloxamer 188 and mannitol After storing at 4 ° C or 37 ° C for 14 days, SEC was performed to measure the amount of residual monomer in the protein solution to evaluate the optimum composition with improved storage stability.
Fig. 12 shows the results of a comparison of the results of the experiment with the results of the experiment. Fig. 12 is a graph showing the results of the experiment. Fig. The SEC was performed at intervals of one day to measure the amount of residual monomer in the protein solution, thereby evaluating the optimum composition with improved storage stability.
FIG. 13 shows the results of a CPE (Cytopathic effect) assay on 20 mM acetic acid buffer solution of pH 3.8, arginine, poloxamer 188, and R27T preparations (F1 to F8) The results are as follows.

The present invention relates to a human interferon beta mutant (R27T) at a concentration of 0.3 to 1.0 mg / mL and an acetic acid buffer at a concentration of 5 to 100 mM, arginine at a concentration of 10 to 150 mM, mannitol at a concentration of 50 to 300 mM ), A Poloxamer 188 at a concentration of 0.1 to 10 mg / mL, and a methionine at a concentration of 0.5 to 5 mM (Methionine).

One of the most fundamental problems in making protein-based preparations is to determine the desired therapeutic protein concentration in solution, which is dependent not only on the protein concentration but also on the pH, temperature, ionic strength and additive concentration of the solution Because. Thus, protein-based preparations should be suitable for stabilizing therapeutic proteins and prevent problems such as aggregation, precipitation, or fragmentation.

R27T is a recombinant human IFN-beta mutant (hereinafter referred to as rhINF-beta) designed by replacing arginine (Arg) at position 27 with threonine (Thr) for further glycosylation at the 25th position of IFN- , DSC, DLS, FT-IR, and SEC were used to develop a formulation for the stabilization of R27T.

It is known that glycation increases the solubility of many proteins, and glycation stability is pH dependent. Therefore, identifying the appropriate pH and buffer is critical to overcoming stability problems and achieving optimum stability at various stages of the development process, including drug production, purification, storage and release.

In the present invention, by preliminarily selecting a solution of pH 2-11 using differential scanning calorimetry (DSC) and dynamic light scattering (DLS), the experimental pI of R27T and the optimum pH range 5.8 and 3.6-4.4, respectively.

In addition, the DoE (Design of Experiment) was used to obtain a basic buffer system in a pH range of 2.9 to 5.7 using various concentrations of acetic acid, histidine, phosphoric acid, and citric acid buffer. The DoE approach was developed as a practical method for formulation studies with pH 2.9-5.7, buffer (phosphate, acetic acid, citrate, histidine), buffer concentration (20 mM and 50 mM) These methods used a weight-based procedure to interpret complex data results and investigate key factors that indicate protein stability. At this time, the factors used are T _m , enthalpy, and helix ratio obtained by DSC and Fourier transform infrared spectroscopy (FT-IR). The weightings varied with these three factors, but the highest objective function was found in 20 mM acetic acid buffer at pH 3.6 among all the scenarios in which evaluation was performed, followed by phosphate buffer at pH 2.9. This means that the thermal stability (T _m and enthalpy) and the secondary structural stability (relative helix content) are optimal for these buffers.

However, since these buffers are too low in pH to cause adverse skin reactions to the patient, the formulation obtained from DoE is not sufficient for subcutaneous injection. Therefore, the pH value of R27T is adjusted with acetic acid buffer considering thermal stability, secondary structural stability, The results of this study are summarized as follows. That is, Size Exclusion Chromatography (SEC) was used to optimize the pH value with acceptable stability.

As a result, the amount of R27T monomer was maintained at 37 DEG C even when the pH was increased from 3.4 to 4.4. Protein aggregation steadily increased until day 7, but both monomers and aggregates decreased on day 11, indicating a loss of R27T from non-native aggregation. The DSC thermogram results showed R27T instability at pH 4.2 and 4.4, and the same problem was observed in SEC, so the secondary structure stability of the sample was analyzed by FT-IR.

rhIFN-β is a 166 amino acid glycoprotein with a 4-helix bundle domain, and an increase in β-sheet content means formation of intermolecular β-sheet that can induce protein aggregation. Analysis of α-helix and β-sheet contents of R27T in the optimum pH range of acetic acid buffer showed that the optimum pH value for thermal stability and secondary structural stability of R27T was pH 3.8 ± 0.2.

That is, the helix ratio and the monomer remaining increased with pH and the highest at pH 4.0, while the helix ratio and thermodynamic stability decreased at pH 4.2 and pH 4.4, which may be due to protein aggregation problems . Thus, the optimized baseline buffer system for R27T was expected to be 20 mM acetic acid buffer at pH 3.8 ± 0.2.

Furthermore, R27T preparations with various excipients mixed or individually added together with 20 mM acetic acid buffer at pH 4.2 were prepared to find the excipients which could further enhance the stability of R27T, and the experiment was conducted through T _m and SEC analysis As a result, Mannitol has an excellent effect of improving the structure and storage stability of R27T, and Arginine, Poloxamer 188, or Polysorbate 20 also has the effect of enhancing the stability of the interferon protein Could know.

Based on the above experimental results, expand the range of excipient to prepare a R27T formulation with a 20 mM acetate buffer, and a variety of excipients R27T, pH 4.2 addition of R27T according to the vehicle type with a T _m and SEC analysis As a result of evaluation of stability, it was confirmed that arginine and poloxamer 188 had an excellent effect for enhancing the stability of R27T. Based on these results, a follow-up experiment for setting the optimal composition of arginine and poloxamer 188- .

Experiments to determine the optimum conditions for the formulation of excipients Through experiments to determine the final conditions of the acetic acid buffer, it was confirmed that the 10 mM or 20 mM acetic acid buffer system at pH 3.8 was the condition for maximizing the storage stability of R27T, the final excipient composition was evaluated under the condition of 20 mM acetic acid buffer solution of pH 3.8 as described above in consideration of the capacity.

Six different compositions of R27T formulation were prepared by adding mannitol, which was found to enhance the stability of interferon in addition to containing arginine, poloxamer 188, and methionine, and a control without excipient and a conventional interferon beta preparation (Rebif ) To determine the optimal composition of the formulation of R27T.

The term " interferon-beta (IFN-beta) " as used herein refers to interferon-beta (IFN-beta) as well as fibroblast interferon of human origin obtained from a biological fluid or obtained by DNA recombinant technology from eukaryotic or prokaryotic host cells, , Variants, analogs and active fractions thereof. A preferred IFN-beta is meant to mean interferon beta-1a.

A "stabilizing agent" is one in which the degree of degradation, denaturation, aggregation, loss of biological activity of the proteins contained therein is tolerably controlled and does not increase unacceptably over time. Preferably, the formulation maintains at least about 60%, preferably at least about 70%, more preferably at least about 80% of the R27T activity by 24 months.

By "buffer" is meant a solution of a compound that has the effect of modulating or maintaining the pH of the formulation to fall within the pH range desired for the formulation. In the present invention, a buffer suitable for controlling the pH is not limited, but may include compounds such as phosphoric acid, acetic acid, citric acid, histidine, preferably 20 mM acetate buffer, and the buffer preferably has a pH of 5 to 100 mM Concentration, more preferably 10 to 30 mM.

 In the present invention, the stabilized pharmaceutical preparation of R27T comprises a step of dialyzing a solution containing R27T, which is a human interferon variant, with a solution containing acetic acid buffer and an excipient at a concentration of 5 to 100 mM; And then filtering the dialysis solution.

In the present invention, the excipient may contain arginine at a concentration of 10 to 150 mM, mannitol at a concentration of 50 to 300 mM, poloxamer 188 at a concentration of 0.1 to 10 mg / mL, and methionine at a concentration of 0.5 to 5 mM, More preferably, arginine at a concentration of 50 to 100 mM, mannitol at a concentration of 150 to 250 mM, poloxamer 188 at a concentration of 0.1 to 1 mg / mL, and methionine at a concentration of 0.5 to 2 mM can be added.

For "liquid formulations" the preferred solvent is water and can be monodose or multidose. The liquid R27T preparations for multidose formulations of the present invention may be formulated with one or more additives selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, It is preferred to include a bacteriostatic agent such as etonium chloride, sodium dehydroacetate and thimerosal. The bacteriostatic can be administered in an amount effective to maintain a sterile formulation (suitable for injection) over a period of from about 12 or 24 hours to about 12 days, preferably about 6 to 12 days, . The bacteriostatic agent is preferably present at a concentration of from about 0.1% to about 2.0% (mass of bacteriostatic agent / mass of solvent).

The formulations of the present invention may optionally further comprise diluents, excipients and carriers as well as physiologically / pharmaceutically acceptable additives such as free-flowing agents, emulsifiers, stabilizers, preservatives, colorants, defoamers and anti-caking agents.

The pharmaceutically acceptable carrier is not particularly limited, but may include physiological saline, polyethylene glycol, ethanol, vegetable oil, and isopropyl myristate, and the like.

The present invention also provides a method for treating a disease such as multiple sclerosis, cancer, autoimmune disease, viral infection disease, HIV infection disease, hepatitis C, rheumatoid arthritis, etc. by administering a stabilized preparation to a subject in a pharmaceutically effective amount .

The term "individual" as used herein refers to a subject in need of treatment for a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, And the like.

It will be apparent to those skilled in the art that the "pharmaceutically effective amount" can vary widely depending on the patient's body weight, age, sex, health condition, diet, time of administration, administration method, excretion rate, and severity of the disease.

The preferred dosage of the agent of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration, and the period of time, but can be appropriately selected by those skilled in the art. However, it is preferably administered at a daily dose of 0.001 to 100 mg / kg body weight, more preferably 0.01 to 30 mg / kg body weight. The administration may be carried out once a day or divided into several times.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. The method of administration is not limited and can be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intra-cerebroventricular injection.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example  1: Experimental method

1-1. dynamic Light scattering (DLS) and dynamic light scattering Zeta potential  analysis

A Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK) was used to evaluate the electrostatic interactions of the interferon beta variant (R27T) samples and the instrument temperature was set at 10 占 폚. Each sample was subjected to a total of five repeated measurements at 30 second intervals to obtain the mean hydrodynamic size, polydispersity index (PdI), and zeta potential.

1-2. Differential Scanning Calorimetry; DSC )

To evaluate the thermodynamic stability of R27T samples, VP-DSC Microcalorimeter (Microcal, Northampton, MA, USA) was used. The experiment was conducted at a heating rate of 1 ° C per minute from 15 ° C to 120 ° C, and was repeated three times in total. The DSC test results were standardized by subtracting the baseline measured using the final measured buffer and calculating the concentration of protein present in the sample.

The R27T measurement results were used to set the baseline for zeroing the result through linear baseline adjustment. At this time, the process of setting the reference line for adjusting the zero point of the sample is complicated because it is disturbed by the coagulation or sedimentation phenomenon caused by heating. In the present invention, in the repeated experiment by selecting the linear baseline option, The most stable results were obtained regardless of the decision of

Final calorimetric recording was made with excess specific heat (cal / mol mol) on the Y axis and temperature on the X axis (° C). From these results, the protein transition temperature (T _m ) and enthalpy (ΔH) were calculated using Multistate model.

1-3. ATR- FTIR  Spectroscopic analysis

Infrared spectra were measured using a Nicolet iS5 spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA) equipped with iD5 diamond ATR accessories. Was measured on iD5 diamond crystal plate by using a sample of about 10㎕, 4000 cm ^-1 ~600 cm in a 4 cm ^{-1 resolution} was carried out a total of 100 times in the ^first wavelength range. The spectrum of the sample was obtained by subtracting the spectrum of the buffer solution and peak evaluation was performed using Nicolet Omnic software.

The ratio of α-helix, β-sheet, β-turn and random coil in protein solution can be obtained by measuring the infrared spectrum in the amide I region. The curve fitting procedure is performed using the Gauss and Lorentz formulas provided by the OMNIC Peak Resolve software at the peaks of the Amide I region, and the regions represented by the respective secondary structures are represented by the ratios of the respective secondary structures in the total region .

1-4. R27T  Experimental design for solution optimization (Design of Experiment; DoE ) And robust design (RD)

(T _m ), enthalpy (? H), and Helix ratio (? /?) Were used to determine the optimum pH at which the protein can be most stably present. The initial data conversion process requires conversion of the measurement results in order to determine the optimum pH conditions in the four buffers, since the three measurement results have different units. As a result, all measurement results are linearly transformed to values between 1 and 2 through the following equations.

y _transformed = yy _min / y _max -y _min + 1

In the above equation, Y _min and Y _max represent the minimum value and the maximum value in the measurement result, and the measured value Y (T _m , Helix ratio (? /?), Enthalpy? A modified Response Surface Methodology (RSM) model is used to express the relation between Y value, buffer concentration and pH and to find the optimal point.

In the above equation, m ₀ , m ₁ , m _2, m ₁₂ and m ₂₂ are coefficients used in the second-order RSM, and RSM is usually a very complex or precise relationship between several output values and their associated input factors It is used to optimize when it is difficult to know the functional relation.

To optimize three measurement values at the same time, we used the robust design principle using the weighted-sum method. This method is the most universal method for producing an effective solution in the robust design. We use the proposed multi-objective robust design optimization model based on weighted sum method with importance level w1 ~ w3, This process can be used to evaluate the optimal R27T solution.

1-5. Size Exclusion Chromatography (SEC)

R27T samples were analyzed using an Agilent high performance liquid chromatography system (Agilent HPLC 1260, Santa Clara, CA, USA) equipped with a TSK-GEL G3000SWXL SEC column (TOSOH Bioscience, PA, USA) and a diode detector (DAD). (0.1% TFA (Trifluoroacetic acid) aqueous solution, 150 mM NaCl) and mobile phase B (0.1% TFA acetonitrile, 150 mM NaCl) at a ratio of 4: 6 at a flow rate of 0.5 mL / min Flow rate. The area analyzed in the sample as the peak of the multimer was calculated along with the area of the water-soluble aggregate. The difference in the total area of the R27T samples (sum of all peak area widths in the chromatogram results) measured after the initial and subsequent measurements was defined to be due to the formation of insoluble aggregates during the measurement. The remaining percentage of each species (insoluble aggregate, monomer, protein fragment) was calculated as the peak area compared with the initial time, and the storage period was plotted as the X axis. The formula for calculating the remaining ratio is as follows.

Remaining amount (%) = (a _{t /} A ₀ ) x 100

In the above equation, at means the width of the protein peak at each time, and A ₀ means the initial width of each protein peak. The error bars mean the standard deviation (SD) over three measurements.

Example  2: stable R27T  Concentration range analysis

2-1. R27T  Solution preparation

The purified R27T (about 24,742 Da) protein was stored in phosphate buffer (pH 2.9) at 4 ° C and the R27T solution was dialyzed at 4 ° C for 24 hours using a cellulose semipermeable membrane (minimum permeation molecular weight: 5000 Da). The buffer used for dialysis was phosphate buffer (pH 2.9, 3.6, 4.3, 5.0), citric acid buffer solution (pH 3.6, 4.3, 5.0, 5.7), acetic acid buffer solution (pH 3.6, 4.3, 5.0, 5.7), histidine buffer , 5.0, 5.7, 6.4), and the respective concentrations were 20 mM or 50 mM. Dialysis was carried out three times at intervals of 8 hours, and filtration was performed to remove the remaining impurity particles after dialysis (0.22 μm cellulose acetate, Advantec, Tokyo, Japan). Protein concentrations in each buffer solution after dialysis were measured at 282 nm using an ultraviolet spectrophotometer (Mecasys, Seoul, Korea).

2-2. R27T  The hydrodynamic size, Zeta potential  And PDI  Measure

To investigate the effects of protein concentration, pH, and buffer concentration on the electrostatic interactions between proteins during the formation of nano - sized aggregates, R27T was subjected to dynamic light scattering (DLS).

As a result, hydrodynamic size, zeta potential, and polydispersity index (PdI) at various R27T concentrations are shown in Table 1 below.

R27T
(mg / mL) Size Std. Zeta potential Std. PDI (nm, Vol%) (mV) 0.80 5.37 (99.99) 0.27 21.16 0.90 0.63 0.50 7.00 (99.99) 0.33 13.24 0.13 0.78 0.30 8.22 (99.80) 0.44 11.84 0.80 0.93 0.10 - - 3.60 1.15 1.00 0.05 - - 3.17 1.80 1.00

As shown in Table 1, the hydrodynamic size of R27T in the storage buffer of 0.80 mg / mL was about 5.37 ± 0.27 nm. As the concentration decreased, the hydrodynamic size increased to 0.10 and 0.05 mg / mL. < / RTI >

In addition, as the R27T concentration decreased, the absolute zeta potential decreased from -21.16 mV to -3.17 mV, because the electrostatic interactions were induced by protein aggregation between neighboring proteins. On the other hand, when the polydispersity index (PDI) is more than 0.7, it means that the polymer sample has a highly polydispersed heterogeneous distribution. In this example, the lower the concentration of R27T, the higher the polydispersity index Protein aggregation has occurred.

2-3. R27T  Depending on concentration Tm  Measure

Differential scanning calorimetry (DSC) was performed to evaluate the thermodynamic stability according to R27T concentration (0.8, 0.5, 0.3, 0.05 mg / mL). Concretely, enthalpy, which represents the energy maintaining the folded tertiary structure at the transition temperature (T _m ), constant pressure and volume, was evaluated as two parameters which play an important role in maintaining the tertiary structure of the protein.

As a result, as shown in the thermogram of FIG. 2, at a concentration of 0.8 mg / mL, the transition temperature (T _m ) of the R27T was 60.07 ° C and an endothermic peak was observed between 40 and 80 ° C. However, as the concentration of R27T decreased from 0.8 mg / mL to 0.05 mg / mL, T _m also decreased from 60.07 ° C to 50.68 ° C, which means that the lower the concentration, the lower the structural stability (T _m value) of R27T.

From the above results of DLS and DSC, it was found that the most stable R27T concentration in the storage buffer was about 0.80 mg / mL.

Example  3: Stable Of R27T  pH range analysis

The physico-chemical stability of R27T (0.8 mg / mL) was evaluated at various pH ranges using DLS-Zeta potential and DSC-transition temperature (T _m ).

First, from the above Example 2, it was confirmed that the high absolute zeta potential value is an important factor for inhibiting protein aggregation, so the change in zeta potential according to pH was measured. As a result, as shown in FIG. 3A, it was found that a relatively high electrical repulsion was observed at an acidic pH, and an experimental isoelectric point (pI) value at which the zeta potential reached zero was 5.84.

In addition, the pH effect on R27T stability (T _m ) was further measured as the protein exposed to extreme pH could lead to structural defects due to internal electrostatic forces and destruction of charge-charge interactions, The pH was adjusted from 2.0 to 11.0 using hydrochloric acid and sodium hydroxide during dialysis. As a result, the optimum pH range was found to be between 3.6 and 4.4 while the highest structural stability (T _m value of about 61 ° C) was shown, as shown in FIG. 3b.

Example  4: Stable Of R27T  Buffer analysis

4-1. Tm  Measure( DSC )

DSC was performed by adding various concentrations of acidic buffer (phosphoric acid, acetic acid, citric acid, histidine) at various pH ranges and the T _m was determined to determine the buffer system in which R27T was most stable.

As a result, as can be seen from the endothermic peak in FIG. 4, the structural stability (T _m ) of R27T varied with pH and buffer, and the highest T _m was observed in 50 mM acetic acid buffer (pH 3.6) 20 mM phosphate buffer (pH 2.9), 50 mM citrate buffer (pH 5.7), and 20 mM acetic acid buffer (pH 3.6). More specific results are shown in Table 2 below.

Buffer Conc. pH T _m
(° C) Enthalpy
(kJ / mol) alpha-helix (%) β-sheet (%) β-turn (%) Rd. Coil (%) α / β Acetate 20mM 3.6 62.59 21.51 48.20 17.24 11.29 23.27 2.80 20mM 4.3 59.84 20.99 30.31 26.33 23.70 19.66 1.15 20mM 5.0 58.52 30.68 29.59 30.59 28.00 12.05 0.97 20mM 5.7 57.74 15.20 25.97 26.56 29.73 17.74 0.98 50 mM 3.6 63.70 13.64 23.65 32.22 28.23 15.90 0.73 50 mM 4.3 59.06 10.63 21.33 32.77 29.82 16.07 0.65 50 mM 5.0 59.71 15.11 32.53 24.49 30.62 12.36 1.33 50 mM 5.7 59.38 11.82 30.43 31.98 30.09 7.50 0.95 Histidine 20mM 4.3 59.64 1.52 22.30 33.97 23.86 19.88 0.66 20mM 5.0 59.20 10.40 22.99 37.99 22.96` 16.06 0.60 20mM 5.7 58.65 12.21 31.16 39.76 19.49 9.59 0.78 20mM 6.4 56.90 11.03 33.45 29.28 23.12 14.12 1.14 50 mM 4.3 58.54 14.05 5.60 31.81 41.53 21.06 0.18 50 mM 5.0 60.15 12.10 20.50 41.58 21.30 16.62 0.49 50 mM 5.7 59.93 13.31 30.38 30.96 26.04 12.61 0.98 50 mM 6.4 57.95 11.61 9.89 29.51 38.87 21.72 0.34 Phosphate 20mM 2.9 63.40 16.07 41.50 26.81 17.35 14.34 1.55 20mM 3.6 60.67 18.09 19.62 23.79 34.35 22.24 0.82 20mM 4.3 60.43 20.60 7.36 24.96 42.56 25.13 0.29 20mM 5.0 59.59 16.56 8.52 24.23 42.63 24.62 0.35 50 mM 2.9 62.00 24.94 38.66 21.51 12.43 27.41 1.80 50 mM 3.6 60.46 17.80 22.72 26.31 30.39 20.57 0.86 50 mM 4.3 61.36 32.02 14.72 23.25 37.20 24.83 0.63 50 mM 5.0 60.99 15.23 21.34 25.07 31.26 22.32 0.85 Citrate 20mM 3.6 55.75 5.36 38.32 27.01 16.83 17.85 1.42 20mM 4.3 58.27 9.47 51.92 21.35 21.12 5.61 2.43 20mM 5.0 60.80 9.00 18.20 20.28 30.57 30.95 0.90 20mM 5.7 60.43 11.31 9.65 26.95 37.14 26.26 0.36 50 mM 3.6 54.51 9.28 19.00 40.70 29.67 10.63 0.47 50 mM 4.3 57.98 10.46 33.15 43.07 23.65 0.13 0.77 50 mM 5.0 61.51 15.48 34.32 40.91 14.91 9.87 0.84 50 mM 5.7 62.84 15.78 18.79 26.68 26.18 28.35 0.70

4-2. Relative content of α-helix (ATR- FTIR )

In addition to the thermodynamic properties obtained from the DSC data of Example 4-1, it is necessary to further consider the structural characteristics of the protein. Therefore, the ATR-FTIR can be used to convert α-helix, β-sheet, , and the ratio of random coil was measured.

Specifically, rhIFN-β is dominant in α-helix, and the increase in β-sheet content means formation of intermolecular β-sheets capable of inducing protein aggregation. The ionization state of amino acid side chains of proteins may cause structural changes The relative content of α-helix to the β structure was determined by performing ATR-FTIR by adding various concentrations of acidic buffers (phosphoric acid, acetic acid, citric acid, histidine) in various pH ranges.

As a result, as shown in Table 2, the highest α-helix content in 20 mM acetic acid buffer (pH 3.6), followed by 20 mM citrate buffer (pH 4.3) and 50 mM phosphate buffer Respectively.

4-3. Robust Design (RD)

Since the three measurement results of the protein transition temperature (T _m ), enthalpy (? H) and Helix ratio (? /?) Obtained in Examples 4-1 and 4-2 have different units, In order to determine optimal pH conditions in acidic buffers (phosphoric acid, acetic acid, citric acid, histidine) environments, it is necessary to convert the measurement results. To do this, a multi-objective robust design (RD) was performed and the results are shown in FIG.

To illustrate the pattern of the four buffers, three reaction and objective functions are plotted. Here, T _m , Helix ratio (α / β), and enthalpy (ΔH) represent the solubilities associated with structural stability, secondary structural stability, and heat capacity, respectively, and all objective functions of the four buffers have different concave patterns Can be seen.

Also, 19 weighting scenarios were investigated in order to select the optimal buffer, buffer concentration, and respective pH values by simultaneously evaluating the three reaction functions, and the results are shown in Table 3 below.

Scenarios T _m Relative helix content Enthalpy w ₁ w ₂ w ₃ One 0.65 0.30 0.05 2 0.65 0.25 0.10 3 0.65 0.20 0.15 4 0.60 0.35 0.05 5 0.60 0.30 0.10 6 0.60 0.25 0.15 7 0.55 0.40 0.05 8 0.55 0.35 0.10 9 0.55 0.30 0.15 10 0.55 0.25 0.20 11 0.50 0.45 0.05 12 0.50 0.40 0.10 13 0.50 0.35 0.15 14 0.50 0.30 0.20 15 0.45 0.40 0.15 16 0.45 0.35 0.20 17 0.45 0.30 0.25 18 0.40 0.35 0.25 19 0.33 0.33 0.33

Since T _m is the most important factor indicating the thermodynamic stability of the protein, it gives the highest weight, selects 19 scenarios of different weights assigned to the three factors, and determines the stability of the protein solution, The correlation was investigated. Finally, the optimization results for the 19 scenarios are shown in Table 4 below.

Optimal solutions Buffers Acetate Citrate Hisitidine Phosphate Acetate Citrate Histidine Phosphate Scenarios C pH C pH C pH C pH Objective functions One 20.00 3.60 50.00 5.67 50.00 5.32 20.00 2.90 1.887 1.659 1.449 1.799 2 20.00 3.60 50.00 5.70 50.00 5.30 20.00 2.90 1.875 1.675 1.463 1.794 3 20.00 3.60 50.00 5.70 50.00 5.29 20.00 2.90 1.864 1.692 1.476 1.789 4 20.00 3.60 50.00 5.59 20.00 5.14 20.00 2.90 1.886 1.623 1.430 1.779 5 20.00 3.60 50.00 5.66 50.00 5.32 20.00 2.90 1.874 1.637 1.441 1.774 6 20.00 3.60 50.00 5.70 50.00 5.30 20.00 2.90 1.863 1.654 1.454 1.769 7 20.00 3.60 50.00 5.51 20.00 5.19 20.00 2.90 1.884 1.588 1.415 1.759 8 20.00 3.60 50.00 5.57 50.00 5.34 20.00 2.90 1.873 1.602 1.419 1.754 9 20.00 3.60 50.00 5.64 50.00 5.32 20.00 2.90 1.862 1.616 1.433 1.750 10 20.00 3.60 50.00 5.70 50.00 5.30 20.00 2.90 1.851 1.632 1.446 1.745 11 20.00 3.60 50.00 5.43 20.00 5.24 20.00 2.90 1.883 1.556 1.401 1.740 12 20.00 3.60 50.00 5.49 20.00 5.26 20.00 2.90 1.872 1.568 1.402 1.735 13 20.00 3.60 50.00 5.55 50.00 5.34 20.00 2.90 1.861 1.581 1.411 1.730 14 20.00 3.60 50.00 5.62 50.00 5.32 20.00 2.90 1.849 1.595 1.424 1.725 15 20.00 3.60 50.00 5.46 50.00 5.36 20.00 2.90 1.860 1.548 1.389 1.710 16 20.00 3.60 50.00 5.53 50.00 5.34 50.00 2.90 1.848 1.560 1.402 1.710 17 20.00 3.60 50.00 5.60 50.00 5.32 50.00 2.90 1.837 1.574 1.416 1.715 18 20.00 3.60 50.00 5.51 50.00 5.34 50.00 2.90 1.836 1.539 1.394 1.705 19 20.00 3.60 50.00 5.49 50.00 5.34 50.00 2.90 1.816 1.516 1.387 1.699

(C: buffer concentration)

By comparing the optimal values of the objective function obtained from the four buffers, it is possible to select the optimum pH value and the buffer solution capable of producing an optimal preparation. Based on the results shown in FIG. 4 and Table 4, mM acetic acid buffer can be used as the optimal formulation.

Example  5: Optimal pH analysis in acetate buffer

Since it was confirmed from Example 4 that the acetic acid buffer solution is the optimum buffer solution for the R27T preparation, further experiments were conducted using acetic acid buffer solution.

5-1. SEC: Amount of monomer  analysis

First, the optimal pH value was 3.6 in the pH range of 3.6, 4.3, 5.0, and 5.7 tested in acetic acid buffer, but the lower pH may cause skin adverse effects in the subcutaneous injection and therefore increase the pH of the preparation for stability There is a need. For this reason, in order to further evaluate the pH effect on the storage stability of the preparation, the R27T preparation was stored at 37 ° C for 11 days in the pH range of 3.4 to 4.4, and then subjected to SEC (size exclusion chromatography) The chromatograms of the monomers were compared daily.

As a result, as shown in FIG. 6, as the pH was increased from 3.4 to 4.4 by 0.2, the residual amount of monomer of R27T increased during the storage period of 11 days. The increased R27T stability results showed low stability at pH 3.4 with only 4% monomer residues on day 11, but as the pH increased from 3.6 to 3.8, 4.0, 4.2, and 4.4, the remaining 11-day monomer remained at 21.35 %, 22.59%, 23.42%, 24.93%, and 25.10%, respectively. The increase in the amount of monomer means the decrease of aggregate, so that the protein stability increases with increasing pH from 3.4 to 4.4.

5-2. DSC  : T _m  analysis

The DSC analysis of R27T was performed while increasing the acetic acid buffer from pH 3.4 to 4.4 by 0.2. As a result, as shown in FIG. 7, it was found that as the pH was increased, the T _m was increased and the structural stability was increased.

Example  6: Additive with excipient R27T  Stability of formulation Promotion  analysis

6-1. R27T  Manufacture of pharmaceutical preparations

To prepare R27T preparations with enhanced stability by adding excipients to R27T protein and buffer solutions, two different formulations (Formulation A and Formulation A) were prepared and then the existing interferon beta formulations Rebif and Stability was compared. To this end, the compositions of the two R27T formulations are shown in Table 5 below and were prepared as follows.

Contents Key excipient Excipients A Formulation 250 mM Mannitol Poloxamer 188 (0.5 g / mL)
Methionine (0.12 g / L),
Benzyl alcohol (4.765 mL / L) B Formulation 150 mM Arginine HCl Polysorbate 20 (0.005%)

First, the purified R27T protein at a concentration of 0.8 mg / mL was stored in phosphate buffer (pH 2.9) at 4 ° C and the R27T solution was incubated at room temperature for 12 hours at room temperature using Cellu Sep ^® H1 cellulosic semipermeable membrane For a period of time. The buffer used for dialysis was 1 L of acetate buffer (pH 4.2) at a concentration of 20 mM. At this time, dialysis was performed by adding buffer and excipient according to the composition shown in Table 5 above. After the completion of the dialysis, the remaining impurity particles were removed by filtration using 0.22 μm cellulose acetate (Advantec, Tokyo, Japan).

6-2. DSC  : T _m  analysis

The structural stability of R27T was evaluated by performing DSC on the two kinds of R27T preparations prepared in Example 6-1 and the existing Rebif preparations of interferon beta, and measuring T _m .

As a result, as shown in FIG. 8A, the T _m value was measured to be higher than the Rebif preparation (reference), and the A formulation was measured to have a lower T _m than the Rebif agent. Higher.

Further, in order to compare the structural stability according to the type of excipient, R27T preparations were prepared in the same manner as described in Example 6-1, except that only one excipient listed in Table 5 was added to the buffer solution to prepare the R27T preparation DSC was performed.

As it is shown in the T _m measurements, Figure 8b, when the addition of benzyl alcohol (Benzyl alcohol) as an excipient is found to decrease T _m value (T _m = 56.74) bore benzyl that of alcohol decreases the stability of the R27T formulation While stability of the preparation was confirmed to be improved when Mannitol, Methionine, Poloxamer 188, or Polysorbate 20 was added. Specific results are shown in Table 6 below.

T _m (° C) SD A formulant 58.08 0.12 B formulations 59.47 0.02 Mannitol 61.06 0.04 Methionine 60.44 0.02 Poloxamer 188 60.71 0.02 Benzyl alcohol 56.74 0.02 Arginine 58.62 0.036 Polysorbate 20 59.84 0.02 Reference 58.88 0.04

6-3. SEC: Amount of monomer  analysis

In addition to the results of the structural stability evaluation by DSC analysis, in order to evaluate the storage stability of the preparation prepared by adding the excipient, the formulations A and B prepared in Examples 6-1 and 6-2 and each excipient Were added to each other, and the amount of the residual monomer was measured every day while the R27T preparation was stored at 40 캜 for 9 days at a concentration of 50 / / mL.

As a result, as shown in FIG. 9, when the A formulations and the B formulations were compared with each other in the same manner as the DSC results, it was confirmed that the residual monomer amount was higher in the B formulations. In addition, in the case of Formulation A, the monomer content of mannitol-added formulations was significantly higher than that of Formulations A and B. In the case of Formulation B, arginine or polysorbate 20 It was confirmed that the amount of monomer remaining in the B formulations prepared by adding them together was lower than that of the added formulations. This suggests that mannitol improves the safety of the R27T preparation and that the mixing of arginine and polysorbate reduces the storage stability of the formulation.

These results suggest that B-formulations of arginine as a major excipient improves stability compared to conventional interferon preparations and that mannitol has an excellent effect of enhancing the stability of interferon. In addition, It was expected to contribute greatly.

Example  7: R27T  Screening of excipients in formulations

7-1. Various excipients added R27T  Manufacture of pharmaceutical preparations

Based on the results obtained in Example 6 above, R27T preparations were prepared by adding a variety of excipients, and their safety was evaluated to screen for optimal excipients. To this end, the types and concentrations of excipients used in the preparation of the R27T preparation are shown in Table 7 below. R27T preparations were prepared according to the following procedure.

Content
Concentration R27T 1 mg / mL Amino acids One Arginine HCl 50, 100, 150 mM 2 Lysine HCl 50, 100, 150 mM 3 Methionine 50, 100, 150 mM Surfactants 4 Poloxamer 188 0.25, 0.5, 0.1 mg / mL Carbohydrates 5 Mannose 50, 150, 250 mM 6 Mannitol 50, 150, 250 mM 7 Sucrose 50, 150, 250 mM 8 Xylitol 50, 150, 250 mM 9 Sorbitol 50, 150, 250 mM

First, the purified R27T protein at a concentration of 1.0 mg / mL was stored in phosphate buffer (pH 2.9) at 4 ° C and the R27T solution was stored at 4 ° C using a Cellu Sep ^® H1 cellulose semipermeable membrane (minimum permeable molecular weight: 5000 Da) Dialyzed while stirring for 12 hours. The buffer used for the dialysis was 1 L of acetate buffer (pH 4.2) at a concentration of 20 mM. At this time, each of the excipients shown in Table 7 was added to the buffer solution at different concentrations and then dialyzed. After the completion of the dialysis, the remaining impurity particles were removed by filtration using 0.22 μm cellulose acetate (Advantec, Tokyo, Japan).

7-2. DSC  : Tm  analysis

Each of the R27T preparations prepared in Example 7-1 was subjected to DSC analysis to evaluate the structural stability of R27T.

As a result, as shown in the following Table 8, when compared with the T _m value of the control without addition of excipient (Control), in the case of the preparation containing arginine and the addition of poloxamer 188 in the amino acid excipient T _m values were measured to be slightly higher, indicating that the excipients slightly enhance the structural stability of the R27T formulation. Among the carbohydrate excipients, it was confirmed that the stability of the preparation prepared by adding sucrose was improved compared with the control.

Samples T _m (° C) Samples T _m (° C) Control 57.82 Mannose 50 mM 57.01 150 mM 57.77 250 mM 57.59 Arginine HCl Mannitol 50 mM 58.13 50 mM 56.67 100 mM 58.02 150 mM 56.28 150 mM 57.94 250 mM 56.98 Lysine HCl Sucrose 50 mM 58.05 50 mM 56.73 100 mM 57.56 150 mM 58.53 150 mM 58.16 250 mM 54.37 Methionone Sorbitol 50 mM 57.88 50 mM 56.93 100 mM 57.88 150 mM 57.61 150 mM 58.49 250 mM 57.02 Poloxamer 188 Xylitol 0.25 mg / mL 58.00 50 mM 56.33 0.5 mg / mL 57.86 150 mM 56.93 1 mg / mL 57.31 250 mM 56.34

7-3. SEC: Amount of monomer  And Aggregate amount  analysis

To evaluate the storage stability of the R27T preparation prepared by adding each excipient, the same R27T formulation as in the DSC analysis sample of Example 7-2 was diluted to a concentration of 1.0 mg / mL at 40 占 폚 for 7 days or 4 占 폚 And the amount of residual monomer and aggregation was measured. The results are shown in Tables 9 to 11. < tb > < TABLE >

40 ℃ Content Concentration / Monomer (%) after 7 days R27T Low Medium High One Arginine HCl 84.33 69.34 67.42 2 Lysine HCl 79.30 64.98 63.00 3 Methionine 75.02 74.84 78.72 4 Poloxamer 188 81.78 76.02 77.42 5 Mannose 62.89 66.17 77.63 6 Mannitol 65.43 67.37 64.24 7 Sucrose 66.89 64.03 86.19 8 Sorbitol 61.33 68.72 53.41 9 Xylitol 68.42 76.08 78.92

40 ℃ Content Concentration / aggregation (%) after 7 days R27T Low Medium High One Arginine HCl 8.85 11.14 14.90 2 Lysine HCl 9.54 13.26 13.37 3 Methionine 7.26 6.80 6.86 4 Poloxamer 188 9.36 7.70 9.01 5 Mannose 9.89 12.80 15.51 6 Mannitol 16.42 12.51 12.37 7 Sucrose 14.13 14.52 22.55 8 Sorbitol 11.77 11.54 9.87 9 Xylitol 11.64 15.29 15.69

4 ℃ Content Concentration / Monomer (%) after 16 days R27T Low Medium High One Arginine HCl 95.01 92.78 91.83 2 Lysine HCl 92.59 92.17 91.64 3 Methionine 95.12 94.45 95.28 4 Poloxamer 188 98.27 98.82 98.92 5 Mannose 91.75 95.53 91.18 6 Mannitol 93.58 92.96 89.65 7 Sucrose 83.3 92.04 124.15 8 Sorbitol 92.82 86.11 68.74 9 Xylitol 93.39 79.79 104.58

As shown in Tables 9 to 11, when the R27T preparation was stored at 4 占 폚 for 16 days, it was confirmed that the R27T protein did not aggregate regardless of the type of excipient added. In addition, when the results according to the above two storage conditions were taken into consideration, it was found that the residual amounts of the monomers in the formulations containing arginine or poloxamer 188 were high, These results corresponded to the DSC analysis results. Among the carbohydrate excipients, the residual monomer content was high when sucrose or xylitol was added at two temperature conditions, but the aggregates were formed at a high rate when the sucrose was added at high temperature of 40 ℃. It was concluded that Mannitol could be substituted by sucrose or xylitol with coagulation inhibitor.

Example  8: R27T  Optimization of stabilizing agent composition

Based on the results of the above examples, an attempt was made to establish an optimum composition of the formulation that could enhance the stability of R27T.

8-1. Optimize buffer conditions

First, it was confirmed through the above Examples 4 and 5 that the buffer solution for improving the stability of the R27T preparation was an acetic acid buffer solution in the range of pH 3.4 to 4.4. The buffer solution concentration and the pH were varied, Respectively. More specifically, the R27T preparation was prepared by varying the concentration of acetic acid buffer at a concentration of 10, 20, 50 mM and varying the pH to 3.8 or 4.2, and incubated at 4 ° C or 25 ° C for 1 week or 2 weeks week, respectively. SEC analysis was performed to determine the residual amount of monomer and aggregate.

As a result, as shown in Fig. 10, when the 10 mM or 20 mM acetic acid buffer solution at pH 3.8 was used, the residual monomer amount was the highest and the residual amount of aggregate was the lowest. Using the acetic acid buffer under the above conditions, It was confirmed that the optimum condition for the improvement of.

8-2. R27T  Optimization of stabilizing agent composition

In order to set the optimum composition of the R27T preparation, the R27T preparation was prepared according to the method of Example 7-1, wherein the buffer solution was adjusted to 20 mM of pH 3.8, which is the optimal condition for enhancing the stability of the preparation, Acetic acid buffer, and arginine HCl, poloxamer 188, and methionine, which were confirmed to enhance the stability of the R27T preparation, were added to the excipients in Examples 6 and 7, and mannitol was added to evaluate the mixing effect of arginine and mannitol Respectively. The composition of the excipients added is shown in Table 12 below.

Formulation Contents F1 (Control) - - - - F2 (Rebif formulation) - - - - F3 Arginine
50 mM Poloxamer 188
0.5 mg / mL Methionine
1 mM F4 Arginine
50 mM Mannitol
150 mM Poloxamer 188
0.5 mg / mL Methionine
1 mM F5 Arginine
50 mM Mannitol
250 mM Poloxamer 188
0.5 mg / mL Methionine
1 mM F6 Arginine
100 mM Poloxamer 188
0.5 mg / mL Methionine
1 mM F7 Arginine
100 mM Mannitol
150 mM Poloxamer 188
0.5 mg / mL Methionine
1 mM F8 Arginine
100 mM Mannitol
250 mM Poloxamer 188
0.5 mg / mL Methionine
1 mM

To evaluate the storage stability of the R27T preparations (F1 to F8) of eight different compositions shown in Table 12 above, each formulation was diluted with either low concentration (100 / / mL) or high concentration (640 / / C for 14 days, and SEC was performed to measure the residual amount of monomer. The results are shown in FIG. 11 and Table 13 below.

Formulation Condition F1 (Control) contrast F2 contrast Condition F1 (Control) contrast F2 contrast F1 (Control) Day14,
4 ℃ 100.00 89.73 Day14,
37 ℃ 100.00 85.61 F2 (Rebif formulation) 111.45 100.00 116.81 100.00 F3 109.38 98.14 101.97 87.30 F4 102.22 91.72 98.10 83.99 F5 115.68 103.80 107.66 92.17 F6 104.66 93.91 93.45 80.00 F7 121.15 108.71 105.92 90.68 F8 112.50 100.95 99.66 85.32

As a result of measurement of residual monomer amount, as shown in Fig. 11, the residual monomer amount was highest at F5 and F7 for low concentration agent and F5 for high concentration agent at 4 DEG C, respectively. Also, as shown in Table 13, it was confirmed that the storage stability of arginine and mannitol-containing F5 and F7 preparations was higher than that of conventional Rebif preparations. However, the stability of the preparation decreased at high temperature (37 ° C) and high concentration (640 μg / ml).

Based on the above results, the stability of the preparations was evaluated after storage for a longer period at low temperature. For this, the formulations of each composition shown in Table 12 were stored at 4 < 0 > C for 28 days, and SEC was performed at 7-day intervals to determine the residual monomer content.

As a result, as shown in Fig. 12 and Table 14, it was confirmed that the formulation with the composition of F5 and F7 had better storage stability than the conventional Rebif preparation (F2) under the low-temperature storage condition.

Formulation Condition F2% (%) F1 (Control) Day28,
4 ℃ 98.85 F2 (Rebif formulation) 100.00 F3 95.96 F4 91.46 F5 101.45 F6 92.73 F7 108.26 F8 97.56

Further, the anti-viral effect of R27T according to each formulation treatment was measured by the CPE (Cytopathic effect) assay in the above-mentioned eight compositions. More specifically, 100 쨉 l of the medium containing 3x10 ⁵ cells / ml of A549 cells in a 96-well plate was treated with 100 쨉 l of each preparation, and the cells were cultured at 37 째 C for 22 hours. On the second day, the supernatant was removed, and 100 μl of encephalomyocarditis virus (EMCV) at a concentration of 1000 TCID50 / mL was treated with the cells and cultured at 37 ° C. for 22 hours. After 3 days, the supernatant was removed, , Absorbance was measured at 570 nm, and the titer to standard was calculated.

As a result, as shown in Fig. 13, it was confirmed that the preparation having the F5 composition had about 40% more activity than the control (control). From the above results, it was confirmed that the composition of F5 is the optimal formulation to enhance the stability and activity of R27T.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (12)

A stabilized pharmaceutical formulation of a human interferon beta variant (R27T) comprising:
(a) a human interferon beta mutant (R27T),
(b) an acetic acid buffer solution at a concentration of 5 to 100 mM,
(c) arginine at a concentration of 10 to 150 mM,
(d) mannitol at a concentration of 50 to 300 mM,
(e) Poloxamer 188 at a concentration of 0.1 to 10 mg / mL, and
(f) Methionine at a concentration of 0.5 to 5 mM.
2. The human interferon beta mutant (R27T) according to claim 1, wherein the human interferon beta mutant (R27T) comprises an N-linked sugar chain in the asparagine residue, which is the 25th amino acid, arginine, which is the 27th amino acid of human interferon beta, ≪ / RTI >
2. The stabilized pharmaceutical preparation according to claim 1, wherein the acetic acid buffer solution is contained at a concentration of 10 to 30 mM.
The stabilized pharmaceutical preparation according to claim 1, wherein the acetic acid buffer has a pH in the range of 3.6 to 4.4.
The stabilized pharmaceutical preparation according to claim 1, wherein the arginine is contained in a concentration of 50 to 100 mM.
The stabilized pharmaceutical preparation according to claim 1, wherein the mannitol is contained at a concentration of 150 to 250 mM.
The stabilized pharmaceutical preparation according to claim 1, wherein the poloxamer 188 is contained at a concentration of 0.1 to 1 mg / mL.
2. The stabilized pharmaceutical preparation according to claim 1, wherein the methionine is contained at a concentration of 0.5 to 2 mM.
2. The stabilized pharmaceutical preparation according to claim 1, wherein the stabilized pharmaceutical preparation has a pH in the range of 3.6 to 4.4.
The pharmaceutical composition according to claim 1, wherein the stabilized pharmaceutical preparation is for the prevention or treatment of diseases selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infection diseases, HIV infection diseases, hepatitis C, and rheumatoid arthritis ≪ / RTI >
2. The stabilized pharmaceutical preparation according to claim 1, wherein the stabilized pharmaceutical preparation is for oral or parenteral administration.
2. The stabilized pharmaceutical preparation according to claim 1, wherein the stabilized pharmaceutical preparation is a liquid or lyophilized formulation.

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