WO2016163764A2 - Stabilized preparation of interferon beta variant - Google Patents

Abstract

The present invention relates to a stabilized pharmaceutical preparation of R27T comprising a human interferon beta variant (R27T), an acetic acid buffer solution, arginine, mannitol, poloxamer 188, and methionine. The stabilized R27T pharmaceutical preparation according to the present invention comprises an acetic acid buffer solution, arginine, mannitol, poloxamer 188, and methionine, and thereby inhibits formation of an aggregate of an R27T protein and enables long-period storage because of an improvement in thermodynamic and structural stability. Thus, it is expected that the stabilized R27T pharmaceutical preparation will be useful in prevention, improvement and treatment of multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C, rheumatoid arthritis, etc.

Description

Stabilizing Agents of Interferon Beta Variants

The present invention relates to a stabilized pharmaceutical formulation 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 exhibit antiviral activity, inhibit cell proliferation and modulate natural immune responses. IFNs are classified into IFN-α, IFN-β, and IFN-γ according to their cell origin (leukocytes, fibroblasts, T cells). Among them, interferon-beta (IFN-β) has five alpha-helix (α-helix). Is a spherical protein with a size of 22 kD and the sugar chains are 18 kD.

Research on the clinical application of IFN-β is actively underway, particularly in the treatment of alleviation, alleviation or treatment of symptoms for multiple sclerosis, as well as antiviral activity, cell growth inhibition, lymphocyte cells. Toxicity enhancing activity, immunomodulatory activity, target cell differentiation induction or inhibition activity, macrophage activation, cytokine production activity, cytotoxic T cell effect increase activity, natural killing cell increase activity, etc. Has been reported to be effective in the treatment of cancer, autoimmune disorders and viral infections, HIV-associated diseases, hepatitis C, and rheumatoid arthritis.

Human IFN-β is also a type of glycoprotein. Since the sugar chain portion linked to the protein plays an important role in the activity of the protein, the glycoprotein may increase its activity when the sugar chain is added. That is, protein glycosylation is known to affect many biochemical properties such as stability, solubility, intracellular trafficking activity, pharmacokinetics and antigenicity.

In this regard, an example of preparing a human IFN-β variant having increased or improved activity or function by introducing a sugar chain into a human glycoprotein, human native IFN-β (Korean Patent Registration 10-0781666) has been reported. R27T is a recombinant human IFN-β variant (hereinafter referred to as rhINF-β) designed by replacing arginine (Arg) at position 27 with threonine (Thr) for further glycosylation at position 25 of IFN-β 1a as shown in FIG. ), Increased stability, decreased protein aggregation tendency, and increased half-life when compared to wild-type INF-β1a (Rebif). In other words, R27T is a biobetter version of rhINF-β produced by additional glycosylation through site-directed mutations.

On the other hand, one of the major challenges in the development of protein pharmaceuticals is to provide products with a long shelf life of more than 24 months by giving them sufficient chemical, physical and biological stability. However, stability is still difficult to achieve due to the inherent susceptibility of the proteolytic pathway and the complexity of protein structures with varying levels of protein macromolecules, secondary, tertiary and quaternary structures.

More than 30 years since insulin was first approved and successfully produced in 1982 as the first recombinant peptide hormone, success stories of numerous recombinant protein / peptide medicines have been reported. However, in the development of biopharmaceuticals, especially in formulation, there are still many challenges due to various factors such as protein aggregation, physicochemical instability, low half-life, low solubility and pharmacokinetic properties.

In particular, protein aggregation is one of the major problems that occur easily in almost all biopharmaceutical processes during storage because therapeutic proteins are structurally / thermodynamically unstable in solution. Since therapeutic proteins are sensitive to structural changes due to a variety of factors during purification, processing and storage, these problems may be exacerbated when the proteins are exposed to high temperatures, peak / low pH, shear strain and surface adsorption. . In addition, protein-based biopharmaceuticals have the potential for physical degradation, such as insoluble granulation due to unfolding, agglomeration, and non-native agglomeration, so as to avoid protein aggregation or physical denaturation and to maximize stability. Formulation system optimization is required, including stable pH ranges, proper buffer systems, and development of excipients.

Conventionally, human growth hormone, human epidermal growth using various biophysical methods such as Dynamic Light Scattring (DLS), Differential Scanning Calorimetry (DSC), Circular Dichroism (CD), Size Exclusion Chromatography (SEC), and ATR-FTIR Efforts have been made to produce protein preparations, such as factors and antibodies, quickly and efficiently, and have been used primarily to observe the diversity of protein structures under various solution conditions such as the pH, buffer, buffer concentration, protein concentration and excipients of the solution. However, because many data can be difficult to interpret due to diversity and complexity, new data analysis methods and stabilization systems are needed to find coherence between derived results and to show meaningful associations to define the physical state of the target protein.

Accordingly, the present inventors have studied to suppress the formation of aggregates of the R27T protein, which is an interferon beta variant, and to improve the thermodynamic and structural stability, thereby improving its usefulness as a stabilized pharmaceutical agent. The invention was completed.

It is therefore an object of the present invention to provide a stabilized pharmaceutical formulation of human interferon beta variant (R27T) of novel composition.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object of the present invention, the present invention is human interferon beta variant (R27T), acetic acid buffer of 5 to 100 mM concentration, arginine of 10 to 150 mM concentration (Arginine), mannitol of 50 to 300 mM concentration (Mannitol), poloxamer 188 at a concentration of 0.1 to 10 mg / mL, and methionine at a concentration of 0.5 to 5 mM, to provide a stabilized pharmaceutical formulation of human interferon beta variant (R27T).

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

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

In another embodiment of the present invention, the acetic acid buffer is characterized in that the pH ranges from 3.6 to 4.4.

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

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

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

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

In another embodiment of the invention, the stabilized pharmaceutical formulation is characterized in that the pH ranges from 3.6 to 4.4.

In another embodiment of the present invention, the stabilized pharmaceutical formulation is prevented or treated for the disease selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C, and rheumatoid arthritis Characterized in that.

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

In another embodiment of the invention, the stabilized pharmaceutical formulation is characterized in that the liquid or lyophilized formulation.

Stabilized R27T pharmaceutical formulations according to the present invention include acetic acid buffer, arginine, mannitol, poloxamer 188, and methionine to inhibit the formation of aggregates of R27T protein and improve long-term storage by improving thermodynamic and structural stability. It is expected to be useful for the prevention, improvement and treatment of sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C, and rheumatoid arthritis.

1 shows the gene and protein sequences of human interferon beta variant (R27T).

FIG. 2 shows the results of evaluating thermodynamic stability according to the concentration of R27T (0.8 mg / mL; 0.5 mg / mL; 0.3 mg / mL; 0.05 mg / mL) using the differential scanning calorimeter (DSC).

3A and 3B show physicochemical stability of R27T (0.8 mg / mL) at various pH ranges using DLS-zeta potential (FIG. 3A) and DSC-transition temperature (FIG. 3B).

Figure 4 shows the results of evaluating the optimal buffer for R27T by adding four acidic buffers (phosphate, acetic acid, citric acid, histidine) and then performing DSC to measure T _m .

FIG. 5 shows the results of evaluating optimal pH conditions in four acid buffers (phosphate, acetic acid, citric acid, histidine) using a multi-objective robust design method (RD).

FIG. 6 shows that the R27T formulation containing 20 mM acetic acid buffer at various pHs was stored at 37 ° C. for 11 days, followed by size exclusion chromatography (SEC) to determine the amount of residual monomer in the protein solution. The results of evaluating the pH effect on the storage stability of the formulation.

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 T _m by performing DSC analysis on the R27T formulation containing 20 mM acetic acid buffer of pH 3.4 to 4.4.

8A and 8B show the T _m measured by DSC analysis for each R27T formulation containing 20 mM acetic acid buffer at pH 4.2 and other types of excipients, with mixed addition of excipients (FIG. 8A) and single The evaluation result of the structural stability of the R27T formulation according to the addition (Fig. (Reference: Existing Rebif Formulations; A Formulation: Formulations containing Mannitol, Poloxamer 188, Methionine, and Benzyl alcohol; B Formulation: Formulations containing Arginine HCl and Polysorbate 20).

FIG. 9 shows a mixed addition of excipients by measuring the residual monomer amount of protein solution by performing SEC while storing each R27T formulation containing 20 mM acetic acid buffer at pH 4.2 and other types of excipients at 40 ° C. for 9 days. Results of evaluating the storage stability of the R27T formulation with addition.

FIG. 10 shows that the R27T stabilizing agent was added at 4 ° C. or 25 ° C. at different concentrations of acetic acid buffer (10, 20, and 50 mM) and pH (3.8, 4.2) to optimize the composition of the R27T stabilizing agent. After storage for 2 weeks, SEC was performed to measure the amount of residual monomer and aggregates in the protein solution, thereby evaluating the concentration and pH effect of the acetic acid buffer on the storage stability.

FIG. 11 shows a low concentration (100 μg / mL) or high concentration (640 μg / mL) of an R27T preparation (F1-F8) containing 20 mM acetic acid buffer, pH 3.8, arginine, poloxamer 188, and mannitol in various compositions. After 14 days of storage at 4 ° C or 37 ° C, SEC was performed to measure the amount of residual monomer in the protein solution, thereby evaluating the optimal composition with improved storage safety.

FIG. 12 shows R27T formulations (F1-F8) containing 20 mM acetic acid buffer, pH 3.8, arginine, poloxamer 188, and mannitol in various compositions at low temperature (4 ° C) for a long time (0-28 days). It is the result of evaluating the optimal composition with improved storage safety by measuring the residual monomer amount of the protein solution by performing SEC at one interval.

FIG. 13 shows a control after performing a CPE (Cytopathic effect) assay on R27T preparations (F1 to F8) to which 20 mM acetic acid buffer pH, pH 3.8, arginine, poloxamer 188, and mannitol were added in various compositions. It is the result of comparing the activity of each formulation on the basis.

The present invention, human interferon beta variant (R27T) at a concentration of 0.3 to 1.0 mg / mL (R27T) and acetic acid buffer of 5 to 100 mM concentration, arginine (Arginine) of 10 to 150 mM concentration, mannitol (Mannitol) of 50 to 300 mM concentration ), Stabilized pharmaceutical formulation of human interferon beta variant (R27T), comprising poloxamer 188 at a concentration of 0.1 to 10 mg / mL, and methionine at a concentration of 0.5 to 5 mM.

One of the most fundamental problems in the preparation of protein-based formulations is the determination of the desired therapeutic protein concentration in the solution, which depends not only on protein stability but also on the pH, temperature, ionic strength and additive concentration of the solution. Because. Thus, protein based formulations should be suitable for stabilizing therapeutic proteins and should avoid problems such as aggregation, precipitation, or fragmentation.

R27T is a recombinant human IFN-β variant (hereinafter referred to as rhINF-β) designed by replacing arginine (Arg) at position 27 with threonine (Thr) for further glycosylation at position 25 of IFN-β 1a, In order to develop a formulation for the stabilization of R27T, various analytical methods such as DSC, DLS, FT-IR, SEC were used.

Glycosylation is known to increase solubility of many proteins, and glycation stability is pH dependent. Therefore, identifying suitable pH and buffers is important to overcome stability issues and to obtain optimal stability at various stages of the development process, including drug production, purification, storage and release.

In the present invention, by using the differential scanning calorimetry (DSC, differential scanning calorimetry) and dynamic light scattering (DLS, dynamic light scattering) in advance to select a solution of pH 2-11, the experimental pI and the optimal pH range of R27T We confirmed that they were 5.8 and 3.6-4.4, respectively.

In addition, Design of experiment (DoE) was used to obtain a basal buffer system in the pH range from 2.9 to 5.7 with various concentrations of acetic acid, histidine, phosphoric acid, and citric acid buffer. The DoE approach was developed as a practical method for formulation studies as a function of pH 2.9-5.7, buffers (phosphate, acetic acid, citric acid, histidine), buffer concentrations (20 mM and 50 mM). This method used a weight-based procedure to interpret complex data results and examine key factors indicative of protein stability. In this case, the factors used are T _m , enthalpy, DSC and helix ratio obtained through Fourier Transform Infrared Spectroscopy (FT-IR). The weights were varied by these three factors, but among all scenarios in which the evaluation was performed, the highest objective function appeared in 20 mM acetic acid buffer at pH 3.6, followed by phosphate buffer at pH 2.9. This means that the thermal stability (T _m and enthalpy) and secondary structural stability (relative helix content) are optimal in these buffers.

However, since these buffers are too low in pH to cause skin side effects in patients, the dosage form obtained from DoE is not sufficient for subcutaneous injection, so the pH value of R27T is adjusted with acetic acid buffer considering thermal stability, secondary structure stability, and storage stability. Further studies that could increase were performed. In other words, Size Exclusion Chromatography (SEC) was used to optimize pH values with acceptable stability.

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

rhIFN-β is a 166 amino acid glycoprotein with a 4-helix bundle domain. Increasing the β-sheet content implies intermolecular β-sheet formation that can induce protein aggregation. As a result of analyzing the α-helix and β-sheet content of R27T in the optimum pH range of acetic acid buffer, it was found that the optimal pH value for the thermal stability and the secondary structural stability of R27T was pH 3.8 ± 0.2.

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

Further, in order to find excipients that can further enhance the stability of R27T, R27T formulations were prepared by mixing or separately adding various excipients with 20 mM acetic acid buffer at pH 4.2 and performing experiments through T _m and SEC analysis. As a result, mannitol (Mannitol) has an excellent effect of improving the structure and storage stability of R27T, arginine, poloxamer 188, or polysorbate 20 (Polysorbate 20) also has the effect of enhancing the stability of the interferon protein Could know.

Based on the above experimental results, the range of excipients was expanded to prepare R27T formulations containing R27T, 20 mM acetic acid buffer at pH 4.2, and various types of excipients, followed by T _m and SEC analysis. As a result of the stability evaluation, it was confirmed that arginine and poloxamer 188 have an excellent effect on the stability of R27T. Based on these results, subsequent experiments to establish the optimal composition of the R27T formulation to which arginine and poloxamer 188 were added were performed. Proceeded.

Prior to experiments evaluating the optimal composition of excipients, experiments to determine the final conditions of acetic acid buffer confirmed that a 10 mM or 20 mM acetic acid buffer system at pH 3.8 was the condition that most enhanced the storage stability of R27T. The final excipient composition was evaluated under 20 mM acetic acid buffer system conditions of pH 3.8 identified above in consideration of capacity.

In addition to containing arginine, poloxamer 188, and methionine, R27T formulations of six different compositions were prepared by the addition of mannitol, which was found to enhance the stability of interferon, and control and excipient-free interferon beta formulations (Rebif) Stability and activity comparisons with) determine the optimal composition of the formulation of R27T.

As used herein, the term “interferon-beta (IFN-β)” refers to a fibroblast interferon of human origin, as well as its salts, functional derivatives, which are obtained from biological fluids or obtained by DNA recombination techniques from eukaryotic or prokaryotic host cells. , Variants, analogs and active fractions. Preferred IFN-beta is intended to mean interferon beta-1a.

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

"Buffer" means a solution of a compound that has the effect of adjusting or maintaining the pH of the formulation to fall within the pH range desired for the formulation. Suitable buffers for adjusting pH in the present invention include, but are not limited to, compounds such as phosphoric acid, acetic acid, citric acid, histidine, preferably 20 mM acetic acid buffer, and the buffer is preferably 5 to 100 mM Concentration, more preferably 10 to 30 mM.

 In the present invention, the stabilized pharmaceutical preparation of R27T comprises dialysis of a solution comprising a human interferon variant R27T with a solution containing an acetic acid buffer and an excipient at a concentration of 5 to 100 mM; It can be prepared including the step of filtering the dialysate.

In the present invention, the excipient may be added 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 the "liquid formulation" the preferred solvent is water, which may be a monodose or a multidose. Liquid R27T formulations for multi-dose formulations of the present invention are phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benz It is preferred to include bacteriostatic agents, such as etonium chloride, sodium dehydroacetate and thimerosal. The bacteriostatic agent is such an amount that can give a concentration effective to maintain a sterile formulation (suitable for injection) necessarily between syringes of the multi-dose formulation, from about 12 or 24 hours to about 12 days, preferably about 6 to 12 days. Used as The bacteriostatic agent is preferably present at a concentration of about 0.1% to about 2.0% (mass of mass / solvent of bactericide).

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, antifoams and anti-caking agents.

There is no particular limitation on the pharmaceutically acceptable carrier, but it may include physiological saline, polyethylene glycol, ethanol, vegetable oil, isopropyl myristate and the like.

The present invention also provides a method for treating diseases such as multiple sclerosis, cancer, autoimmune diseases, viral infectious diseases, HIV infectious diseases, hepatitis C and rheumatoid arthritis by administering to the subject a pharmaceutically effective amount of a stabilizing agent. .

As used herein, "individual" means a subject in need of treatment for a disease, and more specifically human, or non-human primates, mice, rats, dogs, cats, horses, and cattle Means such mammals.

It will be apparent to those skilled in the art that a “pharmaceutically effective amount” may vary in range depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, and severity of the disease of the patient.

Preferred dosages of the formulations of the invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. However, preferably, it is administered at 0.001 to 100 mg / kg body weight per day, more preferably 0.01 to 30 mg / kg body weight. Administration may be administered once a day or may be divided several times.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. The method of administration is not limited, and may be administered by oral, rectal, or intravenous, muscle, subcutaneous, intrauterine dural, or intra cerbroventricular injection.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLE

Example 1 Experimental Method

1-1. Dynamic Light Scattering (DLS) and Zeta Potential Analysis

Zetasizer Nano ZS90 (Malvern Instruments, Worcestershire, UK) was used to evaluate the electrostatic interaction of interferon beta variant (R27T) samples, and the measurement temperature of the instrument was set to 10 ° C. Each sample was subjected to five repeated measurements at 30 second intervals to obtain an average hydrodynamic size, Polydispersity Index (PdI), and zeta potential.

1-2. Differential Scanning Calorimetry (DSC)

To perform thermodynamic stability evaluation of the R27T sample, a VP-DSC Microcalorimeter (Microcal, Northampton, Mass., USA) was used. The experiment was run 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 normalized by subtracting the baseline measured using the finally measured buffer and calculating the concentration of protein present in the sample.

The R27T measurement result was set as the baseline for zero adjustment of the result through the linear baseline adjustment. At this time, the process of setting the baseline for zero adjustment of the sample is complicated because it is disturbed by the aggregation or precipitation phenomenon caused by the heating, in the present invention, the type or user of the sample in the repeated experiment by selecting the linear baseline option The most stable results were obtained regardless of the decision of.

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

1-3. ATR-FTIR Spectroscopy

Infrared spectra were measured using a Nicolet iS5 spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA) equipped with an iD5 diamond ATR accessory. 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 coils in the protein solution can be obtained by measuring the infrared spectrum in the amide I region. The curve fitting process is performed using Gauss and Lorentz formulas provided by OMNIC Peak Resolve software on the peaks in Amide I region, and the area represented by each secondary structure is represented by the ratio of each secondary structure in the total area. It was.

1-4. R27T  Design of Experiment for Solution Optimization; DoE ) And Robust Design (RD)

In order to determine the optimal pH at which the protein can be most stably present, the protein transition temperature (T _m ), enthalpy (ΔH) and Helix ratio (α / β) were evaluated. In the initial data conversion process, since the three measurement results have different units, it is necessary to convert the measurement results in order to find out the optimum pH conditions in the four buffer environments. As a result, all measurement results are linearly converted to values between 1 and 2 using the equation below.

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

In the above formula, Y _min and Y _max represent the minimum and maximum values in the measurement results, and the measured values Y (T _m , Helix ratio (α / β) and enthalpy (ΔH)) by buffer concentration or pH are inserted. In order to express the relationship between Y value, buffer concentration and pH and find the optimal point, a modified Response Surface Methodology (RSM) model defined by the following equation was used.

Where m ₀ , m ₁ , m _2, m ₁₂ , and m ₂₂ are the coefficients used in the secondary RSM, which usually has a complex or exact relationship between several output values and the associated input factors. Used to optimize when functional relationships are difficult to understand.

In addition, in order to optimize the three measured values at the same time, the robust design principle using the weighted-sum method was used. This method is the most common method to create an effective solution in robust design. The proposed multi-objective robust design optimization model is based on the weighting method with importance levels w1 to w3. This procedure allows the evaluation of the optimal R27T solution.

1-5. Size Exclusion Chromatography (SE)

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). In order to separate the water-soluble R27T particles, mobile phase A (0.1% aqueous solution of Trifluoroacetic acid (TFA), 150 mM NaCl) and mobile phase B (0.1% TFA acetonitrile, 150 mM NaCl) were added at a ratio of 0.5 mL / min in a ratio of 4: 6. It flowed at a flow rate. The area analyzed as the peak of the multimer in the sample was calculated along with the area of the water soluble aggregate. The difference in the total area (sum of all peak area areas in the chromatogram results) of the R27T sample measured after the initial measurement was defined as being due to the formation of insoluble aggregates at the time of measurement. The remaining proportion of each species (insoluble aggregates, monomers, protein fragments) was calculated as the peak area compared to the initial time and plotted with the storage period as the X axis. The formula for calculating the remaining ratio is as follows.

Remaining (%) = (a _t ÷ A ₀ ) × 100

Wherein at denotes the width of the protein peak at each time and A ₀ denotes the initial width of each protein peak. Error bars represent the standard deviation (SD) for three measurements.

Example 2: Stable R27T Concentration Analysis

2-1. R27T Solution Preparation

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

2-2. Hydrodynamic magnitude, zeta potential, and PDI measurements with R27T concentration

Dynamic light scattering (DLS) was performed to investigate the effect of protein concentration, pH, and buffer concentration on the electrostatic interactions between proteins in the formation of nano-sized aggregates.

As a result, hydrodynamic size, zeta potential, and polydispersity index (PdI, Polydispersity Index) 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 can be seen in Table 1, the hydrodynamic size of R27T in a storage buffer of 0.80 mg / mL concentration is about 5.37 ± 0.27 nm in diameter, and the hydrodynamic size increases with decreasing concentration, 0.10 and 0.05 mg Protein aggregation was observed at the / mL concentration.

In addition, as the concentration of R27T decreased, the absolute zeta potential value decreased from -21.16 mV to -3.17 mV, because protein aggregation was induced between neighboring proteins, thereby reducing the electrostatic interaction. On the other hand, when the polydispersity index (PDI) is 0.7 or more, it means that the polymer sample has a very polydisperse heterogeneous distribution. In this embodiment, as the concentration of R27T is lower, the polydispersity index (PDI) increases. It means that protein aggregation has occurred.

2-3. Tm measurement according to R27T concentration

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

As a result, as shown in the thermogram of FIG. 2, the transition temperature (T _m ) of R27T at a concentration of 0.8 mg / mL was 60.07 ° C., and one endothermic peak was observed between 40 and 80 ° C. FIG. 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., indicating that the lower the concentration, the lower the structural stability (T _m value) of R27T.

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

Example 3: Analysis of pH Range of Stable R27T

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

First, since it was confirmed from Example 2 that the high absolute zeta potential value is an important factor for inhibiting protein aggregation, the change in zeta potential with pH was measured. As a result, as shown in Figure 3a, it can be seen that the relatively high electrical repulsion at the acidic pH, the experimental isoelectrical point (pI) value of the zeta potential reaches zero was 5.84.

In addition, the effects of pH on R27T stability (T _m ) were further measured, since protein exposure to extreme pH can lead to structural defects due to disruption of internal electrostatic forces and charge-charge interactions. Adjust from 2.0 to 11.0 with hydrochloric acid and sodium hydroxide during dialysis. As a result, as shown in Figure 3b, it was found that the optimum pH range is between 3.6 and 4.4 while showing the highest structural stability (T _m value of about 61 ℃).

Example 4: Stable Buffer Analysis of R27T

4-1. Tm  Measure( DSC )

To determine the buffer system in which R27T can be most stably present, DSC was performed and _Tm was measured by adding various concentrations of acidic buffers (phosphate, acetic acid, citric acid, histidine) at various pH ranges.

As a result, as can be seen from the endothermic peak of FIG. 4, the structural stability (T _m ) of R27T was changed according to 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 citric acid 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 (℃) Enthalpy (kJ / mol) α-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 50mM 3.6 63.70 13.64 23.65 32.22 28.23 15.90 0.73 50mM 4.3 59.06 10.63 21.33 32.77 29.82 16.07 0.65 50mM 5.0 59.71 15.11 32.53 24.49 30.62 12.36 1.33 50mM 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 50mM 4.3 58.54 14.05 5.60 31.81 41.53 21.06 0.18 50mM 5.0 60.15 12.10 20.50 41.58 21.30 16.62 0.49 50mM 5.7 59.93 13.31 30.38 30.96 26.04 12.61 0.98 50mM 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 50mM 2.9 62.00 24.94 38.66 21.51 12.43 27.41 1.80 50mM 3.6 60.46 17.80 22.72 26.31 30.39 20.57 0.86 50mM 4.3 61.36 32.02 14.72 23.25 37.20 24.83 0.63 50mM 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 50mM 3.6 54.51 9.28 19.00 40.70 29.67 10.63 0.47 50mM 4.3 57.98 10.46 33.15 43.07 23.65 0.13 0.77 50mM 5.0 61.51 15.48 34.32 40.91 14.91 9.87 0.84 50mM 5.7 62.84 15.78 18.79 26.68 26.18 28.35 0.70

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

In addition to the thermodynamic properties obtained from the DSC data of Example 4-1, since the structural properties of the protein need to be further considered, α-helix, β-sheet, and β-turn in protein solution using ATR-FTIR , the ratio of the random coil was measured.

Specifically, rhIFN-β is the predominantly α-helix and increasing β-sheet content means intermolecular β-sheet formation that can induce protein aggregation, and the ionization state of the amino acid side chains of the protein can lead to structural changes. Since pH is affected, the relative content of α-helix to β structure was determined by performing ATR-FTIR with various concentrations of acidic buffers (phosphate, acetic acid, citric acid, histidine) at various pH ranges.

As a result, as shown in Table 2, it had the highest α-helix content in 20 mM acetic acid buffer (pH 3.6), followed by 20 mM citric acid buffer (pH 4.3) and 50 mM phosphate buffer (pH 2.9). Appeared.

4-3. Robust Design (RD)

Since the three measurement results of the protein transition temperature (T _m ), enthalpy (ΔH) and Helix ratio (α / β) obtained in Examples 4-1 and 4-2 have different units, To find the optimal pH conditions in an acidic buffer (phosphate, acetic acid, citric acid, histidine) environment, the measurement results need to be converted. To this end, multi-objective robust design (RD) was performed and the results are shown in FIG. 5.

To illustrate the patterns of the four buffers, three response and objective functions are plotted. Where T _m , Helix ratio (α / β), and enthalpy (ΔH) represent structural stability, secondary structural stability, and solubility associated with heat capacity, respectively, and all objective functions of the four buffers have different concave patterns. You can see it.

In addition, 19 weighting scenarios were investigated to select the optimal buffer, buffer concentration, and respective pH values by evaluating three reaction functions simultaneously, 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 in determining the thermodynamic stability of a protein, we gave it the highest weight, and we chose 19 scenarios with different weights assigned to the three factors, and between the stability of the protein solution and the various pH or buffers. Correlation was investigated. Finally, the optimization results of 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 optimal pH value and the buffer that can produce the optimal formulation, the results of FIG. It was found that mM acetic acid buffer could be used in an optimal formulation.

Example 5: Optimal pH Analysis in Acetate Buffer

Since acetic acid buffer was confirmed to be the optimal buffer for the R27T preparation from Example 4, further experiments were further performed using acetic acid buffer.

5-1. SEC: Monomer amount analysis

Initially, among the pH ranges 3.6, 4.3, 5.0, and 5.7 tested in acetic acid buffer, the optimum pH value was pH 3.6, but low pH may cause skin side effects upon subcutaneous injection, thus increasing the pH of the formulation for stability. There is a need. For this reason, in order to further evaluate the pH effect on the storage stability of the formulation, the R27T formulation was stored at 37 ° C. for 11 days at pH 3.4 to 4.4, followed by SEC (size exclusion chromatography) to perform residual Chromatograms of monomers were compared daily.

As a result, as shown in FIG. 6, as the pH increased by 0.2 from 3.4 to 4.4, the monomer residual amount of R27T increased during the 11-day storage period. Increased R27T stability results showed low stability at pH 3.4 with only 4% monomer residue at 11 days, but as the pH increased from 3.6 to 3.8, 4.0, 4.2, and 4.4, the monomer residue for 11 days was also 21.35. %, 22.59%, 23.42%, 24.93%, and 25.10%. Increasing the amount of monomer means a decrease in aggregates, so it can be seen that protein stability increases with increasing pH from 3.4 to 4.4.

5-2. DSC  : Tm  analysis

DSC analysis of R27T was carried out with increasing acetic acid buffer from 0.2 to pH 3.4 to 4.4. As a result, as shown in Figure 7, it can be seen that as the pH is increased T _m increases the structural stability.

Example 6 Analysis of Stability Enhancement of R27T Formulation with Excipients

6-1. Preparation of the R27T Formulation

In order to prepare an R27T formulation having enhanced stability by adding an excipient to a formulation containing an R27T protein and a buffer, two different formulations (A Formulation and B Formulation) were prepared, followed by the conventional interferon beta Rebif formulation. The stability was compared. To this end, the compositions of the two R27T formulations are as shown in Table 5 below, and were prepared according to the following method.

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 a phosphate buffer solution (pH 2.9) at 4 ° C, and the R27T solution was stored at 12 ° C using a Cellu Sep ^® H1 cellulose semipermeable membrane (minimum permeation molecular weight: 5000 Da). Dialysis was carried out with stirring for hours. As the buffer used for dialysis, 1 L of 20 mM acetate buffer (pH 4.2) was used, and dialysis was performed by adding an excipient according to the buffer and the composition shown in Table 5 above. Impurity particles remaining after dialysis was removed by filtration using 0.22 um cellulose acetate (Advantec, Tokyo, Japan).

6-2. DSC  : Tm  analysis

Two types of R27T preparations prepared in Example 6-1 and Rebif preparations, which are conventional interferon beta preparations, were subjected to DSC and T _m was measured to evaluate the structural stability of R27T.

As a result, as shown in Figure 8a, B Formulation was measured to have a higher T _m value than the Rebif formulation (Reference), A Formulation measured a lower T _m value than the Rebif formulation, the structural stability of the B Formulation Confirmed higher.

Furthermore, in order to compare the structural stability according to the type of excipients, R27T formulations were prepared in the same manner as in Example 6-1, but only one excipient described in Table 5 was added to the buffer to prepare R27T formulations. DSC was performed.

As a result of the measurement of T _m , as shown in FIG. 8B, when benzyl alcohol was added as an excipient, the T _m value (T _m = 56.74) decreased, indicating that benzyl alcohol decreased the stability of the R27T formulation. On the other hand, when mannitol (Mannitol), Methionine (Methionine), Poloxamer 188 (Poloxamer 188), or Polysorbate 20 (Polysorbate 20) was added, it was confirmed that the stability of the formulation is improved. Specific results are shown in Table 6 below.

T _m (℃) SD A formulation 58.08 0.12 B formulation 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: Monomer amount  analysis

In addition to the structural stability evaluation results through the DSC analysis, in order to evaluate the storage stability of the formulation prepared by adding an excipient, A Formulation, B Formulation, and each of the excipients prepared in Examples 6-1 and 6-2 The amount of residual monomer was measured every day while storing the R27T preparation prepared by adding them separately at 40 ° C. for 9 days at a concentration of 50 μg / mL.

As a result, as shown in Figure 9, as in the DSC results, when comparing the A Formulation and B Formulation it was confirmed that the higher residual monomer amount in B Formulation. In addition, in the case of A Formulation, as a result of measuring the monomer ratio according to the addition of each excipient, the monomer residual value of the mannitol-added formulation was significantly higher than that of other formulations including A Formulation, and in the case of B Formulation, arginine or polysorbate 20 It was confirmed that the monomer residual amount of B Formulation prepared by adding them together than the added formulation was measured lower. This suggests that Mannitol has the effect of improving the safety of R27T preparations, and that the mixing of arginine and polysorbate reduces the storage stability of the preparations.

Based on the above results, it can be seen that B formulation with arginine added as a major excipient improves stability than conventional interferon preparations, and Mannitol has an excellent effect of enhancing interferon stability. It was expected to contribute greatly.

Example 7: Excipient Screening of R27T Formulations

7-1. Preparation of R27T Formulations with Various Excipients Added

Based on the results obtained in Example 6, a variety of excipients were added to prepare an R27T formulation, and then evaluated for its safety to screen the optimal excipients. To this end, the excipient types and concentrations used in the preparation of the R27T formulation are shown in Table 7 below, and the R27T formulation was prepared according to the following method.

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 1.0 mg / mL concentration was stored in phosphate buffer (pH 2.9) at 4 ° C, and the R27T solution was stored at 4 ° C using Cellu Sep ^® H1 cellulose semipermeable membrane (minimum permeation molecular weight: 5000 Da). Dialysis was carried out with stirring for 12 hours. As the buffer used for dialysis, 1 L of 20 mM acetate buffer (pH 4.2) was used, and the excipients shown in Table 7 above were added to the buffer at different concentrations, followed by dialysis. Impurity particles remaining after dialysis was removed by filtration using 0.22 um cellulose acetate (Advantec, Tokyo, Japan).

7-2. DSC: Tm analysis

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

As a result, as shown in Table 8, when compared to the T _m value of the control (Control) without the excipient, in the case of the formulation with the addition of arginine in the amino acid excipient, and the formulation with the addition of poloxamer 188 A slightly higher T _m value indicates that the excipients somewhat enhance the structural stability of the R27T formulation. In addition, among the carbohydrates excipients, it was confirmed that the structural stability was improved in the preparation prepared by adding sucrose (Sucrose) compared to the control.

Samples Tm (℃) Samples Tm (℃) 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: monomer amount and aggregate amount analysis

In order to evaluate the storage stability of the R27T formulation prepared by the addition of each excipient, each R27T formulation identical to the DSC assay sample of Example 7-2 was subjected to a concentration of 1.0 mg / mL at 40 ° C for 7 days or at 4 ° C. After storage for 16 days at the amount of the monomer (monomer) and aggregation (aggregation) was measured, the results are shown in Tables 9 to 11.

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 a result of the stability evaluation experiment according to the storage conditions, as shown in Tables 9 to 11, when the R27T formulation was stored at 4 ° C. for 16 days, it was confirmed that aggregation of R27T protein did not occur regardless of the type of excipient added. In addition, when comprehensively considering the results of the two storage conditions, it was found that the excipients enhance the storage stability of the R27T preparation, as the monomer residual amount of the arginine or poloxamer 188 added was high. These results corresponded to the DSC analysis results. Among the carbohydrate excipients, monomer residues were higher when Sucrose or Xylitol was added at two temperature conditions, but at a high temperature of 40 ℃, aggregates were formed at a slightly higher rate when sucrose was added. Through this, it could be considered that Mancitol could be substituted by coagulation inhibitor with sucrose or xylitol.

Example 8: Optimization of R27T Stabilizing Formulation Composition

Based on the results of the above examples, it was intended to set the optimal composition of the formulations that could enhance the stability of R27T.

8-1. Optimize buffer conditions

First, it was confirmed through Examples 4 and 5 that the buffer for enhancing the stability of the R27T formulation was an acetic acid buffer in the range of pH 3.4 to 4.4. It was. More specifically, the concentration of acetic acid buffer is changed to 10, 20, 50 mM concentrations, and the pH is changed to 3.8 or 4.2 to prepare an R27T preparation, and at 1 or 2 weeks at 4 ° C or 25 ° C After storage for a week), SEC analysis was performed and the residual amount of monomer and aggregates was measured and compared, respectively.

As a result, as shown in Figure 10, using the 10 mM or 20 mM acetic acid buffer pH 3.8 of the highest monomer residual, the aggregate residual was measured the lowest through the use of acetic acid buffer of the above conditions, the stability of the R27T formulation It was confirmed that it is an optimal condition to enhance the

8-2. Optimization of R27T Stabilizing Formulation Composition

In order to set the optimal composition of the R27T formulation, an R27T formulation was prepared according to the method of Example 7-1, wherein the buffer was 20 mM of pH 3.8, which is an optimal condition for enhancing the stability of the formulation through Example 8-1. Acetic acid buffer was used, and excipients were added arginine HCl, poloxamer 188, and methionine, which were found to enhance stability of the R27T preparation through Examples 6 and 7, and mannitol was added to evaluate the mixing effect of arginine and mannitol. It was. The composition of the added excipients is shown in Table 12 below.

Formulation Contents F1 (Control) - - - - Rebif formulation (F2) - - - - F3 Arginine 50 mM Poloxamer 1880.5 mg / mL Methionine1 mM F4 Arginine 50 mM Mannitol 150 mM Poloxamer 1880.5 mg / mL Methionine1 mM F5 Arginine 50 mM Mannitol 250 mM Poloxamer 1880.5 mg / mL Methionine1 mM F6 Arginine 100 mM Poloxamer 1880.5 mg / mL Methionine1 mM F7 Arginine 100 mM Mannitol 150 mM Poloxamer 1880.5 mg / mL Methionine1 mM F8 Arginine 100 mM Mannitol 250 mM Poloxamer 1880.5 mg / mL Methionine1 mM

In order to evaluate the storage stability of the R27T formulations (F1 to F8) of the eight different compositions shown in Table 12, each formulation was prepared at 4 ° C or 37 at low concentration (100 μg / mL) or high concentration (640 μg / mL). After storage for 14 days at ℃ was carried out SEC to measure the residual monomer monomer, the results are shown in Figure 11 and Table 13.

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 Rebif formulation (F2) 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 measuring the residual amount of monomer, as shown in FIG. 11, the highest monomer residual amount was measured at F5 and F7 for the low concentration formulation and F5 for the high concentration formulation at 4 ° C. In addition, as shown in Table 13, it was confirmed that the storage stability is high in the F5 and F7 formulations to which arginine and mannitol are added compared to the conventional Rebif formulation. However, at high temperature (37 ° C.) and high concentration (640 μg / mL), the stability of the formulation was found to decrease.

Based on the above results, the stability of the formulations after a longer period of storage at low temperatures was to be evaluated. To this end, the formulations of each composition shown in Table 12 were stored at 4 ° C. for 28 days, and SEC was carried out at 7 day intervals to determine the monomer residual.

As a result, as shown in Figure 12 and Table 14, it was confirmed that the formulation of the F5 and F7 composition in the storage stability is superior to the conventional Rebif formulation (F2) under low temperature storage conditions.

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

Furthermore, in the formulations of the above eight compositions, the antiviral effect of R27T according to the treatment of each formulation was measured by CPE (Cytopathic effect) assay. More specifically, 100 μl of the above agent was treated in 100 μl of medium containing A549 cells 3 × 10 ⁵ cells / mL in a 96 well plate, and the cells were incubated at 37 ° C. for 22 hours. On day 2, the supernatant was removed, treated with 100 μl of encephalomyocarditis virus (EMCV) at a concentration of 1000 TCID50 / mL, and incubated at 37 ° C. for 22 hours. The absorbance was measured at 570 nm to calculate the titer against standard.

As a result, as shown in Figure 13, the formulation of the F5 composition was confirmed that the activity was enhanced by about 40% compared to the control (control). The results confirmed that the F5 composition is the optimal formulation composition that can enhance the stability and activity of R27T.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

Claims (12)

Stabilized pharmaceutical formulations of human interferon beta variant (R27T) comprising: (a) human interferon beta variant (R27T), (b) acetic acid buffer at a concentration of 5-100 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 0.5-5 mM. According to claim 1, wherein the human interferon beta variant (R27T) is stabilized, characterized in that the arginine, which is the 27th amino acid of human interferon beta is substituted with threonine to include an N-linked sugar chain at the 25th amino acid asparagine residue. Pharmaceutical preparations. The stabilized pharmaceutical formulation of claim 1, wherein the acetic acid buffer is contained at a concentration of 10 to 30 mM. The stabilized pharmaceutical formulation of claim 1, wherein the acetic acid buffer has a pH in the range of 3.6 to 4.4. The stabilized pharmaceutical formulation according to claim 1, wherein the arginine is contained at a concentration of 50 to 100 mM. The stabilized pharmaceutical formulation of claim 1, wherein the mannitol is contained at a concentration of 150 to 250 mM. The stabilized pharmaceutical formulation of claim 1, wherein the poloxamer 188 is included at a concentration of 0.1 to 1 mg / mL. The stabilized pharmaceutical formulation of claim 1, wherein the methionine is contained at a concentration of 0.5 to 2 mM. The stabilized pharmaceutical formulation of claim 1, wherein the stabilized pharmaceutical formulation has a pH in the range of 3.6 to 4.4. The method of claim 1, wherein the stabilized pharmaceutical formulation is for the prevention or treatment of diseases selected from the group consisting of multiple sclerosis, cancer, autoimmune diseases, viral infections, HIV infectious diseases, hepatitis C, and rheumatoid arthritis Characterized by a stabilized pharmaceutical formulation. The stabilized pharmaceutical formulation of claim 1, wherein the stabilized pharmaceutical formulation is for oral or parenteral administration. The stabilized pharmaceutical formulation of claim 1, wherein the stabilized pharmaceutical formulation is a liquid or lyophilized formulation.

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