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WO2016163764A2 - Préparation stabilisée de variant d'interféron-bêta - Google Patents

Préparation stabilisée de variant d'interféron-bêta Download PDF

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Publication number
WO2016163764A2
WO2016163764A2 PCT/KR2016/003632 KR2016003632W WO2016163764A2 WO 2016163764 A2 WO2016163764 A2 WO 2016163764A2 KR 2016003632 W KR2016003632 W KR 2016003632W WO 2016163764 A2 WO2016163764 A2 WO 2016163764A2
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concentration
formulation
pharmaceutical formulation
acetic acid
buffer
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Korean (ko)
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WO2016163764A3 (fr
Inventor
신영기
박상호
송경
이희정
곽은혜
장은진
정성훈
김남아
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Abion Inc
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Abion Inc
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Priority claimed from KR1020160042320A external-priority patent/KR101781945B1/ko
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/565—IFN-beta

Definitions

  • 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 are a type of cytokine that exhibit antiviral activity, inhibit cell proliferation and modulate natural immune responses. IFNs are classified into IFN- ⁇ , IFN- ⁇ , and IFN- ⁇ according to their cell origin (leukocytes, fibroblasts, T cells). Among them, interferon-beta (IFN- ⁇ ) has five alpha-helix ( ⁇ -helix). Is a spherical protein with a size of 22 kD and the sugar chains are 18 kD.
  • Human IFN- ⁇ 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.
  • R27T is a recombinant human IFN- ⁇ variant (hereinafter referred to as rhINF- ⁇ ) designed by replacing arginine (Arg) at position 27 with threonine (Thr) for further glycosylation at position 25 of IFN- ⁇ 1a as shown in FIG. ), Increased stability, decreased protein aggregation tendency, and increased half-life when compared to wild-type INF- ⁇ 1a (Rebif).
  • rhINF- ⁇ a recombinant human IFN- ⁇ variant designed by replacing arginine (Arg) at position 27 with threonine (Thr) for further glycosylation at position 25 of IFN- ⁇ 1a as shown in FIG.
  • Increased stability decreased protein aggregation tendency
  • increased half-life when compared to wild-type INF- ⁇ 1a
  • 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. .
  • 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.
  • 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.
  • 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).
  • R27T human interferon beta variant
  • acetic acid buffer of 5 to 100 mM concentration
  • arginine of 10 to 150 mM concentration
  • mannitol of 50 to 300 mM concentration
  • poloxamer 188 at a concentration of 0.1 to 10 mg / mL
  • methionine at a concentration of 0.5 to 5 mM
  • 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. .
  • the concentration of acetic acid buffer is characterized in that 10 to 30 mM.
  • the acetic acid buffer is characterized in that the pH ranges from 3.6 to 4.4.
  • the concentration of arginine is characterized in that 50 to 100 mM.
  • the concentration of mannitol is characterized in that 150 to 250 mM.
  • the concentration of poloxamer 188 is characterized in that 0.1 to 1 mg / mL.
  • the concentration of methionine is characterized in that 0.5 to 2 mM.
  • the stabilized pharmaceutical formulation is characterized in that the pH ranges from 3.6 to 4.4.
  • 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.
  • the stabilized pharmaceutical preparation is characterized for oral or parenteral administration.
  • the stabilized pharmaceutical formulation is characterized in that the liquid or lyophilized formulation.
  • Stabilized R27T pharmaceutical formulations 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.
  • R27T human interferon beta variant
  • 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).
  • DSC differential scanning calorimeter
  • FIG. 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 .
  • acidic buffers phosphate, acetic acid, citric acid, histidine
  • 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).
  • RD multi-objective robust design method
  • 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.
  • FIG. 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 ⁇ g / mL) or high concentration (640 ⁇ g / 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.
  • CPE Cytopathic effect
  • human interferon beta variant 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.
  • 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- ⁇ variant (hereinafter referred to as rhINF- ⁇ ) designed by replacing arginine (Arg) at position 27 with threonine (Thr) for further glycosylation at position 25 of IFN- ⁇ 1a,
  • rhINF- ⁇ a recombinant human IFN- ⁇ variant
  • Arg arginine
  • Thr threonine
  • 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.
  • DoE Design of experiment
  • rhIFN- ⁇ is a 166 amino acid glycoprotein with a 4-helix bundle domain. Increasing the ⁇ -sheet content implies intermolecular ⁇ -sheet formation that can induce protein aggregation. As a result of analyzing the ⁇ -helix and ⁇ -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.
  • 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.
  • mannitol Mertol
  • arginine arginine
  • poloxamer 188 arginine
  • polysorbate 20 Polysorbate 20
  • Polysorbate 20 also has the effect of enhancing the stability of the interferon protein could know.
  • 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.
  • interferon-beta 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • physiologically / pharmaceutically acceptable additives such as free-flowing agents, emulsifiers, stabilizers, preservatives, colorants, antifoams and anti-caking agents.
  • the pharmaceutically acceptable carrier 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. .
  • mammals 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.
  • 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.
  • 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.
  • PdI Polydispersity Index
  • 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.
  • 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 ( ⁇ H) were calculated using the Multistate model.
  • Infrared spectra were measured using a Nicolet iS5 spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA) equipped with an iD5 diamond ATR accessory.
  • iD5 diamond ATR accessory was measured on iD5 diamond crystal plate by using a sample of about 10 ⁇ l, 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 ⁇ -helix, ⁇ -sheet, ⁇ -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.
  • the protein transition temperature (T m ), enthalpy ( ⁇ H) and Helix ratio ( ⁇ / ⁇ ) were evaluated.
  • T m protein transition temperature
  • ⁇ H enthalpy
  • ⁇ / ⁇ Helix ratio
  • Y min and Y max represent the minimum and maximum values in the measurement results, and the measured values Y (T m , Helix ratio ( ⁇ / ⁇ ) and enthalpy ( ⁇ H)) by buffer concentration or pH are inserted.
  • Y T m , Helix ratio ( ⁇ / ⁇ ) and enthalpy ( ⁇ H)
  • ⁇ H enthalpy
  • 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.
  • 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.
  • 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).
  • Agilent high performance liquid chromatography system Agilent high performance liquid chromatography system
  • TSK-GEL G3000SWXL SEC column TOSOH Bioscience, PA, USA
  • DAD diode detector
  • mobile phase A 0.1% aqueous solution of Trifluoroacetic acid (TFA), 150 mM NaCl
  • mobile phase B (0.1% TFA acetonitrile, 150 mM NaCl
  • 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 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.
  • 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).
  • DLS Dynamic light scattering
  • 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.
  • 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.
  • the polydispersity index (PDI) is 0.7 or more, it means that the polymer sample has a very polydisperse heterogeneous distribution.
  • the concentration of R27T is lower, the polydispersity index (PDI) increases. It means that protein aggregation has occurred.
  • DSC Differential scanning calorimetry
  • 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.
  • T m transition temperature
  • 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.
  • the concentration of the most stable R27T in the storage buffer was about 0.80 mg / mL.
  • 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 ).
  • DSC was performed and Tm was measured by adding various concentrations of acidic buffers (phosphate, acetic acid, citric acid, histidine) at various pH ranges.
  • acidic buffers phosphate, acetic acid, citric acid, histidine
  • T m structural stability 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.
  • thermodynamic properties obtained from the DSC data of Example 4-1 since the structural properties of the protein need to be further considered, ⁇ -helix, ⁇ -sheet, and ⁇ -turn in protein solution using ATR-FTIR , the ratio of the random coil was measured.
  • rhIFN- ⁇ is the predominantly ⁇ -helix and increasing ⁇ -sheet content means intermolecular ⁇ -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 ⁇ -helix to ⁇ structure was determined by performing ATR-FTIR with various concentrations of acidic buffers (phosphate, acetic acid, citric acid, histidine) at various pH ranges.
  • acidic buffers phosphate, acetic acid, citric acid, histidine
  • T m Helix ratio ( ⁇ / ⁇ ), and enthalpy ( ⁇ H) 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • Example 6 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.
  • 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).
  • Example 7-1 Each R27T formulation prepared in Example 7-1 was subjected to DSC analysis to evaluate the structural stability of R27T.
  • 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.
  • 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.
  • 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
  • each formulation was prepared at 4 ° C or 37 at low concentration (100 ⁇ g / mL) or high concentration (640 ⁇ g / mL). After storage for 14 days at °C 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 °C 100.00 89.73 Day14, 37 °C 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
  • the antiviral effect of R27T according to the treatment of each formulation was measured by CPE (Cytopathic effect) assay. More specifically, 100 ⁇ l of the above agent was treated in 100 ⁇ l 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 ⁇ l 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.
  • CPE Cytopathic effect
  • the formulation of the F5 composition was confirmed that the activity was enhanced by about 40% compared to the control (control).

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Abstract

La présente invention concerne une préparation pharmaceutique stabilisée de R27T comprenant un variant d'interféron beta humain (R27T), une solution tampon d'acide acétique, de l'arginine, du mannitol, du poloxamère 188, et de la méthionine. La préparation pharmaceutique R27T stabilisée selon la présente invention comprend une solution tampon d'acide acétique, de l'arginine, du mannitol, du poloxamère 188 et de la méthionine et, de ce fait, inhibe la formation d'un agrégat d'une protéine R27T et permet un stockage de longue durée, en raison d'une amélioration de la stabilité structurelle et thermodynamique. Ainsi, la préparation pharmaceutique R27T stabilisée sera utile dans la prévention, l'amélioration et le traitement de la sclérose en plaques, du cancer, de maladies auto-immunes, de maladies infectieuses virales, de maladies infectieuses du VIH, de l'hépatite C, de la polyarthrite rhumatoïde, etc.
PCT/KR2016/003632 2015-04-07 2016-04-07 Préparation stabilisée de variant d'interféron-bêta Ceased WO2016163764A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230159605A1 (en) * 2020-04-29 2023-05-25 Abion Inc. Human interferon-beta variant with double mutation and method for improving stability of human interferon-beta variant
US20240270807A1 (en) * 2020-04-29 2024-08-15 Genopharm Inc. Recombinant protein having fused interferon-beta mutein and antibody, and pharmaceutical composition comprising same

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* Cited by examiner, † Cited by third party
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AR044302A1 (es) * 2003-05-13 2005-09-07 Ares Trading Sa Formulaciones con proteinas liquidas estabilizadas en recipientes farmaceuticos
KR20070030855A (ko) * 2004-06-01 2007-03-16 아레스 트레이딩 에스.에이. 단백질을 안정화하는 방법
KR100781666B1 (ko) * 2004-11-02 2007-12-03 신영기 인간 인터페론-베타 변이체
PT2234645E (pt) * 2007-12-20 2012-05-21 Merck Serono Sa Formulações de peg-interferão-beta
US20120269770A1 (en) * 2010-11-22 2012-10-25 Mark Brader Stable Preserved Compositions of Interferon-Beta

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20230159605A1 (en) * 2020-04-29 2023-05-25 Abion Inc. Human interferon-beta variant with double mutation and method for improving stability of human interferon-beta variant
US20240270807A1 (en) * 2020-04-29 2024-08-15 Genopharm Inc. Recombinant protein having fused interferon-beta mutein and antibody, and pharmaceutical composition comprising same

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