Stable proteincoding freeze-dried composition

 

(57) Abstract:

The invention can be used in medicine and pharmacy to create preparations containing proteins. The lyophilisate contains a buffer, alanine and mannitol. The mass ratio of alanine : mannitol 1 : 0.1 to 1. As a protein may contain the enzyme oxidase-lithate, hormone hGH, interleukin - 13. As the buffer is phosphate buffer. Alanine can be in crystalline form, mannitol - amorphous. The protein contained in the composition remains stable at room temperature. The water content of the lyophilized compatible with maintaining the activity of the protein. 7 C.p. f-crystals, 6 tab., 5 Il.

The invention relates to pharmaceutically acceptable compositions are in the form of a lyophilisate containing the protein as an active start. This composition is stable at 25oC and can be converted into liquid form by adding solvent. You can enter parenteral person or an animal or to use in analyzing the set.

It is known that compounds such compositions have a significant detrimental impact on proteins during lyophilization, and also strongly affect their stability in liofilizirovannaya present salts, the type and number of excipients selected type cryoprotection, and the selected temperature, pressure and duration of operations, freezing, sublimation and drying. These variables also affect the physical condition of the resulting lyophilisate, namely amorphous glassy, amorphous soft, crystalline or a combination of both.

The role of each of these variables was investigated separately, but their synergistic effect is still poorly elucidated (Pikai M. J., Dellermann K. M., Roy M. Z., M. Riggin N., Fhe effects of formulation variables on the stability of freeze-dried Human Growth Hormone, Pharm. Research, 1991, 8, N4, 427-236).

The information available on the effect of amino acids and polyhydric alcohols on the properties liofilizarea solutions or liofilizatow allows to draw the following conclusions.

The advantages and disadvantages associated with the presence of amino acids, mannitol, crystalline or amorphous phases are listed below.

The benefits associated with the presence of amino acids.

It is shown that the presence of glycine in the lyophilisate induces crystallization of the molecules present in the solution, during the stage of freezing during lyophilization (Korey D. J. , Schwartz J. B., Effects of excipients on the cristallization of pharmaceutical compounds during lyophylization, J. Parenteral Sci. Tapazole to improve its stability.

In addition, alanine in crystallized form prevents the creation of a freeze-dried in the process of sublimation and drying and allows you to get the lyophilisate with a higher specific surface area, allowing you to achieve faster drying (Pikai M. J., Freeze-drying of proteins, Biopharm. 26 - 30 October 1990).

The disadvantages associated with the presence of amino acids.

The addition of amino acids to sugar or polyhydric alcohol, in lyophilizers solution, usually causes a decrease in the temperature of stelopererad sugar (te Booy MPWH de Ruiter, R. A., de Meere ALJ, Evaluation of the physical stability of freeze-dried sucrose containing formulations by differential scanning calorimetry, Pharm. Research, 1992, 9, 109-114).

However, lowering the temperature of stelopererad usually accompanied by a deterioration in the stability of freeze-dried (Frauks Z., Freeze-drying; from empiricism to predictability. Cryo-Letters, 1990, 11, 93-110).

The benefits associated with the presence of mannitol.

The presence of mannitol in amorphous form in the presence of protein ensures that there is recrystallizations water associated with the protein during freezing, and thus prevents denaturation of the protein. Furthermore, the presence of polyhydric alcohols stabilizes proteins from termoretratil due to hydrophobic vzaimodeistvie, associated with the presence of mannitol.

It is known that mannitol does not allow to preserve the activity of the enzyme at 37oC in contrast to lactose (Ford A. W., Dawson, P. J., The effect of carbo-Rydrate additives in the freeze-drying of alkaline phosphatase, J. Pharm. Pharmacol., 1993, 45 (2), 86 - 93).

The benefits associated with the presence of the crystalline phase.

The presence of crystallized solute in frozen solution favors the stabilization of proteins during drying (Carpenter J. F., and Crowe J. H., Modes of stabilization of a protein by organic solutes during dessication, Cryobiology, 1988, 25, 459 - 470).

The disadvantages associated with the presence of the crystalline phase.

It is shown that the loss of activity of lyophilized protein is directly related to the degree of crystallinity creatasimages molecules (Izutsu K. I., Joshioka S. , Terao, T., Decreased protein-stabilizing effects of cryoprotectants due fo crystallization, Pharm. Research. 1993, 10, N 8, 1232 - 1237).

For medicines containing proteins, it is necessary to avoid crystallization of excipients (Hermansky M., Pesak M., Zyophilization of drugs. VI Amorphous and Crystalline forms. Cesk. Farm., 1993, 42 (2), 95 - 98).

The benefits associated with the presence of amorphous phase.

The presence of additives that are in the amorphous state, stabilizes the activity of some EN zymes is. esearch, 1993, 10, N 8, 1232 - 1237).

Createsite effect excipients explained by the amorphous state of the glycine in the resulting lyophilisate (Pikai M. J., Dellermann K. M., Roy M. Z., M. Riggin N. , The effects of formulation variables on the stability of freeze-dried Human Growth Hormone, Pharm. Research, 1991, 8, N 4, 427 - 436).

The disadvantages associated with the presence of amorphous phase.

In the presence of only one solid amorphous phase, the lyophilisate is deposited at temperatures above the temperature of stelopererad in the process of freezing.

In soft amorphous phase destructive chemical reactions have a much higher kinetics than in the crystalline phase.

Thus, a review of the scientific literature leads to the conclusion about the contradictory nature of the influence of excipients to stabilize proteins. There is no unified theory on the relationship between the structure of lyophilisate and its stability. In addition, does not describe the role of polyhydric alcohols and amino acids, alone or in combination with each other, depending on the totality of their General properties, however, were obtained contradictory results depending on the studied proteins and quantities of excipients.

Currently, the applicant found that there sinergicheskoe impact exists only in the relative concentrations of each of these excipients. Optimal effect takes place for the ratio R, where R denotes the ratio: weight of mannitol/mass alanine present in the lyophilisate, in the interval of 0.1 to 1, particularly 0.2 to 0.8.

In addition, it is shown that when R = 0.1 to 1: lyophilisate is formed of an amorphous phase and a crystalline phase, an amorphous phase is mostly formed by mannitol and protein; crystal phase is mostly formed by alanine.

The effect of the stabilization of proteins is explained according to the proposed hypothesis that at the specified interval ratios: formed amorphous phase critisised protein during freezing; crystalline phase captures the structure of freeze-dried and eliminates its destruction.

This unexpected synergistic effect arising from the coexistence of amorphous phase and crystalline phase, stabilizes the lyophilized protein.

The present invention relates thus to liofilizirovannam forms of compositions containing an effective amount of biologically active protein, a buffer to bring the pH optimum values for the stabilization of the protein, alanine and mannitol, and these latter two eccipienti are mascomposite, remains stable in lyophilized form. The obtained freeze-dried quickly and completely dissolved. The appearance of the lyophilisate is not broken and the water content is compatible with maintaining the activity of the protein.

This composition can be introduced other pharmaceutically acceptable and well-known specialist eccipienti, such as co-solvents, preservatives, antioxidants or chelating agents.

More specifically the present invention relates to stable proteinogram of liofilizatow containing mainly protein, critisising solid amorphous phase, and mannitol, and this amorphous phase in the lyophilisate obtained after sublimation and drying the frozen solution exists simultaneously with the crystalline phase formed mainly by alanine.

Biologically active protein, which is used according to the present invention can be glycosylated or not natural, synthetic, semi-synthetic or recombinant polypeptide such that used in the clinical or laboratory practice. More specifically, the above protein can be, for example, the hormone as a growth hormone, before the can be an enzyme, for example thrombolytic enzyme such as urokinase, PUK, streptokinase, staphylokinase, tissue plasminogen activator (tPA), or such an enzyme, a phosphatase, sulfatase, acyl-transferase, monoamine oxidase, oxidase-lithate.

The protein according to the present invention may also be a cytokine, such as interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6) or interleukin-13 (IL-13). Another class of proteins according to the present invention includes, for example, antibodies, immunoglobulins, immunotoxins. Peptides, such as cholecystokinin (CCK), substance P, neurokinin A, neurokinin B, neurotensin, neuropeptide Y, eledoisin, bombezin, can enter in the composition according to the present invention.

Preferred biologically active protein is hGH (or human growth hormone), oxidase-lithate or interleukin-13.

Human growth hormone is a protein formed by a single polypeptide chain of 191-th amino acids with two disulfide bridges between cysteine residues 53 and 165 and cysteine residues 182 and 189.

Oxidase-lithate is an enzyme that oxidizes uric acid to allantoin and extragere the Oia hyperuricemia in the last twenty years.

kknk (coding deoxyribonucleic acid) encoding this protein, has recently been cloned and expressed in E. coli (R. LEGOUX, etc., J. of Biol. Chem., 1992, 267, 12, 8565 - 8570); Aspergillus flavus and Saccharomyces cerevisiae.

The enzyme is a tetramer of identical subunits of molecular masses close to 3200. The monomer formed by a single polypeptide chain of 301 amino acids, contains no disulfide bonds and acetiminophen at N-end.

Interleukin-13 is a cytokine, an educated one polypeptide chain of 112 amino acids with two disulfide bridges (Minty and other, Nature, 1993, 362, 248 - 250).

The coexistence of amorphous mannitol + protein) phase with crystalline alanine phase does not depend on the presence and concentration of buffer, regulating the pH of the solution, but depends on the above ratio R.

To obtain proteinogenic compositions that could be proposed for wide use as therapeutic agents, it is necessary to release them in a sufficiently stable condition to retain their biological activity between the date of manufacture and the point of use. For example, a composition based growth hormone hGH can be obtained mn the OIC 89/09614; WIPO 92/17200; patent Australia 30771/89; WIPO 91/19773; WIPO 93/19776.

Compositions according to the invention can be stored at room temperature, whereas the compositions containing these proteins and currently available for sale, should be stored at temperatures of 2 to 8oC.

In most cases manufactured by the pharmaceutical form is a freeze-dried, frozen or liquid. They contain protein, buffer, glycine, arginine, mannitol, zinc, surface-active agents, dextran, etc or other eccipienti, but never contains a combination of alanine/mannitol mass ratio R = 0.1 to 1. (EP 0178665, US 5266310, EP 0396777, US 4992419).

Lyophilized or frozen form is used to save the biochemical integrity and biological activity of the molecule. Lyophilized forms before use must be diluted with sterile pharmaceutically acceptable solvents such as distilled water, water with 0.9% solution of sodium chloride or an aqueous 5% glucose, or any other physiologically acceptable solvent may contain antimicrobial preservatives, such as benzyl alcohol, phenol or meta-cresol.

buletinul treatment, as in the case of hormone hGH, it comes in a bottle or in the form of a reusable handle of the syringe.

Such types of packaging can also include the proportioning set.

According to the present invention preference is given to liofilizovannyh form of the composition. This lyophilisate obtained by lyophilization from solution.

The method of obtaining this composition includes the stage of mixing, dissolving, filtration, and lyophilization.

The composition liofilizirovannogo solution is the following: protein, pharmaceutically acceptable buffer for pH control, alanine, mannitol, and the mass ratio R = mass of mannitol/mass alanine is 0.1 - 1; water for injectisome of the drug.

Liofilizirovanny solution is prepared as follows.

Solution get protein on a column of gel filtration, it contains a buffer that supports pH-value in the field that is compatible with the stability of the protein.

To this solution is added the desired amount of buffer, alanine, mannitol and water for dissolving all of excipients. The solution is filtered under sterile conditions and placed into containers, preferably bottles.

The lyophilization of solutions conduct sleduushego volume and capacity which contains the solution. Preferably choose the speed of freezing, close to 2oC/min in liofilizadora Usi-froid (France) type SMH15, or SMJ 100, or SMH2000.

Time, temperature and pressure dry lyophilisate choose depending on volumes liofilizirovannogo solution and the desired residual water in the lyophilisate.

Complete information on cooking methods injectively formulations is given to a person skilled in Remington''s Pharmaceutical Sciences, 1985, 17-e edition; or William N. A. and Polli J. P., The lyophilization of pharmaceuticals: a literature review. J. Parenteral Sci: Tech., 1984, 38 (2), 48-59: or Franks J., Freeze-drying: from empiricism to predictability, Cryo-Letters, 1990, 11, 93-110.

Get the lyophilisate, in which alanine is in crystallized form, and mannitol is in amorphous form. The lyophilisate can be stored at 25oC without compromising the biological activity of the protein.

The present invention is illustrated in the examples with protein hGH or oxidase-lithate. So prepare several solutions containing hGH or oxidase-lithate as a biologically active protein, phosphate buffer at various concentrations with pH 7 for solutions containing hGH (with 4 UI/0.5 ml), and pH 8 for solutions containing oxidase-lithate (30 UAE/ml); only manwoody detail in the examples table. 1 and 2, below. Methods of analysis, and the time and temperature preservation stability are described below.

Analytical methods that are used to determine various parameters, the following.

The content of the dimer and related substances of higher molecular mass.

The content of the dimer and related substances of higher molecular mass determined by exclusive chromatography (SEC-HPLC) using a column SUPERO SE 12 (Pharmacia, Ref. 17-0538-01). Product elute with buffer solution of ammonium phosphate with pH 7.0 (1,38 g of ammonium dihydrophosphate in 1 liter of water while bringing the pH value to 7 using concentrated ammonia solution with a flow rate of 0.4 ml/min). Data collection is carried out at 220 nm (this is the content shown in the analysis results, expressed as% /oligomers + polymer).

Dimers and substances with higher molecular weight can also be determined according to the method described in the monograph of the European Pharmacopoeia "Somatropine pour preparation nizectable", January 1994

Quantitative determination of protein by chromatography reverse phase.

Expressed in mg per vial the amount of protein determined with POM-25). Product elute within 35 minutes gradient method when using a mobile phase flowing from 75 volumes of water with 0.1% triperoxonane acid (by volume) (TFA) and 25 volumes of acetonitrile with 0.08% TFA (by volume) up to 30 volumes of water with 0.1% TFA and 70 volumes of acetonitrile with 0.08% TFA. The flow rate is 1 ml per minute and the determination is carried out at 220 nm.

Quantitative determination of the enzymatic activity of the oxidase of lithate.

The enzymatic activity of the oxidase of lithate in UAE is determined using spectrophotometry in thermostatted cuvette at 30oC, the disappearance of the band of uric acid at 292 nm, according to R. Legoux, Delpech Brunc, X. Dumont, Guillemout J. C. , P. Ramond, Shire D., Caput d, Ferrara p, Loison, G., J. Biol, Chem., 1992, 267 (12), 8565-8570.

The turbidity of the solutions of dissolved substances.

The turbidity of solutions containing hGH lyophilizator, determined by spectrophotometry (Ph. Eur, 2(1), T. 619) at 500 nm on a spectrophotometer Perkin Elmer 554. The results are presented in units of absorbance x 1000.

The turbidity of solutions containing oxidase-lithate in the lyophilisate, determined using turbidimeter Hach Ratio 18900-00. The results of the turbidity values expressed in units of turbidity (NTU) determined according to: Standard mefhods for examinati is given in the European Pharmacopoeia (II), so 6, by comparing the analyzed sample with the control suspension.

Organoleptic criteria liofilizatow.

These criteria examine visually, whereas the color of the lyophilisate, its structure (destroyed or not) and see the possible phase offset between the outer layer and core of lyophilisate.

X-ray structural analysis of powder.

X-ray structural analysis of liofilizatow carried out on a diffractometer SIEMENS D 500 TT: source: CuK1; generator: 40 kV, 25 mA; shielded monochromator; slit: 1/1/1/0,16/0,6; sampling perexodom medium; area scan (sweep): 4 - 40oper minute in the form of 2 theta Bragg.

Differential thermal analysis.

Research liofilizatow using differential thermal analysis carried out in the following conditions:

equipment: DSC 7 Perkin Elmer; calibration: indium and lead,

sampling: between 5 and 10 mg capsule capacity of 50 μl;

initial temperature: 10oC; heating rate of 10oC per minute;

final temperature: 300oC.

Desametasone forms of hGH.

The percentage detalizirovannym forms determined by any syshestvyut using solution A (13.8 g of ammonium dihydrophosphate in 1000 ml of water; pH=7, we would like to inform using a concentrated solution of ammonia and water as solution B, using the following program: 5% solution a for 2 minutes; then the transmission with a 15% solution a for 5 minutes; after that the transmittance of 50% solution a for 20 minutes; finally, the transmission with 100% solution a for 5 minutes and maintaining this last solution for 5 minutes. For elution hGH (transmittance of about 15 minutes) and detalizirovannym forms observed at 220 nm. The flow rate is 1 ml per minute.

The results of these different methods of analysis are presented below.

The content of the dimer and related substances of higher molecular mass.

The total content of oligomers to polymers in lyophilizate, including 4UI hGH absorbed 0.5 ml of water, based on the ratio R (Fig. 1). In Fig. 1 shows that the minimum content of the oligomers to polymers in solutions obtained when R = 0.1 to 1, and the value of 0.1 is obtained by interpolation of the curve.

As an additional example, observe the stability at 25oC and 35oC two parties: hGH concentration 4UI and 8UI. Their excellent stability after storage for 6 months.

The following is the personal time and at different temperatures in comparison with the standards of the European Pharmacopoeia. In these tables, R = 0.45.

Determination of the enzymatic activity of the oxidase of lithate.

The enzymatic activity of liofilizatow containing 30 UAE oxidase-lithate absorbed 1 ml of water, based on the ratio R after 1 month when 35oC (see Fig. 2). In Fig. 2 shows that the initial enzymatic activity of the oxidase of lithate persists after 1 month when 35oC when R = 0.4 to 1.

As an additional example, the composition with R = 0.45 or R = 0.67 completely stable after 3 months at 25oC (residual activity close to 100% activity at time zero).

Turbidity dissolved form of a lyophilisate.

The turbidity of the solutions liofilizatow containing 4 UI hGH absorbed 0.5 ml of water, based on the ratio R (see Fig. 3). In Fig. 3 shows that the minimum turbidity of the solutions is achieved when R = 0 and 1.5.

The turbidity of the solutions liofilizatow containing 30 UEA oxidase-lithate absorbed 1 ml of water, based on the ratio R (see Fig. 4). In Fig. 4 shows that the resulting minimum turbidity of the solutions is achieved when R = 0.2 to 1.

Organoleptic criteria liofilizatow.

Organoleptic criteria each life the phosphate buffer, determined depending on the values of R and these parameters are given in table. 5. In table. 5 shows that lyophilizate have satisfactory organoleptic criteria at R = 0.125 and 1.7. These characteristics do not vary with time.

Diffraction of x-rays (X-rays).

The results of x-ray diffraction analysis powder form obtained liofilizatow containing a mixture of alanine with mannitol ratio R varying from 0 to + presented in table. 5.

The obtained diffraction patterns show that when R = 0 - 1 appear one line lattice alanine, moreover, the deviation from the baseline of the diffraction pattern indicates the presence of amorphous phase formed by mannitol. The higher the R value, the more significant becomes the amorphous phase due to mannitol. For R > 1 mannitol also crystallizes.

For R = 0 - 1 receive amorphous phase formed by mannitol and crystalline phase formed by alanine.

Differential thermal analysis.

Temperature stelopererad of liofilizatow containing 4 UI hGH or 30 UAE oxidase-lithate or contains no protein, and changing the content of phosphate in the morning is ur stelopererad, obtained in the case of liofilizatow containing a mixture of alanine with mannitol, is achieved by (I/R) > 1, i.e., when R = 0 - 1.

Temperature stelopererad freeze-dried represents the maximum temperature of stability of lyophilized. The maximum temperature stability of lyophilized, therefore, is achieved for R = 0 - 1.

Desametasone form.

The results obtained with parties 4 8 UI and UI, show very small changes that are compatible with the provisions of the monograph on the growth of the European Pharmacopoeia. These results suggest that this composition is quite stable at 25oC.

In conclusion, we discovered that each property of liofilizatow is optimal for the interval of values of R, which in General corresponds to the table. 6.

Each analytical criterion /% oligomers + polymers, the stability of hGH, the enzymatic activity of the oxidase of lithate after 1 month when 35oC, the turbidity of the solutions of hGH, the turbidity of the solutions oxidase-lithate, the appearance of liofilizatow, one crystalline alanine, the maximum temperature of stelopererad gives specific optimal interval for each property. In order to obtain an acceptable stood lusterous the invention, without limiting its scope.

1. Stable proteincoding freeze-dried composition comprising a buffer, alanine and mannitol, wherein the mass ratio of alanine : mannitol is 1:0.1 to 1.

2. Stable proteincoding composition under item 1, characterized in that the quality of protein it contains a hormone, enzyme or cytokine.

3. Stable proteincoding freeze-dried composition according to p. 2, characterized in that the quality of protein it contains hormone hGH.

4. Stable proteincoding freeze-dried composition according to p. 2, characterized in that the quality of protein it contains the enzyme oxidase - lithate.

5. Stable proteincoding freeze-dried composition according to p. 2, characterized in that the quality of protein it contains the cytokine interleukin - 13.

6. Stable proteincoding lyophilized composition under item 1, characterized in that it contains alanine in crystalline form, and mannitol - amorphous.

7. Stable proteincoding lyophilized composition under item 1, characterized in that it contains an amorphous phase formed m nadarasa freeze-dried composition for p. 1, characterized in that the buffer contains phosphate buffer.

 

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