JPH03505334A - Lyophilized peptide formulation - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Lyophilized peptide formulation The present invention provides formulations of lyophilized formulations of peptides in a form that is stable for therapeutic administration. relating to things. More particularly, the present invention provides lyophilized titanium in stable dosage form. Concerning the formulation of mopentin.

Technical background of the invention Many peptides, especially those with chain lengths of about 3 to 20 amino acids, are is unstable and therefore typically retains activity for injectable clinical administration. Although it is suitable, it cannot be prepared as a lyophilized form. many short chain lengths Peptides lose their biological activity upon lyophilization. Features in small peptides This loss of activity is due to the loss of water of crystallization that occurs during the lyophilization process. This is due to improper folding of the Long chain peptides should be properly aligned. Because it has a large number of chemical bonds that maintain its position, the activity associated with such freeze-drying is Loss does not occur very often. However, it also experienced loss of activity upon lyophilization. A large number of peptides, including, for example, epidermal growth hormone with a chain length of about 191 amino acids. polypeptides or polypeptides.

One example of a peptide that experiences such a loss of activity is It is a pentapeptide, thymopentin.

U.S. Pat. No. 4,190,646, Goldstein, G. ), Nat-ure (London), 47: 1l-14 (1974); R- 3-Basch and Goldstein, Proc, Natl. Acad, Sci., U.S.A., 71:1474-1478 (1974) M, P, Sci 56:1057-1064 (1982): T, Au dhya) et al., Biochem, 20:6197-6200 (1981); , Venka [asubramanian], etc., Pr. oc, Natl.

Acad, Sci, U, S, A, 83: +3171-3174 (1986 ); M, G, 717~ (Malaise) et al., “Immunoregulat ory UCLA Symposit+m on Mo1ecular and Ce1lular Bio| Rentz et al., Arch-GeschwulstForsch, 5 4(2):113-118 (1948).

Also, US Pat. No. 4,261,886; 4.361.673; 4,420.4 See No. 24 and No. 4,629,723. Regarding the above patents, patent applications, and papers See description of thymopentin.

Lyophilized preparations of thymopentin in clinical (ampoule) quantities are often biologically ineffective. It has been found to be active. This loss of activity is due to the appearance of dry formulations in bulk formulations. It is observed only in a small fraction of the peptide composition located on the surface.

Loss of such activity may result in treatment of patients requiring specific pharmacologically active peptides. May affect treatment. Loss of activity for a dosage unit is too little active Pep This results in Tide being distributed to patients in normal dosage units. Therefore, it is reasonably effective. peptide dosage would not be given to the patient. Where loss of activity is complete If the actual pharmaceutical dosage is less than the It would be impossible to determine. The degree of loss is unknown and many drugs This loss should be avoided, as excessive administration of It is impractical to simply increase the dosage level of the peptide to compensate.

These useless methods of compensating for loss of peptide activity are It would also increase costs.

Therefore, manufacturing pharmaceutical peptides in a manner that retains biological activity in clinical quantities. A technical need exists for a method.

Summary of the invention In one embodiment, the present invention provides clinical quantities of therapeutically active compounds in stable lyophilized form. A method of producing a peptide is provided.

In another aspect of the invention, stable lyophilized Peptide formulations are provided. In one preferred embodiment, the present invention provides stable lyophilization. The present invention provides a formulation of thymobentin. This stable formulation is prepared according to the method of the invention. Manufactured.

Other aspects and advantages of the invention are further described in the detailed description of the invention below. .

Detailed description of the invention The present invention provides lyophilization of pharmacologically desirable peptides to stabilize clinical quantities. provide a method for Any peptide can also be produced by this method. However, the book The method provides about 3 to about 2 doses experiencing loss of biological activity in conventional dosage units. It has been found that the production of short peptides with a chain length of 0 amino acids is particularly advantageous.

Long chain peptides that similarly lose activity upon lyophilization can also be used in the method of the present invention. It can be manufactured by These chain lengths experience a loss of biological activity. An example of a long peptide is epidermal growth factor, which is approximately 91 amino acids long. be.

According to the method of the invention, selected peptides are produced in highly soluble buffered salts. . The term "high solubility" refers to buffer salts that have a solubility in water of 19/IIIQ or higher. means. Buffer salts generally have a higher solubility than that of inorganic molecules such as phosphates. It is characterized by: Buffer salts are used in the manufacture of therapeutic products, preferably for humans. Since the high solubility used in the present invention is used in the production of Buffer salts must be non-toxic and safe for human use. High solubility A large number of buffer salts can be selected by those skilled in the art that meet the criteria of both solubility and human safety. Although optional, a preferred buffer salt according to the invention is citrate.

Surprisingly, low solubility buffer salts such as acetate or phosphate are not susceptible to lyophilization. It has been found that the methods of the present invention are not useful for stabilizing peptides that contain peptides.

Although the present invention is not bound by theory, the present invention provides a method for lyophilizing peptides. Commonly used low-solubility buffer salts can be removed from solution at the low temperatures required for freeze-drying. It is thought that salt separation may occur.

Further in accordance with the invention, the buffered peptide has a chain length of about 5 to 20 amino acids. Short peptides should be produced at moderately regulated pH. these For short-chain peptides such as thymopentin, the pH ranges from about 6.5 to The apH should be in the range of 7.2, a suitable acid and pH that is physiologically safe for humans. Can be adjusted with bases. For example, a suitable base for such PI(v411n) is sodium hydroxide. It is thorium. Acids such as hydrochloric acid may also be used to adjust the pH during this method. can. For long peptides, pH values in this range are generally not required. .

When lyophilization is performed on buffered peptides according to the method of the invention, the peptide Tido requires a carrier. Surprisingly, the present invention A common carrier for the lyophilization process is the lyophilized carrier of the peptide. It was found that this did not contribute to the stabilization of the formulation. For example, common sugar carriers are , it is ineffective when used to stabilize thymopentin in a terminal procedure! be exposed . For example, glycerol, polyethylene glycol, lactose, sucrose, Lucose, mannitol, glycine and raffinose are individually used as carriers. It was recognized that all cases where used as a substitute are invalid. Furthermore, asparagine, glycine and Lysine was also surprisingly unsuitable as a carrier for this process.

Therefore, in the practice of the present invention, suitable Examples of suitable carriers include 0.5 to 2% glycine and 1 to 6% raffinose. It is a combination of Raffinose sugar is generally in the form of D-raffinose pentahydrate. Existing. other amino acids, especially arginine, lysine, aspartic acid or Rutamic acid is also used in combination with D-raffinose instead of glycine. carriers can be provided that are effective for use in the light of light. Preferably amino acids The ratio of raffinose to raffinose is approximately 1:2. This ratio is determined by the pH of the solution and the concentration of the peptide. and may vary based on the buffer salt used. As shown in the examples below, The currently most preferred carrier is 1% glycine and 2% raffinose in a ratio of 1:2. It is.

Another effective carrier useful in the present invention is 1% human serum albumin. . However, due to foreseeable contamination, e.g. viruses that may be present in serum-derived components, Albumin is not suitable because of the

The present invention provides lyophilized peptides with stable biological activity. In this method, the freeze-drying process must be strictly controlled.

Prior to lyophilization, lyophilize at a temperature that avoids the formation of ice crystals that disrupt peptide bonds. The peptide solution must be frozen. Freezing temperature depends on the size of the peptide.

For short peptides of approximately 20 amino acids or less, the freezing temperature is -60°Cff. It must be at a low temperature of 1 degree. Amino i! Long chain peptides of 220 or more The freezing temperature should not be lower than about -30°C. This temperature is each for a sufficient time to freeze the peptide composition. for example, Generally, 1 sou batches are frozen for about 8 to 10 hours.

The temperature of freeze-drying is also important to the practice of this invention. Freeze-drying temperature is above approximately 22°C Must not be allowed. Preferably the freeze drying temperature range is between 5°C and 22°C.

The vacuum conditions used in the freeze-drying process range from 40 mbar to 80 mbar. must be enclosed. Suitable for producing short chain peptides such as thymopentin The vacuum pressure is approximately 60 mbar. excess to obtain a stabilized product. heat or vacuum are undesirable.

These freeze-drying conditions generally vary depending on the size of the batch to be freeze-dried. 1 until the peptide composition to be lyophilized by the method reaches a moisture content of 6% or less. Used for 8 hours or less. Suitable moisture content of freeze-dried products The content range is between 3% and 6%. The water content of peptide preparations is the same as that of conventional curls. - Can be easily measured by Fischer test.

The freeze-drying method of the present invention includes dosage forms of thymoventin, thymolalin, and growth hormone. various therapeutic agents, including but not limited to, encephalin and tumor necrosis factor. Suitable for use in the production of therapeutic peptides. The production and stabilization method of the present invention The selection of the peptides involved and their size are not critical to the invention.

Therefore, this method is not limited to specific peptides, and the biological activity during lyophilization is generally useful in overcoming biological instability of any peptide or protein that is lost. It is for use.

The examples below provide representative peptides, such as thymobentin, for stable lyophilized A method for producing a peptide formulation will be described. These examples are for illustrative purposes only. However, it is not intended to limit the scope of the present invention.

! back! To produce the thymopentin formulation according to the invention, in a 20 g scale batch: Use a combination of ingredients: Peptide content 1; adjusted 1000.0e ( 50,,0+oa/d) of thymoventin; 200. Og (io, 0+ma/gu , 1%) Glycine (USP); 400. Og (2Q, Omg, '+ff, 2% ) D-raffinose pentahydrate; l 76.0 (8,13+g/+d) query water for injection (USP or Ph, Eu　r,).

After lyophilization, the peptide composition was placed into ampoules with a volume of 1.3 sQ per ampoule. It will be done.

The method for producing the formulation using the above ingredients is as follows. Approximately 15Q of water for injection Introduce into a suitable stainless steel or glass container. Added 2009 glycine and stir at maximum speed until dissolved. Add 400g raffinose to the mixture Continue stirring rapidly during this time. Peptide content I; adjusted appropriate amount of thymopenci About 1.163 g of thymobentin was added until all the thymobentin was dissolved and the solution was clear. Continue to stir gently to prevent foaming until .

Check the pH of the resulting solution and adjust it to pH 7.0-7.2 using IN NaOH. make a clause If necessary, the pH can be further adjusted with IN hydrochloric acid.

Further water for injection is added to bring the volume to 2 GQ. Millipore A Pre-prepared using P15 molecular sieves (or equivalent filter media soaked in water for injection). Prefilter to remove bacterial contaminants, dust, or other insoluble materials from the solution. After that The pre-filtered mixture was transferred to a sterile Durapore O-22 micro tube. Refilter through filter media.

The resulting thymopentin solution is placed in an ampoule (volume 1, 311172).

After filling, the Timobentin composition is heated in an ampoule to a temperature of about -60°C for about 8 to 10 minutes. Freeze time. The ampoules were then frozen at a temperature of 22°C and 60 mbar. Place in a conventional freeze dryer with drying conditions for 18 hours.

The resulting ampoules are stable and have sufficient biological activity in conventional thymopentin assays. Contains lyophilized thymoventin. Such analytical methods are known to those skilled in the art. , as disclosed in the U.S. patents and other literature on thymoventin cited above. Ru.

Numerous modifications and variations of the invention are encompassed by the above specification and will be apparent to those skilled in the art. It is expected that there will be. Such modifications and variations of the compositions and methods of the present invention are described below. What is included within the scope of the claims appended hereto.

it is conceivable that.

international search report

Claims (14)

[Claims] 1. A buffer salt with high solubility that allows the peptide to be physiologically tolerated and human serum albumin. and a combination of raffinose and amino acids. The resulting peptide composition is mixed with a carrier; the formation of ice crystals is avoided. freeze at a temperature sufficient to Under conditions of vacuum between bars, the composition retains a moisture content of no more than 6%. lyophilizing the peptide composition for a sufficient period of time to , a method for producing stable lyophilized peptides. 2. The suitable carrier may be glycine, arginine, lysine, aspartic acid or 0.5-2% by weight of amino acids selected from glutamic acid, and 1-6% by weight of 2. The method of claim 1, wherein the method is a mixture of raffinose. 3. 3. The method of claim 2, wherein the amino acid is glycine. 4. Claim 1, wherein the amino acid and raffinose are in a concentration ratio of about 1:2. Method described. 5. 2. A method according to claim 1, wherein said suitable carrier is human serum albumin. Method. 6. Claim 1, wherein the buffer salt has a solubility in water of greater than 1 g/ml. Method described. 7. 7. The method of claim 6, wherein the buffer salt is a citrate buffer. 8. Claim 1, wherein the peptide composition has a pH between 6.5 and 7.2. The method described in section. 9. Claim 1, wherein said temperature of said freeze-drying ranges between 5° and 22°C. The method described in. 10. 2. A method according to claim 1, wherein the freeze-drying vacuum is about 60 mbar. Law. 11. Claims wherein the peptide composition has a water content between 3% and 6%. The method described in Section 1. 12. Physiologically acceptable buffer salts with high solubility and human serum albumin a suitable compound selected from the group consisting of amino acids and combinations of raffinose and amino acids; the resulting peptide composition with a suitable carrier; the formation of ice crystals is avoided. Freeze at a temperature sufficient to Under conditions of vacuum between libars, the composition retains a moisture content of no more than 6%. produced by lyophilizing the peptide composition for a sufficient period of time to A lyophilized peptide or protein that retains sufficient physiological activity. 13. 13. A peptide according to claim 12, comprising thymoventin. 14. Peptides or proteins in highly soluble buffered salts are frozen and lyophilized , an improved way to maintain physiological activity in lyophilized peptides or proteins. In the method, the buffered peptide is combined with human serum albumin and raffinose. in a suitable carrier selected from the class consisting of combinations with amino acids. An improvement method comprising: