Background of the Invention

Many peptides, particularly peptides from about three to about 20 amino acids in length, are unstable during lyophilization and therefore cannot be prepared in the lyophilized form which is usually suitable for maintaining activity for injectable clinical dosages. Many small peptides lose biological activity during lyophilization. This characteristic loss of activity in small peptides may be due to the loss of water of crystallization that occurs during the lyophilization process, resulting in peptides that fold improperly. Because large peptides have a larger number of chemical bonds to retain proper configuration, such activity loss with lyophilization does not occur as frequently. However, there also exist a number of larger peptides or polypeptides which experience loss of activity upon

lyophilization, including, e.g., epidermal growth hormones of approximately 191 amino acids in length.

One example of a peptide which experiences such activity loss is thymopentin, a pentapeptide of proven pharmacological use and significance. See, U. S. Patent

4,190,646 and Goldstein, G. Nature (London) 247; 11-14

(1974); Basch, R.S. and Goldstein, G., Proc. Natl. Acad.

Sci. U.S.A. , 71: 1474-1478 (1974); Scheid, M.P. et al, J.

EXP. Med.. 147: 1727-1743 (1978); Scheid, M.P. et al, Science. 190: 1211-1213 (1975) ; Ranges, G.E. et al, J.

EXTO. Med. , 156: 1057-1064 (1982); T. Audhya et al. ,

Biochem, 20: 6195-6200 (1981) ; Venkatasubramanian, . et al, Plroc. Natl. Acad. Sci. U.S.A.. 83: 3171-3174 (1986) ;

Malaise M.G. et al, in "Immunoregulatory UCLA Symposium on Molecular and Cellular Biology", eds. Goldstein, G., et al (Liss, New York) (1986) ; Sunshine, G.H. et al, J.

Immunol. , 120: 1594-1599 (1978) and E. Rentz et al, Arch.

Geschwulstforsch. 54(2) : 113-118 (1948). See also U.S.

Patents 4,261,886; 4,361,673; 4,420,424; and 4,629,723. Reference is made to the above-described patents, applications and articles for their discussions of thymopentin.

Lyophilized preparations of clinical (ampule) quantities of thymopentin have been frequently found to be biologically inactive. This loss of activity is noted

in bulk preparations only in a small percentage of the peptide composition located on the surface of the dry preparations.

Such activity loss may effect the therapeutic treatment of a patient requiring a particular pharmacologically active peptide. A loss of activity in the dosage unit will result in too little active peptide being delivered to the patient in the normal dosage unit. Thus, the appropriately effective dose of the peptide will not be given to the patient. If the activity loss is less than complete, such a variable loss will render it impossible for a practical pharmaceutical dosage to be accurately determined. Simply raising the dosage level of the peptide to compensate for this loss is not practical because the degree of loss would be unknown and excess dosages of most pharmaceuticals carry an increased risk of serious side effects. Such inefficient methods to compensate for activity loss of the peptide will also increase the cost of the pharmaceutical in question. Therefore a need exists in the art for methods of preparing therapeutic peptides in a manner which will retain the biological activity of clinical quantities thereof.

SUMMARY OF THE INVENTION

As one aspect, the present invention provides a method for preparing clinical quantities of therapeutically active peptides in a stable lyophilized form.

As another aspect of the present invention is provided a stable lyophilized preparation of a peptide produced by the method of the invention. As one preferred embodiment, the invention provides a stable lyophilized preparation of thymopentin. This stable preparation is prepared by the method of this invention.

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

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for stabilizing lyophilized clinical quantities of pharmacologically desirable peptides. Any peptide may be prepared by this method. However, the method has been found to be of particular benefit in the preparation of small peptides of from about 3 to about 20 amino acids in length which experience a biological activity loss in conventional dosage units.

Larger peptides which also exhibit activity loss upon lyophilization may also be prepared according to this method. An example of such a larger peptide which experiences this biological activity loss is epidermal growth factor, which is approximately 191 amino acids in length.

According to the method of the present invention, a selected peptide is prepared in a high solubility buffering salt. By "high solubility" is meant a buffering salt having a solubility greater than one gram/ml in water. In general the buffering salt is characterized by a solubility higher than that of an inorganic molecule such as sodium phosphate. Because the buffering salt is for use in preparing a therapeutic product, desirably for use in humans, the high solubility buffering salt for use in the present invention must be non-toxic and capable of safe use in humans. Although a number of buffering salts which meet both qualifications of high solubility and safety in humans may be selected by one of skill in the art, a preferred buffering salt according to this invention is citrate buffer.

It was surprisingly discovered that low solubility buffering salts, such as acetate or phosphate buffers, are not useful in this method for stabilizing

peptides undergoing lyophilization. While the present invention is not bound by theory, it is speculated that the low solubility buffering salts ordinarily used to lyophilize peptides cause the separation of the salt from the solution at low temperatures essential for lyophilizatio .

Additionally, according to the present invention the buffered peptide, if a small peptide between about 5 to 20 amino acid in length, should be prepared at an appropriately controlled pH. Desirably for these small peptides, like thymopentin, the pH should be in the range of from about 6.5 to about 7.2. The pH may be adjusted with appropriate acids and bases, which are physiologically safe for humans. For example, an appropriate base for such pH adjustments is sodium hydroxide. An acid such as hydrochloric acid may also be employed for pH adjustment during this method. For larger peptides this range of pH values is not generally required. When lyophilization is performed on the buffered peptide according to this method, a carrier is _ required for the peptide. The inventors have surprisingly discovered that many conventionally employed

carriers for lyophilization processes do not contribute to the stabilization of lyophilized preparations of peptides. For example, conventionally employed sugar carriers appear to be ineffective when used to stabilize thymopentin in this process. For example, glycerol, polyethyleneglycol, lactose, sucrose, glucose, mannitol, glycine and raffinose, all used individually as carriers proved ineffective. Additionally, various combinations of asparagine, glucine and lysine were also unexpectedly inadequate as carriers for this process.

Thus, in the practice of this invention, a preferred carrier which facilitated stabilization of the peptide during lyophilization is a combination of 0.5 to 2% glycine and 1 to 6% raffinose. The raffinose sugar is generally present in the form of D-raffinose pentahydrate. Other amino acids, particularly arginine, lysine, aspartic acid or glutamic acid, may also be used in place of glycine for combination with D-raffinose to provide effective carriers for use in this invention. Preferably the ratio of the amino acid to the raffinose is about 1:2. This ratio may vary based on the pH of the solution and the concentration of peptide and buffering salt employed. A presently most preferred carrier, as illustrated in the examples below is 1% glycine and 2% raffinose in a ratio of 1:2.

Another effective carrier useful in this method is 1% human serum albumin. However, it is not preferred due to possible contaminants, e.g., viruses, which may be present in serum-derived components. In this method of the present invention for providing lyophilized peptides having a stable biological activity, the lyophilization procedures must be strictly controlled. Prior to lyophilization, the peptide solution must be frozen at a temperature which avoids the formation of ice crystals which disrupt the peptide bonds. The freezing temperature depends on the size of the peptide. For smaller peptides under about 20 amino acids, the freezing temperature may be as low as -60°C. For larger peptides of greater than 20 amino acids in length, the freezing temperature should be no lower than about -30°C. This temperature is applied for a time sufficient to freeze the batch size of the peptide composition. Generally, for example, a batch size of 1500 liters is frozen for up to about 8 to 10 hours. The temperature of lyophilization is also critical to the performance of this process. The lyophilization temperature must not exceed about 22°C. Preferably the temperature range of lyophilization is between 5°C to 22°C. The vacuum conditions employed in the lyophilization process should range between 40

millibar to 80 millibar. A preferred vacuum pressure for the preparation of small peptides like thymopentin is about 60 millibars. No excess heat or vacuum is desirable in obtaining a resulting stabilized product. These lyophilization conditions are generally applied for a duration of 18 hours or less, depending on the batch size being lyophilized, until the peptide composition being lyophilized according to the method of the present invention reaches a moisture content of less than 6%. A preferred moisture content range for the product of the lyophilization procedure is between 3% to 6%. The moisture content of the peptide preparation is easily determined by means of the conventional Karl- Fischer test. The lyophilization process of the present invention is appropriate for use in preparing dosage forms of a variety of therapeutic peptides, including, but not limited to, thymopentin, thymoralin, growth hormone, encephalin and tumor necrosis factor. The selection of and size of the peptide undergoing this method of preparation and stabilization is not critical to this invention. Therefore this method is not limited to the particular peptide, but is generally useful in overcoming biological instability of any peptide or

protein which loses biological activity upon lyophilization.

The following examples illustrate the method of preparing a stable lyophilized peptide formulation of an exemplary peptide, e.g. thymopentin. These examples are illustrative only and do not limit the scope of the present invention.

EXAMPLE To prepare a thymopentin formulation according to the present invention, the following ingredients are combined in a batch size of 20 liters: 1000.0g (50.0mg/ml) thymopentin adjusted for peptide content; 200.Og (lO.Omg/ml, 1%) glycine (USP); 400.0 g (20.0mg/ml, 2%) D-raffinose pentahydrate; 176.0 g (8.8mg/ml) sodium citrate (2^0, USP) ; and approximately 15 liters of water for injection (USP or Ph. Eur.).

The peptide composition after lyophilization will be .placed in ampules with a fill volume of 1.3 ml per ampule. The process for preparing the formulation using the above ingredients is as follows. Approximately 15 liters of water for injection is introduced into a suitable stainless steel or glass vessel. The 200 g of glycine is added and stirred at maximum speed until

dissolved. Stirring is continued rapidly while the 400 g of raffinose is added to the mixture. The glycine to raffinose ratio is 1:2.

The 176.0 g sodium citrate (2H ₂ 0) is then added and the resulting mixture stirred rapidly until the solution is clear. The appropriate quantity of thymopentin, approximately 1.163 grams, adjusted for peptide content is added, while slow stirring is continued to prevent foaming until all thymopentin is dissolved and the solution is clear.

The pH of the resulting solution is checked and adjusted to pH 7.0-7.2 utilizing IN NaOH. If necessary, the pH may be further adjusted with IN HCl.

Additional water for injection is added to make a volume of 20 liters. The mixture is stirred until completely mixed. The solution is pre-filtered utilizing a Millipore AP 15 molecular sieve (or equivalent filter which has been soaked in water for injection) to remove any bacterial contaminants, dust or other insoluble materials from the solution. Thereafter the pre-filtered mixture is filtered again through a sterile Durapore 0.22 micron filter.

The resulting thymopentin solution is placed into the ampules (1.3 ml fill volume). After fill, the thymopentin compositions are frozen in the ampules to a temperature of approximately -60°C for approximately 8 to 10 hours. The ampules are then placed in a conventional lyophilizer for up to 18 hours with the conditions for lyophilization set for 22°C and 60 millibars.

The resulting ampules contain stable lyophilized thymopentin, which demonstrates full biological activity in conventional thymopentin assays. Such assays are known to one of skill in the art and are disclosed in the U. S. patents and other references on thymopentin cited above.

Numerous modifications and variations of the present invention are included in the above-identified specification and are expected to be obvious to one of skill in the art. Such modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.