Note: Descriptions are shown in the official language in which they were submitted.
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IRIP~37
LYOPHILIZED PEPTIDE FORMULATIONS
The present invention relates to formulations
for lyophilized preparations of peptides in a stable form
for therapeutic administration. More particularly, the
invention relates to a formulation for lyophilizing
thymopentin in stable dosage forms.
8ack~round 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 activlty 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 lyophiliæation does not occur as frequently.
However, there also exist a number of larger peptides or
polypeptides which experience loss of activity upon
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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~ 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.
Exp. Med., 156: 1057-1064 (1982); T. Audhya et al.,
Biochem, 20: 6195-6200 (1981); Venkatasubramanian~ K. et
al, Proc. Natl. Acad. Sci. U.S.A., 83: 3171-3:L74 (1986);
Malaise M.G. et al, in "Immunoregulatory UCLA Symposium
on Molecular and Cellular Biology'i, eds. Goldstein, G.,
et al (Liss, New Yor~) (1986); Sunshine, G.H. et al, J.
Immunol., 120: 1594-1599 (1978) and E. Rentz et al, ~rch.
Geschwulstforsch, 54(2): 113-118 (1948). See also U.S.
Patents 4,261,886; 4,361,673; 4,420,424; and 4,629,723.
Re~erence 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 fre~uently found to
be biologically inactive. This loss of activity is noted
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in bulk prepara-tions only in a small percentage of the
peptide composition located on the surface of the dry
preparations.
Such activity loss may efi-ect 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 wiil 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.
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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 sta~le
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 ~ollowing detailed
description of the present invention~
DETAILE:D 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 o~ 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.
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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
19 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 sa].t 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 me=hod for stabilizing
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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
lyophilization.
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 hases, which
are physiologically safe ~or 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
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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 facilitatecl stabilization of the
peptide during lyophilization is a combination of 0.5 to
2% glycine and 1 to 6% raffinose. The ra~finose sugar is
~5 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.
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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 -60C.
For larger peptides of greater than 20 amino acids in
length, the freezing temperature should be no lower than
about -30C. This temperature is applied for a ~ime
sufficient to freeze the batch sizé of the peptide
composition. Generally, for example, a batch size of
1500 liters is frozen for up to about 8 to lO hours.
The temperature of lyophilization is also
critical to the per~ormance of this process. The
lyophilization temperature must not exceed about 22C.
Preferably the temperature range of lyophilization is
between 5C to 22C. The vacuum conditions employed in
the lyophilization process should range between 40
2 1~
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 i5 not limited
to the particular peptide, but is qenerally useful in
overcoming biological instability of any peptide or
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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: lOOO.Og
(50.0mg/ml) thymopentin adjusted for peptide content;
200.0g tlO.Omg/ml, 1%) glycine (USP); 400.0 g (20.0mg/ml,
2~) D-raffinose pentahydrate; 176.0 g ~8.8mg/ml~ sodium
citrate (2H2O, USP); and approximately 15 liters of water
for injection tUSP or Ph. Eur.).
The peptide composition after lyophilization
will be placed in ampules with a fill volume o~ 1.3 ml
per ampule.
The process for preparing the formulation using
the above ingredients is as follows. Approximately 15
liters of water for injectlon is introduced into a
suitable stainless steel or glass vessel. The 200 g of
glycine is added and stirred at maximum speed until
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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 ~.76.0 g sodium citrate (2H2O) 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 lN NaOH. If necessary,
the pH may be further adjusted with lN 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-~iltered 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.
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The resulting thymopentin solution is placed
into the ampules (1.3 ml fill volume~. After fill, the
thymopentin compositions are frozen ln the ampules to a
temperature of approximately -60C for approximately 8 to
lo hours. The ampules are then placed in a conventional
lyophilizer for up to 18 hours with t:he conditions for
lyophilization set for 22C and 60 millibars.
The resulting ampules contain stable
lyophilized thymopentin, which demonstrates ~ull
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 o~ the
present invention are included in the above-identified
speci~ication and are expected to be obvious to one of
s~ill 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.
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