Note: Descriptions are shown in the official language in which they were submitted.
WO 93/19773 PCl/US93tO3182
s21337?)7
lyoph~ 1 ~ zed somatotropl n formul atl on s
BACKGROUND OF THE I~VENTION
The present invention relates to novel solutions
of somatotropins which remain clear for an ext:ended
period of time and which remain clear when sub~ected to
mechanical agitation, and to kits for makin~ the
solutions.
Somatotropins, also known as growth hormones, are
polypeptide hormones secreted by the pituitary glands
of many animal species. These hormones are valuable
for a number of therapeutic u~es, and compositions
comprising somatotropin can be administered in the
treatment of pituitary deficiency in humans and
gastrointestinal bleeding or to promote the healing of
bone fractures and accelerate t.he healing of contusions
and other wounds. Somatotropins also are useful in
promotin~ meat and milk production in animals when
admini~tered through various drug-releasing d~vices or
by in~ection. (See E.J. Turman, "Some Effects of
Pituitary Anterior Growth Factor~ Thesis: Purdue
University, April, lg53; L.~ Machlin, J. ~nim. Sci.
35: 794-B00 (1972); T.R. Xasser ~t al., J. Anim.. Sci.
53: 420-426; L.J. Machlin, J. Dair~ Sci. 56: 575-580
(1973))
It frequently iQ de~irablQ to work with and
administer protein~ such as somatotropin~ in the form
of a solution. Howev~r, dissolved proteins, such as
WO~3/19773 PCT/US93/03182
'L'!L`3'~ 2
somatotropin, can be adsorbed at hydrophobic
interfaces, thus causing secondary reactions. For
example, "denaturing~', i.e. a change in the shape of
the adsorbed somatotropin molecules can occur. In
addition, aggregation of adsorbed somatotropin
molecules can take place to give soluble or in~oluble
polymeric forms. This aggregation will manifest
itself, for example, as turbidity of the solution or as
biological inactivation of the somatotropin protein on
stirring or shaking of the aqueous solution~ (5ee A.F.
Henson et al., J. Colloid Interface Sci. 32: 162
(1970)).
When the somatotropin precipitates from solution,
the precipitated protein becomes unavailable for
~: 15 administration. Thus, a ~omatotropin solution is
needed which will not become turbid upon ~tirring or
shaking and which will remain clear o~er extended
periods of ~torage.
Summarv of the Invention
In accordance with the present invention, there
are disclosed somatotropin ~olutions which remain clear
for extended periods and which do not become cloudy or
precipitate when sub~ected to mechanical agitation.
The ~omatotropin solutions contain a lyophilized
somatotropin composition containing about 1 part
somatotropin per 2 to 8 parts arginine HCl on a weight
basis, the pH of the composition prior to
lyophilization within the range of about 7.2 to about
8.5, which is dissolved in a diluent. If the pH of the
~omatotropin composition prior to lyophiliz~tion was
less th~n 7.8, the diluent comprises EDTA, a nonionic
~ surf~ctant ~nd ~ buffer. If the pH of tbe somatotropin
::
W093/19773 ~1 3 3 1 ~ 7 PCT/US93/03182
composition prior to lyophilization was 7. a or greater,
the diluent comprises EDTA and a nonionic surfactant
and optionally, further can compriqe a $uitable buffer
or nonbuffering agent if desired. If a non-buffering
agent is added, desirably a buffer is not also added to
the diluent, although both agents can be used.
This invention further provides kit~ for making
the solutions. The kits include a vial containing the
lyophilized somatotropin composition cont~ining about 1
part somatotropin per 2 to 8 part~ arginine HCl on a
weight ba~is. The kit~ al~o include a vial containing
a biocompatible diluent comprising EDTA and a nonionic
surfactant. In addition, as described above, depending
upon the pH of the somatotropin composition prior to
lyophilization, the diluent also can comprise a buffer
or non-buffering agent. If the pH of the somatotropin
-~ composition prior to lyophilization was less than 7.8,
the vial containing the diluent further comprises a
buffer. At a pH of 7.8 or greater th~ vial optionally
~; 20 can contain a buffer or nonbuffering agent. The
diluent can be added to the lyophilized somatotropin
composition and the resultant mixture then shaken to
dissolve the somatotropin.
Detailed Descri~tion of the Invention
This invention is directed to somatotropin
solutions which remain clear for extended periods and
which do not become cloudy or precipitate when
subjected to mechanical agitation such a-~ shaking or
vortex-mixing. A somatotropin ~olution which romains
~clearN is a ~olut~on in which the somatotropin does
not prec~pitste aftor it i8 dissolvod in the diluont.
These somatotropin ~olution~ will re~ain cloar aftor
W093/19773 PCT/US93/03182
~3~ 4
several days of storage, typically at least about S
days. The somatotropin solutions maintain clarity for
at least about 5 days when stored, typically, for
example, at about 25C. At slightly higher storage
temperatures, the somatotropin solution~ maintain
clarity, but possibly for a shorter period of time. A
solution which remains clear will have an apparent
absorbance measurement at 360 nm (O.D. 360) of less
than 0.10.
The somatotropin solutions contain a lyophilized
somatotropin composition, the pH of which prior to
lyophilization was at least about 7.2, generally within
the range of from about 7.2 to about 8.5. If the pH of
the somatotropin composition prior to lyophilization
wa~ less than 7.8, the diluent compri~es EDTA, a
nonionic surfact~nt and a suitable buffer to stabilize
the final somatotropin ~olution. If the pH of the
somatotropin compo~ition prior to lyophilization was
7.8 or higher, the diluent comprises ~DTA and an
nonionic surfactant an~, optionally, a buffer or
nonbufferin~ agent. If a non-buffering agent i8 added
~;~ to the diluent, desirably a buffer i8 not also added to
the diluent, although both agents can be used. Neither
the buffer nor nonbuffering agent need be used if the
pH of the somatotropin composition prior to
lyophilization was at least 7.8, but either can be
provided to adjust or maintain the isotonicity of the
resultant somatotropin solution such that it is less
hypertonic. This is de~irable if the somatotropin
; 30 solution will be in~ected into animals. The choice of
suitable additive can be made based on a number of
factors, includlng, for example, cost, presence on the
FDA GRAS list, autecla~ability, chemical stabillty,
~.
W093/19773 ~ t .~ .~ 7 3 7 pcT/uss3/o3l82
lack of interaction with the somatotropin and
biocompatibility.
The lyophilized somatotropin composition is
prepared by dissolving arginine HCl with somatotropin,
adjusting the pH of the solution to at lea~t 7.2,
preferably from 7.2 to 8.5, and removing any
undi~olved m~terial by filtration or eentrifugation.
The ~omatotropin and arginine HCl solution then is
lyophilized u~ing standard proeedures known in the art.
10The ~olution to be lyophilized generally eontains
about 1 part ~omatotropin per 2 to 8 parts arginine HCl
on a weight ba~i~ and preferably eompri~e~ about 1 part
somatotropin per 3 parts arginine HCl. Thus, the
solutlon preferably contains 10 to 150 mg/ml
15somatotropin and 30 to 450 mg/ml arginine HCl and has a
final pH of at least 7.2. Most preferably, the
solution to be lyophilized eontains 30 mg/ml
somatotropin and 90 mg/ml arginine HCl and has a final
pH of from 7.8 to 8.5.
~0The somatotropin whieh may be employed in the
lyophilized ~omatotropin eomposition of thi~ invention
ean be any ~omatotropin, ineluding natural or
reeombinant bovine, poreine, human, avian, ovine, or
equine somatotropin. Preferably, the somatotropin
employed is porcine somatotropin. As used herein, the
term somatotropin is intended to inelude the full
length natural or reeombinant somatot.ropin as well as
derivatives thereof that have growth-promoting
eapabilities. Derivatives inelude biologieally aetive
fragment~ or analogs of the polypeptid~ hormone, ~ueh
a~ delta 7 poreine ~omatotropin, whieh has an amino
aeid sequence corre~ponding to that of porcine
somatotropin, less the fir~t seven ~mino acid~ of the
mature, full length hormone (descr~bed ~n ~uropean
WO93/19773 PCT/US93/03182
~ ~a~1 3r-l 6
Patent Application Publication No. 0 104 920 to siogen
N.V.). The term biologically active as used herein
means a polypeptide that, following its administration
to a living being, has a demonstrable effect on a
biological process of that living being.
The somatotropins employed in the compositions of
this invention can be metal-associated somatotropins.
Metal-a~sociated somatotropin is produced by the
addition of salts of transition metals to an aqueous
solution containing the somatotropin. The salts of the
transition metals form insoluble complexes with the
somatotropin, thus precipitating the somatotropin out
of the solution. The metal-a~ociated somatotropins
comprise the somatotropin molecules and metal ions such
as Zn , Cu , Co , Mn , Fe or Fe . These metal-
associated somatotropins contain ligand bonds between
the metal ion and the nitrogen atoms of some of the
amino acid residues in the somatotropin molecule. The
metal-associated somatotropins are used as starting
materials for making the lyophilized somatotropin
compositions. The metals likely are not associated
with the somatotropin once the metal-associated
somatotropin is lyophilized, although they are present
in the lyophilized somatotropin composition. The
presence of the transition metal in the product has
been shown to have no significant adverse effect on the
bioactivity of the somatotropin when the product is
administered to a living being.
The somatotropin solutions of this invention are
made by dissolving the lyophilized somatotropin
composition with a diluent such that the pH of the
final solution i8 at least about 7.2, typically from
about 7.2 to about 8.5, and preferably from about 7.2
to about 8.2. If the pH of the 801ution ~8 ~re~tor
WO 93/19773 2 ~ ~ 3 7 .3 7 PCI/U593/03182
than about 8 5, degradation of the protein can occur.
Typically, about 0.5 to about 40 mg lyophilized
somatotropin composition are provided per ml of
diluent. Preferably, the final concentration of the
somatotropin solution is about 10 to 30 mg lyophilized
somatotropin composition per ml of diluent and most
preferably about 20 mg lyophilized somatotropin
-composition per ml of diluent. The weight of the
lyophilized somatotropin composition will be the total
of the weight of the arginine HCl and th~ weight of the
somatotropin contained in the lyophilized ~omatotropin
composition.
If the pH of the somatotropin composition to be
u~ed in making the somatotropin solution wa~ less than
7.8 prior to lyophilization, the diluent compri-Qe~
EDTA, a nonionic ~urfactant and a buffer.
Alternatively, if the somatotropin composition had a pH
of at least 7.8 prior to lyophilization, the diluent
comprises EDTA and a nonionic ~urfactant and optionally
also compri~es a buffer or nonbuffering agent. If the
diluent comprises a buffer, the pH of the final
-Qomatotropin solution will be the s~me a~ the pH of the
diluent. However, if the diluent is not buffered, the
pH of the lyophilized somatotropin composition
di~solved in the diluent will determine the pH of the
final somatotropin solution.
De~irably, when the pH of the final ~omatotropin
solution is between sbout 7.2 and about 7.6, the
concentration of EDTA provided in the diluent is at
least 1.5 mM, and is preferably from about 1.5 mM to
about 10 mM. When the final ~:n~totropin solution pH
i~ greater than 7.6, thQ concentration of EDTA u~d in
the diluent deslrably is at le~st 1.0 mM, and is
preferably from about 1.0 mM to about 10 mM.
W093/19773 PCT/US93/03182
3s3~ 3~ 8
Surfactants which are suitable for use in the
diluent include polyoxyethylene-23 lauryl ether (Brij
35), Tween 80, polyoxyethylene-20 cetyl ether (Lri;
58), and other polyoxyethylene nonionic surfactants
having similar hydrophilic/hydrophobic balance (XLB).
Such nonionic surfactants have been noted in the prior
art as stabilizing and preserving activity in purified
enzymes. (See T. Kitani et al., Eur. J. Biochem. 119:
177-181 (1981); M. Pritchard et al., Biochem. ~io~hvs.
Res. Commun. 100: 1597-1603 (1981); Seely et al.,
Biochemistrv, Vol. 21, No. 14, 3394-3399 (1982)).
Generally, the surfactant i~ pre~ent in a concentration
ranging from about 0.08% to 2.0%. If the surfactant
utilized is Bri; 35, the concentration of the Bri~ 35
is at least 0.08%, and preferably is from about 0.1% to
about 0.2%. If the surfactant is Tween 80 or Bri~ 58,
the concentration preferably is from about 0.1% to
about 1.0%.
If the pH of the somatotropin prior to
lyophilization was at least 7.2 but less than 7.8, a
buffer is provided in the diluent to increasQ the
stability of the somatotropin solution. The buffer can
be Tris HCl, phosphate, or some other neutral, host-
compatible pH buffer. Generally, the buffer is present
in a concentration rangin~ from about 0.2 M to about
0.5 M. If Tris HCl is utilized as the buffer, the Tris
HCl generally has a concentration of at least 0.2 ~,
and is preferably from about 0.2 M to about 0.3 M.
If the pH of the somatotropin composition prior to
lyophilization was at least about 7.8, either a buffer
as described above or a non-buffering agent can be
added to the diluent, if desirQd, to ad~ust or msintain
the isotonicity of the resultant somatotropin solution.
The non-bu~ f ering aqent also is host-compatible. Such
wos3/l9773 PCT/US93/03182
9 ~ 7 ~7
agents include sucrose, trehalose, or NaCl. The non-
buffering agent, if present, generally is provided at a
concentration from about O.OS M to about 0.5 M. When
sucrose or trehalose is utilized as the non-buffering
agent, preferred concentrations are within the ran~e of
from about 0.1 to about 0.3 M. When NaCl is utilized
as the non-buffering agent a concentration of from
O.OS M to O.lS M generally is preferred.
The somatotropin compositions of this invention
can be made using kits containing 8 vial of the
lyophilized somatotropin composition as discussed above
and a vial containing the diluent discussed above. The
diluent can be added to the lyophilized somatotropin
composition and the resultant product shaken to
lS dis~olve the somatotropin.
The somatotropin solution~ of this invention can
be u~ed for further processing or can be admini~tered
directly to animals. The solution generally can be
stored for at least about 5 days at ambient
; 20 temperatures, typically about 25C, without becoming
cloudy. The solutions al~o can be stored at slightly
higher temperatures, although the solutions may
maintain their clarity a shorter period of time. These
solutions also can be sub~ected to mechanical
agitation, such as shaking or mixing, without
pxecipitation of the somatotropin from the solution.
The somatotropin in the~e solutions maintains its
biological activity and can be administered to animals
in accordance with conventional techniques to promote
growth.
The invention is further illustrated by the
following examples, which are not intended to be
limiting.
,. ~
:::
W093/19773 PCT/US93/03182
33~3~
Examples
In the following examples, solution clarity wa~
determined by visual inspection and/or by measuring the
apparent absorbance at~360 nm (O.D. 360); a wavelength
where somatotropins have no intrinsic absorbance. The
somatotropin solution was placed in the sample cuvette
and the O.D. 360 determined using a Shimadzu UVU160
spectrophotometer. A diluent solution which did not
contain somatotropin was used in the reference cuvette.
Quartz cuvetteR were used in all studies. In the
examples described, "clear" solutions are tho-Re which
have an O.D. 360 of less than 0.10.
Exam~le 1
Thirty-three grams of Zn-associated porcine
somatotropin (pST), made in accordance with the
procedures disclosed in published European Patent
Application No. 83300803.9, and 90 grams of arginine
HCl were dissolved in one liter of sterile water. The
pH was adjusted to 7.8 by the addition of aqueous HCl
or NaOH and insolubles were removed by filtration
through a poly~inylidene difluoride membrane. The
clarified ~olution then was lyophilized. The resulting
formulation (termed the ~lyophilized porcine
somatotropin (pST) composition~') contained
approximately 30 g pST per 90 g argilline HCl, due to
the loss of about 10% of the original 33 g of pST as
insoluble during the filtration step.
Exam~le 2
Solid argi nine HCl (1.92 g) was added to a non-Zn
complexed pST solution containing 0. 639 g pST in 21.6
ml of a pH 9.8, 0.46 mM sodium carbonate buffer. The
w093/19773 PCT/US93/03182
')1~3737
arginine was dissolved by stirring and the pH of the
solution was adjusted to 7.8 by the dropwise addition
of 1 M NaOH. The resulting solution contained
approximately 30 mg/ml pST and 90 mg/l arginine.
Aliquots (7.2 ml) of this solution were pipetted into
three 50-ml vials, frozen at -80C, and lyophilized.
This lyophilized pST composition readily di~solved
in 200 mM Tris HCl, 2 mM EDTA, 0.15~ Bri~ 35 (pH 7.8)
at 20 mg~ml tdissolution time ~ 3 minutes). The
resulting solution was clear by vi~ual in~pection.
Exam~le 3
The following solutions were prepared:
1. O.2 M Tris HCl, 2 mM EDTA, 0.15% Brij 35,
pH 7.8
~; 15 2. ~ .......................... n u
pH 7.4
3. 2 mM EDTA, 0.15~ Bri~ 35, pH 7.8
Zinc-associated pST (Zn-pST), non-metal complexed
pST ~non-Zn pST) and the lyophilized pST composition
(made as describèd in Example 1) were dissolved by
shaking for 2 minutes in each of the above solutions 5
mg~ml pST (or 20 mg/ml of the lyophilized pST
composition). After the 2 minute sha~ing, solution
clarity was detarmined by visual observation and by
measuring turbidity at 360nm. As shown in Table 1,
only the lyophilized pST composition gave a clear
~olution after dis~olution under the~e conditions. The
results are shown in Table 1.
~ ,
. : ~
` ~ '
' ~ ~
WO 93/19773 P~r/V~93/03182
3 ~
Table l
PST SOLUTION OD360
Zn-pST #l 0 . 522
" #2 0 . 504
Il #3 ~ 0 . 381
non-Zn pST #l 0 . 242
" #~ O . 347
~' #3 0. 186
lyophilized pST
composition #1 0.019
" #2 0 . 0l9
#3 ~ . 0l9
Exam~le 4
The following diluent solutions were made up:
1. 0.2M 2 mM
Tris HCl EDTA pH 7.8 containing 0.15% Bri~ 35.
2 . " " " " 0 . 125% " "
3. ~' " " " 0.10% " " .
0.08% ~ n
5- " " " " 0.06%
6. " ~ 0.04%
7. ~ 0.00%
Forty mg aliquots of lyophilized pST composition
(made as described in Example 1) were weighed out into
seven 13 X 100 test tubes and dissolved in 2 ml of each
of the above diluents. The solutions then were
30 sub~ected to mechanical agitation by vortexing on a
Vortex-Genie mixer for 30 seconds at a setting of 4.5.
Soluition clarity was determined by visual inspection by
O . D . 3 60. The results are shown on Table 2.
WO93/19773 PCT/US93/03182
13 ~133737
Table 2
Effect of various diluents on mechanical stability
of reconstituted pST
Diluent Solution No. Appearance O.D. 360
1 Clear 0.040
2 Clear 0.047
3 Clear 0.072
4 Clear 0.092
S sl. cloudy 0.257
6 sl. cloudy 0.484
7 v. cloudy 1.708
_ .
* sl. cloudy = slightly cloudy; v. cloudy - very cloudy
As shown in Table 2, the somatotropin solutions
containing a ~ri~ 35 concentration of at least 0.08%
:~ resulted in a clear ~olution after vortexing the
lyophilized pST compo~ition in the diluent under these
conditions. The somatotropin solutions containing a
Bri~ 35 concentration of 1Q8S than 0.08~ did not remain
clear following mechanical agitation by vortQx mixing.
Exam~l~ 5
Forty mg aliquots of lyophilized pST composition
; 25 were weighed into 2 dram glass vials and dissolved in
the diluent containing 200 mM Tris HCl, 0.10% Bri; 35
at the following EDT~ concentrations and final pH:
WO93/19773 3 ~13~ 14 PCT/US93/031R2
1. 2.0 mM EDTA, pH 7.2
2. 0.25 mM ~ , pH 7.4
3. 0.50 mM " , "
4. 1.00 mM " , "
5. 1.50 mM " , "
6. 2.00 mM
7. 0.25 mM " , pH 7.6
8. 0.50 mM
9. 1.00 mM ~
10. 1.50 mN " , "
11. 2.00 mN " , ~
12. 0.25 mM " , pH 7.8
13. 0.50 mN " , "
14. 1.00 mN
15. 1.50 mN " , "
16. 2.00 mN " , "
The~e samples were then placed in a 25C
constant temperature chamber and checked for visual
clarity at various time points. Results are shown in
: 20 Table 3.
.:~
.
;: :
~. ~
WO93/19773 PCT/US93/03182
~1 ~3 .~ 7 .~ 7
Table 3
Effect of various diluents on solution stability of
reconstituted pST
Solution # Day 1 ~ay 5 Day 10
1 clear clear clear
2 sl. cloudy sl. cloudy v. cloudy
3 clear cloudy v. cloudy
4 clear sl. cloudy cloudy
clear clear clear
6 clear clear clear
7 clear cloudy v. cloudy
8 clear sl. cloudy v. cloudy
15 9 clear claar cloudy
clear clear clear
11 clear clear clear
12 clear cloudy v. cloudy
13 clear cle~r cloudy
14 clear clear sl. cloudy
clear clear clear
: 16 clear clear clear
sl. cloudy = slightly cloudy
v. cloudy = very cloudy
As demonstrated in Table 3, somatotropin
solutions, with a pH from 7.2 to 7.6, containing an
EDTA concentration of at least 1.5 mM resulted in a
clear solution for at lesst 10 days after dissolution
of the lyophilized pST composition in the diluent under
these conditions. Further, somatotropin solutions with
a pH of 7.6 or greater, containing an EDTA
concentration of at least 1.O mM, resulted in a clear
solution for at least 5 days after dissolution of the
lyophilized pST in the diluent under these conditions.
Exam~le 6
Forty mg aliquots of lyophilized pST composition
were weighed into 16 two-dram glass ~ials. Two ml of
the following diluent~ were addeds
W0 93/1977~ 3 ~1 16 PCr/US93/0318
1. 300 m~ Tris HCl, 0 .1596Bri j 35,1. 5 mM EDTA,pH 7 . 4
2. 200 mM Tris HCl, " " " " "
3. 100 mM Tris HCl, " " " " "
4 . 5 0 mM Tri s HC l, " " " " "
5. 25 mM Tris HCl, " " " " "
6 . 200 mM Tris HCl, " " 2 . O mM EDTA, pH 7 . 2
7 . ~ , " " 1. 5 mM EDTA,
8 . " ~' " , " " 1. 0 mM EDTA, "
9. 10~ sucrose, 0.15~ Bri~ 35, 2 mM EDTA
10. 5% sucrose, " " , " "
11. 1% sucrose, I~ " , " "
12. 0.2 M NaCl, 0.15% Bri; 35, 2 mM EDTA
13. 0.1 M NaCl, " " , " "
14. 0.0 M NaCl, " " , " "
The final pH of the samples after reconstitution
with diluents 9-14 was -7.8-7.9 (or approximately equal
to the final ad~usted pH of the pST/Arg solution ~ust
prior to lyophilization). These samples were incubated
:~ in a 25C con~tant temperature chamber for six days
after which turbidity (O.D. 360 nm) readings were
: taken. Results are shown in Table 4.
WO93/19773 PCT/US93/03182
17
7 3 7
Table 4
Effect of various diluents on solution stability of
reconstituted pST
Solution # Day 6 (O.D. 360)
1 0.021
2 0.064
3 0.264
4 0-537
0.658
6 0.064
7 0.084
8 0.979
. 9 0.025
0.025
11 0.025
12 0.025
13 ~ 0.03
14 < 0.03
As demonstrated by solutions 1-5 in Table 4, a
Tris HCl concentration of at least 200 mM must be
employed in a diluent containing 0.1.~% Bri~ 35 and 1.5
mM EDTA at a pH of 7.4 in order for the somatotropin
solution to remain clear for six days. Solutions 6-8
in Table 4 illustrated that an EDTA concentration of at
least 1.5 mM was necessary, in addition to a Brij 35
concentration of 0.15% and a Tris HCl concentration of
200 mM at a p~ of 7.2, in order for the somatotropin
solution to remain clear for six days. As demonstrated
by solutivns 9-14, the somatotropin solution remained
clear after six days when the diluent comprised 0.15~
Bri; 35, 2 mM EDTA and either 1%, 5~, or 10% sucrose or
0.2 M, 0.1 M or 0.0 M NaCl and the final pH of the
samples were approximately 7.8-7.9.
