CA2133737A1 - Lyophilized somatotropin formulations - Google Patents
Lyophilized somatotropin formulationsInfo
- Publication number
- CA2133737A1 CA2133737A1 CA002133737A CA2133737A CA2133737A1 CA 2133737 A1 CA2133737 A1 CA 2133737A1 CA 002133737 A CA002133737 A CA 002133737A CA 2133737 A CA2133737 A CA 2133737A CA 2133737 A1 CA2133737 A1 CA 2133737A1
- Authority
- CA
- Canada
- Prior art keywords
- somatotropin
- solution
- buffer
- diluent
- lyophilization
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/27—Growth hormone [GH] (Somatotropin)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
- A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
Abstract
Somatotropin solutions which remain clear for extended periods and which remain clear when subjected to mechanical agitation are prepared by mixing a lyophilized somatotropin composition containing about 1 part somatotropin per 2 to 8 parts arginine HC1 on a weight basis, wherein the pH of the composition prior to lyophilization was about 7.2 to about 8.5, and a biocompatible diluent which comprises EDTA and a nonionic surfactant. If the pH of the somatotropin composition prior to lyophilization was between 7.2 and 7.8, the diluent further comprises a buffer; otherwise the diluent optionally further can comprise a buffer or nonbuffering agent.
Description
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 ~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 ~ ~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 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.
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.
,. ~
:::
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 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.
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 " , "
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 ~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 ~ ~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 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.
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.
,. ~
:::
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 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.
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.
.:~
.
;: :
~. ~
~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.
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.
.:~
.
;: :
~. ~
~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.
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 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.
Solution # Day 6 (O.D. 360) 1 0.021 2 0.064 3 0.264 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.
Claims (34)
1. A somatotropin solution which remains clear for extended periods and when subjected to mechanical agitation comprising a lyophilized somatotropin composition comprising about 1 part somatotropin per 2 to 8 parts arginine HCl on a weight basis, the pH of which was about 7.2 to about 8.5 prior to lyophilization, which is dissolved in a diluent comprising EDTA, a nonionic surfactant, and, if the pH
of the somatotropin composition prior to lyophilization was between 7.2 and 7.8, a buffer.
of the somatotropin composition prior to lyophilization was between 7.2 and 7.8, a buffer.
2. The somatotropin solution of claim 1, wherein the pH of the somatotropin composition prior to lyophilization was at least 7.8 and the diluent further comprises a host compatible non-buffering agent.
3. The somatotropin solution of claim 1, wherein the pH of the somatotropin composition prior to lyophilization was at least 7.8 and the diluent further comprises a host-compatible buffer.
4. The somatotropin solution of claim 1, 2, or 3 wherein the somatotropin is natural or recombinant bovine, porcine, human, avian, ovine, or equine somatotropin or a bioactive fragment or analog thereof.
5. The somatotropin solution of claim 4 wherein the somatotropin is pST.
6. The somatotropin of claim 5 wherein the pST is delta 7 pST.
7. The somatotropin of claim 4 wherein the somatotropin is a metal associated somatotropin.
8. The somatotropin solution of claim 1, 2, or 3 wherein the nonionic surfactant is polyoxyethylene-23 lauryl ether (Brij 35), Tween 80, or polyoxyethylene-20 cetyl ether (Brij 58) present in a concentration of from 0.08% to 2.0%.
9. The somatotropin solution of claim 8 wherein the nonionic surfactant is Brij 35 having a concentration of at least 0.08%.
10. The somatotropin solution of claim 9 wherein the nonionic surfactant is Brij 35 having a concentration of from about 0.1% to about 0.2%.
11. The somatotropin solution of claim 8 wherein the nonionic surfactant is Tween 80 or Brij 58 having a concentration of from about 0.08% to about 1.0%.
12. The somatotropin solution of claim 1 or 2 wherein the buffer is a neutral pH buffer present in a concentration of from 0.2 to 0.5 M.
13. The somatotropin solution of claim 12, wherein the buffer is Tris HCl or phosphate buffer.
14. The somatotropin solution of claim 13 wherein the buffer is Tris HCl having a concentration of from about 0.2 M to about 0.3 M.
15. The somatotropin solution of claim 3 wherein the concentration of the non-buffering component is from about 0.1 M to about 0.5 M.
16. The somatotropin solution of claim 15 wherein the non-buffering component is sucrose, trehalose, or NaCl.
17. The somatotropin solution of claim 1, 2, or 3 wherein the EDTA is present at a concentration of at least 1.5 mM if the pH of the somatotropin solution is less than 7.6, and is present in the diluent at a concentration of at least 1.0 mM of the pH of the somatotropin solution is at least 7.6.
18. A kit for making a somatotropin solution which remains clear when subjected to mechanical agitation and which does not become cloudy for extended periods, comprising a vial containing a lyophilized somatotropin composition which comprises about 1 part somatotropin per 2 to 8 parts arginine HCl on a weight basis, wherein the pH of said somatotropin composition prior to lyophilization was within the range of about 7.2 to about 8.5; and a vial containing a diluent comprising EDTA, a nonionic surfactant, and, if the pH of the somatotropin composition prior to lyophilization was between 7.2 and 7.8, a buffer.
19. A kit in accordance with claim 18, wherein the pH of the somatotropin composition prior to lyophilization was at least 7.8 and the diluent further comprises a biocompatible non-buffering agent.
20. A kit in accordance with claim 18, wherein the pH of the somatotropin composition prior to lyophilization was at least 7.8 and the diluent further comprises a host-compatible buffer.
21. A kit in accordance with claim 18, 19, or 20 wherein from about 0.5 mg to about 40 mg lyophilized somatotropin composition is provided per ml of diluent.
22. A kit in accordance with claim 18, 19, or 20 wherein the somatotropin is natural or recombinant bovine, porcine, human, avian, ovine, or equine somatotropin or a bioactive fragment or analog thereof.
23. A kit in accordance with claim 18, 19, or 20 wherein the surfactant is polyoxyethylene-23 lauryl ether (Brij 35), Tween 80, or polyoxyethylene-20 cetyl ether present in a concentration from 0.08% to 2,0%.
24. A kit in accordance with claim 18 or 20 wherein the buffer is Tris HCl, phosphate, and is present in a concentration of from 0.2 M to 0.5 M.
25. A kit in accordance with claim 19 wherein the non-buffering component is sucrose, trehalose or NaCl having a concentration of from about 0.1 M to about 0.5 M.
26. A process for making a somatotropin solution which remains clear when subjected to mechanical agitation and which remains clear for extended period which comprises the steps of:
(1) preparing a lyophilized somatotropin composition by dissolving arginine HCl with somatotropin in an amount such that there is 1 part somatotropin per 2 to 8 parts arginine HCl, adjusting the pH to within the range of about 7.2 to about 8.5, removing any undissolved material by filtration or centrifugation, and lyophilizing the resultant solution; and (2) dissolving the resulting lyophilized somatotropin composition in a diluent comprising EDTA
and a nonionic surfactant, wherein, if the pH of the somatotropin composition prior to lyophilization was between about 7.2 and about 7.8, the diluent further comprises a buffer.
(1) preparing a lyophilized somatotropin composition by dissolving arginine HCl with somatotropin in an amount such that there is 1 part somatotropin per 2 to 8 parts arginine HCl, adjusting the pH to within the range of about 7.2 to about 8.5, removing any undissolved material by filtration or centrifugation, and lyophilizing the resultant solution; and (2) dissolving the resulting lyophilized somatotropin composition in a diluent comprising EDTA
and a nonionic surfactant, wherein, if the pH of the somatotropin composition prior to lyophilization was between about 7.2 and about 7.8, the diluent further comprises a buffer.
27. A process in accordance with claim 26, wherein the pH of the somatotropin composition prior to lyophilization was at least 7.8 and the diluent further comprises a biocompatible non-buffering agent having a concentration within the range of about 0.1 M to about 0.5 M.
28. A process in accordance with claim 26, wherein pH of the somatotropin composition prior to lyophilization was at least 7.8 and the diluent further comprises a neutral, host-compatible buffer present in a concentration within the range of about 0.2 M to about 0.5 M.
29. A process in accordance with claim 26 or 28, wherein the buffer is Tris HCl or phosphate buffer.
30. A process in accordance with claim 27, wherein the non-buffering component is sucrose, trehalose or NaCl.
31. A process in accordance with claim 26, 27, or 28 wherein the somatotropin is natural or recombinant bovine, porcine, human, avian, ovine, or equine somatotropin or a bioactive fragment or analog thereof.
32. A process in accordance with claim 26, 27, or 28 wherein the nonionic surfactant is polyoxyethylene-23 lauryl ether (Brij 35), Tween 80, or polyoxyethylene-20 cetyl ether present in a concentration from 0.08% to 2.0%.
33. A method for promoting growth which comprises administering a growth-promoting amount of a somatotropin solution which comprises a lyophilized somatotropin composition comprising about 1 part somatotropin per 2 to 8 parts arginine HCl on a weight basis, wherein the pH of the somatotropin composition prior to lyophilization was about 7.2 to about 8.5, dissolved in a diluent comprising EDTA, a nonionic surfactant, and, if the pH of the somatotropin composition prior to lyophilization was less than about 7.8, a buffer.
34. A method for promoting growth which comprises administering a growth-promoting amount of a somatotropin solution which comprises a lyophilized somatotropin composition comprising about 1 part somatotropin per 2 to 8 parts arginine HCl on a weight basis, wherein the pH of the somatotropin composition prior to lyophilization was about 7.2 to about 8.5, dissolved in a diluent comprising EDTA, a nonionic surfactant, and, if the pH of the somatotropin prior to lyophilization was at least 7.8, optionally further comprising a buffer or non-buffering agent.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US86476892A | 1992-04-07 | 1992-04-07 | |
| US864,768 | 1992-04-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2133737A1 true CA2133737A1 (en) | 1993-10-14 |
Family
ID=25344034
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002133737A Abandoned CA2133737A1 (en) | 1992-04-07 | 1993-04-05 | Lyophilized somatotropin formulations |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0639984A1 (en) |
| JP (1) | JPH08502021A (en) |
| CN (1) | CN1089249C (en) |
| AU (1) | AU4278393A (en) |
| CA (1) | CA2133737A1 (en) |
| WO (1) | WO1993019773A1 (en) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5981485A (en) | 1997-07-14 | 1999-11-09 | Genentech, Inc. | Human growth hormone aqueous formulation |
| DE69420872T2 (en) * | 1994-06-17 | 2000-01-13 | Applied Research Systems | PHARMACEUTICAL PREPARATIONS CONTAINING HGH |
| KR100236393B1 (en) * | 1996-02-02 | 1999-12-15 | 나까니시 히로유끼 | A pharmaceutical preparation containing a human growth hormone |
| KR20080011353A (en) | 2000-02-24 | 2008-02-01 | 몬산토 테크놀로지 엘엘씨 | Non-aqueous injectable formulations for extended release of somatotropin |
| WO2002000261A2 (en) | 2000-06-26 | 2002-01-03 | Monsanto Technology Llc | Surfactant-containing formulations for extended release of somatotropin |
| US6664234B1 (en) | 2000-06-30 | 2003-12-16 | Monsanto Technology Llc | Non-aqueous injectable formulation preparation with pH adjusted for extended release of somatotropin |
| EP2805964A1 (en) | 2009-12-21 | 2014-11-26 | Ambrx, Inc. | Modified bovine somatotropin polypeptides and their uses |
| NZ600363A (en) | 2009-12-21 | 2014-07-25 | Ambrx Inc | Modified porcine somatotropin polypeptides and their uses |
| CA2875446A1 (en) | 2012-06-05 | 2013-12-12 | Neuroderm Ltd. | Compositions comprising apomorphine and organic acids and uses thereof |
| ITBO20120368A1 (en) | 2012-07-06 | 2014-01-07 | Alfa Wassermann Spa | COMPOSITIONS INCLUDING RIFAXIMINA AND AMINO ACIDS, RIFAXIMINE CRYSTALS DERIVING FROM SUCH COMPOSITIONS AND THEIR USE. |
| CN103751769B (en) * | 2013-12-25 | 2015-08-19 | 楼秀余 | A kind of lyophilizing recombinant human somatropin capsule and preparation method thereof |
| GB201602880D0 (en) | 2016-02-18 | 2016-04-06 | Ge Healthcare Uk Ltd | Method and composition for biomolecule stabilization |
| CN112618482A (en) * | 2019-09-24 | 2021-04-09 | 江苏恒瑞医药股份有限公司 | Novel protein formulations |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3325223A1 (en) * | 1983-07-13 | 1985-01-24 | Hoechst Ag, 6230 Frankfurt | AGAINST AGAINST DENATURATION, AQUEOUS PROTEIN SOLUTIONS, METHODS FOR THEIR PRODUCTION AND THEIR USE |
| US4774091A (en) * | 1983-10-14 | 1988-09-27 | Sumitomo Pharmaceuticals Company, Ltd. | Long-term sustained-release preparation |
| ES2044941T5 (en) * | 1987-08-21 | 1999-02-16 | Mallinckrodt Group Inc | STABILIZER OF GROWTH PROMOTING HORMONES. |
| US5122512A (en) * | 1988-09-12 | 1992-06-16 | Pitman-Moore, Inc. | Swine growth promoting composition |
| US5008244A (en) * | 1989-05-15 | 1991-04-16 | Pitman-Moore, Inc. | Method for increasing fertility in animals |
-
1993
- 1993-04-05 CA CA002133737A patent/CA2133737A1/en not_active Abandoned
- 1993-04-05 JP JP5517756A patent/JPH08502021A/en active Pending
- 1993-04-05 AU AU42783/93A patent/AU4278393A/en not_active Abandoned
- 1993-04-05 EP EP93912120A patent/EP0639984A1/en not_active Withdrawn
- 1993-04-05 WO PCT/US1993/003182 patent/WO1993019773A1/en not_active Application Discontinuation
- 1993-04-06 CN CN93104030A patent/CN1089249C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| WO1993019773A1 (en) | 1993-10-14 |
| AU4278393A (en) | 1993-11-08 |
| JPH08502021A (en) | 1996-03-05 |
| CN1078910A (en) | 1993-12-01 |
| EP0639984A1 (en) | 1995-03-01 |
| CN1089249C (en) | 2002-08-21 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 20040728 |