CA2148537C

CA2148537C - Stable freeze-dried formulation comprising a protein; assay kit

Info

Publication number: CA2148537C
Application number: CA002148537A
Authority: CA
Inventors: Alain Bayol; Thierry Breul; Patrice Dupin; Philippe Faure
Original assignee: Sanofi Synthelabo SA
Current assignee: Sanofi Aventis France
Priority date: 1994-05-04
Filing date: 1995-05-03
Publication date: 2002-07-16
Anticipated expiration: 2015-05-03
Also published as: FR2719479A1; FI952119A0; EP0682944A1; IL113578A; MX9502034A; CN1088583C; HU220220B; NZ272045A; AU694763B2; DK0682944T3; PL179240B1; CN1116522A; FI952119L; JPH0853361A; KR100273053B1; HU9501276D0; CZ286195B6; PL308416A1; JP2948125B2; AU1777495A

Abstract

The subject of the invention is a formulation which is stable, freeze-dried and pharmaceutically acceptable, comprising a protein, a buffer, alanine and mannitol, in which the ratio R = mass of mannitol/mass of alanine is between 0.1 and 1.
The protein is especially a protein with biologi-cal activity such as a hormone, in particular hGH, an enzyme or a cytoking.
The subject of the invention is also an assay kit comprising a formulation previously defined.

Description

~~.~8~~
The present invention relates to a pharmaceuti-cal~_y acceptable formulation provided in the f.vrm of a freeze-dried product and containing a protein as active Ingredient. This formulation is stable at 25°C and can be reconstituted in liquid form by the addition of a sol-vent . It can be administered parenterally to humans or to animals or used in an assay kit.
It is known that the formulation has a consider able effect on the degradation of proteixls during freeze drying, as well ae a high impact on their stability in freeze-dried form. various formulation variables which affect these parameters are mainly the pH, the quantity of salts present. the type and the quantity of excipientst, the type of cryoprotectiori chosen, as well as the temperatures. pressure sad time which are chosen for the freezing, sublimation and desiccation operations.
These different variables influence the physical state of the freeze-dried product obtained, namely: vitreous ~orphous, soft amorphous, crystalline or a combination of these states.
The role of each of these variables has been studied separately, but their synergistic effect is still poorly elucidated (Pikal MJ., 13e11erman KM., Roy 1~.
Riggin raI., The effects of formulation variables on the stability of freeze--dried Human Growth hormone. pharm.
Research, 1991, 8, No. 4, 427-436).
fa bibliographic revue on the influence of amino acids and polyols on the properties of the solutions to be ~reexe-dried or of the freeze-dried products has made it possible to draw the ~oliowing conclusions:
The advantages and disadvantages linked to the presence of amino acids, mannitol, a crystalline phase or an azaorphoua~ phase are listed below:
Advantages linked to the presex~ce of amino acids.
rt has been destoastrated that the presence of glycine in a freeze-dried product a.nducas crystallization of the molecules pxesent in solution during the freezing stage of the freeze-drying (Korey DJ.r Schwartz J8.

~14~~~'~
Effects of excipiente on the crystallization of pharma-ceutical compounds during lyophiiization, J. Parenteral Sci. Tech., 1989, 43, 2, 80-83). This crystallization of the active ingredient. which is nevertheless not very probable in the cage of proteins, makes it possible to enhance ite stability.
Alanine, is crystallized form, has the advantage of preventing the collapse of the freeze-dried product during sublimation and desiccation and allows the produe-tion of a freeze-dried product with a larger apeeiflc surface area and therefore allows a more rapid desicca-tion (Pikal MJr., Freeze-drying of proteins. Biopharm. 26-30 October 1990).
Disadvantages linked to the pregenaa of amino acids.
The addition of an amino acid to a sugar or to a polyol in a solution to be freeze-dried generally has the effect of decreasing the glass transition temperature of the sugar (te Booy MPWbt de Ruiter RA., de Meere 7~LJ..
$valuation of the physical stability of freeze-dried sucrose containing formulations by differential scanning calorime~try, Pharm. Research., 1992. 9, 109-114). Now, a decrease in glass transition teiaperature is generally synonymous with less stability of a freeze-dried product (Franks F., Freeze-drying: from empiricism to prediet ability, Cryo-letters, 1990. I1, 93-110).
Advantages linked to the presence of mannitol.
The presence of mannitol in amorphous form surrounding the protein guarantees the presence of nonorystallized water lin)Ged to the protein, during freeaing, sad thereby prevents the denaturation of the protein. Furthermore, the presence of polyols stabilizes the proteins against thermal degradations, through hydrophobia interactions (Back JF. Oakenfull D., Smith Ice., Increased thermal stability of proteins in the presence of sugars and polyols, Biochemistry, 1979. 18, 23, 5191-96) .

Disadvantages linked to the presence of mannitol.
It has been reported that mannitol does riot make it pogeible to preserve the activity of an enzyme at 37°C, contrary to lactose (Ford AW, Dawson PJ., The effect of carbohydrate additives in the freeze-drying of alkaline phosphatase, J. Phsrm. Pharmacol., x.993, 45 (2), 8s-93).
Advantages linked to the presence of a crystalline phase.
The presence of a crystallized solute in a fxozen solution is a means of stabilizing the proteins during desiccation (Carpenter JF. ~ Crows .1H., Modes of stabilization of a pxotein by organic solutes during desiccation, Cryobiology, 1988, 25, 459-470).
Disadvantages linked to the presence of a crystalline phase.
It has been damonst~rated that the loss of acti-vity of a freeze-dried protein is dixectly linked to the degree of erystallinity of the cryoprotective molecule (Izutsu KL, Yoshioka 8.. Terao T., Decreased protein-stabilizing effects of cryoprotectants due to axystal lization., Pharm. Regeareh. 1993, 10, No. 8, 1232-1237).
In the formulation of medicinal products contain ing proteins. the Crystallization of exaipients should be avoided according to: (Hermansky M.. Pesak M., Lyophilization of drugs. vI llmorphous and Cristalline forms Cesk. Farm., 1993, 42, (2), 95-98).
Advantages linked to the presence of an. amorphous phase.
The presence of additives in the amorphous state stabilizes the activity of certain enzymes px'oportionally to the concentration of the additive according to (Isutsu KL, Yoahioka S-, Terao T., Decreased protein-stabi.liziag effects of cryoproteetaata due to crystallization, f'harm.
Research. 1993, ID, No. 8. 1232-1237).
The cxyoprotective effect of the exaipients is attributed to the amorphous state of the glycine in the freeze-dried product obtained (Pikal MJ. , Dellermana lmi..

~1~~~~"~

Roy ML. Riggin MN., The affects of formulation variables on the gtabi.lity of freeze-dried Human Growth Hormone, Pharm. Research., 1991, 8, No. 4, 427-435).
Disadvantages linked to the presence of as antorphoua phase.
In the presence of a solid amorphous phase alone, the freeze-dried product collapses at temperatures greater than the glass transition temperature during freezing.
In a soft amoxphous phase, the chemical degra-dation reactions have much more rapid kinetics than in a crystalline phase.
In conCluaion, exhaustive revue of the scient3.fic literature regarding the effect of excipients on the stabilization of proteins makes it possible to find contradictory information on their properties. No theory on the relatiouahips between the structure of a freeze-dried product and its stability is universally accepted.
Likewise, the role of polyola and wino acids. alone or ~.n combination, is not described according to a sat of generalizable propertiem, but has been observed with contradictory results according to the proteins studied sad the quantities of excipients used.
A synergistic effect has therefore now been found, quite surprisingly, between mannitol and alanine on the stabil3.zatioa of freeze-dried proteins. It has been demonstrated that this synergistic effect exists oxlly in a region of relative concentrations of each of these two excip~.ents . The optimum effect is delimited for ratios R, with R representing the mangitol mass/alanine mass present in the fra~aze-dried product, of between 0.1 and l, especially 0.2 and 0.8.
Furthermore, it has been demonstrated that for an R of between 0.1 and l:
The freeze-dried product consists of an amorphous phase and a crystalline phase.
The amorphous phase predominantly consists of ~, ~: 8 ~ ~'~
-mannitol and protein.
The crystalline phase predominantly consists of alanine.
The hypotheses envisaged are that for an R of 5 between 0.1 and 1:
The amorphous phase formed.eryoproteets the protein during freezing.
The crystalline phase fixes the structure of the freeze-dried product and avoids its collapse.
It is this surprising synergistic effect between the aoexiatenee of an amorphous phase and a crystalline phase which stabilizes the free2e-dried protein. The present invention therefore describes the production of this effect for preferred R ratios.
Thus, the present invention relates to freeze-dried pharmsaeutical dosage forma containing an effective quantity of a biologically active protein, a buffer adjusted to the optimum pH for stability of the protein, alanina and mannitol, the latter twa excipienta being is a mass ratio R _ mesa of mannitol/maea of alanine of between 0.1 and 1. The protein included in the said formulation remains stable in freeze-dried form. The dissolution of the freese-dried product obtained is rapid and ca~lete..The structure of the freeze-dxied product Z 5: is not broken or collapsed and its water content is cc~m~atible with the maint-~r~ancx of the act i vi.~-y o f t-~ prote~ n .
Other phsrmaaeutiaally acceptable exaipients.
well knows to persons skilled in they art, can be intro-duced into this formulation, such as for example eoeol-vents, preservatives, antioxidants or chelating agentB.
The object of the present invention therefore aonaists in obtaining stable freeze-dried products aontainirig a protein which is aryoproteated by an amor-phous solid phase dozing freex3.ng, consisting essentially of protein anal mannitol, t~iia~amorphous phase coexisting in they freeze-dried product obtained after subliatatioa ~~.~8~'~

and desiccation of the frozen solution, with a crystal-line phase cons~.~ting essentially of alanina.
The biologically active (or bioactive) protein which i.s formulated according to the present iavention may be a glycosylated or nonglycosylated, natural, synthetic, aemisynthetic or recombinant polypeptide as used in clinical or laboratory practice. More particu-larly, the said protein may be for example a hormone such as a growth hormone, preferably human growth hormone (hGH), a luteinizing hormone (LH-RH), a gonadatrophin.
The protein may also be an enzyme, for example a thrombolytic enzyme such as a urokinase, a prourokinase, a atreptokinase, s staphilokinase, a tissue plasminogen activator (tPA) ar an enzyme such as a phosphatase, a sulphatase, an acyltransferase, a monosuune oxidase, a orate oxidase. Likewise. a protein which is formulated according to the present invention may be a cytokine, such as for example interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6) or intarleukin-13 (IL-13).
Another Class of prote~.us according to the present invention includes for example antibodies, im~tunoglobu lina, immctunotoxins. Peptides such as cholecystakiain (CCK), substance B, neurokinin A, neurokinin B, neurotea sin, neuropeptide Y. eledoisin, bombesin can be fozznu laced aeaording to the present invention.
the biologically active protein is preferably ht~H
(or human growth honaane), orate oxidase or interleukin-13.
Human growth hormone is a pxotein consisting of a single polypeptide chain of 191 amino acids with 2 d3eulphide bridges between the cystein residues 53 and 165 and the cyste~.n residues 182 and 189.
Urate oxidase is an enzyme which oxidizes uric acid into allantain and ~.a extracted from the biomage of Aspergil.Ius flavus (haboureur et al., Bull. Soc. Chim.
Hiol. x.968, 50, 811-825). It has been used for the treatment of hyperuricamias far more than 20 years.
The cDNA encodi.ag this protein hoe been recently cloned and expressed in 8. coli (L$GDUX R at al . , ~. of ~~~~~~7 Hiol. Chem., 1992, 267, 12, 8565-8570), Aspergillus flavus and Saccharomyces eerevisiae. The enzyme is a tetramer with identiaai eubunita of molecular mass in the region of 32,000. The monomer, consisting of a single golypeptz.de chain of 301 amino acids, has no disulphide bridges and is acetylated at the r1-terminal end.
rnterleukin-13 is a c~ttokin consisting of a single polypeptide chain of x.12 amino acids with two disulphide bridges (Minty et al., Nature, 1993, 362, 248 250) .
The obtaining of the coexistence of the amorphous (martnitol + protein) phase with the crystalline aianine phase is independent of the presence arid of the concen tration of a buffer for adjusting the pH of the solution, but it depends on the R ratio defined above.
rn order to gropose protein formulations which are available to the public as therapeutic agents, it is essential to formulate them a.n a sufficiently stable form so as to maintain their biological nativity between the time of the formulation and that of use. Por example, hGFi has been formulated in various ways such as those described in the following patents or patent applica-tions: US 5,096,885; wo 89/09614; w0 92/L7200, Au-30771/89; i~PO 93/19773; WO 93/1977fi.
The formulations according to the invention can be preserved at rooza temperature. whereas currently marketed formulations containing these proteins must be preserved at temperatures of 2°C to 8°G.
In most cases, the pharmaceutical dosage form is freeze-dried, frozen, or is solution. It contains the protein. a buffer, glycine, arginine, mannitol, zinc, surfaotants, dextran, EDTA, or other excipients but never the combination alanine/mannitol in a mass ratio R of between 0.1 and 1. The freeze-dried or frozen forms are used to maintain the bioahemiCal integrity arid the biological activity of the molecule. The freeze-dried formulations should be reconstituted before use by addition of pharmaceutically acceptable sterile solvents suob as distilled water, aqueous solutions of sods"-2~~~~~7 -e_ chloride at 0.9~ or of glucose at 5~ or any other physio-logically acceptable solvent, containing or otherwise antibacterial preservatives such as benzyl alcohol, phenol or metacresol.
A formulation which is stable at room temperature until it is reconstituted is particularly advantageous for an ambulatory treatment as ih the cases of hGH in the form of a bottle or a presentation adapted in the form of a multidose pen.
The formulat3.on thus prepared can also be intro-duced into an assay kit.
The present invention is therefore a preferred composition of a freeze-dried product. This freeze-dried product is obtained by freeze-drying starting with s Solution.
The process of this formulation includes steps of mixing, dissolving, filtering arid freeze-drying.
The composition of the solution to be freeze-dried is the following:
A protein, a pharmaceutically acceptable buffer to adjust the pH, alanine, mannitol where the mas8 ratio R -- mass of mannitol/mass of alanine is between 0.1 and 1, water for injection.
The solutior~ to be freeze-dried is prepared in the following manner:
The pxotein solution is obtained on a gel filtra-tion eoluam and contains a buffer which maintains its pH
in a zone ea~mpatible with the stability of the protein.
The desired quantities of buffer, alaniae, mannitol and water are added to this solution so as to solubilize all the excipients. The solution is sterilely filtered and distributed into containers, preferably vials or carpules.
The freeze-drying of the solutions is carried out a8 follows:
The solution follows a cycle of freezing. then aubliioatiori sad desiccation which is adapt~d to the volume to be freeze-dried and to the container containing the solution. Preferably, a freezing rate of close to -2°C/min is chosen in a Usifroid* freeze-drier (France) of the SMH15 or SMJ100 or SMH2000 type.
The time, temperature and pressure of desiccation of the freeze-dried product are adjusted as a function of the volumes of solution to be freeze-dried and the desired residual- water content. in the freeze-dried product.
Complete information on the techniques for preparing the injecta~>le formulations is available to persons skilled in the art in Remington's Pharmaceutical Sciences, 1985, 17th Edition or in William NA & Polli GP, The lyophilization of pharmaceuticals: a literature review, J.
Parenteral Sci. Tech. , 1984, 38, (2) , 48-59 or in Franks F. , Freeze-drying: from empiricism to predict-ability. Cryo-letters, 1990, 11, 93-110.
A freeze-dried product is thereby obtained in which alanine is in crystallized form and mainnitol is in amorphous form. The freeze-dried product can be preserved at 25°C
without impairing the biological activity of the protein which it contains.
To illustrai~e the present invention, evaluations were made choosing hc3H or urate oxidase as an example of protein. Thus, several solutions containing hGH or urate oxidase as biologically active protein, a phosphate buffer at various concentrations, at pH - 7 for the solutions containing hGH (at 4: IU/0.5 ml), and pH - 8 for those containing urate oxidase (at 30 EAU/ml, mannitol alone, alanine alone, or alanine/mannitol mixtures) were prepared, freeze-dried and analysed.
* trademark 9a The compositions are described in detail in the examples of TABLES 1 and 2 below. The methods of analysis as well as the stabilizing times and temperatures are also described below.

2~9~8~~'~
~'71BLE 1 TABLE 1 below indicates the oompositions o~ the urate oxidase-containing formulae studied.
Batch No. eog ntannitolmg Alaaiaea FDOOphaee buffer rate o7cidwwe _ pH=8; ~O(i) mM

1 33.0 0.0 W 50 3p Z 27.7 6.9 4 30 30 3 X0.8 I0.! 2 30 30 4 13.6 13.6 1.000 40 30 S 10.3 15.3 0.67 40 3p 1 6 7.3 16.0 0.l6 40 30 ~

7 7.3 16.2 0.l5 30 30 8 9.5 21.0 0.45 50 30 9 4.6 18.3 0.25 !0 30 IO 2.5 10.0 0.125 40 30 1 1i 0.0 16.0 0 50 30 ~1~ : 8J1U oeana erisy~ activity unit TABLE 2 below indfcates~ the composition of the hOH-containing formulae studied Hatch ~sg lsennitolmg wlaniasR phoaphatp No. buffer hc'R
pH=T; iU~i) m1t 12 12.5 0.0 + ae 2.50 13 20.0 2.5 a 1.95 ~F

1! 12.5 8.5 2 0.00 4 X5 17.5 6.5 2 2.50 4 2 16 12.5 13.0 1 2.50 4 17 4.T 10.5 0.15 1.95 4 18 0.0 6.5 0 2.50 !

~1~: iU_ intetaskional unit The analytical methods which were used for the 30 determination of the different parameters are the following.
Contents of dimera and related substances of higher molecular iaass The content of dimerg and related substances of 3S higher raolecula3- mass is determined by exclusi.oa chromatography (SEC-HPLC) using a SUPEROSE* 12 column (Pharmacia*, Ref. 17-0538-O1). The product is eluted with an ammonium phosphate buffer solution at pH - 7.0 (1.38 g of ammonium dihydrogen phosphate in 1 litre of water, adjusted to pH - 7.0 with con~~entrated ammonium hydroxide at a flow rate of 0.4 ml/minut:e). The detection is carried out at 220 nm. (This content is noted in the analytical results as percentages of oligomE:rs + polymers).
The content of dimers and substances of higher molecular mass can al~~o be determined by the method described in the European Pharmacopoeia monograph "Somatropine pour preparation injectable" (Somatropin for injectable preparation) of January 1994.
Assay of the protein titre by reversed-phase chromatography.
Expressed in mg per vial, it is determined by reversed-phase chromatography using a C18-300A column - 25 cm, diameter 4.6 mm (SYNCHROM*, ref. CR103-25). The product is eluted in 35 minutes in gradient mode with a mobile phase passing from 75 volumes of water at 0.1% trifluoroacetic acid (V/V) (TFA) and 25 vo_Lumes of acetonitrile at 0.080 TFA (V/V) to 30 volumes of water at 0.1% TFA and 70 volumes of acetonitrile at 0.08% TFA. The flow rate is 1 ml per minute and the detection is carried out at 220 nm.
Assay of the enzymatic activity of urate oxidase.
The enzymatic activity of urate oxidase expressed as EAU is determined by spectrophotometry in a thermo-stated cuvette at 30°C by monitoring the disappearance of uric acid * trademark at 292 nm according to Legoux R, Delpech Bruno, Dumont X, Guillemot JC, Ramond P, Shire D, Caput D, Ferrara P, Loison G, J. Biol. Chem. 199:2, 267 (12), 8565-8570.
Turbidity of the reconstituted solutions.
The turbidity of the hGH-containing freeze-dried products taken up in solution is determined by spectrophotometry (Ph. Eur. 2 (I) V.619) at 500 nm in a Perkin Elmer* 554 spectrophotometer. The results are provided in Absorbance Units x 1000.
The turbidity of the urate oxidase-containing freeze-dried products taken up in solution is determined with the aid of a Ratio Hach* 18900-00 turbidimeter. The turbidity results are expressed in Nephelometric Turbidity units (NTU) defined by: "Manual on industrial water and industrial wastewater, 2nd edition, 1965, printing. American Society for the testing of materi<~ls, Philadelphia, Pa".
The degree of opalescence is also determined according to the method reported in paragraph 2.2.1 concerning the general methods of the Pharmacopee Europeenne - Third edition - (:1952) by comparing the sample to be analysed with a control suspension.
Organoleptic criteria for the freeze-dried products.
These criteria are examined visually and take into account the colour of the freeze-dried product, its structure (collapsed or otherwise), and the observation of a possible phase shift between the crust and the crumb of the freeze-dried product.
* trademarks X-ray diffractometry an powder.
X-ray diffractometric analysis on the freeze-dried products is carried out in a SIEMENS* D500 TT diffraCtometer;
source: CuKal; generator: 40 KV, 25 mA; back monochromator;
slit: 1/1/1/0.16/0.6; sampling on a Pyrex* rack; scanning region: 4° to 40° per minute in 2 Bragg theta.
Differential thermal analysis.
The study of the freeze-dried products by diffe rential thermal analysis is carried out under the following conditions:
Apparatus: L>SC 7 Perkin Elmer*; calibration: indium and lead; sample size: between 5 mg and 10 mg in a 50 ~l dish; initial temperature: 10°C; rate of heating 10°C/minute;
final temperature: 300°C.
Deamidated forms of hGH.
The percentage of deamidated forms is determined by anion-exchange chromatography (AEX-HPLC) using an anion-exchange column (PHARMACIA* mono-Q HR 5/5, ref. 17-0546-Ol).
The elution is performed with a solution A (13.8 g of ammonium dihydrogen phosphate in 1000 ml of water; pH - 7 adjusted with concentrated ammonium hydroxide) and water as solution B, using the following programming: 5% of solution A
for 2 minutes, then passage to 15% of solution A over 5 minutes, then passage to 50% of solution A over 20 minutes, * trademarks 13a finally passage to 100% of solution A over 5 minutes and maintenance of the latter solution for 5 minutes. The elution of hGH (tR about 15 minutes) and of the deamidated forms is monitored at 220 nm. The flow rate is 1 ml per minute.
The analytical results obtained using these different methods are described below.
Content of dimers and related substances of higher molecular mass The content of oligomers plus polymers of the freeze-dried products containing 4 IU of hGH taken up in 0.5 ml of water was determined as a function of the R ratio (Figure 1). Figure 1 indicates that the minimum content of oligomers plus polymers of the solutions is obtained for an R
of between 0.1 and 1, the value 0.1 being interpolated from the curve.
By way of additional example, two batches of hGH at 4 IU and 8 IU were monitored for stability at 25°C and 35°C.
Their stability is excellent after 6 months of preservation.
TABLES 3 anal 4 below indicate the percentages of oligomers and polymer: of deamidated forms at different times and temperatures compared with the European Pharmacopoeia standards.
In these tables, R = 0.45.

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TAHLFs 3: Batch 4 IU per vial Huropeaa pharmaCOpoelaTime at 25C et 36'C

Standard 3 months3 moathe6 m0l:Chs oligomora 6.0 1.0 1,9 3 3.3 and pol ers in i Deamdated 6.6 1.3 1.9 2,A 3 forms g is ~t .

Assay n mg - 1.5A() 1.61() 1.51() 1.55() per vial (~): The titrA does not Vary sigaiffcantly over time given the precision of the assay matho8 (* 5~) TAHLE 4: Batch 8 IU per vi.ai European PbarmacopeeiaTisxe at at 5tsadasd monthsmonths monthsmonths OliQemers E 1.0 1.5 1.2 1.7 !.2 sad olymers in t Deamidated 6.6 1.0 1.5 1.95 2.2 3.3 Eorm is t 1 11say in - 3.0() 3.0() 2.93() 2.95()2.9~(*) 5 mg yr Vial (~1x the titre doss sot vary s~gn~ficaatly over time given the ~reciaioa of the essay method (t 5t) Assay of the enzymatic activity of urate oxi.dase.
The enzymatic activity of the freeze-dried produata containing 30 EAU of urate oxidaee taken up in 1 ml of water was determined as a function of the R ratio after 1 month at 35°C (cf. Figure 2). Figure 2 indicates that the initial enzymatic activity of urate oxidaae is preserved after 1 month at 35°C for an R of between 0.4 and 1.
py way of additional example, a fos~~pula with R = 0.45 or R = 0.6~ is parfeGtly stable after 3 months at 25°C (residual activity alone to 100 of that of tune zero) .
Turbidity of the reconstituted solution.
The turbidity of the freeze-dried products eon-taining 4 IU of hGH taken up in 0.5 ml of water waB
determined ag a function of the R ratio (cf. Figure 3).
Pigure 3 indicateR that the minimum turbidity of the solution( is obtained for an R of betweefl 0 and ~..5.

2~~~~'~~l The turbidity of the freaze-dried products containing 30 EAU of urar_a oxidase taken up in 1 ml of water was determined ac a function of the R ratio (cf.
Figure 4). Figure 4 indicates that the minimum turbidity obtained on the solutions is obtained for an R of between 0.2 and 1.
Organoleptic criteria for the freeze-dried products.
The organolepti.c criteria for each freeze-dried product containing 4 IU of hc~H or 30 sAU of urate oxidase IO or no protein, and various contents of phosphate buffet were determined as a function of the R ratio, arid are provided in TABLB 5. TABLE S indicates that the freeze dried products exhibit satiefaetory organoleptic eri.teria for an R of between 0.125 and 1.7.
Theea characteristics do not vary as a function of tame.
TABLg S
organoleptic criteria for the freeze-dried products and crystallized excipiente as a function of the ratio R = mass of msnnitol/mass of alauine R Organoleptic Crystalline excipients criteria (X-ray diffraction) + oo Broken (or oollap:~ed)Mannitol 10 Br~ ~~ Mannitol + Alanirre 6.469 Brk~ ~~ Mannitol + Alaniue 5 ~~K~ Manriitol + Alatsine 4.014 Bmk~ " Mannitol + Alaniae 2 r'ok~ ~~ Mannitol + Alanine 1.705 Good Max~nitol + Al.anine 1.402 Good Mannitol + Alansne I.25 Good Manaitol + Alanina 1.097 Good Mannitol t Alanine 1 CoOd Alaaine 0.667 Good Alanine 0.456 Good Alauine 21~~~'~
TABLE 5 (continuation) R Organoleptic Crystalline excipients criteria (X-ray diffraction) ---0 . 456 Good Alpine 0.451 Good Alanine 0.452 Good Aianine 0.251 Good Alanina 0.125 Oood ' Alanine 0 Phase shift nlanine (or separation) X-ray diffraction.
The results of the X-ray diffraction analysis on the powder of the freeze-dried products obtained contain-ing Alanine/Mannitol mixtures in R ratios varying from 0 to + oo are provided in TABLE 5.
The diffractograma obtained show that for an R of between 0 and 1, only the linear of the alanine erystal line lattice appear; furthermore, the deviation from the base line of the.diffraetogram indicates the presence of an amorphous phase consisting of. mannitol. The greater R, the greater the amorphous phase due to mannitol. For R >
1, the manaitol also crystallizes.
An amorphous phase is obtained consisting of mannitol and a crystalline phase consisting of alanine for an R of bet'aeen 0 and 1, Differential thermal analysis.
The glass transition temperatures of the fr~aeze-dried products containing 4 IU of hGH or 30 RAU of urate oxidase or no protein, and various contents of phoBphate buffers were determined as a function of the reciprocal of the R ratio (af . Figure 5) . Pigure S indicates that the maximtup glees transition temperature obtained oa the freeze-dried products containing mixtures of alanine and mannitol is obtained for (1/R) > I, that is to say for an R of between 0 and. 1.
It ie the glass transition temperature of the freeze-dried product which indicates the maximum temperature of stability of the freeze-dried product. The maximum temperature of stability of the freeze-dried product is therefore reached :for an R of between 0 and 1.
Deamidated forms The results obtained on the 4 IU and 8 IU batches show only a very small variation which is very acceptable compared with the standards for the somatropin as reported in "Somatropin for injc=ctable preparations (somatropinum ad injecta,bilium) - Pharmacopee Europeenne - Third edition (1952)". They make it possible to think that this formula will be sufficiently :table at 25°C.
In conclusion, it has been proved that each property evaluated on the freeze-dried products exhibits an optimum for an interval of R value which can be summarized in the following manner:
Oligomers + Polymers (hGH st:ability~ R between 0.1 and 1 Enzymatic activity of orate oxidase after 1 month at 35°C R between 0.4 and 1 Turbidity hGH solutions R between 0 and 1.5 Turbidity orate oxidase solutionsR between 0.2 and 1 Appearance of the freeze-dried R between 0.125and 1.7 products Alariine alone crystalline R between 0 and 1 Maximum glass transition temperatureR between 0 and 1 Each analytical criterion (% oligomers + polymers, hGH stability, enzymatic activity of orate oxidase after 1 month at 35°C, turbidity hGH solutions, turbidity orate oxidase solutions, appearance of the freeze-dried products, alanine alone crystalline, maximum glass transition 17a temperature) defines a specific optimum interval per property. To obtain an acceptable formulation, a preferred interval of R is defined, that in to say 0.1 < R < 1.
The following EXAMPLES illustrate the invention without however limiting it.

2148~~'~

87CA.MPLg 1 . Composition of a freeze-dried product of hGH
at 4 IU to be taken up in 0.5 ml of water for injecr_ion CONSTITDSNT9 Unit formula in (mg) h6H 4 IU
Alanine 10.5 mg Mannitol 4.77 mg Dieodium phoephato dodacahydrate . 0,7 lPatar for injection qe 0.5 ml Typo 1 white glass vial of 3 ml 1 Grey ohlorobutyl pillar stopper Blue plfp-off alu dish diameter 13 mm 1 EXA~dPLB Z . Composition of a freeze-dried product of hGH
at 8 IO to be taken up in 1 ml of water for injection CONSTITU~TS Bait formula is (mg) hG~ a IU

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at 18 IU to be taken up in 1 ml of wetter for iri~eation GOt4sTIT0!SgTB tTai t formula iri (mg) hC~Ii 18 It1 30 Alaniae Z1 bsanni tol g , g,4 Disodium phosphate dodaoahydrate 1.4 mg hater for iajactioa qs 1. ml Typo 1 xhitQ glare vial of 3 ml 1 35 Grey ahlorobutyl pillar stopper, diameter 13 mm Slue Plip-off alu dish diamatet~l3 1 ~

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x 10-') polymers Standards < 20 < 1.5 % < 5 % < 6 %

t = 0 1 7.9 0.33 % 1.36 % 0.63 %

1 month 3 7.72 0.35 % - -1 3 months ~ 9 ~ 7.65 ~ 0.59 ~ 1.37 ~ 0.85 %
5 % %

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Claims

1. A formulation which is stable, freeze-dried, and pharmaceutically acceptable comprising a protein, a buffer, alanine, and mannitol, in which the mass ratio of mannitol to alanine ranges from 0.1 to 1.

2. The formulation according to claim 1, in which the protein is a hormone, an enzyme, or a cytokine.

3. A formulation which is stable, freeze-dried, and pharmaceutically acceptable, comprising a protein selected from the group consisting of human growth hormone, urate oxidase, and interleukin-13, a buffer, alanine and mannitol, in which the mass ratio of mannitol to alanine ranges from 0.1 to 1.

4. The formulation according to claim 1, wherein the alanine is in crystallized form and the mannitol is in amorphous form.

5. The formulation according to claim 2, wherein the buffer is a phosphate buffer.

6. A process for the preparation of an injectable solution designed for subcutaneous, intravenous, or intramuscular injection into humans or animals comprising the step of dissolving the formulation according to claim 1, before use, in a pharmaceutically acceptable sterile solvent optionally containing an antibacterial preservative.

7. The process according to claim 6, wherein the pharmaceutically acceptable sterile solvent is selected from the group consisting of distilled water, an aqueous solution of 0.9% sodium chloride, an aqueous solution of 5% glucose, and a mixture thereof.

8. The process according to claim 6, wherein the anti-bacterial preservative is selected from the group consisting of benzylalcohol, phenol, and metacresol.

9. A process for cryoprotecting a protein in a freeze-dried pharmaceutically acceptable formulation comprising the step's of combining a protein, a buffer, alanine, and mannitol, the mass ratio of mannitol to alanine ranging from 0.1 to 1; dissolving the mixture to form a solution; filtering the solution to effect a filtrate; and freeze-drying the filtrate.

10. The process according to claim 9, wherein the buffer is a phosphate buffer.

11. The process according to claim 9, wherein the protein is selected from the group consisting of human growth hormone, urate oxidase, and interleukin-13.

12. An injectable solution comprising the formulation of claim 1, reconstituted with a pharmaceutically acceptable solvent.

13. An injectable solution comprising the formulation of claim 2, reconstituted with a pharmaceutically acceptable solvent.

14. An injectable solution comprising the formulation of claim 3, reconstituted with a pharmaceutically acceptable solvent