CA2201749C

CA2201749C - Stable aqueous alfa interferon solution formulations

Info

Publication number: CA2201749C
Application number: CA002201749A
Authority: CA
Inventors: Pui-Ho C. Yuen; Douglas F. Kline
Original assignee: Schering Corp
Current assignee: Merck Sharp and Dohme LLC
Priority date: 1994-10-11
Filing date: 1995-10-10
Publication date: 1999-06-15
Anticipated expiration: 2015-10-10
Also published as: KR100401401B1; EP0970703A1; US5935566A; CZ110497A3; AU3727995A; JP3978228B2; PT777495E; FI116558B; DE69518084T2; HK1008813A1; NO971633L; EP0777495A1; DK0777495T3; HUT77287A; HU225494B1; US5766582A; JP2006193536A; GR3034619T3; FI971486A0; CA2201749A1

Abstract

Stable aqueous solution formulations containing alfa-type interferon, e.g., interferon alfa-2a and interferon alfa-2b, a buffer to maintain the pH in the range of 4.5-7.1, polysorbate 80 as a stabilizer, edetate disodium as a chelating agent, sodium chloride as a tonicity agent, and m-cresol as an antimicrobial preservative and which maintain high chemical, physical and biological stability of the alfa-type interferon for an extended storage period of at least 24 months are disclosed.

Description

WO96/11018 ~ ~ O 'I 7 4 9 rcr~sg~tl2362 STABLE. A~UEOUS ALFA INTERFERQN SOLUTION
FORMULATIONS

RACK~:ROUND OF THF INVFI~1TION
..
This invention relates to stable, ~ eous solution formulations which are free of products derived from human blood serum and which maintain high biological activity and high chemical and high physical stability of alfa-type interferon for an extendsd period of time.
U. S. Patent 4,496,537 rliscloses biologically stable alfa interferon aqueous solution formulations containing alfa interferon, human serum albumin and alanine or glycine, water, and a buffer system to maintain the -~
pH at 6.5-8Ø The human serum albumin ("HSA") acts as a stabilizer for alfa interferon and prevents losses of alfa interferon from solution by 1 5 coating and/or adsorption of the alfa interferon onto the stainless steel and glass surfaces of compounding vessels, process equipment and storage co"lainers. Solution formulations containing alfa inle,~run and HSA
have maintained the chemical and biological stability of the alfa interferon when such solutions have been stored at 2-8~C for extended periods, i.e., 2 0 more than 2 years.

Recently, the worldwide AIDS epidemic has resulted in health r~g;sl.alion agencies requiring manuf~urers to place wamings on prod~cts, such as alfa interferon, which contain products derived from 2 ~ human blood such as HSA .

There is a need to reformulate alfa-type interferon solution products to obtain a solution formulation free of human blood-derived products such as HSA while maintaining high chemical, high physical stability and 3 0 high biological alfa-type interferon activity in the aqueous solution formulations for exlended storage periods.

-~= ~
~0 ~749 ~:UMMARY OF THF INVF~'ITION

The present invention provides a stable, aqueous solution formulation which maintains high biological alfa-type interferon activity S and is free of human blood~erived products, which comprises:
a. 0.1 x 106 to 100 x 106 lU/mL of alfa-type interferon;

b. a buffer system to maintain a pH in the range of 4.5 to 7.1.

c. an effective amount of a chelating agent;

d. an amount of a sorbitan mono-9-octadecenoate poly (oxy-1,2-ethanediyl) derivative sufficient to stabilize the alfa-type interferon against loss of alfa-type interferon;
e. an effective amount of a tonicity agent;

f. an effective amount of an antimicrobial preservative; and 2 0 9. an amount of water for injection sufficient to prepare a solution of the above-listed ingredients.

The present invention provides a stable, aqueous solution formulation having high alfa-type interferon biological activity and free of 25 human blood-derived products, which comprises:
a. 0.1 x 106 to 100 x 106 lU/mL of alfa-type interferon.
b. a buffer system sufficient to ~"ainlain the pH of the solution in the range of 4.5 to 7.1;
c. about 0.01 to 1 mg/mL of disodium dihydrogen 3 0 ethylenediaminetetraacetate.
d. about 0.01 to 1 mg/mL of a sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivative;
e. about 1 to 9 mg/mL of sodium chloride;
f. an effective amount of an anli",iorubial preservative 3 5 selected from m-cresol, phenol, methylparaben, propylparaben or mixtures thereof; and 9. water for injection q.s. ad. 1 mL.

WO96/11018 2 ~ 0 1 7 4 9 PCI'/US95/12362 In a preferred aspect, the present invention provides a stable, aqueous solution formulation having high biological alfa~type interferon activity and fres of human blood-derived products, which comprises:

m~/ml a. Alfa-2 Interferon 5 x 1 o6 to 50 x 106 IU

b. Sodium Phosphate Dibasic 1.8 Anhydrous c. Sodium Phosphate Monobasic 1.3 Monohydrate d. Disodium Dihydrogen Ethylene- 0.1 diaminetetraacetate e. Polysorbate 80 0.1 f. Methylparaben 1.2 g. Propylparaben 0.12 h. Sodium Chloride; 7.5 and i. Waterforlnjection q.s.ad 1 mL

In another preferred aspect the present invention further provides a stabile aqueous solution formulation having high biological alfa-type interferon activity and free of human blood-derived products, which 1 0 comprises:

mQ/mL
a. Alfa-2 Interferon 5 x 1 o6 to 50 x 106 IU
. .

b. Sodium Phosphate Dibasic 1.8 Anhydrous c. Sodium Phosphate Monobasic 1.3 Monohydrate d. DisodiumDihydrogen 0.1 Ethylenediaminetetraacetate e. Polysorbate 80 0.1 f. m-Cresol 1.5 g. Sodium Chloride; 7.5 and h. Water for Injection q.s. ad 1 mL
The present invention also provides a process of preparing a stable, aqueous solution formulation having high biological alfa-type interferon activity and free of human blood-derived products comprising admixing an effective amount of alfa-type interferon with a buffer system capable of maintaining the pH within the range of 4.5 to 7.1, a chelating agent, a sorbitanmono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivative, a tonicity agent, an antimicrobial preservative and sufficient water to form a solution. In a preferred aspect of the process of the present invention, the solution is prepared and maintained substantially free of dissolved oxygen and a headspace of inert atmosphere above the solution is maintained at a value of less than about 4%
by volume of oxygen.
The formulations of the invention are free of an amount of mannitol effective to act as a bulking agent.
DETAILED DESCRIPTION
We have selected specific amounts of a specific set of ingredients that have allowed us to develop an aqueous alfa-type interferon solution formulation which does not contain human serum albumin yet maintains - -WO96/11018 2 2 0 1 7 4 9 rcr/usg5/l2362 high chemical, biological and physical stability for the alfa-type interferon on storage at 2~ to 8~C for extended periods of at least 24 months.
The term "free of human blood-derived products" as used herein in reference to the formulations of the present invention means that no human blood-derived products such as HSA are used in the preparation of the solution formulations of the present invention.
The term "high chemical stability" as used herein in reference to the alfa-type interferon used in the formulations of the present invention means the alfa-type interferon ~"air,lains at least 85%, preferably 85% to 1 0 100% of its chemical integrity upon storage at 2~ to 8~C for at least 24 months. See Tables 1 and 2. The chemical integrity is determined by measuring the protein content in an HPLC assay such as the one ~closerl by T. L. Nagabhushan, et al., in an article entitled "Characterization of Genetically Engineered ALFA-2 Interferon", pages 1~ 79-88 appearing in Interferon Rese~rch Clini~ ppli~tion. and Re~ul~tory Consider~tion. Zoon, et al., eds., Elsevier Science Publishing Co.,Inc.1984. (See results in Tables 1 to 4).
The term "high biological stability" as used herein in reference to the alfa-type interferon used in the formulations of the present invention 2 0 means the alfa-type interferon in the formulation maintains at least 75%, preferably at least 85%, more preferably 90% to 100% of its biological activity upon storage at 2~ to 8~C for at least 24 months (see results in Tables 1 to 4) as measured in the standard method of inhibition of the cytopathic effect (CPE) of a virus such as the method disclosed by W. P.
2 5 r,uk",an, et al., in J. Clinical Microbiolo~y. (1985), ~, 596-599.

The term "high physical stability~ as used herein in reference to the alfa-type interferon used in the formulations of the present invention means the formulation of the present invention remains clear, i.e., does 3 0 not exhibit haze or visible particulate matter (i.e., particles greater thanabout 60 to 70 microns in diameter) on storage at 2~ to 8~C for at least 24 months. See Tables 1, 2 and 3. The results listed in Tables 1, 2 and 3 are surprising in that most solution formulations containing protein products like alfa-type interferon tend to develop visually observable 3 5 particulate matter (i.e., particles having diameters greater than 60 to 7û
microns) upon extended storage even at 2~ to 8~C. The test method used to determine particulate matter in the solution formulation of this invention WO96/11018 2 2 0 1 7 4 ~ PCI/US95/12362 ~

(see Tables 1 to 4) is described in The United States PharmacopeialThe National Formulary USP 23/NF 18, published by United States Pharmacopsial Convention, Inc., (1995), Rockville, Maryland; see Physical Test ~788~ on pages 1813 to 1816. The method used to 5 determine the visual description of the solution formulations of this invention is also ~Jeso.il~ed in USP 23 as the ~General.Requirement Test and Assays ~1 > Injections~ at pages 16~0 to 1652.

We have found that by adding a chelating agent to the formulations 10 of the present invention, we have been able to avoid visible particulate matter. Typical suit~h!e chelating agents include diso~iium dihydrogen ethylenediamine tetraacetate (EDTA or edetate disodium) or citric acid.
The use of edetate disodi~m is pref~r,~. While we do not wish to be bound by any theory, it is believed that edetate diso~i~ Im effectivsly 1 5 complQxes with trace amounts of metal cations, such as Zn2+, Fe2 l, Cu2+
or Al3+, which ions may be present in excipients and packaging components, e.g., rubber stoppers or gaskets. Since edetate disodium has a higher affinity for these metal cations than the alfa-type interferons, the interaction between metal cations and alfa-type interferon which 2 0 results in formation of insoluble complexes (in the form of, for example, visible particul~te matter) and loss of activity are avoided. The effective amount of the chelating agent is in the range of 0.01 to 1 mg/mL based on 0.1 x 106 to 100 x 1 o6 International Units (~lUn) of alfa-type interferon/mL.
Preferably, 0.1 mg of edetate ~isodium is used for 5 x 1o6 to ~0 X106 IU
2 5 of alfa-2 interferon.

The buffer systems s~it~hle for the formulations of the present invention are those which maintain the pH of the aqueous solution formulation in the range of 4.5 to 7.1, preferably 6.5-7.1 and most 3 0 pl~fer~bly 6.8. The use of a buffer system of sodium phosphate dib~sic and sodium phosphate monob~-sic is preferred. Normally a 0.005 to 0.1 molar buffer of the preferred sodium phosphate monobasic/dibasic buffer system is used for a formulation containing 0.1 x 1 o6 to 100 x 1 o6 lu of alfa-type interferon per mL. Other suitable buffer systems to maintain the 3 5 desired pH range of 4.5 to 7.1 include sodium citrate/citric acid and sodium acetate/acetic acid.

The tonicity agent useful in the present invention is any agent capable of rendering the fomlulations of the present invention iso-osmotic with human serum. Typical suitable tonicity agents include sodium chloride, mannitol, glycine, ~lucose and sorbitol. Use of sodium chloride as a tonicity agent is S pref~rled.

The amount of the tonicity agent used is in the range of 1 to 10 mg/mL
when the formulation of the present invention contains 0.1 x 106 to 100 x 106 IUof alfa-type interferon/mL. The use of 7.5 mg/mL of sodium chloride is prefer,ed1 0 for 5 x 106 to 50 x 106 IU of alfa-type interferon per mL in the formulations of the present invention.

The sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivatives such as polysorbate 80 or polysorbate 20 are useful as a stabilizing agent to 15 prevent a-JsGr~tion of the alfa-type interferon proteins such as alfa-2b interferon onto the stainless steel and glass surfaces of the equipment used to make the indictable formulations containing alfa-type interferon. The amount of polysorbate 20 or 80 effective in the formulation of this invention is in the range of 0.01 to 1.0 mg per mL for a formulation containing 0.1 x 106 to 100 x 106 IU
2 0 of alfa-type interferon per mL. The use of polyso,bate 80 is preferred. The use of 0.1 mg/mL of polysorbate 80 is more preferred in all the solution fommulations of the present invention. When the Concent,atiGns of alfa-type interferon such as alfa-2 interferon is less than about 15 x 106 lU/mL, e.g., 6 x 106 lU/mL, loss of activity due to adsorption of the alfa interferon in the absence of polysorbate 2 5 80 significantly lowers the biological activity of the formulation. Surprisingly, we have found that polysorbate 80 prevents loss of alfa-2b interferon and allows systemic delivery of the alfa-2b interferon without loss of biological activity. In the course of development of the formulation of the present invention, we surprisingly found that polysorbate 80 provided superior chemical and 3 0 biological stability to alfa-2b interferon compared to other non-ionic surfactants, e.g., Pluronic F127 (Trade mark) and Plur~ic F-68 (Trade mark).

The amount of alfa-type interferon useful in the fommulation of the present invention is in the range of 0,1 x 106 to 100 x 106 lU/mL, preferably 5 x 106 to35 50x1061U/mL.

The term "alfa-type interferon" as used herein means the family of highly homologous species-specific proteins that inhibit viral replication and cellular proliferation and modulate immune response. Typical suitable alfa-type interferons include interferon alfa-2a such as ROFERON A (Trade-mark) interferon alfa-2a available from Hoffm~nn-La Roche, Nutley, N.J., interferon alfa-2b such as INTRON A (Trade-mark) interferon alfa-2b available from Schering Corporation, Kenilworth, N.J. interferon alfa-2c such as BEROFOR
(Trade-mark) interferon alfa-2c available from Boehringer Ingelheim Pharmaceutical, Inc., Ridgefield, CT., interferon alfa-nl, a purified blend of natural alfa interferons such as SUMIFERON (Trade-mark) available from Sumitomo, Japan or as WELLFERON (Trade-mark) interferon alfa-nl available from The Wellcome Foundation Ltd., London, Great Britain, or consensus alfa interferon available from Amgen, Inc., Newbury Park, California, or interferon alfa-n3, a mixture of natural alfa interferons, made by Interferon Sciences and available from the Purdue Frederick Co., Norwalk, CT., under the ALFERON (Trade-mark). The use of interferon alfa-2a or alfa-2b is preferred. The use of interferon alfa-2b is more preferred.

The antimicrobial preservatives found useful in the present invention include m-cresol, phenol and methyl- paraben and propylparaben and mixtures of the above-listed preservatives, e.g., phenol-methylparaben mixtures. The effective amount of m-cresol found useful in the present invention is in the range of 0.5 to 2 mg/mL for a formulation containing 0.1 x 106 to 100 x 106 IU/mL of alfa-type interferon. It is preferred to use 1.5 mg/mL of m-cresol for a formulation containing 5 x 106 to 50 x 106 IU/mL of interferon alfa-2b.

- 8a -The effective amount of phenol found useful is in the range of 0.5 to 5 mg/mL for a solution formulation containing 0.1 x 106 to 100 x 106 IU/mL of alfa-type interferon.

The effective amount of methylparaben is in the range of 0.6 to 1.8 mg/mL and the amount of propylparaben is in the range of 0.06 to 0.18 mg/mL
when the formulation of the present invention contains 0.1 x 106 to 100 x 106 IU/mL of alfa-type interferon.

It is preferred to use 1.2 mg/ml of methylparaben in combination with 0.12 mg/mL of propylparaben when the formulation of the present invention contains 0.1 x 1 o6 to 100 x 1 o6 IU/mL of alfa-2b interferon.

~ WO96/11018 ~ 2 0 ~ 7 4 9 PCT/US95/12362 _9_ The use of m-cresol as an antimicrobial preservative is more preferred.
The water used for preparation of the formulations of the present invention is preferably water for injection.
,1 5 During the course of developr"ent of the aqueous solution formulations of the present invention that would maintain high biological activity as well ashigh chemical and high physical stability of the alfa-type interferon over an extended storage period without employing HSA as a st~hili~er, we identified 10 that the amount of a sorbitan mono-9-oct~decenoate poly(oxy-1,2-ethanediyl) derivative such as polysorbate 80 required to act as a stabilizing agent for thealfa-type interferon had a direct effect on the effective amount of the antimicrobial preservative which could be added to the aqueous solution formulation to provide the appropriate antimicrobial protection for said 15 formulation pursuant to various worldwide health registration requirements without causing undesirable haze formation in the solution.

Thus, when the preferred stabilizing agent, polysorbate 80, was present in formulations of the present invention, in the preferred effective amount of 0.1 2 0 mg/mL, the effective amount of the ~,re~er,ed antimicrobial preservative, e.g., m-cresol which could be added without causing hazing of said formulation was found to be critical. For example if the amount of m-cresol added to a formulation which contained 0.1 mg/mL of polysorbate 80, such as shown in Example 3, is increased to greater than 1.7~ mglmL, hazing was observed. A
2 5 similar hazing problem was observed when the amount of polysorbate 80 in the resultant formulation was varied from 0.01 to 1 mg/mL. No hazing was observed when 1.7~ mg/mL or less, preferably about 1.~ mg/mL of m-cresol was added to a formulation prepared in accordance with the procedures of Example 3 which contains 0.1 mglmL of polysorbate 80. This criticality was 3 0 also observed with the parabens and phenol when they were used as antimicrobial preservatives. For formulations of the present invention SU~T~TU~E S~EET (RIJLE 2~) WO96/11018 2 ~ ~ ~ 7 4 9 PCT/US9~/12362 ~

containing 0.01 to 1 mgtmL of polysorbate 80, the effective amount of methylparaben should be no more than about 1.2 mg/mL when used with 0.12 mg/mL of propylparaben to avoid hazing, and the effective amount of phenol (when it is used in place of the parabens) should be in the range 5 of 0.5 to less than about 4 mg/mL to avoid hazing.

Alfa-type interferon formulations are useful for treatment of a variety of ~ise~se states such as renal cell carcinomas, AlDS-related Kaposi's sarcoma, chronic and acute hepatitis B, chronic and acute non-A, non-B/C
1 0 hepatitis. The formulations of the present invention are useful in treating these disease states preferably as injectable aqueous solutions.

FXAMPI FS

The following non-limiting examples illustrate the preparation of the aqueous solutions of alfa-type interferons.

The procedures listed after Example 5 are used to prepare the formulations of the present invention of Examples 1 to 5.
FXAMpl F 1 ActiveSubstance: Interferon alfa-2b 0.1 x 106-100 x 106 lU/mL~
Buffer: Sodium Phosphate 0.005-0.1 M
(monobasic/dibasic) Chelating Agent: Edetate Disodium 0.01 -1 mg/mL
Stabilizer: Polysorbate 80 0.01 -1 mglmL
Tonicity Adjusting Agent: Sodium Chloride 1 -9 mg/mL
Antimicrobial Preservative: m-Cresol 0.5 -1.75 mg/mL
or Phenol 0.5 - <4 mg/mL
or Methylparaben 0.6 -1 .2 mg/mL
Propylparaben 0.06 - 0.12 mg/mL
Solvent: Waterforlnjection q.s. ad 1 mL
~IU-International Units ~ WO96/11018 ~ 2 n 1 7 4 9 rcT/us95ll2362 Interferon alfa-2b 10 X 106 lU/mL
Sodium Phosphate Dibasic Anhydrous 1.8 m~/mL
Sodium Phosphate Monobasic Monohydrate 1.3 mg/mL
Edetate Disodium 0.1 mg/mL
Polysorbate 80 0.1 mg/mL
Methylparaben 1.2 mg/mL
Propylparaben 0.12 mg/mL
Sodium Chloride 7.5 mg/mL
Water for Injection q.s. ad 1 mL --F~ le 3 Interferon alfa-2b 10 x 106 lU/mL
Sodium Phosphate Dibasic Anhydrous 1.8 mg/mL
Sodium Phosphate Monobasic Monohydrate 1.3 mg/mL
Edetate Disodium 0.1 mg/mL
Polysorbate 80 0.1 mg/mL
m-Cresol 1.5 mg/mL
Sodium Chloride 7.5 mg/mL
Water for Injection q.s. ad 1 mL
Stability data on Examples 2 and 3 are summarized in Tables 1 and 2 respectively.

1 5 The formulation of Example 3 was prepared with 6 x 1 o6 lU/mL of alfa-2b interferon in accordance with the method of manufacture detailed herein below using nitrogen sparging of the solution and maintaining no more than about 4% by volume of oxygen in the he~dsp~ce.

WO96/11018 ~ 2 ~ 1 74 ~ PCT/lJS9SI12362 --Vials containing a label volume of 3 mL of solution were stored at 30~, 25~ and 4~C. The results are summarized in Table 3.

FXAMpl F 5 S
The formulation of Example 4 was prepared in accordance with the method of manufacture detailed hereinbelow in all details except no nitrogen was sparged through the solution or overlaid upon it and the oxygen volume in the heAd$pAce was ~20% by volume as found in 10 ambient air.
Vials containing a label volume of 3 mL of solution were stored at 30~, 25~ and 4~C. The results are summarized in Table 4.

Similar results are expected if the interferon alfa-2b in Examples 1 15 to ~ is substituted by an equivalent amount of Roferon A, Wellferon or Sumiferon interferon alfa.
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PI V;ar ~e~ PI C ~C 1Y) ~5~ ~%L.S.) (Jrr~/rrll~) (%Of In;Ua~ ~1/ml ) Inffial 6.48 108 25.7 100 1.47 98.0 6.91 UP 6.00 100 24.8 96.5 1.47 98.0 6.90 INV 6.00 100 24.8 96.5 1.47 98.0 6.90 3 4 UP 6.00 100 23.5 91.4 1.46 97.3 6.88 ~
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6 4 UP 6.00 100 24.6 95.7 1.46 97.3, 6.84 INV 6.00 100 24.3 94.6 1.45 96.7 6.84 UP 5.56 92.3 20.5 79.8 1.45 96.7 6.85 INV 6.00 100 20.4 79.4 1.44 96.0 6.85 12 4 UP 6.00 100 23.4 91.0 1.47 98.0 6.84 ~'' INV 6.00 100 23.4 91.0 1.46 97.3 6.84 UP - UPr;9ht O
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~2 g Z ~ Z ~ _ ~ _ g _ ~ _ ~, ~ ~0 WO96/11018 2 ~ ~ ~ 7 ~ 9 PCI/US95/12362 Method of Nl~nuf~tllre for Fx~mples 1 to 5 A. Compoundin~ P~r~hen-Cont~ining ~lueous Solution J Formul~tions .C~uch ~ Shown in Fx~rnple ~
S
1. Charge approximately 80% of the water for injection at a temperature greater than 70~C into a suitable jacketed compounding vessel equipped with an agitator.

1 0 2. Separately charge approximately 30% of the water for injection into another suitable vessel. Cool and maintain the water temperature between 20~ and 25~C. Begin sparging and overlaying the water which will be used to bring the batch to final volume with filtered nitrogen to maintain a dissolved oxygen level at or below 0.2~ ppm.

3. Charge and dissolve with mixing methylparaben and propylparaben into the compounding vessel in step 1 while maintaining the temperature of the solution between 70~
2 0 and 80~C.

4. Cool the solution in step 3 to a temperature between 20~
and 25~C. Sparge and ~verlay the solution with filtered nitrogen. Maintain a dissolved oxygen level at or below 2 5 0.25 ppm.

5. Charge and dissolve with mixing the following ingredients into the solution in step 4 while maintaining nitrogen sparging and overlaying:
Sodium phosphate dibasic anhydrous Sodium phosphate monobasic monohydrate Edetate disodium Sodium chloride 3~

6. Discontinue nitrogen sparging of the solution from step 5.
Maintain nitrogen overlaying in the compounding vessel.

WOg6/11018 2 ~ 7 4 9 PCI/US95/12362 ~

7. Charge and dissolve polysorbate 80 in approximately 50 mL of water for inje~ion (for a 1-liter size batch) in a separate vessel. Transfer the polysorbate 80 solution into the solution from step 6.

8. Check the pH of the solution. It should be between 6.6 and 7Ø No pH adjustment is required.

1 0 9. Charge interferon alfa-2b bulk drug solution into the solution in step 8 while mixing.

10. Add water for injection that has been sparged with nitrogen (from step 2) to bring batch to final volume. Agitate solution 1 5 gently until homogeneous.

11. Aseptically filter the solution through a sterilized filter that has been washed and tested for integrity. Collect the sterilized solution into a sterilized filling vessel that has 2 0 been overlaid with sterile-filtered nitrogen. Inlegrily test the filter after filtration.

12. Overlay filling vessel in step 11 with sterile-filtered nitrogen and seal.
B. Com~ounding m-Cresol-Containing A~ueous Solution Formul~tions Such as Shown in Fxam~le 3 The manufacturing procedure ùsed to prepare the aqueous solutions containing m~resol as a preservative (such as shown in 3 0 Example 3) is exactly the same as described hereinabove except the temperature of the solution in Step 3 is maintained between 20~ and 25~C
and the m~resol is charged after Step 6.

C. Com,oounding HSA-Free Aqueous Alfa Interferon Solution 3 ~ Formul~tions Under Ambient Air WO96111018 ~ 2 ~ 1 7 4 9 PCr/US95/12362 - 2 1 - ~

The manufacturing procedure used to prepare the HSA-free ~ueous alfa interferon formulations of Examples 1 to 4 was used to prepare the formulations such as that of Example 5 except that all the steps were performed under ambient air; no nitrogen was sparged S through the solution or overlaid upon it and ambient air (normally conlaini-)g about 20% by volume of oxygen) occupiod the he~dsp~e volume.
To ,nainlain high chemical, physical and biological stability, it is preferred that the water used to prepare the ~queo~ ~s alfa interferon solution as well as the so-formed ~r~ueolJs alfa interferon solution be subst~ntially free of dissolved oxygen, and the aqueous solution be made and stored with a he~dsp~ce of an inert atmosphere, such as nitrogen, conlaining no more than about 4% volume of oxygen. By the term "subst~ntially free of dissolved oxygen" as used herein is meant an 1 5 oxygen level of no more than about 0.25 ppm at a water temperature of about 20~ - 25~C. Normally, this preferred dissolved oxygen level of 0.25 ppm is conveniently achieved by sparging an inert at",osphere, e.g.
nitrogen gas into the water used to prepare the aqueous solutions (maintained at a temperature of about 20~ - 25~C) for a time sufficient (e.g.
2 0 about 30 minutes) to lower the dissolved oxygen to a value of no more than about 0.25 ppm. The sparging is continued throughout the manufacturing procedure to maintain the dissolved oxygen level at 0.25 ppm. We have found that aqueous formulations of the present invention which have a dissolved oxygen level of 1 ppm and an oxygen content in 2 5 the he~dspace of 7% by volume demonstrated signi~icanlly greater loss of chemical stability of the alfa interferon after 3 months of storage at 25~C
compared to a similar ~ eous formulation having the preferred dissolved oxygen level of 0.25 ppm and an oxygen content in the headsp~ce of 4%
by volume stored under the same cor,dilions.
3 0 A side-by-side comparison of the alfa-2b interferon solution stabilitydata shown in Tables 3 and 4 shows that there is no significant stability difference between the aqueous solution formulations of the present invention which were prepared under the nitrogen/low oxygen conditions used in Example 4 and those prepared in accordance with Example 5 3 5 under ambient air during storage for 12 months at 4~C. In conl,asl, a comparison of the alfa-2b interferon stability in solutions of Examples 4 and 5 stored at higher temperatures, e.g, 2~~ and 30~C shows the -protective effect achieved by the preferred (safer) use of nitrogen sparging to effect low dissolved oxygen levels in the aqueous solution while simultaneously maintaining an oxygen content in the headspace at a value of no more than about 4% by volume.

The aqueous solution formulation of the present invention may be stored in any suitable washed and sterilized filling vessels or container such as 2-mL
or S-mL Type 1 flint glass vials stoppered with gray butyl rubber closures. The aqueous solution formulations of the present intention may also be stored in prefilled multi-dose syringes such as those useful for delivery of indictable solutions of drugs such as insulin. Typical suitable syringes include systems comprising a prefilled vial attached to a pen-type syringe such as the Novolet Novo Pen (Trade-mark) available from Novo Nordisk. Typical suitable systems include a prefilled, pen-type syringe which allows easy self-injection by the user as well as accurate, reproducible dose settings.

The aqueous solutions formulations of the present invention, such as present in the Examples may also be lyophilized to form a powder for reconstitution. The lyophilized alfa-type interferon powder is expected to maintain its chemical and biological stability when stored at 2~ to 8~C for at least 2 years.

Claims

1. A stable, aqueous formation having high biological alfa-type interferon activity and free of human blood-derived products which comprises:
a. 0.1 x 10 6 to 100 x 10 6 IU/mL of alfa-type interferon;
b. a buffer system to maintain a pH in the range of 4.5 to 7.1;
c. an effective amount of a chelating agent;
d. an amount of a sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivative sufficient to stabilize the alfa-type interferon against loss of alfa-type interferon;
e. an effective amount of a tonicity agent;
f. an effective amount of an antimicrobial preservative; and g. an amount of water for injection sufficient to prepare a solution of the above-listed ingredients;
said formulation being free of an amount of mannitol effective to act as a bulking agent.

2. The formulation of claim 1, wherein the buffer system is sodium dibasic phosphate and sodium monobasic phosphate.

3. The formulation of claim 1, wherein the buffer system is sodium citrate/citric acid.

4. The formulation of claim 1, 2 or 3, wherein the chelating agent is edetate disodium.

5. The formulation of claim 1, 2 or 3, wherein the chelating agent is citric acid.

6. The formulation of claim 1, 2, 3, 4 or 5, wherein the tonicity agent is sodium chloride.

7. The formulation of claim 1, 2, 3, 4, 5 or 6, wherein said preservative is selected from m-cresol, phenol, methylparaben, propylparaben or mixtures thereof.

8. The formulation of claim 1, 2, 3, 4, 5 or 6, wherein said preservative is m-cresol.

9. The formulation of claim 1, 2, 3, 4, 5, 6, 7 or 8, wherein the alfa-type interferon is interferon alfa-2.

10. A stable aqueous solution formulation having high biological interferon alfa-2 activity and free of human serum albumin, which comprises:
mg/mL
a. Interferon alfa-2 5 x 10 6 to 50 x 10 6IU
b. Sodium Phosphate Dibasic Anhydrous 1.8 c. Sodium Phosphate Monobasic Mono-hydrate 1.3 d. Disodium Dihydrogen Ethylenedi-amine tetraacetate 0.1 e. A sorbitan Mono-9-Octadecenoate Poly (Oxy-1,2-Ethanediyl) Derivative 0.1 f. Methylparaben 1.2 g. Propylparaben 0.12 h. Sodium Chloride 7.5 and i. Water for Injection q.s. ad 1 mL
said formulation being free of an amount of mannitol effective to act as a bulking agent.

11. A stable, aqueous solution formulation having high biological interferon alfa-2 activity and free of human serum albumin, which comprises:
mg/mL
a. Interferon alfa-2 5 x 10 6 to 50 x 10 6IU
b. Sodium Phosphate Dibasic Anhydrous 1.8 c. Sodium Phosphate Monobasic Mono-hydrate 1.3 d. Disodium Dihydrogen Ethylenedi-amine tetraacetate 0.1 e. A sorbitan Mono-9-Octadecenoate Poly (Oxy-1,2-Ethanediyl) Derivative 0.1 f. m-Cresol 1.5 g. Sodium Chloride 7.5 and h. Water for Injection q.s. ad 1 mL
said formulation being free of an amount of mannitol effective to act as a bulking agent.

12. An article of manufacture comprising a packaging material and the formulation of any one of claims 1 to 11, wherein said packaging material is a multidose glass vial.

13. An article of manufacture comprising a prefilled syringe containing an effective amount of the formulation of any one of claims 1 to 11.

14 An article of manufacture comprising a packaging material and the formulation of any one of claims 1 to 11, wherein said packaging material is a single-dose vial.

15. An article of manufacture comprising a prefilled, pen-type syringe containing an effective amount of the formulation of any one of claims 1 to 11.

16. A process of preparing a stable, aqueous formulation having high biological alfa-type interferon activity and free of human blood derived products comprising admixing, in the absence of an amount of mannitol effective to act as a bulking agent, an effective amount of alfa-type interferonwith a buffer system capable of maintaining the pH within the range of 4.5 to 7.1, a chelating agent, a sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl)derivative, a tonicity agent, an antimicrobial preservative and sufficient water to make an aqueous solution.

17. The process of claim 16, wherein the aqueous solution is prepared and maintained substantially free of dissolved oxygen.

18. The process of claim 17, wherein the dissolved oxygen is no more than 0.25 ppm.

19. The process of claim 17 or 18, wherein a headspace above the aqueous solution is an inert atmosphere containing no more than 4% by volume oxygen.

20. The process of claim 16, 17, 18 or 19, wherein said buffer system is sodium citrate/citric acid.

21. A sterilized filling vessel containing a formulation prepared by a process as claimed in any one of claims 16 to 20.

22. An article of manufacture comprising a sterilized filling vessel containing a stable, aqueous formulation of alfa interferon having at least 75%
of alfa interferon biological activity and free of human blood-derived products,said formulation comprising 0.1 x 10 6 to 100 x 10 6 IU/mL alfa-interferon with a buffer system capable of maintaining the pH within the range of 4.5 to 7.1, a chelating agent, a sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivative, a tonicity agent, an antimicrobial preservative and sufficient waterto provide an aqueous solution wherein the dissolved oxygen level in said solution is maintained at a value of no more than about 0.25 ppm and a headspace of an inert atmosphere above said solution is simultaneously maintained at a value of less than about 4% oxygen by volume.

23. An article of claim 22, wherein said buffer system is sodium citrate/citric acid.

24. An article according to claim 22 or 23, wherein said chelating agent is disodium dihydrogen ethylene diamine tetraacetate.

25. An article according to claim 22, 23 or 24, wherein said preservative is m-cresol.

26. A process of preparing a stable, aqueous formulation having high alfa-type interferon biological activity and free of human blood-derived products comprising admixing an effective amount of alfa-type interferon with a buffer system capable of maintaining the pH within the range of 4.5 to 7.1, a chelatingagent, a sorbitan mono-9-octadecenoate poly(oxy-1,2-ethanediyl) derivative, a tonicity agent, an antimicrobial preservative and sufficient water to make an aqueous solution wherein the dissolved oxygen level in said solution is maintained at a value of no more than about 0.25 ppm and a headspace of an inert atmosphere above said solution is simultaneously maintained at a value of less than about 4% oxygen by volume by sparging nitrogen into said water and said solution throughout the preparing of the stable, aqueous formulation.

27. An article of manufacture comprising a sterilized filling vessel and an effective amount of the formulation as produced in accordance with the process of claim 26.

28. The article of manufacture of claim 22, 23, 24, 25 or 27, wherein the sterilized filling vessel is a prefilled syringe.

29. The article of manufacture of claim 22, 23, 24, 25 or 27, wherein the sterilized filling vessel is a single-dose vial.

30. The article of manufacture of claim 22, 23, 24, 25 or 27, wherein the sterilized filling vessel is a multi-dose glass vial.

31. The article of manufacture of claim 22, 23, 24, 25 or 27, wherein the sterilized filling vessel is a prefilled pen-type syringe.

32. The article of manufacture of claim 22, 23, 24, 25, 27, 28, 29, 30 or 31, wherein said interferon is alfa-2b in an amount of 10 x 10 6 IU/mL.

33. The article of manufacture of claim 22, 23, 24, 25, 27, 28, 29, 30 or 31, wherein said interferon is interferon alfa-2b.