PL212934B1

Pharmaceutical anti-tnf-alpha antibody formulation

Abstract

This record has no abstract on file.

Term

Term ended

Expired 15 August 2023, 3.1 years ago.

24 claims: 3 independent, 21 dependent

1
Ciekły wodny preparat farmaceutyczny, znamienny tym, że zawiera 30 do 120 mg/ml ludzkiego przeciwciała IgG1 przeciw czynnikowi martwicy nowotworów alfa (TNFa), lub jego części wiążącej antygen, bufor cytrynianowy i fosforanowy, poliol i środek powierzchniowo czynny, który to preparat ma pH 4 do 8, oraz w którym przeciwciało, lub jego część wiążąca antygen, zawiera region zmienny lekkiego łańcucha obejmujący domenę regionu określającego komplementarność (CDR) 1 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO:7: domenę CDR2 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO:5;i domenę CDR3 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO.3 lub SEQ ID NO:3 zmodyfikowaną przez pojedynczą substytucję alaniny w pozycji 1, 4, 5, 7 lub 8 albo przez jedną do pięciu konserwatywnych substytucji aminokwasowych w pozycjach 1, 3, 4, 6, 7, 8 i/lub 9;oraz region zmienny ciężkiego łańcucha obejmujący domenę CDR 1 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO:8;domenę CDR2 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO:6: i domenę CDR3 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO.4 lub SEQ ID NO:4 zmodyfikowaną przez pojedynczą substytucję alaniny w pozycji 2, 3, 4, 5, 6, 8, 9, 10 lub 11 albo przez jedną do pięciu konserwatywnych substytucji aminokwasowych w pozycjach 2, 3, 4, 5, 6, 8, 9, 10, 11 i/lub 12.
2
Ciekły wodny preparat farmaceutyczny według zastrz. 1, znamienny tym, że zawiera około 5 do 20 mg/ml poliolu.
3
Ciekły wodny preparat farmaceutyczny według zastrz. 1 albo 2, znamienny tym, że poliol stanowi alkohol cukrowy.
4
Ciekły wodny preparat farmaceutyczny według zastrz. 5, znamienny tym, że alkohol cukrowy stanowi mannitol.
5
Ciekły wodny preparat farmaceutyczny według zastrz. 1, znamienny tym, że środek powierzchniowo czynny stanowi polisorbat.
6
Ciekły wodny preparat farmaceutyczny według zastrz. 5, znamienny tym, że polisorbat stanowi Polysorbate 80.
7
Ciekły wodny preparat farmaceutyczny według zastrz. 6, znamienny tym, że zawiera około 0,1 do 10 mg/ml Polysorbate 80.
8
Ciekły wodny preparat farmaceutyczny według zastrz. 1 - 7, znamienny tym, że bufor cytrynianowy i fosforanowy zawiera kwas cytrynowy, cytrynian sodu, dihydrat fosforanu disodu i dihydrat diwodorofosforanu sodu.
9
Ciekły wodny preparat farmaceutyczny według zastrz. 8, znamienny tym, że zawiera (a) około 1 do 1,5 mg/ml kwasu cytrynowego, (b) około 0,25 do 0,5 mg/ml cytrynianu sodu, (c) około 1,25 do 1,75 mg/ml dihydratu fosforanu disodu, (d) około 0,7 do 1,1 mg/ml dihydratu diwodorofosforanu sodu i (e) około 6,0-6,4 mg/ml chlorku sodu.
10
Ciekły wodny preparat farmaceutyczny według zastrz. 1 - 9, znamienny tym, że stężenie przeciwciała lub jego części wiążącej antygen wynosi 45-105 mg/ml.
11
Ciekły wodny preparat farmaceutyczny według zastrz. 1 - 10, znamienny tym, że stężenie przeciwciała lub jego części wiążącej antygen jest większe niż 45 mg/ml.
12
Ciekły wodny preparat farmaceutyczny według zastrz. 1 - 11, znamienny tym, że stężenie przeciwciała lub jego części wiążącej antygen wynosi około 50 mg/ml.
13
Ciekły wodny preparat farmaceutyczny według zastrz. 1 - 12, znamienny tym, że jest odpowiedni do jednorazowego podskórnego zastrzyku.
14
Ciekły wodny preparat farmaceutyczny według zastrz. 1 - 13, znamienny tym, że przeciwciało, lub jego część wiążąca antygen, zawiera region zmienny lekkiego łańcucha (LCVR) obejmujący sekwencję aminokwasową SEQ ID NO:1 i region zmienny ciężkiego łańcucha (HCVR) obejmujący sekwencję aminokwasową SEQ ID NO:2.
15
Ciekły wodny preparat farmaceutyczny według zastrz. 1 - 14, znamienny tym, że przeciwciało lub jego część wiążąca antygen jest przeciwciałem D2E7 lub jego częścią wiążącą antygen.
16
Preparat według zastrz. 1, znamienny tym, że poliol stanowi mannitol, a środek powierzchniowo czynny stanowi Polysorbate 80. PL 212 934 B1
17
Preparat według zastrz. 16, znamienny tym, że zawiera 5-20 mg/ml mannitolu i 0,110 mg/ml Polysorbate 80.
18
Ciekły wodny preparat farmaceutyczny, znamienny tym, że zawiera (a) 1-150 mg/ml przeciwciała lub jego części wiążącej antygen, (b) 5-20 mg/ml mannitolu, (c) 0,1-10 mg/ml Tween-80 i (d) układ buforujący zawierający cytrynian i/lub fosforan, mający pH 4 do 8, w którym przeciwciało, lub jego część wiążąca antygen, zawiera region zmienny lekkiego łańcucha obejmujący domenę regionu określającego komplementarność (CDR) 1 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO:7: domenę CDR2 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO:5;i domenę CDR3 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO.3 lub SEQ ID NO:3 zmodyfikowaną przez pojedynczą substytucję alaniny w pozycji 1, 4, 5, 7 lub 8 albo przez jedną do pięciu konserwatywnych substytucji aminokwasowych w pozycjach 1, 3, 4, 6, 7, 8 i/lub 9;oraz region zmienny ciężkiego łańcucha obejmujący domenę CDR 1 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO:8;domenę CDR2 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO:6: i domenę CDR3 obejmującą sekwencję aminokwasową oznaczoną jako SEQ ID NO.4 lub SEQ ID NO:4 zmodyfikowaną przez pojedynczą substytucję alaniny w pozycji 2, 3, 4, 5, 6, 8, 9, 10 lub 11 albo przez jedną do pięciu konserwatywnych substytucji aminokwasowych w pozycjach 2, 3, 4, 5, 6, 8, 9, 10, 11 i/lub 12.
19
Preparat według zastrz. 18, znamienny tym, że pH wybiera się spośród zakresów obejmujących zakres od około 4,5 do około 6,0, od około 4,8 do około 5,5 i od około 5,0 do około 5,2.
20
Ciekły wodny preparat farmaceutyczny według zastrz. 18, znamienny tym, że zawiera (a) około 50 mg/ml przeciwciała, (b) około 12 mg/ml mannitolu i (c) około 1 mg/ml Tween-80.
21
Preparat według zastrz. 18, znamienny tym, że układ buforujący obejmuje (a) około 1,3 mg/ml kwasu cytrynowego, (b) około 0,3 mg/ml cytrynianu sodu, (c) około 1,5 mg/ml dihydratu fosforanu disodu, (d) około 0,9 mg/ml dihydratu diwodorofosforanu sodu i (e) około 6,2 mg chlorku sodu.
22
Ciekły wodny preparat farmaceutyczny według zastrz. 18, znamienny tym, że przeciwciało, lub jego część wiążąca antygen, zawiera region zmienny lekkiego łańcucha (LCVR) obejmujący sekwencję aminokwasową SEQ ID NO:1 i region zmienny ciężkiego łańcucha (HCVR) obejmujący sekwencję aminokwasową SEQ ID NO:2.
23
Preparat według zastrz. 18, znamienny tym, że przeciwciało lub jego część wiążąca antygen jest przeciwciałem D2E7 lub jego częścią wiążącą antygen.
24
Ciekły wodny preparat farmaceutyczny według zastrzeżeń 1-23, znamienny tym, że jest przeznaczony do stosowania u pacjenta cierpiącego na zaburzenie, w którym aktywność TNFa jest szkodliwa, tak że aktywność TNFa u pacjenta jest hamowana.

Independent claims24

346 paragraphs in 12 sections, as filed

Description of the invention

The present invention relates to a liquid aqueous pharmaceutical preparation.

State of the art

Tumor necrosis factor? (TNF?) Is a cytokine produced by numerous cell types including monocytes and macrophages that was originally detected for its ability to induce necrosis of certain tumors in mice (see, e.g., Old, L. (1985) Science 230: 630- 632). Subsequently, it was shown that a factor called cachectic, related to cachexia, has a molecule identical to TNFα. TNF? Mediates shock (see, e.g., Beutler, B. and Cerami, A. (1988) Annu. Rev. Biochem. 57: 505-518; Beutler, B. and Cerami, A. (1989) Annu. Rev. Immunol. 7: 625-655). In addition, TNFα is involved in the pathophysiology of a wide variety of other human diseases and disorders, including sepsis, infection, autoimmune diseases, transplant rejection, and graft versus host reactions (see, e.g., Moeller, A., et al. (1990) Cytokine 2: 162 169, U.S. Patent No. 5,231,024, Moeller et al, European Patent Publication No. 2,60610 B1, Moeller, A., et al. Yasilli, P. (1992) Annu. Rev. Immunol. 10: 411-452; Tracey, KJ. and Cerami, A. (1994) Annu. Rev. Med. 45: 491-503).

Due to the deleterious effects of human TNF? (HTNF?) In a wide variety of human disorders, therapeutic strategies have been devised to inhibit or counteract hTNF? Activity. In particular, antibodies that bind to and neutralize hTNFa are sought as a means to inhibit hTNFa activity. Some of the earliest such antibodies were murine monoclonal antibodies (mAbs), secreted by hybridomas produced from the lymphocytes of mice immunized with hTNF? (See e.g. Hahn T; et al. (1985) Proc Natl Acad Sci USA 82: 3814-3818; Liang, CM., Et al. (1986) Biochem. Biophys. Res. Commun. 137: 847-854; Hirai, M., et al. (1987) J. Immunol. Methods 96: 57-62; Fendly, BM, et al. (1987) Hybridoma 6: 359-370; Moeller, A., et al. (1990) Cytokine 2: 162-169; U.S. Patent No. 5,231,024 to Moeller et al; European Patent Publication No. 186833 B1, Wallach, D .; European Patent Application Publication No. 218,868 A1, Old et al; European Patent Publication No. 260610 B1, Moeller, A., et al.). While these murine anti-hTNFa antibodies often exhibited high affinity for hTNFa (e.g., Kd <10-9 M) and were able to neutralize hTNFa activity, their in vivo use may be limited by problems associated with administering murine antibodies to humans, such as the short plasma half-life, the inability to induce certain effector functions in humans, and the induction of an unwanted immune response against murine antibodies in humans. humans ("human anti-mouse antibody (HAMA) response)).

In an attempt to overcome the difficulties associated with the use of all-murine antibodies in humans, genetic engineering resulted in more "human-like murine anti-hTNF? Antibodies." For example, chimeric antibodies have been prepared in which the variable portions of the antibody chains are of murine origin and the constant portions of the antibody chains are of human origin (Knight, DM, et al. (1993) Mol Immunol. 30: 1443-1453; PCT Publication No. WO 92 (16553, Daddon, PE, et al.). In addition, humanized antibodies have also been produced in which the hypervariable domains of the antibody variable regions are of murine origin and the remainder of the variable and constant regions of the antibodies are of human origin (PCT Publication No. WO 92/11383, Adair, JR, et al.). However, since these chimeric and humanized antibodies still contain some murine sequences, they may still elicit an unwanted immune response, an anti-chimeric antibody response (HACA) in humans, especially when administered over an extended period of time, e.g. as indicated by chronic conditions such as as rheumatoid arthritis (see, e.g., Elliott, MJ, et al. (1994) Lancet 344: 1125-1127; Elliot, MJ, et al. (1994) Lancet 344: 1105-1110).

A more preferred agent than murine mAbs or derivatives thereof (e.g., chimeric or humanized antibodies), an agent that inhibits hTNFa would be a fully human antibody to hTNFa, as such an agent should not induce a HAMA response, even on long-term use. Human monoclonal antibodies against hTNFa have been generated using human hybridoma techniques (Boyle, P., et al. (1993) Cell. Immunol. 152: 556-568; Boyle, P., et al. (1993) Cell. Immunol. 152: 569-581; European Patent Application Publication No. 614,984 A2, Boyle, et al.). However, hybridoma-derived monoclonal autoantibodies were reported to have too little affinity for hTNFa to be calculated by conventional methods, unable to bind soluble hTNFa or abolish hTNFa induced cytotoxicity (see Boyle, et al; supra). Moreover, the success of techniques using human hybridomas depends on the natural presence in humans of peripheral blood lymphocytes producing autoantibodies specific for hTNF ?. In some

Studies have detected plasma autoantibodies against hTNFa in humans (Fomsgaard, A., et al. (1989)

Scand. J. Immunol. 30: 219-223; Bendtzen, K., et al (1990) Prog. Leukocyte Biol 10B: 447-452), while not in other studies (Leusch, HG. Et al. (1991) J. Immunol. Methods 13 9: 145-147).

An alternative to naturally occurring human anti-hTNFa antibodies would be a recombinant hTNFa antibody. To date, recombinant human hTNFa binding antibodies have been described

-7 -2 -1 with relatively weak affinity (i.e., Kd ~ 10-7M) and large dissociation constant (i.e., Koff ~ 10-2 s-1) (Griffiths, AD, et al. (1993) EMBO J. 12: 725-734). However, due to their relatively rapid dissociation kinetics, these antibodies may not be suitable for therapeutic use. In addition, a recombinant human anti-hTNFa antibody has been described that does not neutralize hTNFa activity, but rather increases hTNFa binding to the cell surface and enhances hTNFa internalization (Lidbury, A., et al. (1994) Biotechnol Ther. 5: 27-45; PCT Publication WO 92/03145, Aston, R. et al.)

Recombinant human antibodies that bind soluble hTNFa with high affinity and slow dissociation kinetics, which can neutralize hTNFa activity, including abolition of hTNFa-induced cytotoxicity (in vitro and in vivo) and hTNFa-induced cell activation, have also been described (see US Patent No. 6,090,382. ).

The essence of the invention

There is a need for a stable pharmaceutical formulation in the form of an aqueous solution with a long shelf life containing an antibody that is capable of being used therapeutically to inhibit or counteract the deleterious activity of hTNF ?. There is also a need for a stable pharmaceutical formulation in the form of an aqueous solution with a long shelf life containing an antibody that is therapeutically useful, easy to administer, and contains a high concentration of protein.

The invention relates to a liquid aqueous pharmaceutical preparation, characterized in that it comprises 30 to 120 mg / ml of human IgG1 antibody against tumor necrosis factor alpha (TNFa), or antigen binding portion thereof, citrate and phosphate buffer, polyol and a surfactant, said the formulation has a pH of 4 to 8, and wherein the antibody, or antigen-binding portion thereof, comprises a light chain variable region comprising a complementarity determining region (CDR) 1 domain comprising the amino acid sequence designated SEQ ID NO: 7: a CDR2 domain comprising the amino acid sequence designated SEQ ID NO: 5; and a CDR3 domain comprising the amino acid sequence designated SEQ ID NO.3 or SEQ ID NO: 3 modified by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4 , 6, 7, 8 and / or 9; and a heavy chain variable region comprising a CDR 1 domain comprising the amino acid sequence shown as SEQ ID NO: 8; a CDR2 domain comprising the amino acid sequence designated SEQ ID NO: 6: and a CDR3 domain comprising the amino acid sequence designated SEQ ID NO.4 or SEQ ID NO: 4 modified by a single alanine substitution at position 2, 3, 4, 5, 6, 8 , 9, 10, or 11, or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11, and / or 12.

A liquid aqueous pharmaceutical preparation containing about 5 to 20 mg / ml polyol is preferred.

A liquid aqueous pharmaceutical preparation is preferred, characterized in that the polyol is a sugar alcohol.

Preferably the sugar alcohol is mannitol.

A liquid, aqueous pharmaceutical preparation is preferred, characterized in that the surfactant is a polysorbate.

Preferably the polysorbate is Polysorbate 80.

A liquid aqueous pharmaceutical preparation is preferred, characterized in that it contains about 0.1 to 10 mg / ml of Polysorbate 80.

A liquid pharmaceutical aqueous preparation is preferred, characterized in that the citrate and phosphate buffers contain citric acid, sodium citrate, disodium phosphate dihydrate and sodium dihydrogen phosphate dihydrate.

A liquid aqueous pharmaceutical preparation is preferred, characterized in that it comprises (a) about 1 to 1.5 mg / ml of citric acid, (b) about 0.25 to 0.5 mg / ml of sodium citrate, (c) about 1 25 to 1.75 mg / ml disodium phosphate dihydrate,

(D) about 0.7 to 1.1 mg / mL of sodium dihydrogen phosphate dihydrate and (e) about 6.0-6.4 mg / mL of sodium chloride.

A liquid, aqueous pharmaceutical preparation is preferred, characterized in that the concentration of the antibody or antigen-binding portion thereof is 45-105 mg / ml.

A liquid aqueous pharmaceutical preparation is preferred, characterized in that the concentration of the antibody or antigen-binding portion thereof is greater than 45 mg / ml.

A liquid aqueous pharmaceutical preparation is preferred, characterized in that the concentration of the antibody or antigen-binding portion thereof is about 50 mg / ml.

A liquid aqueous pharmaceutical preparation is preferred, characterized in that it is suitable for a single subcutaneous injection.

A liquid aqueous pharmaceutical formulation is preferred, characterized in that the antibody, or antigen binding portion thereof, comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1 a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO: 2.

A liquid, aqueous pharmaceutical preparation is preferred, characterized in that the antibody or antigen binding portion thereof is the D2E7 antibody or antigen binding portion thereof.

Preference is given to a formulation characterized in that the polyol is mannitol and the surfactant is Polysorbate 80.

A more preferred formulation is characterized in that it contains 5-20 mg / ml mannitol and 0.110 mg / ml Polysorbate 80.

The invention further relates to a liquid aqueous pharmaceutical preparation, characterized in that it comprises (a) 1-150 mg / ml of the antibody or antigen binding portion thereof.

(b) 5-20 mg / ml mannitol, (c) 0.1-10 mg / ml Tween-80, and (d) a citrate and / or phosphate buffer system having a pH of 4 to 8, wherein the antibody, or an antigen-binding portion thereof, comprises a light chain variable region comprising a complementarity determining region (CDR) 1 domain comprising the amino acid sequence designated SEQ ID NO: 7: a CDR2 domain comprising the amino acid sequence designated SEQ ID NO: 5; and a CDR3 domain comprising the amino acid sequence designated SEQ ID NO.3 or SEQ ID NO: 3 modified by a single alanine substitution at position 1, 4, 5, 7 or 8 or by one to five conservative amino acid substitutions at positions 1, 3, 4 , 6, 7, 8 and / or 9; and a heavy chain variable region comprising a CDR 1 domain comprising the amino acid sequence shown as SEQ ID NO: 8; a CDR2 domain comprising the amino acid sequence designated SEQ ID NO: 6: and a CDR3 domain comprising the amino acid sequence designated SEQ ID NO.4 or SEQ ID NO: 4 modified by a single alanine substitution at position 2, 3, 4, 5, 6, 8 , 9, 10, or 11, or by one to five conservative amino acid substitutions at positions 2, 3, 4, 5, 6, 8, 9, 10, 11, and / or 12.

A formulation is preferred, wherein the pH is selected from a range ranging from about 4.5 to about 6.0, from about 4.8 to about 5.5, and from about 5.0 to about 5.2.

A liquid aqueous pharmaceutical preparation is preferred, characterized in that it comprises (a) about 50 mg / ml antibody, (b) about 12 mg / ml mannitol, and (c) about 1 mg / ml Tween-80.

A formulation is preferred, characterized in that the buffering system comprises (a) about 1.3 mg / ml of citric acid, (b) about 0.3 mg / ml of sodium citrate, (c) about 1.5 mg / ml of phosphate dihydrate. disodium, (d) about 0.9 mg / mL of sodium dihydrogen phosphate dihydrate, and (e) about 6.2 mg of sodium chloride.

A preparation is preferred, characterized in that the antibody or antigen binding portion thereof is the D2E7 antibody or antigen binding portion thereof.

PL 212 934 B1

A liquid aqueous pharmaceutical preparation is preferred, characterized in that it is administered to a patient suffering from a disorder in which the activity of TNF? Is deleterious such that the activity of TNF? In the patient is inhibited.

The above-described liquid pharmaceutical formulation in the form of an aqueous solution containing the antibody in a therapeutically effective amount in a buffered solution may have a shelf life of at least 18 months. An aqueous solution pharmaceutical formulation as described above containing the antibody in a therapeutically effective amount in a buffered liquid solution may also have a shelf life of at least 18 months. In one embodiment, the pharmaceutical formulation has increased stability. In a further embodiment, the stability of the formulation according to the invention allows for at least 3 freeze / thaw cycles of the formulation. According to the invention, the antibody is directed against TNFα. In another embodiment, the antibody is directed against human TNFα. In a further embodiment, the antibody is D2E7.

The above-described liquid pharmaceutical formulation in the form of an aqueous solution containing the antibody in a therapeutically effective amount in a buffered solution may also have an enhanced stability of at least 12 months at 2-8 ° C. In one embodiment, the formulation has an increased shelf life of at least 18 months. According to the invention, the antibody is directed against TNFα. In another embodiment, the antibody is directed against human TNFα. In a further embodiment, the antibody is D2E7.

The liquid pharmaceutical preparation described above in the form of an aqueous solution containing the antibody in a therapeutically effective amount in a buffered solution may be a preparation that is easily administered. In one embodiment, the antibody is D2E7.

The pharmaceutical preparation in the form of an aqueous solution may be suitable for injection. Furthermore, the formulation may be suitable for a single subcutaneous injection. In one embodiment, the concentration of antibodies in the preparation is about 50 mg / ml. In addition, the formulation may not be light sensitive.

The liquid pharmaceutical formulation in the form of an aqueous solution may contain the antibody, or antigen-binding portion thereof, that dissociates from human TNF? With a Kd constant of 1 x 10-8 m or -3 -1 less and a Koff constant of 1 x 10-3 s-1 or less, both of which were determined by surface plasmon resonance technique, and canceled the cytotoxicity of human TNFα in standard in vitro L929 assay with an IC50 of 1 x 10-7 M or less. The preparation described above can contain the antibody, or antigen-binding portion thereof, that dissociates from human

-4 -1

TNF? With a Koff constant of 5 x 10-4 s-1 Or less. In a further embodiment, the preparation can contain an antibody, or antigen-binding portion thereof, that dissociates from human TNF? With a Koff constant of

-4 -1 x 10-4 s-1 or less. In yet another embodiment, the formulation described above may contain an antibody, or antigen-binding portion thereof, that descends the cytotoxicity of human TNF? In a standard in vitro L929 assay with an IC50 of 1 x 10-8 m or less. In yet another embodiment, the present formulation comprises an antibody, or antigen-binding portion thereof, that abolishes the cytotoxicity of human TNF? In a standard in vitro L929 assay with an IC50 of 1 x 10-9 M or less. Another embodiment includes a formulation in which the antibody, or antigen binding portion thereof, abolishes the cytotoxicity of human TNF? In a standard in vitro L929 assay -10 with an IC50 of 1 x 10-10 M or less.

The liquid pharmaceutical preparation in the form of an aqueous solution may contain the antibody, or antigen binding portion thereof, that is a recombinant antibody, or antigen binding portion thereof. In another embodiment, the preparation can contain an antibody, or antigen-binding portion thereof, that inhibits human TNFα-induced ELAM-1 expression on human umbilical vein endothelial cells. In yet another embodiment, the present formulation comprises the D2E7 antibody.

In yet another embodiment of the invention, the antibody or antigen binding portion thereof contained in the liquid pharmaceutical formulation in the form of an aqueous solution has a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable region (HCVR) containing the amino acid sequence SEQ ID NO: 2. According to the invention, the antibody, or antigen binding portion thereof, has an IgG1 heavy chain constant region. Optionally, the antibody, or antigen binding portion thereof, may have an IgG4 heavy chain constant region. The antibody, or antigen binding portion thereof, can be a Fab fragment. Optionally, the antibody or antigen binding portion thereof may be a single chain Fv fragment.

PL 212 934 B1

The antibody, or antigen binding portion thereof, can neutralize the activity of human TNFα, chimpanzee TNFα, and TNFα from at least one additional primate selected from the group consisting of baboon TNFα, marmoset TNFα, cynomolgus monkey TNFα, and rhesus TNFα. It can also neutralize murine TNF? And / or porcine TNF? Activity.

In one embodiment of the invention, the antibody, or antigen-binding portion thereof, binding to human TNFα is the D2E7 antibody or antigen-binding portion thereof.

The liquid pharmaceutical formulation in the form of an aqueous solution may also contain about 1.305 mg / mL citric acid, about 0.305 mg / mL sodium citrate, about 1.53 mg / mL disodium phosphate dihydrate, about 0.86 mg / mL sodium dihydrogen phosphate dihydrate, and about 6.165 sodium chloride mg / ml.

Detailed Description of the Invention

This invention relates to a liquid pharmaceutical formulation in the form of an aqueous solution having a pH of about 4 to about 8 containing a high concentration of protein which is antibodies in a concentration ranging from about 1 to about 150 mg / ml and which may have increased stability. The liquid pharmaceutical preparation in the form of an aqueous solution according to the invention is for therapeutic use in an individual suffering from a condition characterized by the harmful effects of TNFα. The formulation of the invention comprises the following components: an antibody that binds human TNF? With high affinity, low dissociation constant and high neutralization potential; buffer including citric acid, sodium citrate, disodium phosphate dihydrate, and sodium dihydrogen phosphate dihydrate; osmotic agents, including mannitol and sodium chloride; a detergent, including Polysorbate 80; and sodium hydroxide to adjust the pH.

Definitions

In order to facilitate the understanding of the invention, certain terms have been defined at the outset.

The term "subject refers to a living organism, eg, prokaryote and eukaryote. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic animals. In specific embodiments, the subject is a human.

The term "pharmaceutical preparation" refers to preparations which are in a form which allows the active ingredients to be substantially biologically effective and which do not contain any additional ingredients which are substantially toxic to the subjects to which the preparation would be administered. Pharmaceutically acceptable excipients (vehicles, additives) are those which can be reasonably administered to a mammal to give an effective dose of the active ingredient to be used.

The term "stable preparation" refers to one in which the antibody retains substantially physical and / or chemical stability and / or biological activity during storage. Various analytical techniques known in the art for measuring protein stability are described, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Life can be measured at a selected temperature over a selected period of time. Preferably, the formulation is stable at room temperature (about 30 ° C) or at 40 ° C for at least 1 month and / or stable at about 2-8 ° C for at least 1 year or for at least 2 years. Furthermore, it is preferred that the formulation is stable after being frozen (down to, e.g., -70 ° C) and thawing, which is hereinafter referred to as a "freeze / thaw cycle."

An antibody "retains physical stability in a pharmaceutical formulation if, when viewed with the naked eye or measured by UV light scattering or size exclusion chromatography, it shows no signs of aggregation, precipitation, and / or denaturation as manifested by a change in color and / or clarity.

An antibody in a pharmaceutical formulation "is chemically stable if the chemical stability at a given point in time is such that the antibody retains biological activity, as defined below. Chemical stability can be determined by detecting and quantifying chemically altered forms of the antibody. A chemical change may include a change in magnitude (e.g. trimming), which can be assessed using, for example, size exclusion chromatography, SDSPAGE, and / or sample-assisted matrix assisted laser desorption and ionization mass spectrometry (MALDI / TOF MS). Other types of chemical changes include a change in charge (e.g., due to deamination), which can be assessed, for example, by ion exchange chromatography.

An antibody in a pharmaceutical preparation "retains biological activity when the antibody in the pharmaceutical preparation is biologically active to an extent compatible with its intended purpose. For example, biological activity is preserved if the biological activity of the antibody

The concentration of the pharmaceutical preparation is in the range of about 30%, about 20%, or about 10% (within the error of measurement) of the biological activity exhibited during the manufacture of the pharmaceutical preparation (e.g., as determined by an antigen binding assay).

The term "isotonic" is known in the art. Isotonic can mean, for example, that a given preparation has essentially the same osmotic pressure as human blood. Isotonic preparations generally have an osmotic pressure of from about 250 to 350 mOsm. Isotonicity can be measured using, for example, a steam-type or ice-freeze osmometer. The term "osmotic agent" means a compound that renders a preparation isotonic.

The term "polyol" means a substance containing multiple hydroxyl groups and includes sugars (reducing and non-reducing sugars), alcohols, and sugar acids. Preferred polyols herein have a molecular weight of less than about 600 kD (e.g., in the range of about 120 to about 400 kD). The term "reducing sugar" means a sugar containing a hemiacetal group, reducing metal ions or covalently reacting with lysine and other protein amino groups, and the term "non-reducing sugar" means a sugar that does not have the property of a reducing sugar. Examples of reducing sugars are fructose, mannose, maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose and glucose. Non-reducing sugars include sucrose, trehalose, sorbose, melesitose, and raffinose. Mannitol, xylitol, erythritol, threitol, sorbitol and glycerol are examples of sugar alcohols. Sugar acids include L-gluconate and its metal salts. In the event that the formulation is required to be stable despite being frozen and thawed, it is preferred that the polyol does not crystallize at freezing temperature (e.g. -20 ° C) which would destabilize the antibodies in the formulation. The polyol can also act as an osmotic agent. In one embodiment of the invention, one component of the formulation is mannitol at a concentration of 5 to 20 mg / ml. The mannitol concentration may preferably be 7.5 to 15 mg / ml. The mannitol concentration may more preferably be 10-14 mg / ml.

The term "buffer as used herein" refers to a buffer solution that counteracts pH changes mediated by the conjugate acid and base components. A buffer in accordance with the invention has a pH ranging from about 4 to about 8; preferably from about 4.5 to about 7; and most preferably, a pH in the range of from about 5.0 to about 6.5. Examples of buffers to control the pH in this range include acetate (e.g. sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffers.

Pharmacologically, in the context of the present invention, the term "therapeutically effective amount or" effective amount of an antibody "refers to an amount effective in preventing or treating the disorder for which the antibody has been found to be effective. The term "disorder" means any condition in which treatment with an antibody provides benefits. It includes chronic and acute disorders or diseases including those pathological conditions which predispose the disorder to occur.

The term "preservative" means a compound that can be included in a formulation to significantly reduce its bacterial activity and thereby facilitate, for example, the preparation of formulations for various uses. Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyldimethyl ammonium chlorides in which the alkyl groups are long chain compounds) and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

The term "treatment" refers to both therapeutic treatment and prophylaxis or preventive measures. Persons in need of treatment include those already suffering from the disorder as well as those for whom it is intended to be prophylaxis for the disorder.

As used herein, the terms "parenteral administration and" parenteral administration mean modes of administration other than enteral and topical administration, usually by injection, which include, without limitation: intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intravenous, intravenous, intravenous, intravenous, intravenous, and intravenous. intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, spinal and intrasternal.

As used herein, the terms "systemic administration," systemically administered, "peripheral administration, and" peripherally administered, e.g., subcutaneously, mean administering a compound, drug, or other agent by any means other than directly to the central nervous system, so that it enters the body. patient, and was thus subjected to metabolism and other similar processes.

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The term "pharmaceutically acceptable carrier is known in the art and includes a pharmaceutically acceptable agent, composition or vehicle suitable for administration to mammals. Carriers include liquid or solid fillers, diluents, excipients, solvents, or encapsulating agents for carrying or transporting an agent from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable, that is, compatible with the other ingredients of the formulation and not injurious to the patient.

As used herein, the term "human TNF? (Represented herein by the abbreviation hTNF? Or simply hTNF) refers to a human cytokine that exists as a 17 kD secreted and 26 kD membrane-bound form, the biologically active form of which consists of a trimer of non-covalently linked molecules 17. kD. The structure of hTNFa is described in more detail e.g. in Pennica, D., et al. (1984) Nature 312: 724729; Davis, JM, et al. (1987) Biochemistry 26: 1322-1326; and Jones, EY, et al. (1989) Nature 338: 225228. The term "human TNFα" includes recombinant human TNFα (rhTNFα), which can be produced by standard recombinant expression methods or purchased by purchase (R&D Systems, Catalog No. 210-TA, Minneapolis, MN).

The term "antibody as used herein" refers to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains, and two light (L) chains linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), alternating with regions of less variability, termed framework regions (FRs). Each VH and VL consists of three CDR regions and four FR regions arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. For example, an antibody may have CDR1, CDR2, and CDR3 sequences, such as those set forth in US Patent Nos. 6,090,382 and 6,258,562, incorporated herein by reference.

The term "antigen-binding portion of an antibody (or simply" antibody portion) as used herein "refers to one or more antibody fragments that retain the ability to specifically bind to an antigen (e.g., hTNFa). It has been shown that fragments of full-length antibodies can act as antibodies by antigen-binding. Examples of binding fragments encompassed by the term "antigen-binding portion of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F (ab ') 2 fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) the Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of single-armed antibodies, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546) which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Moreover, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked, using recombinant methods, by a synthetic linking group that allows them to arise as a single protein chain in which the VL and VH regions join pairing to form a monovalent molecule (known as single chain Fv (scFv), see, e.g., Bird et al (1988) Science 242: 423-426; and Huston et al (1988) Proc. Natl. Acad. Sci. USA 85: 58795883). The term "antigen-binding portion of an antibody" also includes such single chain antibodies. It also includes other forms of single chain antibodies, such as dual specificity antibody fragments (diabodes). Dual specificity antibody fragments are bivalent, dual-antigen-specific antibodies whose VH and VL domains are expressed on a single polypeptide chain with a linking group too short to allow the joining of the two domains of the same chain, causing the domains to join with the complementary ones. domains of other chains, resulting in the formation of two antigen binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). An exemplary antigen-binding fragment is described in US Patent Nos. 6,090,382 and 6,258,562, incorporated herein by reference.

In addition, the antibody, or antigen binding portion thereof, may be part of a larger immunoadhesive molecule, formed by covalently or non-covalently binding the antibody or antibody portion to one or more other proteins or peptides. Examples of such immunoadhesive molecules include the use of the streptavidin core region to generate a tetrameric scFv molecule (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6: 93-101) and the use of a cysteine residue, a peptide marker and a C-terminal poly-histidine tag to generate a bivalent and biotinylated scFv molecule (Kipriyanov, SM, et al. (1994) Mol. Immunol. 3J. ,: 1047-1058). Antibody fragment such as Fab and F (ab ') 2 fragments can be prepared from whole antibodies using conventional techniques, such as digestion of whole antibodies with papain or pepsin. In addition, antibodies, antibody fragments, and immunoadhesive molecules can be obtained using standard recombinant DNA techniques as described.

The term "human antibody," as used herein, includes antibodies with variable and constant regions derived from human germ cell immunoglobulin sequences. Human antibodies of the invention may include amino acid residues not encoded by human germ cell immunoglobulin sequences (e.g., by random or directed mutations in vitro or by somatic mutations in vivo), e.g. in CDRs, and in particular in CDR3. However, as used herein, the term "human antibody does not include antibodies in which CDR sequences derived from the germ cells of other mammalian species, such as a mouse, have been grafted into human framework sequences.

As used herein, the term "recombinant human antibody includes all human antibodies prepared, expressed, produced, or isolated by recombinant methods, such as those expressed using a recombinant expression vector that has been transfected into host cells (as further detailed below in Chapter II). antibodies isolated from a combinatorial library of recombinant human antibodies (as more fully described below in Chapter III), antibodies isolated from an animal (e.g., mouse) that are transgenic for human immunoglobulin genes (see, e.g., Taylor, LD, et al. (1992) Nucl Acids Res. 20: 6287-6295), or antibodies prepared, expressed, produced, or isolated by any other means by assembling human immunoglobulin gene sequences together with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germ cell immunoglobulin sequences. In some embodiments, however, such recombinant human antibodies are mutagenized in vitro (or, using animals transgenic for human Ig sequences, in vivo somatic mutagenesis), and thus, the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, despite that they are derived from and are related to the VH and VL sequences of human germ cells, they may not naturally occur in vivo in the antibody repertoire of human germ cells.

As used herein, the term "isolated antibody refers to antibodies substantially free of other antibodies with distinct antigenic properties (e.g., an isolated antibody that binds specifically to hTNF? Being substantially free of antibodies that specifically bind to antigens other than hTNF?). An isolated antibody that binds specifically to hTNFa may, however, display cross-reactivity to other antigens, such as TNFa molecules from other species.

Moreover, an isolated antibody may be substantially free of other cellular substances and / or chemicals.

The term "neutralizing antibody," (or "antibody neutralizing hTNFa activity) as used herein, refers to antibodies whose binding to hTNFa results in inhibition of hTNFa biological activity. Inhibition of hTNF? Biological activity can be determined by measuring one or more indicators of hTNF? Biological activity, such as hTNF? Induced cytotoxicity (in vitro or in vivo), hTNF? Induced cell activation, and hTNF? Binding to hTNF? Receptors. These markers of hTNFa biological activity can be assessed by one or more of several standard in vitro or in vivo assays known in the art set forth in US Patent Nos. 6,090,382 and 6,258,562, incorporated herein by reference. Preferably, the ability of the antibody to neutralize hTNF? Activity is determined by inhibiting hTNF? Induced cytotoxicity on L929 cells. As an additional or alternative parameter of hTNFα activity, the ability of the antibody to inhibit hTNFα-induced ELAM-1 expression on HUVEC can be determined as an indicator of hTNFα-induced cell activation.

As used herein, the term "surface plasmon resonance technique" refers to an optical phenomenon that enables real-time analysis of specific biological interactions by detecting changes in protein concentration in a biological sensor array, e.g.

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BIAcore (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ). For a more detailed description, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51: 19-26; Jonsson, U., et al. (1991) Biotechniques 11: 620-627; Johnsson, B., et al. (1995) J. Mol Recognit. 8: 125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198: 268-277.

The term "Koff" as used herein means the dissociation constant of an antibody from an antibody / antigen complex.

As used herein, the term "Kd" refers to the dissociation constant of a particular antibody-antigen interaction.

II. Antibodies of the preparation

The invention relates to a liquid pharmaceutical preparation in the form of an aqueous solution containing the antibodies in a therapeutically effective amount in a buffer solution causing the pH of the preparation to be between about 4 and about 8, and the entire preparation may have an extended shelf life, preferably at least about 18 months. The liquid pharmaceutical formulation in the form of an aqueous solution according to the invention may have increased stability. Moreover, the formulation is not sensitive to light. The formulation according to the invention may remain stable after at least 3 freeze / thaw cycles. Furthermore, the pharmaceutical formulation according to the invention may be suitable for single use as a subcutaneous injection.

Antibodies that can be used in the preparation include polyclonal, monoclonal, recombinant, single chain antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, or fragments thereof. Antibody-like molecules having one or two antigen binding sites and an immunoglobulin Fc fragment can also be used. An example of an antibody-like particle is the active ingredient etanercept or infliximab. The preferred antibodies for use in the preparation are human antibodies which are cloned from human cells or from gene archives constituting the human antibody stock. Among human antibodies, antibodies directed against a TNF? Antigen, including human TNF? (Or hTNF?), Are especially preferred.

Optionally, the formulation can contain a combination of antibodies (two or more) or an "antibody cocktail." For example, such a preparation can include the D2E7 antibody and one or more accessory antibodies.

The antibody, or antigen binding portion thereof, can dissociate from human TNF? With a Kd constant of 1 x 10-8 m or less and a Koff constant of 1 x 10-3 s-1 or less, with both constants determined by surface plasmon resonance, and canceling the cytotoxicity of human TNF? in standard in vitro L929 assay with an IC50 of 1 x 10-7 M or less. The antibody, or antigen binding portion thereof, may be as set forth in US Patent Nos. 6,090,382 and 6,258,562, incorporated herein by reference.

In one aspect, a formulation of the invention comprises D2E7 antibodies and antibody fragments. It may also contain D2E7 related antibodies and antibody fragments, and other human antibodies and antibody fragments with properties corresponding to D2E7 such as high affinity binding to hTNFa with slow dissociation kinetics and high neutralization capacity. The human antibody, or antigen binding portion thereof, can dissociate from human TNF? With a Kd constant of 1 x 10-8 M or less and a Koff constant of 1 x 10-3 s-1 or less as determined by surface plasmon resonance techniques, and canceled the cytotoxicity of human TNFα in standard in vitro L929 assay with an IC50 of 1 x 10-7 M or less. More preferably, the isolated human antibody, or antigen binding portion thereof, dissociates from

-4 -1 human TNF? With a Koff constant of 5 x 10-4 s-1 or less, or even more preferably, with a Koff constant of -4-1 of 1 x 10-4 s-1 or less. More preferably, the isolated human antibody, or antigen binding portion thereof, abolishes the cytotoxicity of human TNF? In a standard in vitro L929 assay with an IC50 of 1 x 10-8 M or less, and even more preferably, with an IC50 of 1 x 10-9 M or -10 less, and even more preferably with an IC50 of 5 x 10-10 M or less. The preparation may contain an antibody that is an isolated recombinant human antibody, or an antigen-binding portion thereof. Preferably, the antibody also neutralizes TNF? Induced cell activation as assessed using a standard assay for detecting ELAM-1 expression on human umbilical vein endothelial cells (HUVEC) induced by TNF? In vitro.

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III. Preparation of the preparation

The invention relates to antibody formulations with superior properties compared to formulations known in the art. For example, the formulations of the invention have a longer shelf life and / or greater shelf life than those formulated in the art. In a preferred aspect, the preparations according to the invention contain the protein at a high concentration, e.g. a protein concentration of greater than about 45 mg / ml, a protein concentration of greater than about 50 mg / ml, a protein concentration of greater than about 100 mg / ml, or a protein concentration of greater than about 150 mg / ml. Antibodies constitute the protein in the preparation. In a further preferred embodiment, the antibody is D2E7 antibody. The invention also relates to an aqueous pharmaceutical composition comprising a polyol, a surfactant and a buffering system containing citrate and / or phosphate at a pH of about 4 to 8, in an amount sufficient to produce an antibody for therapeutic use at a concentration greater than about, e.g., 45 mg / ml.

Production of the given antibody is performed by standard methods known in the art. In a preferred embodiment of the invention, the antibody used in the preparation is expressed in CHO cells and then purified by a series of standard chromatography steps. In a further preferred embodiment, the antibody is directed against hTNFa and is prepared according to the methods set forth in US Patent Nos. 6,090,382 and 6,258,562, incorporated herein by reference.

After producing a given antibody, a pharmaceutical preparation containing that antibody is prepared. The therapeutically effective amount of the antibody present in a formulation is determined, e.g., with the desired dose volume and the mode (s) of administration. In one embodiment of the invention, the concentration of the antibody in the preparation is between about 1 to about 150 mg of the antibody per ml of the liquid preparation. Thus, the concentration of the antibody in the preparation may be between about 5 to about 80 mg per ml, preferably between about 25 to about 50 mg / ml. The preparation is particularly suitable for high doses of antibodies exceeding 15 mg / ml. In a preferred embodiment, the antibody concentration is 50 mg / ml.

Thus, the antibody concentration in the formulation may be about 1-150 mg / ml, about 5-145 mg / ml, about 10-140 mg / ml, about 15-135 mg / ml, about 20-130 mg / ml, about 25- 125 mg / ml, about 30-120 mg / ml, about 35-115 mg / ml, about 40-110 mg / ml, about 45-105 mg / ml, about 50-100 mg / ml, about 55-95 mg / ml, about 60-90 mg / ml, about 65-85 mg / ml, about 70-80 mg / ml, or about 75 mg / ml. The invention also includes the intermediate ranges of the stated concentrations, e.g., about 6-144 mg / ml. For example, the invention encompasses any range of values that is a combination of any of the above values as an upper and / or lower limit.

In one embodiment, the invention relates to a formulation with an extended shelf life comprising an antibody as an active ingredient in combination with mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, Polysorbate 80, water, and sodium hydroxide. The formulation according to the invention may have an extended shelf life in the liquid state of at least about 18 months. Freezing the formulation according to the invention can also be used to further extend the shelf life.

An aqueous solution formulation is made by placing the antibody in a buffer solution. The buffer of the invention maintains the pH in the range of from about 4 to about 8, preferably from about 4.5 to about 6.0, more preferably from about 4.8 to about 5.5, and most preferably, maintains the pH between about 5.0 to about 6.0. about 5.2. Ranges intermediate to the pH values mentioned are also within the scope of the invention. For example, the invention also encompasses value ranges that use a combination of any of the above-mentioned values as upper and / or lower limit. Examples of buffers that adjust the pH in this range include acetate (e.g., sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate, and other organic acid buffers.

In a preferred embodiment of the invention, the formulation comprises a buffering system comprised of citrate and phosphate to maintain the pH in the range of about 4 to about 8. In a further preferred embodiment, the pH range is from about 4.5 to about 6.0, more preferably from about pH 4.8 to about 5.5, and most preferably from about 5.0 to about 5.2. In a further preferred embodiment, the buffering system comprises citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and / or sodium dihydrogen phosphate dihydrate. In a further preferred embodiment, the buffer system comprises about 1.3 mg / ml citric acid (e.g. 1.305 mg / ml), about 0.3 mg / ml sodium citrate (e.g. 0.305 mg / ml), about 1.5 mg / ml. mL disodium phosphate dihydrate (e.g. 1.53 mg / mL), approximately 0.9 mg / mL sodium dihydrogen phosphate dihydrate (e.g. 0.86), and approximately 6.2 mg / mL sodium chloride (e.g. 6.165 mg / mL) . In other preferred embodiments, the buffer12 system

The solution contains 1-1.5 mg / ml of citric acid, 0.25 to 0.5 mg / ml of sodium citrate, 1.25 to 1.75 mg / ml of disodium phosphate dihydrate, 0.7 to 1, 1 mg / ml sodium dihydrogen phosphate dihydrate and 6.0 to 6.4 mg / ml sodium chloride. In a further embodiment, the pH of the formulation is adjusted with sodium hydroxide.

Also included in the formulation is a polyol which acts as an osmolarity regulator and may stabilize the antibody. The polyol is added to the formulation in an amount that depends on the desired degree of isotonicity of the formulation. Preferably, the aqueous solution formulation is isotonic. The amount of polyol added may also depend on the molecular weight of the polyol. For example, a smaller amount of monosaccharide (e.g. mannitol) may be added compared to a disaccharide (such as trehalose). In a preferred embodiment of the invention, the polyol used in the formulation as osmotic agent is mannitol. In a preferred embodiment of the invention, the mannitol concentration is about 5 to 20 mg / ml. In a further preferred embodiment of the invention, the mannitol concentration is about 7.5 to 15 mg / ml. In a more preferred embodiment of the formulation according to the invention, the concentration of mannitol is about 10-14 mg / ml. In a most preferred embodiment, the mannitol concentration is about 12 mg / ml. In another embodiment of the invention, the polyol in the formulation is sorbitol.

A detergent or surfactant is also added to the antibody preparation. Exemplary detergents include nonionic detergents such as polysorbates (e.g., Polysorbate 20, 80, etc.) or poloxamers (e.g., Poloxamer 188). The amount of detergent is added to reduce antibody aggregation and / or minimize particle formation in the formulation and / or reduce adsorption. In a preferred embodiment of the invention, the formulation comprises a surfactant which is a polysorbate. In a further preferred embodiment of the invention, the formulation comprises a detergent which is Polysorbate 80 or Tween 80. Tween 80 is the term used for polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th edition, 1996). In a preferred embodiment, the formulation contains between about 0.1 and about 10 mg / ml of Polysorbate 80, more preferably between about 0.5 and about 5 mg / ml. Alternatively, approximately 0.1% of Polysorbate 80 is present in the formulation of the invention.

The formulation may be a 0.8 ml vial solution containing the ingredients shown in Table 1, below.

Table 1

One vial of 0.8 ml solution for injection1) includes:

<td>Ingredient</td><td>Quantity</td><td>Function</td>

<td>active ingredient: antibody (D2E7) 2)</td><td>40.0 mg</td><td>the active substance</td>

<td>excipients:</td><td></td><td></td>

<td>mannitol</td><td>9.6 mg</td><td>osmotic agent</td>

<td>citric acid monohydrate citric acid</td><td>1.044 mg</td><td>buffer</td>

<td>sodium citrate sodium citrate</td><td>0.244 mg</td><td>buffer</td>

<td>disodium phosphate dihydrate dibasic sodium phosphate dihydrate</td><td>1.224 mg</td><td>buffer</td>

<td>Sodium dihydrogen phosphate dihydrate, monobasic sodium phosphate dihydrate</td><td>0.688 mg</td><td>buffer</td>

<td>sodium chloride</td><td>4.932 mg</td><td>osmotic agent</td>

<td>Polysorbate 80</td><td>0.8 mg</td><td>detergent</td>

<td>water for injection</td><td> 759,028-</td><td>solvent</td>

<td>water for injection</td><td>759.048 mg</td><td></td>

<td>sodium hydroxide3)</td><td>0.02-0.04 mg</td><td>pH adjustment</td>

<td>together</td><td>817.6 mg</td><td></td>

1) Solution density: 1.022 g / ml

2) It is used as a concentrate

3) Added as a 1M solution

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The formulation contains the substances listed above (i.e. antibody, buffer, polyol and detergent) and is substantially free of one or more preservatives such as benzyl alcohol, phenol, m-cresol, chlorobutanol and benzethonium chloride. Optionally, the formulation may contain a preservative, especially when the formulation is a multi-dose formulation. The formulation may contain one or more other pharmaceutically acceptable carriers, excipients or stabilizers such as those described in Remington.'s Pharmaceutical Sciences 16th Edition, Ed. Osol, A. (1980), provided that they do not significantly adversely affect the desired properties of the formulation. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include: additional buffers; co-solvents; antioxidants including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g. Zn-protein complexes); biodegradable polymers such as polyesters; and / or salt-forming counterions such as sodium.

As required by the particular condition to be treated, the present formulation may also be combined with one or more other therapeutic agents, preferably complementary, not detrimental to the antibodies of the formulation. Such therapeutic agents are formulated in an amount that is effective for its intended purpose. Additional therapeutic agents that may be combined with the formulation of the invention are described in more detail in US Patent Nos. 6,090,382 and 6,258,562, each of which is herein incorporated by reference.

Preparations intended for in vivo administration must be sterile. This is readily accomplished by filtration through sterilizing filter membranes before or after preparation of the formulation.

IV. Administration of the preparation

The formulation of the invention can be used with indications similar to those set forth in US Patent Nos. 6,090,382 and 6,258,562, both of which are hereby incorporated by reference, and detailed below.

The term "effective amount of a preparation" means an amount necessary or sufficient to inhibit TNF? Activity, e.g., prevent various morphological and somatic symptoms of a disorder caused by the deleterious effects of TNF ?. In another embodiment, the effective amount of the formulation is the amount necessary to obtain the desired effect. For example, an effective amount of a formulation is an amount sufficient to inhibit the deleterious activity of TNF ?. In another example, the effective amount of the formulation is 0.8 ml of a 40 mg antibody formulation as shown in Table 1. The effective amount may vary, depending upon factors such as the size and weight of the subject, or the type of disease. For example, the selection of a preparation that inhibits TNF? Activity can affect the "effective amount." Considering the above-mentioned factors, one skilled in the art can, without undue experimentation, determine an effective amount of a TNF? Inhibitory preparation.

The schedule of administration can influence the effective amount. The TNF? Inhibitory preparation can be administered to the subject either before or after the onset of the deleterious effects of TNF ?. In addition, several sub-doses as well as staggered doses may be administered daily or sequentially, or the dose may be continuously infused or injected as a bolus. Moreover, depending on the exigencies of the therapeutic or prophylactic situation, the doses of the TNF? Inhibitory preparation may be proportionally increased or decreased.

The terms "treated," treatment, or "method of treatment" mean the reduction or amelioration of at least one symptom associated with or caused by the condition, disorder or disease treated. For example, treatment can mean the reduction of one or more symptoms of the disorder, or the elimination of the disorder completely.

The actual dosage levels of the active ingredients (antibodies) in a pharmaceutical formulation of the invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic effect in a given patient, for a given composition and mode of administration, without being toxic to the patient.

The dosage level selected will depend on a variety of factors including the activity of the antibodies in the formulation, route of administration, time of administration, rate of excretion of the particular compound employed, duration of treatment, other drugs, compounds, and / or substances used in conjunction with the compound, age, the sex, weight, condition, general health, and medical history of the treated patient, and similar factors well known in the art.

A physician or veterinarian of average experience can readily determine and prescribe the required effective amount of the pharmaceutical formulation of the invention. For example, a doctor or veterinarian

The dosage of the compounds contained in the pharmaceutical formulation of the invention may be initiated at a dose lower than that necessary to achieve the desired therapeutic effect, and then gradually increased until the desired effect is achieved.

In general, a suitable daily dose of a formulation of the invention will be that amount of the formulation which is the lowest dose effective to produce a therapeutic effect. Such effective dose will generally depend on the factors described above. An effective amount of a formulation according to the invention is an amount which inhibits TNF? Activity in an individual suffering from a disorder wherein the TNF? Activity is s. harmful. In a preferred embodiment, the formulation provides an effective dose of the active ingredient of 40 mg of antibody per injection. In another embodiment, the formulation provides an effective dose of the antibody which ranges from about 1 to 150 mg. If necessary, an effective daily dose of the pharmaceutical preparation may be administered in two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally in unit dosage forms.

For example, the dose of antibodies in the formulation is between about 5 and about 80 mg. In another embodiment, the dose of the antibodies in the formulation is between about 25 and about 50 mg. The preparation is particularly suitable for use in high doses of the antibody exceeding 15 mg. In a preferred embodiment, the formulation of the invention provides antibodies in a dose of about 40 mg. In a most preferred embodiment, the antibody is D2E7.

In one embodiment of the invention, the dose of the antibody in the formulation is between about 1-150 mg, thus it can also be about 5-145 mg, about 10-140 mg, about 15-135 mg, about 20-130 mg, about 25-125 mg. , about 30-120 mg, about 35-115 mg, about 40-110 mg, about 45-105 mg, about 50-100 mg, about 55-95 mg, about 60-90 mg, about 65-85 mg, about 70-80 mg or about 75 mg. For example, the dose of the antibody is 40 mg. According to the invention, the antibody is directed against TNFα. In a most preferred embodiment, the antibody is D2E7. Intermediate ranges of the doses mentioned, e.g. about 2-149 mg, are also part of the invention. For example, the invention also includes ranges of values using a combination of any of the above-mentioned values as upper and / or lower limit.

It is worth noting that the size of the dose may vary depending on the severity of the disorder to be alleviated. Furthermore, it is to be understood that the particular dosing regimen for an individual subject should be adapted over time as needed and by the expert judgment of the person administering or supervising the administration of the composition, and that the dosage ranges provided herein are merely exemplary and should not be taken as limiting the scope or use of that composition. .

The invention provides a pharmaceutical formulation with an extended shelf life that, in one embodiment, is used to inhibit TNF? Activity in individuals suffering from a disorder wherein TNF? Activity is deleterious. This use consists in administering to an individual the antibody or antibody portion in a formulation of the invention so as to inhibit TNF? Activity in the individual. Preferably, the subject is a human. Alternatively, the subject may be a mammal that expresses TNFα with which an antibody of the invention cross-reacts. In addition, the subject may be a mammal into an organism with hTNFa (eg, by administration of hTNFa or by expressing the hTNFa transgene). The formulation of the invention can be administered to humans for therapeutic purposes (detailed below). In one embodiment of the invention, the liquid pharmaceutical preparation is easy to administer, and the preparation, e.g. is self-administered by the patient. In a preferred embodiment, the formulation according to the invention is administered by subcutaneous injection, preferably single injection. In addition, the formulation of the invention may be administered to a non-human mammal that expresses TNFα with which the antibody cross-reacts (e.g., in a primate, porcine, or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter application, such animal models may be useful for assessing the therapeutic efficacy of the antibodies in a formulation of the invention (e.g., in dose and timing testing).

The term "disorder in which TNFα activity is deleterious" as used herein includes diseases and other disorders in which the presence of TNFα in the subject suffering from the disorder has been shown or suspected to be responsible for the pathophysiology of the disorder or as a factor contributing to the aggravation of the disorder. Accordingly, a disorder in which TNF? Activity is deleterious is a disorder in which inhibition of TNF? Activity is expected to alleviate the symptoms and / or the progress of the disorder. This can be demonstrated, for example, by the observation of an increase in TNFα concentration in the physiological fluids of a subject suffering from a given disorder (e.g.

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TNF? In the serum, plasma, synovial fluid, etc. of a subject) which can be detected as described above, e.g. using an anti-TNF? Antibody.

There are numerous examples of disorders in which TNFα activity is detrimental. Examples of disorders in which TNFα activity is deleterious are set out in US Application No. 60/397275, incorporated herein by reference. Examples of the deleterious activity of TNFα are also shown in U.S. Patent Nos. 6,015,557, 6,177,077, 6,379,666, 6,419,934, 6,419,944, 6,423,321, and 6,428,787; US Application Nos. US2001 / 0016195, US2001 / 0004456, and US2001 / 026801; WO 00/50079 and WO 01/49321, each of which is incorporated herein by reference.

The use of the antibodies and antibody fragments of the invention in the treatment of specific disorders is discussed in more detail below:

A. Sepsis

Through biological actions including blood pressure lowering, myocardial inhibition, vascular hyperpermeability syndrome, organ necrosis, stimulation of the release of toxic secondary mediators, and activation of the coagulation cascade, tumor necrosis factor plays a specific role in the pathophysiology of sepsis (see, e.g., Tracey, KJ and Cerami, A. (1994) Annu. Rev. Med. 45: 491-503; Russell, D and Thompson, RC (1993) Curr. Opin. Biotech. 4: 714-721). Accordingly, the formulation of the invention can be used to treat sepsis in any clinical form, including septic shock, endotoxin shock, gram-negative sepsis, and toxic shock syndrome.

In addition, in the treatment of sepsis, the formulation of the present invention may be co-administered with one or more additional therapeutic agents, the presence of which may better ameliorate sepsis, including an interleukin-1 inhibitor (such as those described in PCT Publication No. WO 92/16221). and WO 92/17583), the cytokine interleukin-6 (see, e.g., PCT publication No. WO 93/11793), or a platelet activating factor antagonist (see, e.g., European Patent Application Publication No. EP 374510).

Moreover, in a preferred embodiment, the formulation of the invention can be administered to sepsis patients whose serum or plasma IL-6 concentration exceeds 500 pg / ml, and more preferably 1000 pg / ml during treatment (see PCT Publication No. WO 95/20978, p. Daum, L., et al.).

B. Autoimmune diseases

Tumor necrosis factor plays a role in the pathophysiology of a variety of autoimmune diseases. For example, TNF? Is believed to induce tissue inflammation and damage joints in rheumatoid arthritis (see, e.g., Tracey and Cerami, supra; Arend, WP and Dayer, JM. (1995) Arth. Rheum. 38: 151-160; Fava, RA, et al. (1993) Clin. Exp. Immunol. 94: 261-266). TNFα is also thought to cause pancreatic islet cell death and mediate insulin resistance in diabetes (see e.g. Tracey and Cerami, supra; PCT Publication No. WO 94/08609). TNF? Is also believed to mediate oligodendrocyte cytotoxicity and induction of inflammatory plaques in multiple sclerosis (see, e.g., Tracey and Cerami, supra). Chimeric and humanized murine anti-hTNFα antibodies have been studied in clinical trials for the treatment of rheumatoid arthritis (see, e.g., Elliott, MJ, et al. (1994) Lancet 344: 1125-1127; Elliot, MJ, et al. (1994) Lancet 344: 1105-1110; Rankin, EG, et al. (1995) Br. J. Rheumatol. 34: 334-342).

The preparation according to the invention can be used in the treatment of autoimmune diseases, in particular those related to inflammation, including rheumatoid arthritis, ankylosing spondylitis, osteoarthritis and gouty arthritis, allergy, multiple sclerosis, diabetes caused by autoimmune disease, autoimmune disease. uveitis and nephrotic syndrome. Typically, the formulation is administered systemically, however, in some disorders, local administration of the antibody or antibody portion at the site of inflammation may be beneficial (e.g., topical administration to joints in rheumatoid arthritis or topical administration to diabetic ulcers, alone or in combination. with an ylidene derivative of cyclohexane as described in PCT Publication No. WO 93/19751).

C. Infectious diseases

Tumor necrosis factor is believed to mediate the biological effects observed in a wide variety of infectious diseases. For example, TNF? Is believed to mediate encephalitis as well as capillary thrombosis and infarction in malaria (see, e.g., Tracey and Cerami, supra). TNFα is also believed to mediate encephalitis, induce damage to the blood-brain barrier, induce septic shock syndrome, and activate venous embolism in meningitis (see, e.g., Tracey and Cerami, supra). TNFa

It also induces cachexia, stimulates viral proliferation, and mediates central nervous system damage in acquired immune deficiency syndrome (AIDS) (see, e.g., Tracey and Cerami, supra). Accordingly, the antibodies and antibody fragments of the invention can be used in the treatment of infectious diseases, including bacterial meningitis (see e.g. European Patent Application Publication No. EP 585,705), cerebral complications in malaria, AIDS and AIDS related syndrome (ARC) (see, e.g., European Patent Application Publication No. EP 230574), as well as cytomegalovirus infection following transplantation (see, e.g., Fietze, E ., et al. (1994) Transplantation 58: 675-680). The formulation of the invention may also be used to relieve symptoms associated with infectious diseases, including fever and muscle pain due to infections (such as influenza) and cachexia due to infection (e.g., AIDS or ARC).

D. Transplantation

Tumor necrosis factor is assumed to be a key mediator of allograft rejection and graft versus host response (GVHD), and to mediate the deleterious response that has been observed when the rat antibody OKT3, directed against the CD3 receptor complex of T cells, is used to inhibit renal graft rejection (see e.g., Tracey and Cerami, supra; Eason, JD, et al. (1995) Transplantation 59: 300-305; Suthanthiran, M. and Strom, TB (1994) New Engl. J. Med. 331: 365-375). Accordingly, the formulation of the invention can be used to inhibit graft rejection, including allograft and heterogeneous graft rejection, and to inhibit GVHD. Although the antibody or antibody portion may be used alone, it is more preferred to use in combination with one or more other agents that suppress the immune response to allograft or inhibit GVHD. For example, in one embodiment, the formulation of the invention may be used in combination with OKT3 to inhibit reactions induced by OKT3. In another embodiment, a formulation of the invention can be used in combination with one or more antibodies to other target molecules involved in the regulation of the immune response, such as CD25 cell surface molecules (interleukin-α receptor-2), CD11a (LFA-1). , CD54 (ICAM-1), CD4, CD45, CD28 / CTLA4, CD80 (B7-1) and / or CD86 (B7-2). In yet another embodiment, the formulation of the invention can be used in combination with one or more general immunosuppressants such as cyclosporin A or FK506.

E. Malignant neoplasms

Tumor necrosis factor is believed to induce cachexia, stimulate tumor growth, increase the likelihood of metastasis, and play a role in cytotoxicity in malignant tumors (see, e.g., Tracey and Cerami, supra). Accordingly, the formulation according to the invention may be used in the treatment of malignant neoplasms, for inhibiting tumor growth or metastasis, and / or for ameliorating cachexia caused by a malignant tumor. The preparation can be administered systemically or locally to the tumor.

F. Pulmonary disorders

Tumor necrosis factor is believed to be involved in the pathophysiology of adult respiratory distress syndrome, including stimulation of leukocyte and endothelial cell activation, cytotoxicity to pneumocytes, and induction of vascular hyperpermeability syndrome (see, e.g., Tracey and Cerami, supra). Accordingly, the formulation of the invention can be used in the treatment of a variety of pulmonary disorders, including adult respiratory distress syndrome (see e.g. PCT Publication No. WO 91/04054), shock lung, chronic pulmonary inflammatory disease, pulmonary sarcoidosis, pulmonary fibrosis and silicosis. The preparation can be administered systemically or locally to the lungs, e.g. as an aerosol.

G. Intestinal disorders

Tumor necrosis factor is believed to be involved in the pathophysiology of inflammatory bowel disorders (see, e.g., Tracy, KJ, et al (1986) Science 234: 470-474; Sun, XM., Et al (1988) J. Clin. Invest. 81: 1328-1331; MacDonald, TT, et al. (1990) Clin. Exp. Immunol. 81; 301-305). For the treatment of Crohn's disease, chimeric murine anti-hTNFa antibodies (van Dullemen, HM, et al. (1995) Gastroenterology 109: 129-135) have been tested in clinical trials. The preparation according to the invention can also be used in the treatment of intestinal disorders, such as idiopathic inflammatory bowel diseases, among which we distinguish between two disease entities: Crohn's disease and ulcerative colitis.

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H. Cardiac disorders

The formulation according to the invention can also be used in the treatment of various cardiac disorders including cardiac ischemia (see e.g. European Patent Application Publication No. EP 453 898) and heart failure (myocardial weakness) (see e.g. PCT Publication No. WO 94/20139).

And other

The pharmaceutical preparation according to the invention can also be used in the treatment of various other disorders in which TNF? Activity is deleterious. Examples of other diseases and disorders in the pathophysiology mediated by TNF? Activity and thus treatable with the formulation of the invention include: bone inflammatory disorders and bone resorption diseases (see, e.g., Bertolini, DR, et al. (1986) Nature 319: 516-518; Konig, A., et al. (1988) J. Bone Miner. Res. 3: 621627; Lerner, UH and Ohlin, A. (1993) J. Bone Miner. Res. 8: 147-155; and Shankar, G. and Stern, PH (1993) Bone 14: 871-876), hepatitis, including alcoholic hepatitis (see, e.g., McClain, CJ and Cohen, DA (1989) Hepatology 9: 349-351; Felver , ME, et al (1990) Alcohol Clin Exp. Res. 14: 255259; and Hansen, J., et al. (1994) Hepatology 20: 461-474) and viral hepatitis (Sheron, N., et al (1991) J. Hepatol 12: 241-245 and Hussain, MJ, et al (1994) J. Clin Pathol. 47: 1112-1115), coagulation disorders (see, e.g., van der Poll, T., et al. (1990) N. Engl. J. Med. 322: 1622-1627; and van der Poll, T., et al. (1991) Prog. Clin. Biol. Res. 367: 55-60), burns (see, e.g., Giroir, BP, et al. (1994) Am.

J. Physiol 267: H118-124; and Liu, XS, et al. (1994) Burns 20: 40-44), tissue damage by reperfusion (see, e.g., Scales, WE, et al. (1994) Am. J. Physiol. 267: G1122-1127; Serrick, C., et al. (1994) Transplantation 58: 1158-1162; and Yao, YM, et al (1995) Resuscitation 29: 157-168), keloid formation (see, e.g., McCauley, RL, et al. (1992) J. Clin. Immunol. 12: 300-308), scar tissue formation; fever; periodontal disease; obesity and the harmful effects of radiation.

Other disorders in which TNFα activity is detrimental include, but are not limited to: Still's disease in adults, Alzheimer's disease, ankylosing spondylitis, asthma, cancer and cachexia, atherosclerosis, chronic atherosclerosis, chronic fatigue syndrome, liver failure, chronic liver failure, obstructive pulmonary disease, chronic obstructive pulmonary disease, congestive heart failure, dermatomyositis and polymyositis, diabetic macroangiopathy, endometriosis, periodic familial fever, fibrosis, hemodialysis, Jarisch-Herxheimer reaction, juvenile arthritis, Kawasaki syndrome, myelodysplastic syndrome, myocardial infarction, a disorder called panciaticular vulgaris, periodontitis, peripheral neuropathy, polyarthritis, polymyositis, progressive kidney failure, psoriasis, psoriatic arthritis, Rehen's syndrome sarcoidosis, scleroderma, spondyloarthropathies, Still's disease, stroke, therapy-related syndrome, therapy-induced inflammatory syndrome, inflammatory syndrome due to administration of IL-2, thoracic aortic aneurysm (TAAA) surgery, Vasulo-Behcet disease, yellow fever vaccine, type 1 diabetes, type 2 diabetes, neuropathic pain, sciatica, cerebral edema, spinal edema and / or around the spinal cord, vasculitis, Wegener's granulomatosis, temporal arteritis, myalgia rheumatoid type, Takayasu's arteritis, polyarteritis nodosa, microscopic polyangiitis, Churg-Strauss syndrome, Felta syndrome'ego, Sjogren's syndrome, mixed connective tissue disease, recurrent polychondritis, gout-like inflammation, denture loosening, autoimmune hepatitis, sclerosing cholangitis, acute pancreatitis, chronic pancreatitis, glomerulonephritis, post-streptococcal glomerulonephritis or post-streptococcal glomerulonephritis IgA nephropathy, rheumatic heart disease, cardiomyopathy, orchitis, pyoderma gangrenosum, multiple myeloma, TNF Receptor Periodic Syndrome [TRAPS], atherosclerosis, steroid-dependent giant cell arteritis, uveitis, and drug reactions.

The following examples, which should not be construed as limiting, illustrate the invention. The content of all references, pending patent applications, and published patent descriptions cited in this application are expressly incorporated herein by reference.

Examples

Example 1: Preparation of the formulation

The pharmaceutical preparation according to the invention was prepared according to the following protocol.

The substances used in the preparation include: mannitol, citric acid monohydrate (citric acid), sodium citrate, disodium phosphate dihydrate (sodium phosphate dihydrate dibasic), sodium dihydrogen phosphate dihydrate (sodium phosphate dihydrate monobasic), sodium chloride, polysorbate

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80, water for injection, sodium hydroxide, which was used as a 1M solution for pH control, and a protein concentrate (e.g., antibody concentrate).

Preparation of 20 l buffer (equivalent to 20.180 kg - solution density: 1.009 g / ml)

The ingredients were weighed as follows: 240.0 g of mannitol, 26.1 g of citric acid monohydrate, 6.1 g of sodium citrate, 30.6 g of disodium phosphate dihydrate, 17.2 g of sodium dihydrogen phosphate dihydrate, 123.3 g of sodium chloride, 20, 0 g of Polysorbate 80 and 19715.7 to 19716.1 g of water.

The sodium hydroxide solution was prepared by combining 40.0 g of sodium hydroxide with 1000.8 g of water for injection.

Then, a buffer was prepared by dissolving the following pre-weighed ingredients (described above) in approximately 90% water for injection: mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate, sodium chloride, and Polysorbate 80. The order of addition of the ingredients was found to be buffer was irrelevant, so it could be freely chosen.

After all buffer components had been added, the pH of the solution was adjusted with 1M sodium hydroxide which was prepared as described above. Finally, after adding sodium hydroxide, water was added. Thereafter, the buffer solution was filtered through a sterilized filter (hydrophilic polyvinylidene difluoride, 0.22 µm pore diameter) into a sterilized container. Filtration was performed in a sterile nitrogen environment.

Production of 40 l of the preparation (equivalent to 40.88 kg)

Then, the filtered buffer solution was added to the thawed and pooled antibody concentrate (active ingredient of pharmaceutical formulation) prepared in the following manner. Before starting the pharmaceutical preparation, the antibody (concentrate) was thawed in a water bath. 34.207 g of the antibody concentrate were used which for a protein concentrate of 60 mg protein / ml corresponds to 2.0 kg protein. The density of the concentrate was 1.0262 g / ml. Any protein concentrate can be used in the range from 25,655 to 37,316, which corresponds to a protein concentration in the protein concentrate of 55 to 80 mg / ml. The buffer was added with stirring until the final weight of the total solution was reached.

Thereafter, the formulation, containing all ingredients, was sterilized by filtration as described above, except that the formulation was filtered through two sterile 0.22 µm pore membrane filters. After sterilization, the formulation was packaged in either vials or prefilled syringes.

It is also appreciated in the art that weights and / or mass / volume ratios given herein can be converted into moles and / or molar concentrations using art-known molecular weights of the listed components. Weights exemplified herein (e.g. in grams or kilograms) correspond to the volumes stated (e.g. in buffer or pharmaceutical formulation). It is appreciated in the art that weights can be converted on a proportional basis for the various formulation volumes required. For example, 32L, 20L, 10L, 5L or 1L formulations contain 80%, 50%, 25%, 12.5% or 2.5%, by weight of examples exemplified herein, respectively.

Example 2:

Freeze-thaw studies

After selecting the buffer for the D2E7 antibody, the drug substance was formulated on the same template as the finished product.

The behavior of the drug substance containing the D2E7 antibody at a protein concentration of 63 mg / ml upon freezing and thawing was assessed by alternating the frozen state of the drug substance into the liquid state 3 times. Table N shows the results of an experiment evaluating the effects of three cycles of fast and slow freeze-thaw in the presence and absence of 0.1% Polysorbate 80 starting at -80 ° C or -30 ° C, respectively.

As shown in Table 2, the drug substance containing the D2E7 antibody can be alternately thawed and frozen at least 3 times without any deleterious effects on both chemical properties (cation exchange HPLC, HPLC exclusion chromatography, color, pH), physicochemical properties (particles below the limit of visibility, clarity) and for biological activity (in vitro TNF neutralization test). Table 2 also shows that the incorporation of Polysorbate 80 improves the physicochemical properties of the drug substance containing the D2E7 antibody as evidenced by the lower number of particles below the limit of visibility, regardless of whether a slow or fast freeze / thaw cycle was used (see shaded areas in Table 2).

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Table 2: Effect of freezing and thawing on drug substance containing D2E7 antibody with / without Polysorbate 80

<td>Criteria test</td><td>Polysorbate (0.1%)1)</td><td>Number of freeze / thaw cycles</td><td>-30 ° C slow defrosting in the refrigerator</td><td>Quick defrost -30 ° C in a water bath</td><td>-80 ° C slow defrosting in a refrigerator</td><td>Quick defrost -80 ° C in a water bath</td>

<td>clarity</td><td> -</td><td> 25,0</td><td> 22,5</td><td> 25,3</td><td> 25,8</td><td> 25,6</td>

<td>coloring</td><td> -</td><td><B9</td><td><B9</td><td><B9</td><td><B9</td><td><B9</td>

<td>Particles below</td><td> -</td><td> 42</td><td> 600</td><td> 303</td><td> 1891</td><td> 303</td>

<td>border</td><td></td><td> 2</td><td> 4</td><td> 5</td><td> 8</td><td> 0</td>

<td>visibility</td><td> +</td><td> 0</td><td> 5</td><td> 1</td><td> 0</td><td> 8</td>

<td>chromatography</td><td> -</td><td> 99,8</td><td> 99,8</td><td> 99,8</td><td> 99,8</td><td> 99,8</td>

<td>HPLC exclusions</td><td> +</td><td> 99,8</td><td> 99,8</td><td> 99,8</td><td> 99,8</td><td> 99,8</td>

<td>cation exchange</td><td> -</td><td> 87,1</td><td> 87,0</td><td> 87,2</td><td> 86,9</td><td> 86,9</td>

<td>Neutralization test</td><td> -</td><td> 118,0</td><td> 123,8</td><td> 118,0</td><td> 103,3</td><td> 120,5</td>

<td>TNF in vitro</td><td> +</td><td> 111,8</td><td> 96,2</td><td> 100,9</td><td> 96,7</td><td> 95,8</td>

1) + = preparation containing 0.1% Polysorbate 80;

- = preparation without 0.1% Polysorbate 80

Example 3: Tests on the proliferation of microorganisms

Tests were carried out to determine whether the preparation allowed for the multiplication of microorganisms. The results of these experiments showed that the preparation does not allow the multiplication of microorganisms if it is stored for 14 days at a temperature of 20-25 ° C. This result was obtained directly by sowing microorganisms (e.g. Staphylococous aureus, ATCC #: 6538P, Candida albicans, ATCC #: 10231, Aspergillus niger, ATCC #: 16404, Pseudomonas aeruginosa, ATCC #: 9027, ambient bacterial sample) at low concentration (NMT 100 colony forming units / ml) per a sterile preparation. Thereafter, the total multiplication of the microorganisms, e.g. changes in turbidity, is examined in the inoculated preparations. The lack of turbidity found in the inoculated containers after 14 days indicated a lack of multiplication. Moreover, no microorganisms could be isolated from the containers. Thus, it was concluded that the formulation did not allow the multiplication of the microorganisms under these conditions.

Literature references

All references and patents cited herein are hereby incorporated by reference in their entirety.

Equivalent solutions

Those skilled in the art will be able to recognize or ascertain, based solely on routine testing, of many equivalents to those described in the present invention. Such equivalent arrangements are intended to be included within the scope of the following claims.

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SEQUENCE LIST <110> Abbot Biotechnology Ltd <12 0> Liquid Aqueous Pharmaceutical Preparation <130? BBC-166 <140> 10/222140 <141> 2002-08-16 <160> 34 <170> FastSSQ for Windows version 4.0 <210> 1 <211> 107 <212> PRT <213> Dummy <220>

<223> mutant human antibody

<td>Asp</td><td>How much</td><td>Gin</td><td>Underworld</td><td>Thr</td><td>Gin</td><td>Cheese</td><td>Pro</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Ala Ser</td><td>Val</td><td>Gly</td>

<td>Asp</td><td>Arg</td><td>Val</td><td>Thr</td><td>How much</td><td>Thr</td><td>Cys</td><td>Arg</td><td>Ala</td><td>Cheese</td><td>Gin</td><td>Gly</td><td>How many Arg</td><td>Asn</td><td>Tyr</td>

<td rowspan="2">Tyr</td><td>Ala</td><td>Ala</td><td>Cheese</td><td>Thr</td><td>Leu</td><td>Gin</td><td>Cheese</td><td>Gly</td><td>Val</td><td>Pro</td><td>Cheese</td><td>Arg Phe</td><td>Cheese</td><td>Gly</td>

<td>Cheese</td><td rowspan="2">Gly</td><td>Cheese</td><td>Gly</td><td>Thr</td><td>Asp</td><td>Phe</td><td>Thr</td><td>Leu</td><td>Thr</td><td>How much</td><td>Sar</td><td>Leu cheese</td><td>Gin</td><td>Pro</td>

<td> <210> 2</td><td rowspan="4">artificial</td>

<td><213> sequence</td>

<td><220> <223> mutated</td><td>human antibody</td>

<td>Leu Arg Leu cheese 20</td><td>Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 25 30</td>

PL 212 934 B1

<td>Glu</td><td>Gly</td><td>Arg</td><td>Phe</td><td>Thr</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>Asn</td><td>Ala</td><td>Lys</td><td>Asn</td><td>Cheese</td><td>Leu</td><td>Tyr</td>

<td>Leu</td><td>Gin</td><td>Underworld</td><td>Asn</td><td>Cheese</td><td>Leu</td><td>Arg</td><td>Ala</td><td>Glu</td><td>Asp</td><td>Thr</td><td>Ala</td><td>Val</td><td>Tyr</td><td>Tyr</td><td>Cys</td>

<td>Ala</td><td>Lys</td><td>Val</td><td>Cheese</td><td>Tyr</td><td>Leu</td><td>Cheese</td><td>Thr</td><td>Ala</td><td>Cheese</td><td>Cheese</td><td>Leu</td><td>Asp</td><td>Tyr</td><td>Trp</td><td>Gly</td>

<td>Gin</td><td>Gly</td><td>Thr</td><td>Leu</td><td>Val</td><td>Thr</td><td>Val</td><td>Cheese</td><td>Cheese</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<210> 3 <211 = · 9 <212> PRT <213> Dummy <220>

<223> mutant human antibody <20>

<221> VARIANT <222> 9 <223> Xaa = Thr or Ala <4C0> 3

Gin Arg Tyr Asn Arg Ala Pro Tyr Xaa 1 5

<td><210 = 4 <211 = 12 <212> PRT <213> Sequence <220></td><td>artificial</td><td></td>

<td><223 = · mutant <220> <221> VARIANT <222> 12</td><td>human antibody</td><td></td>

<td><400 = 4 Val Ser Tyr Leu</td><td>Cheese Thr Ala Ser Ser</td><td>Leu Asp Xaa</td>

<td><210> 5 <211> 7 <212> PRT <213> Sequence</td><td>artificial</td><td></td>

<td><223> mutated</td><td>human antibody</td><td></td>

<210=· 6 <211> 17

PL 212 934 B1 <212> PRT <213> Dummy <220>

<223> mutant human antibody <400> 6

Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu 15 10 15

Gly <210> 7 <211> 11 <212> PRT <213> Dummy <220>

<223> mutant human <40Q> 7 antibody

Arg Ala Ser Gin Gly Ile ArgAsn Tyr Leu Ala

10 <210> 8 <211> 5 <212> PRT <213> Dummy <220>

<223> mutant human antibody <400> 8

Asp Tyr Ala Met His

5 <210> 9 <400> 9 000

<td> <213></td><td>Sequence</td><td>artificial</td>

<td> <223></td><td>mutated</td><td>human antibody</td>

Gin Lys Tyr Asn Ser Ala Pro Tyr Ala 1 5 <210> 12

PL 212 934 B1 <211 9 <212? PRT <213? Artificial sequence <220?

<223? mutant human antibody <400 12

Gin Lys Tyr Asn Aig Ala Pro Tyr Ala 1 5 <210? 13 <211? 9 <212? PRT <213? Artificial sequence <220?

<223? mutant human antibody <400 13

Gin Lys Tyr Gin Arg Ala Pro Tyr Thr 1 5 <210? 14 <211? 9 <212? PRT <213? Artificial sequence <220?

<223? mutant human antibody <400 14

Gin Lys Tyr Ser Ser Ala Pro Tyr Thr 1 5 <210? 15 <211? 9 <212? PRT <213? Artificial sequence <220?

<223? mutant human antibody <400 15

Gin Lys Tyr Asn Ser Ala Pro Tyr Thr 1 5

<td> <210? <211? <212? <213?</td><td>1S 9 PRT Sequence</td><td>artificial</td>

<td> <220? <223?</td><td>mutated</td><td>human antibody</td>

PL 212 934 B1 <400-16

Gin Lys Tyr Asn Arg Ala Pro Tyr Thr 1 5 <210> 17 <211 »9 <212» PRT <213> Artificial sequence <220 »<223» mutant human antibody <400 »17

Gin Lys Tyr Asn Ser Ala Pro Tyr Tyr 1 5 <210 »18 <211» 9 <212 »PRT <213» Artificial sequence <220 »<223> mutant human antibody <400» 18

Gin Lys Tyr Asn Ser Ala Pro Tyr Asn 1 5 <210 »19 <211» 9 <212 »PRT <213» Artificial sequence <220 »<223» mutant human antibody <400 »19

Gin Lys Tyr Thr Ser Ala Pro Tyr Thr 1 5 <210 »20 <211» 9 <212 »PRT <213» Artificial sequence <220 »<223» mutant human antibody <400 »20

Gin Lys Tyr Asn Arg Ala Pro Tyr Asn 1 5 <210 »21 <211» 9 <212 »PRT

Artificial sequence <220>

<223> mutant human antibody <400> 21

Gin Lys Tyr Asn Ser Ala Ala Tyr Ser 1 5 <210> 22 <211> 9 <212> PRT <213> Dummy <220>

<223> mutant human antibody <400> 22

Gin Gin Tyr Asn Ser Ala Pro Asp Thr 1 5 <210> 23 <211> 9 <212> PRT <213> Dummy <220>

<223> mutant human antibody <400> 23

Gin Lys Tyr Asn Ser Asp Pro Tyr Thr 1 5 <210> 24 <211> 9 <212> PRT <213> Dummy <220>

<223> mutant human antibody <400> 24

Gin Lys Tyr Ile Ser Ala Pro Tyr Thr 1 5 <210> 25 <211> 9 <212> PRT <213> Dummy <220>

<223> mutant human antibody <400> 25

Gin Lys Tyr Asn Arg Pro Eto Tyr Thr 1 5

PL 212 934 B1 <210 »26 <211» 9 <212 »PRT <213> Artificial sequence <220>

<223 »mutant human antibody <400» 26

Gin Arg Tyr Asn Arg Ala Pro Tyr Ala 1 5 <210 »27 <211» 12 <212 »PRT <213> Artificial sequence <220» <223> mutant human antibody <400 »27

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn 15 10 <210 »26 <211» 12 <212> PRT <213 »Artificial sequence <220» <223> mutant human antibody <400 »28

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Lys 15 10 <210 »29 <211» 12 <212 »PRT <213» Artificial sequence <220 »<223» mutant human antibody <400 »29

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Tyr 15 10

<td> <213»</td><td>Artificial sequence</td>

PL 212 934 B1 <2 '.3> mutant human antibody <400 thirty

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asp

<td> <210? <211? <212? <213?</td><td>31 12 PRT Artificial sequence</td>

<td> <220? <223?</td><td>mutant human antibody</td>

<400? 31

Ala Ser Tyr Leu Ser Thr Ser Phe Ser Leu Asp Tyr

<td> <210? <211? <212? <213?</td><td>32 12 PRT Artificial sequence</td>

<td> <220? <223?</td><td>mutant human antibody</td>

Ala Ser Tyr Leu Ser Thr Ser Ser Ser Sar Leu His Tyr

<td> <210? <211? <212? <213?</td><td>33 12 PRT Artificial sequence</td>

<td> <220? <223?</td><td>mutant human antibody</td>

Ala Ser Phe Leu Ser Thr Ser Ser Ser Leu Glu Tyr 1 5 10

<td> <210? <211? <212? <213?</td><td>34 12 PRT Artificial sequence</td>

<td> <220? <223?</td><td>mutant human antibody</td>

Ala Ser Tyr Leu Ser Thr Ala Ser Ser Leu Glu Tyr

138 members in 27 offices

This recordMember, by publication datePriority claim17 more offices are in the list below

Priority claims4

Priority claims
Document	Office	Kind	Date
22214002	United States of America	A	2002-08-16
22214002	United States of America	A	2002-08-16
10222140	–	–	–
US20020222140	–	–	–

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US2004033228A1	United States of America	A1	2004-02-19
CA2494756A1	Canada	A1	2004-02-26
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KR20050067140A	Republic of Korea	A	2005-06-30
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Pharmaceutical anti-tnf-alpha antibody formulation

Abstract

Term

PL 212 934 B1

PL 212 934 B1

PL 212 934 B1

PL 212 934 B1

PL 212 934 B1

PL 212 934 B1

PL 212 934 B1

PL 212 934 B1

PL 212 934 B1

PL 212 934 B1

PL 212 934 B1

PL 212 934 B1

Priority claims4

Members138

Events

Numbers

Titles2

Classification