HK1117076A - Stabile isotonic lyophilized protein formulation - Google Patents

Description

Stable isotonic lyophilized protein formulations

This application is a divisional application of the patent application having application number 200410030256.3, filed on 1996, 7/23, entitled "Stable isotonic lyophilized protein formulation".

Technical Field

The present invention relates to a lyophilized protein formulation. More particularly, the present invention relates to a stable lyophilized protein formulation which can be reconstituted with a diluent to produce a stable reconstituted formulation suitable for subcutaneous administration.

Prior Art

In the last decade, advances in biotechnology have made it possible to use recombinant DNA technology to produce a variety of proteins for pharmaceutical use. The formulation of proteins poses special problems, since they are larger and more complex than the commonly used organic and inorganic drugs (i.e. have multiple functional groups in addition to a complex three-dimensional structure). In order for a protein to be biologically active, the formulation must ensure the conformational integrity of at least the protein's amino acid core sequence, while at the same time protecting the protein's multiple functional groups from degradation. Pathways for protein degradation include chemical instability (i.e., any modification of a protein by bond formation or cleavage to produce a new chemical species) or physical instability (i.e., a change in the higher order structure of a protein). Chemical instability can be caused by deamidation, racemization, hydrolysis, oxidation, beta elimination, or disulfide exchange. Physical instability is caused by, for example, denaturation, aggregation, precipitation or adsorption. The three most common protein degradation pathways are protein aggregation, deamidation and oxidation (Cleland et al, clinical Reviews in Therapeutic Drug carriers Systems 10 (4): 307-377 (1993)).

Lyophilization is a commonly used technique for preserving proteins that removes water from the protein preparation of interest. The freeze-drying or lyophilization process involves freezing the material to be dried and then subliming the frozen material under vacuum to remove the ice or frozen solvent. Excipients may be included in the formulation prior to lyophilization to improve stability during lyophilization and/or to improve the stability of the lyophilized product upon storage (Pikal, M.Biopharm.3(9)26-30(1990) and Arakawa et al, pharm.Res.8 (3): 285-291 (1991)).

It is an object of the present invention to provide a lyophilized protein formulation that is stable upon storage and transport. It is also an object to provide a stable reconstituted protein formulation for subcutaneous administration. In certain instances, it is an object of the present invention to provide a multiple use formulation that is stable for at least the time of administration to a patient.

Summary of The Invention

The present invention is based on the discovery that stable lyophilized protein formulations can be prepared using a lyoprotectant (1yoprotectant), preferably a saccharide such as sucrose or trehalose, which can be reconstituted to form stable reconstituted formulations having a protein concentration that is much higher (e.g., about 2-40 times higher, preferably 3-10 times higher, and more preferably 3-6 times higher) than the protein concentration in the pre-lyophilized formulation. Specifically, if the protein concentration in the formulation before lyophilization is 5mg/mL or less, the protein concentration in the reconstituted formulation is typically 50mg/mL or more. Such high protein concentrations in reconstituted formulations are believed to be particularly useful when the formulations are intended for subcutaneous administration. Despite the very high protein concentration in the reconstituted formulation, the reconstituted formulation was found to be stable (i.e., the protein did not exhibit significant or unacceptable chemical or physical instability) at 2-8 ℃ for about at least 30 days. In some examples, the reconstituted formulation is isotonic. Although lower concentrations of lyoprotectant are used to achieve such an isotonic formulation upon reconstitution, it has been found that the proteins in the lyophilized formulation substantially retain their physical and chemical stability and integrity upon lyophilization and storage.

When reconstituted with a diluent comprising a preservative (e.g. bacteriostatic agent for injection, BWFI), the reconstituted formulation may be used as a multiple use formulation. Such formulations are useful, for example, when the patient requires frequent subcutaneous protein administration to treat chronic conditions. The advantage of a multiple use formulation is that it can be more easily used by the patient, waste can be reduced by using the entire contents of the vial, and significant cost savings can be made to the manufacturer since several doses are contained in one vial (reducing filling and shipping costs).

In accordance with the discoveries described herein, one aspect of the invention is to provide a stable, isotonic reconstituted formulation comprising a protein in an amount of at least about 50mg/mL and a diluent, the reconstituted formulation prepared from a lyophilized mixture of the protein and a lyoprotectant, wherein the concentration of the protein in the reconstituted formulation is about 2-40 times greater than the concentration of the protein in the mixture prior to lyophilization.

In another example, the invention provides a stable reconstituted formulation comprising an antibody in an amount of at least about 50mg/mL and a diluent, the reconstituted formulation prepared from a lyophilized mixture of the antibody and a lyoprotectant, wherein the concentration of the antibody in the reconstituted formulation is about 2-40 times greater than the concentration of the antibody in the mixture prior to lyophilization.

The lyoprotectant to protein ratio in the lyophilized formulation of the preceding paragraph is related to, for example, the protein selected and the lyoprotectant and the desired protein concentration and isotonicity in the reconstituted formulation. In the preparation of an isotonic reconstituted formulation with high protein concentration using full-length antibody (as protein) and trehalose or sucrose (as lyoprotectant), the ratio may be, for example, about 100-1500 moles of trehalose or sucrose per 1 mole of antibody.

Typically, the pre-lyophilized formulation of protein and lyoprotectant (pre-lyophilized) may also include a buffer to provide the formulation with a suitable pH based on the protein of the formulation. For this purpose, it was found to be advantageous to use a histidine buffer as described below, which exhibits lyoprotectant properties.

The formulation may also include a surfactant (e.g., a polysorbate), which has been found to reduce aggregation of the reconstituted protein and/or to reduce particle formation in the reconstituted formulation. The surfactant may be added to the pre-lyophilized formulation, the lyophilized formulation and/or the reconstituted formulation (but preferably the pre-lyophilized formulation) as desired.

The present invention also provides a method of preparing a stable isotonic reconstituted formulation comprising reconstituting a lyophilized mixture of a protein and a lyoprotectant in a diluent such that the protein concentration in the reconstituted formulation is at least 50mg/mL, wherein the protein concentration in the reconstituted formulation is about 2-40 times greater than the protein concentration in the mixture prior to lyophilization.

In yet another embodiment, the present invention provides a method of preparing a formulation comprising: (a) lyophilizing a mixture of a protein and a lyoprotectant; and (b) reconstituting the mixture of step (a) in a diluent such that the reconstituted formulation is isotonic stable and has a protein concentration of at least about 50 mg/mL. For example, the protein concentration in the reconstituted formulation may be about 80mg/mL to about 300 mg/mL. Typically, the protein concentration in the reconstituted formulation is about 2-40 times higher than the protein concentration in the mixture prior to lyophilization.

The present invention also provides a product comprising: (a) a container containing a lyophilized mixture of a protein and a lyoprotectant; and (b) instructions for reconstituting the lyophilized mixture in a diluent to a protein concentration of at least about 50mg/mL in the reconstituted formulation. The product also includes another container containing a diluent, such as bacteriostatic for injection (BWFI) including aromatic alcohol.

The invention also provides a method of treating a mammal comprising administering to the mammal a therapeutically effective amount of the reconstituted formulation disclosed herein, wherein the mammal has a disease requiring treatment with the protein in the formulation. For example, the formulation may be administered subcutaneously.

A useful pre-lyophilized formulation of an anti-HER 2 antibody was found in the examples detailed below, which included anti-HER 2 in an amount of about 5-40mg/mL (e.g., 20-30mg/mL) and sucrose or trehalose in an amount of about 10-100mN (e.g., 40-80mM), a buffer (e.g., histidine, pH6 or succinate buffer, pH5) and a surfactant (e.g., polysorbate). The lyophilized formulation was found to be stable for at least 3 months at 40 ℃ and at least 6 months at 30 ℃. The anti-HER 2 formulation is reconstituted with a diluent to form a formulation suitable for intravenous administration comprising anti-HER 2 in an amount of about 10-30mg/mL and is stable for at least about 30 days at 2-8 ℃. When higher concentrations of the anti-HER 2 antibody are desired (e.g., when subcutaneous delivery of the antibody is the mode of administration to a patient), the lyophilized formulation can be reconstituted to produce a reconstituted formulation having a stable protein concentration of 50mg/mL or more.

One desirable formulation of an anti-IgE antibody found herein, prior to lyophilization, comprises an anti-IgE in an amount from about 5 to about 40mg/mL (e.g., 20 to 30mg/mL) and sucrose or trehalose in an amount from about 60 to about 300mM (e.g., 80 to 300mM or 80 to 170mM), a buffer (preferably histidine, pH6), and a surfactant (e.g., polysorbate). Lyophilized anti-IgE may remain stable for at least 1 year at 30 ℃. The formulation is reconstituted to produce a formulation having an anti-IgE content of about 15-45mg/mL (e.g., 15-25mg/mL), which is suitable for intravenous administration and is stable for at least 1 year at 2-8 ℃. Alternatively, where higher concentrations of anti-IgE are desired in the formulation, the lyophilized formulation may be reconstituted to produce a formulation with an anti-IgE concentration of 50mg/mL or more.

The present invention provides a stable isotonic reconstituted formulation comprising a protein in an amount of at least about 50mg/mL and a diluent, the reconstituted formulation being prepared from a lyophilized mixture of the protein and a lyoprotectant, wherein the protein concentration in the reconstituted formulation is about 2-40 times greater than the protein concentration in the mixture prior to lyophilization, preferably the lyoprotectant is sucrose or trehalose, preferably the formulation further comprises a buffer, more preferably the buffer is histidine or succinate buffer, preferably the formulation further comprises a surfactant.

The invention also provides the use of the above formulation for the manufacture of a medicament for the treatment of a mammal suffering from a condition for which treatment with a protein in the formulation is desired, preferably for subcutaneous administration.

The present invention also provides a stable reconstituted formulation comprising an antibody in an amount of at least about 50mg/mL and a diluent, the reconstituted formulation being prepared from a lyophilized mixture of the antibody and a lyoprotectant, wherein the concentration of the antibody in the reconstituted formulation is about 2-40 times greater than the concentration of the antibody in the mixture prior to lyophilization, preferably wherein the antibody in the formulation is an anti-IgE antibody or an anti-HER 2 antibody. Preferably the formulation is isotonic.

The present invention also provides a method of preparing a stable reconstituted formulation comprising reconstituting a mixture of a protein and a lyoprotectant in a diluent such that the protein concentration in the reconstituted formulation is at least about 50mg/mL, wherein the protein concentration in the reconstituted formulation is about 2-40 times greater than the protein concentration in the mixture prior to lyophilization.

The invention also provides a method of preparing a formulation comprising the steps of:

(a) freeze-drying the mixture of protein and lyoprotectant; and

(b) reconstituting the lyophilized mixture of step (a) in a diluent such that the reconstituted formulation is isotonic, stable, and has a protein concentration of at least about 50 mg/mL. Preferably, the method comprises a concentration of protein in the reconstituted formulation of from about 80mg/mL to about 300mg/mL, preferably the concentration of protein in the reconstituted formulation is about 2-40 times greater than the concentration of protein in the mixture prior to lyophilization, and preferably the lyophilization is performed while maintaining a shelf temperature of about 15-30 ℃ throughout the lyophilization process.

The invention also provides a product comprising:

(a) a container containing a lyophilized mixture of a protein and a lyoprotectant; and

(b) instructions for reconstituting the lyophilized mixture in a diluent to a protein concentration of at least about 50mg/mL in the reconstituted formulation.

Preferably, the product further comprises another container filled with a diluent, and preferably, the diluent in the product is bacteriostatic agent for injection BWFI containing aromatic alcohol.

The invention also provides a formulation comprising a lyophilized mixture of a lyoprotectant and a monoclonal antibody, wherein the molar ratio of lyoprotectant to monoclonal antibody is about 100 to 1500 moles lyoprotectant to 1 mole antibody.

The invention also provides a formulation comprising an anti-HER 2 antibody in an amount of about 5-40mg/mL, sucrose or trehalose in an amount of about 10-100mM, a buffer and a surfactant, preferably further comprising a bulking agent. Preferably, the formulation is lyophilized and stable at 30 ℃ for at least 6 months, and further preferably the formulation is reconstituted with a diluent such that the concentration of antibody in the reconstituted formulation is about 10-30mg/mL, wherein the reconstituted formulation is stable at 2-8 ℃ for at least about 30 days.

The present invention also provides a formulation comprising an anti-IgE antibody in an amount of about 5-40mg/mL, sucrose or trehalose in an amount of about 80-300mM, a buffer and a surfactant, preferably the formulation is lyophilized and stable at 30 ℃ for at least 1 year.

The present invention relates to:

1. a stable isotonic reconstituted formulation comprising a protein in an amount of at least 50mg/mL and a diluent, the reconstituted formulation being prepared from a lyophilized mixture of the protein and a lyoprotectant wherein the molar ratio of lyoprotectant to protein in the mixture is 100 to 600 moles lyoprotectant: 1 molar protein, wherein the protein concentration in the reconstituted formulation is 2-40 times higher than the protein concentration in the mixture prior to lyophilization.

2. The formulation of claim 1, wherein the lyoprotectant is sucrose or trehalose.

3. The formulation of claim 1, further comprising a buffer.

4. The formulation of claim 3, wherein the buffer is histidine or succinate.

5. The formulation of any one of claims 1-4, further comprising a surfactant.

6. A stable reconstituted formulation comprising an antibody in an amount of at least 50mg/mL and a diluent, the reconstituted formulation being prepared from a lyophilized mixture of the antibody and a lyoprotectant, wherein the molar ratio of lyoprotectant to antibody in the mixture is 100-600 mol lyoprotectant: 1 mol antibody, and wherein the concentration of antibody in the reconstituted formulation is 2-40 times higher than the concentration of antibody in the mixture prior to lyophilization.

7. The formulation of claim 6, wherein the antibody is an anti-IgE antibody or an anti-HER 2 antibody.

8. The formulation of claim 6 or7, which is isotonic.

9. A method of preparing a formulation comprising the steps of:

(a) freeze-drying a mixture of a protein and a lyoprotectant, wherein the molar ratio of lyoprotectant to protein in the mixture is 100-600 mol lyoprotectant: 1 mol protein; and

(b) reconstituting the lyophilized mixture of step (a) in a diluent such that the reconstituted formulation is isotonic, stable, and has a protein concentration of at least 50 mg/mL.

10. The method of 9, wherein the concentration of protein in the reconstituted formulation is 2-40 times greater than the concentration of protein in the mixture prior to lyophilization.

11. The method of 10, wherein the protein concentration in the reconstituted formulation is 80mg/mL to 300 mg/mL.

12. The method of 10 or 11, wherein the concentration of protein in the reconstituted formulation is 2-40 times higher than the concentration of protein in the mixture prior to lyophilization.

13. The method according to 10, wherein the freeze-drying is performed at a shelf temperature maintained at 15-30 ℃ throughout the freeze-drying process.

14. A product, comprising:

(a) a container containing a lyoprotectant mixture comprising a protein and a lyoprotectant, wherein the mixture comprises 100 moles lyoprotectant to protein and 600 moles lyoprotectant: 1 mole of protein; and

(b) instructions for reconstituting the lyophilized mixture in a diluent to a protein concentration of at least 50mg/mL in the reconstituted formulation.

15. The product of claim 14, further comprising another container containing a diluent.

16. The product of claim 15, wherein the diluent is an injectable bacteriostatic agent comprising an aromatic alcohol.

17. A formulation comprising a lyophilized mixture of a lyoprotectant and an antibody, wherein the molar ratio of lyoprotectant to antibody is 100 to 600 moles of lyoprotectant to 1 mole of antibody.

18.1 the use of the formulation for the manufacture of a medicament for the treatment of a mammal suffering from a condition which requires treatment with a protein in the formulation.

19. The use according to 18, wherein the formulation is for subcutaneous administration.

20. A formulation comprising an anti-HER 2 antibody in an amount of 5-40mg/mL, sucrose or trehalose in an amount of 10-100mM, a buffer and a surfactant.

21. The formulation of claim 20, further comprising a bulking agent.

22. The formulation of claim 20 or 21, wherein the formulation is lyophilized and stable for at least 6 months at 30 ℃.

23. The formulation of 20, reconstituted with a diluent such that the concentration of anti-HER 2 antibody in the reconstituted formulation is 10-30mg/mL, wherein the reconstituted formulation is stable for at least 30 days at 2-8 ℃.

24. A preparation comprises an anti-IgE antibody in an amount of 5-40mg/mL, sucrose or trehalose in an amount of 80-300mM, a buffer and a surfactant.

25. The formulation of claim 24, which is lyophilized and stable at 30 ℃ for at least 1 year.

Brief Description of Drawings

Figure 1 shows the effect of reconstitution volume on the stability of lyophilized rhuMAb HER 2. The lyophilized formulation was prepared from a mixture comprising 25mg/mL protein, 60mM trehalose, 5mM sodium succinate pH5.0 and 0.01% Tween 20^TMThe preparation before lyophilization of (1). The lyophilized cake was incubated at 40 ℃ and then reconstituted with 4.0mL (o) or 20.0mL (●) of BWFI. The fraction of intact protein in the reconstituted formulation was determined by non-denaturing size exclusion chromatography and was determined by the peak area of the non-denatured protein relative to the total peak area including the aggregates.

Figure 2 depicts the effect of trehalose concentration on the stability of lyophilized rhuMAb HER 2. 25mg/mL protein was lyophilized in 5mM sodium succinate, pH5.0 (circles) or 5mM histidine, pH6.0 (squares) and trehalose at a concentration of 60mM (360 molar) to 200mM (1200 molar). The lyophilized proteins were incubated at 40 ℃ for 30 days (filled) or 91 days (empty). The amount of intact protein was determined after reconstitution of the lyophilized protein with 20mL BWFI.

FIG. 3 depicts trehalose concentrations versus lyophilized rhuMAb HER2 deposited at 40 deg.CThe long-term stability of (c). 25mg/mL protein in 5mM sodium succinate, pH5.0, 0.01% Tween 20^TMAnd 60mM trehalose (■) or 5mM histidine, pH6.0, 0.01% Tween 20^TMAnd 60mM trehalose (□) lyophilized or 21mg/mL protein in 10mM sodium succinate, pH5.0, 0.2% Tween 20^TMAnd 250mM trehalose (●). The lyophilized protein was incubated at 40 ℃ and then reconstituted with 20 mLBWFI. The amount of intact protein was determined after reconstitution.

FIG. 4 shows the results of the analysis of the expression levels of mannitol (7mg/mL) at 38.4mM, sucrose (7mg/mL) at 20.4mM, histidine at 5mM, pH6.0, Tween 20 at 0.01%^TMLyophilized rhuMAb HER2 stability. The lyophilized protein was incubated at 40 ℃ and then reconstituted with either 4.0mL (. smallcircle.) or 20.0mL (●) of BWFI. The amount of intact protein was determined after reconstitution.

FIG. 5 shows rhuMAb HER2 in 5mM sodium succinate, pH5.0, 60mM trehalose, 0.01% Tween 20^TMStability of reconstitution after lyophilization. The sample was reconstituted with 4.0mL (squares) or 20.0mL (circles) of BWFI (20 mL: 0.9% benzyl alcohol; 4 mL: 1.1% benzyl alcohol) and then stored at 5 deg.C (solid) or 25 deg.C (open). The% non-denatured protein was determined by the peak area of the non-denatured (native) (undegraded) protein relative to the total peak area, which was determined by cation exchange chromatography.

FIG. 6 shows the stability of rhuMAb HER2 reconstitution after lyophilization in 5mM histidine, pH6.0, 60mM trehalose, 0.01% Tween 20. The sample was reconstituted with 4.0mL (squares) or 20.0mL (circles) of BWFI (20 mL: 0.9% benzyl alcohol; 4 mL: 1.1% benzyl alcohol) and then stored at 5 deg.C (solid) or 25 deg.C (open). The% non-denatured protein was determined by the peak area of the non-denatured (undegraded) protein relative to the total peak area, which was determined by cation exchange chromatography.

FIG. 7 shows the reconstitution stability of rhuMAb HER2 after lyophilization in 5mM histidine, pH6.0, 38.4mM mannitol, 20.4mM sucrose, 0.01% Tween 20. The sample was reconstituted with 4.0mL (squares) or 20.0mL (circles) of BWFI (20 mL: 0.9% benzyl alcohol; 4 mL: 1.1% benzyl alcohol) and then stored at 5 deg.C (solid) or 25 deg.C (open). The% non-denatured protein was determined by the peak area of the non-denatured (undegraded) protein relative to the total peak area, which was determined by cation exchange chromatography.

FIG. 8 shows the stability of rhuMAb HER2 reconstitution after lyophilization in 10mM sodium succinate, pH5.0, 250mM trehalose, 0.2% Tween 20. The sample was reconstituted with 20.0mL BWFI (0.9% benzyl alcohol) and then stored at 5 deg.C (●) or 25 deg.C (. largecircle.). The% non-denatured protein was determined by the peak area of the non-denatured (undegraded) protein relative to the total peak area, which was determined by cation exchange chromatography.

FIG. 9 shows the aggregation of rhuMAb E25 added to a buffer with a pH of 5 to 7, a buffer concentration of 10mM, and an antibody concentration of 5 mg/mL. Samples were lyophilized and assayed for aggregation after 0, 4 weeks, 8 weeks and 52 weeks at 2-8 ℃. The buffer solution is: potassium phosphate ph7.0(°); sodium phosphate ph7.0(□); histidine ph7.0 (); sodium succinate ph6.5(●); sodium succinate pH6.0(■); sodium succinate pH5.5 (. diamond-solid.) and sodium succinate pH5.0 (. tangle-solidup.).

FIG. 10 shows the aggregation of rhuMAb E25 lyophilized in 5mM histidine buffer at pH6 and pH7 as determined on the following deposits. The buffer solution is: pH6.0, deposited at 2-8 ℃ (. o); pH6, preserved at 25 ℃ (□); pH6, deposited at 40 ℃ (o); pH7, preserved at 2-8 deg.C (●); pH7, preserved at 25 ℃ (■); and pH7, stored at 40 ℃. (. diamond-solid.).

FIG. 11 shows the aggregation of 5mg/mL rhuMAb E25 formulated in 10mM sodium succinate at pH5 and an added lyoprotectant concentration of 275mM (isotonic). The freeze-drying protective agent is: control, no lyoprotectant (∘); mannitol (□); lactose (); maltose (●); trehalose (■); and sucrose (. diamond.). Samples were lyophilized and assayed for aggregation after 0, 4,8 and 52 weeks storage at 2-8 ℃.

FIG. 12 shows the aggregation of 5mg/mL rhuMAb E25 formulated in 10mM sodium succinate at pH5 and an added lyoprotectant concentration of 275mM (isotonic). The freeze-drying protective agent is: control, no lyoprotectant (∘); mannitol (□); lactose (); maltose (●); trehalose (■); and sucrose (. diamond.). Samples were lyophilized and assayed for aggregation after 0, 4,8 and 52 weeks at 40 ℃.

FIG. 13 shows the hydrophobic interaction chromatography results of 20mg/mL rhuMAb E25 reconstituted after lyophilization in histidine buffer at lactose isotonic concentration (i.e., 275mM), pH6, and storage at 2-8 deg.C, 25 deg.C, or 40 deg.C for 24 weeks.

FIG. 14 shows the hydrophobic interaction chromatography results of 20mg/mL rhuMAb E25 reconstituted after lyophilization in histidine buffer pH6 and storage at 2-8 deg.C, 25 deg.C or 40 deg.C for 24 weeks.

FIG. 15 shows the hydrophobic interaction chromatography results of 20mg/mL rhuMAb E25 reconstituted after lyophilization in histidine buffer at sucrose isotonic concentration (i.e., 275mM), pH6, and storage at 2-8 deg.C, 25 deg.C, or 40 deg.C for 24 weeks.

FIG. 16 shows the effect of sugar concentration on 20mg/mL rhuMAbE25 formulated in 5mM histidine (pH 6.0). Sucrose (●) and trehalose (□) were added to the formulation at a molar ratio of 0 to 2010 (isotonic) (see table 1 below). Samples were lyophilized and assayed for aggregation after 12 weeks at 50 ℃.

TABLE 1

Molar ratio of sugar to E25 antibody	Sugar concentration (mM)
		0	0
260	34.4
		380	51.6
510	68.8
		760	103.1
1020	137.5
		1530	206.3
2010	275

FIG. 17 reveals that the concentration of sucrose (. smallcircle.) was 85mM at 5mM histidine (pH 6); 85mM trehalose (□); agglomeration was prepared at 25mg/mL rhuMAb E25 in 161mM sucrose (. diamond-solid.) or 161mM trehalose (. tangle-solidup.). Samples were lyophilized and stored at 2-8 ℃ and then reconstituted with 0.9% benzyl alcohol to form isotonic (340mM) and hypertonic (644mM) sugar concentration formulations with an antibody concentration of 100mg/mL, histidine of 20mM, pH6.

FIG. 18 reveals that at 5mM histidine (pH6), 85mM sucrose (. smallcircle.); 85mM trehalose (□); agglomeration was prepared at 25mg/mL rhuMAb E25 in 161mM sucrose (. diamond-solid.) or 161mM trehalose (. tangle-solidup.). Samples were lyophilized and stored at 30 ℃ and then reconstituted with 0.9% benzyl alcohol to isotonic (340mM) and hypertonic (644mM) sugar concentration formulations with an antibody concentration of 100mg/mL, histidine of 20mM, pH6.

FIG. 19 reveals that at 5mM histidine (pH6), 85mM sucrose (. smallcircle.); 85mM trehalose (□); agglomeration was prepared at 25mg/mL rhuMAb E25 in 161mM sucrose (. diamond-solid.) or 161mM trehalose (. tangle-solidup.). Samples were lyophilized and stored at 50 ℃ and then reconstituted with 0.9% benzyl alcohol to make up formulations with isotonic (340mM) and hypertonic (644mM) sugar concentrations of 100mg/mL antibody, 20mM histidine, pH6.

Detailed description of the preferred embodiments

I. Definition of

"protein" refers to an amino acid sequence having a chain length sufficient to produce high levels of tertiary and/or quaternary structure. This is in contrast to "polypeptides" or other small molecular weight drugs that do not have such a structure. Generally, the molecular weight of the proteins herein is at least about 15 to about 20kD, preferably at least about 20 kD.

Examples of proteins encompassed by the definition herein are mammalian proteins, e.g., growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; a thyrotropin; a lipoprotein; alpha-1-antitrypsin; an insulin a chain; insulin B chain; proinsulin; a follicle stimulating hormone; a calcitonin; luteinizing hormone; glucagon; coagulation factors, such as factor VIIIC, factor IX, tissue factor and von willebrand factor; anti-coagulation factors such as protein C; atrial natriuretic peptides; a pulmonary surfactant; plasminogen activators, such as urokinase or tissue-type plasminogen activator (t-PA); bombesin; thrombin; tumor necrosis factor-alpha and-beta; enkephalinase; the chemokine RANTES (which is regulatable upon activation and is usually expressed and secreted by T cells); human macrophage inflammatory protein (MIP-1-alpha); serum albumin such as human serum albumin; (ii) a muller inhibitor; a relaxin a chain; a relaxin B chain; (ii) prorelaxin; a murine gonadotropin-associated peptide; deoxyribonuclease; a statin; an activin; vascular Endothelial Growth Factor (VEGF); receptors for hormones or growth factors; an integrin; protein A or D; rheumatoid factor; neurotrophic factors such as Bone Derived Neurotrophic Factor (BDNF), neurotrophin-3, -4, -5 or-6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factors such as NGN-beta; platelet Derived Growth Factor (PDGF); fibroblast growth factors such as aFGF and bFGF; epidermal Growth Factor (EGF); transforming Growth Factors (TGF) such as TGF-alpha and TGF-beta, including TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4 or TGF-beta 5; insulin-like growth factors-I and-II (IGF-I and IGF-ID; des (1-3) -IGF-I (brain IGF-D; insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19 and CD 20; Erythropoietin (EPO); platelet growth factor (TPO); osteoinductive factors (osteopontic factors); immunotoxins; Bone Morphogenetic Proteins (BMPs); interferons such as interferon-alpha, -beta and-gamma; Colony Stimulating Factors (CSFs) such as M-CSF, GM-CSF and G-CSF; Interleukins (IL) such as IL-1 to IL-10; superoxide dismutase; T cell receptor; membrane surface proteins; Decay Accelerating Factor (DAF); viral antigens such as part of AIDS; envelope transporters; homing receptors; regulatory proteins; immunoadhesins; antibodies; and any of the above polypeptides A biologically active fragment or variant of (a).

The protein of the formulation is preferably substantially pure, and preferably substantially homogeneous (i.e., free of contaminating proteins, etc.). By "substantially pure" protein is meant that the component comprises at least about 90% by weight protein (based on the total weight of the component), preferably at least about 95% by weight protein. By "substantially homogeneous" protein is meant that the component comprises at least about 99% by weight protein, based on the total weight of the component.

In some examples, the protein is an antibody. For example, an antibody can bind to any of the molecules described above. Typical target molecules for antibodies of the invention include CD proteins such as CD3, CD4, CD8, CD19, CD20 and CD 34; a member of the HER receptor family, such as the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules, such as LFA-1, Mol, p150, 95, VLA-4, ICAM-1, VCAM, and α v/β 3 integrin, including its α or β subunit (e.g., anti-CD 11a, anti-CD 18, or anti-CD 11b antibody); growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; a lipid (OB) receptor; protein C, and the like.

The term "antibody" is intended to be very broad and encompasses, inter alia, monoclonal antibodies (including full length antibodies having an immunoglobulin Fc region), antibody compositions having multiple epitope specificities, bispecific antibodies, diabodies and single chain molecules, and antibody fragments (e.g., Fab, F (ab')₂And Fv).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies included in the population are identical, except for a small number of possible natural mutations. Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, i.e. preparations which typically comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against only a single determinant of the antigen. In addition to specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma incubation and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody. For example, monoclonal antibodies useful in the present invention can be prepared by the hybridoma method described by Kohler et al (Nature, 256: 495(1975)), or by recombinant DNA techniques (see U.S. Pat. No.4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques such as those described by Clackson et al (Nature, 352: 624-628(1991)) and Marks et al (J.mol.biol.222: 581-597 (1997)).

Monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from or belonging to a particular antibody class or subclass, while the remaining chains are identical or homologous to corresponding sequences in antibodies derived from another class or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)).

"humanized" antibodies of non-human origin (e.g., murine) are chimeric immunoglobulins, immunoglobulin chains, or fragments comprising minimal sequence obtained from non-human immunoglobulins (e.g., Fv, Fab ', F (ab')₂Or other antibody sequences that bind to an antigen). Humanized antibodies are mostly human immunoglobulins (recipient antibody) in which residues from a Complementarity Determining Region (CDR) of the recipient are replaced by CDR residues from a non-human antibody of the desired specificity, affinity and capacity, e.g. a murine, rat or rabbit antibody (donor antibody). In some instances, Fv Framework Region (FR) residues of a human immunoglobulin are substituted for corresponding non-human residues. Furthermore, humanized antibodies may include residues that are not found in either the acceptor antibody or the imported CDR or framework sequences. These modifications can further refine and optimize the performance of the antibody. In general, a humanized antibody will comprise substantially at least one, and typically two, variable domains in which all or substantially all of the CDR regions corresponding to non-human immunoglobulins and all or substantially all of the FR regions are sequences of human immunoglobulins. The humanized antibody may also optionally comprise at least a portion of the constant region (Fc) of an immunoglobulin, typically of human origin. More detailed descriptions are given in Jones et al Nature (321: 522-525(1986)), Reichmann et al Nature (332: 323-329(1988)) and Presta in curr. Op. struct. biol. (2: 593-596 (1992)). Humanized antibodies include Primatized^TMAn antibody, wherein the antigen binding region of the antibody is obtained by immunizing macaque (macaque) monkeys with an antigen of interest to produce the antibody.

By "stable" formulation is meant a formulation in which the protein substantially maintains its physical and chemical stability and integrity upon storage. There are a number of analytical techniques in the art for determining Protein stability, which are summarized in Peptide and Protein Drug Delivery (Peptide and Protein Drug Delivery, 247. sup. 301, edited by VincentLee, Marcel Dekker Inc., New York Press (1991)) and Jones in A.Adv.drug Delivery Rev. (10: 29-90 (1993)). Stability can be measured at selected temperatures and times. For rapid screening, the formulations can be stored at 40 ℃ for 2 weeks to 1 month while stability is being determined. When the formulation is to be stored at 2-8 ℃, typically the formulation should be stable at 30 ℃ or 40 ℃ for at least 1 month and/or at 2-8 ℃ for at least 2 years. When the formulation is to be stored at 30 ℃, the formulation should generally be stable for at least 2 years at 30 ℃ and/or stable for at least 6 months at 40 ℃. The degree of aggregation, for example after lyophilization and storage, can be used as an indicator of protein stability (see examples herein). For example, a "stable" formulation is one in which less than about 10%, and preferably less than about 5%, of the protein in the formulation is in the form of aggregates. In another example, the increase in aggregate of a formulation is determined after lyophilization and storage of the lyophilized formulation. For example, a "stable" lyophilized formulation means that the amount of coagulant in the lyophilized formulation should be less than about 5%, preferably less than about 3%, when the lyophilized formulation is stored at 2-8 ℃ for at least 1 year. In another example, the stability of a protein formulation can be determined using a bioactivity assay (see example 2 below).

By "reconstituted" formulation is meant that the protein is dispersed in the reconstituted formulation by dissolving the lyophilized protein formulation in a diluent. In some examples of the invention, reconstituted formulations suitable for administration to a patient in need of treatment with a desired protein (e.g., parenteral administration) may be suitable for subcutaneous administration.

By "isotonic" is meant that the formulation of interest is substantially equal to the osmotic pressure of human blood. The osmolality of an isotonic formulation is typically about 250 to 350 mOsm. Isotonicity can be measured, for example, by using a pneumatic or refrigerated osmometer.

"lyoprotectant" refers to a molecule that, when bound to a protein of interest, prevents or reduces chemical and/or physical instability of the protein upon lyophilization and subsequent storage. Typical lyoprotectants include sugars such as sucrose or trehalose; amino acids such as monosodium glutamate or histidine; methylamines such as betaine (betaine); readily soluble salts such as magnesium sulfate; polyhydric alcohols such as trihydric or higher sugar alcohols, such as glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol; pluronic; and combinations thereof. Preferred lyoprotectants are non-reducing sugars, such as trehalose or sucrose.

The amount of lyoprotectant added to the pre-lyophilized formulation "lyoprotecting amount" means that the protein substantially retains its physical and chemical stability and integrity upon lyophilization and storage in the presence of the lyoprotecting amount of lyoprotectant.

The "diluent" of interest herein is pharmaceutically acceptable (safe and non-toxic for human administration) and can be used to prepare reconstituted formulations. Typical diluents include sterile water, bacteriostatic for injection (BWFI), pH buffers (e.g., phosphate buffer), sterile saline solution, Ringer's solution, or dextrose solution.

A "preservative" is a compound added to a diluent to substantially reduce the bacterial action in the reconstituted formulation, thereby enabling multiple use of the reconstituted formulation. Examples of useful preservatives include octadecyl dimethyl phenyl ammonium chloride, hexane methyl ammonium chloride, phenylalkali chloride (a mixture of alkyl phenyl dimethyl ammonium chlorides, where the alkyl group is a long chain compound), and phenethyl ammonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butanol and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexane, 3-pentanol and m-cresol. The most preferred preservative here is benzyl alcohol.

A "bulking agent" is a compound that is added to a lyophilization mixture to form the physical structure of a lyophilized cake (e.g., to facilitate the manufacture of a substantially uniform lyophilized cake that maintains an open cell structure). Typical bulking agents include mannitol, glycerol. Polyethylene glycol and sorbitol.

"treatment" refers to both medical treatment and prophylactic measures. Those in need of treatment include those already with the disease and those in which the disease is prevented.

"mammal" in need of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals and zoos, stadium animals, or pets, such as dogs, horses, cats, cattle, etc. Preferably the mammal is a human.

"disease" is a disease that can benefit from protein therapy. It includes chronic and acute diseases or diseases that are pathological conditions that predispose a mammal to disease. Non-limiting examples of diseases to be treated herein include cancer and allergies.

Embodiments of the invention

A. Protein production

The proteins to be formulated are prepared by techniques well established in the art, including synthetic techniques (e.g., recombinant techniques and peptide synthesis or a combination of such techniques) or isolated from endogenous proteins. In some examples of the invention, the protein of choice is an antibody. Techniques for producing antibodies are as follows.

(i) Polyclonal antibodies

Polyclonal antibodies are typically obtained by multiple subcutaneous (sc) or intraperitoneal injections of the relevant antigen and adjuvant in the animal. It may be advantageous to couple the relevant antigen to an immunogenic protein in the species to be immunized (e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor) with a bifunctional or derivatizing reagent (e.g., sulfosuccinimidyl maleimidobenzoyl ester (via cysteine residue coupling reaction), N-hydroxysuccinimidyl (via lysine residue), glutaraldehyde, succinic anhydride, SOCl2, or R1N ═ C ═ NR (where R and R1 are different alkyl groups)).

Animals were immunized with antigen, immunogenic conjugate or derivative obtained by conjugation of 1mg or 1ug of peptide or conjugate (rabbit or murine respectively) with 3 volumes of Freund's complete adjuvant. Animals were boosted 1 month later by subcutaneous injection of an amount of 1/5 to 1/10 of the original peptide or conjugate in Freund's complete adjuvant. After 7 to 14 days, the animals were bled and the antibodies in the serum were titrated. Animals were boosted until titers leveled off. Preferably, the animal can be boosted with a conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking agent. Conjugates can be prepared as protein fusions in recombinant cell culture. Also, coagulants such as alum are suitable for enhancing immune responses.

(ii) Monoclonal antibodies

Monoclonal antibodies can be obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies included in the population are identical except for a small number of natural mutations that may occur. Thus, the modifier "monoclonal" refers to a mixture of antibodies which is not specifically characterized.

For example, monoclonal antibodies can be made by the hybridoma method first proposed by Kohler et al (Nature, 256: 495(1975)), or by recombinant DNA methods (U.S. Pat. No.4,816,567).

In the hybridoma method, a mouse or other suitable host animal, such as a hamster, can be immunized by the methods described above to elicit the production of lymphocytes or antibodies that specifically bind to the protein for use in the immune response. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to produce hybridoma cells (Goding, monoclonal antibodies: principles and Practice, pp.59-103(Academic Press)).

The hybridoma cells thus obtained are then seeded into a suitable culture medium to grow, preferably containing one or more substances that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), then typically the culture medium used for the hybridomas will contain hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibits the growth of HGPRT-deficient cells.

Preferred myeloma cells are those that are sufficiently fused to produce antibodies highly stably in selective antibody-producing cells and are sensitive to, for example, HAT medium. Among them, preferred myeloma Cell lines are murine-derived myeloma Cell lines, such as those derived from MOPC-21 and MPC-11 murine tumors (obtained from the Salk Institute Cell Distribution Center, san Diego, Calif.) and SP-2 cells (obtained from the American Type Culture Collection, Rochville, Md.). Human myeloma and murine-human heteromyeloma cell lines are also described in the Production of human Monoclonal antibodies (Kozbor, J.Immunol., 133: 3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51-63(Marcel Dekker, Inc., New York, 1987)).

The media used for growth of the hybridoma cells is assayed to produce monoclonal antibodies to the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or an in vitro binding assay, such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis (Munson et al, anal. biochem., 107: 220 (1980)).

After identification of hybridoma cells producing Antibodies of the desired specificity, affinity and/or activity, clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: principles and Practice, pp.59-103(Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-164G medium. Hybridoma cells can grow in animals as ascites tumors.

Monoclonal antibodies secreted by the subclone line may be suitably isolated from culture medium, ascites fluid, or serum using conventional immunoglobulin purification procedures, such as protein A sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

DNA encoding the monoclonal antibody can be readily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed in an expression vector, which is then transfected into a host cell such as an E.coli cell line, a monkey COS cell line, a Chinese Hamster Ovary (CHO) cell line, or a myeloma cell that does not otherwise produce immunoglobulin protein, to allow synthesis of the monoclonal antibody in the recombinant host cell. Literature on recombinant expression of DNA encoding antibodies in bacteria is Skerra et al in curr. opinion in immunol, 5: 256-charge 262(1993) and Pluckthun in Immunol.Revs., 130: 151-.

In another example, antibodies can be isolated from antibody phage libraries using techniques described in McCafferty et al (Nature, 348: 552-554 (1990)). Clackson et al (Nature, 352: 624-628(1991)) and Marks et al (J.mol.biol., 222: 581-597(1991)) describe methods for isolating murine and human antibodies using phage libraries, respectively. Thereafter, the literature discloses the generation of high affinity (in the nM range) humanized antibodies using chain shuffling (Marks et al, Bio/Technology, 10: 779-783(1992)), as well as the construction of large phage libraries using combinatorial infection and in vivo recombination (Waterhouse et al, Nuc. acids. Res., 21: 2265-2266 (1993)). Therefore, these techniques are promising approaches different from conventional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

The DNA may also be modified by replacing the coding sequences for the constant regions of the heavy and light chains of human origin with homologous murine-derived sequences (U.S. Pat. No.4,816,567; Morrison et al, Proc. Natl Acad. Sci. USA, 81: 6851(1984)) or by covalently binding all or part of the sequence encoding the non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

Typically, such non-immunoglobulin polypeptides are used in place of the constant regions of an antibody, or the variable regions of an antibody at a site that binds an antigen, to form a chimeric bivalent antibody having one antigen binding site that specifically binds an antigen and another antigen binding site that specifically binds a different antigen.

Chimeric or hybrid antibodies can also be prepared in vitro using methods known in protein synthesis chemistry, including those using cross-linking agents. For example, immunotoxins may be constructed by disulfide exchange reactions or formation of thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrylidene urethane.

(iii) Humanized and humanized antibodies (human antibodies)

Humanization of non-human antibodies is well known in the art. Typically, humanized antibodies have one or more amino acid residues introduced into them that are of non-human origin. These non-human amino acid residues are often referred to as "import" residues, which are typically removed from the "import" variable region. Humanization can be performed essentially according to the method of Winter and co-workers (Jones et al, Nature, 321: 522-525 (1986); Reichman et al, Nature, 332: 323-327 (1988); Verhoeyen et al, Science, 239: 1534-1536(1988)), by replacing the corresponding sequences of a human antibody with murine CDRs or CDR sequences. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.4,816567) in which substantially less than the entire human variable region is replaced by a corresponding sequence that is not of human origin. In practice, humanized antibodies are typically of the human type in which some CDR residues and FR residues are replaced by residues from the same site in a murine antibody.

The human variable regions of the heavy and light chains used to generate the humanized antibody are important to reduce antigenicity. In the so-called "most suitable" method, the variable region sequences of murine antibodies are screened against all known variable region sequences of human origin. The most similar human-derived type sequences to the murine type were then used as the humanizing Framework (FR) in the humanized antibody (Sims et al, J.Immunol., 151: 2296 (1993); Chothia et al, J.mol.biol., 196: 901 (1992)). Another approach employs specific frameworks derived from consensus sequences of specific subclasses of all human antibodies' light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al, J.Immunol., 151: 2623 (1993)).

It is also important that the antibody after humanization still have a high affinity for the antigen and other advantageous biological properties. To achieve this, according to a preferred method, humanized antibodies are prepared by analyzing the parental sequences and various humanized products produced using three-dimensional models of the parental and humanized sequences. Three-dimensional models of immunoglobulins are generally available and are well known to those skilled in the art. Computer programs can be used to describe and display the possible three-dimensional conformational structures of selected immunoglobulin sequences. By observing these indications, one can analyze the likely role residues may play in the selected immunoglobulin sequence, i.e., the effect of residues on the ability of the immunoglobulin to be selected to bind to its antigen. In this way, FR residues can be selected from the recipient and import sequences to achieve desired antibody properties, such as improved affinity for the target antigen. Usually the CDR residues have a direct, large influence on the binding to the antigen.

Alternatively, transgenic animals (e.g., mice) can now be generated that produce all the components of the human antibody when immunized with a deletion in the endogenous immunoglobulin production. For example, it is described that chimeric and germline mutated murine antibody heavy chain binding region (JH) genes will completely suppress endogenous antibody production. The arrangement of human germline immunoglobulin gene transfer in such germline mutant mice will result in its production of antibodies of human origin upon antigen challenge. See, e.g., Jakobovits et al, proc.natl.acad.sci.usa, 90: 2551 (1993); jakobovits et al, Nature, 362: 255-258 (1993); bruggermann et al, Yeast in immunity, 7: 33(1993). Human antibodies can also be obtained from phage display libraries (Hoogenboom et al, J.mol.biol., 277: 381 (1991); Marks et al, J.mol.biol., 222: 581: 597 (1991)).

(iv) Bispecific antibodies

Antibody bispecific antibodies (BsAbs) are antibodies that can specifically bind at least two different epitopes. Such antibodies can be obtained from full-length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies).

Methods for making bispecific antibodies are known in the art. The conventional generation of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, the two chains differing in specificity from each other (Millstein et al, Nature, 305: 537-539 (1983)). Due to the random assignment of the heavy and light chains of immunoglobulins, these four-body hybridomas (quadromas) produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule (usually by an affinity chromatography step) is rather cumbersome and the product amount is low. The same procedure is described in WO93/08829 and Traunecker et al, EMBO J., 10: 3655-3659 (1991).

According to another method, the variable region of an antibody with the desired specificity (antibody-antigen binding site) is fused to an immunoglobulin constant region sequence. Preferably to an immunoglobulin heavy chain constant region, which constant region comprises at least part of the hinge, CH2 and CH3 regions. Preferably, in at least one of the fusions, the heavy chain constant region (CH1) contains the site required for light chain binding. DNA encoding a fusion of an immunoglobulin heavy chain (and if desired an immunoglobulin light chain) is inserted into a separate expression vector and then co-transfected into a suitable host organism. In the example, this method allows flexibility in adjusting the ratio between the three polypeptide fragments when constructed with three polypeptide chains in different ratios to provide optimal yields. However, when the expression of at least two polypeptides in the same ratio results in high yield, or when the ratio is not particularly affected, the coding sequences for two or all three polypeptide chains can be inserted into one expression vector.

In a preferred embodiment of this method, the bispecific antibody is composed of a hybrid immunoglobulin heavy chain (having a first binding specificity in one arm) and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity on the other arm). It was also found that this asymmetric structure makes the desired bispecific compound easier to isolate from unwanted immunoglobulin chain compositions, since the immunoglobulin light chain present in only half of the bispecific molecule provides an easy method for isolation. This method is disclosed in WO94/04690, published 3.3.1994. For more detailed details on the generation of bispecific antibodies see, for example, Suresh et al, Methods in Enzymology, 121: 210 (1986).

Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heterologous conjugate may be coupled to avidin and the other to biotin. Such antibodies are designed, for example, to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980) and to treat HIV infection (WO91/00360, WO 92/200373). Heteroconjugate antibodies can be made by any convenient cross-linking method. Suitable crosslinking agents are well known in the art and a number of crosslinking techniques are disclosed in U.S. Pat. No.4,676,980.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. The following techniques can also be used to generate bivalent, but not necessarily bispecific, antibody fragments. For example, Fab' fragments obtained from E.coli can be chemically combined in vitro to form bivalent antibodies. See, Shalaby et al, j.exp.med., 175: 217-225(1992).

Various techniques for the preparation and isolation of bivalent antibody fragments directly from recombinant cell culture have been described. For example, leucine zippers can produce bivalent heterodimers (Kostelny et al, J.Immunol., 148 (5): 1547-1553 (1992)). The leucine zipper peptide fragment proteins from Fos to Jun were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers at the hinge region are reduced to monomers and then reoxidized to antibody heterodimers. The "diabody" technology described by Hollinger et al (Proc. Natl. Acad. Sci. USA, 90: 6444-. The fragment contains a heavy chain variable region (VH) which is linked to a light chain variable region (VL) by a very short linker such that no pairing between the two regions on the same chain is possible. Thus the VH and VL domains on one fragment are forced to complement the V on the other fragment_LAnd V_HThe segments pair, thereby forming two antigen binding sites. In addition, the preparation of bispecific antibodies using single chain fv (sFv) has been reportedThe idea of a hetero/bivalent antibody fragment (see Gruber et al, J. Immunol., 152: 5368 (1994)).

B. Preparation of lyophilized preparation

After the protein of interest is prepared as described above, a "pre-lyophilized formulation" can be prepared. The protein content of the formulation prior to lyophilization can be determined based on the desired dosage volume, mode of administration, and the like. When the protein of choice is an intact antibody (e.g., anti-IgE or anti-HER 2 antibody), the initial protein concentration will typically be about 2mg/mL to about 50mg/mL, preferably about 5mg/mL to about 40mg/mL, and most preferably about 20-30 mg/mL. Proteins are typically present in solution. For example, the protein may be in a pH buffer at a pH of about 4 to about 8, preferably at a pH of about 5 to about 7. Typical buffers include buffers of histidine, phosphate, Tris, citrate, succinate and other organic acids. The buffer concentration is about 1mM to about 20mM, or about 3mM to about 15mM, as determined by, for example, isotonicity of the buffer and the desired formulation (e.g., reconstituted formulation). The preferred buffer is histidine, which is shown below to have lyoprotectant properties. Succinate buffer is also a useful buffer.

The lyoprotectant is added to the pre-lyophilized formulation, and in a preferred embodiment, is a non-reducing sugar such as sucrose or trehalose. The amount of lyoprotectant in the formulation prior to lyophilization is such that the formulation obtained upon reconstitution is isotonic. However, hypertonic reconstituted formulations are also suitable. Furthermore, the amount of lyoprotectant should also not be so low that an unsatisfactory amount of protein degradation/aggregation occurs upon lyophilization. When the lyoprotectant is a carbohydrate (e.g., sucrose or trehalose) and the protein is an antibody, the concentration of lyoprotectant in the formulation prior to lyophilization is typically from about 10mM to about 400mM, preferably from about 30mM to about 300mM, and most preferably from about 50mM to about 100 mM.

The ratio of protein to lyoprotectant is chosen for each combination of protein and lyoprotectant. When the protein of choice is an antibody and the lyoprotectant is a carbohydrate (e.g., sucrose or trehalose) used to produce an isotonic reconstituted formulation of the protein at a high concentration, the ratio of lyoprotectant to antibody is from about 100 to about 1500 moles of lyoprotectant to 1 mole of antibody, preferably from about 200 to about 1000 moles of lyoprotectant to 1 mole of antibody, for example from about 100 to about 600 moles (more preferably from about 200 to about 600 moles of lyoprotectant to 1 mole of antibody).

In the preferred embodiment of the invention, it has been found desirable to add a surfactant to the formulation prior to lyophilization. Alternatively, surfactants may also be added to the lyophilized and/or reconstituted formulations. Typical surfactants include non-ionic surfactants such as polysorbates (e.g., polysorbate 20 or 80); poloxamers (such as poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl, myristyl, linoleyl or octadecyl sulphobetaines; lauryl, myristyl, linoleyl or octadecylsarcosine; linoleyl, tetradecyl or hexadecylbetaine; lauramidopropyl, cocamidopropyl, linoleamidopropyl, tetradecamidopropyl, hexadecylamidopropyl or isostearyl amidopropyl betaine (e.g., lauramidopropyl); tetradecylamidopropyl, hexadecylamidopropyl or isooctadecanylamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and MONAQUA^TMSeries (mona industries, inc., Paterson, New Jersey), polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.). The surfactant is added in an amount such that protein aggregation upon reconstitution is reduced and the tendency of granulation of the reconstituted formulation is minimized. For example, the amount of surfactant in the formulation prior to lyophilization is about 0.001-0.5%, preferably about 0.005-0.05%.

In some embodiments of the invention, a mixture of a lyoprotectant (e.g., sucrose or trehalose) and a bulking agent (e.g., mannitol or glycine) may be used in the preparation of the formulation prior to lyophilization. The bulking agent can produce a homogeneous lyophilized cake without excessive open cells therein.

Other pharmaceutically acceptable carriers, excipients or stabilizers may be included in the formulation prior to lyophilization (and/or lyophilized and/or reconstituted formulations), as described in Remington's Pharmaceutical Sciences (16th edition, Osol, a.ed. (1980)), so long as they have no negative effect on the desired properties of the formulation. Acceptable carriers, excipients, or stabilizers are employed at dosages and concentrations which are non-toxic to the recipient and include other buffers, preservatives, co-solvents, antioxidants such as ascorbic acid and methionine, chelating agents such as EDTA, metal complexes such as Zn-protein complexes, biodegradable polymers such as polyesters, and/or salt-forming counterions such as sodium.

The formulations herein may also contain a plurality of proteins for a particular therapeutic indication, preferably such proteins having complementary activities that do not have a negative effect on other proteins. For example, two or more antibodies that bind to HER2 receptor or IgE may be provided in one formulation. Furthermore, anti-HER 2 and anti-VEGF antibodies may be used in combination in the same formulation. These proteins may be present in the combination in an amount effective for the purpose intended.

Formulations for in vivo administration must be sterile. This can be achieved by filtration through a sterile filtration membrane before or after lyophilization and reconstitution. Alternatively, the entire mixture may be rendered sterile by, for example, autoclaving the components other than the protein at about 120 ℃ for about 30 minutes.

After the protein, lyoprotectant, and any other components are mixed, the formulation may be lyophilized. Different freeze dryers can be used for this purpose, such as Hull50^TM(Hull, USA) or GT20^TMFreeze dryers of the type (Leybold-Heraeus, Germany). Lyophilization is the freezing of the formulation followed by sublimation of the frozen ice at a temperature suitable for primary drying. Under these conditions, the product temperature is below the eutectic or disintegration point of the formulation. Typically the shelf temperature for primary drying is about-30 ℃ to 25 ℃ (the product should remain frozen during primary drying), with a suitable pressure of about 50 to 250 mTorr. The time required for drying is determined mainly by the formulation, the size and type of container (e.g. glass bottle) in which the sample is placed and the volume of liquid, and can range from a few hours to several hoursWithin a few days (e.g., 40-60 hours). The secondary drying step may be carried out at about 0-40 c, depending primarily on the type and size of container used and the type of protein. However, it has been found that a secondary drying step is not necessary here. For example, the temperature of the shelf from which the entire aqueous phase of the lyophilizate is removed is about 15-30 deg.C (e.g., about 20 deg.C). The time and pressure required for secondary drying should be such that a suitable lyophilized cake is formed, which is dependent on, for example, temperature and other parameters. The secondary drying time is determined by the desired residual moisture level in the product and typically takes at least about 5 hours (e.g., 10-15 hours). The pressure may be the same as the pressure used in the primary drying step. The freeze-drying conditions may vary depending on the formulation and the size of the vial.

In some instances, it is desirable to lyophilize the protein formulation in a container, where reconstitution of the protein can be performed to avoid the transfer step. The container in this example may be, for example, a 3, 5, 10, 20, 50 or 100cc bottle.

Typically, lyophilization should result in a lyophilized formulation having a moisture content of less than about 5%, preferably less than about 3%.

C. Reconstitution of lyophilized formulations

Typically, when administration to a patient is desired, the desired step is to reconstitute the lyophilized formulation with a diluent such that the protein concentration in the reconstituted formulation is at least 50mg/mL, for example, from about 50mg/mL to about 400mg/mL, more preferably from about 80mg/mL to about 300mg/mL, and most preferably from about 90mg/mL to about 150 mg/mL. Such high protein concentrations in reconstituted formulations are considered particularly useful when subcutaneous delivery of the reconstituted formulation is desired. However, for other routes of administration, such as intravenous administration, it may be desirable to have a somewhat lower concentration of protein in the reconstituted formulation (e.g., a concentration of protein in the reconstituted formulation of about 5-50mg/mL, or about 10-40 mg/mL). In some examples, the concentration of protein in the reconstituted formulation is much higher than the concentration in the formulation prior to lyophilization. For example, the protein concentration in the reconstituted formulation is about 2-40 times, preferably 3-10 times, and most preferably 3-6 times (e.g., at least 3 times or at least 4 times) that of the formulation prior to lyophilization.

Reconstitution is usually carried out at about 25 ℃ to ensure complete hydration, although other temperatures may be used as desired. The time of reconstitution is related to, for example, the type of diluent, excipients and the amount of protein. Typical diluents include sterile water, bacteriostatic for injection (BWFI), pH buffers (e.g., phosphate buffer), sterile saline solution, ringer's solution, or dextrose solution. The diluent may also contain a preservative, typically as described above, preferably an aromatic alcohol such as phenyl or a phenolic alcohol. The amount of preservative used is determined by measuring the compatibility with the protein and preservative efficacy at different preservative concentrations. For example, if the preservative is an aromatic alcohol (e.g., benzyl alcohol), it can be present in an amount of about 0.1-2.0%, preferably about 0.5-1.5%, but most preferably about 1.0-1.2%.

Preferably, there should be less than 6000 particles of 10 μm or more in particle size per vial of reconstituted formulation.

D. Administration of reconstituted formulations

The reconstituted formulation can be administered to a mammal, preferably a human, in need of treatment with the protein by known methods, such as intravenous administration, e.g., bolus injection or continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, intraoral, dermal or inhalation routes.

In a preferred embodiment, the reconstituted formulation is administered to the mammal subcutaneously (i.e., beneath the skin). For this purpose, the preparation is injected using a syringe. However, other devices for administering the drug may be used, such as injection devices (e.g. injection-ease)^TMAnd Genjec^TMA device); injection pen (e.g. GenPen)^TM) (ii) a Needleless devices (e.g. mediJecter)^TMAnd BioJector^TM) And subcutaneous delivery systems.

The appropriate dosage of protein ("therapeutically effective amount") is related to, for example, the disease to be treated, the severity and course of the disease, whether the protein is administered for prophylaxis or therapy, previous therapy, patient history and response to the protein, the type of protein used, and the discretion of the attending physician. The protein may be administered to the patient at once or over a series of courses, and may be administered at any time based on previous diagnosis. The proteins may be used alone or in combination with other drugs or therapies useful for treating diseases.

When the protein of choice is an antibody, whether administered separately, e.g., one or more times, the initial selected dose administered to the patient is about 0.1-20 mg/kg. However, other dosage regimens may be used. The progress of the therapy is readily monitored by conventional techniques.

In the case of an anti-HER 2 antibody, a therapeutically effective amount of the antibody should be capable of treating or preventing a cancer characterized by overexpression of the HER2 receptor. Reconstituted formulations of anti-HER 2 antibodies are believed to be useful in the treatment of breast, ovarian, gastric, endometrial, salivary gland, lung, kidney, colon and/or bladder cancer. For example, the anti-HER 2 antibody can be used to treat Ductal Carcinoma In Situ (DCIS). Typical doses of anti-HER 2 antibody in one or more divided doses are 1-10 mg/kg.

The anti-IgE formulations are useful in the treatment or prevention of IgE-mediated allergic diseases, such as parasitic infections, interstitial cystitis, and asthma. A therapeutically effective amount of an anti-IgE antibody (e.g., about 1-15mg/kg) may be administered to a patient depending, for example, on the disease being treated.

E. The product produced

Another aspect of the invention is to provide a product comprising the lyophilized formulation of the invention and instructions for its reconstitution and/or use. The product comprises a container. Suitable containers include, for example, glass bottles, vials (e.g., dual chamber vials), syringes (e.g., dual chamber syringes), and test tubes. The container may be made of various materials such as glass or plastic. The container contains the lyophilized formulation and the label on or associated with the container indicates the method of reconstitution and/or administration. For example, the label may indicate reconstitution of the lyophilized formulation to the protein concentration described above. The label may also indicate that the formulation is suitable for subcutaneous injection. The container holding the formulation may be a multiple use vial which may be repeatedly dosed (e.g. 2-6 times) with the reconstituted formulation. The product may also comprise another container with a suitable diluent (e.g., BWFI). Upon mixing the diluent and the lyophilized formulation, the final concentration of protein in the reconstituted formulation is at least 50 mg/mL. From a commercial and consumer perspective, the product may also include other items, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The present invention may be more fully understood with reference to the following examples. However, they should not be construed as limiting the scope of the invention. All documents are incorporated by reference.

Example 1

anti-HER 2 formulations

HER2 protooncogene product (p 185)^HER2) Is associated with various invasive human malignancies. The murine monoclonal antibody, designated muMAb4D5, acts directly on p185^HER2The extracellular region (ECD). The muMAb4D5 molecule was humanized in an attempt to improve clinical efficacy by reducing its immunogenicity and allowing it to support human effector functions (see WO 92/22653). This example describes the development of a lyophilized formulation comprising the full length humanized antibody huMAb4D5-8 described in WO 92/22653.

In developing lyophilized formulations, excipients and buffers are first screened by determining the stability of the protein after lyophilization and reconstitution. Further stability studies were performed on the lyophilized protein in each formulation to determine the stability of the protein over its shelf life. These further studies are usually carried out at the storage conditions temperatures given above and the activation energy required for the degradation reaction is then estimated from data on Arrhenius kinetics (Cleland et al, clinical Reviews in Therapeutic drug Carrier System 10 (4): 307-377 (1993)). The activation energy is then used to calculate the expected shelf life of the protein preparation under the given storage conditions.

In an early screening study, the stabilization of some lyophilized recombinant humanized anti-HER 2 antibody (rhuMAbHER2) formulations was studied after incubation at 5 ℃ (given storage conditions) and 40 ℃ (conditions for further stability studies)And (4) sex. In the liquid state, rhuMAB HER2 was found to degrade by deamidation (asparagine at position 30 of the light chain) and isoaspartic acid (aspartic acid at position 102 of the heavy chain) by the cyclic imine intermediate succinimide. At pH5.0 the deamidation reaction is minimal, resulting in degradation to essentially succinimide. Higher amounts of deamidation reactions were found in the liquid protein formulations at ph 6.0. Thus, the lyophilized formulation was studied at (a)5 or 10mM succinate buffer, pH5.0 and (b)5 or 10mM histidine buffer, pH 6.0. The buffers all contained the surfactant polysorbate 20 (Tween 20)^TM) It can be used to reduce the tendency of the reconstituted protein to agglomerate and minimize particle formation after reconstitution. These buffers may be used with or without various sugars. The protein was dosed to the buffer at a concentration of 5.0, 21.0 or 25.0 mg/mL. The formulations were then lyophilized and the stability of the protein was determined after storage at 5 ℃ and 40 ℃ for 2 weeks. In the freeze-dryer, vials were frozen at a shelf temperature of about-55 ℃ for about 5 hours, then primary dried at a shelf temperature of 5 ℃ for 30 hours at a pressure of 150mTorr, and secondary dried with a shelf temperature of 20 ℃ for 10 hours to dry to 1-2% residual moisture. The major degradation pathway of this protein upon lyophilization is aggregation, and therefore protein stability can be determined by determining the yield of intact non-denatured protein using non-denaturing size exclusion chromatography (see table 2 below for% intact protein).

The effect of various lyoprotectant carbohydrates on the stability of lyophilized proteins was determined in 10mM sodium succinate, pH5.0 (Table 2). At high sugar concentration (250-275mM) and low protein concentration (5.0mg/mL), trehalose and lactose stabilized the lyophilized protein when stored at 40 ℃ for 2 weeks and prevented it from clumping. However, lactose is a reducing sugar which is found to react with proteins when stored for long periods at 40 ℃. A formulation containing 5.0mg/mL protein of sorbitol or mannitol resulted in protein aggregation after 2 weeks at 40 ℃. At high protein concentrations (21.0mg/mL), formulations of mannitol or mannitol in combination with sorbitol or glycine contained aggregated protein after lyophilization storage under both conditions. In contrast, trehalose and sucrose prevented aggregation under both storage conditions.

The long term stability of the formulations containing 250mM trehalose and 250mM lactose is now determined. The percent intact protein in the trehalose-containing formulations did not change after 9 months of storage at 40 ℃ or 12 months of storage at 5 ℃. Whereas for the lactose formulation the percentage of intact protein remains constant (as it was) after 3 months of storage at 40 ℃ or after 6 months of storage at 25 ℃. Trehalose-containing formulations can be stored for 2 years at controlled room temperature (15-30 ℃) without significant changes in the percentage of intact protein.

The formulation with histidine and mannitol at pH6.0, 10mM contained less aggregated protein than the formulation with succinate and mannitol at pH5.0, 10mM after storage at 40 ℃ for 2 weeks. This result may be due to the stabilizing effect contributed by the mono-amino acid. However, there was significant agglomeration in the histidine alone or in the histidine/mannitol formulations after 2 weeks at 40 ℃. Addition of sucrose in an amount equal to mannitol (10mg/mL) to the histidine formulation stabilizes the protein against aggregation under both storage conditions. The use of glycine and mannitol did not improve the stability of the protein, whereas the sucrose/glycine formulation provided the same stability as the sucrose/mannitol formulation. The results further indicate that sucrose helps prevent aggregation of the lyophilized protein upon storage.

TABLE 2

Components before lyophilization		Percentage of intact proteina
					Proteinb(mg/mL)	Formulation of	Liquid (5 ℃ C.)	Freeze-drying (2 weeks, 5 ℃ C.)	Freeze-drying (2 weeks, 40 ℃ C.)
	10mM sodium succinate, pH5.0
					5.0	275mM trehalose, 0.01% Tween 20TM	98.9	99.1	98.9
5.0	275mM lactose, 0.01% Tween 20TM	96.8	96.5	96.6
					5.0	275mM sorbitol, 0.01% Tween 20TM	99.4	99.3	95.4
5.0	250mM mannitol, 0.01% Tween 20TM	100.0	99.9	98.8
					5.0	250mM trehalose, 0.01% Tween 20TM	100.0	99.9	100.0
5.0	250mM lactose, 0.01% Tween 20TM	100.0	100.0	100.0
					21.0	250mM trehalose, 0.2% Tween 20TM	99.3	99.1	99.1
21.0	250mM sucrose, 0.2% Tween 20TM	99.6	99.6	99.7
					21.0	250mM mannitol, 0.01% Tween 20TM	100.0	94.6	94.0
21.0	188mM mannitol/63 mM sorbitol,	99.8	98.6	96.5

	0.01% Tween 20TM
					21.0	250mM mannitol/25 mM glycine, 0.01% Tween 20TM	99.5	96.5	96.4
	10mM histidine, pH6.0
					21.0	Sugar free, 0.01% Tween 20TM	100.0	99.9	98.9
21.0	54.9mM mannitol, 0.01% Tween 20TM	100.0	99.9	99.2
					21.0	29.2mM sucrose/266.4 mM glycine, 0.01% Tween 20TM	100.0	100.0	99.6
21.0	54.9mM mannitol/266.4 mM glycine, 0.01% Tween 20TM	100.0	99.8	98.9
					21.0	54.9mM mannitol/29.2 mM sucrose, 0.01% Tween 20TM	99.8	100.0	99.7

a. The fraction of intact protein was determined by non-denaturing size exclusion HPLC and the value of the peak area of the non-denatured protein relative to the total peak area including the aggregates (TSK3000 SW XL column, TosoHaas, flow rate 1.0 mL/min; elution with phosphate buffer; detection at 214 and 280 nm). Protein formulations were analyzed before lyophilization (liquid, 5 ℃), after lyophilization, and after storage at 5 ℃ or 40 ℃ for 2 weeks.

b. The preparation containing 5mg/mL protein was reconstituted with distilled water (20mL, 5.0mg/mL protein) and the preparation containing 21mg/mL protein was reconstituted with bacteriostatic for injection (BWFI, 0.9% benzyl alcohol; 20mL, 20mg/mL protein).

Delivery of high protein concentrations is often required due to volume limitations (< 1.5mL) and dosage requirements (> 100mg) for subcutaneous administration. However, high protein concentrations (. gtoreq.50 mg/mL) are often difficult to achieve in a production process due to the tendency of the protein to coagulate during processing at high concentrations, which is difficult to handle (e.g.draw-off) and sterile filtration. Additionally the lyophilization process provides a method that allows for protein concentration. For example, proteins are filled into vials at a volume (Vf) and then lyophilized. The lyophilized protein is then reconstituted with a smaller volume (Vr) (e.g., Vr ═ 0.25Vf) of water or a preservative (e.g., BWFI) than the original volume, resulting in a higher protein concentration in the reconstituted solution. The method also concentrates the buffers and excipients. For subcutaneous administration, it is desirable that the solution be isotonic.

The amount of trehalose in lyophilized rhuMAB HER2 was reduced to form an isotonic solution upon reconstitution into 100mg/mL protein. The stabilizing effect of trehalose was determined as a function of concentration at pH5.0, 5mM sodium succinate and pH6.0, 5mM histidine, 25.0mg/mL protein (Table 3). At trehalose concentrations from 60 to 200mM, no significant aggregation of the lyophilized protein was observed after 4 weeks of incubation at 40 ℃. These formulations were reconstituted in 20mL of bacteriostatic for injection (BWFI, USP, 0.9% benzyl alcohol). A50 mM trehalose preparation (5mM sodium succinate) was reconstituted in 4mL BWFI (100mg/mL protein) after 4 weeks incubation at 40 ℃ to yield a slightly increased amount of aggregation of the resulting preparation. The preserved reconstituted formulation provides the advantage of being removable from the same vial multiple times without contamination. When using an inoculating needle, the preparation in this one vial can be taken out several times.

TABLE 3

Components before lyophilization		Percentage of intact proteina
					Protein (mg/mL)	Formulation of	Liquid (5 ℃ C.)	Freeze-drying (4 weeks, 5 ℃ C.)	Freeze-drying (4 weeks, 40 ℃ C.)
	5mM sodium succinate, pH5.0
					25.0	50mM trehalose, 0.01% Tween 20TMb	100.0	100.0	99.5
25.0	60mM trehalose, 0.01% Tween 20TM	100.0	100.0	99.9
					25.0	60mM trehalose, 0.01% Tween 20TM	100.0	100.0	99.2
25.0	100mM trehalose, 0.01% Tween 20TM	100.0	100.0	99.7
					25.0	150mM trehalose, 0.01% Tween 20TM	100.0	100.0	99.8
25.0	200mM trehalose, 0.01% Tween 20TM	100.0	100.0	100.0
						5mM histidine, pH6.0
25.0	38.4mM mannitol/20.4 mM sucrose, 0.01% TweenTM	100.0	100.0	99.3
					25.0	38.4mM mannitol/20.4 mM sucrose, 0.01% TweenTM c	100.0	100.0	99.4
25.0	60mM trehalose, 0.01% Tween 20TM d	100.0	100.0	99.8
					25.0	60mM trehalose, 0.01% Tween 20TM	100.0	100.0	99.4
25.0	100mM trehalose, 0.01% Tween 20TM	100.0	100.0	99.6
					25.0	150mM trehalose, 0.01% Tween 20TM	100.0	100.0	100.0
25.0	200mM trehalose, 0.01% Tween 20TM	100.0	100.0	100.0

a. The fraction of intact protein was determined by non-denaturing size exclusion HPLC and was defined as the value of the peak area of the non-denatured protein relative to the total peak area including the aggregates (TSK3000 SW XL column, TosoHaas, flow rate 1.0 mL/min; elution with phosphate buffer; detection at 214 and 280 nm). Protein formulations were analyzed before lyophilization (liquid, 5 ℃), after lyophilization, and after storage at 5 ℃ or 40 ℃ for 4 weeks. The preparation was reconstituted with injectable bacteriostatic agent (BWFI, USP, 0.9% w/w benzyl alcohol; 20mL, 22mg/mL protein).

b. The protein concentration was reconstituted with 4mL BWFI (0.9% benzyl alcohol) to 100 mg/mL.

c. The protein concentration was reconstituted with 4mL BWFI (1.1% benzyl alcohol) to 100 mg/mL.

d. The samples were incubated at 5 ℃ or 40 ℃ for 2 weeks and then reconstituted with 20mL BWFI (0.9% benzyl alcohol) to a protein concentration of 22 mg/mL.

Currently, rhuMAb HER2 is being investigated as a drug for the treatment of breast cancer. To giveThe patient took 2mg/kg protein weekly. Since the average body weight of these patients was 65kg, the average weekly dose was 130mg rhumab HER 2. For subcutaneous administration, an injection volume of 1.5mL or less is acceptable, and thus, once a week the concentration of rhuMAb HER2 protein administered subcutaneously is about 100mg/mL (130mg average dose/1.5 mL). As mentioned above, such high protein concentrations are difficult to produce and maintain stable. To achieve high protein concentrations, rhuMAb HER2 can be formulated at a protein concentration of 25mg/mL into: (a)5mM sodium succinate, pH5.0 or (b)5mM histidine, pH6.0 and 60mM trehalose, 0.01% Tween 20^TMNeutralized and lyophilized. 18mL of the protein formulation was dispensed into 50cc vials for lyophilization. In the freeze dryer, vials were frozen at a shelf temperature of about-55 ℃ for about 5 hours, then primary dried at a shelf temperature of 5 ℃ at a pressure of 150mTorr for 30 hours, and secondary dried with a shelf temperature of 20 ℃ for 10 hours to dry to 1-2% residual moisture. A thermocouple placed in the formulation containing the ineffective control (protein-free formulation) indicated that the product in the vial remained below-10 ℃ throughout the primary drying. Sequential hold studies in lyophilization show that residual moisture after primary drying is typically less than 10%.

The lyophilized protein is then reconstituted with 4 or 20mL BWFI (0.9 or 1.1% benzyl alcohol) to produce a concentrated protein solution:

(a)4mL of: 102mg/mL rhuMAb HER2, 245mM trehalose, 21mM sodium succinate, pH5.0 or 21mM histidine, pH6.0, 0.04% Tween 20^TM；

(b)20mL of: 22mg/mL rhuMAb HER2, 52mM trehalose, 4mM sodium succinate, pH5.0 or 4mM histidine, pH6.0, 0.009% Tween 20^TM。

After the lyophilized preparation was stored at 40 ℃ for 4 weeks, a protein concentration of 22mg/mL was established, and the amount of protein aggregation showed a slight increase as the trehalose concentration decreased. The stability of the lyophilized protein is not affected by the reconstituted volume. As shown in FIG. 1, the amount of intact protein after reconstitution with 4 or 20mL BWFI after incubation of lyophilized protein (60mM trehalose, 5mM sodium succinate, pH5.0, 0.01% Tween 20. TM.) at 40 ℃ was the same.

The results in Table 3 show a relationship between trehalose concentration and protein stability. To further confirm this relationship, sodium succinate or histidine preparations containing trehalose at various formulation concentrations were incubated for 91 days at 40 ℃. The stability as a function of the molar ratio of trehalose to protein at each trehalose concentration was then determined. As shown in fig. 2, there was a significant decrease in protein stability in both formulations as the trehalose concentration was decreased. There was no significant difference between the two different buffers used in these formulations, succinate and histidine, indicating that the primary stabilizer under these conditions was trehalose. Furthermore, the reduction in intact protein found in both formulations is acceptable even for the low trehalose concentration formulations stored at 2-8 ℃ during their shelf life. However, if room temperature stabilization is to be controlled (maximum temperature of 30 ℃), a higher trehalose concentration may be required (trehalose: protein ratio ≧ 600: 1) according to the product stability criteria (criteria for the amount of intact protein remaining after 2 years of storage). Generally, controlled room temperature storage conditions for stable storage at 40 ℃ for 6 months are equivalent to storage at 30 ℃ for 2 years.

As shown in FIG. 3, the 250mM trehalose formulation was preserved at 40 ℃ for 6 months without change, while the 60mM trehalose formulation was less stable. If the end of the shelf life of the product is standardized to greater than 98% intact protein, as determined by non-denaturing size exclusion chromatography, for example, then a formulation with a trehalose concentration of 60mM will require cryopreservation.

In previous screening studies, sucrose was also found to prevent aggregation of rhuMab HER2 after lyophilization and upon subsequent storage. To obtain an isotonic solution for subcutaneous administration (approximately 4-fold concentration of formulation components and proteins) after reconstitution, the sucrose concentration must be greatly reduced. In screening studies, the use of sucrose and mannitol (bulking agent) at the same mass concentration prevented protein aggregation. Lower concentrations of sucrose and mannitol (same mass concentrations) are now selected for an effective subcutaneous formulation of rhuMAb HER 2. Lyophilized protein solution (25mg/mL protein, 5mM group)Amino acid, pH6.0, 38.4mM (7mg/mL) mannitol, 20.4mM (7mg/mL) sucrose, 0.01% Tween 20^TM) The procedure used was the same as for the preparation of 60mM trehalose except that the primary drying period was extended to 54 hours. After 4 weeks at 40 ℃, the amount of aggregates increased slightly upon reconstitution with 4.0 or 20.0mL BWFI (Table 3). The amount of protein aggregation was the same as that obtained for reconstitution to a protein concentration of 22 or 100mg/mL (FIG. 4). Like the 60mM trehalose formulation, the mannitol/sucrose formulation will form less intact protein mass when stored at 40 ℃ for a certain period of time. The molar ratio of sucrose to protein in this formulation was 120 to 1, indicating that the mannitol/sucrose combination is more effective than trehalose alone at the same molar ratio of stabilizing sugars (fig. 2 and 4).

In the previous examples, the stability of lyophilized rhuMAb HER2 formulations was determined as a function of temperature. These studies demonstrated that trehalose and mannitol/sucrose formulations can prevent protein degradation in the lyophilized state at high temperatures (40 ℃). However, these tests do not determine the stability of the protein after reconstitution and storage. Lyophilized formulations of rhuMAb HER2 are useful for multiple administrations when reconstituted with BWFI. In particular, the vial capacity (450mg of rheMAb HER2) was designed to provide an average of three patients with drug (130mg of rhuMAb HER2 per dose). Since the drug was administered once a week, the vials had to be stored for at least three weeks after reconstitution, and to confirm that rhuMAb HER2 remained stable after reconstitution, stability studies were now conducted on the reconstituted rhuMAb HER2 formulations at 5 ℃ and 25 ℃.

For subcutaneous administration, the formulation can be reconstituted to a protein concentration of 100mg/mL (4mL BWFI). At this high protein concentration, the intravenous formulation of protein reconstituted to 22mg/mL protein (20mL BWFI) is more prone to aggregation. The four rhuMAb HER2 preparations of the previous example were tested for aggregation (loss of intact protein). As shown in tables 4 to 6, there was no difference in the stability of the formulations reconstituted to protein concentrations of 22 and 100 mg/mL. Moreover, these formulations can retain the protein intact for 90 days at 5 ℃ and 30 days at 25 ℃, indicating that the reconstituted formulations can be stored frozen for at least 90 days. Unlike the lyophilized protein stability of the previous example, trehalose concentration in this formulation had no effect on protein stability (table 7).

TABLE 4

Protein concentration of 25mg/mL rhuMAb HER2 in 5mM sodium succinate, pH5.0, 60mM trehalose, 0.01% Tween 20^TMIn the form of lyophilized preparation

Time (days)	Percentage of intact protein
					22mg/mL protein		100mg/mL protein
	5℃	25℃	5℃	25℃
					0	99.9	99.9	99.7	99.7
14	ND	100.0	ND	100.0
					30	100.0	100.0	100.0	100.0
91	99.8	ND	100	ND

The sample was reconstituted with 4.0mL or 20.0mL BWFI (1.1% or 0.9% benzyl alcohol) and then stored at 5 ℃ or 25 ℃. The percentage of intact protein was determined by non-denaturing size exclusion chromatography, defined as the fraction of the non-denaturing peak area (fraction). ND is not measured.

TABLE 5

Protein concentration of 25mg/mL rhuMAb HER2 in 5mM histidine, pH6.0, 60mM trehalose, 0.01% Tween 20^TMIn the form of lyophilized preparation

Time (days)	Percentage of intact protein
					22mg/mL protein		100mg/mL protein
	5℃	25℃	5℃	25℃
					0	100.0	100.0	100.0	100.0
14	ND	100.0	ND	100.0

31	99.3	99.7	100.0	100.0
					61	100.0	ND	ND	ND

The sample was reconstituted with 4.0mL or 20.0mL BWFI (1.1% or 0.9% benzyl alcohol) and then stored at 5 ℃ or 25 ℃. The percentage of intact protein was determined by non-denaturing size exclusion chromatography, defined as the fraction of non-denaturing peak area. ND is not measured.

TABLE 6

Protein concentration of 25mg/mL rhuMAb HER2 in 5mM histidine, pH6.0, 38.4mM mannitol, 20.4mM sucrose, 0.01% Tween 20^TMIn the form of lyophilized preparation

Time of flightTime (days)	Percentage of intact protein
					22mg/mL protein		100mg/mL protein
	5℃	25℃	5℃	25℃
					0	99.7	99.7	99.8	99.8
14	ND	100.0	ND	99.8
					31	100.0	100.0	100.0	100.0
92	100.0	ND	100	ND

The sample was reconstituted with 4.0mL or 20.0mL BWFI (1.1% or 0.9% benzyl alcohol) and then stored at 5 ℃ or 25 ℃. The percentage of intact protein was determined by non-denaturing size exclusion chromatography, defined as the fraction of non-denaturing peak area. ND is not measured.

TABLE 7

Protein concentration of 21mg/mL rhuMAb HER2 in 10mM sodium succinate, pH5.0, 250mM trehalose, 0.2% Tween 20^TMIn the form of lyophilized preparation

Time (days)	Percentage of intact protein 21mg/mL protein
			5℃	25℃
	0	99.8			99.8
14			ND	100.0
	31	99.9			99.4
92			99.8	ND

The sample was reconstituted with 20.0mL BWFI (0.9% benzyl alcohol) and stored at 5 ℃ or 25 ℃. The percentage of intact protein was determined by non-denaturing size exclusion chromatography, defined as the fraction of non-denaturing peak area. ND is not measured.

As described above, the major degradation pathway of rhuMAb HER2 in aqueous solution is deamidation or succinimide formation. The four reconstituted rhuMAb HER2 preparations were now assayed for non-denatured protein lost due to deamidation or succinimide formation.

Deamidation and succinimide formation of rhuMAb HER2 can be analyzed by cation exchange chromatography. The flow rate of a Bakerboom Wide-Pore Carbon Sulfon (CSX) column (4.6X 250mm) was 1 mL/min. The mobile phase buffer was (A)0.02M sodium phosphate, pH6.9 and (B)0.02M sodium phosphate, pH6.9, 0.2M NaCl. Chromatography was carried out at 40 ℃ in the following manner:

TABLE 8

Time (minutes)	Percentage of buffer B
		0	10
55	45
		57	100
62	100
		62.1	10
63	10

The peak was detected at 214nm and the amount of protein loaded was 75. mu.g per assay.

Likewise, there was no difference in the stability of the formulations reconstituted to protein concentrations of 22mg/mL and 100mg/mL (FIGS. 5 to 7). The protein degradation rate at 25 ℃ was faster than 5 ℃ for each formulation, while the degradation rate was similar for all formulations deposited at 5 ℃. The degradation rate of the histidine containing formulation was slightly faster than the succinate formulation at 25 ℃. Trehalose concentrations in the formulations had no effect on the degradation rate at both temperatures (fig. 5 to 8). These results demonstrate that the degradation rates of these four formulations are acceptable under refrigerated conditions (5 ℃) during the intended use period (within 30 days after reconstitution with BWFI).

Multiple use formulations must pass the preservative test as described in the United States Pharmacopeia (USP) for use in the united states. Adding protein 25mg/mL, histidine 5mM, pH6.0, and sea water 60mMTrehalose, 0.01% tween 20^TMThe lyophilized formulation of rhuMAb HER2 was reconstituted with 20mL of benzyl alcohol at a concentration w/w of 0.9 to 1.5%. When the concentration was equal to or greater than 1.3% w/w, the reconstituted formulation became cloudy after overnight incubation at room temperature (. about.25 ℃). Reconstitution with a standard BWFI formulation (0.9% benzyl alcohol) will not allow the solution to consistently pass the preservative test. However, formulations reconstituted with 1.0 or 1.1% formulation compatible concentrations of benzyl alcohol can pass the preservative test. The product specifications require a solution concentration within 10%, so the lyophilized formulation is reconstituted with 1.1% benzyl alcohol (1.1 + -0.1%).

A single step freeze-drying process of rhuMAb HER2 formulation is now investigated. In a single step freeze drying process, 25mg/mL rhuMAb HER2, 60mM trehalose, 5mM histidine (pH6), and 0.01% polysorbate 20 were lyophilized at a shelf temperature of 20 ℃ under a pressure of 150 mTorr. After 47 hours, the residual moisture content of the lyophilized cake was less than 5%. This freeze drying process is considered advantageous because it eliminates the secondary drying step, simplifying the manufacturing process.

Example 2

anti-IgE formulations

IgE antibodies bind to specific high affinity receptors on mast cells, causing the mast cells to degranulate and release mediators such as histamine which produce symptoms of allergy, and thus anti-IgE antibodies which block the binding of IgE to high affinity receptors are of high therapeutic value for the treatment of allergic diseases. These antibodies also fail to bind IgE after binding of IgE to the receptor, as this triggers the release of histamine. This example describes the development of a lyophilized formulation containing the full length humanized anti-IgE antibody MaE11(J.immunology, 151: 2623-2632(1993)) as described by Presta et al.

Materials: highly purified rhuMAb E25 (recombinant humanized anti-IgE antibody MaE11), which contained no Tween 20 in the formulations used below^TM. Spectra/Por7 dialysis membranes were purchased from Spectrum (Los Angeles, Calif.). All other reagents in this study were commercially available and of analytical grade. Formulation buffer andthe chromatographic mobile phase can be prepared by mixing the appropriate amounts of buffer and salt with Milli-Q grade water in a volumetric flask.

Preparation: the E25S sepharose pool was dialyzed against a specific formulation buffer. Dialysis was achieved by exchanging 4X 2L of minimal exchange buffer at 2-8 ℃ for 48 hours. After dialysis, lyoprotectants were added to some of the formulations as needed to form isotonic concentrations. The protein concentration after dialysis was measured with a 1.60 molar absorption coefficient uv spectrophotometer. The dialyzed protein was diluted to the desired formulation concentration with the appropriate formulation buffer, sterile filtered with a 0.22 μm Millex-GV filter (Millipore), and dispensed into previously washed and autoclaved glass vials. The vials were fitted with silanized teflon stoppers and then freeze dried under the following conditions: the E25 formulation was frozen at a rate of 80 ℃/hour to-55 ℃ and the vial contents were kept frozen for 4 hours. The temperature was then raised to 25 ℃ at a rate of 10 ℃/hour for primary drying. Primary drying was carried out at 25 ℃ under 50 μ chamber pressure (primer vacuum pressure) for 39 hours, so that the residual moisture content in the lyophilized cake was 1-2%. After freeze-drying, one vial of each formulation was removed and analyzed for t-0, while the remaining vials were analyzed at different temperatures, e.g., -70 ℃, 2-8 ℃, 25 ℃, 30 ℃ (controlled room temperature), 40 ℃ and 50 ℃.

And (3) chromatography: at Bio-Rad Bio-Select^TMNon-denaturing size exclusion chromatography was performed on a SEC 250-5 column (300X 7.8 mm). The column was flow washed with PBS at a flow rate of 0.5mL/min in equilibrium and using a Hewlett Packard model 1090L HPLC equipped with a diode-array detector. The column was calibrated with molecular weight standards (Bio-Rad, Inc.) consisting of thyroglobulin (670kd), gamma-globulin (158kd), ovalbumin (44kd) and vitamin B12(1.35 kd). The sample loading was 25 μ g and protein was obtained by monitoring uv absorbance at 214nm using Turbochrom 3 software (PE Nelson, Inc.).

Hydrophobic interaction chromatography: the F (ab') 2 fragment of the E25 antibody was chromatographed on a TosoHass Butyl-NPR column (3.5X 4.6mm) and a Hewlett Packard model 1090L HPLC equipped with a diode array detector. Elution buffer a was: 20mM Tris, 2M ammonium sulfate, 20% (v/v) glycerol, pH8.0, and elution buffer B is 20mM Tris, 20% (v/v) glycerol, pH 8.0. The column was equilibrated with 10% elution buffer B at a flow rate of 1.0mL/min for at least 20 minutes. The sample loading was 5 μ g and protein was obtained by monitoring uv absorbance at 214nm using Turbochrom 3 software (PE Nelson, Inc.). After sample injection, the column was maintained in 10% buffer B for 1 min, then eluted with a linear gradient of buffer B from 10% to 62% over 20 min. The column was washed with 100% buffer B for 5 minutes and then re-equilibrated with 10% buffer B for at least 20 minutes between successive sample injections.

Antibody binding activity: the IgE receptor binding inhibition assay (IE 25: 2) was performed on samples diluted to 20. mu.g/mL and 30. mu.g/mL with assay diluents (phosphate buffer, 0.5% BSA, 0.05% polysorbate 20, 0.01% Thimerosol). The assay was then repeated three times for each dilution and the results multiplied by the appropriate dilution factor to generate the active concentration. The results of 6 measurements were averaged. This assay measures the ability of rhuMAb E25 to competitively bind IgE thereby preventing IgE binding to high affinity receptors immobilized on ELISA plates. The result is divided by the antibody concentration measured by ultraviolet absorbance spectrophotometry to obtain the specific activity value. Previous tests have shown that this measurement can be indicative of stability.

Granulation test: the reconstituted formulation of lyophilized rhuMAb E25 was combined to obtain a volume of approximately 7 mL. The number of particles having a particle size of between 2 and 80 μm in 1mL of sample was determined using a Hiac/Royo type 8000 counter. The counter was first washed three times with 1mL of sample and then the measurement of 1mL of sample was repeated three times. The instrument measures the number of particles having a particle size of 10 μm or more and the number of particles having a particle size of 25 μm or more per ml.

The first step in the preparation of anti-IgE antibodies is to determine the appropriate buffer and pH at which the product is lyophilized and stored. The antibody was formulated at a concentration of 5.0mg/mL into 10mM succinate buffer at pH5.0 to 6.5 and sodium, potassium and histidine phosphate buffer at pH 7.0. Figure 9 shows the increase in the amount of antibody coacervate in the higher pH formulations before and after lyophilization. One exception was the histidine preparation at pH7, which was found to show no increase in the amount of coacervate when stored at 2-8 ℃. FIG. 10 shows the aggregation of rhuMAb E25 after lyophilization in 5mM histidine buffer at pH6 and pH7 and storage at 2-8 deg.C, 25 deg.C and 40 deg.C for 1 year. The amount of aggregate in the formulation at pH6 was less at each assay time point and storage temperature than in the antibody formulation at pH7. This result indicates that histidine at pH6 is particularly suitable for use as a buffer system to prevent antibody aggregation.

To achieve screening for lyoprotectants, anti-IgE antibodies can be formulated into sodium succinate at pH5 with or without lyoprotectant. Effective lyoprotectants added to isotonic concentrations can be divided into 3 classes:

(a) non-reducing monosaccharides (i.e., mannitol);

(b) reducing disaccharides (i.e., lactose and maltose); and

(c) non-reducing disaccharides (i.e., trehalose and sucrose).

The aggregation of the formulations after 1 year storage at 2-8 ℃ and 40 ℃ is shown in FIGS. 11 and 12. The aggregation rate of the monosaccharide formulation (mannitol) was the same as in the buffer control when stored at 2-8 ℃, while the disaccharide containing formulation was very effective in controlling aggregation (fig. 11). The results were the same when stored at 40 ℃ except that the sucrose formulation had a rapid aggregation (which correlates to a brownish yellow reaction in the lyophilized cake (fig. 12)). This will later be shown to be caused by degradation of sucrose when stored at acidic pH and high temperatures.

Hydrophobic interaction chromatography of the antibody formulated into histidine buffer at pH6 and lactose showed that the antibody would change after 6 months of storage at 40 ℃ (fig. 13). The chromatographic peak broadens and the retention time decreases. As shown in fig. 14 and 15, these changes were not observed when the buffer control and sucrose formulation were stored under similar conditions. And isoelectric focusing showed acidic shifts in pI of the lactose formulated antibody stored at 25 ℃ and 40 ℃. This suggests that reducing sugars are not suitable for use as lyoprotectants for antibodies.

FIG. 16 shows the aggregation of lyophilized formulations of anti-IgE at 20mg/mL in 5mM histidine buffer, pH6, and various concentrations of sucrose and trehalose, after 12 weeks at 50 ℃. When the sugar concentration per mole of antibody is more than 500 moles, the effect of preventing aggregation of both sugars is similar. Based on these results, further studies were conducted on isotonic and hypertonic formulations of sucrose and trehalose. The formulation is added to a lower concentration of antibody prior to lyophilization and the lyophilized product is reconstituted in an amount less than the amount of bacteriostatic for injection (BWFI) containing 0.9% benzyl alcohol. This allows the antibody to be concentrated prior to subcutaneous delivery and includes a preservative for multiple use formulations while avoiding the effects of protein and preservative on long term storage.

Isotonic preparation: the concentration of anti-IgE formulated in 5mM histidine buffer pH6 was 25mg/mL with 500 moles of sugar per mole of antibody, i.e., the sugar concentration was equal to 85 mM. This preparation was reconstituted with BWFI (0.9% benzyl alcohol) 4-fold less than the original addition volume. This resulted in an antibody concentration of 100mg/mL in 20mM histidine at pH6, and an isotonic sugar concentration of 340 mM.

Hypertonic preparations: the concentration of anti-IgE formulated in 5mM histidine buffer pH6 was 25mg/mL, with 1000 moles of sugar per mole of antibody, i.e., the sugar concentration was equal to 161 mM. This preparation was reconstituted with BWFI (0.9% benzyl alcohol) 4-fold less than the original addition volume. This resulted in an antibody concentration of 100mg/mL in 20mM histidine at pH6 and a hypertonic sugar concentration of 644 mM.

A comparison of antibody aggregation for the isotonic and hypertonic formulations up to 36 weeks after storage is shown in figures 17 to 19. No agglomeration change was observed in either the hypertonic or isotonic formulations stored at 2-8 deg.C (FIG. 17). Whereas no increase in aggregates was observed in hypertonic solutions when stored at controlled room temperature (30 ℃), there was an increase of aggregates of about 1 to 2% in isotonic formulations (FIG. 18). Finally, a small increase in coagulum was found in the hypertonic formulations, 4% in the isotonic trehalose formulations and 12% in the isotonic sucrose formulations when stored at 50 ℃ (fig. 19). These results indicate that the isotonic formulation should contain the minimum amount of sugar necessary to maintain stability of the antibody stored at up to 30 ℃.

The binding activity of anti-IgE in isotonic and hypertonic formulations can be determined by IgE receptor inhibition assays. It was found that the binding activity of isotonic and hypertonic sucrose and trehalose formulations was substantially unchanged after 36 weeks of storage at-70 deg.C, 2-8 deg.C, 30 deg.C and 50 deg.C.

Lyophilized formulations are known to contain insoluble aggregates or particles (Cleland et al, Critical reviews in Therapeutic Drug Carrier Systems, 10 (4): 307-377 (1993)). Thus, the granulation assay was performed on lyophilized antibody at a concentration of 25mg/mL in 5mM histidine at pH6 with 85mM and 161mM sucrose and trehalose added. Polysorbate 20 was added to concentrations of 0.005%, 0.01% and 0.02%. The samples were lyophilized and reconstituted to 20mM histidine, pH6 with an antibody concentration of 100mg/mL and sugar concentrations of 340mM and 644 mM. The concentration of polysorbate 20 after reconstitution was 0.02%, 0.04%, and 0.08%, respectively.

Table 9 below shows the number of particles having a particle size of 10 μm or more and the number of particles having a particle size of 25 μm or more in the formulations of sucrose and trehalose, both isotonic and hypertonic. Polysorbate 20 was added to the formulation to concentrations of 0.005%, 0.01% and 0.02% prior to lyophilization. The results show that Tween (Tween) is added into the preparation^TMThe number of particles in each tested particle size range can be greatly reduced. The standard of the United States Pharmacopeia (USP) for small injected doses is: the number of particles having a particle size of 10 μm or more in each container is not more than 6000 and the number of particles having a particle size of 25 μm or more is not more than 600 (Cleland et al, supra). Both hypertonic and isotonic formulations passed the test after addition of polysorbate 20.

TABLE 9

Preparation	Polysorbate 20	Number of particles per ml
				≥10μm	≥25μm
		Isotonic sucrose	Is not provided with			16122	28
	0.005％			173	2
			0.01％			224	5
	0.02％			303	6
		Hypertonic sugar	Is not provided with			14220	84
	0.005％			73	6

	0.01％	51	0
					0.02％		6
Isotonic trehalose	Is not provided with	33407	24
					0.005％	569	4
	0.01％	991	16
					0.02％	605	9
Hypertonic trehalose	Is not provided with	24967	28
					0.005％	310	11
	0.01％	209	6
					0.02％	344	6

Table 10 below shows a formulation of an anti-IgE antibody (i.e., a 143mg bottled rhuMAb E25 isotonic formulation) that is believed to be suitable for subcutaneous delivery of the antibody. 5.7mL of 25mg/mL rhuMAb E25 was added to a 10cc vial, and rhuMAb E25 was formulated in 5mM histidine at pH6 and 0.01% polysorbate 20. Sucrose was added as lyoprotectant to a concentration of 85mM, corresponding to a molar ratio of 500 to 1 sugar to antibody. The vial was freeze dried and reconstituted with 0.9% benzyl alcohol to 1/4 or 1.2mL of the original additive volume. The final concentration of the formulation components increased 4-fold, resulting in a rhuMAb E25 concentration of 100mg/mL in 20mM histidine, pH6, 0.04% polysorbate 20, 340mM sucrose (isotonicity), and 0.9% benzyl alcohol. The formulation contained histidine buffer at pH6, as it was demonstrated to prevent antibody aggregation. Sucrose was added as a lyoprotectant due to previous use in the pharmaceutical industry. The sugar concentration is chosen so that upon reconstitution it is an isotonic formulation. Finally, polysorbate 20 may be added to prevent the formation of insoluble aggregates.

Watch 10

Preparation before lyophilization (5.7 mL in 10cc bottle)	Reconstituted formulation (1.2mL 0.9% benzyl alcohol)
		25mg/mL rhuMAb E25	100mg/mL rhuMAb E25
5mM histidine, pH6.0	20mM histidine, pH6.0
		85mM sucrose	340mM sucrose
0.01% polysorbate 20	0.04% polysorbate 20
		-	0.9% benzyl alcohol

Claims (41)

1. A formulation comprising a lyophilized mixture of a recombinant humanized anti-HER 2 monoclonal antibody, a lyoprotectant, and a buffer.

2. The formulation of claim 1, wherein the recombinant humanized anti-HER 2 monoclonal antibody is present at a concentration of 2-50mg/mL and the lyoprotectant is a sugar present at a concentration of 10-400 mM.

3. The formulation of claim 2, wherein the formulation has a pH of 5-7.

4. The formulation of claim 3, wherein the formulation has a pH of 6.

5. The formulation of any one of claims 1-4, wherein the buffer is a buffer of histidine, phosphate, Tris, citrate, succinate or other organic acid.

6. The formulation of claim 5, wherein the buffer is a histidine buffer.

7. The formulation of claim 6, wherein the buffer is a succinate buffer.

8. The formulation of any one of claims 1-7, wherein the antibody is huMAb4D 5-8.

9. The formulation of any one of claims 1-8, further comprising a surfactant.

10. The formulation of claim 9, wherein the surfactant is a non-ionic surfactant.

11. The formulation of claim 10, wherein the surfactant is a polysorbate.

12. The formulation of any one of claims 1-11, wherein the lyoprotectant is selected from the group consisting of: non-reducing sugars, monosodium glutamate, histidine, betaine, magnesium sulfate, trihydric or higher sugar alcohols, propylene glycol, polyethylene glycol, Pluronic, and combinations thereof.

13. The formulation of any one of claims 1-12, which is stable for at least 6 months at 30 ℃.

14. The formulation of any one of claims 1-13, wherein the humanized anti-HER 2 monoclonal antibody is at a concentration of about 5-40 mg/mL.

15. The formulation of claim 14, wherein the humanized anti-HER 2 monoclonal antibody is at a concentration of about 20-30 mg/mL.

16. The formulation of any one of claims 1-15, which is in lyophilized form.

17. A lyophilized formulation comprising a recombinant humanized anti-HER 2 monoclonal antibody at a concentration of about 5-40mg/mL, such as 20-30mg/mL, sucrose or seaweed at a concentration of about 10-100mM, such as 40-80mM, a buffer and a surfactant.

18. The lyophilized formulation of claim 17, wherein the water content in the formulation is less than 5%, preferably less than 3%.

19. A vial containing the formulation of any one of claims 1-18.

20. The vial of claim 19, which is a 3, 5, 10, 20, 50 or 100cc vial.

21. The vial of claim 19 or 20, wherein the antibody is huMAb4D 5-8.

22. The vial of any one of claims 19-21, wherein the formulation has a pH of 5-7.

23. The vial of claim 22, wherein the formulation has a pH of 6.

24. The vial of any one of claims 19-23, wherein the buffer is a buffer of histidine, phosphate, Tris, citrate, succinate or other organic acid.

25. A reconstituted formulation prepared by diluting the formulation of claim 16 or the lyophilized formulation of any one of claims 17-18 with a diluent.

26. The reconstituted formulation of claim 25, which has a pH of 5-7.

27. The reconstituted formulation of claim 26, which has a pH of 6.

28. The reconstituted formulation of any one of claims 23-27, wherein the humanized anti-HER 2 monoclonal antibody is at a concentration of about 5-50 mg/mL.

29. The reconstituted formulation of claim 28, wherein the humanized anti-HER 2 monoclonal antibody is at a concentration of about 10-40mg/mL

30. The reconstituted formulation of any one of claims 25-29, wherein the humanized anti-HER 2 monoclonal antibody is huMAb4D 5-8.

31. The recombinant formulation of any one of claims 25-30, wherein the diluent is selected from the group consisting of: sterile water, bacteriostatic agent for injection (BWFI), pH buffer solution, sterile salt solution, ringer's solution or glucose solution.

32. Use of a formulation comprising a recombinant humanized anti-HER 2 monoclonal antibody in the manufacture of a medicament for the treatment of breast cancer that is overexpressed from HER2, wherein the formulation comprises a lyophilized mixture of the recombinant humanized anti-HER 2 monoclonal antibody, a lyoprotectant, and a buffer, wherein the lyophilized mixture is reconstituted with a diluent to produce a reconstituted formulation.

33. The use of claim 32, wherein the formulation has a pH of 5-7.

34. The use of claim 33, wherein the formulation has a pH of 6.

35. The use according to any one of claims 32-34, wherein the buffer is a buffer of histidine, phosphate, Tris, citrate, succinate or other organic acid.

36. The use of any one of claims 32-35, wherein the concentration of humanized anti-HER 2 monoclonal antibody in the reconstituted formulation is about 5-50 mg/mL.

37. The use of any one of claims 32-36, wherein the concentration of humanized anti-HER 2 monoclonal antibody in the reconstituted formulation is about 10-40 mg/mL.

38. The use according to any one of claims 32-37, wherein the antibody is huMAb4D 5-8.

39. The use according to any one of claims 32-38, wherein the reconstituted formulation has a pH of 6.

40. The use according to any one of claims 32-39, wherein the diluent is sterile water.

41. The use of any one of claims 32-39, wherein the diluent is bacteriostatic for injection (BWFI).