EXCIPIENT FOR BIOTHERAPEUTICS

Abstract

The present invention relates to excipients for stabilizing active agents, in particular peptides, polypeptides, nucleic acids, viruses, virus-like particles, proton pump inhibitors and antibiotics. The excipient reduces aggregate and/or particle formation in preparations comprising said agents. The excipient is a diamide of a dicarboxylic acid comprising at least one N—H amido group, at least one unsubstituted or substituted N-hydroxyethylamido group and/or at least one unsubstituted N-hydroxymethylamido group. In particular, the excipient is N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide.

Claims

1. A method for stabilizing an active agent, comprising combining a diamide of a dicarboxylic acid wherein said diamide comprises at least one N—H amido group, at least one unsubstituted or substituted N-hydroxyethyl amido group and/or at least one unsubstituted or substituted N-hydroxymethyl amido group with an active agent, wherein the active agent is selected from the group consisting of peptides, polypeptides, nucleic acids, viruses or virus-like particles, proton pump inhibitors and antibiotics.

2. The method of claim 1, wherein the diamide is a compound of Formula (I): ##STR00011## wherein each R.sup.1 is independently selected from H and C.sub.1-C.sub.10 hydrocarbon residues, said hydrocarbon residues optionally comprising at least one heteroatom, and wherein two R.sub.1 together may form a ring, with the proviso that at least one R.sup.1, e.g. 1, 2, 3 or 4 of R.sup.1 is H, a group of Formula (II) or a group of Formula (III): ##STR00012## wherein each R.sup.2 is independently selected from H and C.sub.1-8 hydrocarbon residues, particularly from C.sub.1-C.sub.2 hydrocarbon residues, said hydrocarbon residues optionally comprising at least one heteroatom, and wherein A is selected from linear, branched or cyclic C.sub.1-C.sub.24 hydrocarbon residues, particularly from linear or branched C.sub.1-C.sub.6 hydrocarbon residues or cyclic C.sub.3-C.sub.6 residues, said hydrocarbon residues optionally comprising at least one heteroatom.

3. The method of claim 1, wherein the active agent is stabilized in a liquid preparation, particularly in an aqueous solution, particularly wherein (i) the concentration of the active agent in the preparation is in the range from about 0.01 mg/ml to about 300 mg/ml, of about 0.1 mg/ml to about 200 mg/ml or of about 1 mg/ml to about 150 mg/ml, and/or wherein (ii) the concentration of the diamide in the preparation is in the range of about 1 μmol/l to about 1 mol/l, of about 100 mmol/l to about 500 mmol/l, of about 1 mmol/l to about 250 mmol/l or of about 10 mmol/l to about 100 mmol/l.

4. The method of claim 1, wherein the active agent is selected from antibodies such as IgG antibodies, antibody derivatives, immunoglobulin fusion proteins, interferons, interleukins, interleukin receptors, agonists and antagonists of interleukins and interleukin receptors, agonists and antagonists of members of the TNF family and TNF family receptors, cytokines, and enzymes.

5. The method of claim 4, wherein the active agent is selected from trastuzumab, rituximab, omalizumab, interferon-alpha, G-CSF and anakinra.

6. The method of claim 1, further comprising a buffer suitable for use in the field of medicine, cosmetics, diagnostics, and/or analytics.

7. The method of claim 1, wherein the diamide has a solubility in water of at least about 0.02% (w/v), of at least about 0.05% (w/v), of at least about 0.1% (w/v), of at least about 0.5% (w/v) or of at least about 1% (w/v) at 20° C.

8. The method of use of claim 1, wherein the diamide is N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide.

9. The method of claim 1, wherein said active agent is stabilized against aggregation and/or particle formation, particularly against aggregation and/or particle formation from freezing/thawing stress, shaking stress and/or stirring stress.

10. A preparation comprising an active agent and a compound of Formula (I): ##STR00013## wherein each R.sup.1 is independently selected from H and C.sub.1-C.sub.10 hydrocarbon residues, said hydrocarbon residues optionally comprising at least one heteroatom, and wherein two R.sub.1 together may form a ring, with the proviso that at least one R.sup.1 e.g. 1, 2, 3 or 4 of R.sup.1 is H, a group of Formula (II) or a group of Formula (III): ##STR00014## wherein each R.sup.2 is independently selected from H and C.sub.1-8 hydrocarbon residues, e.g. 1, 2, 3 or 4 of R.sup.1 is H, said hydrocarbon residues optionally comprising at least one heteroatom, wherein A is selected from linear, branched or cyclic C.sub.1-C.sub.24 hydrocarbon residues, particularly from linear or branched C.sub.1-C.sub.6 hydrocarbon residues or cyclic C.sub.3-C.sub.6 residues, said hydrocarbon residues optionally comprising at least one heteroatom, and wherein the active agent is selected from peptides, polypeptides, nucleic acids, viruses or virus-like particles, proton pump inhibitors and antibiotics.

11. The preparation of claim 10, which is a liquid preparation, particularly an aqueous solution.

12. The preparation of claim 10, wherein the compound of Formula (I) is N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide.

13. The preparation of claim 10, wherein the active agent is selected from the group consisting of antibodies, antibody derivatives, immunoglobulin fusion proteins, interferons, interleukins, interleukin receptors, agonists and antagonists of interleukins and interleukin receptors, agonists and antagonists of members of the TNF family and TNF family receptors, cytokines, and enzymes.

14. The preparation of claim 10, which does not contain a surfactant, particularly a surfactant selected from polysorbates, poloxamers, solutol HS15 or SDS.

15. The preparation of claim 10, further comprising a buffer suitable for use in medicine.

Description

FIGURE LEGENDS

[0129] FIG. 1: Amount of particles after 3 freezing/thawing cycles. 50 mM phosphate buffer, pH 7.0; 5 mg/ml antibody IgG trastuzumab.

[0130] FIG. 2: Amount of particles after stirring stress. 50 mM phosphate buffer, pH 7.0; 5 mg/ml antibody IgG trastuzumab.

[0131] FIG. 3: Amount of particles after 3 freezing/thawing cycles. 50 mM phosphate buffer, pH 7.0; 1 mg/ml interferon-alpha-2a.

EXAMPLES

Example 1: Materials and Methods

[0132] Production of Test Solutions: The buffer solution consisted of 50 mM sodium phosphate at pH 7.0. Protein stock solutions were liberated from other excipients by IEX chromatography to remove potential impurities and surfactants and by dialysis over 24 h in buffer solution 100-200 times of their volumes. The buffer solution was renewed after 3 hours and after 14 hours. Stock solutions of test excipients were produced by dissolving 500 mM of the excipient in 90% of the required amount of buffer solution. After that, the pH was adjusted and the remaining volume of buffer solution was added. Then, the solution was filtered with a 0.22 μm filter. The corresponding amount of protein stock solution was added. Sufficient homogenization was provided.

[0133] Freeze/Thaw-Cycle: The protein-containing solutions were filled into cleaned 2R vials and crimped. The samples were frozen from 20° C. to −50° C. in 3 cycles at a rate of 2K/min in a Christ 2D-6 freeze dryer and then thawed at room temperature until the entire sample had reached the liquid state, before the cycle was started again.

[0134] Stirring Stress: The protein-containing solutions were filled into cleaned 2R vials and crimped. Afterwards, the samples were stirred for 2 h at 200 rpm with a magnetic stirrer (Variomag Poly 15, Thermofisher, 3 mm polytetrafluoroethylene—coated stirring bars).

[0135] Flow Imaging Microscopy: 165 μl of the sample solution were measured at 10× magnification using a flow imaging microscope (FlowCam, Fluid Imaging Technologies, Inc., Scarborough, Me., USA).

[0136] Size Exclusion Chromatography (Antibodies): The samples were analyzed by a Dionex Summit Chromatography system. As solid phase, a Superdex 200 Increase 10/300 GL Column (GE Healthcare) was used. The mobile phase was a 50 mM phosphate solution with 200 mM NaCl at pH 7.0. The monomer elution was detected by means of absorption of the UV signal at a wavelength of 280 nm. Recovery was defined as the proportion of the areas under the absorption curve of the monomer peak before and after stress.

[0137] Size Exclusion Chromatography (Interferon-alpha-2a): The samples were analyzed by a Dionex Summit Chromatography system. As solid phase, a Superose 12 10/300 GL Column (GE Healthcare) was used. The mobile phase was a 50 mM phosphate solution with 200 mM NaCl at pH 7.0. The protein concentration was detected by means of UV at a wavelength of 280 nm.

Example 2: Stabilization of an IgG Antibody (Trastuzumab) with N,N,N′,N′-tetrakis-(2-hydroxyethyl) Adipinic Acid Amide

[0138] N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide (Ark Pharm Inc.), L-arginine (J. T. Baker) or D(+) trehalose (Sigma-Aldrich), respectively, was added as a test excipient to an aqueous solution containing the recombinant IgG antibody trastuzumab so that the resulting solution had an IgG concentration of 5 mg/ml and contained 50 mM of excipient. The resulting solutions were subjected to freezing/thawing stress and stirring stress.

[0139] It was shown that stressed formulations comprising N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide (compound A) contain less particles than formulations comprising standard excipients like, glycerol, NaCl, or D(+) trehalose or formulations without any excipients, and similar to L-arginine. Polysorbate 20 leads to even less particles after freeze/thaw experiments, but this class of excipients is not preferred (FIGS. 1 and 2).

Example 3: Stabilization of interferon-alpha-2a by N,N,N′,N′-tetrakis-(2-hydroxyethyl) Adipinic Acid Amide

[0140] N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide was added to an aqueous solution containing interferon-alpha-2a so that the resulting solution had a protein concentration of 1 mg/ml and contained 50 mM of excipient. The resulting solutions were subjected to freeze/thawing stress. It was shown that stressed formulations comprising N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide (compound A) contain less particles than formulations without any excipient and formulations with arginine or polysorbate 20 and similar amounts as trehalose (FIG. 3).

Example 4: Stabilization of the IgG Antibodies Omalizumab and Rituximab with N,N,N′,N′-tetrakis-(2-hydroxyethyl) Adipinic Acid Amide

[0141] Production of Test Solutions: The buffer solution consisted of 20 mM histidine buffer pH 6.0. Protein stock solutions were liberated from other excipients by dialysis over 24 h in buffer solution 100-200 times of their volumes. The buffer solution was renewed after 6 hours and after 20 hours. Stock solutions of test excipients were produced by dissolving 500 mM of the excipient in 90% of the required amount of buffer solution. After that, the pH was adjusted and the remaining volume of buffer solution was added. Then, the solution was filtered with a 0.22 μm filter. The corresponding amount of protein stock solution was added. Sufficient homogenization was provided.

[0142] Freeze/Thaw-Cycle: The protein-containing solutions were filled into cleaned 6R vials and crimped. The samples were frozen from 20° C. to −70° C. in 5 cycles in a freezer and then thawed at room temperature until the entire sample had reached the liquid state, before the cycle was started again.

[0143] Stirring Stress: The protein-containing solutions were filled into cleaned 6R vials and crimped. Afterwards, the samples were stirred for 2 h at 200 rpm with a magnetic stirrer (Variomag Poly 15, Thermofisher, 5 mm polytetrafluoroethylene—coated stirring bars).

[0144] Visual inspection: 3 Vials of each formulation were visually inspected according to the method described in the European Pharmacopoeia. Particular emphasis was given to the detection of turbidity.

[0145] N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide (Ark Pharm Inc.), D(+) trehalose (Sigma-Aldrich), sucrose and mixtures of trehalose or sucrose with methionine (Sigma-Aldrich) respectively, were added as a test excipient to an aqueous solution containing an recombinant IgG antibody, either rituximab or omalizumab. The resulting solutions had an IgG concentration of 10 mg/ml and contained 75 mM of excipient. In the case of sucrose or trehalose mixtures with methionine, the concentration of the sugar was 50 mM, and the concentration of methionine was 20 mM. The resulting solutions were subjected to freezing/thawing stress and stirring stress.

[0146] It was found that stressed formulations comprising N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide (compound A) contained less particles and were less turbid than formulations comprising standard excipients like, D(+) trehalose or sucrose, or formulations with mixtures of either trehalose with methionine or sucrose with methionine.

Example 5: Stabilization of the IgG Antibodies Rituximab and Omalizumab with Mixtures of N,N,N′,N′-tetrakis-(2-hydroxyethyl) Adipinic Acid Amide and Further Excipients

[0147] The production of test solutions, the freeze/thaw-cycle, the measurement of stirring stress and the visual inspection were performed as described in Example 4.

[0148] Mixtures of N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide (Ark Pharm Inc.) at a concentration of 30 mM with either D(+) trehalose (Sigma-Aldrich) at a concentration of 30 mM, or sucrose at a concentration of 30 mM respectively, were prepared as aqueous test excipient solutions and a recombinant IgG antibody either rituximab or omalizumab in 20 mM histidine buffer pH 6.0 was dialyzed into these solutions. As a comparison, solutions containing either trehalose or sucrose, both either in a concentration of 30 mM or 60 mM were prepared and the antibody in a 20 mM histidine buffer pH 6.0 was dialyzed into these solutions. The resulting solutions had an IgG1 concentration of 10 mg/ml and contained 60 mM of excipients and were subjected to freezing/thawing stress and stirring stress.

[0149] It was found that stressed formulations comprising N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide (compound A) in mixture with trehalose or sucrose contained less particles and were less turbid than formulations comprising standard excipients like, D(+) trehalose or sucrose alone.

Example 6: Stabilization of G-CSF by N,N,N′,N′-tetrakis-(2-hydroxyethyl) Adipinic Acid Amide

[0150] The production of test solutions, the freeze/thaw-cycle, the measurement of stirring stress and the visual inspection were performed substantially as described in Example 4.

[0151] The buffer solution consisted of 10 mM acetate buffer pH 4.5 and was renewed after 4 hours and after 16 hours. For the freeze/thaw cycle and the measurements of stirring stress, 2R vials were used.

[0152] N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide (Ark Pharm Inc.), D(+) trehalose (Sigma-Aldrich), sucrose, and mixtures of trehalose or sucrose with methionine (Sigma-Aldrich) respectively, was added as a test excipient to an aqueous solution containing recombinant G-CSF. The resulting solutions had a G-CSF concentration of 1 mg/ml and contained 50 mM of excipients. In the case of sucrose or trehalose mixtures with methionine, the concentration of the sugar was 50 mM, and the concentration of methionine was 20 mM. The resulting solutions were subjected to freezing/thawing stress and stirring stress.

[0153] It was found that stressed formulations comprising N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide (compound A) contained less particles and were less turbid than formulations comprising standard excipients like, D(+) trehalose, or sucrose, or formulations with mixtures of either trehalose with methionine or sucrose with methionine.

Example 7: Stabilization of Anakinra by N,N,N′,N′-tetrakis-(2-hydroxyethyl) Adipinic Acid Amide

[0154] The production of test solutions, the freeze/thaw-cycle, the measurement of stirring stress and the visual inspection were performed substantially as described in Example 4.

[0155] The buffer solution consisted of 20 mM citrate buffer pH 6.5. The samples were stirred for 3 h.

[0156] N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide (Ark Pharm Inc.), D(+) or trehalose (Sigma-Aldrich), or polysorbate 80, was added as a test excipient to an aqueous solution containing recombinant anakinra. The resulting solution had an anakinra concentration of 50 mg/ml and contained 50 mM of excipient. In the case of polysorbate 80 its concentration was 0.05%. The resulting solutions were subjected to freezing/thawing stress and stirring stress.

[0157] It was found that stressed formulations comprising N,N,N′,N′-tetrakis-(2-hydroxyethyl) adipinic acid amide (compound A) contained less particles and were less turbid than formulations comprising trehalose, and contained about equally low amounts of particles and were about equally low in turbidity than formulations containing polysorbate 80.

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