AQUEOUS COMPOSITION COMPRISING AT LEAST ONE PROTEIN AND ONE SOLUBILIZING AGENT, PREPARATION THEREOF AND USES THEREOF

Abstract

The present invention relates to a liquid composition which comprises, in an aqueous medium, one or more protein(s) and one or more solubilizing agent(s) chosen from the group consisting of anionic compounds of non-saccharide structure, said structure of which contains at least one aromatic nucleus comprising at least 6 ring members (6 atoms) and at least one carboxylic acid group in salified form, and which has, in its acid form, a molar mass of between 130 and 500 g/mol.

It also relates to the use of said solubilizing agent(s) for preparing compositions according to the invention.

It also relates to a process for solubilizing one or more protein(s), wherein at least one solubilizing agent chosen from the group consisting of anionic compounds of non-saccharide structure, said structure of which contains at least one aromatic nucleus comprising at least 6 ring members (6 atoms) and at least one carboxylic acid group in salified form, and which has, in its acid form, a molar mass of between 130 and 500 g/mol, is added to an aqueous protein preparation in order to solubilize the protein.

Claims

1. A process for solubilizing one or more protein(s) in water, wherein at least one solubilizing agent chosen from the group consisting of anionic compounds of non-saccharide structure, said structure of which contains at least one aromatic nucleus comprising at least 6 ring members (6 atoms) and at least one carboxylic acid group in salified form and which, in its acid form, has a molar mass of between 130 and 500 g/mol, is added to an aqueous protein composition.

2. A process for solubilizing proteins within an aqueous composition, of at least one solubilizing agent chosen from the group consisting of anionic compounds of non-saccharide structure, said structure of which contains at least one aromatic nucleus comprising at least 6 ring members (6 atoms) and at least one carboxylic acid group in salified form, and which has, in its acid form, a molar mass of between 130 and 500 g/mol.

3. The process for solubilizing agent(s) correspond(s) to general formula (I) below: ##STR00019## with: Ar denoting an aromatic nucleus comprising at least 6 ring members; X denoting a saturated or unsaturated, linear or branched divalent radical, the main chain of which consists of 1 to 4 carbon atoms and optionally 1 or 2 heteroatoms chosen from nitrogen and oxygen atoms, it being possible for said main chain to optionally bear one or more substituents; Y1 and Y2 denoting, independently of one another: a hydrogen atom; an OH group; an OR1 group with R1 denoting an alkyl radical containing from 1 to 6 carbon atoms or a hydroxyalkyl radical containing from 1 to 6 carbon atoms; and preferably Y1 and Y2 denote, independently of one another, a hydrogen atom or an OH group; and M denoting a cation.

4. The process as claimed in claim 3, wherein, in formula (I), Ar denotes a benzene nucleus or an indole nucleus.

5. The process as claimed in claim 3, wherein the solubilizing agent(s) correspond(s) to formula (Ia) below: ##STR00020## with X, Y1, Y2 and M as defined in claim 3.

6. The process as claimed in claim 3, wherein the solubilizing agent(s) correspond(s) to formula (Ib) below: ##STR00021## with X, Y1, Y2 and M as defined in claim 3.

7. The process as claimed in claims 3, wherein, in formula (I), (Ia) or (Ib), the divalent radical X bears, on its main chain, one or more substituents corresponding to the general formula:
-L-Z with: L denotes a single bond or a group chosen from an amide group NHCO, a carbamate group NHCOO or a urea group NHCONH; and Z denotes a hydroxyl group (OH); a saturated or unsaturated, linear or branched radical comprising from 1 to 4 carbon atoms, said carbon atoms bearing at least one hydroxyl group [CH2]X-[OH]Y; a salified carboxylic acid group (COOM with M=Na+or K+); or a saturated or unsaturated, linear or branched radical comprising from 1 to 12 carbon atoms and optionally one or more heteroatoms.

8. The process as claimed in claim 7, wherein L denotes a single bond or an amide group.

9. The process as claimed in claim 1, wherein the solubilizing agent is resulting from a natural or synthetic amino acid bearing an aromatic ring, chosen from phenylalanine, tyrosine and tryptophan, and phenylalanine.

10. The process as claimed in claim 1, wherein the solubilizing agent is chosen from the group consisting of N-hydroxyacetylphenylalanine and N-hydroxyacetyltryptophan, corresponding to the formulae below: ##STR00022## used in the form of a sodium salt or of a potassium salt.

11. The process as claimed in claim 1, wherein the solubilizing agent is resulting from a phenol.

12. The process as claimed in claim 1, wherein the solubilizing agent is chosen from the following compounds, used in the form of sodium salts or of potassium salts: TABLE-US-00005 Name Structure Phenylacetic acid Mandelic acid Hydrocinnamic acid Trans-cinnamic acid 2-Phenoxypropionic acid 3-Phenyllactic acid Phenylsuccinic acid Alpha-hydroxyhippuric acid

13. The process as claimed in claim 1, wherein said aqueous composition comprises the solubilizing agent(s) in a total concentration of between 1 g/l and 100 g/l.

14. The process as claimed in claim 1, wherein said aqueous composition contains a total concentration of protein(s) of between 0.5 and 400 mg/ml.

15. The process as claimed in claim 1, wherein the molar ratio between the total amount of solubilizing agent(s) and the total amount of protein(s) in the composition is greater than or equal to 20.

16. The process as claimed in claim 1, wherein said aqueous composition is intended to be administered by intravenous injection, by subcutaneous injection or by intramuscular injection.

17. A liquid composition comprising, in an aqueous medium, one or more protein(s) chosen from proteins containing at least one antibody fragment from monoclonal antibodies (mAbs), polyclonal antibodies, fusion proteins, nanobodies, bispecific antibodies and antibodies coupled to cytotoxic active ingredients (ADCsantibody-drug conjugates) and one or more solubilizing agent(s), wherein the solubilizing agent(s) correspond(s) to general formula (I) below: ##STR00031## with: Ar denoting an aromatic nucleus comprising at least 6 ring members; X denoting a saturated or unsaturated, linear or branched divalent radical, the main chain of which consists of 1 to 4 carbon atoms and optionally 1 or 2 heteroatoms chosen from nitrogen and oxygen atoms, it being possible for said main chain to optionally bear one or more substituents; Y1 and Y2 denoting, independently of one another: a hydrogen atom; an OH group; an OR1 group with R1 denoting an alkyl radical containing from 1 to 6 carbon atoms or a hydroxyalkyl radical containing from 1 to 6 carbon atoms; and preferably Y1 and Y2 denote, independently of one another, a hydrogen atom or an OH group; and M denoting a cation.

18. The composition as claimed in claim 17, wherein, in formula (I), Ar denotes a benzene nucleus or an indole nucleus.

19. The composition as claimed in claim 17, wherein the solubilizing agent(s) correspond(s) to formula (Ia) below: ##STR00032## with X, Y1, Y2 and M as: X denoting a saturated or unsaturated, linear or branched divalent radical, the main chain of which consists of 1 to 4 carbon atoms and optionally 1 or 2 heteroatoms chosen from nitrogen and oxygen atoms, it being possible for said main chain to optionally bear one or more substituents; Y1 and Y2 denoting, independently of one another: a hydrogen atom; an OH group; an OR1 group with R1 denoting an alkyl radical containing from 1 to 6 carbon atoms or a hydroxyalkyl radical containing from 1 to 6 carbon atoms; and preferably Y1 and Y2 denote, independently of one another, a hydrogen atom or an OH group; and M denoting a cation.

20. The composition as claimed in claim 17, wherein the solubilizing agent(s) correspond(s) to formula (Ib) below: ##STR00033## with X, Y1, Y2 and M as: X denoting a saturated or unsaturated, linear or branched divalent radical, the main chain of which consists of 1 to 4 carbon atoms and optionally 1 or 2 heteroatoms chosen from nitrogen and oxygen atoms, it being possible for said main chain to optionally bear one or more substituents; Y1 and Y2 denoting, independently of one another: a hydrogen atom; an OH group; an OR1 group with R1 denoting an alkyl radical containing from 1 to 6 carbon atoms or a hydroxyalkyl radical containing from 1 to 6 carbon atoms; and preferably Y1 and Y2 denote, independently of one another, a hydrogen atom or an OH group; and M denoting a cation.

21. The composition as claimed in claim 17, wherein, in formula (I), (Ia) or (Ib), the divalent radical X bears on its main chain one or more substituents corresponding to the general formula:
-L-Z with: L denotes a single bond or a group chosen from an amide group NHCO, a carbamate group NHCOO or a urea group NHCONH; and Z denotes a hydroxyl group (OH); a saturated or unsaturated, linear or branched radical comprising from 1 to 4 carbon atoms, said carbon atoms bearing at least one hydroxyl group [CH2]X-[OH]Y; a salified carboxylic acid group (COOM with M=Na.sup.+ or K.sup.+); or a saturated or unsaturated, linear or branched radical comprising from 1 to 12 carbon atoms and optionally one or more heteroatoms.

22. The composition as claimed in claim 21, wherein L denotes a single bond or an amide group.

23. The composition as claimed in claim 17, wherein the solubilizing agent is resulting from a natural or synthetic amino acid bearing an aromatic ring, chosen from phenylalanine, tyrosine and tryptophan, and phenylalanine.

24. The composition as claimed in claim 17, wherein the solubilizing agent is chosen from the group consisting of N-hydroxyacetyl-phenyl-alanine and N-hydroxyacetyltryptophan, corresponding to the formulae below: ##STR00034## used in the form of a sodium salt or of a potassium salt.

25. The composition as claimed in claim 17, wherein the solubilizing agent is resulting from a phenol.

26. The composition as claimed in claim 17, wherein the solubilizing agent is chosen from the following compounds, used in the form of sodium salts or of potassium salts: TABLE-US-00006 Name Structure Phenylacetic acid Mandelic acid Hydrocinnamic acid Trans-cinnamic acid 2-Phenoxypropionic acid 3-Phenyllactic acid Phenylsuccinic acid Alpha-hydroxyhippuric acid

27. The composition as claimed in claim 17, which comprises the solubilizing agent(s) in a total concentration of between 1 g/l and 100 g/l.

28. The composition as claimed in claim 17, which contains a total concentration of protein(s) of between 0.5 and 400 mg/ml.

29. The composition as claimed in claim 17, wherein the molar ratio between the total amount of solubilizing agent(s) and the total amount of protein(s) in the composition is greater than or equal to 20.

30. The composition as claimed in claim 17, which is intended to be administered by intravenous injection, by subcutaneous injection or by intramuscular injection.

Description

EXAMPLES

Part A: Synthesis

Example A1

Molecule A1

[0246] The molecule A1 or N-(2-hydroxyacetyl)-L-phenylalanine is obtained from the methyl ester of L-phenylalanine, hydrochloride salt (Bachem) and from glycolic acid (Alfa Aesar) according to the process described in the article Pratt R. F. et al. Biochemistry, 2006, 45, 13074-13082.

[0247] Yield: 7.5 g (77%)

[0248] .sup.1H NMR (DMSO-d.sub.6, ppm): 3.00-3.20 (2H); 3.80 (1H); 4.55 (1H); 5.60 (1H); 7.15-7.50 (5H); 7.70 (1H); 12.90 (1H).

Example A2

Molecule A2

[0249] The molecule A2 or N-(2-hydroxyacetyl)-L-tryptophan is obtained from the methyl ester of L-phenylalanine, hydrochloride salt (Bachem) and from glycolic acid (Alfa Aesar) according to the process described in the article Pratt R. F. et al. Biochemistry, 2006, 45, 13074-13082.

[0250] Yield: 2.1 g (42%)

[0251] .sup.1H NMR (DMSO-d.sub.6, ppm): 3.25 (2H); 3.80 (2H); 4.60 (1H); 5.55 (1H); 6.99-7.55 (5H); 7.65 (1H); 10.90 (1H); 12.75 (1H).

Part B: Preparation of the Solutions of Compounds used in the Following Examples

TABLE-US-00002 CAS Refer- Stock solution number Supplier ence concentration Molecule A1 20917-41-3 Adocia 315 mg/ml Sucrose 57-50-1 Sigma S3929 800 mM L-histidine 71-00-1 Sigma H6034 200 mM Mandelic acid 90-64-2 Aldrich M2101 1000 mM Acetic acid 64-19-7 Roth 3738.1 1000 mM Phenylacetic 103-82-2 Aldrich P16621 900 mM acid 2-Phenoxy- 940-31-8 Aldrich 197149 1125 mM propionic acid Molecule A2 70134-21-3 Adocia 315 mg/ml

Example B1

Preparation of a Solution of the Molecule A1 at 315 mg/ml

[0252] The solid form of the molecule Al is solubilized in sodium hydroxide at 1 mol/l, and then by adding sodium hydroxide at 10 mol/l, so as to obtain a solution at 315 mg/ml at pH 5.1.

Example B2

Preparation of a Solution of Sucrose at 800 mM

[0253] The sucrose (CAS 57-50-1, Sigma ref S3929) is solubilized in water at a concentration of 800 mM.

Example B3

Preparation of a Solution of L-Histidine at 200 mM

[0254] The L-histidine (CAS 71-00-1, Sigma ref H6034) is solubilized in water at a concentration of 200 mM. The solution obtained has a pH of 6.5.

Example B4

Preparation of the Solution of Mandelic Acid at 1000 mM

[0255] The mandelic acid (CAS 90-64-2, Aldrich ref M2101) is solubilized in sodium hydroxide at 1 mol/l, and then by adding sodium hydroxide at 10 mol/l, so as to obtain a solution at 1000 mM at pH 5.1.

Example B5

Preparation of the Solution of Acetic Acid at 1000 mM

[0256] The acetic acid (CAS 64-19-7, Roth ref 3738.1) is diluted in water to 1000 mM.

Example B6

Preparation of the Solution of Phenylacetic Acid at 900 mM

[0257] The phenylacetic acid (CAS 103-82-2, Aldrich ref P16621) is solubilized in sodium hydroxide at 1 mol/l so as to obtain a solution at 900 mM at pH 5.9.

Example B7

Preparation of the Solution of 2-phenoxypropionic Acid at 1125 mM

[0258] The 2-phenoxypropionic acid (CAS 940-31-8, Aldrich ref 197149) is solubilized in sodium hydroxide at 1 mol/l, and then by adding sodium hydroxide at 10 mol/l, so as to obtain a solution at 1000 mM at pH 12.5.

Example B8

Preparation of a Solution of the Molecule A2 at 315 mg/ml

[0259] The solid form of the molecule A2 is solubilized in sodium hydroxide at 1 mol/l, and then by adding sodium hydroxide at 10 mol/l, so as to obtain a solution at 315 mg/ml at pH 5.1.

Part C: Solubilization of Proteins at their Isoelectric Points

Example C1

Solubilization of Human Insulin at its Isoelectric Point

[0260] Human insulin has an isoelectric point (pI) of 5.3. At the pH of 5.3, human insulin precipitates at a concentration of greater than or equal to 10 IU/ml. A test of solubility at the pI of human insulin with various compounds is carried out.

[0261] A solution of human insulin at 500 IU/ml is prepared. Solutions of compounds at various concentrations in water are prepared as described in examples B1 to B4. Mixing between a solution of human insulin and the solution of compound is carried out in order to obtain a solution containing 100 IU/ml of human insulin and the desired concentration of compound. The pH of the various solutions is adjusted to pH 5.3 by adding hydrochloric acid or sodium hydroxide depending on the pH achieved following the mixing between the compound and the solution of human insulin.

[0262] The appearance of the solution is documented. If the solution is cloudy, the compound at the concentration tested does not allow total solubilization of human insulin at its isoelectric point. If the solution is clear, the compound allows total solubilization of human insulin at the concentration tested. In addition, the mixtures are centrifuged at 4000 rpm for 10 minutes in a Hereaus Biofuge Pico centrifuge (Rotor #3328) and then filtered through 0.22 m in order to remove the precipitate. The resulting soluble fractions are then assayed by RP-HPLC (column: Sunfire C18, Waters ref:186003417; mobile phase: sodium phosphate/acetonitrile gradient; detection: UV at 276 nm) with an external insulin range in order to quantify the percentage of soluble insulin at the pI. The results obtained (appearance and soluble percentages) are given in table 1.

TABLE-US-00003 TABLE 1 Soluble Molar ratio Compound insulin (compound/ concentration Visual recovery Mixtures insulin) (mmol/l) appearance (%) Human Cloudy 11 insulin control Molecule A1 500 300 Clear 100 Mandelic acid 1250 750 Clear 100 Sucrose 250 150 Cloudy 67 Sucrose 500 300 Cloudy 35 Histidine 125 75 Cloudy 48 Molecule A2 200 120 Clear 100

[0263] The examples with the molecule A1, with the molecule A2 and with mandelic acid (according to the invention) demonstrate a very strong improvement in the solubility of human insulin at its pI. Indeed, they result in clear solutions of insulin at its isoelectric point with an insulin concentration above its maximum solubility at the pI.

Example C2

Reduction in the Aggregation of a Formulation of Human Immunoglobulins (Nanogam) at its Isoelectric Point

[0264] The Nanogam formulation is a formulation of human immunoglobulins at 50 mg/ml and at pH 4.3 containing various IgG subclasses (IgGl: 54-70%, IgG2: 29-45%, IgG3: 1-4%, IgG4: 0-0.5%, IgA: at most 6 g/ml). The isoelectric point of this composition is approximately 8.5. At this pH of 8.5, the immunoglobulins have a tendency to aggregate. A test with various compounds is therefore carried out at the isoelectric point in order to identify the compounds which make it possible to reduce this aggregation phenomenon.

[0265] A commercial solution of Nanogam at 50 mg/ml is used. Solutions of compounds at various concentrations are prepared as described in the examples B1-2 and B3-B7. Mixing between the solution of Nanogam and one of the solutions of compound is carried out in order to obtain a solution containing 40 mg/ml of human immunoglobulins and the desired concentration of compound. The pH of the various solutions is adjusted to pH 8.5 by adding hydrochloric acid or sodium hydroxide depending on the pH achieved following the mixing between the compound of interest and the solution of human immunoglobulins.

[0266] The mixtures are then analyzed by light scattering on a Malvern NanoZS instrument. The results obtained (scattered intensities at 12.8 standardized, i.e. Iscat 12.8 Mixture/Iscat 12.8 Nanogam at pH 4.3) are given in table 2.

[0267] The scattered intensities are measured at 12.8. This angle of measurement is selected since it is sensitive to the largest nanoparticles/microparticles in suspension, such as the fibrils which appear at the isoelectric point of the Nanogam.

TABLE-US-00004 TABLE 2 Molar ratio Compound (COMPOUND/ concentration Iscat 12.8 Mixtures NANOGAM) (mmol/l) standardized Nanogam control 65.31 Compound A1 320 85 23.5 Mandelic acid 675 27.5 22.15 Phenylacetic acid 675 180 20.082 2-Phenoxy- 675 180 6.38 propionic acid Histidine 150 40 80.82 Acetic acid 675 180 200.18

[0268] The examples with the molecule Al, mandelic acid, phenylacetic acid and 2-phenoxypropionic acid show a very strong improvement of the solubility of Nanogam at its isoelectric point, whereas the examples with histidine and acetic acid do not demonstrate any improvement of the solubilization of Nanogam at its isoelectric point.