US20150335751A1

US20150335751A1 - Aqeous composition comprising at least one protein and one solubilizing agent, preparation thereof and uses thereof

Info

Publication number: US20150335751A1
Application number: US14/712,328
Authority: US
Inventors: Rémi Soula
Original assignee: Adocia SAS
Current assignee: Adocia SAS
Priority date: 2014-05-14
Filing date: 2015-05-14
Publication date: 2015-11-26
Also published as: US20180214556A1; FR3020947B1; WO2015173377A1; FR3020947A1; US10525133B2

Abstract

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

Description

The present invention relates to an aqueous composition comprising one or more protein(s) and at least one particular solubilizing agent. The invention also relates to the preparation of such a composition, and to the uses thereof in particular in the pharmaceutical and veterinary fields.
Finally, the invention relates to the use of particular compounds, in order to improve the solubilization of protein(s) within an aqueous composition.
The use of proteins of natural or synthetic origin in the form of solutions in a liquid medium is common, in particular in the pharmaceutical and veterinary fields, where it is necessary to be able to have liquid compositions containing protein active ingredients, intended to be administered to humans or to animals for therapeutic and/or prophylactic purposes. These liquid compositions must, as far as possible, be able to be formulated using water as solvent.
However, proteins can have a low solubility in an aqueous, or biological, medium and can give rise to unsatisfactory solubility, and in particular to unwanted precipitation phenomena.
The solubility of a protein in water depends to a large extent, on the one hand, on its structure and, on the other hand, on the pH. Indeed, depending on the pH, the protein may be in a more or less ionized form, which is capable of varying its solubility in water. The point where the solubility of a protein in water is the lowest is the isoelectric point (pI) of this protein, i.e. the pH at which the overall charge of the protein is zero.
Thus, when compositions comprising at least one protein are brought into contact with an aqueous medium, in particular when the pH of this medium corresponds to the isoelectric point of the protein, there is a need to improve the solubility of said protein, in particular in order to limit or avoid the precipitation thereof. This is particularly useful in the case of injection of the composition, in particular subcutaneous injection.
Quite particularly, there is a need to improve the solubility of proteins which have an isoelectric point around physiological pH (approximately 7.4) and which have problems of solubility in biological fluids, such as serum, blood, the subcutaneous space, etc.
Moreover, there is also a need to be able to formulate stable aqueous compositions containing proteins, which do not give rise to precipitation phenomena, whatever the pH of the composition, including at the isoelectric point of the proteins under consideration. For this, it has been proposed in the prior art to solubilize the proteins in water by means of water-soluble polymers such as, in particular, polysaccharides, which have the effect of interacting with the protein and promoting the solubilization thereof in water.
Thus, patent applications WO 2008/038111 and WO 2010/041119, filed in the name of Adocia, describe polysaccharides and/or oligosaccharides which have the property of creating interactions with active ingredients, in particular protein active ingredients.
These polymers consist of chains of which the lengths are statistically variable, and which are highly rich in possible sites of interaction with protein active ingredients. This multiple interaction potential could, however, create a lack of specificity in terms of interaction, whereas a smaller and better defined molecule could make it possible to be more specific in this respect.
Moreover, a polymer chain can interact with various sites present on a protein ingredient, but can also, owing to the length of the chain, interact with several protein ingredients, thereby leading to a bridging phenomenon. This bridging phenomenon may, for example, result in unwanted protein aggregation.
Furthermore, the use of polymeric compounds as solubilizing agents is not always desirable, in particular in the pharmaceutical field, since the elimination of such compounds by the organism can sometimes prove to be lengthy, or difficult. In addition, the use of such polymeric compounds often has the effect of increasing, sometimes considerably, the viscosity of the aqueous composition, which can be particularly problematic, in particular in the case of solutions intended to be administered by injection, in particular by subcutaneous injection.
In addition, polymers have the drawback of not being easily traceable (by mass spectrometry, for example) in biological media during pharmacokinetics or ADME (administration, distribution, metabolism, excretion) experiments, and generally give a diffuse signal with a high background noise in mass spectrometry.
Moreover, some solubilizing agents can be expensive and/or can require numerous synthesis, and optionally purification, steps.
Continuing its research in the field of the formulation of aqueous compositions containing proteins, and more particularly for the administration of protein active ingredients, the applicant has now demonstrated that, surprisingly, the use of certain non-polymeric compounds of particular structure makes it possible to significantly improve the solubility of proteins in an aqueous medium, while at the same time remedying all or some of the drawbacks of the prior art compounds and methods.
A subject of the present invention is thus a liquid composition comprising, in an aqueous medium, one or more protein(s) and one or more solubilizing agent(s), wherein said solubilizing agent(s) is (are) 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.
By virtue of their particular structure, said solubilizing agents interact with proteins and particularly notably increase their solubilization in water, thereby enabling the preparation of aqueous solutions containing proteins. These solutions may be clear, optionally including at the isoelectric point, or “pI”, of the proteins under consideration.
The term “clear” is intended to mean devoid of any light-scattering object, said objects leading to a loss of recovery (measured by separative, for example electrophoretic or chromatographic, analytical techniques such as RP-HPLC) and/or leading to an increase in scattered intensities by DLS measurement.
The recovery by separative analytical techniques can be measured in the manner presented in the examples.
The scattered intensities can be measured in the manner presented in the examples.
The term “nonclear” is intended to mean the presence of light-scattering objects and/or the presence of macroscopic cloudiness evaluated by eye. This can be measured by the loss of recovery by separative analytical techniques, said objects being separable either by centrifugation or by filtration.
In the case of a nonclear liquid composition, the recovery by separative analytical techniques is less than 99% and/or the scattered light intensity at 173° and/or at 12.8° increases by more than 5%.
The proteins under consideration exhibit a decrease in their maximum solubility at the pI. This decrease in maximum solubility at the pI can be measured by means of the methods presented in the examples.
In particular, the process according to the invention makes it possible to substantially increase the concentrations at which the proteins can be solubilized in water at their isoelectric point.
In particular, the compositions obtained according to the invention are homogeneous with good solubilization of protein active agents, and are stable over time.
In addition, the solubilizing agents according to the invention are small compounds, which makes it possible to limit the increase in the viscosity of the aqueous composition. In particular, and this constitutes a particularly surprising aspect of the invention, the applicant has demonstrated that it is not necessary to use compounds of polymeric structure, in particular saccharide structure, in order to improve protein solubilization. Indeed, it was generally considered up until now that polymeric structures were preferable, whereas there was a risk with small compounds of there being too few sites of interaction with protein active ingredients.
The present invention has a particularly advantageous application in the pharmaceutical and veterinary fields since it provides solubilizing agents which allow the stabilization, administration and delivery of protein active ingredients in an aqueous solution, by methods that are simple to carry out.
The solubilizing agents according to the invention can exhibit a biodegradability that is sufficiently rapid and suitable for their use in the preparation of a wide category of pharmaceutical formulations, including for medicaments intended for chronic and/or high-frequency administration. These compounds can also comply with the constraints established by pharmaceutical regulations, in particular in terms of their stability under normal preservation and storage conditions, in particular in solution.
A subject of the present invention is also the preparation of the composition above, and the use thereof in the pharmaceutical or veterinary field.
Other subjects, characteristics, aspects and advantages of the invention will emerge even more clearly on reading the description and the examples which follow.
In what follows, and unless otherwise indicated, the limits of a range of values are included in said range, in particular in the expression “between”.
Moreover, the expression “at least one” used in the present description is equivalent to the expression “one or more”.
The solubilizing agents used in the invention are compounds of non-saccharide structure.
The term “non-saccharide structure” is intended to mean that these compounds do not contain in their structure any saccharide unit, whether in cyclic or open and reduced or oxidized form.
The term “saccharide unit” denotes pentoses, hexoses, uronic acids, and N-acetylhexosamines in cyclic or open and reduced or oxidized form.
The solubilizing agents used in the invention are anionic compounds. The term “anionic compound” denotes a chemical compound containing only negative charges, and no positive charge. In particular, in the case where the compound comprises one or more nitrogen atoms in its structure, said nitrogen atoms do not carry a positive charge.
The solubilizing agents used in the invention contain in their structure one or more aromatic nucleus or nuclei comprising at least 6 ring members, i.e. an aromatic ring or heterocycle comprising at least 6 atoms chosen from carbon, nitrogen, sulfur or oxygen. This or these aromatic nucleus or nuclei can be advantageously chosen from optionally substituted benzene nuclei and optionally substituted indole nuclei, and preferably optionally substituted benzene nuclei.
The aromatic nucleus or nuclei may be substituted or unsubstituted. The substituent(s) may be linear or branched, saturated or unsaturated, and cyclic or noncyclic. It/They may also be condensed or polycyclic, but must comprise at least one aromatic ring or heterocycle comprising at least 6 atoms chosen from carbon, nitrogen, sulfur or oxygen. These rings comprising at least 6 atoms chosen from carbon, nitrogen, sulfur or oxygen are defined in the present application as aromatic nuclei comprising at least 6 ring members.
The substituent(s) may in particular be chosen from —OH, and —OR₁groups with R₁denoting an alkyl or hydroxyalkyl radical containing from 1 to 6 carbon atoms.
Preferably, the aromatic nucleus is not substituted.
The solubilizing agents used in the invention also comprise in their structure one or more carboxylic acid group(s), in salt form, i.e. one or more groups of structure:

- with M₁ ⁿ⁺ representing a cation, preferably a pharmaceutically acceptable cation, and
- n is an integer equal to 1 or 2.

According to one preferred embodiment, M₁ ⁿ⁺ denotes a cation chosen from Ca²⁺, Mg²⁺, Na⁺ or K⁺ and more preferentially M₁ ⁿ⁺denotes Na⁺ or K⁺.
The solubilizing agents used in the invention have a molar mass of between 130 and 500 g/mol.
This molar mass corresponds to the acid form of the solubilizing agent, i.e. when the carboxylic acid group(s) is (are all) in acid form:
Preferably, the molar mass of the solubilizing agent(s) is between 130 and 450 g/mol, and preferentially between 130 and 400 g/mol.
According to one preferred embodiment, the solubilizing agents used in the invention are water-soluble. The term “water-soluble” is intended to mean that these agents have, in water at a pH of 7 and at 25° C., a minimum solubility of 50 mmol/l, preferably a minimum solubility of 100 mmol/l and more preferentially of 250 mmol/l.
According to one preferred embodiment, the solubilizing agent(s) used in the invention correspond(s) to general formula (I) below:
with:

- Ar denotes an aromatic nucleus comprising at least 6 ring members;
- X denotes 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;
- Y₁and Y₂denote, 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 Y₁and Y₂denote, independently of one another, a hydrogen atom or an OH group; and
- M denotes a cation such as, in particular, Na⁺ or K⁺.

Preferably, Ar denotes a benzene nucleus or an indole nucleus.
According to a first particularly preferred embodiment, Ar denotes a benzene nucleus. In this embodiment, the solubilizing agent(s) correspond(s) to formula (Ia) below:

- with X, Y₁, Y₂and M as defined above.

According to a second preferred embodiment, Ar denotes an indole nucleus. In this embodiment, the solubilizing agent(s) preferably correspond(s) to formula (Ib) below:

- with X, Y₁, Y₂and M as defined above.

In formulae (I), (Ia) and (Ib) above, the divalent radical X comprises a main chain, consisting of 1 to 4 carbon atoms and optionally 1 or 2 heteroatoms chosen from nitrogen and oxygen atoms. The carbon atoms constituting the main chain may be, independently of one another, saturated or unsaturated. The term “main chain” denotes a series of atoms comprising from 1 to 4 carbon atoms and linking, linearly, the aromatic group Ar to a carboxylate group —COOM.
As set out above, this main chain may bear one or more substituents, i.e. one or more atoms or groups of atoms other than a hydrogen atom.
The substituent(s) then advantageously correspond(s) 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, such as, in particular, one or more oxygen atoms.

Preferably, L denotes a single bond or an amide group.
Preferably, Z is chosen from a hydroxyl group (—OH) or a saturated or unsaturated, linear or branched radical comprising from 1 to 4 carbon atoms which can optionally bear one or more hydroxyl groups (—OH) and/or salified carboxylic acid groups (—COOM″ with M″=Na⁺ or K⁺).
According to one preferred embodiment, the solubilizing agent(s) correspond(s) to formula (Ia) above in which:

- Y₁and Y₂both denote a hydrogen atom;
- X denotes a saturated or unsaturated, linear divalent radical, the main chain of which consists of 1 to 4 carbon atoms and optionally one or two heteroatom(s) chosen from nitrogen and oxygen atoms, said chain not bearing any substituent other than hydrogen atoms.

In this embodiment, the main chain preferably consists of 1 or 2 carbon atoms and optionally a heteroatom chosen from nitrogen and oxygen atoms, and more preferentially the main chain consists of 1 or 2 carbon atoms.
According to another preferred embodiment, the solubilizing agent(s) correspond(s) to formula (Ia) above in which:

- Y₁and Y₂both denote a hydrogen atom;
- X denotes a saturated or unsaturated, branched divalent radical, the main chain of which consists of 1 to 4 carbon atoms and optionally one or two heteroatom(s) chosen from nitrogen and oxygen atoms, said chain bearing one or more substituents -L-Z as defined above.

In this embodiment, the main chain preferably consists of 1 or 2 carbon atoms and optionally a heteroatom chosen from nitrogen and oxygen atoms, and more preferentially the main chain consists of 1 or 2 carbon atoms.
In this embodiment, according to a first preferred variant, the main chain bears one or more, and preferably one, substituent(s) -L-Z, with L denoting an amide group and Z denoting a saturated or unsaturated, linear or branched radical comprising from 1 to 4 carbon atoms which can optionally bear one or more hydroxyl groups (—OH) and/or one salified carboxylic acid group (—COOM″ with M″=Na⁺ or K⁺).
In this embodiment, according to a second variant which is likewise preferred, the main chain bears one or more substituent(s) -L-Z, L denoting a single bond and Z denoting a hydroxyl group (—OH); 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 bearing one or more hydroxyl groups (—OH) and/or one salified carboxylic acid group (—COOM″ with M″=Na⁺ or K⁺).
In this second variant, Z preferably denotes a hydroxyl group (—OH) or a salified carboxylic acid group (—COOM′ with M′=Na⁺ or Kr⁺).
The main chain of the divalent radical X may then, for example, bear:

- one or more hydroxyl group(s) (—OH); or
- one or more salified carboxylic acid groups (—COOM′) and preferably one salified carboxylic acid group; or else
- one or more hydroxyl groups (—OH) and one or more salified carboxylic acid groups (—COOM′) and preferably one hydroxyl group and one salified carboxylic acid group.

According to another preferred embodiment, the solubilizing agent(s) correspond(s) to formula (Ia) above in which:

- at least one of Y₁and Y₂denotes an —OH group, and preferably Y₁denotes an —OH group and Y₂denotes a hydrogen atom;
- X denotes a saturated or unsaturated, linear divalent radical, the main chain of which consists of 1 to 4 carbon atoms and optionally one or two heteroatom(s) chosen from nitrogen and oxygen atoms, said chain not bearing any substituent other than hydrogen atoms.

- at least one of Y₁and Y₂denotes an —OH group, and preferably Y₁denotes an —OH group and Y₂denotes a hydrogen atom;
- X denotes a saturated or unsaturated, branched divalent radical, the main chain of which consists of 1 to 4 carbon atoms and optionally one or two heteroatom(s) chosen from nitrogen and oxygen atoms, said chain bearing one or more substituents -L-Z as defined above.

In this embodiment, the main chain preferably consists of 1 or 2 carbon atoms and optionally a heteroatom chosen from nitrogen and oxygen atoms, and more preferentially the main chain consists of 1 or 2 carbon atoms.
In this embodiment, according to a first preferred variant, the main chain bears one or more, and preferably one, substituent(s) -L-Z, with L denoting an amide group and Z denoting a saturated or unsaturated, linear or branched radical comprising from 1 to 4 carbon atoms which can optionally bear one or more hydroxyl groups (—OH) and/or one salified carboxylic acid group (—COOM″ with M″=Na⁺ or K⁺).
In this embodiment, according to a second variant which is likewise preferred, the main chain bears one or more substituent(s) -L-Z, L denoting a single bond and Z denoting a hydroxyl group (—OH); 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 bearing one or more hydroxyl groups (—OH) and/or one salified carboxylic acid group (—COOM″ with M″=Na⁺ or K⁺).
In this second variant, Z preferably denotes a hydroxyl group (—OH) or a salified carboxylic acid group (—COOM′ with M′=Na⁺ or K⁺).
The main chain of the divalent radical X can then, for example, bear:

According to one likewise preferred embodiment of the invention, the compound of formula (I) is resulting from a natural or synthetic amino acid bearing an aromatic ring.
Among the natural amino acids, the use of alpha-amino acids, such as phenylalanine, tyrosine and tryptophan, is quite particularly preferred.
According to one particularly preferred embodiment, the compound of formula (I) is resulting from phenylalanine.
According to one particularly preferred embodiment, the compound of formula (I) is resulting from tryptophan.
According to one embodiment, the compound of formula (I) is resulting from a synthetic amino acid and, in one embodiment, the synthetic amino acid is phenylglycine.
The amino acids can be used in the form of either of their optical isomers (L or D forms), or in the form of a mixture of such isomers, and in particular in racemate form.
Preferably, the solubilizing agent according to the invention is resulting from an amino acid of which the amine group has been converted into a group chosen from an amide group, a carbamate group or a urea group, or substituted.
Said amide, carbamate or urea group can be linked to a hydrogen atom or to a hydrocarbon-based substituent containing from 1 to 6 carbon atoms and optionally one or more oxygen atoms.
When the amine function is substituted, it can be substituted with a substituent chosen from the group consisting of C₂to C₄hydroxycarboxyls, in particular the hydroxyacetyl group.
Preferably, the solubilizing agent, and in particular the compound of formula (I), is resulting from an amino acid of which the amino group has been converted into an amide group. Preferably, said amide group is substituted with a hydrocarbon-based radical containing from 1 to 6, and preferably from 1 to 4, carbon atoms, and which can optionally bear one or more hydroxyl groups (—OH).
Two particularly preferred compounds are N-hydroxyacetylphenylalanine and N-hydroxyacetyltryptophan, corresponding to the formulae below:
and used in sodium salt or potassium salt form.
According to one likewise preferred embodiment, the solubilizing agent, and in particular the compound of formula (I), is resulting from a phenol.
The composition according to the invention advantageously comprises the solubilizing agent(s) as described above in a total concentration of between 1 g/I and 100 g/I.
The invention also relates to 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 has, in its acid form, a molar mass of between 130 and 500 g/mol, is added to an aqueous protein composition in order to solubilize the protein.
The invention also relates to the use, in order to improve the solubilization of one or more protein(s) 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.
The following embodiments apply both to the process for solubilizing one or more protein(s) within an aqueous composition and/or to their use.
In one embodiment, the process or the use according to the invention is one wherein at least one solubilizing agent corresponds to general formula (I) below:
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 atom(s) and optionally 1 or 2 heteroatom(s) chosen from nitrogen and oxygen atoms, it being possible for said main chain to optionally bear one or more substituent(s);
- Y₁and Y₂denoting, 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 atom(s); and preferably Y₁and Y₂denote, independently of one another, a hydrogen atom or an —OH group; and
- M denoting a cation such as, in particular, Na⁺ or K⁺.

In one embodiment, the process or the use according to the invention is one wherein, in formula (I), Ar denotes a benzene nucleus or an indole nucleus.
In one embodiment, the process or the use according to the invention is one wherein the solubilizing agent(s) correspond(s) to formula (Ia) below:
with:

- X denoting a saturated or unsaturated, linear or branched divalent radical, the main chain of which consists of 1 to 4 carbon atom(s) and optionally 1 or 2 heteroatom(s) chosen from nitrogen and oxygen atoms, it being possible for said main chain to optionally bear one or more substituent(s);
- Y₁and Y₂denoting, 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 atom(s) or a hydroxyalkyl radical containing from 1 to 6 carbon atom(s); and preferably Y₁and Y₂denote, independently of one another, a hydrogen atom or an OH group; and
- M denoting a cation such as, in particular, Na⁺ or K⁺.

In one embodiment, the process or the use according to the invention is one wherein the solubilizing agent(s) correspond(s) to formula (Ib) below:
with:

- X denoting a saturated or unsaturated, linear or branched divalent radical, the main chain of which consists of 1 to 4 carbon atom(s) and optionally 1 or 2 heteroatom(s) chosen from nitrogen and oxygen atoms, it being possible for said main chain to optionally bear one or more substituent(s);
- Y₁and Y₂denoting, independently of one another: a hydrogen atom; an OH group; an OR₁group with R₁denoting an alkyl radical containing from 1 to 6 carbon atom(s) or a hydroxyalkyl radical containing from 1 to 6 carbon atom(s); and preferably Y₁and Y₂denote, independently of one another, a hydrogen atom or an OH group; and
- M denoting a cation such as, in particular, Na⁺ or K⁺.

In one embodiment, the process or the use according to the invention is one 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 such as, in particular, one or more oxygen atoms.

In one embodiment, the process or the use according to the invention is one wherein L denotes a single bond or an amide group.
In one embodiment, the process or the use according to the invention is one wherein the solubilizing agent is resulting from a natural or synthetic amino acid bearing an aromatic ring, preferably chosen from phenylalanine, tyrosine and tryptophan, and more preferentially phenylalanine or tryptophan.
In one embodiment, the process or the use according to the invention is one wherein the solubilizing agent is chosen from the group consisting of N-hydroxyacetylphenylalanine and N-hydroxyacetyltryptophan, corresponding to the formulae below:
used in sodium salt or potassium salt form.
In one embodiment, the process or the use according to the invention is one wherein the solubilizing agent is resulting from a phenol.
In one embodiment, the process or the use according to the invention is one wherein the solubilizing agent is chosen from the following compounds, used in sodium salt or potassium salt form:


	Name	Structure

	Phenylacetic acid

	Mandelic acid

	Hydrocinnamic acid

	Trans-cinnamic acid

	2-Phenoxypropionic acid

	3-Phenyllactic acid

	Phenylsuccinic acid

	Alpha-hydroxyhippuric acid

In one embodiment, the process or the use according to the invention is one wherein said aqueous composition comprises the solubilizing agent(s) in a total concentration of between 1 g/I and 100 g/I.
In one embodiment, the process or the use according to the invention is one wherein said aqueous composition contains a total concentration of protein(s) of between 0.5 and 400 mg/nil, preferably between 50 and 350 mg/ml.
In one embodiment, the process or the use according to the invention is one 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, preferably greater than or equal to 35, more preferentially greater than or equal to 45, even more preferentially greater than or equal to 100, more preferentially greater than or equal to 150, and even better still greater than or equal to 200.
In one embodiment, the process or the use according to the invention is one wherein said aqueous composition is intended to be administered by intravenous injection, by subcutaneous injection or by intramuscular injection, and preferably by subcutaneous injection.
The composition according to the invention also contains one or more protein(s).
The term “protein” denotes, in a manner known per se, a macromolecule composed of one or more chains of amino acids linked to one another by peptide bonds.
The proteins used in the invention may be of natural or synthetic origin.
The invention is quite particularly suitable for the solubilization of proteins containing at least 10, and preferably at least 50, amino acids.
Preferably, the proteins involved in the present invention have an isoelectric point of between 4 and 9, more preferentially between 4.5 and 8.5, and more particularly between 5.5 and 8.
The invention applies quite particularly to proteins which exhibit at their isoelectric point a decrease in their maximum solubility in water of at least 2%, preferably at least 5%, or even at least 10%. For such proteins, it is noted in particular by simple visual observation that an aqueous solution containing them goes from clear to cloudy when the pH of the solution approaches the isoelectric point of the protein.
According to one preferred embodiment of the invention, the protein(s) is (are) chosen from therapeutic proteins.
According to one preferred embodiment of the invention, the protein(s) is (are) chosen from proteins containing at least one antibody fragment.
The term “protein comprising at least one antibody fragment” is intended to mean a protein chosen from monoclonal antibodies (mAbs), polyclonal antibodies, fusion proteins, nanobodies, bispecific antibodies and antibodies coupled to cytotoxic active ingredients (ADCs—antibody-drug conjugates).
According to one embodiment of the present invention, the protein comprising at least one antibody fragment is a monoclonal antibody.
The term “monoclonal antibody” is intended to mean a complete antibody, an antibody fragment or an antibody derivative which has an identical and unique specificity, i.e. which recognizes just one type of epitope on a given antigen.
According to the present invention, a monoclonal antibody may also be called an immunoglobulin (hereinafter Ig).
The term “complete antibody” is intended to mean an antibody composed of two identical heavy chains (“HC”) and of two identical light chains (“LC”) which are linked by a disulfide bridge. Each chain consists, in the N-terminal position, of a variable region (or domain) (encoded by the rearranged V-J genes for the light chains and the rearranged V-D-J genes for the heavy chains) specific for the antigen against which the antibody is directed, and, in the C-terminal position, of a constant region, consisting of a single CL domain for the light chains or of several domains for the heavy chains. Each variable region comprises three segments called “complementarity determining regions” (“CDRs”) or “hypervariable regions”, which are mainly responsible for the binding to the epitope of an antigen. The two heavy (H) chains and the two light (L) chains are identical to one another. The light chain is composed of 2 domains, a variable domain V and a constant domain C, folded in space independently of one another. They are called VL and CL. The heavy chain also comprises a domain V denoted VH and 3 or 4 domains C denoted from CH1 to CH4. Each domain comprises approximately 110 amino acids and is structured comparably. The 2 heavy chains are linked by disulfide bridges and each heavy chain is linked to a light chain, also by a disulfide bridge. The region which determines the specificity of the antibody for the antigen is carried by the variable parts, while the constant parts can interact with the Fc receptors of effector cells or of molecules such as complement in order to mediate various functional properties. The term “VH” refers to the variable regions of an immunoglobulin heavy chain of an antibody, including the heavy chains of an Fv, scFv, dsFv, Fab, Fab′ or F(ab)′ fragment. The term “VL” refers to the variable regions of an immunoglobulin light chain of an antibody, including the light chains of an Fv, scFv, dsFv, Fab, Fab′ or F(ab)′ fragment. The term “CDR regions” or “CDRs” is intended to denote the hypervariable regions of the heavy and light chains of immunoglobulins as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th ed., U.S. Department of Health and Human Services, NIH, 1991, and later editions). There are 3 heavy-chain CDRs and 3 light-chain CDRs. The term CDR or CDRs is used herein to denote, as appropriate, one of these regions or several, or even all, of these regions which contain the majority of the amino acid residues responsible for the affinity binding of the antibody for the antigen or the epitope that it recognizes. The most conserved regions of the variable domains are called FR (for “framework”) regions or sequences and there are 4 of them (FR1 to FR4).
Antibodies are subdivided into 5 classes or isotypes: IgG, IgA, IgM, IgE and IgD according to the structure of the heavy-chain constant domains, i.e. respectively γ, α, μ, ε and δ chains.
The IgG and IgA classes are, moreover, subdivided into subclasses according in particular to the size of the hinge regions and also the number and position of disulfide bridges between heavy chains.
The IgG class is subdivided into 4 subclasses, i.e. IgG1, IgG2, IgG3 and IgG4.
The IgA class is, for its part, subdivided into 2 subclasses, i.e. IgA1 and IgA2.
Preferably, the protein comprising at least one antibody fragment is a monoclonal antibody chosen from IgGs, IgAs, IgMs, IgEs and IgDs. The IgAs can be chosen from IgA1s and IgA2s, and the IgGs can be chosen from IgG1s, IgG2s, IgG3s and IgG4s.
In one embodiment, the monoclonal antibody is an IgG.
In one embodiment, the monoclonal antibody is an IgA.
In one embodiment, the monoclonal antibody is an IgM.
In one embodiment, the monoclonal antibody is an IgE.
In one embodiment, the monoclonal antibody is an IgD.
In one embodiment, the monoclonal antibody is an IgG1.
In one embodiment, the monoclonal antibody is an IgG2.
In one embodiment, the monoclonal antibody is an IgG3.
In one embodiment, the monoclonal antibody is an IgG4.
In one embodiment, the monoclonal antibody is an IgA1.
In one embodiment, the monoclonal antibody is an IgA2.
The term “antibody fragment” is intended to mean any functional antibody fragment, e.g. Fab (Fragment, antigen binding), Fv, scFv (single chain Fv), Fc (Fragment, crystallizable), F(ab′)2, Fab′, scFv-Fc, synthetic polypeptides containing the sequences of one or more CDRs, which generally have the same binding specificity as the antibody from which they are derived.
The antibody fragments used in the invention can be obtained from the antibodies by methods such as digestion with enzymes, for instance pepsin or papain, and/or by disulfide-bridge cleavage by chemical reduction. The enzymatic digestion of antibodies with papain generates 2 identical fragments, which are called “Fab fragment” (Fragment, antigen binding), and an Fc fragment (Fragment, crystallizable). The Fc fragment is the support for the effector functions of immunoglobulins. Digestion with pepsin generates an F(ab′)2 fragment, where the two Fab fragments remain linked by two disulfide bridges, and the Fc fragment is split up into several peptides. The F(ab′)2 fragment is made up of two Fab′ fragments, linked by inter-chain disulfide bridges so as to form one F(ab′)2.
Thus, the monoclonal antibody or antibodies according to the invention can advantageously contain one or more of these fragments, and all the combinations between the abovementioned fragments can be used in the context of the present invention.
The term “antibody derivative” is intended to mean any antibody, it being possible for this antibody to comprise one or more mutations, substitutions, deletions and/or additions of one or more amino acid residues. Such an addition, substitution or deletion can be located at any position in the molecule. In the case where several amino acids have been added, substituted or deleted, any combination of addition, substitution or deletion can be considered, provided that the resulting antibody still has at least the advantageous properties of the antibody of the invention.
According to the invention, the monoclonal antibody can advantageously be a chimeric antibody or a humanized antibody. The term “chimeric antibody” is intended to mean an antibody of which the heavy- and light-chain variable regions, or at least one domain or fragment of these regions, belong to a species different than the species to which the constant regions of the light chains and of the heavy chains belong. The term “humanized antibody” is intended to mean an antibody which contains mainly human immunoglobulin sequences. This term generally refers to a non-human immunoglobulin which has been modified by incorporation of human sequences or of residues found in human sequences.
The antibodies described above can, for example, be obtained using the standard recombinant DNA techniques well known to those skilled in the art, for example using the techniques for constructing chimeric antibodies described, for example, in Morrison et al., Proc. Natl. Acad. Sci. U.S.A., 81, pp. 6851-55 (1984), where recombinant DNA technology is used to replace the constant region of a heavy chain and/or the constant region of a light chain of an antibody originating from a non-human mammal, with the corresponding regions of a human immunoglobulin. Such antibodies and the method for preparing them have also been described in patent application EP 173 494, in the document Neuberger, M. S. et al., Nature 312 (5995): 604-8 (1985), and also in document EP 125 023, for example. Methods for generating chimeric antibodies are widely available to those skilled in the art. For example, the heavy and light chains of the antibody can be expressed separately using a vector for each chain, or else can be integrated into a single vector.
By way of example, among the commercially available monoclonal antibodies, mention will be made of the following monoclonal antibodies: Muromonab-CD3 (sold under the name Orthoclone Okt3®), Abciximab (sold under the name Reopro®), Rituximab (sold under the names MabThera® and Rituxan®), Basiliximab (sold under the name Simulect®), Daclizumab (sold under the name Zenapax®), Palivizumab (sold under the name Synagis®), Infliximab (sold under the name Remicade®), Trastuzumab (sold under the name Herceptin®), Alemtuzumab (sold under the names MabCampath®, Campath-1H®), Adalimumab (sold under the name Humira®), Tositumomab-I131 (sold under the name Bexxar®), Efalizumab (sold under the name Raptiva®), Cetuximab (sold under the name Erbitux®), Ibritumomab tiuxetan (sold under the name Zevalin®), Omalizumab (sold under the name Xolair®), Bevacizumab (sold under the name Avastin®), Natalizumab (sold under the name Tysabri®), Ranibizumab (sold under the name Lucentis®), Panitumumab (sold under the name Vectibix®), Eculizumab (sold under the name Soliris®), Certolizumab pegol (sold under the name Cimzia®), Golimumab (sold under the name Simponi), Canakinumab (sold under the name Ilaris®), Catumaxomab (sold under the name Removab®), Ustekinumab (sold under the name Stelara®), Tocilizumab (sold under the names RoActemra®, and Actemra®), Ofatumumab (sold under the name Arzerra®), Denosumab (sold under the name Prolia®), Belimumab (sold under the name Benlysta®), Raxibacumab (not yet marketed), Ipilimumab (sold under the name Yervoy®) and Pertuzumab (sold under the name Perjeta®).
According to one embodiment of the present invention, the protein comprising at least one antibody fragment is a polyclonal antibody.
The term “polyclonal antibody” is intended to mean a mixture of whole antibodies, a mixture of antibody fragments or a mixture of antibody derivatives, as described above, recognizing various types of epitopes on a given antigen.
According to one embodiment of the present invention, the protein comprising at least one antibody fragment is a fusion protein.
The term “fusion protein” is intended to mean a construction which contains several proteins or polypeptides of different origin. This fusion protein is encoded by a nucleic acid obtained by recombinant DNA techniques well known to those skilled in the art. According to the present invention, the fusion protein is made up of a monoclonal antibody fragment as previously described and a fragment of a protein of interest.
By way of example, mention will be made of the fusion protein made up of a monoclonal antibody fragment which is the Fc region of an IgG1 immunoglobulin and a fragment of a protein of interest which is the extracellular domain of the CTLA-4 (Cytotoxic T-Lymphocyte Antigen 4) protein receptor, this fusion protein, i.e. abatacept, being sold under the name Orencia®.
By way of example, mention may also be made of the fusion protein made up of a monoclonal antibody fragment which is the Fc region of an IgG1 and a fragment of a protein of interest which is the P75 fraction of the soluble TNF-alpha receptor, this fusion protein, i.e. etanercept, being sold under the name Enbrel®.
By way of example, mention will also be made of the fusion protein made up of a monoclonal antibody fragment which is the Fc region of an IgG1 and a fragment of a protein of interest which is the extracellular portions of IL-1R1 (interleukin-1 receptor component) and of IL-1RAcP (IL-1 receptor accessory protein), this fusion protein, i.e. rilonacept, being sold under the name Arcalyst®.
By way of example, mention will also be made of the fusion protein made up of a monoclonal antibody fragment which is the IgG1 hinge, C(H)2 and C(H)3 regions, and a fragment of a protein of interest which is the extracellular domain of LFA-3, this fusion protein, i.e. alefacept, being sold under the name Amevive®.
According to one embodiment of the present invention, the protein comprising at least one antibody fragment is a nanobody.
The term “nanobody” is intended to mean any unique variable domain of immunoglobulin heavy chains. Nanobodies are more widely described in the publication D. Saerens and S. Muyldermans (eds.) Single Domain Antibodies: Methods and Protocols, Methods in Molecular Biology, vol. 911; and Med Microbiol Immunol (2009).
According to one embodiment of the present invention, the protein comprising at least one antibody fragment is a bispecific antibody.
The term “bispecific antibody” (also called bifunctional antibody or “diabody”) is intended to mean any immunoglobulin fragment comprising 2 antigen-presenting sites. Bifunctional antibodies are more widely described in the publication Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).
According to one embodiment of the present invention, the protein comprising at least one antibody fragment is an antibody coupled to a cytotoxic active ingredient.
The expression “antibody coupled to a cytotoxic active ingredient” is intended to mean a monoclonal antibody as previously described, coupled to a cytotoxic active ingredient.
By way of example of a cytotoxic active ingredient, mention may in particular be made of vedotin.
An example of an antibody coupled to a cytotoxic active ingredient is the antibody brentuximab coupled to the cytotoxic active ingredient vedotin. This antibody coupled to this cytotoxic active ingredient is sold under the name Adcetris®.
According to one likewise preferred embodiment of the invention, the protein(s) is (are) chosen from hormones.
Mention may in particular be made of: insulin; growth factors such as BMPs (Bone Morphogenetic Proteins), PDGFs (Platelet-Derived Growth Factors), coagulation factors and parathyroid hormones.
The protein(s) is (are) present in the composition according to the invention in solubilized form.
Generally, the composition according to the invention contains a total concentration of protein(s) of between 0.5 and 400 mg/ml.
Preferably, the total concentration of protein(s) is between 50 and 350 mg/ml, in particular between 80 and 250 mg/ml, preferably between 80 and 200 mg/ml, more preferentially between 100 and 200 mg/ml, better still between 120 and 200 mg/ml, and even better still between 120 and 180 mg/ml.
According to one particularly advantageous embodiment of the invention, the concentration of the protein in the composition is greater than the maximum concentration of the same protein in an aqueous solution at its isoelectric point, at a temperature of 25° C.
Typically, the protein is present in the composition at an osmolality of less than or equal to 700 mosmol/l, in particular less than or equal to 500 mosmol/l, or even less than or equal to 350 mosmol/l.
Typically, the protein is present in the composition at an osmolality of greater than or equal to 150 mosmol/l, in particular greater than or equal to 200 mosmol/l, or even greater than or equal to 250 mosmol/l.
Typically, the protein is present in the composition at an osmolality of between 150 and 700 mosmol/l, in particular of between 200 and 500 mosmol/l, or even of between 250 and 350 mosmol/l.
The osmolality can be measured using a Foske Micro-Osmometer instrument—Model 210.
In addition, the molar ratio between the total amount of solubilizing agent(s) and the total amount of protein(s) in the composition is advantageously greater than or equal to 20, preferably greater than or equal to 35, and more preferentially greater than or equal to 45.
More particularly, 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 100, preferably greater than or equal to 150, and more preferentially greater than or equal to 200.
The composition according to the invention comprises an aqueous medium, i.e. it comprises water as main constituent. Advantageously, the composition comprises more than 50% by weight of water, preferably at least 70% by weight of water, more preferentially at least 80% by weight of water and even better still at least 90% by weight of water, relative to its total weight.
The water used in the composition may in particular be sterile water for injection or bacteriostatic water for injection.
Generally, the pH of the composition according to the invention may range from 4 to 8.
According to one embodiment of the invention, the pH of the composition is between 5 and 6.5.
According to another embodiment, the pH is between 5 and 8, preferably between 6 and 7.5, and more preferentially between 6 and 7.
The pH of the composition can be adjusted in a manner known per se by the addition of acids, of bases and/or of buffer systems, which are preferably pharmaceutically acceptable.
The composition according to the invention advantageously has a viscosity, measured at 25° C. and at atmospheric pressure, of less than or equal to 20 cP.
According to one embodiment, the composition according to the invention comprises one or more pharmaceutically acceptable acid(s).
These acids can in particular be chosen from hydrochloric acid, phosphoric acid, citric acid, acetic acid, ascorbic acid, ethylenediaminetetraacetic acid (also called EDTA) and tartaric acid.
According to one embodiment, the composition according to the invention comprises one or more pharmaceutically acceptable base(s).
These bases can in particular be chosen from inorganic bases formed from metals such as sodium, potassium, calcium or magnesium, and in particular from the group consisting of sodium hydroxide (NaOH), potassium hydroxide (KOH), and magnesium hydroxide (Mg(OH)₂).
Additionally, the pharmaceutically acceptable acids and/or bases include those resulting from amino acids, for instance histidine, arginine or glycine.
According to one embodiment, the composition according to the invention comprises a pharmaceutically acceptable buffer system. These pharmaceutically acceptable buffer systems include those which are resulting from the salts of the abovementioned acids and bases or from the combination thereof.
The buffer system can in particular be chosen from the following combinations: monobasic sodium phosphate (also called monosodium phosphate)/dibasic sodium phosphate (also called disodium phosphate), monobasic potassium phosphate (also called monopotassium phosphate)/dibasic sodium phosphate (also called disodium phosphate)/sodium salt, acetic acid/sodium acetate, citric acid/sodium citrate, L-histidine hydrochloride/histidine, glycine hydrochloride/glycine.
The composition according to the invention may also comprise one or more inorganic salt(s), preferably chosen from pharmaceutically acceptable inorganic salt(s).
Such salts may in particular be chosen from sodium chloride, potassium chloride and tin(II) chloride.
The composition according to the invention may comprise the protein(s) as sole therapeutic active agent. It may also comprise other therapeutic active agents in addition to the protein(s).
The composition according to the invention may also comprise any additive, adjuvant or excipient, which is preferably pharmaceutically acceptable.
Those skilled in the art will take care to select this or these optional additional compound(s) and/or active agent(s) in such a way that the advantageous properties intrinsically associated with the composition according to the invention are not, or not substantially, impaired by the envisioned addition(s).
Such additives may generally be present in an amount, for each of them, of between 0 and 10% by weight relative to the total weight of the composition.
In particular, the composition according to the invention may also comprise at least one preservative.
The preservative(s) may in particular be chosen from benzyl alcohol, phenol, m-cresol and povidone.
The composition according to the invention may also comprise at least one surfactant.
The surfactant(s) may be, for example, chosen from polysorbate 20 (also called PS20 or Tween 20), polysorbate 80 (also called PS80 or Tween 80), Pluronic F-68, the “Brij” products and also alkylglucosides such as n-dodecyl-a-D-maltoglucoside (DDM).
The composition according to the invention may also comprise a lyoprotectant and/or a pharmaceutically acceptable sugar.
The lyoprotectant and the pharmaceutically acceptable sugar may for example be chosen from α-trehalose, saccharose (also called sucrose), maltose, mannitol, sorbitol and dextran. Use may also be made, as lyoprotectant, of amino acids such as histidine.
According to one particularly preferred embodiment, the composition according to the invention is intended for therapeutic use, in humans or animals.
The composition according to the invention is then a pharmaceutical or veterinary composition, preferably a pharmaceutical composition.
In this embodiment, the composition according to the invention is preferably intended for systemic administration. It is in particular an injectable composition, intended to be administered, for example, by intravenous injection, by subcutaneous injection or by intramuscular injection, and more preferentially by subcutaneous injection.
Particularly preferably, the composition according to the invention is intended for therapeutic use in human beings.
A subject of the present invention is also the composition as described above, for use as a medicament.
According to one preferred embodiment, a subject of the invention is the composition as described above, for use in preventing and/or treating one or more pathological conditions in humans or animals.
The composition is particularly of use for treating all human pathological conditions involving the administration, to the patient, of one or more therapeutic proteins. In particular, and in a nonlimiting manner, the composition according to the invention may be used for treating the various forms of cancer, diabetes, autoimmune diseases, Alzheimer's disease, Crohn's disease, cardiovascular diseases, anemias, graft rejections, scleroses and rheumatoid arthritis.
The composition according to the invention can be prepared by simple mixing of its ingredients in water, with stirring.
It can in particular be prepared by mixing the solubilizing agent(s) and the protein(s) in water, at a pH which is preferably different than the isoelectric point of the protein(s) under consideration. The pH can then be adjusted if required.
Finally, the invention relates to the use, in order to improve the solubilization of proteins within an aqueous composition, of a solubilizing agent consisting of an anionic compound of non-saccharide structure, which contains at least one aromatic nucleus comprising at least 6 ring members 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.
Everything which has been described above regarding the composition according to the invention applies by analogy to the use according to the invention.
The following examples serve to illustrate the invention without, however, being limiting in nature.

EXAMPLES

Part A: Synthesis

Example A1

Molecule A1

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.
Yield: 7.5 g (77%)
¹H NMR (DMSO-d₆, 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

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.
Yield: 2.1 g (42%)
¹H NMR (DMSO-d₆, 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


			Stock solution
CAS number	Supplier	Reference	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 acid	103-82-2	Aldrich	P16621	900	mM
2-Phenoxypropionic	940-31-8	Aldrich	197149	1125	mM
acid
Molecule A2	70134-21-3	Adocia		315	mg/ml

Example B1

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

The solid form of the molecule A1 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

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

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

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

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

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

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

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

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.
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.
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 μm 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 1

	Molar ratio	Compound		Soluble
	(compound/	concentration	Visual	insulin
Mixtures	insulin)	(mmol/l)	appearance	recovery (%)

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

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

The Nanogam formulation is a formulation of human immunoglobulins at 50 mg/ml and at pH 4.3 containing various IgG subclasses (IgG1: 54-70%, IgG2: 29-45%, IgG3: 1-4%, IgG4: 0-0.5%, IgA: at most 6 μg/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.
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.
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.
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 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

The examples with the molecule A1, 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.

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. The solubilization of 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:

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;

Y₁and Y₂denoting, independently of one another: a hydrogen atom; an —OH group; an —OR₁group with R₁denoting an alkyl radical containing from 1 to 6 carbon atoms or a hydroxyalkyl radical containing from 1 to 6 carbon atoms; and preferably Y₁and Y₂denote, 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:

with X, Y₁, Y₂and 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:

with X, Y₁, Y₂and M as defined in claim 3.

7. The process as claimed in claim 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:

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:

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

14. The process as claim 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 claim 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 claim 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 (ADCs—antibody-drug conjugates) and one or more solubilizing agent(s), wherein the solubilizing agent(s) correspond(s) to general formula (I) below:

with:

Ar denoting an aromatic nucleus comprising at least 6 ring members;

Y₁and Y₂denoting, independently of one another: a hydrogen atom; an OH group; an —OR₁group with R₁denoting an alkyl radical containing from 1 to 6 carbon atoms or a hydroxyalkyl radical containing from 1 to 6 carbon atoms; and preferably Y₁and Y₂denote, 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 1, wherein the solubilizing agent(s) correspond(s) to formula (Ia) below:

with X, Y₁, Y₂and M as:

Y₁and Y₂denoting, independently of one another: a hydrogen atom; an —OH group; an —OR₁group with R₁denoting an alkyl radical containing from 1 to 6 carbon atoms or hydroxyalkyl radical containing from 1 to 6 carbon atoms; and preferably Y₁and Y₂denote, independently of one another, a hydrogen atom or an —OH group; and

M denoting a cation.

20. The composition as claimed in claim 1, wherein the solubilizing agent(s) correspond(s) to formula (Ib) below:

with X, Y₁, Y₂and M as;

M denoting a cation.

21. The composition as claimed in claim 1, 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:

22. The composition as claimed in claim 1, wherein L denotes a single bond or an amide group.

23. The composition 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.

24. The composition 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:

used in the form of a sodium salt or of a potassium salt.

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

26. The composition 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:

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 1, which comprises the solubilizing agent(s) in a total concentration of between 1 g/1 and 100 g/l.

28. The composition as claimed in claim 1, which contains a total concentration of protein(s) of between 0.5 and 400 mg/ml.

29. The composition 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.

30. The composition as claimed in claim 1, which is intended to be administered by intravenous injection, by subcutaneous injection or by intramuscular injection.