Pharmaceutical formulations for subcutaneous administration of proteins

Abstract

The present invention relates to a pharmaceutical formulation of a pharma- ceutical active protein comprising an effective amount of a protein or a combination of proteins, chitosan A, and at least one statistical polysaccharide B. Furthermore, the present invention relates to a kit comprising at least one first container comprising chitosan A and statistical polysaccharide B, and at least one second container comprising a protein or a combination of proteins. Furthermore, the present invention relates to a pharmaceutical formulation or kit for use in the treatment of cancer, typically breast cancer or gastric cancer.

Description

[Background technology]

[0001] The pharmaceutical use of therapeutic proteins, such as antibodies, has increased over the past few years. Such therapeutic proteins are primarily administered via the intravenous (IV) route.

[0002] Another route of administration includes subcutaneous injection.

[0003] The advantage of subcutaneous injection is that it can be performed by a healthcare professional with a relatively short intervention time for the patient. Furthermore, patients can be trained to administer subcutaneous injections themselves. Such self-administration is particularly useful during maintenance dosing, as it avoids the need for hospital care (reducing the use of healthcare resources) and can have a positive impact on the patient's quality of life.

[0004] Although IV administration allows for the administration of large volumes of drugs directly into the bloodstream without volume limitations, parenteral drug injection into subcutaneous tissue is generally limited to volumes of less than 2 ml due to the viscoelastic resistance of the subcutaneous (SC) tissue, the back pressure generated during injection, and the perception of pain, thus limiting the amount that can be injected into the extracellular matrix of the SC tissue.

[0005] Subcutaneous (SC) formulations of proteins therefore require high protein concentrations in the final infused solution.

[0006] HERCEPTIN™ (trastuzumab) is a monoclonal antibody against the HER2 receptor (anti-HER2 antibody) that is sold in Europe as a 150 mg lyophilized powder (containing the antibody, α,α-trehalose dihydrate, L-histidine and L-histidine hydrochloride, and polysorbate 20) that must be reconstituted for injection with water for injection to produce an injection volume of approximately 21 mg / ml.

[0007] ROCHE has developed a subcutaneous formulation of HERCEPTIN® (hereafter referred to as HERCEPTIN® SC). This SC formulation contains recombinant human hyaluronidase PH20 (rHuPH20), which induces local and temporary modification of the SC space by degrading hyaluronic acid, a naturally occurring glycosaminoglycan found throughout the body that creates resistance to bulk fluid flow in the subcutaneous extracellular matrix and limits high-volume SC drug delivery. By degrading hyaluronic acid at the local injection site, hyaluronidase allows SC bulk fluid flow and facilitates high-volume SC delivery.

[0008] However, large amounts of hyaluronidase carry the risk of causing irritation at the injection site. The HERCEPTIN® SC formulation contains hyaluronidase, which can cause irritation if administered in large amounts, so there is a limit to the amount that can be precisely injected: the maximum volume is approximately 6 mL.

[0009] Therefore, injections of the HERCEPTIN® SC formulation must be repeated approximately every two weeks.

[0010] Furthermore, using circulating antibodies from immunized cynomolgus monkeys, it has been demonstrated that circulating antibodies that specifically recognize PH-20 bind to the surface of macaque sperm. Therefore, we cannot exclude the effect of rHuPH20 antibodies in attenuating human fertility, since antisperm antibodies could theoretically inhibit sperm maturation, motility, and in women, antisperm antibodies could prevent passage through the cervix and uterus, etc. Although nonclinical studies performed on SC rHuPH20, including commercial drugs, have not shown reproducible effects in several species, reversible infertility has been observed in male and female guinea pigs immunized to produce antibodies against hyaluronidase. The potential risk to reproducibility remains very important, especially for medicines used long-term in young non-cancer patients (e.g. multiple sclerosis, inflammatory bowel disease).

[0011] Another solution for local administration of antibodies is to use a suitable support material to achieve long-term release of the antibody. Several hydrogels based on synthetic or natural polymers have been proposed for long-term release of antibodies. Hydrogels are physical or chemical gels composed of polymer chains swollen with a large amount of water (about 70% of the total volume of the hydrogel). For example, hydrogel implants based on poly(ethylene-vinyl acetate copolymer (EVAc) have been proposed. However, these implants are formed with harsh solvents that may degrade the antibodies, and the polymer is not biodegradable, requiring patients to undergo additional surgery to remove the implant. An angiogenic antibody called bevacizumab (trade name Avastin®, Genentech), which targets vascular endothelial growth factor (VEGF), has been explored for various clinical applications, including treatment of various cancers and use in the eye for macular degeneration and other pathologies. One study investigated the delivery of bevacizumab from a blended glycol chitosan and oxidized alginate hydrogel for its potential use in intraocular drug delivery, but release was complete after 3 days. Additionally, several biopolymers, such as alginate-chitosan hydrogels, were loaded with bevacizumab and a control IgG. However, these hydrogels have only been evaluated in vitro, and biodegradability and biocompatibility have not been examined.

[0012] Thus, there remains a need to provide a pharmaceutical composition comprising a protein or combination of proteins that overcomes the shortcomings of the prior art, can be easily injected by an operator, and can provide sustained release of the protein with little or no irritation at the injection site.

[0013] The reduced inflammation is advantageous because it allows for a shorter interval between two injections and the selection of injection sites (such as the abdominal wall) that are usually avoided with HERCEPTIN® SC formulations due to the presence of hyaluronidase. Another objective is to provide an SC formulation of HERCEPTIN® or a similar antibody (such as a biosimilar of Herceptin® or other anti-HER2 antibodies) that does not require the presence of hyaluronidase and therefore does not bear the risk of inflammation and toxicity associated with this enzyme. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Flecher et al., Materials Science and Engineering:C, 2016, 59.806-809 Summary of the Invention

[0015] Embodiment E1 of the present disclosure is a pharmaceutical formulation of a pharma- ceutical active protein, comprising: an effective amount of a protein or a combination of proteins; chitosan A containing 90-100 mol % D-glucosamine and 0-15 mol % N-acetyl-D-glucosamine; - at least one statistical polysaccharide B comprising D-glucosamine, N-acetyl-D-glucosamine and at least one saccharide unit of formula I: [ka] (In the formula, Rc is a hydrophilic group, Z is a linker which is a single bond or a hydrocarbon chain containing 1 to 12 carbon atoms, said chain being linear or branched, saturated or unsaturated, and optionally containing one or more heteroatoms (preferably selected from nitrogen, oxygen and sulfur); and - one or more pharma- ceutically acceptable excipients The present invention relates to a pharmaceutical formulation of a pharma- ceutical active protein comprising:

[0016] Embodiment E2 of the present disclosure is a pharmaceutical formulation according to E1, wherein the pharmaceutical formulation forms a hydrogel at physiological pH and osmolality.

[0017] Embodiment E3 of the present disclosure is a pharmaceutical formulation according to E1, wherein Rc is a group having acidic properties, typically a carboxyl group (-COOH), a sulfonic acid group (-SO 2 OH), phosphonate group (-PO(OH) 2 ), a thiol group (-SH), an alcohol group (-OH) and a group containing a chelating agent.

[0018] Embodiment E4 of the present disclosure is a pharmaceutical formulation according to E3, wherein Rc is a group comprising a chelator, the chelator being selected from the group consisting of DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid), DOTA ... TAGA (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanedioic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), NOTAM (1,4,7-tetrakis(carbamoylmethyl)-1,4,7-triazacyclononane), DOTP (1,4 Rc is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonate)), NOTP (1,4,7-tetrakis(methylenephosphonate)-1,4,7-triazacyclononane), TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid), TETAM (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetrakis(carbamoylmethyl)), DTPA (diethylenetriaminopentaacetic acid), DFO (deferoxamine), Bz-DFO, and bispidine ([3,7]-diazabicyclo[3.3.1]nonane), preferably DOTAGA, DFO, Bz-DFO, DOTAM, DTPA, and bispidine, more preferably DOTAGA.

[0019] Embodiment E5 of the present disclosure is a pharmaceutical composition according to E1, wherein the statistical polysaccharide B is a polysaccharide of general formula II: [ka] (In the formula, Rc is a hydrophilic group, Z is a linker which is a single bond or a hydrocarbon chain containing from 1 to 12 carbon atoms, said chain being linear or branched, saturated or unsaturated, and optionally containing one or more heteroatoms (preferably selected from nitrogen, oxygen and sulfur), x is between 0.01 and 0.5. y is between 0.05 and 0.5, the ratio y / x is greater than 0.2, preferably greater than 1; · The sum of x+y is greater than 0.1.

[0020] Embodiment E6 of the present disclosure is a pharmaceutical formulation according to E5, wherein Rc is a group having acidic properties, typically a carboxyl group (-COOH), a sulfonic acid group (-SO 2 OH), phosphonate group (-PO(OH) 2 ), a thiol group (-SH), an alcohol group (-OH) and a group containing a chelating agent.

[0021] Embodiment E7 of the present disclosure is a pharmaceutical formulation according to E6, wherein Rc is a group comprising a chelator, the chelator being selected from the group consisting of DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid), DOTA ... TAGA (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanedioic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), NOTAM (1,4,7-tetrakis(carbamoylmethyl)-1,4,7-triazacyclononane), DOTP (1,4 Rc is selected from 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonate)), NOTP (1,4,7-tetrakis(methylenephosphonate)-1,4,7-triazacyclononane), TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid), TETAM (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetrakis(carbamoylmethyl)), DTPA (diethylenetriaminopentaacetic acid), DFO (deferoxamine), Bz-DFO, and bispidine ([3,7]-diazabicyclo[3.3.1]nonane), preferably DOTAGA, DFO, Bz-DFO, DOTAM, DTPA, and bispidine, more preferably DOTAGA.

[0022] Embodiment E8 of the present disclosure is a pharmaceutical formulation according to E1, wherein the statistical polysaccharide B is a polysaccharide of general formula III: [ka] (In the formula, - R c1 and R c2 are not the same and are hydrophilic groups, - Z1 and Z2 are linkers, whether identical or not, which are a single bond or a hydrocarbon chain containing from 1 to 12 carbon atoms, said chain being linear or branched, saturated or unsaturated, and optionally containing one or more heteroatoms (preferably selected from nitrogen, oxygen and sulfur); - x is comprised between 0.01 and 0.5, preferably between 0.01 and 0.1, and more preferably between 0.05 and 0.1; - y is 0.01 to 0.5, preferably 0.05 to 2; - z is comprised between 0 and 0.2, the ratio y / x is greater than 0.2, preferably greater than 1; - The sum of x+y is greater than 0.1.

[0023] Embodiment E9 of the present disclosure is a pharmaceutical formulation according to E8, wherein Rc1 and Rc2 are groups having acidic properties, typically a carboxyl group (-COOH), a sulfonic acid group (-SO 2 OH), phosphonate group (-PO(OH) 2 ), a thiol group (-SH), an alcohol group (-OH) and a group containing a chelating agent.

[0024] Embodiment E10 of the present disclosure is a pharmaceutical formulation according to E9, wherein Rc1 and / or Rc2 are chelating agents, preferably DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4, 7-diacetic acid), DOTAGA (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanedioic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), NOTAM (1,4,7-tetrakis(carbamoylmethyl)-1,4,7-triazacyclononane), DOTP (1,4, 7,10-Tetraazacyclododecane-1,4,7,10-tetrakis(methylenephosphonate)), NOTP (1,4,7-tetrakis(methylenephosphonate)-1,4,7-triazacyclononane), TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid), TETAM (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetate) Rc1 is selected from DOTAGA, DFO, Bz-DFO, DOTAM, DTPA and bispidine, more preferably Rc1 is DOTAGA and Rc2 is DFO or Bz-DFO.

[0025] Embodiment E11 of the present disclosure is a pharmaceutical formulation according to any of the preceding embodiments, wherein Z, Z1 and Z2 are independently selected from the group consisting of a single bond, a hydrocarbon chain comprising 1 to 12 carbon atoms, which chain is linear or branched, saturated or unsaturated, and optionally containing one or more heteroatoms, preferably selected from nitrogen, oxygen and sulfur, an alkyl chain comprising 1 to 12 carbon atoms, which chain is linear or branched, and an alkenyl chain comprising 2 to 12 carbon atoms, which chain is linear or branched, and wherein the alkyl and alkenyl chains are selected from the group consisting of C 6 ~C 10 and / or one or more heteroatoms or groups selected from the group consisting of -O-, -S-, -C(O)-, -NR'-, -C(O)NR'-, -NR'-C(O)-, -NR'-C(O)-NR'-, -NR'-C(O)-O-, -OC(O)NR', ​​-C(S)NR'-, -NR'-C(S)-, -NR'-C(S)-NR', and the alkyl and alkenyl chains may be interrupted by one or more heteroatoms or groups selected from the group consisting of -OR', -COOR', -SR', -NR'2 (wherein each R' is independently H or a C1-C6 alkyl group). each of Z, Z1 and Z2 is independently a single bond, an alkyl chain containing 1 to 12 carbon atoms, said chain being linear or branched, said alkyl chain being one or more aryl groups of C6 to C10, and / or -O-, -S-, -C(O)-, -NR'-, -C(O)NR'-, -NR'-C(O)-, -C(S)NR'-, -NR'-C(S)-, -NR'-C(S)-NR' (each R' is independently H or C 1 ~C 6 and n is an integer from 1 to 3. The alkyl groups may be selected from the group consisting of aryl, aryl, aryl and alkyl.

[0026] Embodiment E12 of the present disclosure is the pharmaceutical formulation according to E11, wherein each of Z, Z1 and Z2 is an alkyl chain containing 1 to 12 carbon atoms.

[0027] Embodiment E13 of the present disclosure is the pharmaceutical formulation according to E11, wherein each of Z, Z1 and Z2 is a polyethylene glycol (PEG) moiety (fraction).

[0028] Embodiment E14 of the present disclosure is a pharmaceutical formulation according to any one of E1 to E13, wherein the statistical polysaccharide B has an average molecular weight of 100 kDa to 1000 kDa, more preferably 200 kDa to 750 kDa, even more preferably 250 kDa to 500 kDa, and most preferably 300 kDa to 400 kDa.

[0029] Embodiment E15 of the present disclosure is a pharmaceutical formulation according to any one of E1-E14 for subcutaneous administration.

[0030] Embodiment E16 of the present disclosure is the pharmaceutical formulation according to any one of E1-E15, wherein the hydrogel releases protein over a period of 10-100 days, preferably 20-65 days from the date of administration.

[0031] Embodiment E17 of the present disclosure is a pharmaceutical formulation according to any one of E1-E16, wherein the viscosity of the formulation is measured by rotational rheometry in a plane cone geometry at room temperature at a shear rate of 0.01 to 0.001 s -1 , e.g. 0.001 seconds -1 or 0.01 seconds -1 , especially 0.01 seconds -1 It is measured in the range of 10 to 500 Pa·s.

[0032] Embodiment E18 of the present disclosure is a pharmaceutical formulation according to any one of E1-E17, wherein the formulation has a pH of 5.0-6.5, preferably 5.5-6.0.

[0033] Embodiment E19 of the present disclosure is a pharmaceutical formulation according to any one of E1-E18, wherein the osmolality of the formulation is comprised between 50-600 mOsm / L, preferably between 100-600 mOsm / L, more preferably between 250-450 mOsm / L, or between 50-300 mOsm / L, preferably between 50-250 mOsm / L.

[0034] Embodiment E20 of the present disclosure is a pharmaceutical formulation according to any one of E1-E19, - 5 to 200 g / l, preferably 10 to 200 g / l, more preferably 10 to 150 g / l of chitosan A and 10 to 200 g / l, preferably 20 to 100 g / l of statistical polysaccharide B, and - 1 g / l to 200 g / l, preferably 50 to 100 g / l of protein or a combination of proteins Includes.

[0035] Embodiment E21 of the present disclosure is a pharmaceutical formulation according to any one of E1 to E20, wherein the protein has a molecular weight comprised between 10 kDa and 250 kDa, preferably between 20 kDa and 250 kDa, preferably between 100 kDa and 250 kDa, preferably between 120 kDa and 250 kDa, preferably between 150 kDa and 250 kDa.

[0036] Embodiment E22 of the present disclosure is the pharmaceutical formulation of any one of embodiments E1-E21, wherein the protein or combination of proteins is selected from the group consisting of an antibody, an enzyme, a fusion protein, and combinations thereof.

[0037] Embodiment E23 of the present disclosure is a pharmaceutical formulation according to any one of E1-E22, wherein the formulation does not contain a hyaluronidase enzyme.

[0038] Embodiment E24 of the present disclosure is the pharmaceutical formulation of any one of E1-E23, wherein the protein or combination of proteins comprises an antibody or an antigen-binding fragment of an antibody.

[0039] Embodiment E25 of the present disclosure is a pharmaceutical formulation according to E24, wherein said antibody or antibody combination is selected from the group consisting of: (i) a chimeric antibody, a human antibody, or a humanized antibody, and (ii) an antibody-drug conjugate.

[0040] Embodiment E26 of the present disclosure is a pharmaceutical formulation according to any one of E1-E25, wherein the protein or combination of proteins is an antibody or combination of antibodies that binds to an antigen selected from the group of HER2 and CD20.

[0041] Embodiment E27 of the present disclosure is the pharmaceutical formulation according to E27, wherein the antibody is selected from the group consisting of trastuzumab, rituximab and pertuzumab or a combination thereof.

[0042] Embodiment E28 of the present disclosure is a pharmaceutical formulation according to any one of E27 or E28, wherein the formulation comprises a combination of trastuzumab and pertuzumab.

[0043] Embodiment E29 of the present disclosure is a pharmaceutical formulation according to any one of E1-E28, further comprising at least one pharma- ceutically acceptable excipient selected from solvents, stabilizers, surfactants, buffers, antimicrobial preservatives, protectants, antioxidants, chelating agents, and bulking agents.

[0044] Embodiment E30 of the present disclosure is a pharmaceutical formulation according to any one of E1-E30, comprising: - 1 g / l to 200 g / l, preferably 50 to 100 g / l of an antibody or a combination of antibodies, in particular trastuzumab, rituximab, pertuzumab or daratumumab, or a combination of trastuzumab and pertuzumab, - 5 to 200 g / l, preferably 10 to 200 g / l, more preferably 10 to 150 g / l of chitosan A, and 20 to 400 g / l, preferably 20 to 300 g / l, of statistical polysaccharide B, and At least one excipient selected from the following: a 1-100 mM buffer, preferably a histidine buffer, more preferably a histidine chloride buffer such as L-histidine hydrochloride monohydrate, having a pH of 5.0-6.5, preferably 5.0-6.4, more preferably 5.5-6.00; 1 to 500 mM of a stabilizer, preferably trehalose, such as α,α-trehalose dihydrate, or 5-25 mM antioxidant, preferably methionine Includes.

[0045] Embodiment E31 of the present disclosure comprises: At least one first container comprising chitosan A and statistical polysaccharide B as defined in any one of E1 to E12; At least one second container containing a protein or combination of proteins as defined in any one of E22 to E29. The kit comprises:

[0046] Embodiment E32 of the present disclosure is a kit according to E31 for preparing a ready-to-use injectable formulation according to the present disclosure.

[0047] Embodiment E33 of the present disclosure is a pharmaceutical formulation according to any one of claims E1 to E31, or a kit according to any one of E32 or E33, for use in the treatment of cancer, typically breast or gastric cancer, or a non-malignant disease.

[0048] Embodiment E34 of the present disclosure is a method of treating cancer in a subject in need thereof, comprising subcutaneously administering to the subject a therapeutically effective amount of an antibody or combination of antibodies that binds to an antigen selected from the group of HER2 and CD20 as described in any one of E27 to E29. [Brief description of the drawings]

[0049] [Figure 1] FIG. 1 shows the gel formed in Hemosol B0 from the MEX-CD2-I-tmb solution. [Diagram 2] FIG. 2 shows the in situ gelation of a fluorescently labeled MEX-CD2-I-tmb solution (FIG. 2A) and the resulting gel after complete gelation in physiological serum (FIG. 2B). [Diagram 3]FIG. 3 shows the stability range of MEX-CD2-I solutions as a function of pH and osmolality. [Figure 4] Figure 4 shows a time follow-up of mouse weight after subcutaneous injection of 200 μL of MEX-CD2-I-tmb solution spiked with 100 g / L trastuzumab antibody. N=9 mice. [Diagram 5] Figure 5 shows hematoxylin and eosin (H&E) labeling 20 days after subcutaneous injection of 200 μL of MEX-CD2-I-tmb solution (equivalent to a 15 mg trastuzumab dose). [Figure 6] FIG. 6 shows the time course of the presence of trastuzumab antibodies in plasma following subcutaneous injection of 200 μL of MEX-CD2-I-tmb solution (equivalent to a 15 mg dose of trastuzumab) and following subcutaneous injection of the Roche SC HERCEPTIN® formulation (equivalent to a 15 mg dose of trastuzumab). [Figure 7] FIG. 7 shows monitoring the presence of fluorescently labeled solution MEX-CD2-I after injection. [Figure 8] FIG. 8 shows the time course monitoring of the degradation of antibody-loaded hydrogels by fluorescence imaging. [Figure 9] FIG. 9 shows the percentage of fluorescence compared to the initial fluorescence of the hydrogel over time. [Figure 10] FIG. 10 shows the percentage of gadolinium present at each organ / site compared to the maximum amount of gadolinium initially complexed in the gel for solutions with and without trastuzumab. [Figure 11] FIG. 11 shows (A) T1MRI signals of trastuzumab-loaded hydrogels implanted in the neck (200 μL) of mice (n=3) (dashed lines represent the contours of the hydrogels) and (B) volume curves of the hydrogels with varying volumes and ratios of loaded trastuzumab. [Figure 12]Figure 12 shows a comparison of the injected volume of trastuzumab-loaded hydrogels implanted subcutaneously. 50 μL and 200 μL of trastuzumab-loaded hydrogels (100 G / L trastuzumab) implanted subcutaneously (n=3) show the same degradation profile. [Figure 13] FIG. 13 shows immunofluorescence staining of proinflammatory neutrophils localized at the injection site 5 days after implantation of trastuzumab-loaded hydrogels. [Figure 14] FIG. 14 shows the cumulative release of monoclonal antibodies (trastuzumab or rituximab) and a combination of monoclonal antibodies (trastuzumab and pertuzumab) in PBS at 37° C. [Figure 15] FIG. 15 shows the results for the cumulative release of monoclonal antibody fragments (Fab'2 and VHH) in PBS at 37°C. [Figure 16] FIG. 16 shows SEM images at various magnifications of gels formed in PBS solution from polymer solutions with and without trastuzumab.

[0050] Detailed Description The first aspect of the present disclosure is a therapeutically effective amount of a protein or combination of proteins; chitosan A containing 90-100 mol % D-glucosamine and 0-15 mol % N-acetyl-D-glucosamine; - at least one statistical polysaccharide B comprising D-glucosamine, N-acetyl-D-glucosamine and at least one saccharide unit of formula I: [ka] (In the formula, Rc is a hydrophilic group, Z is a linker which is a single bond or a hydrocarbon chain containing 1 to 12 carbon atoms, said chain being linear or branched, saturated or unsaturated, and optionally containing one or more heteroatoms (preferably selected from nitrogen, oxygen and sulfur); and - one or more pharma- ceutically acceptable excipients A pharmaceutical formulation comprising:

[0051] polysaccharide According to the present disclosure, "chitosan" refers to a naturally occurring polymer of polysaccharide type of D-glucosamine (GlcN) or of copolysaccharide type of D-glucosamine and N-acetyl-D-glucosamine (GlcNAc) linked by glycosidic bonds of the β(1→4) type in a random (statistical copolysaccharide) or non-random (block copolysaccharide) distribution.

[0052] According to the present disclosure, the average molecular weight Mw of polysaccharides such as chitosan A and polysaccharide B is measured by steric exclusion chromatography, the method of which is described in “Physico-chemical studies of the gelation of chitosan in a hydroalcoholic medium” A. MONTEMBAULT, C. VITON, A. DOMARD, Biomaterials, 26(8), 933-943, 2005.

[0053] The proportion of N-acetyl-D-glucosamine was determined according to the Hirai method (A. Hirai, H Odani, A. Nakajima, Polymer Bulletin, 26(1), 87-94, 1991). 1 Calculated using NMR.

[0054] According to the present disclosure, crystallinity refers to the percentage of crystalline material. For polysaccharides, crystallinity is often measured using X-ray diffraction (Alexander, LE, 'X-ray Diffraction Methods in Polymer Science', Wiley-Interscience, New York, 1969, p. 137).

[0055] Chitosan A The pharmaceutical formulation of the present disclosure comprises chitosan A. The formulation comprises: - a crystallinity of at least 10% by weight of chitosan A relative to the total dry mass of the formulation, and - Chitosan A crystallite size less than 20 nm

[0043]

[0056] The crystallinity of chitosan A may be 10 to 25%. The crystallites of chitosan A function as physical cross-linking sites essential for forming a gel. The size of the crystallites is, for example, 1 to 20 nm.

[0057] Advantageously, the chitosan A has an average molecular weight Mw between 100 kg / mol and 1000 kg / mol, preferably between 200 kg / mol and 700 kg / mol.

[0058] According to a preferred embodiment, chitosan A contains 1 mol% to 9 mol% N-acetyl-D-glucosamine, preferably 2 mol% to 8 mol%, more preferably 3 mol% to 7 mol%, even more preferably 4 mol% to 6 mol%, and most preferably 5 mol%.

[0059] statistical polysaccharide B The preparation contains statistical polysaccharide B.

[0060] Advantageously, the statistical polysaccharide B has an average molecular weight of between 100 kDa and 1000 kDa, more advantageously between 200 kDa and 750 kDa, even more advantageously between 250 kDa and 500 kDa, and most advantageously between 300 kDa and 400 kDa.

[0061] Preferably, Rc is a group having acidic properties, typically a carboxyl group (-COOH), a sulfonic acid group (-SO 2 OH), phosphonate group (-PO(OH) 2 ), a thiol group (-SH), an alcohol group (-OH) and a group containing a chelating agent.

[0062] In certain embodiments where Rc is a chelator, Rc is selected from the group consisting of DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid, DOTAGA (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane-1-yl)pentanedioic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), NOTAM (1,4,7-tetrakis(carbamoylmethyl)-1,4,7-triazacyclononane), DOTP (1,4,7,10-tetraazacyclododecane), Rc may be selected from 1,4,7,10-tetrakis(methylenephosphonate)), NOTP (1,4,7-tetrakis(methylenephosphonate)-1,4,7-triazacyclononane), TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid), TETAM (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetrakis(carbamoylmethyl)), DTPA (diethylenetriaminopentaacetic acid), DFO (deferoxamine), Bz-DFO and bispidine ([3,7]-diazabicyclo[3.3.1]nonane), preferably selected from DOTAGA, DFO, Bz-DFO, DOTAM, DTPA and bispidine, more preferably Rc is DOTAGA.

[0063] The chelating agent of polysaccharide B preferably participates in the gelation of the polysaccharide mixture and provides swelling properties to the hydrogel. Furthermore, the chelating agent preferably promotes the formation of a hydrogel containing non-crystalline regions, which may result in good biodegradability of the hydrogel.

[0064] Chitosan A promotes the formation of crystals that contribute to the gel state of the material in contact with body fluids.

[0065] According to the present disclosure, the expression "polysaccharide mixture" refers to chitosan A and polysaccharide B.

[0066] In one embodiment, the statistical polysaccharide B is a polysaccharide of the following general formula II: [ka] (In the formula, Rc is a hydrophilic group, Z is a linker which is a single bond or a hydrocarbon chain containing from 1 to 12 carbon atoms, said chain being linear or branched, saturated or unsaturated, and optionally containing one or more heteroatoms (preferably selected from nitrogen, oxygen and sulfur), x is between 0.01 and 0.5. y is between 0.05 and 0.5, the ratio y / x is greater than 0.2, preferably greater than 1; · The sum of x+y is greater than 0.1.

[0067] In some embodiments, the statistical polysaccharide B comprises two different units of formula I. In these embodiments, the statistical polysaccharide B is a polysaccharide of the following general formula III: [ka] (In the formula, - Rc 1 , Rc 2 are not the same and are hydrophilic groups, - Z1 and Z2 are linkers, whether identical or not, which are a single bond or a hydrocarbon chain containing from 1 to 12 carbon atoms, said chain being linear or branched, saturated or unsaturated, and optionally containing one or more heteroatoms (preferably selected from nitrogen, oxygen and sulfur); - x is comprised between 0.01 and 0.5, preferably between 0.01 and 0.1, and more preferably between 0.05 and 0.1; - y is comprised between 0.01 and 0.5, preferably between 0.05 and 2; - z is comprised between 0 and 0.2, the ratio y / x is greater than 0.2, preferably greater than 1; - The sum of x+y is greater than 0.1.

[0068] Preferably, Rc 1 and Rc 2 is a group having acidic properties, typically a carboxyl group (-COOH), a sulfonic acid group (-SOOH), a phosphonate group (-PO(OH) 2 ), a thiol group (-SH), an alcohol group (-OH) and a group containing a chelating agent.

[0069] In embodiments in which Rc1 and / or Rc2 are chelators, Rc1 and / or Rc2 are preferably selected from the group consisting of DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid), DOTAGA (2 -(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanedioic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), NOTAM (1,4,7-tetrakis(carbamoylmethyl)-1,4,7-triazacyclononane), DOTP (1,4,7,10-tetraaza Cyclododecane 1,4,7,10-tetrakis(methylenephosphonate)), NOTP (1,4,7-tetrakis(methylenephosphonate)-1,4,7-triazacyclononane), TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid), TETAM (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetrakis(ca Rc1 is selected from DOTAGA, DFO, Bz-DFO, DOTAM, DTPA and bispidine, more preferably, Rc1 is DOTAGA and Rc2 is DFO or Bz-DFO.

[0070] The precursor of the Bz-DFO chelating group is p-NCS-Bz-DFO (Cas number: 1222468-90-7), whose NCS group reacts with amines to generate thioureas and is therefore no longer NCS when grafted onto molecules such as polysaccharide B.

[0071] Chelating groups Rc, Rc 1 and Rc 2 As described above, the Rc groups in formula I and formula II, and the Rc groups in formula III 1 and Rc 2 The group may be a chelating group. In embodiments, R, R 1 and R.C. 2 is a chelating group, Rc, Rc 1 and Rc 2 is capable of chelating one or more metals by forming a complex.

[0072] In one embodiment, less than 10%, preferably less than 5%, of the chelating groups are chelated with cations, especially metal cations. When the chelating groups are in free form, the metals are well captured and polysaccharide B also has a strong hydrophilic behavior, which means good swelling properties.

[0073] Rc, Rc 1 and Rc 2 Each of Rc, Rc may contain one or more coordination sites. Preferably, the coordination sites are nitrogen or oxygen atoms. Advantageously, Rc, Rc 1 and Rc 2 Each of Rc, Rc preferably contains 4 to 8 coordination sites, more preferably 6 to 8 coordination sites, and even more preferably Rc, Rc 1 and Rc 2 Each of these contains eight coordination sites.

[0074] In the present disclosure, coordination site means a specific functional group that can chelate a metal. For example, an amine functional group represents one coordination site by forming a coordination bond between the nitrogen of the amine and the metal, and a hydroxamic acid functional group also represents one coordination site by forming a coordination bond between the oxygen of the carbonyl moiety and the metal, and also by forming a covalent bond between the oxygen of the N-oxide moiety and the very same metal, and the coordination site forms a 5-link ring.

[0075] Z, Z 1 、Z 2 As mentioned above, Z, Z1 and Z 2 is a linker that is a single bond or a hydrocarbon chain containing from 1 to 12 carbon atoms, said chain being linear or branched, saturated or unsaturated, and optionally containing one or more heteroatoms (preferably selected from nitrogen, oxygen and sulfur).

[0076] Z, Z 1 , and Z 2 The choice of is essentially Rc, Rc 1 , and Rc 2 , as well as the metal that is ultimately chelated. In fact, for steric reasons in particular, Rc, Rc 1 , and Rc 2 can be more or less close to a 6 links ring.

[0077] In some embodiments, Z, Z 1 and Z 2 are independently selected from the group consisting of a single bond, a hydrocarbon chain containing 1 to 12 carbon atoms, which chain is linear or branched, saturated or unsaturated, and optionally containing one or more heteroatoms, preferably selected from nitrogen, oxygen and sulfur, an alkyl chain containing 1 to 12 carbon atoms, which chain is linear or branched, and an alkenyl chain containing 2 to 12 carbon atoms, which chain is linear or branched, and the alkyl and alkenyl chains are selected from the group consisting of 6 ~C 10 and / or one or more heteroatoms or groups selected from the group consisting of -O-, -S-, -C(O)-, -NR'-, -C(O)NR'-, -NR'-C(O)-, -NR'-C(O)-NR'-, -NR'-C(O)-O-, -OC(O)NR', ​​-C(S)NR'-, -NR'-C(S)-, -NR'-C(S)-NR', and said alkyl and alkenyl chains may be interrupted by one or more heteroatoms or groups selected from the group consisting of -OR', -COOR', -SR', -NR' 2 (Each R' is independently H or C 1 ~C 6 and n is an integer from 1 to 3. The alkyl group may be substituted with one or more atoms or groups selected from the group consisting of:

[0078] Advantageously, Z, Z 1 and Z 2 each is independently a single bond, an alkyl chain containing 1 to 12 carbon atoms, said chain being linear or branched, and said alkyl chain being 6 ~C 10 and / or one or more aryl groups of -O-, -S-, -C(O)-, -NR'-, -C(O)NR'-, -NR'-C(O)-, -C(S)NR'-, -NR'-C(S)-, -NR'-C(S)-NR' (each R' is independently H or C 1 ~C 6 and n is an integer from 1 to 3. The alkyl group may be any of the alkyl groups selected from the group consisting of aryl, aryl, aryl and aryl.

[0079] In certain embodiments, Z, Z 1 , Z 2 Each of is an alkyl chain containing 1 to 12 carbon atoms.

[0080] In another particular embodiment, Z, Z 1 and Z 2 Each of the is a polyethylene glycol (PEG) moiety (fraction).

[0081] Preparation process of polysaccharide B Polysaccharide B can be obtained by a process comprising three successive steps: - Step 1: Chitosan is solubilized in an acidic solution with a pH of 4-5; - Step 2: Partially acetylate the amine functional groups of the chitosan solubilized in step 1; Step 3: Functionalizing at least a portion of the amine functions remaining after step 2.

[0082] Step 3 is especially Z, Z 1 , and / or Z 2 When is a hydrocarbon chain as defined above, it may be divided into several sub-steps.

[0083] Z, Z 1 , and / or Z 2 In the embodiment where R is a hydrocarbon chain as defined above, step 3 comprises the sub-step 3-1 of grafting said hydrocarbon chain onto at least a portion of the amine functional groups remaining after step 2, followed by the sub-step 3-2 of grafting R, R 1 , and / or Rc 2 Alternatively, step 3 does not include a sub-step. In this alternative, the hydrocarbonated chain may include a sub-step 3-2 consisting of grafting Rc, Rc 1 , and Rc 2 In this case, the step 3 is 1 or / and with a molecule comprising said hydrocarbonated chain.

[0084] Alternatively, polysaccharide B can be obtained starting from chitosan having an appropriate number of N-acetyl-D-glucosamine units. In this alternative, it is not necessary to carry out step 2 above, therefore, in this alternative, the process for obtaining polysaccharide B comprises at least the following two successive steps: - Step 1b: Chitosan containing N-acetyl-D-glucosamine units is solubilized in an acidic solution of pH 4-5; - step 2b: functionalizing at least a portion of the amine functions of the chitosan comprising N-acetyl-D-glucosamine units solubilized in step 1.

[0085] As in step 3, step 2b is particularly 1 , and / or Z 2 When is a hydrocarbon chain as defined above, it may be divided into several sub-steps.

[0086] Z, Z 1 , and / or Z 2 In the embodiment where R is a hydrocarbon chain as defined above, step 2b comprises a sub-step 2b-1 consisting of grafting said hydrocarbon chain onto at least some of the amine functional groups, and then grafting said hydrocarbon chain with R, R 1, and / or Rc 2 Alternatively, step 2b does not include a sub-step. In this alternative, the hydrocarbonated chain may comprise Rc, Rc 1 and Rc 2 In this case, the step 2b is combined with Rc, Rc 1 or / and with a molecule comprising said hydrocarbonated chain.

[0087] Polysaccharide content The content of chitosan A in the pharmaceutical preparation of the present disclosure is preferably 5 to 200 g / l, preferably 10 to 200 g / l, more preferably 10 to 150 g / l.

[0088] The content of statistical polysaccharide B in the pharmaceutical preparation of the present disclosure is preferably comprised of statistical polysaccharide B in the range of 10 to 200 g / l, preferably 20 to 100 g / l.

[0089] The weight ratio of chitosan A / statistical polysaccharide B is preferably in the range of 5 / 1 to 1 / 5, preferably 1 / 1 to 1 / 5, preferably 1 / 1 to 1 / 3.

[0090] In one embodiment, the weight ratio of chitosan A to statistical polysaccharide B is about 1 / 2, preferably 1 / 2.

[0091] In one embodiment, the pharmaceutical composition comprises chitosan A and statistical polysaccharide B in a weight percentage relative to the weight of the pharmaceutical composition of 2% to 10%, preferably 2%, 3%; 4%, 5%, 6%, 7%, or 8% to 10%, more preferably 3% to 8%.

[0092] viscosity The pharmaceutical formulations of the present disclosure have rheological properties that allow them to be easily injected into tissues, while in vivo they can form biodegradable hydrogels that encapsulate proteins and allow for controlled, long-term release in tissues.

[0093] The pharmaceutical formulations of the present disclosure are preferably in the form of a liquid formulation, more preferably in the form of a liquid injectable formulation.

[0094] In this disclosure, "liquid injectable formulation" means a liquid formulation that has a sufficiently low viscosity to allow good injectability through a gauge needle.

[0095] The term "injectability" or "syringeability" as generally used herein refers to the ability to inject a pharmaceutical formulation of the present disclosure through a syringe equipped with an 18-32 gauge needle, preferably a 20-27 gauge needle.

[0096] The pharmaceutical formulations of the present disclosure have a melting rate of 0.01 to 0.001 seconds at room temperature by rotational rheometry in a plane cone geometry. -1 , e.g. 0.001 seconds -1 or 0.01 seconds -1 , preferably 0.01 seconds -1 The viscosity, measured at a shear rate of 10 to 500 Pa·s, preferably 10 to 300 Pa·s, and more preferably 15 to 250 Pa·s.

[0097] In one embodiment, the viscosity of the pharmaceutical formulation is comprised between 10 and 80 Pa·s, preferably between 10 and 50 Pa·s, more preferably between 10 and 30 Pa·s, or the viscosity is comprised between 80 and 250 Pa·s, preferably between 80 and 120 Pa·s, such as about 100 Pa·s.

[0098] The term "viscosity" refers to the resistance of a substance, usually a liquid, to flow. Viscosity is related to the concept of shear; it can be understood as the effect that different layers of a fluid exert on each other or on other surfaces as they move against each other. The unit of viscosity is Ns / m 2 and is known as the pascal second (Pa s).

[0099] The viscosity of the pharmaceutical formulations of the present disclosure is typically measured by rotational rheometry in a plane-cone geometry using a viscometer with a deformation imposed such as the Advanced Rheometer AR2000 from TA instruments. Briefly, a fluid is subjected to shear between two surfaces, one fixed and the other rotating around its axis. In practice, these two surfaces are typically a cone and a plane. The shear gradient is determined on the one hand by the geometry of the surfaces and on the other hand by the rotation speed. The shear stress is calculated from the measurement of the torque transmitted by the sample to be characterized. Details of this method are described in El Kissi et al., Rheology, 10, 13-39 (2006). In the present invention, the measurements are performed at room temperature (e.g., about 20°C, preferably about 25°C) and typically with a cone-plane geometry of 4° and 25 mm, with a shear stress of 0.01 to 0.001 s -1 , for example 0.01s -1 or 0.001s -1 , especially 0.01 s-1 (Viscosity measurement method (i)). Alternatively, the measurement is performed using a C35 / 2° TiL cone-plate geometry at room temperature (e.g., about 20° C., preferably about 25° C.) for 10 -2 From 10 3 s -1 The viscosity may be measured by a flow sweep linearity test, scanning a shear rate of 100 Pa·s (viscosity measurement method (ii)). Viscosity measurement method (i) is preferably performed using an Advanced Rheometer AR2000 from TA instruments, with a 4° and 25 mm flat cone geometry. Viscosity measurement method (ii) is preferably performed using a Thermo Scientific HAAKE RheoStress600 rheometer, with a C35 / 2° TiL cone-plate geometry. Viscosity measurement method (i) allows the measurement of viscosity values ​​at relatively low shear rates. However, for low viscosity solutions, e.g. solutions having a viscosity lower than 20 Pa·s, the viscosity may be measured at a shear rate of about 0.001 s. -1It should be noted that the viscosity values ​​measured according to viscosity measurement method (i) at shear rate values ​​of 0.001 s may be distorted since they correspond to the measurement limits of the machine. For such low viscosity solutions, the viscosity measurements are preferably performed according to viscosity measurement method (i) at shear rate values ​​of 0.001 s -1 At higher shear rates, typically about 0.01 s -1 At a shear rate of 0.01 s -1 or according to viscosity measurement method (ii).

[0100] Prior to the measurement, zero deviation is set and the inertial and rotational mapping of the instrument is calibrated. The sample is then spread on a plate and the deviation from the cone-plane geometry is set to 116 microns for viscosity measurement method (i) and 105 microns for viscosity measurement method (ii). Advantageously, excess sample is removed with a spatula to reduce edge effects.

[0101] The higher the viscosity of the pharmaceutical formulation, the more localized it will be within the tissue at the injection site before forming a hydrogel.

[0102] In the pharmaceutical formulation of the present disclosure, the mixture of chitosan A and polysaccharide B is preferably present in the pharmaceutical formulation in dissolved form, meaning that at least 90%, preferably at least 95%, of the mixture is in dissolved form (% represents the weight of the mixture in dissolved form relative to the total weight of the mixture in the pharmaceutical formulation).

[0103] The presence of a mixture of polysaccharides in dissolved form in the pharmaceutical formulation of the present disclosure allows the pharmaceutical formulation to be in the form of a viscous solution that is fluid enough to be injected through a wide range of needles commonly used in the medical field.

[0104] In other words, the pharmaceutical formulations of the present disclosure are not in the form of a solid hydrogel, as is the case for implantable hydrogels.

[0105] Biodegradable and biocompatible hydrogels The polysaccharide mixture of the pharmaceutical formulation of the present disclosure is capable of gelling in situ under physiological conditions, particularly at physiological pH and osmolality, thereby forming a hydrogel.

[0106] In the pharmaceutical formulation of the present invention, chitosan A, polysaccharide B, and a protein or combination of proteins are intimately mixed, and thus, the protein or combination of proteins present in the pharmaceutical formulation of the present disclosure is encapsulated in a hydrogel by in situ gelation of the polysaccharide mixture.

[0107] The hydrogels formed within the tissue are biodegradable and biocompatible, and are capable of releasing proteins in a controlled, prolonged, and substantially constant manner, particularly while avoiding the phenomenon of rapid initial release known as "burst release."

[0108] According to the present disclosure, the term "gel" refers to a non-flowable polymer network swollen with a solvent.

[0109] According to the present disclosure, the term "hydrogel" refers to a viscoelastic material containing at least 60% water by weight, a physical or chemical gel. Hydrogels are physical gels. In such physical gels, the driving force for gel formation is not a chemical reaction forming covalent bonds, but more a physical phenomenon such as van der Waals interactions and / or hydrogen bonds and / or electrostatic interactions.

[0110] The hydrogel contains approximately 50% to 95% by weight of water, and preferably 50% to 80% by weight of water, based on the total weight of the hydrogel.

[0111] According to the present disclosure, the term "biodegradable" referring to in situ formed hydrogels means that the hydrogels will naturally degrade under physiological conditions, particularly under the action of macrophages. Reactions involved during biodegradation may include hydrolysis reactions, i.e., cleavage of covalent bonds by reaction with water. These reactions may be catalyzed by the action of enzymes naturally present at the injection site.

[0112] In one embodiment, when the hydrogel is placed under physiological conditions, typically after subcutaneous injection into a subject, greater than 50% by weight, preferably greater than 80% by weight, preferably greater than 95% by weight of the hydrogel degrades within a period comprised between 10 and 100 days, preferably between 20 and 65 days.

[0113] In particular, the hydrogel of the present disclosure can substantially release protein over a period comprised between 10 and 100 days, preferably between 20 and 65 days, from the date of administration of the pharmaceutical formulation of the present invention.

[0114] The longer degradation time has the advantage that the time between two injections can be increased, which is particularly advantageous compared to prior art preparations using, for example, hyaluronidase, which must be injected preferably about every two weeks.

[0115] According to the present disclosure, the term "biocompatible" with respect to an in situ formed hydrogel refers to a hydrogel that has the ability to not degrade the biological environment in which it is placed, and in particular, the hydrogel produces little or no inflammatory response even when exposed to the biological environment for an extended period of time.

[0116] In one embodiment, the hydrogel causes little or no inflammation for 10 to 100 days, preferably 20 to 65 days, from the date of administration of the pharmaceutical formulation of the present invention.

[0117] Osmolarity The pharmaceutical formulations of the present disclosure preferably have a pH and / or osmolality close to but different from physiological conditions, in particular lower than physiological conditions. When placed under physiological conditions in contact with body fluids, the pharmaceutical formulations of the present disclosure form a gel due to the change in pH and / or osmolality due to equilibration with the physiological medium.

[0118] The pharmaceutical preparation preferably has an osmolality comprised between 50 and 600 mOsm / L, preferably between 100 and 600 mOsm / L, more preferably between 250 and 450 mOsm / L, or between 50 and 300 mOsm / L, preferably between 50 and 250 mOsm / L.

[0119] According to the present disclosure, osmolality (also known as osmolality) refers to the concentration of a solute per unit volume of a solution. Osmolality is similar to molarity, but includes the total number of moles of dissolved species in a solution. Osmolality is defined as the number of osmoles (Osm) of solute per liter (L) of solution (osmol / L or Osm / L). An osmole is the number of particles in the solution that is equal to Avogadro's number (6.0221×10 23 osmolality is the amount of a substance that must be dissolved to produce a molarity (Osmolality). For substances that do not dissociate, molar concentration and osmolality are the same, but for substances that ionize, osmolality is molar concentration multiplied by the number of dissociated parts (e.g., for sodium chloride, osmolality is doubled). Osmolality is the number of osmoles of solute per kg of solvent. Physiological osmolality usually ranges from about 280 mOsm / L to about 310 mOsm / L, typically about 300 mOsm / L.

[0120] The osmolality of the pharmaceutical formulations of the present disclosure is typically measured using an osmometer, such as Camlab's Loser Micro Osmometer MOD200 Plus. Prior to measurement, zero is set using 50 μL of distilled water, and then the instrument is calibrated against 25 μL of 300 mOsm / kg water standard. Samples are measured by taking the same volume of 25 μL frozen at -6.0°C. Alternatively, zero is set using 15 μL of distilled water, and then the instrument is calibrated against 15 μL of 300 mOsm / kg water standard. Samples are measured by taking the same volume of 15 μL frozen at -6.2°C.

[0121] pH The pharmaceutical preparation preferably has a pH of 5.0 to 6.5, preferably 5.0 to 6.4, and more preferably 5.5 to 6.0.

[0122] The pH of the pharmaceutical formulations of the present disclosure is measured according to methods known to those skilled in the art, for example using a pH meter such as a Mettler Toledo SevenCompact S210 pH meter. According to the present disclosure, the phrase "physiological pH" refers to a pH in the range of 6.8 to 7.6, typically around 7.4.

[0123] Proteins in pharmaceutical formulations The pharmaceutical formulations of the present disclosure contain a therapeutically effective amount of a protein or combination of proteins.

[0124] According to the present disclosure, a "therapeutically effective amount" is the minimum concentration required to achieve a measurable improvement or prevention of any symptom or a particular condition or disorder, to achieve a measurable increase in life expectancy, or to generally improve the quality of life of a patient. The therapeutically effective amount depends on the particular biologically active molecule and the particular condition or disorder being treated.

[0125] The content of the protein or protein combination in the pharmaceutical formulation is typically comprised between 1 g / l and 200 g / l, for example between 50 g / l and 100 g / l of the protein or protein combination.

[0126] Therapeutically effective amounts of many proteins, such as the antibodies described herein, are well known in the art. Therapeutically effective amounts of proteins that have not yet been established, or that are known proteins, such as antibodies, that treat a particular disease and are clinically applicable to treat additional diseases, can be determined by standard techniques that are within the skill of a physician or other skilled artisan.

[0127] According to the present disclosure, the term "protein" refers to a polymer of amino acid residues (natural or non-natural) linked together, most often by peptide bonds. Proteins include naturally occurring proteins and recombinant proteins. Proteins may be functionally linked (e.g., by chemical bonds, non-covalent bonds or other methods) to one or more other molecules, such as small molecules, polymers (e.g., polyethylene glycol) or other proteins. The term "protein" may refer to a single molecule or a multi-molecular complex, such as a dimer, trimer or tetramer. It may also include single-chain or multi-chain proteins, such as antibodies. Disulfide bonds are common in multi-chain proteins. The term protein may also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs or corresponding natural amino acids.

[0128] In the present disclosure, protein molecular weight can be determined using standard methods known to those skilled in the art, including but not limited to mass spectrometry (e.g., ESI, MALDI, SDS-PAGE), or calculation from known amino acid sequence and glycosylation. Proteins can be naturally occurring, non-naturally occurring, synthetic, or semi-synthetic. Proteins or protein combinations can be derived, particularly from human plasma.

[0129] The protein may have a molecular weight comprised between 10 kDa and 250 kDa, preferably between 20 kDa and 250 kDa, preferably between 100 kDa and 250 kDa, preferably between 120 kDa and 250 kDa, preferably between 150 kDa and 250 kDa.

[0130] In certain embodiments of the pharmaceutical formulation of the present disclosure, the protein has a molecular weight comprised preferably between 40 kDa and 250 kDa, advantageously between 100 kDa and 250 kDa, more advantageously between 120 kDa and 250 kDa, and even more advantageously between 150 kDa and 250 kDa.

[0131] In certain preferred embodiments, the protein or combination of proteins is selected from the group consisting of antibodies, enzymes, fusion proteins, and combinations thereof, in particular antibodies or combinations thereof.

[0132] The pharmaceutical formulations of the disclosure preferably do not contain recombinant human hyaluronidase PH20 (rHuPH20), and preferably do not contain enzymes capable of degrading hyaluronic acid.

[0133] antibody The protein or combination of proteins is preferably an antibody or combination of antibodies.

[0134] According to the present disclosure, the term "antibody" refers to an immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds to an antigen. Thus, the term "antibody" encompasses not only whole antibody molecules, but also antibody fragments, and variants (including derivatives) of antibodies and antibody fragments.

[0135] The combination of antibodies may be derived from human plasma. In one embodiment, the antibodies are derived from randomly extracted blood donor plasma. Such antibodies are known as normal (i.e. non-specific) immunoglobulins, usually abbreviated as HNI or HNIg. This may be used to provide antibodies to patients with primary immunodeficiency (PID), hypogammaglobulinemia, primary immune thrombocytopenia, Guillain-Barre syndrome, Kawasaki disease, multifocal motor neuropathy, chronic inflammatory demyelinating polyneuropathy, or secondary (i.e. acquired) immunodeficiency diseases. In another embodiment, the antibodies are derived from the plasma of selected blood donors. Such antibodies are known as "hyperimmune immunoglobulins." These are prepared in a similar manner to normal human immunoglobulins, except that the blood donor has a high titer of antibodies to a particular organism or antigen in the plasma. Pathogens for which hyperimmune globulins may be used include hepatitis B, rabies, tetanus toxoid, varicella zoster, etc.

[0136] The antibody may be a monoclonal or polyclonal antibody.

[0137] According to the present disclosure, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies. That is, the individual antibodies that make up the population bind to the same epitope, except for possible variants that may arise during the production of the monoclonal antibody (such variants are generally present in minor amounts). In contrast to polyclonal antibody preparations, which usually contain different antibodies against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are not contaminated by other immunoglobulins. The term "monoclonal" refers to the character of the antibody as being obtained from a population of substantially homogeneous antibodies, and is not to be construed as requiring production of the antibody by a particular method. Specific examples of monoclonal antibodies in this specification include chimeric antibodies, humanized antibodies, and human antibodies.

[0138] According to the present disclosure, a "chimeric" monoclonal antibody is an antibody in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence of an antibody from another species or belonging to another antibody class or subclass, so long as it exhibits the desired biological activity. Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences from a non-human primate (e.g., baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences. An example of a chimeric antibody is rituximab.

[0139] According to the present disclosure, a "humanized" form of a non-human (e.g., murine) antibody is a chimeric antibody that contains minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in the recipient antibody or the donor antibody. These changes are made to further improve antibody performance. In general, a humanized antibody will contain substantially all of at least one, and usually two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence, except for the FR substitutions noted above. A humanized antibody may optionally contain at least a portion of an immunoglobulin constant region, typically a human immunoglobulin constant region. An example of a humanized antibody is trastuzumab.

[0140] According to the present disclosure, a "full-length antibody" is an antibody that comprises an antigen-binding variable region, as well as a light chain constant domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, a full-length antibody has one or more effector functions. Rituximab and trastuzumab are examples of full-length antibodies.

[0141] According to the present disclosure, an "antibody fragment" includes a portion of a full-length antibody, such as the antigen-binding region and / or the variable region of the full-length antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; single domain antibodies. F(ab')2, Fab, Fab', and Fv are antigen-binding fragments that can be generated from the variable regions of IgG and IgM. F(ab')2 fragments contain two antigen-binding regions hinged together via disulfides. F(ab')2 fragments usually lack most, but not all, of the Fc region. Fab' fragments can be formed by reduction of F(ab')2 fragments. Fab' fragments contain free sulfhydryl groups that can be alkylated or used to bind enzymes, toxins, or other proteins of interest. Fab' fragments may contain only a small portion of Fc, since they are derived from F(ab')2. Fab is a monovalent fragment generated from IgG and IgM, consisting of the VH, CH1 and VL, CL regions, linked by intramolecular disulfide bonds. Fv fragment refers to the smallest fragment generated from IgG and IgM that contains an intact antigen-binding site. Fv fragments have identical binding properties and similar three-dimensional binding characteristics as Fab. The VH and VL chains of the Fv fragment are held together by non-covalent interactions. "Single-domain antibodies", also known as "nanobodies", are monomeric antigen-binding fragments of antibodies that offer various advantages over other antibody fragments as "building blocks" for bsAbs. They exist in nature as the antigen-binding portion of heavy chain antibodies in camelid species (called VHH) and cartilaginous fish (called VNAR), or can be generated from conventional IgG by obtaining or engineering monomeric stable VH or VL domains.

[0142] Exemplary antibodies that can be formulated according to the present disclosure include, but are not limited to, abatacept, adalimumab, alemtuzumab, alirocumab, amivantamab, anifrolumab, atezolizumab, avelumab, balstilimab, basiliximab, belatacept, belimumab, benralizumab, besilesomab, bevacizumab, bezlotoxumab, bimekizumab, Blinatumab, Brodalumab, Burosumab, Canakinumab, Carotuxomab, Cemiplimab, Cetuximab, Concizumab, Crizanlizumab, Daratumumab, Denosumab, Dinutuximab beta, Dostallimab, Dupilumab, Durvalumab, Eculizumab, Elotuzumab, Epratuzumab, Erenumab, Evolocumab, Fab Ig Antidigitalique Ovin, Fremanezumab, Galcanezumab, Golimumab, Guselkumab, Ibalizumab, Ig Anti-human Lymphocytes (Lapine), Ig Anti-human Thymocytes (Horse), Ig Anti-human Thymocytes (Lapine), Infliximab, Iph 4102, Ipilimumab, Isatuximab, Ixekizumab, Lactamab, Lag525, Lanadelumab, McLa-128, Mepolizumab, Natalizumab, Naxitamab, Nimotuzumab, Nivolumab, Obinutuzumab, Ocrelizumab, Ofatumumab, Omalizumab, Palivizumab, Panitumumab, Pembrolizumab, Pertuzumab, Ramucirumab, Ranibizumab, Reslizumab, Risankizumab, Rituximab Cimab, Sarilumab, Secukinumab, Seribantumab, Siltuximab, Spartalizumab, Tafasitamab, Teprotumumab, Tildrakizumab, Tocilizumab, Tralokinumab, Trastuzumab, Urelumab, Ustekinumab, Vedolizumab, Zalifrelimab, and combinations thereof.

[0143] Exemplary combinations of antibodies that can be formulated according to the present disclosure include, but are not limited to, casirivimab and imdevimab, pertuzumab and trastuzumab, or tixagevimab and silgavimab.

[0144] An antibody can be functionally linked (e.g., by chemical conjugation, genetic fusion, non-covalent bonding, etc.) to one or more other molecules to increase half-life or stability, or to otherwise improve the antibody.

[0145] For example, the antibody may be linked to one of a variety of nonproteinaceous polymers (e.g., polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol). The antibody may be operatively linked to poly(ethylene glycol), e.g., the antibody is certolizumab pegol. The antibody may be linked to any of a variety of nonproteinaceous polymers, such as DOTA (1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NODAGA (1,4,7-triazacyclononane-1-glutaric acid-4,7-diacetic acid), DOTAGA (2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraaza cyclododecan-1-yl)pentanedioic acid), DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), NOTAM (1,4,7-tetrakis(carbamoylmethyl)-1,4,7-triazacyclononane), DOTP (1,4,7,10-tetraazacyclododecane 1,4,7,10-tetrakis(methylene The chelating agent may be functionally linked to a chelating agent such as 1,4,7-tetrakis(methylenephosphonate)-1,4,7-triazacyclononane, TETA (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetraacetic acid), TETAM (1,4,8,11-tetraazacyclotetradecane-N,N',N'',N'''-tetrakis(carbamoylmethyl)), DTPA (diethylenetriaminopentaacetic acid), DFO (deferoxamine), Bz-DFO, bispitine ([3,7]-diazabicyclo[3.3.1]nonane), and is preferably functionally linked to a chelating agent selected from DOTAGA, DFO, Bz-DFO, DOTAM, DTPA, and bispitine.

[0146] The antibody may also be functionally linked to a cytotoxic agent, such as calicheamicin, monomethylauristatin F (MMAF, also known as mafodotin), monoethylauristatin E (MMAE, also known as vedotin), emtamsine (DM1), Exatecan derivative (Dxd), N-acetyl-gamma calicheamicin, and the like, thereby forming an antibody-drug conjugate. Exemplary antibody-drug conjugates (ADCs) that can be formulated according to the present disclosure include, but are not limited to, belantamab mafodotin, brentuximab vedotin, depatuxizumab mafodotin, enfortumab vedotin, gemtuzumab ozogamicin, inotuzumab ozogamicin, moxetumomab pasudotox, sacituzumab govitecan, trastuzumab deruxtecan, trastuzumab emtansine, and the like.

[0147] An antibody may be operatively linked to one or more antibodies or antibody fragments to create a bispecific or multispecific molecule. Exemplary bispecific antibodies that can be formulated according to the present disclosure include, but are not limited to, Glofitamab, KN046, IBI318, IBI318, Emicizumab, Epcoritamab, Tebotelimab, Tebentafusp, Teclistamab, Faricimab, Amivantamab, Mosunetuzumab, Zanidatamab, Flotetuzumab, APVO436, Zenocutuzumab, TNB383B, and combinations thereof. In one embodiment, the bispecific antibody that is formulated is epcoritamab.

[0148] In one embodiment, the protein or combination of proteins is an antibody or combination of antibodies that binds to an antigen selected from the group of HER2 and CD20. CD20 antibodies may be used to treat B-cell malignancies (such as non-Hodgkin's lymphoma or chronic lymphocytic leukemia) or autoimmune diseases (such as rheumatoid arthritis and vasculitis); HER2 antibodies may be used to treat cancer (such as breast cancer and gastric cancer). The phrases "antibody that binds to an antigen", "antibody that recognizes an antigen" and "antibody with specificity for an antigen" have the same meaning and are used interchangeably herein.

[0149] In one embodiment, the antibody that is formulated binds to HER2. In a preferred embodiment, the antibody that is formulated is trastuzumab.

[0150] In one embodiment, the antibody that is formulated binds to CD20. In a preferred embodiment, the antibody that is formulated is rituximab.

[0151] In one embodiment, the antibody formulated binds to HER2. In a preferred embodiment, the antibody formulated is pertuzumab.

[0152] In one embodiment, the antibody that is formulated binds to CD38. In a preferred embodiment, the antibody that is formulated is daratumumab.

[0153] In one embodiment, the antibody combination that is formulated is a combination of antibodies that bind to two different epitopes of HER2. In a preferred embodiment, it comprises the combination of trastuzumab and pertuzumab.

[0154] Excipients The pharmaceutical formulations of the present disclosure may further comprise one or more pharma- ceutically acceptable excipients selected from solvents, stabilizers, surfactants, buffers, antimicrobial preservatives, protectants, antioxidants, chelating agents, and bulking agents.

[0155] According to the present disclosure, a "solvent" is any pharma- ceutically acceptable (i.e., safe and non-toxic for administration to humans or other mammals) ingredient useful in the preparation of liquid formulations (such as aqueous formulations). Exemplary solvents include water, such as sterile water for injection (WFI) or bacteriostatic water for injection (BWFI), a pH buffered solution (e.g., phosphate buffered saline), sterile saline, Ringer's solution or dextrose solution, and combinations thereof. Preferably, the solvent is for disclosure sterile water or bacteriostatic water for injection (BWFI).

[0156] According to the present disclosure, a stabilizer is a compound that increases the stability of a protein, in particular its stability against unfolding and aggregation. Preferably, the stabilizer is recognized by authorities as a suitable additive or excipient in pharmaceutical formulations.

[0157] The stabilizer may be a sugar. As used herein, "sugar" refers to a sugar having a general composition (CH 2 O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. Examples of sugars in this specification include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, siritol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, etc.

[0158] Preferably, the formulation comprises as a stabilising agent a non-reducing disaccharide such as a sugar selected from the group of trehalose (eg in the form of α,α-trehalose dihydrate) and sucrose.

[0159] The concentration of the stabilizer in the pharmaceutical formulation of the present disclosure is preferably 1-500 mM, 15-250 mM, or 150-250 mM, or about 210 mM.

[0160] According to the present disclosure, "surfactant" refers to a surface-active agent. Surfactants are generally added to protein formulations to reduce the exposure of hydrophobic regions and to reduce protein-protein interactions and interface-induced aggregation that are also prevented by competition for adsorption sites.

[0161] Examples of surfactants herein include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodium methyl oleyl-taurate (sodium methyl cocoyl-, or disodium methyl oleyl-taurate). oleyl-taurate; polyethyl glycol, polypropyl glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). Other examples of pharma-ceutically acceptable surfactants include polyoxyethylene sorbitan fatty acid esters (Tween), polyethylene-polypropylene glycol, polyoxyethylene stearates, polyoxyethylene alkyl ethers (e.g., polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ethers (Triton-X), polyoxyethylene-polyoxypropylene copolymers (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). The most preferred polyoxyethylene sorbitan fatty acid esters are polysorbate 20 (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™).

[0162] The most preferred polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. The most preferred polyoxyethylene alkyl ethers are those sold under the trademark Brij. The most preferred alkylphenol-polyoxyethylene ethers are those sold under the trade name Triton-X.

[0163] The surfactant is preferably a non-ionic surfactant, preferably a polysorbate (eg selected from the group of polysorbate 20, polysorbate 80 and polyethylene polypropylene copolymer).

[0164] The concentration of the surfactant in the pharmaceutical formulation of the present disclosure is preferably comprised between 0.01 and 0.1% (w / v), or between 0.01 and 0.08% (w / v), or between 0.025 and 0.075% (w / v).

[0165] According to the present disclosure, the term "buffer" refers to an agent that provides a solution containing it with resistance to changes in pH by the action of its acid / base conjugate components. Examples of buffers that control pH in this range include acetate, succinate, gluconate, histidine, citrate, glycylglycine and other organic acid buffers.

[0166] A preferred buffer in the present disclosure is a histidine buffer.

[0167] A "histidine buffer" is a buffer containing the amino acid histidine. Examples of histidine buffers include histidine chloride (e.g., L-histidine hydrochloride monohydrate), histidine acetate, histidine phosphate, histidine sulfate, and the like.

[0168] A "preservative" is a compound that can be added to the formulation herein to reduce contamination and / or the action of bacteria, fungi, or other infectious agents. The addition of a preservative can, for example, facilitate the manufacture of a multi-use (multiple-dose) formulation. Examples of possible preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl group is long chain), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl, and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and w-cresol.

[0169] A "protectant," as generally used herein, is a substance that, when combined with a protein, significantly reduces the chemical and / or physical instability of the protein upon lyophilization and / or subsequent refrigerated storage.

[0170] Exemplary protective agents include sugars and their corresponding sugar alcohols, such as sucrose, lactose, trehalose, dextran, erythritol, arabitol, xylitol, sorbitol, and mannitol; amino acids, such as arginine or histidine; lyotropic salts, such as magnesium sulfate; polyols, such as propylene glycol, glycerol, poly(ethylene glycol), or poly(propylene glycol); and combinations thereof. Further exemplary protective agents include gelatin, dextrin, modified starch, and carboxymethylcellulose. Preferred sugar alcohols are compounds obtained by reduction of monosaccharides and disaccharides, such as lactose, trehalose, maltose, lactulose, and maltulose. Further examples of sugar alcohols include glucitol, maltitol, lactitol, and isomaltulose.

[0171] The protecting agent may be added to the formulation prior to lyophilization in a "lyoprotective amount," meaning that after the protein is lyophilized in the presence of a lyoprotective amount of the protecting agent, the protein essentially retains its physical and chemical stability and integrity.

[0172] As generally used herein, an "antioxidant" is a pharma- ceutically acceptable excipient that is commonly used to limit oxidation reactions and maintain protein stability and safety. Examples of antioxidants include ascorbic acid, sodium metabisulfite, histamine, methionine, ascorbic acid, glutathione, vitamin E, and polyethyleneimine.

[0173] The antioxidant is preferably methionine, in particular L-methionine.

[0174] The concentration of the antioxidant in the pharmaceutical preparation of the present disclosure is preferably 5 to 25 mM, more preferably 5 to 15 mM.

[0175] A "chelating agent," as used generally herein in the context of an excipient, is a pharma- ceutically acceptable excipient commonly used to maintain protein stability.

[0176] Examples of chelating agents include disodium edetate, diethylenetriaminepentaacetic acid, citric acid, hexaphosphate, thioglycolic acid, zinc, and the like.

[0177] A "bulking agent," as generally used herein, is a pharma- ceutically acceptable excipient that is commonly used to add mass to a lyophilized mixture and contribute to the physical structure of the lyophilized cake (e.g., facilitating the production of an essentially uniform lyophilized cake that maintains an open pore structure). Exemplary bulking agents include mannitol, glycine, lactose, modified starch, polyethylene glycol, and sorbitol.

[0178] Specific Pharmaceutical Formulations In one embodiment, the pharmaceutical formulation of the present disclosure preferably comprises a protein or a combination of proteins, preferably an antibody or a combination of antibodies as defined herein, in particular trastuzumab, rituximab, pertuzumab, daratumumab or a combination of trastuzumab and pertuzumab, a mixture of polysaccharides as defined herein, and preferably a buffering agent, stabilizer, or antioxidant as defined herein At least one of Includes.

[0179] In one embodiment, the pharmaceutical formulation of the present disclosure preferably comprises: - 1 g / l to 200 g / l, preferably 100 to 150 g / l, of an antibody or a combination of antibodies, in particular trastuzumab, rituximab, pertuzumab, daratumumab or a combination of trastuzumab and pertuzumab, - 5 to 200 g / l, preferably 10 to 200 g / l, more preferably 10 to 150 g / l of chitosan A, - 10 to 200 g / l, preferably 20 to 100 g / l, of statistical polysaccharide B, - and preferably a 1-100 mM buffer, preferably a histidine buffer, more preferably a histidine chloride buffer such as L-histidine hydrochloride monohydrate, having a pH of 5.0-6.5, preferably 5.0-6.4, more preferably 5.5-6.00; 1 to 500 mM of a stabilizer, preferably trehalose, such as α,α-trehalose dihydrate, or 5-25 mM antioxidant, preferably methionine At least one of Includes.

[0180] In one embodiment, the pharmaceutical formulation of the present disclosure preferably comprises: - trastuzumab from 1 g / l to 200 g / l, preferably from 100 to 150 g / l, - 5 to 200 g / l, preferably 10 to 200 g / l, more preferably 10 to 150 g / l of chitosan A, - 10 to 200 g / l, preferably 20 to 100 g / l, of statistical polysaccharide B, - and preferably a 1-100 mM buffer, preferably a histidine buffer, more preferably a histidine chloride buffer such as L-histidine hydrochloride monohydrate, having a pH of 5.0-6.5, preferably 5.0-6.4, more preferably 5.5-6.00; 1 to 500 mM of a stabilizer, preferably trehalose, such as α,α-trehalose dihydrate, or 5-25 mM antioxidant, preferably methionine At least one of Includes.

[0181] In one embodiment, the pharmaceutical formulation of the present disclosure preferably comprises: - rituximab from 1 g / l to 200 g / l, for example from 5 to 20 g / l, preferably from 100 to 150 g / l; - 5 to 200 g / l, preferably 10 to 200 g / l, more preferably 10 to 150 g / l of chitosan A, - 10 to 200 g / l, preferably 20 to 100 g / l, of statistical polysaccharide B, - and preferably a 1-100 mM buffer, preferably a histidine buffer, more preferably a histidine chloride buffer such as L-histidine hydrochloride monohydrate, having a pH of 5.0-6.5, preferably 5.0-6.4, more preferably 5.5-6.00; 1 to 500 mM of a stabilizer, preferably trehalose, such as α,α-trehalose dihydrate, or 5-25 mM antioxidant, preferably methionine At least one of Includes.

[0182] In one embodiment, the pharmaceutical formulation of the present disclosure preferably comprises: - 1 g / l to 200 g / l, for example 30 to 50 g / l or about 40 g / l, preferably 100 g / l to 150 g / l of trastuzumab, - Pertuzumab from 1 g / l to 200 g / l, for example from 50 to 100 g / l or about 80 g / l, preferably from 100 to 150 g / l, - 5 to 200 g / l, preferably 10 to 200 g / l, more preferably 10 to 150 g / l of chitosan A, - 10 to 200 g / l, preferably 20 to 100 g / l, of statistical polysaccharide B, - and preferably a 1-100 mM buffer, preferably a histidine buffer, more preferably a histidine chloride buffer such as L-histidine hydrochloride monohydrate, having a pH of 5.0-6.5, preferably 5.0-6.4, more preferably 5.5-6.00; 1 to 500 mM of a stabilizer, preferably trehalose, such as α,α-trehalose dihydrate, or 5-25 mM antioxidant, preferably methionine At least one of Includes.

[0183] In one embodiment, the pharmaceutical formulation of the present disclosure preferably comprises: - Pertuzumab from 1 g / l to 200 g / l, for example from 50 to 100 g / l, preferably from 100 to 150 g / l, - 5 to 200 g / l, preferably 10 to 200 g / l, more preferably 10 to 150 g / l of chitosan A, - 10 to 200 g / l, preferably 20 to 100 g / l, of statistical polysaccharide B, - and preferably a 1-100 mM buffer, preferably a histidine buffer, more preferably a histidine chloride buffer such as L-histidine hydrochloride monohydrate, having a pH of 5.0-6.5, preferably 5.0-6.4, more preferably 5.5-6.00; 1 to 500 mM of a stabilizer, preferably trehalose, such as α,α-trehalose dihydrate, or 5-25 mM antioxidant, preferably methionine At least one of Includes.

[0184] In one embodiment, the pharmaceutical formulation of the present disclosure preferably comprises: - 1 g / l to 200 g / l, for example 50 to 100 g / l, preferably 100 to 150 g / l daratumumab; - 5 to 200 g / l, preferably 10 to 200 g / l, more preferably 10 to 150 g / l of chitosan A, - 10 to 200 g / l, preferably 20 to 100 g / l, of statistical polysaccharide B, - and preferably a 1-100 mM buffer, preferably a histidine buffer, more preferably a histidine chloride buffer such as L-histidine hydrochloride monohydrate, having a pH of 5.0-6.5, preferably 5.0-6.4, more preferably 5.5-6.00; 1 to 500 mM of a stabilizer, preferably trehalose, such as α,α-trehalose dihydrate, or 5-25 mM antioxidant, preferably methionine At least one of Includes.

[0185] temporal stability The pharmaceutical formulations of the present disclosure may be stored for an extended period of time, typically for at least six months, preferably comprised between six months and one year.

[0186] Ready to use In one embodiment, the pharmaceutical formulation of the present disclosure is in the form of a ready-to-use (i.e., immediately administered) injectable formulation comprising a polysaccharide mixture, a protein or a combination of proteins, and one or more pharma- ceutically acceptable excipients, as defined in this disclosure.

[0187] In that embodiment, the pharmaceutical formulation may be supplied, for example, in a pre-filled syringe.

[0188] Preparation method In a second aspect, the present disclosure relates to a process for preparing a pharmaceutical formulation as defined in the first aspect of the present disclosure, the process comprising at least the step of mixing a polysaccharide mixture, a protein or a combination of proteins as defined in the first aspect of the present disclosure, and one or more pharmaceutical excipients.

[0189] In one embodiment, the process comprises the following steps: a) providing a solution comprising a polysaccharide mixture as defined in the first aspect of the present disclosure, b) providing a solution comprising a protein or a combination of proteins as defined in the first aspect of the present disclosure, c) mixing the solution of step a) with the solution of step b) to obtain a homogenous solution; d) optionally adjusting the osmolality of the solution obtained in step c) to an osmolality comprised between 50 and 300 mOsm / L, preferably between 50 and 250 mOsm / L; e) optionally adjusting the pH of the solution obtained in step c) to between 5.0 and 6.5, preferably between 5.0 and 6.4, more preferably between 5.5 and 6.0. Includes.

[0190] The solutions in steps a) and b) are preferably aqueous solutions.

[0191] The solution in step b) may be prepared by dissolving the protein in powder form (e.g. lyophilized form), typically by mixing the powder with a solvent, in particular a solvent suitable for injection, to form a solution containing the protein or combination of proteins.

[0192] Step c) may be carried out by centrifugation. For example, centrifugation may be carried out at 2000-8000 rpm for 5-30 minutes, preferably at about 5000 rpm for 10 minutes, or at 50-200 rpm for 1-4 hours, preferably at about 100 rpm for 1 hour. Alternatively, centrifugation may be carried out after step c) to remove air bubbles from the solution obtained in step c).

[0193] In one embodiment, step c) may be performed by a static mixer. In that embodiment, the solution of step a) is contained in a first container and the solution of step b) is contained in a second container, which are connected to a static mixer to provide a system for mixing the solutions of steps a) and b) by injecting the contents of one solution into the other and vice versa. Such injection may be repeated 100-500 times, typically 100-300 times.

[0194] Step c) is preferably carried out in a static mixer.

[0195] In one embodiment, step c) may be carried out by mixing the solutions of step a) and step b) directly in the reactor by means of a static mixer.

[0196] Any adjustment of the osmolality in step d) may be carried out by appropriate dialysis of the solution obtained in step c) against water or a solution containing a salt such as NaCl, optionally maintaining the pH by addition of a base solution.

[0197] The optional pH adjustment in step e) may be carried out by suitable addition of a base or an acid, preferably a base such as NaOH.

[0198] Steps d) and e) may be performed in any order, i.e., step d) precedes step e), or step e) precedes step d), or simultaneously, e.g., osmolality may be adjusted by dialysis while maintaining pH, or vice versa.

[0199] Part Kit A third aspect of the present disclosure is - at least one first container comprising chitosan A and a statistical polysaccharide B, i.e. a polysaccharide mixture as defined in the first aspect of the present disclosure, - at least one second container comprising a protein or a combination of proteins as defined in the first aspect of the present disclosure The present invention relates to a kit comprising:

[0200] The kit is for preparing a pharmaceutical formulation according to the present disclosure, in particular a ready-to-use injectable formulation according to the present disclosure.

[0201] In one embodiment, the polysaccharide mixture in the first container is in the form of a powder or a liquid solution.

[0202] In one embodiment, the protein or protein combination in the second container is in the form of a powder or a liquid solution.

[0203] In one embodiment, the polysaccharide mixture in the first container and the protein or protein combination in the second container are in the form of a powder or liquid solution.

[0204] In one embodiment, the first and second containers may be connected to a static mixer that provides a mechanism for mixing the polysaccharide mixture with the protein or combination of proteins, for example, the first and second connectors may include luer lock adapters and be connected via luer lock connectors.

[0205] The first and / or second container is preferably a syringe, more preferably a ready-to-use syringe. In one embodiment, the first and second containers are luer lock vials or syringes connected by a luer lock connector.

[0206] The kits of the present disclosure allow for the preparation of pharmaceutical compositions from any source of sterile, commercially available protein, thereby providing great versatility.

[0207] Additionally, the kits of the present disclosure allow for the preparation of the formulation immediately prior to injection.

[0208] When the kit includes excipients other than the solvent and a buffer as a stabiliser, these are preferably placed in a second container.

[0209] Administration The pharmaceutical formulations of the present disclosure are preferably suitable for injection, preferably subcutaneous injection.

[0210] "Injectability" or "syringeability" as generally used herein refers to the ability to inject a pharmaceutical formulation through a syringe equipped with an 18-32, preferably 20-27 gauge needle.

[0211] According to the present disclosure, a "subcutaneous injection" refers to an injection administered into the subcutaneous tissue, which is the layer of skin just below the dermis and epidermis (collectively referred to as the cutis). Subcutaneous administration may be abbreviated as SC, SQ, sub-cu, sub-Q, SubQ, or subcut.

[0212] Subcutaneous injections can be given at any site suitable for subcutaneous injections, such as the thigh, abdomen (particularly at the navel), upper arm (particularly the back or side of the lower upper arm), back, lower waist, and buttocks (particularly the upper outer area of ​​the buttocks).

[0213] Multiple subcutaneous injections can be given simultaneously at various sites suitable for subcutaneous injection.

[0214] Subcutaneous injection volume may comprise 1-20 ml, preferably 2-15 ml.

[0215] As shown in the experimental section of this application, the injection amount of the pharmaceutical composition does not affect the degradation rate of the hydrogel formed after injection. Furthermore, it has also been shown that the release of the hydrogel scaffold formed after injection and the protein encapsulated therein can be adjusted depending on, for example, the weight ratio of chitosan A / statistical polysaccharide B.

[0216] In one embodiment, the subcutaneous injection volume is comprised between 1 and 5 ml, preferably between 1 and 3 ml, preferably about 2 ml, in which embodiment the subcutaneous injection is preferably administered into the subcutaneous tissue of the thigh, upper arm, back and lower back.

[0217] In one embodiment, the subcutaneous injection volume is comprised between 5 and 20 ml, preferably between 5 and 10 ml, or between 10 and 20 ml, more preferably about 15 ml. In that embodiment, the subcutaneous injection is preferably administered into the subcutaneous tissue of the abdominal region.

[0218] The pharmaceutical formulations may also be administered by intraperitoneal, intraarticular, intrathecal, or intraocular injection.

[0219] According to this disclosure, "intraocular injection" (also known as intravitreal injection) refers to a route of administration in which the drug is injected into the interior of the eye through the vitreous, the gel-like substance that fills the eyeball.

[0220] According to the present disclosure, "intra-articular injection" refers to a route of administration by injection into a joint. Intra-articular injection can be useful not only in the treatment of arthritis, but also in chemotherapy.

[0221] According to the present disclosure, "intrathecal administration" refers to an administration route that is injected into the spinal canal or intrathecal space to reach the cerebrospinal fluid (CSF). Intrathecal administration can be a useful chemotherapy.

[0222] The pharmaceutical formulation may also be administered by intratumoral injection.

[0223] According to the present disclosure, "intratumoral injection" refers to direct injection into a tumor. Intratumoral injection can be considered for any tumor whose primary lesion or its metastasis is accessible percutaneously (i.e., needle puncture of the skin) by direct injection or by specific procedures such as colonoscopy, cystoscopy, bronchoscopy, thoracoscopy, laparoscopy, or even surgery.

[0224] Injections may be administered with an 18 to 32 gauge needle, preferably a 20 to 27 gauge needle. The formulations can be administered using small gauge needles, for example 20 to 30 gauge, typically 27, 28, 29, or 30 gauge.

[0225] In one embodiment, the pharmaceutical formulation does not cause significant irritation when administered no more than twice a day, no more than once a day, no more than twice a week, no more than once a week, no more than once every two weeks, or no more than once a month.The pharmaceutical formulation of the present disclosure is preferably administered once every two weeks without causing significant irritation at the injection site.

[0226] In one embodiment, the pharmaceutical formulation shows increased bioavailability when administered by subcutaneous injection compared to the formulation containing the same protein. "Bioavailability" refers to the extent and speed at which a protein, such as an antibody, reaches the circulation or site of action.One method of measuring bioavailability is to compare the "area under the curve" (AUC) of plasma concentration as a function of time.

[0227] The AUC can be calculated, for example, using the linear trapezoidal rule. 0 " refers to the area under the plasma concentration curve from time zero to the time when the plasma concentration returns to the baseline level. o-t " refers to the area under the plasma concentration curve from time zero to a subsequent time t, e.g., to the time baseline is reached. Time is usually measured in days, but hours can also be used as the context dictates. For example, the AUC can be increased by more than 10%, 20%, 30%, 40%, or 50% compared to the same formulation administered in the same manner without the viscosity-reducing water-soluble organic dye.

[0228] According to the present disclosure, "t max " refers to the time after administration at which maximum plasma concentration is achieved.

[0229] According to this disclosure, "C max" refers to the maximum plasma concentration after administration and before the next dose.

[0230] According to this disclosure, "C min " refers to the minimum plasma concentration after one dose and before the next dose.

[0231] In one embodiment, an advantageous feature of the pharmaceutical formulation of the present disclosure is the t max However, in preparations that do not contain the polysaccharide mixture (typically containing recombinant human hyaluronidase PH20 (rHuPH20) or, more generally, enzymes capable of degrading hyaluronic acid), max and this increase is at least 1.5-fold, preferably at least 3-fold.

[0232] Without wishing to be bound by any theory, the inventors believe that the increase in Tmax observed in Example 8 supports a slower release of trastuzumab compared to the formulation ROCHE HERCEPTIN® SC.

[0233] The pharmaceutical formulations of the present disclosure may allow for greater dosing flexibility and less frequent dosing compared to pharmaceutical formulations that do not include polysaccharide mixtures, for example, by increasing the dosage per injection multiple-fold, in some embodiments, the dosing frequency can be reduced from once every two weeks to once every six weeks.

[0234] Therapeutic Use In a fourth aspect, the present disclosure relates to the pharmaceutical formulation of the first aspect of the disclosure or the kit of the second aspect of the disclosure for use as a medicament, preferably for use in the treatment of cancer.

[0235] The terms "cancer" and "cancerous" refer to or describe a physiological condition in mammals that is typically characterized by uncontrolled cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma (including medulloblastoma and retinoblastoma), sarcoma (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumor, gastrinoma and islet cell carcinoma), mesothelioma, schwannoma (including acoustic neuroma), meningioma, adenocarcinoma, melanoma, and leukemia or lymphoid malignancies (including Waldenstrom's macroglobulinemia), multiple myeloma or myeloid tumors. More specific examples of such cancers include lung cancer such as squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer such as gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, liver cancer, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, biliary tract tumor, and head and neck cancer.

[0236] The pharmaceutical formulation of the first aspect of the disclosure or the kit of the second aspect of the disclosure may also be used to treat non-malignant diseases, in particular autoimmune diseases (e.g. psoriasis); endometriosis; scleroderma; restenosis; polyps such as colonic, nasal or gastrointestinal polyps; fibroadenoma; respiratory diseases; cholecystitis; neurofibromatosis; polycystic kidney disease; inflammatory diseases; skin disorders including psoriasis and dermatitis; vascular diseases; conditions involving abnormal proliferation of vascular epithelial cells; gastrointestinal ulcers; Ménétrier's disease, secretory adenoma or protein-losing syndrome; renal disorders; angiogenic disorders; eye diseases such as age-related macular degeneration, presumed ocular histoplasmosis, retinal neovascularization due to proliferative diabetic retinopathy, retinal angiogenesis, diabetic retinopathy or age-related macular degeneration. macular degeneration; bone-related pathologies such as osteoarthritis, rickets and osteoporosis; injury following cerebral ischemic events; fibrotic or edematous diseases such as liver cirrhosis, pulmonary fibrosis, sarcoidosis, thyroiditis, systemic hyperviscosity syndrome, Osler-Weber-Rendu disease, chronic obstructive pulmonary disease or edema following burns, trauma, radiation, stroke, anoxia or ischemia; skin hypersensitivity reactions; diabetic retinopathy and diabetic nephropathy; Guillain-Barre syndrome; graft-versus-host disease or transplant rejection; Paget's disease; bone or joint inflammation; photoaging (caused, for example, by UV exposure of human skin); benign prostatic hyperplasia; adenoviruses, hantaviruses, Borrelia burgdorferi, Yersinia spp. and Bordetella pertussis; thrombi caused by platelet aggregation; reproductive conditions such as endometriosis, ovarian hyperstimulation syndrome, preeclampsia, dysfunctional uterine bleeding, or dysfunctional uterine bleeding; synovitis; atheroma; acute and chronic nephropathy (including proliferative glomerulonephritis and diabetes-induced kidney disease); eczema; hypertrophic scar formation; endotoxic shock and fungal infections; familial adenomatous polyposis; neurodegenerative diseases (e.g., Alzheimer's disease, AIDS-related dementia, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, retinitis pigmentosa, spinal muscular atrophy, and cerebellar degeneration); myelodysplastic syndromes; aplastic anemia; ischemic injury; pulmonary, renal, or hepatic fibrosis; T-cell mediated hypersensitivity disorders; infantile hypertrophic pyloric stenosis; ureteral obstruction syndrome; psoriatic arthritis; and Hashimoto's thyroiditis.Examples of non-malignant indications for treatment herein include psoriasis, endometriosis, scleroderma, vascular disease (e.g., restenosis, arteriosclerosis, coronary artery disease, or hypertension), colonic polyps, fibroadenomas, or respiratory diseases (e.g., asthma, chronic bronchitis, bronchial asthma, bronchiectasis, or cystic fibrosis), inflammatory bowel disease (IBD), primary immune deficiencies (PIDs), hypogammaglobulinemia, primary immune thrombocytopenia, Guillain-Barre syndrome, Kawasaki disease, multifocal motor neuropathy, chronic inflammatory demyelinating polyneuropathy, and the like.

[0237] In this disclosure, the term "treatment" includes curative treatment or prophylactic treatment. The term "curative treatment" refers to treatment aimed at curing a disease or ameliorating symptoms associated with a disease. The term "prophylactic treatment" refers to treatment aimed at preventing a disease or ameliorating symptoms associated with a disease.

[0238] In one embodiment, the pharmaceutical formulation of the first aspect of the disclosure or the kit of the second aspect of the disclosure comprises an antibody or a combination of antibodies.

[0239] In one embodiment, the pharmaceutical formulation of the first aspect of the disclosure or the kit of the second aspect of the disclosure comprises an antibody or combination of antibodies that binds to an antigen selected from the group of HER2, such as trastuzumab, pertuzumab or a combination thereof, for use in the treatment of breast cancer, gastric cancer, B-cell malignancies (such as non-Hodgkin's lymphoma or chronic lymphocytic leukemia) or autoimmune diseases (such as rheumatoid arthritis and vasculitis).

[0240] In one embodiment, the pharmaceutical formulation of the first aspect of the disclosure or the kit of the second aspect of the disclosure comprises an antibody or combination of antibodies that bind to an antigen selected from the group of CD20 antibodies, such as rituximab, for use in the treatment of rheumatoid arthritis, non-Hodgkin's lymphoma (NHL), leukemia such as chronic lymphocytic leukemia, granulomatosis with polyangiitis (Gpa or Wegener's disease), microscopic polyangiitis (Mpa) or pemphigus vulgaris.

[0241] In one embodiment, the pharmaceutical formulation of the first aspect of the disclosure or the kit of the second aspect of the disclosure comprises an antibody or combination of antibodies that bind to an antigen selected from the group of CD38 antibodies, such as daratumumab, for use in the treatment of multiple myeloma or myeloid tumors.

[0242] In a fifth aspect, the present disclosure relates to a method of treating a cancer or a non-malignant disease as defined in the fourth aspect in a subject in need of such treatment, the method comprising subcutaneously administering to the subject a therapeutically effective amount of a pharmaceutical formulation of the present disclosure.

[0243] In a sixth aspect, the present disclosure relates to the use of a pharmaceutical formulation as disclosed herein in the manufacture of a medicament for treating cancer or a non-malignant disease as defined in the fourth aspect of the present disclosure.

[0244] According to this disclosure, the singular forms "a," "an," and "the" include both the singular and plural referents unless the context clearly dictates otherwise.

[0245] In accordance with the present disclosure, the terms "about" or "approximately" as used herein when referring to a measurable value of a parameter, amount, time duration, and the like, are meant to encompass a variation of no more than ±5%, more preferably no more than ±2%, and even more preferably no more than ±1% of the specified value, to the extent that such variations are appropriate for the practice of the disclosed invention. EXAMPLES

[0246] The present disclosure is further illustrated by the following examples.

[0247] The materials used in the examples are as follows: Non-functionalized precursor chitosan (chitosan A) For the synthesis of MEX-CD2-I-tmb, a medical grade, animal-derived, non-functionalized precursor chitosan (chitosan A) was used. The weight-average molar mass and number-average molar mass (Mw=2,583.105 g / mol, Mn=1,323.105 g / mol, respectively) were measured by size-exclusion chromatography combined with refractive index and multi-angle laser light scattering measurements (Schatz et al., Biomacromolecules, 2003;4(3):641-8). The degree of acetylation (percentage of N-acetyl-D-glucosamine units) of such crude chitosan (chitosan A) was measured by 1H NMR spectroscopy using the Hirai method (Hirai et al., Polymer Bulletin, 26, 87-94.1991) and is estimated to be 6±0.5%.

[0248] Polysaccharide MEX-CD2 (Polysaccharide B) This synthesis of MEX-CD2-I-tmb also includes the polysaccharide MEX-CD2 (polysaccharide B) obtained after chemical modification of chitosan A with DOTA-GA anhydride, the synthesis of which has been described in the previous patent application FR2110474. Briefly, 60 g of chitosan was placed in a 10 L reactor together with 4 L of ultrapure water and 50 mL of acetic acid, and the mixture was mechanically stirred at 500 rpm. After the chitosan was completely dissolved (3 h), 4 L of 1,2-propanediol was added to the medium and the mixture was kept stirring until homogenization (2 h). Then, 120 g of DOTA-GA anhydride was added and the mixture was kept stirring overnight. The synthesis product was then filtered through a Sartocon® Slice PESU cassette (polyethersulfone membrane, cut-off: 100 kDa, filtration area: 0.1 m 2The polysaccharide B was purified by tangential filtration according to the diafiltration-concentration model against 200 L of 0.1 M acetic acid solution, followed by 200 L of 5 mM acetic acid solution, using a Sartoflow® Advanced device equipped with a diafiltration filter. The degree of acetylation of polysaccharide B is identical to that of the starting chitosan A, and the degree of substitution of polysaccharide B (percentage of N-DOTAGA-D-glucosamine units) is measured by the copper chelation method (Natuzzi et al., Nature Scientific Reports, 2021, 11, 19948) and is estimated to be 15±0.5%.

[0249] Trastuzumab The synthesis of MEX-CD2-I-tmb involves the antibody trastuzumab. Trastuzumab was obtained from Trazimera® powder, which is commercially available as a concentrate for infusion solutions. One vial contains 420 mg of trastuzumab and excipients: L-histidine hydrochloride monohydrate, L-histidine, sucrose, and polysorbate 20 (E432).

[0250] In vivo studies will use trastuzumab and the HERCEPTIN® SC formulation, which contains recombinant human hyaluronidase (rHuPH20) as the main excipient. The HERCEPTIN® SC formulation is in a 600 mg / 5 mL single-dose vial. It is a ready-to-use solution for injection that does not require dilution.

[0251] Example 1: Preparation of the formulation according to the invention MEX-CD2-I-tmb (also called MEX-CD2-I-HER2) by method 1 According to method (1), MEX-CD2-I-tmb was prepared from a viscous solution of a composition called MEX-CD2-I solution with an α / β% ratio (α is the mass concentration (w / v) of chitosan A in the target formulation and β is the mass concentration (w / v) of polysaccharide B).

[0252] Briefly, in a 50 mL reactor, 3.0 g of polysaccharide B (MEX-CD2) and 1.5 g of chitosan A were dispersed in 28.93 mL of Milli-Q water and 1.07 mL of ultrapure acetic acid was added under mechanical stirring at 50 rpm. The mixture was left under stirring for 24 h until complete dissolution and homogenization of the medium.

[0253] The resulting solution was collected and introduced into a suitable fluid dispenser, then centrifuged at 4000 rpm for 10 minutes to obtain a bubble-free solution containing the composition. MEX-CD2-I solution was obtained.

[0254] In method (1), 1.5 mL of this 5 / 10% MEX-CD2-I solution was thoroughly mixed in a reactor with 1.5 mL of 420 mg of Trazimera® solution (a commercially available trastuzumab solution formulated with excipients including histidine buffer and surfactant) previously reconstituted at 250 g / L in 1.68 mL of Milli-Q water.

[0255] The mixture was centrifuged at 5000 rpm for 10 min and then mixed again to homogenize the solution. This step was repeated three times until a homogenous opaque white viscous solution was obtained.

[0256] The mixture was introduced into a SpectraPor® Float-A-Lyzer® dialysis tube (cutoff: 8-10 kDa) and dialyzed against Milli-Q water for 24 hours, with the pH maintained at 6.5 by adding 0.1 mol / L NaOH aqueous solution. The mass inside the dialysis tube was confirmed by weighing before dialysis. The dialysis solution was then changed to 8 g / L sodium chloride solution, the pH was maintained at 6.5 by adding 0.1 mol / L NaOH solution, and dialysis was continued for 48 hours. At the end of dialysis, the dialysis tube was weighed again to estimate the dilution factor of the medium (C トラスツズマブ = 88g / L;C MEX-CD2 =52g / L).

[0257] The pH and osmolality of the mixtures were measured using a Mettler Toledo SevenCompact S210 pH meter and a Camlab Loser Micro MOD200 Plus osmometer, respectively (pH = 5.6; osmolality = 268 mOsm / L).

[0258] Injection tests using different needle diameters and 1 mL syringes were performed to qualitatively evaluate the injectability of the solutions. The ability to inject through the needle was given a score from 1 to 10, with 1 being a score given to a solution that was not injectable or very difficult to inject; 10 being a score given to a solution that was very easy to inject.

[0259] The solution gelling test was also performed using a buffered hemodialysis solution (Hemosol B0, 1.75 mmol / L Ca 2+ , 0.5mmol / L Mg 2+ , 140mmol / L Cl - , 3 mmol / L lactate, and 32 mmol / L HCO 3 - A hemodialysis / hemofiltration test was performed in a hemodialysis / hemofiltration solution containing 0.1% ethanol (H2O2) and the strength of the formed hydrogels was qualitatively evaluated. The resistance of the gels in the medium was given a score of 1 to 10, with 10 being a score given to a very resistant gel. The results are shown in Table 1 and examples of the obtained hydrogels are shown in Figure 1.

[0260] [Table 1]

[0261] These results show that the synthesized solution is easily injectable by the operator and is compatible with a wide range of needles commonly used in the medical field for subcutaneous injection of active substances or various pharmaceuticals. Moreover, in situ gelation of the solution was observed after injection in a medium with physiological conditions (pH = 7.4, osmolality = 300 mOsm / L).

[0262] Example 2: Preparation of the formulation according to the invention MEX-CD2-I-tmb (also called MEX-CD2-I-HER2) by method 2 According to method (2), MEX-CD2-I-tmb was prepared from a viscous solution of a composition called MEX-CD2-I solution with an α / β% ratio (α is the mass concentration (w / w) of chitosan A in the target formulation and β is the mass concentration (w / w) of polysaccharide B).

[0263] Briefly, in a 50 mL reactor, 1.33 g of polysaccharide B (MEX-CD2) and 0.67 g of chitosan A were dispersed in 19.87 mL of Milli-Q water and 150 μL of ultrapure acetic acid was added under mechanical stirring at 100 rpm. The mixture was stirred for 2 h until complete dissolution and homogenization of the medium. The resulting solution was collected and introduced into a suitable fluid dispenser, followed by centrifugation at 4000 rpm for 10 min to obtain a bubble-free solution containing the composition. The pH and osmolality of the mixture were measured using a Mettler Toledo SevenCompact S210 pH meter and a Camlab Loser Micro MOD200 Plus osmometer, respectively (pH = 5.9; osmolality = 503 mOsm / L).

[0264] According to method (2), two 420 mg vials of Trazimera® were reconstituted with 8.4 mL of Milli-Q water to 50 g / L, and the trastuzumab solution was homogenized using a vortex shaker at 1000 rpm for 5 min. The trastuzumab solution was then introduced into a Sartorius Vivaspin™ 20 centrifugal concentrator (polyethersulfone membrane, cut-off: 100 kDa) and centrifuged at 4000 rpm for 4 h. This allows the separation of the trastuzumab antibody from the excipients contained in the Trazimera® commercial solution. The retentate was collected and the trastuzumab concentration was monitored by measuring the absorbance at 275 nm using a Varian Cary® 50 UV-Vis spectrophotometer. The trastuzumab concentration was readjusted to 200 g / L by adding Milli-Q water, and the pH and osmolality of the trastuzumab solution were measured (pH = 6.19; osmolality = 129 mOsm / L).

[0265] In one method (2a), 2 mL of the trastuzumab solution was introduced into a Luer-lock syringe and 2 mL of the 3.3 / 6.7% solution was introduced into another Luer-lock syringe. The two syringes were connected with a Luer-lock connector and the contents of one solution were injected into the other and vice versa repeatedly (approximately 200 times) and thoroughly mixed. The pH and osmolality of the solutions were measured (pH = 5.77; osmolality = 228 mOsm / L).

[0266] In another method (2b), 2 mL of trastuzumab solution and 2 mL of 3.3 / 6.7% solution were thoroughly mixed in a reactor by mechanical stirring at 100 rpm with a bladed rotor for 2 h. The pH and osmolality of the solution were measured (pH = 5.77; osmolality = 243 mOsm / L).

[0267] Injection tests using different needle diameters and 1 mL syringes were performed to qualitatively evaluate the injectability of the solutions. The ability to inject through the needle was given a score from 1 to 10, with 10 being a score given to a solution that was very easy to inject. Solution gelation tests were also performed in physiological serum solution to qualitatively evaluate the strength of the formed hydrogel. The resistance of the gel in the medium was given a score from 1 to 10, with 10 being a score given to a very resistant gel. The results are shown in Table 2.

[0268] [Table 2]

[0269] These results demonstrate the feasibility of formulating a solution that is easily injectable by the operator and capable of gelling in situ under physiological conditions, even after purification of the excipients originally contained in the commercial solution of trastuzumab (Trazimera®). This study also shows that it is possible to mix the antibody and polymer solutions in different ways without affecting the injectability and gelling ability of the final solution.

[0270] Example 3: Preparation of a formulation according to the invention, MEX-CD2-I-tmb (also called MEX-CD2-I-HER2), with a fluorescent label According to method (3), polysaccharide B was chemically prelabeled with cyanine 5.5 (Cy5.5). According to this method, trastuzumab was also chemically labeled with fluorescein by grafting fluorescein isothiocyanate (FITC).

[0271] Labeling of polysaccharide B with cyanine 5.5 was achieved by adding the fluorophore directly to the modified chitosan solution. During the synthesis and purification steps, the solution was protected from light by aluminum foil. Briefly, 100 mg of cyanine 5.5 was dissolved in 20 mL of anhydrous DMSO to prepare a 5 g / L cyanine 5.5 solution.

[0272] In parallel, a solution of polysaccharide B was prepared by completely dissolving 1.1 g of polysaccharide B in 100 mL of Milli-Q water. The pH of this solution was adjusted to pH = 6.02 by adding 750 μL of 1 mol / L NaOH solution while stirring. Cyanine 5.5 solution was added dropwise to the polysaccharide B solution while stirring at 200 rpm to release the 27 free NH 2 One of the functional groups was grafted with 5.5 molecules of cyanine (molar ratio = 0.037). The reaction mixture was left in the dark for 24 hours with stirring at 100 rpm. The synthesis product was then filtered through a Sartocon® Slice PESU cassette (polyethersulfone membrane, cutoff: 100 kDa, filtration area: 200 cm 2 The product was purified by tangential filtration according to the diafiltration-concentration model against 10 L of 5 mM acetic acid solution using a Sartoflow® Smart device equipped with a diafiltration-concentration model against 10 L of 5 mM acetic acid solution. The product was then lyophilized for 48 h.

[0273] Labeling of trastuzumab with fluorescein (sample 3a) was also performed by adding the fluorophore directly to a Trazimera® solution from which excipients had previously been removed (compound 3b). First, two vials of 420 mg of Trazimera® were reconstituted to 50 g / L with 8.4 mL of Milli-Q water and the solution was left at 4° C. for 24 hours. The solution was then introduced into a Sartorius Vivaspin™ 20 centrifugal concentrator (polyethersulfone membrane, cut-off: 100 kDa) and centrifuged at 4000 rpm for 7 hours. The retentate was collected and the concentration of trastuzumab was monitored by measuring the absorbance at 275 nm using a Varian Cary® 50 UV-Vis spectrophotometer. The trastuzumab concentration was measured to be 247 g / L.

[0274] 50 μL of this purified trastuzumab solution (compound 3b) was diluted with 1.95 mL of carbonate-bicarbonate buffer at pH=9.06 to make a 5 g / L trastuzumab solution. In parallel, 1.9 mg of FITC was dissolved in 1.9 mL of anhydrous DMSO. This FITC solution was then added dropwise to the purified trastuzumab solution while stirring at 200 rpm, so that one molecule of trastuzumab was grafted with 3.7 molecules of fluorescein. The reaction mixture was left in the dark for 3 hours while stirring at 200 rpm. The resulting solution was introduced into a SpectraPor® Float-A-Lyzer® dialysis tube (cutoff: 5 kDa) and dialyzed against 800 mL of 0.1 M phosphate buffer at pH=7.4 for 18 hours. The resulting solution was finally dialyzed against 800 mL of Milli-Q water for 2 hours.

[0275] According to this method, a viscous solution was prepared, called composition 3.3 / 6.7% in the ratio α / β% (α is the mass concentration (w / w) of chitosan A and β is the mass concentration (w / w) of cyanine 5.5 labeled polysaccharide B) (compound 3c). Briefly, in a 50 mL reactor, 667 mg of cyanine 5.5 labeled polysaccharide B (MEX-CD2) and 333 mg of chitosan A were dispersed in 8.92 mL of Milli-Q water and 75 μL of ultrapure acetic acid were added under mechanical stirring at 100 rpm. The mixture was stirred for 2 hours until complete dissolution and homogenization of the medium. The resulting solution was collected and introduced into a suitable fluid dispenser, then centrifuged at 4000 rpm for 10 minutes to obtain a bubble-free solution containing the composition. In parallel, a 3.3 / 6.7% solution identical to that described in Example 2 was synthesized (compound 3d).

[0276] The fluorescently labeled MEX-CD2-I-tmb solution was finally prepared as follows: first, 0.9 mL of a 3.3 / 6.7% mixture based on unlabeled polysaccharide B (compound 3d) was introduced into a Luer-lock syringe, and 0.1 mL of a 3.3 / 6.7% mixture made from cyanine 5.5-labeled polysaccharide B was introduced into another Luer-lock syringe. The two syringes were connected with a Luer-lock connector and thoroughly mixed by repeatedly injecting the contents of one into the other and vice versa (compound 3e). In parallel, 0.2 mL of a 5 g / L fluorescein-labeled trastuzumab solution (compound 3a) was added to 0.8 mL of purified 247 g / L trastuzumab (compound 3b) while stirring at 100 rpm. The resulting solution was collected and inserted into the appropriate Luer-lock syringe (compound 3f). The syringes containing 3e and 3f were finally connected with a Luer lock connector and thoroughly mixed by repeatedly injecting the contents of one into the other and vice versa (100–200 times). The pH and osmolality of the mixture were measured using a Mettler Toledo SevenCompact S210 pH meter and a Camlab Loser Micro MOD200 Plus osmometer, respectively (pH = 5.69; osmolality = 285 mOsm / L).

[0277] Gelation and injection tests of this solution were performed by direct injection into 0.9% (w / w) NaCl and 0.1 M PBS (phosphate buffered saline) solutions using a 1 mL syringe fitted with a 22 G needle. The solution was easily injected by the operator and gelled immediately upon contact with physiological serum or PBS. Furthermore, the gel was qualitatively much more resistant in PBS than in physiological serum.

[0278] The in situ gelation of fluorescently labeled MEX-CD2-I-tmb solution is shown in Figure (2A) and the resulting gel after complete gelation is shown in Figure (2B).

[0279] This work demonstrates the feasibility of easily and independently labeling polymers or antibodies with different fluorophore probes without significantly altering the physicochemical properties of the resulting solutions, whose pouring and gelling abilities are also comparable to those obtained without pre-labeling of the reagents.

[0280] Example 4: Study of the rheological properties of MEX-CD2-I solutions (before mixing with antibody) as a function of pH and osmolality (not according to the invention) The objective of this study was to determine the physicochemical parameters of the precursor polymer solution (before mixing with the antibody) at which the compound would remain stable in solution and would not induce significant gelation of the system. Therefore, solutions of 5% (w / w) MEX-CD2-I were prepared at various osmolalities and pH and their rheological properties were measured to establish the range of osmolality and pH that would lead to gelation of the solution.

[0281] To prepare samples of varying osmolality, viscous solutions were made with a composition called 1.7 / 3.3% in the ratio α / β% (α is the mass concentration (w / w) of chitosan A and β is the mass concentration (w / w) of polysaccharide B).

[0282] Briefly, in a 50 mL reactor, 1.67 g of polysaccharide B (MEX-CD2) and 0.83 g of chitosan A were dispersed in 47.33 mL of Milli-Q water and 162 μL of ultrapure acetic acid was added under mechanical stirring at 100 rpm. The mixture was stirred for 2 hours until complete dissolution and homogenization of the medium. The resulting solution was collected and introduced into a suitable fluid dispenser, followed by centrifugation at 4000 rpm for 10 minutes to obtain a bubble-free solution containing the composition. The pH and osmolality of the mixture were measured using a Mettler Toledo SevenCompact S210 pH meter and a Camlab Loser Micro MOD200 Plus osmometer, respectively (pH = 5.75; osmolality = 57 mOsm / L). 2 mL of this viscous solution was introduced into an adapted Luer-Lok syringe and a large amount of NaCl was introduced into another Luer-Lok syringe. The contents of the two syringes are thoroughly mixed by injecting from one syringe to the other and vice versa using a Luer lock connector to obtain samples with identical pH of 5.75 and osmolality ranging from 57 mOsm / L to 352 mOsm / L.

[0283] For preparation of samples with varying pH, samples were obtained from the same 1.7 / 3.3% solution as above.

[0284] 2 ml of this solution was added to a SpectraPor® Float-A-Lyzer® dialysis tubing (cut-off: 50 kDa). Prior to dialysis, the dialysis device was washed in a 10% (V / V) ethanol solution for 30 min, followed by Milli-Q water for 30 min.

[0285] Next, in order to keep the solution concentration inside the dialysis membrane stable during the experiment, dialysis was performed against an isotonic NaCl solution (1.9 g / L NaCl, equivalent to an osmolality of 57 mOsm / L).

[0286] After 3 hours, the pH of the dialysis solution was adjusted to pH = 6.0 by addition of NaOH and left for a period ranging from 18 to 72 hours depending on the desired pH of the sample. Samples with osmolalities ranging from 55 to 98 mOsm / L and pHs ranging from 5.75 to 6.4 were obtained.

[0287] The rheological properties of each sample were measured using a TA Instruments AR2000 rheometer. Specifically, measurements were performed using 4° and 25mm flat cone geometries. Prior to the measurements, zero deviation was set and the inertial and rotational mapping of the instrument was calibrated. The samples were then spread on the stage and the deviation from the geometry was set to 116 microns. Excess sample was removed with a spatula to reduce edge effects.

[0288] A preliminary study was carried out to determine the limits of the material's linear deformation region by performing a 10 Hz amplitude sweep with amplitude varying from 0.1% to 100% at an operating temperature of 25 °C.

[0289] First, the storage modulus G' and loss modulus G'' of each sample were measured at 25°C and a fixed strain of 10% (6.9.10 -3 rad) and angular frequency from 1 to 100rad.s -1 The viscosity of each sample was then measured by sweeping the frequency in the range of 10 to 150 Hz, without removing the sample from the tray, at an operating temperature of 25 °C and a shear rate of 10. -2 From 10 2 s -1 The storage modulus G' characterizes the elasticity of the material, increases during the gel state of the system and characterizes the flow behavior of the solution. The loss modulus G'' characterizes the viscous behavior of the system. The results obtained for each sample are shown in Table 3. The material exhibits a solution state (if G''>G') or a soft gel state (if G'>G').

[0290] [Table 3]

[0291] For samples with an osmolality below 352 mOsm / L, the storage modulus G' is lower than the loss modulus G''. Thus, above 350 mOsm / L the system tends to gel. The gel point can be determined when the moduli G' and G'' are equal. At pH 5.75, the gel point appears to occur at an osmolality of around 300 mOsm / L. Thus, gelation of the system at pH=5.75 occurs when the osmolality exceeds 300 mOsm / L.

[0292] The range of stability of MEX-CD2-I solutions measured as a function of pH and osmolality is summarized in FIG.

[0293] The results obtained with samples with varying pH show heterogeneity of the rheological properties due to the heterogeneity of the medium caused by the dialysis method used. However, in the osmolality range of 50-100 mOsm / L, gelation of the system was qualitatively observed when the pH was higher than 6.4.

[0294] Example 5: Study of the physicochemical properties of MEX-CD2-I-tmb (also called MEX-CD2-HER2) solutions as a function of trastuzumab concentration The objective of this study was to determine the optimal concentration of trastuzumab in MEX-CD2-I-tmb solution at which the compound has suitable physicochemical properties for subcutaneous injection of the product. Samples of MEX-CD2-I-HER2 containing 5% (w / w) polymer, purified and unpurified from commercial trastuzumab solution excipients, were prepared at varying concentrations ranging from 12.5 g / L to 75 g / L. For each sample, the solution pH and osmolality were measured using a Mettler Toledo SevenCompact S210 pH meter and a Camlab Loser Micro MOD200 Plus osmometer, respectively.

[0295] Briefly, a sample was made from a viscous solution of the same composition 3.3 / 6.7% as described in Example 2 (Sample 5a). In parallel, a 420 mg vial of Trazimera® was reconstituted to 100 g / L with 4.2 mL of Milli-Q water and the solution was homogenized for 5 min at 1000 rpm using a vortex mixer. This solution is called trastuzumab solution with excipients (Sample 5b). Additionally, two 420 mg vials of Trazimera® were reconstituted to 50 g / L with 8.4 mL of Milli-Q water and the solution was homogenized for 5 min at 1000 rpm using a vortex mixer. This solution was then introduced into a Sartorius Vivaspin™ 20 centrifugal concentrator (polyethersulfone membrane; cut-off: 100 kDa) and purified using the same protocol as described in Example 2. The resulting solution is diluted to 100 g / L with Milli-Q water and referred to as excipient-free trastuzumab solution (compound 5c).

[0296] MEX-CD2-I-tmb samples were prepared by introducing an appropriate amount of the 3.3 / 6.7% solution into a 2 mL Luer-lock syringe. In parallel, different volumes of trastuzumab solution (purified or unpurified) were introduced into a 2 mL Luer-lock syringe to obtain final MEX-CD2-I-tmb solutions with trastuzumab concentrations ranging from 12.5 g / L to 75 g / L. The two syringes were connected with a Luer-lock connector and thoroughly mixed by repeatedly injecting the contents of one into the other and vice versa (approximately 200 times). The pH and osmolality of the solutions were then measured.

[0297] For example, the synthesis of a 50 g / L sample of trastuzumab unpurified from excipients was performed as follows: 1 mL of the 3.3 / 6.7% solution was introduced into a 2 mL Luer lock syringe. 1 mL of the unpurified trastuzumab solution (compound 5b) was introduced into a 2 mL Luer lock syringe. The two syringes were connected with a Luer lock connector and thoroughly mixed manually by repeatedly injecting the contents of one into the other and vice versa (approximately 200 times).

[0298] The results obtained for each sample are shown in Table 4.

[0299] [Table 4]

[0300] This study shows the biocompatibility of MEX-CD2-I-tmb solutions for subcutaneous injection as a function of trastuzumab concentration. Samples obtained from purified solutions of excipients show a very slight increase in pH as a function of trastuzumab concentration. This increase is due to the higher pH of the purified trastuzumab solution compared to the 3.3 / 6.7% solution (pH = 6.19). Also, the osmolality increases as a function of trastuzumab concentration due to the contribution of osmotically active species contained in the trastuzumab solution. This trend is even more pronounced in samples obtained from unpurified trastuzumab solutions.

[0301] Example 6 - Study of the gelling ability of solutions (not according to the invention) as a function of the MEX-CD2 / Chitosan A ratio The aim of this study was to determine the gelling ability of MEX-CD2-I solutions as a function of the starting MEX-CD2 / chitosan A weight ratio. Solutions were prepared in the ratios 3.5 / 0%, 0 / 7% and 2.5 / 2.5%, respectively, with the ratio α / β% (α is the mass concentration (w / w) of chitosan A and β is the mass concentration (w / w) of polysaccharide B). For each sample, the pH and osmolality of the solutions were measured using a Mettler Toledo SevenCompact S210 pH meter and a Camlab Loser Micro MOD200 Plus osmometer, respectively.

[0302] Briefly, in a 50 mL reactor, 350 mg of chitosan A was dissolved in 10 mL of Milli-Q water to produce a 3.5 / 0% solution. Glacial acetic acid was added (100 μL) with mechanical stirring until a clear mixture was obtained in which all the chitosan was solubilized. The mixture was left stirring for 2 hours until complete dissolution and the medium was homogenized. The resulting solution was collected and introduced into a suitable fluid dispenser (pH=5.64; osmolality=101 mOsm / L).

[0303] A 0 / 7% solution was prepared by dissolving 700 mg of MEX-CD2 in 10 mL of Milli-Q water in a 50 mL reactor. Glacial acetic acid was added (50 μL) with mechanical stirring until a clear mixture was obtained in which all the chitosan was solubilized. The mixture was left stirring for 2 hours until complete dissolution and the medium was homogenized. The resulting solution was collected and introduced into a suitable fluid dispenser (pH = 4.79; osmolality = 157 mOsm / L). The resulting solution was collected and introduced into a suitable fluid dispenser (pH = 4.79; osmolality = 157 mOsm / L).

[0304] A 2.5 / 2.5% solution was prepared by dissolving 250 mg of chitosan A and 250 mg of MEX-CD2 in 10 mL of Milli-Q water in a 50 mL reactor. Glacial acetic acid was added (80 μL) with mechanical stirring until a clear mixture was obtained in which the entire amount of polymer was solubilized. The mixture was left under stirring for 2 hours until complete dissolution and the medium was homogenized. The resulting solution was collected and introduced into a suitable fluid dispenser (pH = 5.18; osmolality = 117 mOsm / L).

[0305] 300 μL of each solution was injected into 20 mL of physiological serum solution (NaCl 9 g / l) or 20 mL of commercial phosphate-buffered saline (PBS) 10 mmol / L using a 1 mL syringe and a 25 G needle. For each injection test, the gelation of the solution was qualitatively evaluated after 30 min. A score of 1 to 10 was assigned to the ability to inject through a 25 G needle, with 10 being a score given to a very easy to inject solution. A score of 1 to 10 was given to the resistance of the gel in the medium, with 10 being a score for a very resistant gel. The results are shown in Table 5.

[0306] [Table 5]

[0307] The gelation of the solution in physiological serum solution is mainly due to the presence of MEX-CD2 in the solution. In PBS, chitosan A tends to precipitate and form a very soft gel with little cohesive strength, but still contributes to the gelation of the system. Solutions with a high percentage of MEX-CD2 have a higher tendency to gel while chitosan is added to obtain crystalline zones, as described in our previous patent application FR2110474.

[0308] Example 7: Preparation of the formulation according to the invention MEX-CD2-I-HER2 (also called MEX-CD2-tmb) by method (4) 7.1. General procedure for preparation of MEX-CD2-I-HER2 by method (4) According to method (4), MEX-CD2-I-HER2 was prepared from a viscous solution of a composition called 3.3 / 6.7% MEX-CD2-I solution with a ratio of α / β% (α is the mass concentration (w / w) of chitosan A in the formulation in question and β is the mass concentration (w / w) of polysaccharide B).

[0309] Briefly, in a 50 mL reactor, 667 mg of polysaccharide B (MEX-CD2) and 133 mg of chitosan A were dispersed in 8.92 mL of Milli-Q water and 72 μL of ultrapure acetic acid were added under mechanical stirring at 100 rpm. The mixture was stirred for 2 h until complete dissolution and homogenization of the medium. The resulting solution was collected and introduced into a suitable fluid dispenser, followed by centrifugation at 4000 rpm for 10 min at room temperature to obtain a bubble-free solution containing the composition. The mixture was then introduced into a glass syringe and sterilized in an autoclave at 121 °C for 20 min. The pH and osmolality of the mixture were measured using a Mettler Toledo SevenCompact S210 pH meter and a Camlab Loser Micro MOD200 Plus osmometer, respectively (pH = 5.7; osmolality = 410 mOsm / L). The Newtonian viscosity of the resulting solutions was measured on a Thermo Scientific HAAKE RheoStress 600 sensor system using a C35 / 2° Ti L cone-plate geometry. The viscosity was measured at a temperature of 25°C and a gradient of 10 -2 From 10 3 s -1 The viscosity was measured by a flow sweep linearity test, scanning the shear rate from 0.01 to 0.01. The measurement time for each value was 10 seconds, and 10 points per decade were recorded. The Newtonian viscosity was η 0 = 2023 Pa·s and η 0 =3260 Pa s.

[0310] According to method (4), a 420 mg vial of Trazimera® is reconstituted to 200 g / L in 2.1 mL of Milli-Q water and left at 4° C. for 24 hours until a completely dissolved and homogenized trastuzumab solution is obtained.

[0311] According to this method (4), 1.2 mL of trastuzumab solution was introduced into a Luer-lock syringe and 1.2 mL of 3.3 / 6.7% solution was introduced into another Luer-lock syringe. The two syringes were connected using a Luer-lock connector and thoroughly mixed by repeatedly injecting the contents of one solution into the other and vice versa (approximately 50-200 times). The pH, osmolality and Newtonian viscosity of this solution were measured (pH = 5.81; osmolality = 564 mOsm / L; η 0 =126.7 Pa·s). The injectability of this MEX-CD2-I-HER2 solution was precisely measured using a Shimadzu AG-X Plus Force Machine. The solution was introduced into a BD Hylok™ 1 mL prefillable glass syringe fitted with a Terumo Agani 25G needle (0.5×16 mm). Injectability was measured as the force (in Newtons) required to expel the solution at a constant plunger speed of 1 mm / s. F e = 46.7 N of ejection force was obtained, which means that the system can be manually injected by the practitioner (Terobinson et al., Filling the Gap: A Correlation between Objective and Subjective Measures of Injectability, Adv. Healthc. Mater., vol. 9, no 5, pp. 1901521, 2020).

[0312] A gelation test of this solution was performed by injecting it directly into a 10 mM PBS (phosphate buffered saline) solution using a 1 mL syringe equipped with a 25 G needle, and the solution gelled immediately upon contact with PBS.

[0313] 7.2. Preparation procedure for gadolinium-labeled MEX-CD2-I-HER2 According to the method described below, polysaccharide B is grafted with gadolinium (Gd) on its pre-grafted chelating group. 3+ It was chemically labelled with gadolinium by ionic complexation.

[0314] Labeling of polysaccharide B with gadolinium was achieved by adding a 1 mol / L solution of gadolinium directly to the modified chitosan solution. Briefly, 8.74 mL of Gd 3+ A 1 mol / L solution of was added to 1 L of a 7 g / L solution of polysaccharide B under stirring. Then, the pH was adjusted from pH=3.6 to pH=5.6 by adding 1 mol / L sodium hydroxide solution. The solution was stirred at 60° C. for 48 hours. The synthesis product was then filtered through a Sartocon® Slice PESU cassette (polyethersulfone membrane, cut-off: 100 kDa, filtration area: 200 cm). 2 The synthesis was purified by tangential filtration according to the diafiltration-concentration model against 10 L of 5 mM acetic acid solution using a Sartoflow® smart device equipped with a diafiltration column. The synthesis was then freeze-dried for 48 h. The amount of gadolinium complexed to the grafted chelating groups of polysaccharide B was determined by ICP-MS performed on a PerkinElmer NexION 2000. 158 It was estimated that 70% of all DOTAGA groups were complexed with gadolinium, as determined by Gd analysis.

[0315] According to this method, a viscous solution was produced, called composition 3.3 / 6.7%, in the ratio α / β% (α is the mass concentration (w / w) of chitosan A and β is the mass concentration (w / w) of the total polysaccharide B (including unmodified and Gd-labeled compounds). Briefly, in a 50 mL reactor, 601.9 mg of polysaccharide B, 66.2 mg of gadolinium-labeled polysaccharide B, and 332.9 mg of chitosan A were dispersed in 8.92 mL of Milli-Q water and 76 μL of ultrapure acetic acid were added under mechanical stirring at 100 rpm. The mixture was left under stirring for 2 hours until complete dissolution and homogenization of the medium. The resulting solution was collected and introduced into a suitable fluid dispenser, followed by centrifugation at 4000 rpm for 10 minutes to obtain a bubble-free solution containing the composition. The mixture was then introduced into a glass syringe and sterilized in an autoclave at 121 °C for 20 minutes.

[0316] According to this method, a 420 mg vial of Trazimera® is reconstituted to 200 g / L in 2.1 mL of Milli-Q water and left at 4° C. for 24 hours until a completely dissolved and homogenized trastuzumab solution is obtained.

[0317] According to this method, 1 mL of trastuzumab solution was introduced into a Luer-lock syringe and 1 mL of 3.3 / 6.7% solution was introduced into another Luer-lock syringe. The two syringes were connected using a Luer-lock connector and the contents of one solution were injected into the other and vice versa repeatedly (approximately 50-200 times) until thoroughly mixed.

[0318] 7.3. Preparation procedure for fluorescently and gadolinium-labeled MEX-CD2-I-HER2 According to the method described below, polysaccharide B was chemically prelabeled with cyanine 5.5 (Cy5.5). According to this method, polysaccharide B also had gadolinium (Gd) attached to its pregrafted chelating groups. 3+ It is chemically labelled with gadolinium by forming an ionic complex.

[0319] Labeling of polysaccharide B with cyanine 5.5 was achieved using the same procedure as described in Example 3.

[0320] Labeling of polysaccharide B with gadolinium was achieved using the same procedure as described in Example 7.2.

[0321] According to this method, a viscous solution was produced, referred to as composition 3.3 / 6.7%, in the ratio α / β% (α being the mass concentration (w / w) of chitosan A and β being the mass concentration (w / w) of the total polysaccharide B (including unmodified, Cy5.5-labeled and Gd-labeled compounds). Briefly, in a 50 mL reactor, 318 mg of polysaccharide B, 7.63 mg of cyanine 5.5-labeled polysaccharide B, 17.2 mg of gadolinium-labeled polysaccharide B and 167.5 mg of chitosan A were dispersed in 4.46 mL of Milli-Q water and 38 μL of ultrapure acetic acid were added under mechanical stirring at 100 rpm. The mixture was left under stirring for 2 hours until complete dissolution and homogenization of the medium. The resulting solution was collected and introduced into a suitable fluid dispenser, followed by centrifugation at 4000 rpm for 10 minutes to obtain a bubble-free solution containing the composition. This mixture is then introduced into a glass syringe and sterilized in an autoclave at 121° C. for 20 minutes.

[0322] According to this method, a 420 mg vial of Trazimera® is reconstituted to 200 g / L in 2.1 mL of Milli-Q water and left at 4° C. for 24 hours until the trastuzumab solution is completely dissolved and homogenized.

[0323] According to this method, 0.5 mL of the trastuzumab solution was introduced into a Luer-lock syringe and 0.5 mL of the 3.3 / 6.7% solution was introduced into another Luer-lock syringe. The two syringes were connected using a Luer-lock connector and the contents of one solution were injected into the other and vice versa repeatedly (approximately 50-200 times) until thoroughly mixed.

[0324] Example 8: In vivo study of MEX-CD2-I-tmb (also called MEX-CD2-I-HER2 solution) according to the present invention Unless otherwise stated, the MEX-CD2-I-tmb solutions used in this example were prepared according to Example 1.

[0325] 8.1. In vivo toxicity testing Sixteen mice were injected subcutaneously (in the neck) with 200 μL of a MEX-CD2-I solution (not according to the invention) and nine mice were injected with 250 μL of a MEX-CD2-I-tmb solution loaded with 100 g / L trastuzumab antibody (corresponding to a dose of 15 mg of anti-HER2 antibody) prepared according to Example 1.

[0326] The body weight of the mice was monitored. The results show that no significant toxicity occurred in the mice. Body weight measurements after subcutaneous injection of 200 μL of MEX-CD2-I-HER2 solution carrying 100 g / L trastuzumab antibody are shown in Figure 4.

[0327] Hematoxylin and eosin (H&E) immunohistochemical studies were performed in three mice 20 days after injection, and the results are shown in Figure 5. No cell recruitment (macrophages) was observed, confirming the absence of inflammation.

[0328] 8.2. In vivo study of antibody release from hydrogels In this study, the plasma presence of anti-HER2 antibody (trastuzumab) released from an in situ formed hydrogel following subcutaneous injection of 200 μL of MEX-CD2-I-tmb solution loaded with 100 g / L trastuzumab antibody (corresponding to a dose of 15 mg trastuzumab antibody) was quantified by Homogeneous Time Resolved Fluorescence (HTRF) over a period of 20 days and compared to the controlled release of the same antibody following subcutaneous injection of 200 μL of the HERCEPTIN® SC formulation (corresponding to a dose of 15 mg trastuzumab antibody per injection).

[0329] In this study, nine mice were used per group, with one blood sample taken from three mice every three days, and the results are shown in Figure 6.

[0330] The results show that the time to maximum abundance (Cmax) shifted from 1 day (HERCEPTIN® SC formulation) to 3 days for our MEX-CD2-I-tmb solution. Furthermore, at the end of the experiment (26 days after SC injection), there was no trace of hydrogel at the injection site, confirming the biodegradability of the formulation.

[0331] 8.3. In vivo studies on the biodegradability of hydrogels The polymer was functionalized with a cyanine 5.5 fluorescent probe to track its biodegradation after subcutaneous injection. Three mice were injected with 200 μL of such fluorescently labeled MEX-CD2-I-tmb, prepared according to Example 3, and monitored over time by whole-body fluorescence imaging (IVIS, PerkinElmer) every three days.

[0332] The results are shown in Figure 7.

[0333] With this formulation of cyanine 5.5-labeled hydrogel, a stable signal plateau was observed up to 26 days after implantation, confirming the possibility of improving the hydrogel formulation to extend its subcutaneous longevity.

[0334] Example 9: In vivo biodegradability and toxicity study of MEX-CD2-I-HER2 solutions according to the present invention 9.1. Fluorescence-based study of biodegradability in vivo A solution of MEX-CD2-I-HER2 was prepared according to the process of Example 7.3.

[0335] A portion of the MEX-CD2 used in the preparation was labeled with the fluorescent cyanine 5.5 (Cy5.5) dye, and another portion was labeled with the gadolinium ion Gd3 +The complex was complexed with . 150 μL of the final solution loaded with 100 g / L trastuzumab was injected into nude mice (n=3). Fluorescence and brightfield images were acquired with a back-thinned CCD cooled camera ORCAIIBT-512G (Hamamatsu Photonics Deutschland GmbH, Herrsching am Ammersee, Germany) using a colored glass long-pass RG 665 filter (Melles Griot, Voisins les Bretonneaux, France). Optical excitation was performed at 633 nm, and emission wavelength was detected at 680 nm. Exposure times were set at 30 s for optical imaging and 0.05 s for brightfield imaging. Fluorescence images were acquired at different time points to monitor the disappearance of fluorescence over time, which resulted in the degradation of the hydrogel.

[0336] The results are shown in Figures 8 and 9.

[0337] After 8 days, approximately 55% of the initial fluorescence intensity was observed, implying that significant degradation of the formed hydrogel occurred during this period.

[0338] 9.2. Biodistribution studies by ICP-MS Following the experimental details in section 9.1, mouse organs were collected after dissection (21 days after injection) and mineralized with concentrated nitric acid in an Anton Paar Multiwave5000. Organs were divided and digested according to their weight to ensure homogeneity of digestion throughout the procedure. Standard 69% HNO 3A ROTIPURAN Supra (provided by Roth) and ultrapure water (18.2 MΩcm) were used for all digestions and sample preparations. The reactor was washed with a programmed cleaning method (4 mL water and 6 mL 69% HNO3, 10 min ramp from 0 °C to 180 °C, 10 min at 180 °C) before and after the sample cycle. After the cycle, it was rinsed with ultrapure water. Samples were digested using the Bio-Organic Digestion Method with the following parameters: 69% HNO3, 10 min ramp from 0 to 100 °C, 10 min at 100 °C, 10 min ramp from 100 to 200 °C, and 10 min at 200 °C (40 min cycle).

[0339] ICP-MS analysis was performed on a PerkinElmer NexION 2000 inductively coupled plasma mass spectrometer (ICP-MS). Prior to use, the instrument was primed with 10% HNO3 for 20 min, followed by 1% HNO 3 The ICP samples were washed continuously with 0.5% HNO for 1.5 hours. All system parameters were optimized according to the instrument protocol (mass calibration, torch alignment, QID in KED and standard mode, nebulizer gas flow, and dual detector calibration) before the analysis started. Once all parameters were optimized, a final performance check was performed with standard and KED. An internal standard of 2 ppb indium was used for all ICP samples to ensure consistent measurements and reliability. The base solvent was 0.5% HNO 3 The mixture consisted of 0.7% tertiary butanol, 0.7% tertiary butanol, and water. Tertiary butanol was included to minimize carbon matrix effects, as sample digestion included proteins and other carbon-rich biomolecules. Gadolinium (158Gd) was analyzed.

[0340] The results are shown in Figure 10.

[0341] Twenty-one days after injection, approximately 40% of the hydrogel was found at the injection site and approximately 20% in the kidney, revealing an excretion route for the hydrogel after degradation. The hydrogel remaining at the injection site was in the form of already degraded hydrogel, slightly attached to the neck tissue.

[0342] 9.3. MRI study of biodegradability in vivo A solution of MEX-CD2-I-HER2 was prepared according to the process of Example 7.2.

[0343] The MEX-CD2 used in this formulation is a compound in which a portion of the pre-grafted chelating agent is gadolinium (Gd 3+ These include those complexed with Gd ions. 200 μL of such Gd-complexed MEX-CD2-I-HER2 solutions were injected into the neck of three mice and 40 μL into three other mice to evaluate the degradation profile according to the gel volume administered. 200 μL of a solution without antibody (not according to the invention) was also injected into three mice to evaluate the degradation profile with and without antibody loading. Furthermore, drug-loaded solutions were prepared according to the process described in Example 7.2. using viscous solutions of 2 / 4% and 0 / 10% respectively in the ratio α / β% (α is the mass concentration (w / w) of chitosan A and β is the mass concentration (w / w) of total polysaccharide B (including unmodified and Gd-labeled compounds)) as precursor solutions before mixing with the antibody solution. Such solutions were injected (200 μL) into the neck of three mice, respectively for each solution.

[0344] The results are shown in Figure 11.

[0345] The hydrogels formed by the MEX-CD2-I-HER2 solution (carrying the antibody) had different t values ​​compared to the hydrogels formed by the reference solution (not according to the invention) not carrying the antibody. 1 / 2 values ​​(30 vs. 20dd) and significantly different T 1 The presence of trastuzumab reduces the size and T 1 This affects both the change in

[0346] The antibody-loaded scaffolds obtained from the 3% polymer solution had a smaller starting volume than the other scaffolds, even when injected in the same amount. This is probably due to the fact that more water is resorbed soon after injection. The size decreases quickly (t 1 / 2 The decrease in scaffold concentration (from 5% to 3%) leads to a decrease in size and T 1 Changes in both affect

[0347] Scaffolds obtained from polymer solutions composed only of polysaccharides (i.e., 0 / 10% polymer precursor solution) start to increase in size and then disappear very quickly from the injection site. At t=3 days, the scaffold is no longer present. The change in scaffold composition is reflected by a decrease in size and T 1 This affects both the change in

[0348] Therefore, the hydrogel scaffold obtained from the MEX-CD2-I-HER2 solution appears to be the best formulation. The presence of trastuzumab accelerates water loading (T 1 Changes in and T 1w and T 2w (It can be detected by heterogeneous signals on MRI.)

[0349] Moreover, results obtained with other formulations demonstrate the ability to tailor the hydrogel scaffold formed after injection and the release of the encapsulated protein (here, trastuzumab) as needed.

[0350] 9.4. Conclusions regarding biodegradability Importantly, once normalized, the injection volume did not affect the degradation rate of the hydrogel, such that 50 μL and 200 μL of trastuzumab-loaded hydrogels (n=3) implanted subcutaneously exhibited the same degradation profile, as shown in Figure 12.

[0351] 9.5. Research on inflammatory cells (neutrophils) The MEX-CD2-I-HER2 solution used in this example was prepared according to the process detailed in Example 7.1.

[0352] Ex-vivo quantification of IL6, TNFa and mub-40 levels was assessed by cytometry using digested samples 24 and 120 hours after implantation (n=3 per group). Results were compared to negative (untreated) and positive controls (neutrophils preactivated with LPS) (data not shown). After implantation, a slight inflammation was initially observed - i.e., an increase in the amount of proinflammatory neutrophils localized at the injection site, elevated TNFa levels. However, by day 5 after implantation, the total amount of proinflammatory neutrophils was significantly reduced and returned to basal levels, as was TNFa. The cytokine IL6 was low on day 1 and increased on day 6 (2.5% and 13%). However, these levels were significantly lower than positively activated neutrophils (54%), confirming the biocompatibility of the hydrogel formulation.

[0353] The absence of proinflammatory neutrophils at the transplant site was further verified by immunofluorescence staining with anti-mub40 antibody (day 5 post-transplant, n = 3). The results are shown in Figure 13.

[0354] 9.6. Conclusions regarding the safety profile of monoclonal antibody-loaded hydrogel formulations The results show that the presence of the gel under the skin does not induce inflammation, and is much less irritating than prior art gels, especially those containing alginate.

[0355] Example 10: In vitro study of the release of various proteins by hydrogels Materials and Methods The MEX-CD2-I solution used in this example was prepared according to Example 7.1, except that the solution was loaded with Trastuzumab (Roche), Rituximab (Roche), Trastuzumab, HER2 Fab'2 and HER2 VHH. The mixture of solutions was prepared overnight before placing the samples in 1 mL Eppendorf tubes filled with PBS. At each time point, an aliquot was taken from the milieu and fresh PBS was added to the solution. A Bradford assay was performed to quantify the amount of protein in each aliquot and to quantify the amount of antibody / fragment / protein.

[0356] 10.2.Results The results for cumulative release of monoclonal antibodies alone (trastuzumab or rituximab) and in combination (trastuzumab and pertuzumab) in PBS at 37° C. are shown in FIG.

[0357] The results regarding the cumulative release of monoclonal antibody fragments (Fab'2 and VHH) in PBS at 37°C are shown in FIG.

[0358] Example 11: SEM imaging of the formulation according to the invention, MEX-CD2-I-HER2 A solution of MEX-CD2-I-HER2 was prepared according to the process of Example 7.2.

[0359] The MEX-CD2 used in this formulation contains several pre-grafted chelators, including gadolinium (Gd 3+ions. 500 μL of such Gd-complexed MEX-CD2-I-HER2 solution was injected into 100 mL of 10 mmol / L PBS solution (phosphate buffered saline). 500 μL of a solution without antibody (not according to the invention) was also injected into 100 mL of 10 mmol / L PBS solution, and the effect of antibody loading on the surface morphology of the formed hydrogel was further evaluated by SEM imaging. Both solutions were injected with a Terumo agani 25G×16 mm needle with an inner diameter of 284 μm as measured by optical microscopy. After 1 h of gelation, the PBS was replaced with 100 mL of 10% (V / V) EtOH / H 2 The solution is replaced with another solution in which the percentage of ethanol is increased by 20% (V / V) every hour up to 100% EtOH. The samples then undergo critical point drying (CPD) involving 12 cycles of 120 s at 18 °C, followed by the drying of the samples from 100% EtOH with gaseous CO. 2 The dehydrated hydrogel is placed on a metal STUB and metallized by evaporating a thin layer of 10 nm copper onto the polymer scaffold surface. The resulting scaffold is observed with a SEM FEI QUANTA 250 FEG at 2 kV or 3 kV.

[0360] The results are shown in Figure 16.

[0361] The measured gel diameter in all cases (average 260 μm) was slightly smaller than the inner diameter of the needle used for injection (284 μm). The hydrogel scaffolds obtained with the trastuzumab-loaded solutions exhibit a significantly thicker and more pronounced network compared to the scaffolds obtained from the solutions without antibody. A rougher network was obtained, which is a result of the gelation of the system in the presence of high antibody concentrations. This difference in surface morphology indicates the gels' ability to encapsulate drugs and release them after gelation.

Claims

1. 1. A pharmaceutical formulation of a pharmaceutically active protein, comprising: an effective amount of a protein or a combination of proteins, chitosan A containing 90-100 mol% D-glucosamine and 0-15 mol% N-acetyl-D-glucosamine, at least one statistical polysaccharide B comprising D-glucosamine, N-acetyl-D-glucosamine and at least one saccharide unit of formula I: 【Chemistry 1】 (In the formula, Rc is a hydrophilic group, Z is a linker that is a single bond or a hydrocarbon chain containing from 1 to 12 carbon atoms, said chain being linear or branched, saturated or unsaturated, and optionally containing one or more heteroatoms (preferably selected from nitrogen, oxygen and sulfur); and one or more pharmaceutically acceptable excipients 1. A pharmaceutical formulation of a pharmaceutically active protein comprising:

2. Rc is a group having acidic properties, typically a carboxyl group (—COOH), a sulfonic acid group (—SO ２ OH), phosphonate group (-PO(OH) ２ 2. The pharmaceutical formulation of claim 1, wherein the aryl group is selected from the group comprising a thiol group (-SH), an alcohol group (-OH) and a group containing a chelating agent.

3. 2. The pharmaceutical composition according to claim 1, wherein the statistical polysaccharide B is a polysaccharide of general formula II: 【Chemistry 2】 (In the formula, Rc is a hydrophilic group, Z is a linker which is a single bond or a hydrocarbon chain containing from 1 to 12 carbon atoms, said chain being linear or branched, saturated or unsaturated, and optionally containing one or more heteroatoms (preferably selected from nitrogen, oxygen and sulfur); ・x is comprised of 0.01 to 0.5, ・y is comprised between 0.05 and 0.5, the ratio y / x is greater than 0.2, preferably greater than 1; - The sum of x + y is greater than 0.

1.

4. The viscosity of the formulation was determined to be 0.001 s by rotational rheometry in a flat cone geometry at room temperature. －１ ~0.01 seconds －１ , for example 0.001 seconds －１ and 0.01 seconds －１ , especially 0.01 seconds －１ 2. The pharmaceutical formulation of claim 1, wherein the viscosity is 10 to 500 Pa s measured at a shear rate of 100 Pa s.

5. The pharmaceutical formulation according to claim 1, wherein the pH of the formulation is comprised between 5.0 and 6.5, preferably between 5.5 and 6.

0.

6. 2. The pharmaceutical formulation of claim 1, wherein the osmolality of the formulation is comprised between 50 and 600 mOsm / L, preferably between 100 and 600 mOsm / L, more preferably between 250 and 450 mOsm / L, or between 50 and 300 mOsm / L, preferably between 50 and 250 mOsm / L.

7. - 5 to 200 g / l, preferably 10 to 200 g / l, more preferably 10 to 150 g / l of chitosan A and 10 to 200 g / l, preferably 20 to 100 g / l of statistical polysaccharide B, and - 1 g / l to 200 g / l, preferably 50 to 100 g / l of protein or a combination of proteins 2. The pharmaceutical formulation of claim 1, comprising:

8. 2. The pharmaceutical formulation according to claim 1, wherein said protein has a molecular weight comprised between 10 kDa and 250 kDa, preferably between 20 kDa and 250 kDa, preferably between 100 kDa and 250 kDa, preferably between 120 kDa and 250 kDa, preferably between 150 kDa and 250 kDa.

9. 2. The pharmaceutical formulation of claim 1, wherein the protein or combination of proteins is selected from the group consisting of an antibody, an enzyme, a fusion protein, and combinations thereof.

10. 10. The pharmaceutical formulation of claim 1, wherein the protein or combination of proteins comprises an antibody or an antigen-binding fragment of an antibody.

11. 11. The pharmaceutical formulation of claim 10, wherein the antibody is selected from the group consisting of trastuzumab, rituximab and pertuzumab, or a combination thereof.

12. 10. The pharmaceutical formulation of claim 1, further comprising at least one pharmaceutically acceptable excipient selected from a solvent, a stabilizer, a surfactant, a buffer, an antimicrobial preservative, a protectant, an antioxidant, a chelating agent, and a bulking agent.

13. The formulation - 1 g / l to 200 g / l, preferably 50 to 100 g / l, of an antibody or combination of antibodies, in particular trastuzumab, rituximab, pertuzumab or daratumumab, or a combination of trastuzumab and pertuzumab, - 5 to 200 g / l, preferably 10 to 200 g / l, more preferably 10 to 150 g / l of chitosan A, and 20 to 400 g / l, preferably 20 to 300 g / l, of statistical polysaccharide B, and below: 1-100 mM of a buffering agent, preferably L-histidine hydrochloride monohydrate, to give a pH of 5.5-6.5; - 1 to 500 mM of a stabilizer, preferably α,α-trehalose dihydrate, 5-25 mM of an antioxidant, preferably methionine At least one excipient selected from 2. The pharmaceutical formulation of claim 1, comprising:

14. A kit comprising: - at least one first container containing chitosan A and statistical polysaccharide B as defined in claim 1; - at least one second container containing a protein or a combination of proteins as defined in claim 8 A kit comprising:

15. 15. The pharmaceutical formulation of claim 1 or the kit of claim 14 for use in the treatment of cancer, typically breast cancer or gastric cancer.

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