WO2020002650A1 - Formulation - Google Patents

Formulation Download PDF

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Publication number
WO2020002650A1
WO2020002650A1 PCT/EP2019/067428 EP2019067428W WO2020002650A1 WO 2020002650 A1 WO2020002650 A1 WO 2020002650A1 EP 2019067428 W EP2019067428 W EP 2019067428W WO 2020002650 A1 WO2020002650 A1 WO 2020002650A1
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WIPO (PCT)
Prior art keywords
formulation
csf
variants
formulations
buffer
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PCT/EP2019/067428
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English (en)
Inventor
Jon Amund Eriksen
Einar JONSBU
Berit IVERSEN
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Targovax Asa
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Publication of WO2020002650A1 publication Critical patent/WO2020002650A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions

Definitions

  • the present invention provides formulations comprising a GM-CSF protein and the use of the formulations as a medicament, particularly in the treatment of cancer.
  • Granulocyte macrophage colony-stimulating factor is monomeric glycoprotein secreted by macrophages, T-cell, mast cell, natural killer T-cells, endothelial cells and fibroblasts. It functions as a cytokine, and is an immune system stimulator and hematopoietic growth factor.
  • GM-CSF is produced as a response to immune or inflammatory stimuli and plays an important role in regulating the proliferation, differentiation, survival and activation of hematopoietic cells such as granulocytes and monocytes, neutrophils, basophils and eosinophils, erythroid cells, megakaryocytes and T cells.
  • GM-CSF stimulates stem cells to produce granulocytes and monocytes, as well as having effects on mature cells of the immune system, such as inhibiting neutrophil migration.
  • GM-CSF has also been shown to be involved in the maturation, mobilisation and antigen presentation of myeloid dendritic cells (DCs), in vivo or ex vivo.
  • DCs myeloid dendritic cells
  • Arellano and Lonial (Biologies: Targets and Therapy, 2008:2(1 ), 13-27,“Clinical uses of GM-CSF: a critical appraisal and update”) and van de Laar et al.
  • GM-CSF GM-CSF
  • APCs functionally active antigen presenting cells
  • Naturally-occurring, mature, wild-type, human GM-CSF is a 127 amino acid residue-protein, consisting of amino acid residues 18-144 of the sequence shown in UniProtKB data base entry P04141 (http://www.uniprot.org/uniprot/P04141 ) and SEQ ID NO: 1.
  • the amino acid sequence of mature, wild-type human GM-CSF is shown in SEQ ID NO: 2.
  • Amino acid residues 1-17 of the sequence shown in UniProtKB data base entry P04141 and SEQ ID NO: 1 are a signal sequence, and do not form part of the mature GM-CSF product.
  • Mature wild-type human GM- CSF is glycosylated at positions 22, 24, 26, 27, 44 and 54 of the amino acid sequence of SEQ ID NO: 1 , which corresponds to amino acid residues 5, 7, 9, 10, 27 and 37 of the amino acid sequence of SEQ ID NO: 2.
  • Pharmaceutical formulations of GM-CSF have been developed for raising white blood cell counts and boosting the immune system after chemotherapy, for mobilisation of hematopoietic progenitor cells and myeloid reconstitution after transplantation of autologous peripheral blood progenitor cells, and for myeloid reconstitution after autologous or allogenic bone marrow transplantation.
  • Pharmaceutical formulations of GM-CSF are also used for improving the immune response to peptide vaccines.
  • GM-CSF attracts dendritic cells.
  • a further use of pharmaceutical formulations of GM-CSF is the acceleration of myeloid recovery following bone marrow transplantation.
  • Molgramostim is GM-CSF produced by E. coil.
  • the resulting GM-CSF product has 128 amino acid residues, has a molecular weight of approximately 14,600, is non-glycosylated and has an additional N-terminal methionine which is only partially cleaved (i.e. the N-terminal methionine is cleaved from some but not all molecules).
  • the amino acid sequence of Molgramostim is shown in SEQ ID NO: 3.
  • Molgramostim contains, before lyophilisation, 0.15mg/ml (targeted value of 1.64x10 6 lll/ml) Molgramostim, 50mg/ml mannitol, 19.4mg/ml Magrocol 4000, 1 mg/ml human serum albumin (HSA), 0.99mg/ml disodium phosphate and 0.42mg/ml potassium dihydrogen phosphate.
  • HSA human serum albumin
  • Sargramostim is GM-CSF produced by yeast, and has the amino acid sequence shown in SEQ ID NO: 4.
  • Sargramostim has an amino acid substitution at position 23 of the amino acid sequence of mature wild-type human GM-CSF (SEQ ID NO: 2), replacing arginine with leucine. Due to being produced by yeast, Sargramostim is hyper-glycosylated as compared to mature wild-type human GM-CSF, by about 100-150 times.
  • Sargramostim consists of three molecular species having relative molecular weights of 19,500, 16,800 and 15,500, due to the different levels of glycosylation, and these forms are present in an amount of 25-42%, 14- 32% and 35-53%, in order of elution (U.S.
  • Sargramostim is marketed in the USA as Leukine® (approved by the FDA in March 1995), which contains 40mg/ml mannitol, 10mg/ml sucrose and 1.2mg/ml tromethanine, and is available in either liquid or lyophilised form.
  • the liquid form of Leukine® contains 500pg (2.8x10 6 IU) Sargramostim and 1.1% benzyl alcohol in a 1 ml solution, and has a pH of 6.7-7.7.
  • the lyophilised form contains 250pg (1.4x10 6 IU) Sargramostim per vial, and has a pH of 7.1- 7.7.
  • GM-CSF has several pharmaceutical uses, there is currently no approved formulation of GM-CSF available on the European market.
  • Leukine® has been used off-label, in Europe, for clinical studies, as an immune-stimulator for cancer vaccines.
  • HSA human serum albumin
  • rHA recombinant human albumin
  • SEQ ID NO: 5 The amino acid sequence of immature HSA is shown in SEQ ID NO: 5, wherein amino acid residues 1-18 are a signal sequence, amino acid residues 19-24 are a pro-peptide, and amino acid residues 25-609 forms the mature protein. It has been used in therapeutic applications to replace lost fluid (such as after blood loss), as well as a pharmaceutical excipient.
  • HSA human albumin
  • rHA recombinant human albumin
  • yeast, bacteria or plant-based expression systems rHA has the same amino acid sequence as HSA (shown in SEQ ID NO: 5), but may have a different glycosylation pattern from HSA.
  • yeast and plants are animal-free, rHA do not present risks commonly associated with animal-derived products (www.albuminbio.com/safety).
  • the quality of rHA differs depending on the source, due to the difference in formation of dimers and multimers.
  • both HSA and rHA are no longer desirable in pharmaceutical formulations. It has been found that both HSA and rHA form as monomers, dimers and multimers, which are undesirable in pharmaceutical compositions, and mask impurities in the formulations, such that the purity measurements of existing formulations may not be accurate. HSA and rHA may even cause some of these impurities. As a result of HSA and rHA masking impurities in the formulation, it is difficult to control the drug product, in terms of a safe biological dose, control of degradation products and control of purity.
  • HSA is also undesirable from a regulatory point of view.
  • HSA is difficult to produce and human donors result in contaminants being present in the samples.
  • the use of human donors also means that HSA must be certified BSA and TSA-free, which requires a lot of testing.
  • human donors causes variance in potency between batches of HSA.
  • regulatory agencies are restricting the use of HSA because of the concerns about potential infectious agents in animal-derived products (Ohtake et al.,“Interactions of formulation excipients with proteins in solution and in the dried state”, Adv. Drug Deliv. Rev. (201 1 ), doi: 10.1016/j.addr.2011.06.011 ).
  • the glycosylation of GM-CSF means that the molecular weight of the drug is 20% greater than the molecular weight of Molgramostim, such that, as mentioned above, Sargramostim has only half the potency of Molgramostim, mol/mol. This means that twice the amount of Sargramostim is required for the same therapeutic effect. Thus, treatment with Sargramostim is more expensive than with Molgramostim (because a greater amount of Sargramostim is required for the same effect).
  • treatment with Sargramostim is less convenient than with Molgramostim, because a longer interval is required between administration of GM-CSF and a subsequent pharmaceutical, such as a vaccine, in order to allow the blister created by Sargramostim to reduce in size, and because injection of the greater volume of Sargramostim is more painful than injection of Molgramostim. It is also not known whether a solution of Sargramostim, having double the volume but only half the potency of a corresponding solution of Molgramostim, has the same effect in vivo as a corresponding solution of Molgramostim. Furthermore, yeast are more difficult to use, to express proteins such as GM-CSF, than E. coli, and the use of yeast produces a risk of severe allergic reactions and anaphylaxis in patients having a hypersensitivity to yeast-derived products.
  • Treatment using pharmaceutical formulations of GM-CSF with peptide vaccines requires intradermal injection of the formulation. This creates a blister or bleb, and the peptide vaccine is administered to the same site after a period of about 20 minutes. This waiting time allows the recruitment of dendritic cells to the injection site, in order to ensure an immune response, and allows the blister to reduce in size somewhat before the peptide vaccine is injected. If the blister is not allowed to recede, then the subsequent injection of the vaccine into the same site can be very painful for the subject, and, in addition, may result in leakage from the blister. It is considered amongst some in the art that the reduction of the blister to avoid pain is a more important reason for the interval between injections of the GM-CSF formulation and the peptide vaccine than the recruitment of dendritic cells.
  • G-CSF granulocyte-colony stimulating factor
  • Leukine® is still on the market in the US, it is a marginal product compared to G-CSF products such as Amgen’s Neulasta® (pegylated G-CSF; pegfilgrastim).
  • G-CSF stimulates bone marrow to produce granulocytes and stem cells, and release them into the blood stream.
  • G-CSF also stimulates the survival, proliferation, differentiation and function of neutrophil precursors and mature neutrophils.
  • G-CSF would be suitable for promoting the presentation of peptide vaccines by dendritic cells.
  • G-CSF and GM-CSF target, and have significantly different effects on the mobilisation and differentiation of, different subsets of dendritic cells (DC).
  • DC1 type-1 DCs
  • GM-CSF This increase in DC1 content and activity, following local administration of GM-CSF, supports a role for GM-CSF as an immune stimulant and vaccine adjuvant in cancer patients (Arellano & Lonial; Biologies: Targets and Therapy, 2008:2(1 ), 13-27,“Clinical uses of GM-CSF: a critical appraisal and update”).
  • G-CSF preferentially enhances the differentiation of type-2 DCs (DC2), which do not induce an immune response.
  • the present invention provides solutions to the problems discussed above because it has now, surprisingly, been found that formulations of a GM-CSF protein, wherein the formulations contain no or substantially no HSA or rHA, have at least the same potency as previous formulations. It has been found that the formulations of the present invention are more controllable, of better quality and are safer than previous formulations, because the purity of the formulations of the invention can be tested more accurately.
  • the formulations of the invention have good potency and stability, and have improved purity.
  • a formulation comprising a GM-CSF protein having at least 60% sequence identity to the polypeptide sequence of SEQ ID NO: 2 and a stabiliser comprising a polymer or a surfactant.
  • the formulation substantially does not comprise and human serum albumin or recombinant human albumin, for example, no more than 0.2wt% of human serum albumin or recombinant human albumin.
  • the GM-CSF protein has at least 70%, 80%, 90%, 95% or 99% sequence identity to the polypeptide sequence of SEQ ID NO: 2.
  • the stabiliser further comprises a disaccharide or arginine.
  • the disaccharide or arginine is present in an amount of 10.0-99.0wt%, preferably 30.0-99.0wt%, most preferably 95.0-98.0wt%, excluding water.
  • the disaccharide is sucrose and/or mannitol.
  • the polymer or the surfactant is present in an amount of 0.5-3.0wt%, preferably 1.0-2.5wt%, excluding water.
  • the polymer is a poloxamer, more preferably P188.
  • the surfactant is polysorbate 20 or polysorbate 80.
  • the formulation further comprises a buffer.
  • the buffer is present in an amount of 0.5-5.0wt%, preferably 1.0-2.5wt%, excluding water.
  • the buffer is potassium phosphate.
  • the formulation does not comprise more than 0.02wt% of human serum albumin or recombinant human albumin.
  • the GM-CSF protein is present in an amount of 0.001 -10.0wt%, preferably 0.01-10.0wt%, most preferably 0.1-10.0wt%, excluding water.
  • the GM-CSF protein has at least 60%, 70%, 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 3.
  • no more than 25% of the amino acid residues of the GM-CSF protein are glycosylated.
  • no more than 5% of the amino acid residues of the GM-CSF protein are glycosylated.
  • the formulation is liquid.
  • the formulation has a pH in the range of 6.5 to 9.0.
  • the formulation has a pH in the range of 7.3 to 8.1 , preferably wherein the formulation has a pH of 7.3 or 8.1 , and more preferably a pH of 8.1.
  • the formulation is a frozen liquid.
  • the formulation comprises water in an amount of 10.0-99.0wt%, preferably 50.0- 99.0wt%, and most preferably 90.0-99.9wt%.
  • the formulation is freeze dried and contains less than 5% residual water.
  • the formulation has a potency in the range of 0.26x10 6 to 8.33x10 6 IU, preferably in the range of 0.26x10 6 to 0.42x10 6 IU.
  • the formulation has a purity of at least 95% when measured after 12 weeks of storage, preferably at least 97% when measures after 12 weeks of storage.
  • the formulation is for use as a medicament.
  • the formulation is for use simultaneously or sequentially with a peptide or protein vaccine.
  • the percentage “identity” between two sequences may be determined using the BLASTP algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997),“Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, nucleic Acids Res. 25:3389-3402) using default parameters.
  • the BLAST algorithm can be accessed on the internet using the URL http://www.ncbi.nlm.nih.gov/blast/.
  • GM-CSF protein is, in some embodiments, a protein having at least 60% sequence identity to the mature monomeric glycoprotein having the amino acid sequence of SEQ ID NO: 2.
  • Naturally-produced, wild-type GM-CSF is secreted by macrophages, T-cell, mast cell, natural killer T-cells, endothelial cells and fibroblasts, and functions as a cytokine and a hematopoietic growth factor, to stimulate the immune system.
  • the amino acid sequence of naturally-produced, wild-type, human GM-CSF consists of amino acid residues 18-144 of UniProtKB database entry P04141 (i.e. SEQ ID NO: 1 ), and is shown in SEQ ID NO: 2.
  • a GM-CSF protein corresponds to the mature monomeric glycoprotein having the amino acid sequence of SEQ ID NO: 2.
  • a GM-CSF protein must have at least 1% of the cytokine activity of mature, wild-type GM-CSF (i.e. SEQ ID NO: 2).
  • Cytokine activity means that a protein is capable of affecting the differentiation and behaviour of other, nearby, cells.
  • cytokine activity means that GM-CSF is capable of attracting and activating the maturation process of local dendritic cells (DCs) into mature antigen-presenting cells (APCs).
  • GM-CSF functions as a cytokine by binding to the GM-CSF receptor in the DCs (Arellano and Lonial, Biologies: Targets and Therapy, 2008:2(1 ), 13-27,“Clinical uses of GM-CSF: a critical appraisal and update”; and van de Laar et ai, Blood, 12 April 2012:1 19(15),“Regulation of dendritic cell development by GM-CSF; molecular control and implications for immune homeostasis and therapy”).
  • a suitable assay for determining the cytokine activity of a protein is an in vitro bioassay as described in “European Pharmacopeia 7.0, 01/2008:1641 ; Monograph of Molgramostim Concentrated Solution”.
  • this assay involves stimulating TF-1 cells with GM-CSF, and subsequently measuring the conversion of stains such as tetrazolium bromide (MTT). MTT is converted to purple formazan, which is measured spectrophotometrically.
  • a GM-CSF protein In order to be considered to have cytokine activity, a GM-CSF protein must have a minimum activity 0.7x10 5 lU/mg, as measured by the above-mentioned bioassay in “European Pharmacopeia 7.0, 01/2008:1641 ; Monograph of Molgramostim Concentrated Solution”.
  • A“stabiliser”, as used herein, is a molecule used to stabilise proteins by maintaining protein structure, integrity and biological activity, to suppress protein aggregates, to prevent degradation, to reduce surface adsorption or to provide physiological osmolality.
  • Stabilisers are well-known in the art and include sugars, salt, polymers, surfactants and amino acids.
  • A“surfactant”, as used herein, is a compound which lowers the surface tension between two liquids, or a liquid and a solid.
  • Surfactants are well known in the art, and include, for example, polysorbate 20 (P20), polysorbate 80 (P80), N-octyl glucoside, Pluronic, Brij 35, Cru 30, Triton X-10, and sodium dodecyl sulfate (SDS), amongst others.
  • A“poloxamer”, as used herein, refers to a non-ionic co-polymer of polyoxypropylene and polyoxyethylene.
  • these co-polymers are named with a“P” (for poloxamer) followed by a three-digit number, wherein the first two digits multiplied by 100 give the approximate molecular mass of the polyoxypropylene core, and the last digit multiplied by 10 gives the percentage polyoxyethylene content.
  • P188 indicates that the poloxamer has an approximate molecular mass of 1800g/mol, and a 80% polyoxyethylene content.
  • Human serum albumin is the most abundant protein found in human plasma. It is a soluble and monomeric protein, and is produced by the liver. It is produced by fractionating human plasma, and is used as a stabiliser for drugs and vaccines, as well as in imaging and cell culture, and therapeutic uses. Immature wild-type HSA has the amino acid sequence shown in SEQ ID NO: 5.
  • Recombinant human albumin is human serum albumin which has been produced by genetic recombination, typically using Saccharomyces cerevisiae. Recombinant human albumin has the same amino acid sequence as human serum albumin (SEQ ID NO: 5).
  • “Potency”, as used herein, is a measure of biological efficacy and the amount of drug required to produce an effect of a given intensity, and is measured in International Units (IU).
  • IU International Units
  • the potency of GM-CSF is tested by incubating TF-1 cells with varying dilutions of test and reference preparations of GM-CSF, and then incubating the TF-1 cells with a solution of MTT. MTT is converted to purple formazan by the TF-1 cells, which is measured spectrophotometrically, and compared to dilutions of the appropriate International Standard of GM-CSF as defined by the WHO.
  • Figure 1 is a schematic illustration of a binary CG-MALS experiment, showing the concentrations in each gradient step of sample 1 (AUX-I) and sample 2 (AUX-II).
  • Figure 2 is an exemplary thermogram of the nanoDSC measurement of variant #1 of the pre- screening with different buffering agents.
  • Figure 3 is a diagram showing the main effect for A2 from the first DoE matrix.
  • Figure 4 is a diagram showing the main effect for T on set from the first DoE matrix.
  • Figure 5 is a photograph of sub-variant #2 (PS20, N 2 ) after light stress.
  • multimer RT - 3.7 min. Measurement at 214 nm.
  • Figure 8 is a graph showing the minimum purity of test variants, by showing the minimum relative GM-CSF areas cumulative, throughout all stress conditions determined by SE-HPLC. Peaks of rHA and human serum albumin (“control”), in rHA / HSA containing samples, were not integrated.
  • Figure 9 is an overlay of IEX-HPLC chromatograms of injections of 15 pL GM-CSF working standard (1 mg/mL) throughout the entire forced degradation study. Chromatograms are in chronological order starting at the black chromatogram at the bottom (beginning of FD study) and going up to the dark green one at the top (end of FD study).
  • Figure 10 is a representative IEX-HPLC chromatogram of 15 pL of a human serum albumin containing sample (variant "control"). Measurement at 214 nm.
  • Figure 1 1 is two representative chromatograms of test variant #2 by RP-HPLC.
  • A test variant #2 with PS20 and air after light stress;
  • B test variant #2 with rHA and air after light stress. Measurement at 214 nm.
  • Figure 12 is a graph showing the minimum purity, by showing the relative GM-CSF areas, throughout all stress conditions determined by RP-HPLC. Monomeric peaks of rHA and human serum albumin (HSA) (“control”) were not integrated.
  • Figure 13 is an overview of the measurement principle of the DynaPro Plate reader II (dynamic light scattering).
  • Figure 14 is a schematic illustration of the position of a thermocouple inside a vial.
  • the dot close to the base of the vial displays the point where the temperature was measured.
  • the position of the sensor (dot) between vial bottom and fill surface might vary from vial to vial.
  • Figure 15 is exemplary photographs of representative vials after the GM-CSF drug product lyophilization feasibility test run. Vials are shown from side view (upper row) and bottom view (lower row) in comparison to current lyophilized drug product #C (“control”).
  • Figure 16 is an SE-HPLC chromatogram recorded using 100mI_ of 0.3mg/ml_ GM-CSF Control Sample 1 diluted with running buffer (A), and an enlargement of the chromatogram (B). Measurement at 214nm. GM-CSF main peak at 33 minutes retention time, fragments detectable at 38 minutes retention time.
  • Figure 17 ia a SE-HPLC chromatogram recorded using 100pL of 0.3mg/mL final formulation variant #1 (representative for all final formulation variants #1 , #2, #1 d, #2d), measured at 214nm (A), and an enlargement of the chromatogram (B).
  • Figure 18 is a SE-HPLC chromatogram recorded using 100pL of 0.3mg/mL of lyophilized existing commercial formulation (“control” (#C)), measured at 214nm (A), and an enlargement of the chromatogram (B).
  • control #C
  • A lyophilized existing commercial formulation
  • B an enlargement of the chromatogram
  • Figure 19 is a RP-HPLC chromatogram recorded using 100 pL of 0.3 mg/mL GM-CSF Control Sample 2 diluted with dilution buffer (A), and an enlargement of the chromatogram (B). Measurement at 214 nm. GM-CSF main peak at 25.2 minutes retention time.
  • Figure 20 is a RP-HPLC chromatogram recorded using 100 pL of 0.3 mg/mL GM-CSF drug product variant #1 (representative for all variants #1 , #2, #1 d, #2d), measured at 214 nm (A), and an enlargement of the chromatogram (B). GM-CSF main peak at 25.2 minutes retention time.
  • Figure 21 is a RP-HPLC chromatogram recorded using 100 pL of 0.3 mg/mL lyophilized existing commercial formulation (“control” (#C)), measured at 214 nm (A), and an enlargement of the cromatogram (B). GM-CSF main peak at 25.3 minutes retention time, HSA peak at 16.1 minutes.
  • Figure 22 is a chromatogram of GM-CSF Control Sample 3 generated positive control showing the recorded UV-signal (continuous line) as well as the weight average molecular weight by MALS (dots).
  • Figure 23 is an overlay of SE-HPLC chromatograms of the three different GM-CSF Control Samples (A), and an enlargement of the overlay (B). Measurement at 214 nm.
  • Figure 24 is an overlay of SE-HPLC chromatograms of GM-CSF Control Sample 3 and an existing commercial formulation of Molgramostim (A), and an enlargement of the overlay (B). Both measurements used the drug substance (i.e. not formulated). Measurement at 214 nm.
  • Figure 25 is an overlay of SE-HPLC chromatograms of GM-CSF Control Sample 3 and lyophilizates of drug products produced in Example 5 (A), and an enlargement of the overlay (B). Measurement at 214 nm.
  • Figure 26 is an overlay of SE-HPLC chromatograms of final formulation variant #1 drug product produced in Example 5 to its corresponding placebo solution without GM-CSF drug substance (A), and an enlargement of the overlay (B). Measurement at 214nm.
  • Figure 27 is an overlay of SE-HPLC chromatograms of final formulation variant #2 produced in Example 5 to its corresponding placebo solution without GM-CSF drug substance (A), and an enlargement of the overlay (B). Measurement at 214nm.
  • Figure 28 is an overlay of SE-HPLC chromatograms of GM-CSF Control Sample 3 and a positive control stressed at 55 °C for 60 hours (A), and an enlargement of the overlay (B). Measurement at 214nm.
  • Figure 29 is an overlay of RP-HPLC chromatograms of the three different GM-CSF Control Samples (A), and an enlargement of the overlay (B). Measurement at 214 nm.
  • Figure 30 is an overlay of RP-HPLC chromatograms of GM-CSF drug substances from GM- CSF Control Sample 3 and from an existing commercial formulation (A), and an enlargement of the overlay (B). Measurement at 214nm.
  • Figure 31 is an overlay of RP-HPLC chromatograms of GM-CSF Control Sample 3 and drug product lyophilizates produced in Example 5 (A), and an enlargement of the overlay (B).
  • Figure 32 is an overlay of RP-HPLC chromatograms of lyophilized final formulation variant #1 produced in Example 5 to its corresponding placebo solution without drug substance GM-CSF (A), and an enlargement of the overlay (B). Measurement at 214 nm.
  • Figure 33 is an overlay of RP-HPLC chromatograms of lyophilized final formulation variant #2 produced in Example 5 to its corresponding placebo solution without drug substance GM-CSF (A), and an enlargement of the overlay (B). Measurement at 214 nm.
  • Figure 34 is an overlay of RP-HPLC chromatograms of GM-CSF Control Sample 3 and a positive control stressed at 60 °C for 18 hours (A), and an enlargement of the overlay (B).
  • Figure 35 is an overlay of SE-HPLC chromatograms of lyophilized final formulation variant #1 (TGWP7b/1-170814), lyophilized final formulation variant #2 (TGWP7b/2-170814), and lyophilized existing commercial formulation (A), and an enlargement of the overlay (B). Measurement at 214nm.
  • Figure 36 is an overlay of RP-HPLC chromatograms of lyophilized final formulation variant #1 (TGWP7b/1-170814), lyophilized final formulation variant #2 (TGWP7b/2-170814), and lyophilized existing commercial formulation (A), and an enlargement of the overlay (B). Measurement at 214nm.
  • Figure 41 is a graph showing the relative main peak areas of samples stored at 25 °C detected by SE-HPLC at 214nm.
  • Figure 42 is a graph showing the relative main peak areas of samples stored at 40°C detected by SE-HPLC at 214nm.
  • Figure 44 is a graph showing the relative main peak areas of samples stored at 25°C detected by RP-HPLC at 214nm.
  • Figure 45 is a graph showning the relative main peak areas of samples stored at 40°C detected by RP-HPLC at 214nm.
  • Figure 46 is a microscope photograph of inflammation at the injection site of the existing commercial formulation in animal no. 2. Overview, HE, lens x4.
  • Figure 47 is a microscope photograph of inflammation at the injection site of the existing commercial formulation in animal no. 2. Overview, HE, lens x40.
  • Figure 48 is a microscope photograph of inflammation at untreated control site 6 in animal no. 2. Overview, HE, lens x4.
  • Figure 49 is a microscope photograph of inflammation at untreated control site 6 in animal no. 2. Overview, HE, lens x40.
  • Figure 50 is a microscope photograph of inflammation at the final formulation variant #1 injection site in animal no. 6. Overview, HE, lens x4.
  • Figure 51 is a microscope photograph of inflammation at the final formulation variant #1 injection site in animal no. 6. Overview, HE, lens x40.
  • Figure 52 is a microscope photograph of inflammation at the untreated control site 6 in animal no. 6. Overview, HE, lens x4.
  • Figure 53 is a microscope photograph of inflammation at the untreated control site 6 in animal no. 6. Overview, HE, lens x40.
  • Figure 54 is a photograph of representative vials after the final formulation lyophilization feasibility test run, using sucrose and mannitol. Vials are shown from side view (upper row) and bottom view (lower row).
  • Figure 55 is an SE-HPLC chromatogram recorded using 100mI_ of 0.3mg/ml_ GM-CSF DS (Control Sample 3) diluted with running buffer (A), and an enlargement of the chromatogram (B). Measurement at 214nm. GM-CSF main peak at 33 minutes retention time.
  • Figure 56 is a SE-HPLC chromatogram overlay recorded using 100pL of 0.3mg/mL of each of final formulation variants #1-m, #2-m, #1 d-m, #2d-m (A), and an enlargement of the overlay (B). Measurement at 214nm. GM-CSF main peak at 33 minutes retention time, fragments detectable at 38 minutes retention time, aggregates detectable at 28 minutes.
  • Figure 57 is a RP-HPLC chromatogram recorded using 100pL of 0.3mg/mL GM-CSF DS (Control Sample 3) diluted with dilution buffer (A), and an enlargement (B). Measurement at 214 nm. GM-CSF main peak at 25.5 minutes retention time.
  • Figure 58 is a RP-HPLC chromatogram overlay recorded using 100pL of 0.3mg/mL of each of final formulation variants #1-m, #2-m, #1 d-m, #2d-m (A), and an enlargement (B). Measurement at 214nm. GM-CSF main peak at 26 minutes retention time.
  • Figure 59 is a graph showing the relative main peak area by RP-HPLC, detected at 214nm, of the Norm-Ject tuberculin syringe at 2-8°C.
  • Figure 60 is a graph showing the relative main peak area by RP-HPLC, detected at 214nm, of the Norm-Ject tuberculin syringe at 30°C.
  • Figure 61 is a graph showing the relative main peak area by RP-HPLC, detected at 214nm, of the Micro-Fine insulin syringe at 2-8°C.
  • Figure 62 is a graph showing the relative main peak area by RP-HPLC, detected at 214nm, of the Micro-Fine insulin syringe at 30°C.
  • SEQ ID NO: 1 shows the amino acid sequence of immature wild-type human GM-CSF.
  • SEQ ID NO: 2 shows the amino acid sequence of mature wild-type human GM-CSF.
  • SEQ ID NO: 3 shows the amino acid sequence of Molgramostim.
  • SEQ ID NO: 4 shows the amino acid sequence of Sargramostim.
  • SEQ ID NO: 5 shows the amino acid sequence of immature Human Serum Albumin (HSA).
  • the present invention is based on a formulation of a GM-CSF protein which does not, or substantially does not, contain human serum albumin (HSA) or recombinant human albumin (rHA), but which has the same, or improved, properties as existing formulations of GM-CSF.
  • HSA human serum albumin
  • rHA recombinant human albumin
  • the invention relates, in general terms, to a formulation of a GM-CSF protein and a surfactant, which contains no, or substantially no, HSA or rHA.
  • the formulation of various embodiments of the invention has at least the same potency as previously known formulations of GM-CSF proteins, a higher purity than previously known formulations of GM-CSF proteins, and has good stability.
  • the purity of the formulation of the invention can be measured more accurately, such that the purity and degradation products can be controlled more easily. This provides a much safer and higher quality formulation than previously available. Moreover, the absence of HSA and rHA overcomes problems with obtaining regulatory approval for these excipients. In some embodiments, the formulation of the present invention has an improved potency compared to previous formulations.
  • the units“mg/ml”,“w/v”,“mM” and “III” are calculated when the formulation of the invention is a liquid or frozen liquid. Unless otherwise stated, the units“wt%” are calculated excluding water or WFI, except for residual water after lyophilisation.
  • the GM-CSF protein used in preferred embodiments of the formulations of the present invention corresponds to mature, wild-type GM-CSF.
  • the GM-CSF protein has at least 60% sequence identity to the amino acid sequence set out in SEQ ID NO: 2.
  • the GM-CSF protein has at least 60%, 70%, 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 2.
  • the GM-CSF protein has an amino acid substitution at position 23 of SEQ ID NO: 2, to replace arginine with leucine.
  • the GM-CSF protein has at least 60%, 70%, 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 4.
  • the GM-CSF protein has the amino acid sequence of mature wild-type GM-CSF, as described above, with an additional N-terminal methionine residue.
  • the GM-CSF protein has at least 60%, 70%, 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 3, preferably 100% sequence identity to SEQ ID NO: 3.
  • the N-terminal methionine may be partially cleaved, meaning that the N-terminal methionine is not cleaved from every GM-CSF molecule.
  • the GM-CSF protein used in the formulations of the present invention may be non-glycosylated, partially glycosylated, fully glycosylated or hyper-glycosylated, as compared to the mature, wild- type GM-CSF.
  • no more than 25%, no more than 15%, no more than 12.5%, no more than 10%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% of the amino acid residues of the GM-CSF protein are glycosylated.
  • no more than 25% of the amino acid residues of the GM-CSF protein are glycosylated.
  • no more than 12.5% of the amino acid residues of the GM-CSF protein are glycosylated. In yet further preferred embodiments, no more than 5% of the amino acid residues of the GM-CSF protein are glycosylated. In particularly preferred embodiments, none of the amino acid residues of the GM- CSF protein are glycosylated. In some embodiments, the percentage glycosylation discussed above is an average of the percentage glycosylation of the GM-CSF protein.
  • the GM-CSF protein is glycosylated but has a different glycosylation pattern from mature, wild-type GM-CSF.
  • the GM-CSF protein may have a relative molecular weight of approximately 19,500, 16,800 or 15,500, or the GM-CSF protein may be a mixture of one or more of these three glycosylated forms of GM-CSF (i.e. a mixture of GM-CSF proteins having the above three molecular weights).
  • the GM-CSF protein may be a mixture of 25-42% of molecular weight 19,500 GM-CSF, 14-32% of molecular weight 16,800 GM-CSF and 35-53% of molecular weight 15,500 GM-CSF.
  • the GM-CSF protein has at least 60%, 70%, 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 2, but is not glycosylated. In some embodiments, the GM-CSF protein has at least 60%, 70%, 80%, 90%, 95%, 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, has an additional N-terminal methionine as compared to SEQ ID NO: 2, which may be partially cleaved, and is not glycosylated.
  • GM-CSF used in the formulations of the present invention has at least 60%, 70%, 80%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 3, preferably 100% sequence identity to SEQ ID NO: 3, and is not glycosylated.
  • the GM-CSF protein used in the formulations of the present invention may comprise no more than 127 or 128 amino acid residues. In preferred embodiments, the GM-CSF protein has 128 amino acid residues. In some embodiments, the GM-CSF protein may be part of a fusion protein, wherein the fusion protein comprises more that 127 or 128 amino acid residues.
  • the GM-CSF protein used in the formulations of the present invention has cytokine activity, and is capable of activating local Dendritic Cells.
  • the cytokine activity of GM- CSF can be measured by an in vitro bioassay, as described in“Monograph of Molgramostim Concentrated Solution” (European Pharmacopeia, 01/2008:1641 ).
  • the biological activity of GM-CSF is determined by its stimulation of proliferation of TF-1 cells.
  • the GM-CSF protein used in the formulations of embodiments of the present invention has at least 1% of the cytokine activity of mature, wild-type GM-CSF. In some embodiments, the GM-CSF protein has at least 10% of the cytokine activity of mature, wild-type GM-CSF.
  • the GM-CSF protein used in the formulations of the present invention may be produced by host cells including bacteria, yeast or Chinese Hamster Ovary (CHO) cells.
  • the GM-CSF protein is produced by E. coli.
  • the GM-CSF protein is produced by yeast, such as Saccharomyces cerevisiae.
  • the concentration of GM-CSF protein in the formulations of certain embodiments of the present invention may be adjusted to achieve a particular potency of the formulation for a given dose.
  • the concentration of the GM-CSF protein is such that a given dose of the formulation has a potency in the range of 0.26x10 6 to 8.33x10 6 IU.
  • the concentration of the GM-CSF protein is such that a given dose of the formulation has a potency in the range of 0.26x10 6 to 0.42x10 6 IU.
  • the concentration of the GM- CSF protein is such that a given dose of the formulation has a potency of 0.33x10 6 IU.
  • a 100mI dose of the formulation has a potency in the range of 0.26x10 6 to 0.42x10 6 IU, preferably a potency of 0.33x10 6 IU.
  • the concentration of GM-CSF is such that a given dose has a maximum potency of 3.3x10 6 IU.
  • the GM-CSF protein may be present in the formulations at a concentration in the range of 0.001-10mg/ml, 0.01-10mg/ml, 0.05-10mg/ml, 0.1-10mg/ml, 0.05-5mg/ml, 0.05-2.5mg/ml, 0.05- 1.5mg/ml, 0.1-1 mg/ml, 0.1-0.5mg/ml or 0.1-0.4mg/ml.
  • the GM-CSF protein is present at a concentration of between 0.15 and 0.3mg/ml.
  • the GM-CSF protein is present in the formulations at a concentration of 0.15mg/ml or 0.3mg/ml.
  • the formulation of the present invention comprises 0.3mg/ml of the GM-CSF protein.
  • a 100mI dose of the formulation has a potency of 0.33x10 6 IU.
  • the formulations of embodiments of the present invention include no, or substantially no, HSA or rHA.
  • the formulations contain less than or equal to 0.2mg/ml, 0.15mg/ml, 0.1 mg/ml, 0.05mg/ml, 0.02mg/ml or 0.01 mg/ml HSA or rHA.
  • the formulations of the invention contain less than or equal to 0.02mg/ml HSA or rHA.
  • the formulations contain no HSA or rHA (i.e. O.Omg/ml HSA or rHA).
  • the formulations of embodiments of the present invention have the advantage that the purity thereof can be accurately measured because of the lack, or substantial lack, of HSA or rHA.
  • the formulations of embodiments of the present invention are a higher quality product than previously-known formulations, because there is good control of the purity of the formulations.
  • the absence of HSA or rHA from the formulations of embodiments of the present invention means that the formulations avoid any regulatory difficulties that arise with the use of HSA and rHA.
  • the formulations of the invention include a stabiliser which comprises a polymer or a surfactant.
  • the polymer or surfactant may be selected from polymers and surfactants already known in the art, such as polyethylene glycol (PEG), gelatin, hydroxyethyl starch, dextran, dextran sulfate, polyethyleneimine (PEI), hydroxypropyl b-cyclodextrin, polyvinylpyrrolidone (PVP), hydroxyethylcellulose (HEC), poloxamers, Pluronic, N-octyl glucoside.
  • the polymer may act as a surfactant.
  • the polymer is a poloxamer, and, in some embodiments, the polymer is Poloxamer 188 (P188).
  • the surfactant is Polysorbate 20 (P20) or Polysorbate 80 (P80).
  • the stabiliser comprises P188, as Example 2 and Figure 8 show that formulations containing P188 showed the best results in the forced degradation studies. In particular, Figure 8 shows that the monomer content of formulations with P188 only drops below 99% for one variant, namely formulation #2 without air (sub-variant #2 (P188) N2). This high level of purity was also detected using RP-HPLC, as shown in Example 2 and Figure 12, with P188 providing the greatest stability.
  • the stabiliser comprising the polymer or the surfactant is present in an amount in the range of 0.005-10%w/v, 0.005-5%w/v, 0.01-10%w/v, 0.02-5%w/v, or 0.05-2%w/v, 0.05-1.5%w/v or 0.05-1 %w/v. In some embodiments, the stabiliser comprising the polymer or the surfactant is present in an amount of 0.05%w/v. In other embodiments, the stabiliser comprising the polymer or the surfactant is present in an amount of 0.1 %w/v.
  • the polymer or the surfactant is present in an amount in the range of 0.01- 10%w/v, 0.02-5%w/v, or 0.05-2%w/v, 0.05-1.5%w/v or 0.05-1 %w/v. In preferred embodiments, the polymer or surfactant is present in an amount of 0.05%w/v or 0.1 %w/v. In particularly preferred embodiments, the polymer is P188 or the surfactant is P20 or P80, present in an amount of 0.05%w/v or 0.1%w/v.
  • the stabiliser comprising the polymer or the surfactant is present in an amount of 0.1-5mg/ml, 0.2-2.5mg/ml, 0.2-1.5mg/ml, or 0.5-1.0mg/ml. In some embodiments, the stabiliser comprising the polymer or the surfactant is present in an amount of 0.5mg/ml. In other embodiments, the stabiliser comprising the polymer or the surfactant is present in an amount of 1.0mg/ml. In some embodiments, the polymer or the surfactant is present in an amount of 0.1- 5mg/ml, 0.2-2.5mg/ml, 0.2-1.5mg/ml, or 0.5-1.0mg/ml.
  • the polymer or the surfactant is present in an amount of 0.5mg/ml or 1.0mg/ml.
  • the polymer is P188 or the surfactant is P20 or P80, present in an amount of 0.5mg/ml or 1.0mg/ml.
  • the stabiliser may comprise one or more further components, for example, further components known in the art to act as stabilisers.
  • the stabiliser may further comprise a disaccharide or an amino acid.
  • the disaccharide is sucrose, trehalose, lactose, inositol and/or mannitol, and the amino acid is arginine and/or glycine.
  • the disaccharide is mannitol.
  • the amino acid is arginine.
  • the disaccharide is sucrose.
  • the stabilizer further comprises sucrose and a second further component, which may be a disaccharide and/or an amino acid as defined above.
  • the further components are sucrose and mannitol, sucrose and glycine, sucrose and trehalose, or sucrose and arginine.
  • sucrose and mannitol sucrose and glycine
  • sucrose and trehalose sucrose and arginine.
  • the use of mannitol, glycine, trehalose or arginine in combination with sucrose may improve the lyo cake resulting from lyophilisation, by reducing the amount of shrinkage of the lyo cake.
  • the stabiliser further comprises arginine
  • the formulation had a high T onset and A 2 , as shown in Table 4, indicating a good ability to stabilise the GM-CSF protein.
  • Example 2 shows that formulations containing arginine have good results with regard to chemical degradation and aggregate status.
  • the disaccharide is a mixture of sucrose and mannitol.
  • the presence of mannitol reduces the amount of shrinkage of the lyo cake, following lyophilisation of the formulation, as discussed in Example 12.
  • An improved lyo cake has the advantage that the correct dosage is more reliably reconstituted because, for example, a more solid lyo cake has a reduced risk of fragmentation, wherein fragmentation can affect the dose in vials with a low fill volume.
  • a more solid lyo cake has a reduced risk of fragments or powder from the cake sticking to the vial stopper.
  • Example 14 shows that such formulations are suitable for use as a lyophilized drug product.
  • the one or more further components may be present at a concentration in the range of 5- 1000mM, 5-500mM, 50-500mM, 100-500mM, 100-400mM or 200-300mM.
  • the formulation is isotonic to the physiological conditions.
  • sucrose is present in the formulation at a concentration of 5-1000mM, 5- 500mM, 10-400mM, 50-400nM, 100-400mM, 100-300mM or 200-300mM. In preferred embodiments, sucrose is present in the formulation at a concentration of 260mM. In other embodiments, sucrose is present in the formulation at a concentration of 130mM. Mannitol may be present in the formulation at a concentration of 5-1000mM, 5-500mM, 50- 500mM, 100-500mM, 100-400mM, 100-300mM or 100-250mM. In some embodiments, mannitol is present in the formulation at a concentration of 220mM. In other embodiments, mannitol is present at a concentration of 1 10mM.
  • Arginine may be present in the formulation at a concentration of 5-1000mM, 5-500mM, 20- 400mM, 100-400mM, 100-300mM or 100-250mM. In some embodiments, arginine is present in the formulation at a concentration of 260mM. In other embodiments, arginine is present at a concentration of 130mM.
  • Glycine may be present in the formulation at a concentration of 5-1000mM, 5-500mM, 50- 500mM, 100-500mM, 100-400mM, 100-300mM or 100-250mM. In some embodiments, glycine is present in the formulation at a concentration of 220mM. In other embodiments, glycine is present at a concentration of 1 10mM.
  • the stabiliser contains more than one further component
  • the total concentration of the further components does not exceed 500mM in some embodiments, and the formulation is isotonic to the physiological conditions.
  • the ratio of sucrose to the second further component may be 1 :2.
  • sucrose is present in the formulation at a concentration of 5-500mM
  • the second further component is present in the formulation at a concentration of 5-500mM.
  • sucrose is present in the formulation at a concentration of 10-300mM, 10- 250mM, 10-200, 10-150 or 10-100mM
  • the second further component is present at a concentration of 50-300mM, 50-250mM or 100-250mM.
  • sucrose is present in the formulation at a concentration of 58mM and the second further component is present at a concentration of 220mM.
  • sucrose is present in the formulation at a concentration of 29mM and the second further component is present at a concentration of 1 10mM.
  • the formulations of embodiments of the present invention may comprise a buffer, such as potassium phosphate, disodium phosphate, potassium dihydrogen phosphate or Tris.
  • the buffer maintains the pH of the formulations of the invention in the range of 6.5 to 9.5, 7.0 to 8.5 or, more preferably, in the range of 7.3 to 8.1.
  • the buffer is potassium phosphate.
  • potassium phosphate also acts as a stabilizer, and Table 2 shows that potassium phosphate was found to be good at stabilising the GM-CSF protein, with weaker aggregation (i.e. a less negative A 2 ) than other buffers.
  • Table 2 also shows that formulations comprising potassium phosphate as the buffer had a high T on set, and delta H .
  • the GM-CSF drug substance i.e. unformulated
  • potassium phosphate as a buffer
  • the buffer may be Tris.
  • Table 2 shows that Tris provides weak aggregation (i.e. a positive A 2 ), and that formulations comprising Tri had a high T on set, and delta H.
  • the concentration of the buffer is at least 0.1 mM, at least 1 mM, at least 2mM, at least 3mM, at least 4mM, at least 5mM, at least 6mM, at least 7mM, at least 8mM, at least 9mM, at least 10mM, at least 1 1 mM, at least 12mM, at least 13mM, at least 14mM or is at least 15mM.
  • the concentration of the buffer is no more than 40mM, 30mM, 20mM or 10mM.
  • the buffer is present at a concentration between 1 mM and 40mM, between 5mM and 40mM or between 5mM and 15mM. More preferably, the concentration of the buffer is 5mM or 10mM. In a preferred embodiment, the buffer is potassium phosphate at a concentration of 5mM or 10mM.
  • the concentration of the buffer is at least 0.1 mg/ml, at least 0.2mg/ml, at least 0.3mg/ml, at least 0.4mg/ml, at least 0.5mg/ml, at least 0.6mg/ml, at least 0.7mg/ml, at least 0.80mg/ml, at least 0.85mg/ml, at least 0.9mg/ml, at least 0.95mg/ml, at least 0.96mg/ml, at least 0.97mg/ml, at least 0.98mg/ml, at least 0.99mg/ml or is at least 1.0mg/ml. In preferred embodiments, the concentration of buffer is 0.98mg/ml. pH
  • the formulation of the present invention may have a pH of at least 6.5, at least 6.6, at least 6.7, at least 6.8, at least 6.9, at least 7.0, at least 7.1 , at least 7.2, at least 7.3, at least 7.4, at least 7.5, at least 7.6, at least 7.7, at least 7.8, at least 7.9, at least 8.0, at least 8.1 , at least 8.2, at least 8.3, at least 8.4, at least 8.5, at least 8.6, at least 8.7, at least 8.8, at least 8.8 or at least 9.0, and a maximum pH of 9.5, 9.4, 9.3, 9.2, 9.1 , 9.0, 8.9, 8.8, 8.8, 8.6 or 8.5.
  • the formulation has a pH in the range of 6.5 to 9.5, 7.0 to 8.5, and more preferably in the range of 7.3 to 8.2.
  • the formulation has a pH of 7.3, 8.1 or 8.2, most preferably 7.3 or 8.1.
  • the pH is measured after reconstitution of the formulation in WFI.
  • Example 2 From the pre-screening in Example 1 (Table 2), it was determined that a more basic environment had a more beneficial effect on colloidal stability, while the first and second DoE matrices of Example 1 showed that formulations having a pH of 7.3 or 8.1 have good colloidal and thermodynamic stability.
  • Example 3 also shows this particularly good colloidal stability and thermodynamic property of formulations having a pH of 7.3 or 8.1 , which have excellent results in the nanoparticle analysis.
  • the formulation has pH 8.1.
  • Figures 3 and 4 show that the A 2 value increases (indicating less aggregation) with increasing pH and that T onset increase with increasing buffer molality.
  • Tables 4 and 28 show that a pH of 8.1 provides for less aggregation
  • Figures 8 and 12 show that formulations having pH 8.1 (i.e. sub-variants derived from test variants #3 and #4) have the best purity.
  • formulations of the present invention having a pH in the range of 7.0-8.5 have good purity.
  • Table 24 shows that more than 99% of nanoparticles in the formulation had a radius in the size range of 0.1 -10nm, even when temperature-stressed.
  • Examples 10 and 1 1 also show that such formulations have good potency, which at least meets the acceptance criterion for minimal potency of human GM-CSF (Tables 85 and 95).
  • Example 9 shows that formulations of the invention, and particularly formulations having pH8.1 , are less of an irritant than previously known formulations of GM-CSF.
  • Example 9 shows that, at day 24 after injection of the formulation, formulations of the present invention having pH8.1 were less irritating than an existing commercial formulation and formulations having pH7.3.
  • the pH of the formulation is preferably 7.3 or 8.1 , preferably 8.1.
  • formulations having these pHs have good colloidal and thermodynamic stability, low aggregation and good purity, and formulations having pH 8.1 show the best properties.
  • the pH of the formulation is preferably 8.1.
  • formulations comprising arginine and having a pH of 8.1 show a good purity, with a relative main peak area of between 70% and 90%.
  • formulations of the present invention can be adjusted during preparation to the requisite pH as required, for example using NaOH, KOH or HCI. wt% of each component
  • GM-CSF is present in the range of 0.001-10wt%, 0.01-10wt%, 0.05-10wt%, 0.1- 10wt%, 0.05-5wt%, 0.05-2.5wt%, 0.05-1.5wt%, 0.1-1wt%, 0.1-0.7wt% or 0.3-0.7wt%. In some embodiments, GM-CSF is present in the range of 0.3-0.5wt%. In some embodiments, GM-CSF is present at 0.33wt%, 0.48wt%, 0.60wt% or 0.63wt%.
  • the stabiliser is present in the range of 0.005-99.0wt%, 0.01-99.0wt%, 0.05-99.0wt%, 0.1-99.0wt%, 0.5-99.0wt%, 1.0-99.0wt%, 1.0-98.0wt%, 1.0-97.5wt%, 1.1-98wt% or 1.1-97.5wt%.
  • the polymer or surfactant is present in the range of 0.005-10.0wt%, 0.01- 10.0wt%, 0.05-10.0wt%, 0.05-5.0wt%, 0.5-5.0wt%, 0.5-3.0wt%, or 1.0-3.0wt%.
  • the polymer or surfactant is present in the range of 1.0-2.5wt%, and preferably in the range of 1 0-2.0wt%. In some embodiments, the polymer or surfactant is present at 1 1wt%, 1 6wt%, 2.0wt% or 2.1wt%.
  • the one or more further components such as a disaccharide or arginine
  • the one or more further components may be present in total in the range of 10.0- 99.0wt%, 20.0-99.0wt%, 30.0-99.0wt%, 40.0-99.0wt%, 50.0-99.0wt%, 60.0-99.0wt%, 70.0- 99.0wt%, 80.0-99.0wt%, 90.0-99.0wt%, 90.0-98.0wt%, 92.0-98.0wt% or 95.0-98.0wt%.
  • the one or more further components is present in the range of 96.0-98.0wt%.
  • sucrose When the stabiliser comprises sucrose, sucrose may be present in the range of 10.0-99.0wt%, 20.0-99.0wt%, 30.0-99.0wt%, 40.0-99.0wt%, 50.0-99.0wt%, 60.0-99.0wt%, 70.0-99.0wt%, 80.0- 99.0wt%, 90.0-99.0wt%, 90.0-98.0wt%, 92.0-98.0wt% or 95.0-98.0wt%. In some embodiments, sucrose is present in the range of 97.0-98.0wt%. In some embodiments, sucrose is present at 97.5wt%.
  • mannitol When the stabiliser comprises mannitol, mannitol may be present in the range of 10.0-99.0wt%, 20.0-99.0wt%, 30.0-99.0wt%, 40.0-99.0wt%, 50.0-99.0wt%, 60.0-99.0wt%, 70.0-99.0wt%, 80.0- 99.0wt%, 90.0-99.0wt%, 90.0-98.0wt%, 92.0-98.0wt% or 95.0-98.0wt%. In some embodiments, mannitol is present in the range of 95.0-96.0wt%. In some embodiments, mannitol is present at 95.4wt%.
  • each of sucrose and mannitol may be present in the range of 10.0-99.0wt%, 20.0-99.0wt%, 30.0-99.0wt%, 40.0-99.0wt%, 50.0- 99.0wt%, 60.0-99.0wt%, 70.0-99.0wt%, 80.0-99.0wt%, 90.0-99.0wt%, 90.0-98.0wt%, 92.0- 98.0wt% or 95.0-98.0wt%.
  • sucrose is present in the range of 15.0- 50.0wt%, 25.0-35.0wt%, 27.0-33.0wt% or 28.0-32.0wt%, and mannitol is present in the range of 45.0-85.0wt%, 60.0-70.0wt%, 60.0-67.0wt%, or 62.0-67.0wt%.
  • sucrose is present in the range of 30.0-32.0wt%, and mannitol is present in the range of 64.0-65.0wt%.
  • sucrose is present at 31 7wt% and mannitol is present at 64.8wt%.
  • arginine may be present in the range of 10.0-99.0wt%, 20.0-99.0wt%, 30.0-99.0wt%, 40.0-99.0wt%, 50.0-99.0wt%, 60.0-99.0wt%, 70.0-99.0wt%, 80.0- 99.0wt%, 90.0-99.0wt%, 90.0-98.0wt%, 92.0-98.0wt%, 93.0-98.0wt%, 94.0-98.0wt% or 95.0- 98.0wt%. In some embodiments, arginine is present in the range of 95.0-97.0wt% or 95.0- 96.0wt%. In some embodiments, arginine is present at 95.3wt%.
  • the buffer is present in the range of 0.1 -10.0wt%, 0.1 -5.0wt%, 0.1 - 3.0wt%, 0.5-5.0wt%, 0.5-2.5wt%, 1.0-2.5wt% or 1 .0-2.0wt%. In some embodiments, the buffer is present in the range of 1 .0-2.0wt%. In some embodiments, the buffer is present at 1.0wt%, 1 5wt%, 1 9wt% or 2.0wt%.
  • the formulations of embodiments of the present invention contain HSA or rHA in an amount less than or equal to 0.20wt%, 0.15wt%, 0.10wt%, 0.050wt%, 0.030wt%, 0.020wt%, 0.010wt%, 0.005wt%, 0.003wt%, 0.002wt% or 0.001wt%.
  • HSA or rHA is present in the formulations of the invention in an amount less than or equal to 0.02wt%.
  • the formulations of the invention contain no (i.e. 0.0wt%) HSA or rHA.
  • water may be present at at least 10.0wt%, 20.0wt%, 30.0wt%, 40.0wt%, 50.0wt%, 60.0wt%, 65.0wt%, 70.0wt%, 75.0wt%, 80.0wt%, 85.0wt%, 90.0wt%, 91.0wt%, 92.0wt%, 93.0wt%, 94.0wt%, 95.0wt%, 95.5wt%, 96.0wt%, 96.5wt%, 97.0wt%, 97.5wt%, 98.0wt%, 98.5wt% or 99.0wt%.
  • water may be present in the range of 10.0-99.0wt%, 20.0-99.0wt%, 30.0-99.0wt%, 40.0-99.0wt%, 50.0-99.0wt%, 60.0-99.0wt%, 70.0- 99.0wt%, 80.0-99.0wt%, 85.0-99.0wt%, 90.0-99.0wt%, 91 .0-99.0wt%, 91 .0-98.5wt% or 91.0- 98.0wt%.
  • water is present in the range of 85.0-97.5wt%, 90.0-97.5wt%, 91.0-97.5wt%, 91 .0-96.0wt%, 91.0-95.5wt% or 91.5-95.5wt%. In other embodiments, water is present in the range of 85.0-99.0wt%, 90.0-99.0wt%, 93.0-99.0wt%, 95.0-99.0wt%, 95.0- 98.0wt% or 95.0-98.0wt%. In some embodiments, water is present at 91.6wt%, 94.1wt%, 95.2wt% or 95.4wt%. In other embodiments, water is present at 95.6wt%, 97.0wt%, 97.6wt% or 97.7wt%.
  • the formulation comprises GM-CSF in the range of 0.1 -2.5wt%, P188 in the range of 0.5-5.0wt%, sucrose in the range of 70.0-99.0wt% and potassium phosphate in the range of 0.1 -2.5wt%.
  • the formulation comprises GM-CSF in the range of 0.30-0.36wt%, P188 in the range of 1 .0-1.2wt%, sucrose in the range of 87.5-97.8wt% and potassium phosphate in the range of 0.9-1 2wt%.
  • water may be in the range of 82.4-99.0wt% or in the range of 86.0-99.0wt%.
  • the formulation comprises GM-CSF in the range of 0.1 -2.5wt%, P188 in the range of 0.5-5.0wt%, mannitol in the range of 70.0-99.0wt% and potassium phosphate in the range of 0.1 -3.0wt%.
  • the formulation comprises GM-CSF in the range of 0.5-0.7wt%, P188 in the range of 1.9-2.1wt%, mannitol in the range of 85.9-95.9wt% and potassium phosphate in the range of 1 7-2.1wt%.
  • water may be in the range of 85.7-99.0wt% or in the range of 87.8-99.9wt%.
  • the formulation comprises GM-CSF in the range of 0.1 -2.5wt%, P188 in the range of 0.5-5.0wt%, sucrose in the range of 15.0-50.0wt%, mannitol is present in the range of 45.0-85wt% and potassium phosphate is present in the range of 0.1 -3.0wt%.
  • the formulation comprises GM-CSF in the range of 0.4-0.5wt%, P188 in the range of 1.4-1 8wt%, sucrose in the range of 28.6-35.0wt%, mannitol in the range of 58.1- 71.0wt% and potassium phosphate in the range of 1.4-1.7wt%. When water is present, it may be in the range of 84.7-99.0wt% or in the range of 87.3-99.0wt%.
  • the formulation comprises GM-CSf in the range of 0.1-2.5wt%, P188 in the range of 0.5-5.0wt%, arginine in the range of 70.0-99.0wt% and potassium phosphate in the range of 0.1-3.0wt%.
  • the formulation comprises GM-CSF in the range of 0.6-0.7wt%, P188 in the range of 1.9-2.3wt%, arginine in the range of 85.7-95.7wt% and potassium phosphate in the range of 1.8-2.2wt%.
  • water may be present in the range of 85.9-99.0wt% or in the range of 87.9-99.0wt%.
  • Example 2 Figure 7
  • Figures 35 and 36 show that HSA masks the potential aggregates of GM-CSF, and that the results from samples containing rHA cannot be considered exact as the main aggregation peaks from rHA and the main aggregation peaks from GM-CSF were not separated.
  • Figure 1 1 shows that rHA masks the impurities in formulations containing rHA, such that these formulations appear to be purer than they actually are (see Example 2).
  • the purity of the formulation was also tested after reconstitution from lyophilisates, and was shown to be very good for formulations of the present invention.
  • Table 40 shows that formulations of embodiments of the present invention had an aggregate content of less than 2%
  • Table 41 shows that formulations of embodiments of the present invention have a purity of at least 97%.
  • Table 56 shows that formulations of the invention had an aggregate content of less than 2%
  • Table 57 shows that formulations of the invention had a purity of over 96%, even after having been held at 40°C for 12 weeks.
  • the formulation of embodiments of the present invention has a purity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100%, when measured after 8, or 12 weeks of storage.
  • the formulations of the present invention have the vast majority of their nanoparticles in the smallest size range measured (i.e. 0.1-10nm), even after reconstitution from a lyophilisate, thereby indicating that the risk of aggregation of the formulations of the present invention is low.
  • Table 42 shows that, in nearly all reconstituted formulations of the present invention, more than 98% of the nanoparticles had a radius in the range of 0.1-10nm, and had an average radius of about 2.0nm.
  • the reconstituted existing commercial formulation (#C) showed only 95% of nanoparticles being in the range of 0.1-10nm, with average radius which was twice that of the formulations of the present invention, namely, 4.3nm.
  • the formulations of the present invention continue to have the vast majority of nanoparticles in the size range of 0.1-10nm (Example 8, Table 58).
  • Table 58 shows that, even after storage at 25°C and 40°C for 8 or 12 weeks, the formulations had a mean mass fraction in the size range of 0.1-10nm of more than 93%.
  • the formulations of the present invention have a very good purity, and this indicates that the European Pharmacopoeia requirement for the number of sub-visible particles (maximum 6000 particles of 10pm and maximum 600 particles of 25pm, in a volume of 25ml or at least 25ml) is expected to be met, even when the process of producing the formulations of the present invention is scaled up.
  • the formulations of the present invention have an aggregate content of nanoparticles having a radius greater than 10nm of less than 4.5%, less than 3.5%, less than 2.5% or, preferably, less than 2%.
  • the formulations have an average nanoparticle radius in the range of 0.1-10nm, 0.5-10nm, 0.5-5nm, 1-5nm, or, preferably, 1-3nm. In some embodiments, the average nanoparticle diameter is about 2nm.
  • the formulation of embodiments of the present invention also provides the advantage that, after lyophilisation, there is a reduced amount of residual water compared to previously known formulations, thereby reducing the risk of spoilage of the formulation.
  • the residual water in formulations of the present invention was well below 1.0%, with the maximum residual water being 0.5%, while the control formulation (an existing commercial formulation) had 0.6% residual water.
  • Table 54 shows that the residual water in the lyophilised product remained well below 1% even after forced degradation using temperature.
  • the formulation has a maximum residual water content, immediately after lyophilisation, of 1 .0% or 0.5%.
  • the formulation has a maximum residual water content, after storage at 25°C for 12 weeks, of 1 % or 0.6%.
  • lyophilizates of the formulations of the present invention can be reconstituted in WFI in less than 10 seconds, which is more rapid than existing commercial formulations of GM-CSF (Example 8).
  • the formulation of the present invention is stored in vials, at a volume in the range of 0.1 -2.0ml.
  • the volume of formulation per vial is in the range of 0.1 -1 .0ml, 0.2-0.9ml, or 0.3-0.8ml.
  • the vials are filled to a volume of 0.1 ml.
  • the volume of formulation per vial is 0.33ml.
  • the volume of formulation per vial is 0.66ml.
  • the volume of formulation per vial is 1 ml.
  • each vial contains the same amount of each ingredient, except that each vial contains twice the amount of water for injection (WFI), such that the formulations in the vials filled with 0.66ml of formulation are twice as dilute compared to the formulation in the vials filled with 0.33ml of formulation.
  • WFI water for injection
  • the formulation comprises 0.3mg/ml GM-CSF protein
  • vials containing 0.33ml of the formulation will contain 0.1 mg of GM-CSF protein.
  • Vials containing 0.66ml of this same formulation will also contain 0.1 mg of GM-CSF protein, but will contain twice as much WFI as corresponding vials having a fill volume of 0.33ml.
  • each vial contains a maximum dose of GM-CSF of 2.0mg, 1.0mg, 0.5mg, 0.4mg, 0.3mg, 0.2mg, 0.1 mg or 0.05mg.
  • each vial contains no more than 1 .0mg of GM-CSF.
  • the vial is a syringe
  • the dose or fill volume is the same as described above. This provides the advantage that the risk of transferring an incomplete dose from vial to syringe is avoided.
  • the formulation consists of a GM-CSF protein having at least 60% sequence identity, and preferably 100% sequence identity, to SEQ ID NO: 3, potassium phosphate, sucrose and P188.
  • the formulation is made up to a final volume with WFI.
  • the liquid formulation has a pH of 7.3 or 8.1 , preferably 8.1.
  • the formulation consists of a GM-CSF protein having at least 60% sequence identity, and preferably 100% sequence identity, to SEQ ID NO: 3, potassium phosphate, arginine/ H3PO4 and P188.
  • the formulation is made us to a final volume with WFI.
  • the liquid formulation has a pH of 7.3 or 8.1 , preferably 8.1.
  • the formulation consists of a GM-CSF protein having at least 60% sequence identity, and preferably 100% sequence identity, to SEQ ID NO: 3, potassium phosphate, mannitol and P188.
  • the formulation is made up to a final volume with WFI.
  • the liquid formulation has a pH of 7.3 or 8.1 , preferably 8.1.
  • the formulation consists of a GM-CSF protein having at least 60% sequence identity, and preferably 100% sequence identity, to SEQ ID NO: 3, potassium phosphate, sucrose, mannitol and P188.
  • the formulation is made us to a final volume with WFI.
  • the liquid formulation has a pH of 7.3 or 8.1 , preferably 8.1.
  • the formulation consists of 0.3mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 10mM potassium phosphate, 260mM sucrose, 0.1 % w/v P188, and has a pH of 7.3.
  • a liquid formulation is made up to 0.33ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 29.4mg sucrose and 0.33mg P188, and the liquid formulation is made up to 0.33ml with WFI.
  • the pH is adjusted to 7.3 using potassium hydroxide.
  • the formulation consists of 0.15mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 5mM potassium phosphate, 130mM sucrose, 0.05% w/v P188, and has a pH of 7.3.
  • the liquid formulation is made up to 0.66ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 29.4mg sucrose and 0.33mg P188, and the liquid formulation is made up to 0.66ml with WFI.
  • the pH is adjusted to 7.3 using potassium hydroxide. As discussed above, it was found that the increased dilution of the formulation decreased the amount of aggregation in the formulation (Example 4; Table 28) and that vials could be more accurately filled when using a greater fill volume.
  • the formulation consists of 0.3mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 10mM potassium phosphate, 260mM sucrose, 0.1% w/v P188, and has a pH of 8.1.
  • the liquid formulation is made up to 0.33ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 29.4mg sucrose and 0.33mg P188, and the liquid formulation is made up to 0.33ml with WFI.
  • the pH is adjusted to 8.1 using potassium hydroxide.
  • the formulation consists of 0.15mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 5mM potassium phosphate, 130mM sucrose, 0.05% w/v P188, and has a pH of 8.1.
  • the liquid formulation is made up to 0.66ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 29.4mg sucrose and 0.33mg P188, and the liquid formulation is made up to 0.66ml with WFI.
  • the pH is adjusted to 8.1 using potassium hydroxide. As discussed above, it was found that the increased dilution of the formulation decreased the amount of aggregation in the formulation (Example 4; Table 28) and that vials could be more accurately filled when using a greater fill volume.
  • the formulation consists of 0.3mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 10mM potassium phosphate, 260mM Arginine/H 3 P0 4 , 0.1 %w/v P188, and has a pH of 7.3.
  • the liquid formulation is made up to 0.33ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 15.1 mg Arginine/H 3 P0 4 and 0.33mg P188, and the liquid formulation is made up to 0.33ml with WFI.
  • the pH is adjusted to 7.3 using potassium hydroxide.
  • the formulation consists of 0.15mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 5mM potassium phosphate, 130mM Arginine/H 3 P0 4 , 0.05% w/v P188, and has a pH of 7.3.
  • the liquid formulation is made up to 0.66ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 15.1 mg Arginine/H 3 P0 4 and 0.33mg P188, and the liquid formulation is made up to 0.66ml with WFI.
  • the pH is adjusted to 7.3 using potassium hydroxide.
  • the formulation consists of 0.3mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 10mM potassium phosphate, 260mM Arginine/H 3 P0 4 , 0.1% w/v P188 and has a pH of 8.1.
  • the liquid formulation is made up to 0.33ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 15.1 mg Arginine/H 3 P0 4 and 0.33mg P188, and the liquid formulation is made up to 0.33ml with WFI.
  • the pH is adjusted to 8.1 using potassium hydroxide.
  • the formulation consists of 0.15mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 5mM potassium phosphate, 130mM Arginine/H 3 P0 4 , 0.05% w/v P188, and has a pH of 8.1.
  • the liquid formulation is made up to 0.66ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 15.1 mg Arginine/H 3 P0 4 and 0.33mg P188, and the liquid formulation is made up to 0.66ml with WFI.
  • the pH is adjusted to 8.1 using potassium hydroxide.
  • the formulation consists of 0.3mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 10mM potassium phosphate, 260mM mannitol, 0.1 %w/v P188 and has a pH of 7.3.
  • the liquid formulation is made up to 0.33ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 15.6mg mannitol and 0.33mg P188, and the liquid formulation is made up to 0.33ml with WFI.
  • the pH is adjusted to 7.3 using potassium hydroxide.
  • the formulation consists of 0.15mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 5mM potassium phosphate, 130mM mannitol, 0.05% w/v P188 and has a pH of 7.3.
  • the liquid formulation is made up to 0.66ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 15.6mg mannitol and 0.33mg P188, and the liquid formulation is made up to 0.66ml with WFI.
  • the pH is adjusted to 7.3 using potassium hydroxide.
  • the formulation consists of 0.3mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 10mM potassium phosphate, 260mM mannitol, 0.1 % w/v P188, and has a pH of 8.1.
  • the liquid formulation is made up to 0.33ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 15.6mg mannitol and 0.33mg P188, and the liquid formulation is made up to 0.33ml with WFI.
  • the pH is adjusted to 8.1 using potassium hydroxide.
  • the formulation consists of 0.15mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 5mM potassium phosphate, 130mM mannitol, 0.05% w/v P188, and has a pH of 8.1.
  • the liquid formulation is made up to 0.66ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 15.6mg mannitol and 0.33mg P188, and the liquid formulation is made up to 0.66ml with WFI.
  • the pH is adjusted to 8.1 using potassium hydroxide.
  • the formulation consists of 0.3mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 10mM potassium phosphate, 220mM mannitol, 58mM sucrose, 0.1 % w/v P188, and has a pH of 7.3.
  • the liquid formulation is made up to 0.33ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 13.5mg mannitol, 6.6mg sucrose and 0.33mg P188, and the liquid formulation is made up to 0.33ml with WFI.
  • the pH is adjusted to 7.3 using potassium hydroxide.
  • the formulation consists of 0.15mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 5mM potassium phosphate, 110mM mannitol, 29mM sucrose, 0.05% w/v P188, and has a pH of 7.3.
  • the liquid formulation is made up to 0.66ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 13.5mg mannitol, 6.6mg sucrose and 0.33mg P188, and the liquid formulation is made up to 0.66ml with WFI.
  • the pH is adjusted to 7.3 using potassium hydroxide.
  • the formulation consists of 0.3mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 10mM potassium phosphate, 220mM mannitol, 58mM sucrose, 0.1 % w/v P188, and has a pH of 8.1.
  • the liquid formulation is made up to 0.33ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 13.5mg mannitol, 6.6mg sucrose and 0.33mg P188, and the liquid formulation is made up to 0.33ml with WFI.
  • the pH is adjusted to 8.1 using potassium hydroxide.
  • the formulation consists of 0.15mg/ml GM-CSF protein having 100% sequence identity to SEQ ID NO: 3, 5mM potassium phosphate, 110mM mannitol, 29mM sucrose, 0.05% w/v P188, and has a pH of 8.1.
  • the liquid formulation is made up to 0.66ml with WFI. More specifically, in this embodiment, the formulation consists of 0.1 mg GM-CSF protein, 0.32mg potassium phosphate, 13.5mg mannitol, 6.6mg sucrose and 0.33mg P188, and the liquid formulation is made up to 0.66ml with WFI.
  • the pH is adjusted to 8.1 using potassium hydroxide.
  • the formulation of the present invention can be in one of several physical states.
  • the formulation may be a liquid, a frozen liquid, or a freeze-dried lyophilizate.
  • the components of the formulation are as described above, with the addition of WFI to the required volume.
  • the liquid formulation may be prepared by adding WFI to the components of the formulation, or by adding water to a lyophilised formulation.
  • the formulation comprises the components are as described above, with the addition of WFI, in frozen form.
  • the lyophilizate preferably contains less than 5% residual water, and preferably less than 1% residual water.
  • the lyophilized formulation comprises the components aside from water as described above, and can be reconstituted for use by the addition of WFI to the required volume.
  • the formulations of the present invention are useful as a medicament.
  • the formulations of the present invention are used for boosting the immune system of a patient after chemotherapy, raising white blood cell counts, and/or for improving the immune response to peptide vaccines.
  • the formulations of the present invention are for use in the treatment and/or prophylaxis of a patient, particularly a human.
  • the formulation is for use as an immune-modulatory agent.
  • the formulation may be used in the treatment and/or prophylaxis of cancer.
  • the cancer is one or more of adrenal gland, autonomic ganglia, biliary tract, bladder, bone, breast, central nervous system, cervical, colorectal, endometrial, haematopoietic, lymphoid, kidney, large intestine, liver, lung, non-small cell lung, oesophagus, ovarian, pancreatic, prostate, salivary gland, skin, small intestine, stomach, testicular, thymus, thyroid, upper aerodigestive tract and urinary tract cancer and/or malignant melanoma.
  • the cancer is colorectal, lung and pancreatic cancer.
  • the cancer is cancer that arises due to a mutation in the RAS protein.
  • the formulations of the present invention may also be used for the reduction of neutropenia in patients receiving chemotherapy.
  • a further use of the formulations of the present invention is to accelerate myeloid recovery in patients following bone marrow transplant, or for the treatment of super-orphan lung diseases such as pulmonary alveolar proteinosis (PAP).
  • PAP pulmonary alveolar proteinosis
  • the use of the formulations of the present invention as a medicament comprises use with a peptide vaccine.
  • a peptide vaccine is particularly useful for the treatment and/or prophylaxis of cancer.
  • the peptide vaccine is a RAS peptide vaccine, which is used for the treatment and/or prophylaxis of cancer arising due to a mutation in the RAS protein, such as RAS peptide vaccines described in WO2015/086590, WO2015/169804, and WO2016/202937, incorporated herein by reference.
  • the peptide vaccine is as described in WO00/02581 , which is incorporated herein by reference.
  • the formulations of the present invention are for use simultaneously or sequentially with a peptide vaccine.
  • the formulation of the present invention and the peptide vaccine are administered one after another.
  • the time between administration of the formulation and the peptide vaccine may be about 5, 10, 15, 20, 25 or 30 minutes.
  • the formulation and the peptide vaccine are administered simultaneously.
  • Simultaneous administration may involve administering the formulation and the peptide vaccine one immediately after the other, with no, or substantially no, delay between the administration of the formulation and the peptide vaccine.
  • simultaneous administration may involve administrating both the formulation of the present invention and the peptide vaccine as a single dose, for example, by a single injection.
  • Formulations are prepared for administration to a patient by reconstituting the lyophilised formulation in sterile WFI, according to procedures known in the art.
  • one vial of the lyophilised formulation containing 0.1 mg of the GM-CSF protein is reconstituted with 0.33ml of sterile WFI, to give a concentration of the GM-CSF protein of 0.3mg/ml.
  • the date and time of reconstitution is recorded on the vial label, and the vial is gently rotated (to minimise foaming).
  • the vial is allowed to rest for 10 minutes before preparation of a syringe for administering the formulation.
  • the reconstituted formulation is inspected for visible particle, and is rotated again if any particles are visible.
  • the vial is then allowed to rest for a further 10 minutes, after this second rotation. (If particles are still visible after the second rotation, then the vial is not used and is be reported as an IMP deviation.)
  • the reconstituted formulation is stored at 2-8°C, away from sunlight, and is used within 6 hours of reconstitution. In some instances, the reconstituted formulation is stored at 2-8°C, away from sunlight, for up to 12 hours before use.
  • formulations of the present invention may be administered to the patient by any suitable delivery technique known to those skilled in the art.
  • the formulation of the present invention may be administered to a subject by intradermal injection, subcutaneous injection, intra-muscular injection, intravenous infusion, or nasal administration or inhalation.
  • GM-CSF Control Sample 1 An existing formulation (20 mM K-phosphate, pH 7.3) of GM-CSF drug substance (GM-CSF Control Sample 1 ) served as the basis for the development. Suitable buffer systems and excipients were selected and tested with respect to their relevance for increasing the colloidal as well as the thermodynamic stability of GM-CSF.
  • Nano DSC is specifically designed to determine the denaturation temperature and thermal denaturation enthalpy of proteins and other macromolecules in solution with the versatility and precision to perform molecular stability screenings.
  • a buffer scan was conducted prior to each sample run to generate a baseline.
  • Samples were dialyzed against the corresponding formulation buffer (see 3.4) and diluted to 1 mg/mL.
  • the corresponding dialysis buffer was used as buffer scan and buffer reference.
  • Differential scanning calorimetry measures the amount of heat that is absorbed or released from biomolecules in solution during heating or cooling.
  • Native proteins respond to heating by unfolding (thermal denaturation) at a characteristic temperature (Tm).
  • Tm characteristic temperature
  • CG-MALS Composition gradient multi-angle light scattering
  • A2 is characteristic for a substance and its solvent, and describes molecular interactions between the dissolved components.
  • a negative A 2 indicates attractive interactions between molecules of the dissolved substance whereas a positive A 2 is characteristic for repulsive interactions between the dissolved protein molecules (Valent et al. “Colloidal behaviour of proteins: Effects of the second virial coefficient on solubility, crystallization and aggregation pf proteins in aqueous soluntion”, Current Pharmaceutical Biotechnology, 2005, 6, 427-436).
  • the CG-MALS technique is based on a series of differently concentrated macromolecular solutions which are directly injected into the flow cell of a multi-angle light scattering detector. After each injected concentration step the flow is stopped to permit the reaction to reach equilibrium for signal stabilization and subsequent detection.
  • the apparent weight average molecular weight (Mw app ) is determined for each step in the concentration gradient by analyzing the light scattering and concentration data by the following equation:
  • R(q) ⁇ describes the angular dependence of the scattered light, and can be related to the rms radius
  • Samples were dialyzed against the corresponding formulation buffer (see part 3.4) and used undiluted.
  • the corresponding dialysis buffer was used as diluent for the CG-MALS experiment.
  • the samples were passed over a 0.1 pm syringe filter.
  • concentration of the sample was determined by UV-absorption measurement (see part 3.3). Sample and buffer were loaded into the Calypso II system and the measurement was started.
  • a Calypso II CG-MALS system was used to supply the MALS detector with the concentration gradient of the analyte.
  • the sample was loaded on one syringe pump of the system and the dialysis buffer on another syringe pump of the system.
  • Ten equidistant concentration steps from approx. 3.5 mg/mL to 0.5 mg/mL were applied in each measurement by diluting the sample with dialysis buffer.
  • At each gradient step 1 mL of sample was injected into the MALS detector.
  • the resulted light scattering signal was recorded over a time period of 180 seconds.
  • the concentration of each gradient step was recorded using a refractive index detector. Control of the system and analysis of the data via Zimm plot analysis was performed with Calypso software version 2.1.5.
  • the two protein samples of interest were loaded on syringe pump 1 and syringe pump 2 of the system and the dialysis buffer was loaded on syringe pump 3.
  • the binary CG-MALS measurement consists of three steps. In the first step, a concentration gradient of sample 1 ranging from 10 % to 100 % was applied to determine the self-virial coefficient of sample 1 in the formulation.
  • the second step consists of a cross-over gradient, in which the concentration of sample 1 was reduced from 90 % to 10 % while the concentration of sample 2 was increased from 10 % to 90 % in ten steps. This step was conducted to determine the cross-virial coefficient.
  • FIG. 1 displays schematically the steps of a binary CG-MALS experiment to detect the self-virial coefficient of the two molecules as well as the cross-virial coefficient which measures the interactions between the two protein species.
  • At each gradient step 1 ml. of sample was injected into the MALS detector.
  • Zimm plot analysis was performed with Calypso software version 2.1.5.
  • the concentration of GM-CSF in solution was determined using a Cary 60 UV-spectrometer (Agilent Technologies, Santa Clara, USA) by adsorption measurement at 280nm. Samples were measured at a concentration of about 1 mg/ml_ using plastic cuvettes with an optical path thickness of 1.0cm. The concentration was calculated according to Lambert-Beer's law using an extinction coefficient of 0.974ml_/(mg * cm) as provided by Wacker Biotech for GM-CSF.
  • the predictive formulations analytics comprised a screening experiment consisting of variants with different pH, buffer components, and additional stabilizers to identify suitable formulation conditions for GM-CSF with respect to thermodynamic (measurement by nanoDSC) and colloidal stability (measurement by CG-MALS).
  • the pH was modulated from 6.0 to 8.2 in order to accommodate an intradermal administration close to physiologic pH.
  • suitable buffer components were selected to achieve a sufficient buffer capacity.
  • the buffer components were selected from commonly used pharmaceutically accepted substances.
  • L-histidine/HCI citric acid/NaOH, potassium phosphate and Tris/HCI were selected.
  • Potassium phosphate buffer was used because the GM-CSF drug substance (DS) was supplied in it by default and also as it covers a broad pH range.
  • L-histidine/HCI is often used for antibody formulations and covers neutral to slightly acidic pH ranges.
  • Citric acid/NaOH was chosen as an intermediate buffer system between His/HCL and potassium phosphate.
  • Tris/HCI is active at slightly basic pH values due to its basic pKs. During the first pre-screening (part 4.1 ) only the pH and buffer systems were varied.
  • formulations were supplemented with potential protein stabilizers and bulking agents in the second DoE matrix (part 4.3) to detect any beneficial effects of these excipients.
  • Variant #1 showed some strong aggregation of insoluble aggregates visible after dialysis, a consolidated precipitation was observed.
  • T onset (the onset temperature of thermal denaturation, i.e. the starting point of the unfolding transition) of variants #2 - #4 (neutral towards basic pH) is above 50 °C and quite distant from any commonly used storage and handling temperature, thus lies in the non-critical range.
  • T on set of pH 6.0 variant #1 is clearly lower towards more critical range.
  • the positive effect of increasing pH on protein stability is also confirmed by continuously increasing enthalpies DH for thermal denaturation.
  • An exemplary thermogram is shown in Figure 2.
  • the second DoE matrix was designed to test stabilizing effects of several excipients on GM- CSF (variants #13 - #21 ).
  • a combination of excipients suitable for liquid and/or lyo formulations was selected to keep both options open, which decision will be subject to chemical stability data that will be obtained in a forced degradation study.
  • Variant #5 was selected as a root variant facilitating a liquid and freeze-dried presentation
  • variant #6 was selected as preferred root variant for a liquid DP presentation only
  • variant #10 was selected as a preferred root variant for a lyophilized DP presentation.
  • Excipient concentration was selected to facilitate isotonicity (-300 mM).
  • Arg/Phosphate ionic strength, bulking agent, protein stabilizer, stability and solubility enhancer
  • Glycerol non-ionic protein stabilizer, a smaller polyol, for liquid only, cannot be freeze- dried
  • Proline hydrotropic substance with aggregation protection effects e.g. also applied for protein refolding, but cannot be lyophilized as it does not provide a stable lyo matrix
  • thermodynamic stability (T on set and T m ) dropped some distinct °C units, which is less favourable.
  • the Tris/HCI buffer thus represents a compromise between more beneficial colloidal stability (A 2 ) and unfavourable thermodynamic stability.
  • the lyo formulation variants #19 - #21 seem to present the more stable formulations for GM-CSF with regard to maximizing colloidal and thermodynamic stability at the same time (highest value for A 2 , highest value for T on set) - any of the tested isotonic liquid/lyo or liquid formulations would always account for a compromise between higher thermodynamic stability, but lowered colloidal stability.
  • thermodynamic properties and colloidal stability of GM-CSF in different formulation variants were evaluated.
  • pH, buffering agents, ionic strength, and excipients the most beneficial variants for either lyo or liquid formulation were determined successively.
  • thermodynamic properties seem to benefit from higher ionic strength in every scenario.
  • the aim of this study was to find the most promising formulation variants for a GM-CSF lyophilized drug product by applying five different harsh stress conditions and subsequent analysis with regard to aggregate status and potential chemical degradation.
  • the two most promising formulation candidates were selected for further development and analysis.
  • the four test variants displayed in Table 6 were selected for this study.
  • test variants were prepared with a GM-CSF target concentration of 0.3 mg/ml_ and spiked with purified water (w/o), polysorbate 20 (PS20), poloxamer 188 (P188), and recombinant human albumin (rHA), respectively, in order to evaluate the effect of these additives on the stability of GM-CSF. Furthermore, these resulting 16 sub-variants were enclosed in lyo vials with either air atmosphere or nitrogen atmosphere, respectively, resulting in a total of 32 sub- variants.
  • purified water w/o
  • PS20 polysorbate 20
  • P188 poloxamer 188
  • rHA recombinant human albumin
  • a“control” forced degradation run was performed using a reconstituted lyophilizate of an existing commercial formulation of GM-CSF (“current DP”), containing human serum albumin (HSA), mannitol, macrogol 4000, dibasic sodium phosphate and monobasic potassium phosphate.
  • current DP human serum albumin
  • control lyophilizates (0.1 mg/vial of GM-CSF) were reconstituted with 330 pl_ of water for injection (WFI) and pooled up to a total volume of 700 mI_ (hereinafter described as“control”). Subsequently the solution was filled into a 2R glass vial, stoppered with a serum stopper, and crimped using a 13 mm crimp cap before the same stress procedure was applied to it as to the formulation variants (see section 3.1.2 for details).
  • 2R glass vials were washed with purified water and subsequently depyrogenized and sterilized by dry heat at 300 °C for 2 hours. Stoppers were autoclaved at 134 °C for 10 minutes and subsequently dried at 70 °C for 8 hours.
  • Table 8 Preparation of formulation buffers and detergent/stabilizer stock solutions.
  • the dialysis of GM-CSF drug substance was accomplished in three independent dialysis steps to achieve a quantitative buffer exchange.
  • the protein sample was removed from the Slide-A-Lyzer cassettes and processed further.
  • Each dialysis step represents a 1/83 fold buffer exchange leading to a calculated buffer exchange factor of 1 :5.7x10 s in total. All dialysis steps were performed at 2-8 °C.
  • the GM-CSF solution was analyzed via UV absorption measurement at 280 nm in order to determine the GM-CSF concentration.
  • the solution was then diluted to the target concentration for the forced degradation study of 0.3mg/ml_ using formulation buffer. Subsequently the solution was spiked with the respective detergent/stabilizer stock solution to obtain a concentration of 0.1% (w/w) detergent/stabilizer.
  • the respective test variant without detergent/stabilizer was spiked with the same amount of purified water.
  • the readily compounded formulations were sterile filtered using a 0.1 pm PES syringe filter and filled into sterile 2R glass vials by aliquots of 700 pL using a multistep pipette. Subsequently the vials for air/nitrogen exchange were partially stoppered with lyo stoppers and placed in a freeze dryer. Sub-variants to be evaluated with air atmosphere were stoppered with serum stoppers and crimped with 13 mm crimp caps. All compounding/filling steps were performed under a laminar flow bench to avoid microbial contamination of the product.
  • Vials to be tested with nitrogen atmosphere were partially stoppered with 13 mm lyo stoppers and placed in a freeze dryer.
  • the air in the freeze drying chamber was removed by applying a vacuum of 100 mbar and subsequent venting of the chamber with pure nitrogen. This procedure was carried out three times.
  • the vials were closed under nitrogen atmosphere and a pressure of 900 mbar absolute.
  • the vials were then unloaded of the freeze dryer and crimped with 13 mm crimp caps.
  • the crimp caps and stoppers were removed from the respective vials before the formulation was transferred into 1.5 ml. Eppendorf reaction vessels. From these vessels the respective volumes of samples were taken for the five different analytical methods: 60 pL for IEX-HPLC, 130 pL for SE-HPLC, 130 pL for RP-HPLC, 150 pL for UV absorption measurements, and 40 pL for SDS-PAGE. A compilation of all tested stress conditions and sampling points is provided in section 4, Table 20.
  • Vials were treated in the sun tester at 750 W/m 2 and 25 °C for 7.5 hours in upside-down position.
  • Vials were treated on a shaker at 200 rpm and 25 °C for 5 days in upside-down position.
  • Samples were analyzed using a Cary 60 UV-spectrometer (Agilent Technologies, Santa Clara, CA, USA) by absorption measurement at 280 nm for concentration determination as well as at 350 nm and 510 nm for the evaluation of potential turbidity caused by the stress treatment. Samples were measured at a concentration of about 1 mg/ml_ (dilution with formulation buffer) or undiluted if the concentration was below 1 mg/ml_ using plastic cuvettes with an optical path length of 1.0 cm. The concentration was calculated according to Lambert-Beer ' s law using an extinction coefficient of 0.974 ml_/(mg * cm) as provided by Wacker Biotech for GM-CSF. 3.2.2 SE-HPLC (size exclusion chromatography) with Acquity BEH SEC
  • UV-detection 214 nm and 280 nm
  • Injection volume 100 pL at 0.3 mg/mL GM-CSF
  • Injection volume 100 mI_ at 0.5 mg/ml_
  • GM-CSF concentration was 0.3mg/ml_ for all variants.
  • concentrations determined by UV absorption measurement at 280 nm showed rather high fluctuations (see e.g. sub-variant #2 PS20 at 40 °C). This was most probably caused by evaporation of water in the small sample volume leading to a concentration of the solutes in the small sample volume of roughly -200 mI_ used for absorption measurements (repeat measurement was not possible as remainder 500 mI_ were used for other analytical evaluation). However, the concentrations never significantly dropped below the target GM-CSF concentration of 0.3 mg/ml_.
  • the aggregate status of stressed samples was evaluated by SE-HPLC and SDS-PAGE, respectively.
  • HSA human serum albumin
  • HSA masks potential GM-CSF aggregates and, thus, the SE-HPLC method was not a good tool for the aggregation status evaluation of GM-CSF in GM-CSF current formulation HSA-containing samples (existing commercial formulation; “current DP”). Interestingly, the aggregate content decreased by applying stress (Figure 8) which might be due to precipitation.
  • the graph displayed in Figure 8 shows the minimum relative GM-CSF peak areas (without integration of the rHA monomeric peaks) of all stress conditions throughout the entire forced degradation study, to compress and visualize the huge amount of data.
  • the sub-variants without additive (w/o) and the sub-variants containing poloxamer 188 (P188) showed the best results overall. Due to a high dimer content and the described masking effect of the HSA/rHA main peak, the results of the“control” as well as of the rHA-containing variants are not comparable with the sub-variants without HSA/rHA.
  • the values for rHA and“control” in Figure 8 are only shown for completeness of the data.
  • the monomer content in the sub-variants without additive and with P188 only drops below 99% in sub-variant #2 (without additive with air, as well as with P188 without air) and once without additive in sub-variant #3 (without air).
  • the sub-variants containing polysorbate 20 (PS20) and rHA, respectively, show monomer contents of less than 99% in each scenario for at least one stress condition and, hence, are considered inferior to sub-variants without additive and with P188.
  • the aggregate status of stressed GM-CSF samples was further evaluated by SDS-PAGE. None of the gels showed any aggregate bands, which indicates rather low aggregate contents. Merely in the rHA-containing sub-variants very light/barely visible bands at around 150 kDa can be seen. These bands probably represent the rHA dimer fraction that was also observed by SE- HPLC. The“control” however showed well visible aggregate bands in the >150 kDa area confirming again the SE-HPLC results. The samples contain aggregates that are most probably dimeric and multimeric human serum albumin (HSA) molecules in a much higher concentration than in the stressed test variants.
  • HSA human serum albumin
  • the protein bands were slightly more retarded in the reduced samples which accounts for disulfide bonds being reduced by the DTT, thus enlarging the molecule by unfolding.
  • test variant #3 (10 mM potassium phosphate + 260 mM sucrose, pH 8.1 , with and without air) showed the best results overall, whereas test variant #2 (10 mM potassium phosphate + 260 mM L-arginine, pH 7.3, with and without air) showed the lowest purities.
  • the respective rHA-containing sub-variant does in fact show a similar purity as P188 in test variants #1 and #3, but this sub-variant is actually not comparable to the other sub- variants as impurities of GM-CSF get masked by rHA (as described in section 4.2.1 for the SE- HPLC results, and also clearly visible in Figure 1 1 with regard to RP-HPLC analysis). If the detected purity of the rHA sub-variants had been much higher than for the others, only then it would have been necessary to investigate rHA-containing sub-variants further.
  • test root-variant #4 (10 mM potassium phosphate + 260 mM L-arginine, pH 8.1 , with and without air) should be treated with caution.
  • test root-variant #4 Taking into account this high pH sensitivity in the presence of L-arginine and a compounding tolerance at the CMO of probably around 0.2 pH units (plus/minus), the very good results of test root-variant #4 become somewhat overshadowed as a slight difference in pH may significantly negatively affect the product’s stability.
  • test variant #4 was the minimum purity in the rHA- containing sub-variant lower than in the“control”. The minimum detected purities cumulative throughout all stress conditions are shown in Figure 12.
  • GM-CSF forced degradation study four test root-variants with 0.3 mg/mL GM- CSF and four different additives were tested in the presence and, respectively, in the absence of oxygen resulting in a total of 32 sub-variants. Additionally, a reconstituted GM-CSF lyophilizate containing HSA as a stabilizer underwent the same treatment to serve as a reference (“control”). The tested additives in the sub-variants were polysorbate 20 (PS20), poloxamer 188 (P188), rHA, and water as a baseline. All samples were stressed by light, shaking, elevated temperature at 40 °C, and multiple freeze/thaw cycles in order to discriminate the different formulation variants with respect to the stability of GM-CSF.
  • PS20 polysorbate 20
  • P188 poloxamer 188
  • rHA rHA
  • test root-variant #2 did show absorption > 0.05 AU indicating the inferiority of this formulation compared to the others.
  • Test variant #2 seems to be the weakest formulation amongst the four formulations, test variant #3 the best candidate formulation.
  • Test variant #1 seems to be quite comparable to test variant#3 and, thus, test variant #1 would be preferred on second place.
  • test variant #4 would also not be considered one of the preferred candidates as it would“only” be different from test variant #2 in pH and this pH sensitivity/dependence weakens the performance of test variant #4, as it could bear some risks with regard to product stability.
  • Test variant #2 yielded extremely bad results and should be dismissed completely from any further tests.
  • Test variants #1 , #3, and #4 all yielded good results, whereby #3 was superior over the others.
  • test variants #1 , #3, and #4 containing P188 and no additive were champions of this investigation.
  • Test variant #4 is the same formulation as the very unfavorable test variant #2 but at a higher pH (8.1 compared to 7.3) which might lead to complications during production where a compounding tolerance of about 0.2 pH units usually has to be granted. This tolerance in combination with the observed high pH sensitivity poses a risk to the GM-CSF stability.
  • rHA-containing variants were not bad overall but were potentially overrated due to the masking effect of the rHA peak in chromatograms. Furthermore, the use of rHA as an additive is rather inconvenient regulatory-wise as well as supply-wise.
  • test variants #1 and #3 containing P188 and no additive, respectively yielded the overall best results and should be looked further into for formulation development.
  • the objective of this development work was to evaluate the presence of nanoparticles by dynamic light scattering (DLS) measurements in selected sub-variants from the forced degradation study of Example 2.
  • DLS dynamic light scattering
  • a DynaPro Plate Reader (Wyatt Technology) was to be used for DLS measurements of nanoparticles in a microwell plate by measurement of the hydrodynamic radius (applicable range: from 0.1 nm to 1000 nm).
  • Table 23 lists substances and materials used during nanoparticle analysis.
  • Samples were stored at -70 °C and were thawed at room temperature prior use. Each sample constituted a 2R vial with a fill volume of 0.7 ml. and 0.3 mg/ml_ GM-CSF. Samples were filled into the wells of the measurement plate by portions of 30 mI_ and subsequently overlaid with silicone oil to avoid evaporation of liquid. No further treatment was applied before measurement.
  • a DLS plate reader was used for determination of nanoparticles in selected samples from Example 2 (Table 22). Each sample was measured in triplicate, whereby the procedure was repeated once resulting in a total of six values per sample (each value was generated by 10 acquisitions). To simplify the complex set of data generated with the DLS plate reader, three ranges of particle sizes were defined (i.e. 0.1 -10 nm, 10-100 nm, and 100-1000 nm) and the mass fraction of molecules covered by the specific range was calculated. Table 24 shows the mean radius measured in the specific range as well as the related mass fraction.
  • GM-CSF is a rather small molecule, where the mean particle radius would be expected in the measured range between 1 - 10 nm, which seems to correlate well with the high amount of mass fraction detected in this range.
  • samples #1 and #3, and stressed samples #2 and #4 do all show the same cluster pattern within the detectable range of this method, it is not obvious that any treatment towards samples #1-#4 so far changed the nano- particle level of the GM-CSF Control Sample 2 in the range of 0.1 - 1 ,000 nm.
  • samples #1 and #3 (sub-variants 1 and 3) seem to provide a stable matrix for GM-CSF with regard to nano-particle formation up to 1000 nm.
  • samples #1 and #3 and stressed samples #2 and #4 do all show the same cluster pattern within the detectable range of this method, it is not obvious that any treatment towards samples #1 -#4 so far changed the nano- particle level of the BDS in the range of 0.1 - 1000 nm.
  • samples #1 and #3 seem to provide a stable matrix for GM-CSF with regard to nano-particle formation up to 1000 nm.
  • the objective of the overall development work was to develop a lyophilized formulation for GM- CSF drug product for intradermal application primarily in glass vial (30 pg in 100 pi injection volume, cone. ⁇ 0.3 mg/mL GM-CSF, fill volume -330 pL (100 pi injection volume incl. 220 pi overfill) in standard 2R type I clear glass vial.
  • Two final formulation variants determined from Example 2 sub-variant results were selected for subsequent development (Table 25, final formulation variants #1 and #2).
  • the concentration of GM-CSF in solution was determined using a Cary 60 UV-spectrometer (Agilent Technologies, Santa Clara, CA, USA) by adsorption measurement at 280 nm. Samples were measured at a concentration of about 1 mg/ml_ using plastic cuvettes with an optical path thickness of 1.0 cm. The concentration was calculated according to Lambert-Beer ' s law using an extinction coefficient of 0.974 ml_/(mg * cm) as provided by Wacker Biotech for GM-CSF.
  • Nano DSC is specifically designed to determine the denaturation temperature and thermal denaturation enthalpy of proteins and other macromolecules in solution with the versatility and precision to perform molecular stability screenings.
  • a buffer scan was conducted prior to each sample run to generate a baseline.
  • Differential scanning calorimetry measures the amount of heat that is absorbed or released from biomolecules in solution during heating or cooling.
  • Native proteins respond to heating by unfolding (thermal denaturation) at a characteristic temperature (T m ).
  • T m characteristic temperature
  • CG-MALS Composition gradient multi-angle light scattering
  • a 2 is characteristic for a substance and its solvent, and describes molecular interactions between the dissolved components.
  • a negative A 2 indicates attractive interactions between molecules of the dissolved substance whereas a positive A 2 is characteristic for repulsive interactions between the dissolved protein molecules.
  • the CG-MALS technique is based on a series of differently concentrated macromolecular solutions which are directly injected into the flow cell of a multi-angle light scattering detector. After each injected concentration step the flow is stopped to permit the reaction to reach equilibrium for signal stabilization and subsequent detection.
  • the apparent weight average molecular weight (Mw app ) is determined for each step in the concentration gradient by analyzing the light scattering and concentration data by the following equation:
  • concentration c It is directly proportional to the intensity of the excess light scattered by the solute and the light scattered by the pure solvent.
  • P(0) describes the angular dependence of the scattered light, and can be related to the rms radius
  • Samples were dialyzed against the corresponding final formulation variant buffer (see section 3.1.2) and used undiluted.
  • the corresponding dialysis buffer was used as diluent for the CG- MALS experiment.
  • the samples were passed over a 0.1 pm syringe filter.
  • concentration of the sample was determined by UV-absorption measurement (see section 3.2). Sample and buffer were loaded into the Calypso II system and the measurement was started.
  • a Calypso II CG-MALS system was used to supply the MALS detector with the concentration gradient of the analyte.
  • the sample was loaded on one syringe pump of the system and the dialysis buffer on another syringe pump of the system.
  • Ten equidistant concentration steps from approx. 3.5 mg/mL to 0.5 mg/mL were applied in each measurement by diluting the sample with dialysis buffer.
  • At each gradient step 1 mL of sample was injected into the MALS detector.
  • the resulted light scattering signal was recorded over a time period of 180 seconds.
  • the concentration of each gradient step was recorded using a refractive index detector. Control of the system and analysis of the data via Zimm plot analysis was performed with Calypso software version 2.1.5.
  • final formulation variant #1 showed a negative A 2 indicating unfavorable attractive molecule-molecule interactions in solution
  • counterpart formulation with more basic pH final formulation variant #2
  • final formulation variant #2 showed a positive A 2 indicating repulsive molecule- molecule interactions in solution, lowering the tendency for aggregation events.
  • the halving of the buffer molarity and of the excipient concentration led to improved intermolecular interactions as A 2 values were both clearly positive.
  • the rather high A 2 value for final formulation variant #2d should not be overrated as measurements were extremely noisy and thus hard to evaluate. 5.
  • Example 1 onset of thermal denaturation was determined for final formulation variant #1 and #2 (corresponding to variants #rep. 13 and #19 in Table 4) to be at -54 °C, a comfortable range far away from common handling and storage temperatures.
  • Example 5
  • 2R glass vials were washed with purified water and subsequently depyrogenized and sterilized by dry heat at 300 °C for 2 hours.
  • the dialysis of GM-CSF drug substance was accomplished in three independent dialysis steps to achieve a quantitative buffer exchange.
  • the protein sample was removed from the Slide-A-Lyzer cassettes and processed further.
  • Each dialysis step represented a 1/83 fold buffer exchange leading to a calculated buffer exchange factor of 1 :5.7x10 s in total. All dialysis steps were performed at 2-8 °C.
  • the GM-CSF solution was analyzed via UV absorption measurement at 280 nm to determine the GM-CSF concentration.
  • the solution was then diluted to a concentration of 0.3 mg/ml_ using formulation buffer before it was spiked with the required amount of poloxamer 188 to obtain a concentration of 0.1 % (w/v) detergent resulting in final formulation variants #1 and #2.
  • portions of the readily compounded final formulation variants #1 and #2 were diluted with the same volume of water (1v+1v).
  • Variant #2v (vehicle/placebo formulation) was formulation buffer #2 with 0.1 % (w/v) poloxamer 188 but without GM-CSF.
  • the readily compounded lyo solutions were sterile filtered using a 0.1 pm PES syringe filter and filled into sterile 2R glass vials by aliquots of 330 pL (final formulation variants #1 , #2, and #2v) and 660 pL (final formulation variants #1d and #2d), respectively, using a multistep pipette. Subsequently the vials were partially stoppered with lyo stoppers in lyo position and placed in a freeze dryer. All compounding/filling steps were performed under a laminar flow bench to avoid microbial contamination of the product.
  • the filled vials with lyo-stoppers attached to the vials in“lyo-position” were positioned on the shelves of the pilot freeze dryer.
  • 160 vials were loaded per final formulation variant #1 and #2, 60 vials per final formulation variant #1 d and #2d, and 40 vials of formulation variant #2v.
  • the process parameters applied for lyophilization are listed in Table 34.
  • a so called radiation cage was employed to actively cool the walls of the lyo chamber in order to simulate large scale conditions.
  • thermocouples were inserted into product vials as displayed in Figure 14. Pressure was controlled during lyophilization by using a capacitive pressure sensor (MKS). Pressure regulation was managed via vacuum and dosing valve (nitrogen injection). After completion of secondary drying vials were closed at a pressure of 800 mbar under nitrogen atmosphere. Vials were unloaded from the freeze dryer and crimp capped (using a hand crimping tool).
  • MKS capacitive pressure sensor
  • Samples were analyzed using a Cary 60 UV-spectrometer (Agilent Technologies, Santa Clara, CA, USA) by absorption measurement at 280 nm for concentration determination. Samples were measured at a concentration of about 1 mg/ml_ (dilution with formulation buffer) or undiluted if the concentration was below 1 mg/ml_ using plastic cuvettes with an optical path length of 1.0 cm. The concentration was calculated according to Lambert-Beer’s law using an extinction coefficient of 0.974 ml_/(mg * cm).
  • UV-detection 214 nm and 280 nm
  • Injection volume 100 mI_ at 0.5 mg/ml_
  • Each sample constituted a 2R vial with lyophilized GM-CSF drug product with a target GM-CSF amount of 0.10 mg per vial. After reconstitution with 0.33 ml. WFI, samples were filled into the wells of the measurement plate by portions of 30 mI_ and subsequently overlaid with silicone oil to avoid evaporation of liquid. No further treatment was applied before measurement. 3.3.5 Process analytics of the lyophilization
  • the process data of the lyophilization (time, pressure and temperature readings including in-vial product temperature measurements), were logged by a controlling computer and visualized in a graph.
  • Lyophilizate-containing vials were examined visually. Photographs were taken for documentation.
  • the residual moisture of the samples was determined via Karl Fischer titration using a 756 Karl Fischer coulometer equipped with a 774 oven sample processor (Metrohm).
  • the vials were reconstituted by the addition of 330 pl_ WFI using a volumetric pipette after removing the cap and stopper from the vial. Subsequently the vial was gently shaken until complete dissolution of the lyo cake.
  • 330mI_ WFI was added to the vial, and gently swirled for 2 minutes by hand, then allowed to stand at ambient temperature for 11 minutes.
  • the lyophilized cake should reconstitute to a clear solution. Analysis can commence after the standing time - the length of time from reconstitution until start of analysis was noted in the test documentation.
  • the chamber pressure was monitored by capacitive pressure sensor and Pirani pressure sensor and recorded via on-line data acquisition to detect the end of sublimation.
  • the lyophilization program was completed as planned.
  • the recorded temperature and pressure profile complied with the predefined process parameters.
  • the product temperatures measured via thermo couples indicated the end of the sublimation phase after about 9 hours total process time for final formulation variants #1 and #2, and after about 13 hours for final formulation variants #1d and #2d, respectively.
  • the diluted final formulation variants had a longer sublimation phase due to the higher fill volume of the vials (0.66 ml. as opposed to 0.33 ml_). The entire cycle lasted 69 hours.
  • 160 vials were freeze dried per final formulation variant #1 and #2, 60 vials per final formulation variant #1 d and #2d, and 40 vials of formulation variant #2v.
  • the freshly thawed GM-CSF working standard showed a small peak of 0.36% relative peak area eluting after the main peak (99.64% relative peak area).
  • the eluting order indicated a smaller Mw of those“fragments” than of the GM-CSF molecule.
  • All reconstituted drug product samples showed the same peak pattern of one peak of larger aggregates eluting before the main peak and one peak of smaller fragments eluting after the main peak.
  • the impurity peak profile in all reconstituted GM-CSF drug product final formulation variants #1 , #2, #1 d, #2d as well as in the working standard was the same and as shown in Figure 19 and Figure 20. Peaks eluting before the main peak represented fragments that are more hydrophilic than GM-CSF whereas the peak eluting after the main peak represented fragments that are more hydrophobic than GM-CSF. Likewise as for SE-HPLC, the chemical degradation of GM-CSF in the existing commercial formulation #C could not be determined as potential impurity peaks get masked by the HSA peak. Again, the respective chromatographic data should be considered informational only.
  • GM-CSF Control Sample 3 was not analyzed on SE-HPLC and RP-HPLC before lyophilization in Example 5. However, this analysis was found to be missing in order to relate obtained results of Example 5 against a non-freeze-dried reference of GM-CSF Control Sample 3.
  • Example 6 and Example 7 are the same as listed in section 0 of Example 5.
  • GM-CSF containing DS solutions were diluted to a concentration of 0.3 mg/ml_ using elution buffer (for SEC-MALS) or dilution buffer (for RP-HPLC), respectively. Lyophilizates were reconstituted prior to analysis using 0.33 ml. WFI.
  • Example 6 Analytical methods used in Example 6 and Example 7 were described in sections 0 and 0 of Example 5.
  • Peak identification was performed using a stressed“new” BDS sample of lot -/ 095 (Control Sample 3).
  • the BDS was stressed at 55 °C for 60 hours before the aggregate status was determined by SEC-MALS.
  • the chromatogram is shown in Figure 22.
  • the Mw in the main peak was determined as 14 kDa.
  • the MALS and UV signals of suspected fragments was too low for the proper calculation of molecular weight.
  • Example 5 The BDS material used for Examples 5 and 8 (lot. no. 170118/1167/095, Control Sample 3) was compared to reconstituted lyophilizates produced in Example 5 (TGWP7b/1-170814, final formulation variant #1 ; and TGWP7b/2-170814, final formulation variant #2) using the described SE-HPLC method for aggregate status determination. An overlay of the three chromatograms is shown in Figure 25. A slight increase in aggregate and fragment content in the lyophilized samples is visible.
  • Final formulation variant #2 (TGWP7b/2-170814) has slightly less impurity content than final formulation variant #1 (TGWP7b/1-170814).
  • a positive control was produced by incubating“new” BDS of lot -/095 (Control Sample 3) at 55 °C for 60 hours. The positive control was compared to freshly thawed“new” BDS of the same batch using the described SE-HPLC method for aggregate status determination. An overlay of the two chromatograms is shown in Figure 28.
  • Example 5 The BDS material used for Example 5 (lot. no. 1701 18/1 167/095, Control Sample 3) was compared to reconstituted drug product lyophilizates produced in Example 5 (TGWP7b/1- 170814, final formulation variant #1 ; and TGWP7b/2-170814, final formulation variant #2) using the described RP-HPLC method for purity analysis.
  • An overlay of the three chromatograms is shown in Figure 31.
  • Example 5 The reconstituted drug product lyophilizates produced in Example 5 (TGWP7b/1-170814, final formulation variant #1 ; and TGWP7b/2-170814, final formulation variant #2) were compared to their respective placebo formulations without drug substance GM-CSF using the described RP- HPLC method for purity analysis. Overlays are shown in Figure 32 and Figure 33.
  • a positive control was produced by incubating BDS of lot -/095 (Control Sample 3) at 60 °C for 18 hours.
  • the positive control was compared to freshly thawed BDS of the same lot using the described RP-HPLC method for purity analysis.
  • An overlay of the two chromatograms is shown in Figure 34.
  • Example 5 The lyophilizates obtained in Example 5 (GM-CSF lyophilizate #1 (final formulation variant #1 )and GM-CSF lyophilizate #2 (final formulation variant #2)) were compared to an existing commercial formulation by overlaying the respective chromatograms.
  • the chromatograms obtained by SE-HPLC analysis are shown in Figure 35.
  • the main peak area detected in the existing commercial formulation was between 10% and 15% higher than in the two lyophilizates obtained in Example 5. This may be due to an overage applied by the manufacturer to overcome extraction issues (i.e. not the complete liquid volume can be extracted after reconstitution).
  • the peak pattern is not comparable as the existing commercial formulation contains HSA and its aggregates masking the relevant retention time range for GM-CSF aggregates.
  • the main peak area detected in the existing commercial formulation was higher (up to almost 20% here) than in the lyophilizates obtained in Example 5.
  • the relative differences between SE-HPLC and RP-HPLC may be due to the HSA peak interfering with the GM-CSF peak differently in the two methods.
  • the main peak eluted about one minute earlier than in the two lyophilizates. This is a phenomenon commonly observed in RP- HPLC deriving from slight differences in composition of the elution buffer.
  • the large HSA peak in the current drug product again masks a broad range of retention time making a proper evaluation of impurities impossible by this method.
  • Example 8 was aimed to evaluate the stability of the four different final formulation variants of lyophilized GM-CSF drug product. Lyophilized samples that entered this study were manufactured using the same procedure as in Example 5, using a conservative, non-optimised lyo-cycle.
  • Lyo samples obtained in Example 5 were stored at 25 °C and 40 °C, respectively, for up to 12 weeks (pull points were set to T8 weeks and T12 weeks). Formulation variants stored are listed in Table 47.

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Abstract

L'invention concerne une formulation comprenant une protéine GM-CSF ayant au moins 60% d'identité de séquence avec la séquence polypeptidique de SEQ ID NO: 2. La formulation comprend un stabilisant, le stabilisant comprenant un poloxamère présent en une quantité de 0,5 à 3,0 % en poids, à l'exclusion de l'eau. La formulation ne comprend pas plus de 0,2 % en poids d'albumine sérique humaine ou d'albumine humaine recombinante.
PCT/EP2019/067428 2018-06-29 2019-06-28 Formulation WO2020002650A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2021185921A1 (fr) * 2020-03-17 2021-09-23 Drugrecure Aps Formulation liquide de gm-csf pour inhalation

Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005799A1 (fr) * 1991-09-24 1993-04-01 Farmitalia Carlo Erba S.R.L. Compositions pharmaceutiques stables et lyophilisees contenant un facteur de stimulation de colonies de granulocytes et de macrophages
WO1997013502A2 (fr) * 1995-10-12 1997-04-17 Immunex Corporation Liberation prolongee de facteurs stimulant les colonies de granulocytes-macrophages (gm-csf)
WO2000002581A1 (fr) 1998-07-08 2000-01-20 Norsk Hydro Asa Peptides antigenes derivees de la telomerase
WO2002069999A1 (fr) * 2001-03-05 2002-09-12 Schering Aktiengesselschaft Solutions aqueuses stables de facteur de stimulation des granulocytes et macrophages
WO2005013933A1 (fr) * 2003-07-31 2005-02-17 Universite De Liege Composition mucoadhesive comprenant du polyacrylate et une substance de chimio-attraction
EP1598074A1 (fr) * 2003-02-28 2005-11-23 Chugai Seiyaku Kabushiki Kaisha Preparation stabilisee contenant une proteine
WO2008122415A1 (fr) * 2007-04-05 2008-10-16 Sandoz Ag Formulations aqueuses stables de g-csf
KR20130020760A (ko) * 2009-04-22 2013-02-28 (주)비씨월드제약 유전자 재조합 과립구-대식세포 집락 촉진인자를 함유하는 정제
WO2015086590A2 (fr) 2013-12-09 2015-06-18 Targovax As Mélange de peptides
WO2015169804A1 (fr) 2014-05-06 2015-11-12 Targovax As Vaccin peptidique comprenant un peptide ras mutant et agent chimiothérapeutique
WO2015177379A2 (fr) * 2014-05-23 2015-11-26 Reponex Pharmaceuticals Aps Compositions destinées à favoriser la cicatrisation des plaies
WO2016202937A1 (fr) 2015-06-16 2016-12-22 Targovax Asa Fragments mutés de la protéine ras

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005799A1 (fr) * 1991-09-24 1993-04-01 Farmitalia Carlo Erba S.R.L. Compositions pharmaceutiques stables et lyophilisees contenant un facteur de stimulation de colonies de granulocytes et de macrophages
WO1997013502A2 (fr) * 1995-10-12 1997-04-17 Immunex Corporation Liberation prolongee de facteurs stimulant les colonies de granulocytes-macrophages (gm-csf)
WO2000002581A1 (fr) 1998-07-08 2000-01-20 Norsk Hydro Asa Peptides antigenes derivees de la telomerase
WO2002069999A1 (fr) * 2001-03-05 2002-09-12 Schering Aktiengesselschaft Solutions aqueuses stables de facteur de stimulation des granulocytes et macrophages
EP1598074A1 (fr) * 2003-02-28 2005-11-23 Chugai Seiyaku Kabushiki Kaisha Preparation stabilisee contenant une proteine
WO2005013933A1 (fr) * 2003-07-31 2005-02-17 Universite De Liege Composition mucoadhesive comprenant du polyacrylate et une substance de chimio-attraction
WO2008122415A1 (fr) * 2007-04-05 2008-10-16 Sandoz Ag Formulations aqueuses stables de g-csf
KR20130020760A (ko) * 2009-04-22 2013-02-28 (주)비씨월드제약 유전자 재조합 과립구-대식세포 집락 촉진인자를 함유하는 정제
WO2015086590A2 (fr) 2013-12-09 2015-06-18 Targovax As Mélange de peptides
WO2015169804A1 (fr) 2014-05-06 2015-11-12 Targovax As Vaccin peptidique comprenant un peptide ras mutant et agent chimiothérapeutique
WO2015177379A2 (fr) * 2014-05-23 2015-11-26 Reponex Pharmaceuticals Aps Compositions destinées à favoriser la cicatrisation des plaies
WO2016202937A1 (fr) 2015-06-16 2016-12-22 Targovax Asa Fragments mutés de la protéine ras

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"Monograph of Molgramostim Concentrated Solution", EUROPEAN PHARMACOPEIA, vol. 7.0, January 2008 (2008-01-01), pages 1641
"UniProtKB", Database accession no. P04141
ALTSCHUL, STEPHEN F.THOMAS L. MADDENALEJANDRO A. SCHAFFERJINGHUI ZHANGZHENG ZHANGWEBB MILLERDAVID J. LIPMAN: "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402, XP002905950, DOI: doi:10.1093/nar/25.17.3389
ARELLANOLONIAL: "Clinical uses of GM-CSF: a critical appraisal and update", BIOLOGICS: TARGETS AND THERAPY, vol. 2, no. 1, 2008, pages 13 - 27
CHUANGOTAGIRI, DRUGS TODAY (BARE, vol. 43, no. 8, 2007, pages 547 - 561
OHTAKE ET AL.: "Interactions of formulation excipients with proteins in solution and in the dried state", ADV. DRUG DELIV. REV., 2011
VALENT ET AL.: "Colloidal behaviour of proteins: Effects of the second virial coefficient on solubility, crystallization and aggregation pf proteins in aqueous soluntion", CURRENT PHARMACEUTICAL BIOTECHNOLOGY, vol. 6, 2005, pages 427 - 436
VAN DE LAAR ET AL.: "Regulation of dendritic cell development by GM-CSF; molecular control and implications for immune homeostasis and therapy", BLOOD, vol. 119, no. 15, 12 April 2012 (2012-04-12), XP055380210, DOI: doi:10.1182/blood-2011-

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021185921A1 (fr) * 2020-03-17 2021-09-23 Drugrecure Aps Formulation liquide de gm-csf pour inhalation
CN115297844A (zh) * 2020-03-17 2022-11-04 德拉格雷丘尔公司 用于吸入的gm-csf的液体制剂

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