JP2019043961A - New stabilizing agent for pharmaceutical protein - Google Patents

Abstract

To provide a method for stabilizing a human blood protein or human blood plasma protein with a molecular weight of greater than 10 KDa.SOLUTION: The human blood protein or human plasma protein having a molecular weight of greater than 10 KDa is stabilized by addition of melezitose to a solution containing the human blood protein or human blood plasma protein having a molecular weight of greater than 10 KDa.SELECTED DRAWING: None

Description

  The present invention relates to a method of stabilizing human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa, solid state or liquid state compositions of human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa, and human blood proteins or human The use of melezitose to stabilize plasma proteins.

  Stabilization of therapeutic proteins is a major challenge for pharmaceutical scientists in the pharmaceutical industry today. There are many types of stresses that can cause both reversible and irreversible changes in proteins, such as aggregation, precipitation, or denaturation. These difficulties require agents that stabilize these delicate proteins. Formulation development is an important step and requires careful selection of excipients during the purification and formulation process as well as for high protein activity in the final product. This applies in particular to human blood proteins and human plasma proteins.

  The most widely used stabilizer of protein formulations is carbohydrates, also called sugars. Carbohydrates are composed of linked basic carbohydrate components, called monosaccharides, and can be of various lengths and thus have various characteristics.

  Since the two most commonly used stabilizers, sucrose and trehalose, are both disaccharides, they are composed of two monosaccharides.

  Melisitose is a trisaccharide, whereas sucrose and trehalose, two of the most commonly used carbohydrate stabilizers, are disaccharides. It is generally indicated that the first option for stabilizing proteins in both solution and lyophilization states is the disaccharide (Non-patent Documents 1 and 2). Some disaccharides such as lactose or maltose are reducing sugars that can degrade proteins by the Maillard reaction during storage in the solid state. It is suggested in the literature that the use of larger saccharides as stabilizers in lyophilised preparations is less efficient due to steric hindrance of protein-stabilizer interactions (Non-patent Document 2) .

A non-patent document 1 which is a review article, among others, maltose, glucose and maltotriose can increase the recovery of catalase activity at 1 mg · ml ⁻¹ but maltopentaose, maltohexaose and maltoheptaose do not It discloses that it is not effective. The absence of the effect of larger saccharides suggests that protein stabilization by saccharides may depend on the length of glucoside side chains of saccharides that may interfere with the intermolecular hydrogen bonding between the saccharide and protein being stabilized. Be done. This review paper recommends disaccharides as stabilizers (page 9/10).

  US Patent No. 5,648, 544 discloses the use of stachyose, melezitose and carbohydrates containing various mono- and disaccharides to prepare an intranasally administrable polypeptide preparation. The sugars function as agents that reduce the effects of shear stress during spraying.

  WO 03/087200 discloses, inter alia, the chromatographic purification of factor VIII using Melezitose as a hydration additive during the chromatography process.

  Patent Document 3 discloses a method for preserving delicate biological substances or organic compounds under (a) dry condition and / or (b) high temperature and / or (c) irradiation, which comprises In a system comprising the compound, (i) a non-reducing glycoside of a polyhydroxy compound selected from sugar alcohols and other linear polyalcohols, or (ii) a non-reducing oligo selected from raffinose, stachyose and melezitose Including introducing a sugar or sugar derivative selected from sugars.

  Non-patent document 3 reports on the stability of insulin in a solid preparation containing melezitose and starch.

International Publication No. 2003/0864443 A brochure WO 86/04486 A brochure WO 91/18091 A pamphlet

Wang W, Lyophilisation and development of solid protein pharmaceuticals, Int J Pharm 203, 1-60, 2000 Carpenter J. F., Chang B. S., Garzon-Rodriguez W., Randolph T. W., Rational design of stable lyophilized protein formulations: Theroy and practice, chapter 5, ed Carpenter and Manning, Kluiwer Academic / Plenum Publishers, New York 2002 Mollmann, S. H. et al, Drug Dev. Ind. Pharm. 2006 Jul; (6): 765-78

  The present invention provides a method for stabilizing human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa by adding mereditose to a solution containing human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa.

  Preferably, the molecular weight of said human blood protein or human plasma protein is more than 10 KDa. More preferably, the molecular weight is 10 KDa to 300 KDa. Most preferably, the molecular weight is between 20 kDa and 200 kDa. It may be beneficial that the molecular weight of human blood proteins or human plasma proteins is 50 KDa to 100 KDa, 100 KDa to 150 KDa, or 150 KDa to 200 KDa.

  In particular, human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa are proteins of pharmaceutical or biological significance. The human blood proteins or human plasma proteins of pharmaceutical or biological significance which have a molecular weight of more than 10 KDa that can be stabilized according to the invention may be recombinantly produced human blood proteins or human plasma proteins.

  The term "protein" includes chemically synthesized proteins, naturally occurring synthetic proteins encoded by genes of cultured cells and recombinant proteins secreted by cells. A recombinant protein is one encoded by a transgene introduced into cells by molecular biological techniques. The proteins may be modified by chemical methods or by enzymes during post-translational processes.

  In the present invention, "protein" includes human proteins, particularly those produced by cell culture, but also proteins and muteins of other sources such as plants and insects, artificial proteins, synthetic proteins, fusion proteins Or chimeric proteins.

  The term "human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa" is, in particular, human blood coagulation factors such as fibrinogen, fibrin monomers, prothrombin, thrombin, FV, FVa, FX, FXa, FIX, FIXa, FVII, FVIIa , FVIII, FXI, FXIa, FXII, FXIIa, FXIII, von Willebrand factor, ADAMTS13 etc., transport proteins such as albumin, transferrin, ceruloplasmin, haptoglobin, hemoglobin, hemopexin etc, protease inhibitors such as 6-anti Thrombin, α-antithrombin, α2-macroglobulin, C1 inhibitor, tissue factor pathway inhibitor (TFPI), heparin cofactor II, protein In C inhibitor (PAI-3), protein C, protein S, protein Z etc., immunoglobulin, such as polyclonal antibody (IgG), monoclonal antibody, IgG1, IgG2, IgG3, IgG4, IgA, IgA1, IgA2, IgM, IgE, IgD, Bence Jones protein etc., cell-related plasma proteins such as fibronectin, thromboglobulin, platelet factor 4 etc., apolipoproteins such as apoA-I, apoA-II, apoE, complement factors such as factor B, factor D, factor H, factor I, C3b inactivation factor, properdin, C4 binding protein, etc., growth factors such as platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor alpha (TGF-α), transformation Growth factor beta (T F-β), fibroblast growth factor (FGF) and hepatocyte growth factor, anti-angiogenic proteins such as latent-antithrombin and prelatent-antithrombin, hyperglycosylated proteins such as alpha-1-acid glycoprotein Antichymotrypsin, inter-α-trypsin inhibitor, α-2-HS glycoprotein, C-reactive protein, and other human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa, such as histidine-rich glycoprotein, mannan-binding lectin, C4 Binding protein, fibronectin, GC-globulin, plasminogen, alpha-1 microglobulin, C-reactive protein, blood factor such as erythropoietin, interferon, tumor factor, tPA, g SF as well as their derivatives and muteins. Of particular interest are factor IX, factor VIII, G-CSF, vWF, antithrombin (AT), hepatocyte growth factor (HGF), polyclonal IgG, alpha-1 antitrypsin, factor H, factor I, C1-. Esterase inhibitors, factor VII and combinations thereof. However, since insulin simply has a molecular weight of about 5,700 Da, polypeptides such as insulin are not included in the term "human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa".

  The terms "human blood protein" and "human plasma protein" include derivatives, in particular molecules that have been modified to have an increased half-life. Modifications for half-life extension include, but are not limited to, fusion proteins, proteins modified by mutagenesis and proteins covalently or non-covalently linked to conjugates. According to the invention, human plasma proteins or human blood proteins may be covalently linked to hydroxyethyl starch (HES) molecules, in particular to bring the molecular weight of the molecules to 20 to 200 KDa.

  When referring to molecular weight, this refers to the molecular weight of the compound (ie, including the molecular weight of any chemical substance covalently linked to the protein).

  The invention in particular provides a method of transferring a solution to a solid state.

  The present invention relates to the discovery of novel stabilizers of human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa for pharmaceuticals.

Surprisingly, as a stabilizer for human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa, such as recombinant factor VIII (170 KDa), factor IX (55 kDa) and HES-modified G-CSF (120 kDa) It has been found that it can be used. Human blood proteins and human plasma proteins of similar molecular weight are expected to have similar stabilization requirements. For example, Factor IX is a vitamin K dependent human plasma protein and has biochemical similarities to all other vitamin K dependent human plasma proteins. The Gla domain is a structural feature common to all these vitamin K dependent proteins, and immediately behind the Gla domain each protein (except prothrombin) has one or more EGF-like domains. Vitamin K-dependent proteins require Ca ²⁺ ions to exert their physiological functions, and the calcium binding site contains at least a Gla domain and an EGF-like domain. Calcium binding allows these proteins to bind to phospholipids / cell membranes, thus fully exerting their biological activity. Seven human plasma proteins are known to be vitamin K dependent on their biosynthesis. These include prothrombin (factor II, 72 kDa), factor VII / factor VIIa (50/50 kDa), factor IX (55 kDa) or factor IXa, factor X (59 kDa) or factor Xa, protein C ( 62 KDa), protein S (69 KDa) and protein Z (62 KDa).

  It has surprisingly been found that human blood proteins and human plasma proteins covalently linked to hydroxylethyl starch (HES) molecules with a molecular weight of 20 to 200 KDa are suitable for stabilization with Melezitose.

  Melezitose showed an excellent ability to maintain protein activity in both lyophilised formulations and solutions.

  In the present invention, the step of transferring the solution to the solid state is lyophilization after addition of the mesylate. Melezitose may be used in combination with other sugars such as trehalose or sucrose. Melezitose (melezitose or melicitose) is a non-reducing trisaccharide produced in an enzymatic reaction by many insects that eat the sap of plants, including aphids, such as Cinara pilicornis. The IUPAC name is (2R, 3R, 4S, 5S, 6R) -2-[[(2S, 3S, 4R, 5R) -4-hydroxy-2,5-bis (hydroxymethyl) -3-[[(2R, 3R, 4S, 5S, 6R) -3,4,5-trihydroxy-6- (hydroxylmethyl) -2-tetrahydropyranyl] oxy] -2-tetrahydrofuranyl] oxy] -6- (hydroxylmethyl) -tetrahydro Pyrane-3,4,5-triol.

  The molecular weight of Melezitose is 504.44 g / mol.

  A representative structure is represented by the following formula.

  Typically, melezitose is present in amounts up to about 1000 mM. The lower limit depends on the amount of melezitose that provides a sufficient stabilizing effect on the protein of interest. Suitable amounts can be readily determined by one of ordinary skill in the art using the methodology of the example and ordinary knowledge of the person of ordinary skill in the art. The possible range is, for example, 10 mM to about 200 mM or about 10 mM to about 100 mM in relation to the final formulation.

  Preferably, the amount is more than 20 mM or more than 30 mM.

  Preferably, the amount of Meregitose to the amount of human blood protein or human plasma protein is 1:10 to 1: 5000, preferably 1:50 to 1: 150 or 1: 1000 to 1: 3000, calculated on a weight basis That is, the amount of melezitose is greater than the amount of protein.

  In a preferred embodiment, 10 to 100 mg of melezitose are included in the pharmaceutical single dose of protein.

  The subject of the present invention is a composition comprising human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa and Melezitose. The composition may exist in liquid or solid state.

  In one embodiment of the present invention, the composition of the present invention further comprises a bulking agent, a surfactant, a buffer, an additional stabilizer and / or a tonicity modifier.

  The surfactant according to the present invention is a compound that adsorbs to the surface and interface, thereby suppressing the loss of protein activity due to adsorption. This type of loss of activity can occur throughout the pharmaceutical process and during handling of the reconstituted product prior to and during administration to a patient. Commonly used surfactants are polysorbate 80, polysorbate 20 and poloxamer, in particular poloxamer 188. Furthermore, proteins such as albumin, especially recombinant albumin, can also be used as surfactants. Recombinant albumin can also be used in accordance with embodiments of the present invention.

  pH buffer refers to a compound that has the ability to buffer in the optimal pH range of the protein to be formulated. The invention relates to sodium citrate, maleic acid, histidine, 2- (4- (2-hydroxy-ethyl) -1-piperazinyl) -ethanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid as appropriate. (MOPS), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), or Tris (Tris (hydroxymethyl) aminomethane) at pH As a buffer. The buffer is present in an amount that maintains the pH within the range in which the functionality of the protein is maintained. This depends on the protein. The preferred range of each protein, in particular the preferred range of human blood proteins or human plasma proteins having a molecular weight of more than 10 KDa, is known to the person skilled in the art. As an example, sodium citrate maintains the pH at 6.5-7.5. The preferred form of sodium citrate is the dihydrate form. In general, the composition according to the invention may be in lyophilised form but also in solution form such as a solution to be lyophilised, a solution reconstituted from a lyophilised composition.

  An osmotic pressure regulator refers to a compound present in a formulation to balance osmotic pressure. The present invention optionally includes sodium chloride, arginine, glycine, potassium chloride, sugar or sugar alcohol as an osmotic pressure regulator.

  Melezitose exhibits lyophilization protection properties, but additional lyophilization protection (cryo / lyoprotectant) may be present. This is a compound present in the formulation to further reduce or prevent the loss of protein activity during the freezing and drying steps of the lyophilization process and subsequent storage of the lyophilised product. The invention optionally includes non-reducing disaccharides such as sucrose and trehalose and reducing disaccharides such as maltose and lactose as further lyoprotectants.

  Bulking agent refers to the excipients present in the formulation to mechanically support the lyophilised cake and to increase the dry weight. The bulking agent may be crystalline, such as sodium chloride, or amorphous, such as arginine. The amount of bulking agent can be up to 10% by weight with respect to the final formulation. The invention optionally comprises sodium chloride, glycine, mannitol, sucrose or arginine as a bulking agent.

  A further subject matter of the present invention is a medicament for stabilizing long-term, preferably at least 12 months, preferably at least 12 months, more preferably at least 18 months, even more preferably 24 months, dry proteins such as lyophilised formulations. The use of

  The invention is further illustrated by the following non-limiting examples.

Active analysis-Factor VIII activity was measured using a factor VIII chromogenic assay or a one stage assay and the factor VIII unit was expressed in international units (IU).

  The chromogenic assay is the method described in the European Pharmacopoeia. This method is a two-step photometric method that measures the biological activity of factor VIII as a cofactor. Factor VIII activates factor X to factor Xa, which is enzymatically cleaved to yield a product which can be quantified spectrophotometrically.

  A one-stage clotting assay is based on the ability of factor VIII-containing samples to correct the clotting time of factor VIII deficient plasma in the presence of phospholipids, contact activators and calcium ions . The time to appearance of the fibrin clot is measured in one step.

Active analysis- International unit defined by the current WHO factor IX concentrate standard (IU), which measures the biological activity of factor IX using factor IX one-step coagulation assay and / or chromogenic assay The unit of factor IX was expressed in).

  One-step clotting assays are the methods described in the European Pharmacopoeia. The principle of the assay is based on the ability of factor IX containing samples to correct the clotting time of factor IX deficient plasma in the presence of phospholipids, contact activators and calcium ions. The time to appearance of the fibrin clot is measured in one step. Factor IX activity is inversely proportional to clotting time.

  The chromogenic assay is a two-step photometric method. In the first step, activated factor XI (XIa) activates factor IX to factor IXa in the presence of thrombin, phospholipids and calcium. Factor IXa forms an enzyme complex with thrombin activated factor VIII (VIIIa), which activates factor X to form factor Xa in the presence of phospholipids and calcium. In the second step, factor Xa hydrolyses the factor Xa-specific chromogenic substrate, thereby liberating the chromophore group pNA which can be quantified spectrophotometrically. Factor IX activity is directly proportional to the amount of factor Xa generated.

Analysis-Recombinant HES-ized G-CSF
Resource S HPLC analysis of HES-G-CSF: Dilute the sample to 0.1 mg / mL with eluent A. 20 μg are injected onto a Resource S 1 mL column (GE Healthcare, Munich, Germany).
Eluent A: 20 mM sodium acetate, pH 4.0
Eluent B: 20 mM sodium acetate, 0.5 M NaCl, pH 4.0
Flow rate: 1 mL / min Gradient:
0% to 8%, 1.8 to 2.0 minutes 8% to 52%, 2.0 to 13.0 minutes 52% to 100%, 13.0 to 13.6 minutes

  Since it is shown that aggregated HES-G-CSF has a large peak width, the peak width of HES-G-CSF is taken as a quality standard. The increase in peak width is defined as the difference in HES-G-CSF peak width before and after thermal stress or shear stress.

Example
The factor VIII used for the recombinant factor VIII experiment is a recombinant human B domain deleted factor VIII protein produced in the human cell line HEK293F according to the process described in European Patent Application Publication No. 1739179A (Schroder et al) It is. The purification process consists of five chromatography steps, and a high purity factor VIII protein preparation showing human glycosylation-like pattern (Sandberg et al, International Application No. PCT / EP2009 / 060829) was obtained (Winge et al. al, WO 2009/156430 A).

Plasma-Derived Factor IX The material used for these experiments is derived from the commercially available product Nanotov®, which is a high purity Factor IX concentrate that has been SD-treated and nanofiltered. Prior to use in these experiments, the material was further purified using a gel filtration column, and the monomer peak of factor IX was used in subsequent experiments.

Recombinant HES-modified G-CSF
The cell line used is a cell line derived from human embryonic kidney cells 293 (HEK 293) adapted for serum-free growth. This host, HEK293F, was stably transfected with an expression cassette carrying the G-CSF cDNA coding sequence. A strong promoter was used for the cassette. The general process is also described in EP 1739179 (Schroder et al).

  The purification process consisted of four chromatography steps to obtain a high purity G-CSF protein preparation. G-CSF protein was coupled to a hydroxylethyl starch (HES) derivative with a molecular weight of about 100 KDa. Finally, HES-G-CSF was purified from unreacted HES derivative and G-CSF by one-step chromatography to obtain a molecule with an overall molecular weight of about 120 KDa.

Example 1: Stabilization preparation of rFVIII by Melezitose in solution Recombinant factor VIII (rFVIII) was prepared as described in the above experimental section. In this experiment, the stabilizing effect of Melezitose on rFVIII in solution was compared to the commonly used stabilizing agent trehalose. The rFVIII concentration was 100 IU / ml. The composition of the formulation examined in this experiment is shown in Table 1.

  The formulations were stored at + 25 ° C. for 7 days to assess protein activity over time. Samples were taken at regular intervals and analyzed in the chromogenic assay as described in the experimental section above. The results are summarized in Table 2 as a percentage of the initial value.

Conclusion of Example 1: This experiment surprisingly shows that Melezitose, despite the lower molar concentration, has a stabilizing effect equivalent to trehalose on rFVIII in solution.

Example 2 Stabilized Preparation of rFVIII by Melezitose in Lyophilized Form Recombinant factor VIII (rFVIII) was prepared as described in the experimental section above. In this experiment, the stabilizing effect of Melezitose during the lyophilization process and in the lyophilised formulation was compared to the commonly used stabilizing agent trehalose. The rFVIII concentration was 100 IU / ml. The composition of the preparation examined in this experiment is shown in Table 3.

  The solution was lyophilized at 1.5 ml aliquots on a laboratory scale lyophilizer. The protein recovery of the lyophilization step was 93% for formulation 2B and 86% for formulation 2A. Lyophilized samples were stored at + 25 ° C. and + 40 ° C. for up to 4 weeks to assess protein activity over time. The samples were reconstituted with 1.5 mL of distilled water for injection and analyzed in the chromogenic assay described in the experimental section above. The results are summarized in Table 4.

Conclusion of Example 2: Surprisingly, this experiment shows that in the lyophilization step, Melezitose can protect rFVIII at lower molar concentrations than Trehalose, and that Melezitose stabilizes rFVIII better than Trehalose during storage ing.

Example 3 Stabilized Preparation of rFVIII by Melezitose in Lyophilized Form Recombinant factor VIII (rFVIII) was prepared as described in the above experimental section. In this experiment, the stabilizing effects of the lyophilization process and the concentration of Melezitose in the lyophilised formulation were compared, and the stabilizing effect was further compared to the tetrasaccharide stachyose. The rFVIII concentration was 170 IU / ml. The composition of the preparation investigated in this example is shown in Table 5.

  The solution was lyophilized at 1.5 ml aliquots on a laboratory scale lyophilizer. The protein recovery rate of the lyophilization step was 91 to 100% for the preparation containing Melezitose, and 84% for preparation 3D containing stachyose as a stabilizer. Lyophilized samples were stored at + 25 ° C. and + 40 ° C. for up to 12 months to assess protein activity over time.

  The samples were reconstituted with 1.5 mL of distilled water for injection and analyzed in the chromogenic assay described in the experimental section above. The results are summarized in Table 6.

Conclusion of Example 3: This experiment shows that Melezitose performs very well as a lyophilization step and as a stabilizer of rFVIII in lyophilised form. This experiment further shows that stachyose is not a preferred stabilizer for a lyophilised formulation, as it shows less than satisfactory results during storage at both 25 ° C. and 40 ° C. as compared to Melezitose containing formulations. It shows.

Example 4: Stabilized preparation of plasma factor IX by Melezitose in lyophilised form Plasma derived factor IX (pFIX) was prepared as described in the experimental section above. This experiment examines the stabilization effect of meditose on pFIX. The pFIX concentration was 100 IU / ml. The composition of the formulation examined in this experiment is shown in Table 7.

  The solution was lyophilized at 1.5 ml aliquots on a laboratory scale lyophilizer. Lyophilized samples were stored at + 5 ° C., + 25 ° C. and + 40 ° C. for up to 6 months to assess protein activity over time. The samples were reconstituted with 1.5 ml of water for injection and analyzed in the chromogenic assay described in the experimental section above.

Results The protein recovery of the lyophilization step was about 100%. The results of the stability test are shown in Table 8.

Conclusion of Example 4: This experiment surprisingly shows that Melezitose functions well as a stabilizer of factor IX in lyophilized form.

Example 5 Stabilized Preparation of HESed Recombinant G-CSF by Melezitose in Lyophilized Form Recombinant HESed G-CSF (rHES-G-CSF) was prepared as described in the experimental section above. In this experiment, the stabilizing effect of Melezitose on rHES-G-CSF in lyophilised form was compared with the commonly used stabilizer trehalose. The rHES-G-CSF concentration was 0.3 mg / ml, and the composition of the investigated formulation is shown in Table 9.

  The solution was lyophilized at 1.5 ml aliquots on a laboratory scale lyophilizer. After 4 weeks storage at + 40 ° C., protein recovery was determined using the Resource S method described in the experimental section above. The results are summarized in Table 10.

Conclusion of Example 5: This experiment shows that Melezitose has a better stabilizing effect on rHES-G-CSF than equimolar concentrations of the commonly used stabilizing agent trehalose. There is.

Example 6: Stabilized Preparation of Plasma Factor IX by Melezitose in a Lyophilization Step Plasma derived Factor IX (pFIX) was prepared as described in the experimental section above. In this experiment, we investigated the stabilizing effect of Merezitose on pFIX in the lyophilization step compared to the tetrasaccharide stachyose. The pFIX concentration was 100 IU / ml. The compositions of the formulations investigated in this experiment are shown in Table 11.

  The solution was lyophilized at 1.5 ml aliquots on a laboratory scale lyophilizer. Samples were analyzed before and after the lyophilization step in the chromogenic assay described in the experimental section above.

Results The protein recovery for the lyophilization step was approximately 100% for formulation 6A, but 84% for formulation 6B.

Conclusions of Example 6: This experiment shows that Mereditose functions well as a stabilizer of factor IX in the lyophilization step. However, stachyose is not a good candidate as a stabilizing agent, as significant loss of activity occurs in the lyophilization step.

Conclusions of Example 6: This experiment shows that Mereditose functions well as a stabilizer of factor IX in the lyophilization step. However, stachyose is not a good candidate as a stabilizing agent, as significant loss of activity occurs in the lyophilization step.
(Supplementary Note) The present disclosure includes the following aspects.
<1> Pharmaceutical or biological significance selected from the group consisting of Factor VII, Factor VIIa, Factor IX, Factor VIII, and HES-modified G-CSF, and their mutant proteins or derivatives A method for stabilizing human plasma protein, which is human plasma protein or recombinant human plasma protein, and which has a molecular weight of 50 KDa to 200 KDa,
Adding Melezitose to a solution containing human plasma protein having a molecular weight of 50 KDa to 200 KDa so as to be 10 mM to 200 mM;
Adding at least one bulking agent selected from the group consisting of sodium chloride, arginine and glycine,
Adding at least one surfactant selected from the group consisting of poloxamer 188, polysorbate 80, and polysorbate 20,
Sodium citrate, maleic acid, histidine, 2- (4- (2-hydroxy-ethyl) -1-piperazinyl) -ethanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid (MOPS), 2- At least one selected from the group consisting of (N-morpholino) ethanesulfonic acid (MES), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), and Tris (tris (hydroxymethyl) aminomethane) Adding one pH buffer,
A method of stabilizing human plasma proteins, wherein said molecular weight is between 50 kDa and 200 kDa, further comprising
<2> The method according to <1>, wherein the solution is transferred to a solid state.
<3> The method according to <1>, wherein the solution is transferred to a solid state by lyophilization.
<4> The method according to <1>, wherein the human plasma protein is stabilized in a solution.
<5> The method according to any one of <1> to <4>, wherein the melezitose is added to a solution containing human plasma protein having a molecular weight of 50 kDa to 200 kDa so as to be 10 mM to 100 mM. .
<6> The method according to any one of <1> to <5>, wherein the at least one surfactant comprises poloxamer 188.
<7> The method according to any one of <1> to <6>, wherein the at least one surfactant comprises polysorbate 80.
<8> The method according to any one of <1> to <7>, wherein the at least one surfactant comprises polysorbate 20.
<9> The method according to any one of <1> to <8>, wherein the at least one pH buffer comprises sodium citrate.
<10> The method according to any one of <1> to <9>, wherein the at least one extender comprises sodium chloride.
<11> The method according to any one of <1> to <10>, wherein the at least one extender comprises arginine.
The method as described in any one of <1>-<11> which further includes adding at least 1 sort (s) of stabilizer other than <12> melezitose, and at least 1 sort (s) of osmotic pressure regulator.
<13> Human plasma selected from the group consisting of Factor VII, Factor VIIa, Factor IX, Factor VIII, and HES-modified G-CSF, and their mutant proteins or derivatives, having a molecular weight of 50 kDa to 200 kDa With proteins
With 10 mM to 200 mM Melezitose,
At least one bulking agent selected from the group consisting of sodium chloride, arginine and glycine;
At least one surfactant selected from the group consisting of poloxamer 188, polysorbate 80, and polysorbate 20;
Sodium citrate, maleic acid, histidine, 2- (4- (2-hydroxy-ethyl) -1-piperazinyl) -ethanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid (MOPS), 2- At least one selected from the group consisting of (N-morpholino) ethanesulfonic acid (MES), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), and Tris (tris (hydroxymethyl) aminomethane) One pH buffer,
A composition comprising:
<14> The composition according to <13>, which is in a liquid or solid state.
The composition as described in <14> which is a <15> liquid state.
<16> The composition according to any one of <13> to <15>, further comprising a stabilizer other than melezitose, and / or an osmotic pressure regulator.
<17> Human plasma selected from the group consisting of Factor VII, Factor VIIa, Factor IX, Factor VIII, and HES-modified G-CSF, and their mutant proteins or derivatives, having a molecular weight of 50 kDa to 200 kDa. To stabilize the protein during in-process stabilization, during the lyophilization process, in solution, during purification, or during production, or for long-term stabilization that is at least 6 months,
10 mM to 200 mM Melezitose;
At least one bulking agent selected from the group consisting of sodium chloride, arginine and glycine;
At least one surfactant selected from the group consisting of poloxamer 188, polysorbate 80, and polysorbate 20;
Sodium citrate, maleic acid, histidine, 2- (4- (2-hydroxy-ethyl) -1-piperazinyl) -ethanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid (MOPS), 2- At least one selected from the group consisting of (N-morpholino) ethanesulfonic acid (MES), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), and Tris (tris (hydroxymethyl) aminomethane) One pH buffer,
Use of.
<18> The use according to <17>, wherein the human plasma protein is stabilized in solution.
<19> The use according to <17> or <18>, wherein the production is production in cell culture.
<20> The use according to any one of <17> to <19>, wherein the long-term stabilization is performed to extend the effective period.
<21> The use according to any one of <17> to <20>, wherein the long-term stabilization is performed, and the long-term is at least 12 months.

Claims (21)

Human plasma proteins of pharmaceutical or biological significance selected from the group consisting of Factor VII, Factor VIIa, Factor IX, Factor VIII, and HES-modified G-CSF, and mutant proteins or derivatives thereof Or a recombinant human plasma protein, which has a molecular weight of 50 kDa to 200 kDa and is a method for stabilizing human plasma protein,
Adding Melezitose to a solution containing human plasma protein having a molecular weight of 50 KDa to 200 KDa so as to be 10 mM to 200 mM;
Adding at least one bulking agent selected from the group consisting of sodium chloride, arginine and glycine,
Adding at least one surfactant selected from the group consisting of poloxamer 188, polysorbate 80, and polysorbate 20,
Sodium citrate, maleic acid, histidine, 2- (4- (2-hydroxy-ethyl) -1-piperazinyl) -ethanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid (MOPS), 2- At least one selected from the group consisting of (N-morpholino) ethanesulfonic acid (MES), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), and Tris (tris (hydroxymethyl) aminomethane) Adding one pH buffer,
A method of stabilizing human plasma proteins, wherein said molecular weight is between 50 kDa and 200 kDa, further comprising   The method of claim 1, wherein the solution is transferred to a solid state.   The method according to claim 1, wherein the solution is transferred to a solid state by lyophilization.   The method of claim 1, wherein the human plasma protein is stabilized in solution.   The method according to any one of claims 1 to 4, wherein the melezitose is added to a solution containing human plasma protein having a molecular weight of 50 KDa to 200 KDa to be 10 mM to 100 mM.   6. The method of any one of the preceding claims, wherein the at least one surfactant comprises poloxamer 188.   The method according to any one of the preceding claims, wherein the at least one surfactant comprises polysorbate 80.   8. The method of any one of claims 1-7, wherein the at least one surfactant comprises polysorbate 20.   9. The method of any one of the preceding claims, wherein the at least one pH buffer comprises sodium citrate.   10. The method of any one of claims 1-9, wherein the at least one bulking agent comprises sodium chloride.   11. The method of any one of claims 1 to 10, wherein the at least one bulking agent comprises arginine.   12. The method according to any one of the preceding claims, further comprising adding at least one stabilizer other than mellisitose, and at least one osmo-regulator. A human plasma protein having a molecular weight of 50 KDa to 200 KDa selected from the group consisting of Factor VII, Factor VIIa, Factor IX, Factor VIII, and HES-modified G-CSF, and muteins or derivatives thereof,
With 10 mM to 200 mM Melezitose,
At least one bulking agent selected from the group consisting of sodium chloride, arginine and glycine;
At least one surfactant selected from the group consisting of poloxamer 188, polysorbate 80, and polysorbate 20;
Sodium citrate, maleic acid, histidine, 2- (4- (2-hydroxy-ethyl) -1-piperazinyl) -ethanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid (MOPS), 2- At least one selected from the group consisting of (N-morpholino) ethanesulfonic acid (MES), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), and Tris (tris (hydroxymethyl) aminomethane) One pH buffer,
A composition comprising:   The composition according to claim 13, which is in the liquid or solid state.   The composition according to claim 14, which is in a liquid state.   The composition according to any one of claims 13 to 15, further comprising a stabilizer other than mellisitose, and / or an osmotic pressure regulator. A human plasma protein having a molecular weight of 50 KDa to 200 KDa selected from the group consisting of Factor VII, Factor VIIa, Factor IX, Factor VIII, and HES-modified G-CSF, and muteins or derivatives thereof, To stabilize during in-process stabilization, during lyophilization process, in solution, during purification, or during production, or for long-term stabilization that is at least 6 months,
10 mM to 200 mM Melezitose;
At least one bulking agent selected from the group consisting of sodium chloride, arginine and glycine;
At least one surfactant selected from the group consisting of poloxamer 188, polysorbate 80 and polysorbate 20; and sodium citrate, maleic acid, histidine, 2- (4- (2-hydroxy-ethyl) -1-piperazinyl ) -Ethanesulfonic acid (HEPES), 3- (N-morpholino) propanesulfonic acid (MOPS), 2- (N-morpholino) ethanesulfonic acid (MES), piperazine-N, N'-bis (2-ethanesulfone) At least one pH buffer selected from the group consisting of: acid) (PIPES), and Tris (Tris (hydroxymethyl) aminomethane),
Use of.   The use according to claim 17, wherein the human plasma protein is stabilized in solution.   19. The use according to claim 17 or claim 18, wherein said production is production in cell culture.   20. The use according to any one of claims 17-19, wherein the long term stabilization is performed to extend the shelf life. 21. The use according to any one of claims 17 to 20, wherein the long term stabilization is performed and the long term is at least 12 months.