CN101262918B - Method for the determination of poloxamers - Google Patents
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Abstract
The invention relates to the analytical determination of poloxamers in a liquid protein sample.
Description
Technical field
The present invention relates to belong in the liquid protein quality sample analyzing and testing field of the surfactant of poloxamer class (poloxamers).
Background of invention
Poloxamer class (poloxamers) is the non-ionic block copolymer of oxirane (EO) and expoxy propane (PO).They are used as surfactant, emulsifying agent, solubilizer and dispersant in pharmaceutical preparation.
The analytical method of an evaluation poloxamer (poloxamer) of knowing is a calorimetry, this method detect compound that poloxamer and cobalt thiocyanate (II) form 320 and the UV of 620nm absorb.
(Journal of Pharmaceutical and Biomedical Analysis such as Yun Mao, 35 (2004), 1 127) size exclusion chromatography (SEC) (using chromatographic column, is the phase that flows with THF) and the application of refractive index (RI) analysis on poloxamer detects have been described.This method once was used for pharmaceutical preparation Avapro, Gabapentin (Neurontin) and the pseudoephedrine hydrochloride (Sudafed) that active component is " a little molecule ".By SEC, little molecule can separate with high molecular weight material poloxamer class easily.
Size exclusion chromatography (size-exclusion chromatography) (SEC) is also referred to as gel permeation chromatography (GPC), adopts porous particle to separate the molecule of different volumes.Be generally used for the molecular weight and the molecular weight distribution of isolating polymer molecule and definite polymer.Molecule less than the aperture can enter particle, so its path and transit time all are longer than the big molecule that can not enter particle.All molecules greater than the aperture will can not be retained and together by wash-out.The molecule that can enter in the hole depends on bulk of molecule and shape in intragranular average retention time.Therefore, different molecular is by total transit time difference of pillar.
At present also have no idea quantitatively to detect the poloxamer class in the protein example, because wherein the molecular weight of protein is suitable with the poloxamer class.
Therefore, especially need to develop the method for poloxamer class in the quantitative detection protein example, the molecular weight of wherein said protein is between 5-70kDa, between the preferred 20-70kDa.
Summary of the invention
The present invention relates to quantitatively to detect the method for (especially in the liquid protein quality sample, in liquid pharmaceutical formulation) poloxamer in the protein example.Specifically, the invention provides the method for poloxamer concentration in a kind of quantitative detection protein example.Therefore, in any time of protein formulation in about 2 years shelf life, can detect the amount of poloxamer in the preparation.
Quantitatively the method for poloxamer comprises the step of described sample being carried out following processing in the tracer liquid protein example:
(a) separate the separating step of the composition in the described sample with the SEC post; With
(b) the analytical refraction rate is to detect the detection step of poloxamer.
Preferably, the present invention relates to the method for poloxamer in the quantitative tracer liquid protein example, comprise the step of described sample being carried out following processing:
(a) separating step of employing SEC post;
(b) elution step of the mobile phase of employing; With
(c) randomly, detect the detection step of poloxamer.
Therefore, the size exclusion chromatography in conjunction with the phase elution step that flows can separate poloxamer and all the other compositions.
In the description of another step, detect poloxamer mutually by analyzing wash-out, for example, adopt RI (refractive index) detection system.
The accompanying drawing summary
Fig. 1 is the chromatogram that quantitatively detects poloxamer 188 in a kind of hCG preparation.The elution time of poloxamer 188 (peak area) is about 14-16 minute (retention time can be used as the function of flow velocity and changes) among this embodiment.Area under a curve is the poloxamer 188 in this hCG sample quantitatively.
Abbreviation
Used following abbreviation in the specification of the present invention:
FSH: follicle-stimulating hormone (FSH);
R-FSH; R-LH; R-hCG; R-GH; R-IFN β, r-TSH: RECFSH, LH, hCG, GH, INF β, TSH;
HFSH: people FSH;
R-hFSH: restructuring people FSH
RI: refractive index
KD or Kd or kDa: kilodalton
SEC: size exclusion chromatography
RT: room temperature
WFI: water for injection
The synonym of the Pluronic F68 of poloxamer 188:BASF company
Detailed Description Of The Invention
The present invention relates to a kind of method that can quantitatively detect surfactant poloxamer in the protein example easily.Preferably, described protein example is the liquid protein quality sample.Described liquid protein quality sample can be any liquid preparation form, preferably the liquid pharmaceutical formulation as describing below.In a specific embodiment, described liquid medicine protein example splendid attire is used for single dose or multiple dose administration in bottle.
In another embodiment, protein example to be analyzed is cryodesiccated, before detection it is dissolved in the suitable aqueous solvent.
Quantitatively the method for poloxamer comprises the step of described sample being carried out following processing in the tracer liquid protein example:
(a) separate the separating step of described sample composition with SEC post (specifically being the SE-HPLC post); With
(b) the analytical refraction rate is to detect the detection step of poloxamer.
Preferably, the invention provides the method for poloxamer in a kind of quantitative tracer liquid protein example, comprise the step of described sample being carried out following processing:
(a) separating step of employing SEC post;
(b) elution step of the mobile phase of employing; With
(c) randomly, detect the detection step of poloxamer.
Usually, the SEC post is the SE-HPLC post.
In one embodiment, described poloxamer is a poloxamer 188.
One preferred embodiment in, protein molecular weight and poloxamer in the liquid protein quality sample are suitable.
In a particularly preferred embodiment, the protein molecular weight in the liquid protein quality sample is between 5-70kDa, more preferably between the 20-70kDa.
The mass ratio of protein and each poloxamer is preferably 1: 3-10: between 1, and more preferably 1: 2-7: between 1.
According to the present invention, the example of protein comprises mammiferous protein, and for example growth hormone comprises human growth hormone (HGH) and BGH; Somatotropin releasing factor; Parathormone; Thyrotropic hormone; Lipoprotein; The a-1-antitrypsin; INSULIN A-chain; Insulin B-chain; Proinsulin; Follicle-stimulating hormone (FSH); Human chorionic gonadtropin; Calcitonin; Metakentrin; Hyperglycemic factor; Clotting factor, for example Factor IX C, factors IX, tissue factor and von Willebrand factor (vonWillebrands factor); Anticoagulin, for example protein C; ANF; Pulmonary surfactant; Plasminogen activator, for example dextren sulfate kinases (asurokinase) or tissue-type plasminogen activator (t-PA); Bombazine; Fibrin ferment; Tumor necrosis factor-alpha and-β; Enkephalinase; RANTES (normal T cellular expression and regulation of secretion activation factor); Human macrophage inflammatory protein (MIP-1-α); Seralbumin, for example human serum albumins; Mullerian inhibiting substance; Relaxain A-chain; Relaxain B-chain; Relaxation precipitinogen; Mouse promoting sexual gland hormone connection peptide; Deoxyribonuclease; Inhibin; Activin; VEGF (VEGF); Hormone or growth factor receptors; The whole albumen that connects; Albumin A or D; Rheumatoid factor; Neurotrophic factor, for example bone derived neurotrophic factor (BDNF), neurotrophic factor-3 ,-4 ,-5 or-6 (NT-3, NT-4, NT-5 or NT-6) or nerve growth factor, for example NGF-P; The growth factor (PDGF) in blood platelet source; Fibroblast growth factor, for example aFGF and bFGF, especially FGF-18; EGF (EGF); TGF (TGF), for example TGF-α and TGF-β comprise TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4 or TGF-β 5; Insulin-like growth factor I and-II (IGF-I and IGF-ll); Des (I-3)-IGF-1 (brain IGF-I); Insulin-like growth factor binding protein; CD albumen, for example CD3, CD4, CD8, CD19 and CD20; Hematopoietin (EPO); TPO (TPO); Bone-inducing factor; Osteopontin; The immunotoxin class; Bone morphogenetic protein (BMP); Interferon, for example interferon-' alpha ' ,-β and-γ; Colony stimulating factor (CSFs), for example M-CSF, GM-CSF and G-CSF; Interleukins class (ILs), for example IL-1 to IL-10; Superoxide dismutase; T-cell receptors; Surface membrane protein; Decay accelerating factor (DAF); Viral antigen, for example AIDS coating part; The transport protein class; Homing receptor; Addressin; Regulate albumen; Immunoadhesin; Antibody; And the bioactive fragment or the variant of any top listed polypeptide.
Preferably, protein according to the present invention is selected from follicle-stimulating hormone (FSH) (FSH), human chorionic gonadtropin (CG), metakentrin (LH), interferon beta (IFN-β), polyethylene glycol interferon beta (PEG-IFN-β), growth hormone (GH), thyrotropic hormone (TSH), desmocyte growth factor-21 8 (FGF-18) or osteopontin.
FSH, CG, LH and TSH are the glycoprotein that belongs to gonadotrophins.Gonadotrophins is used for the treatment of sterility.
IFN-β is the glycoprotein that belongs to the interleukins class.IFN-β is used for the treatment of multiple sclerosis.
PEG-IFN-β is the IFN-β of polyglycol chain derivatization, and by contrast, its stability is higher.
Growth hormone is non-glycosylated protein.It is used for the treatment of children or adult's GHD.
FGF-18 is used for the treatment of osteoarthritis.
Osteopontin matter is glycosylated single chain polypeptide.
In a preferred specific embodiment, described protein is heterodimer, for example gonadotropic hormone class (FSH, LH, CG, TSH and variant thereof).In another embodiment, described protein is growth hormone (GH) or interferon beta (IFN-β or variant, for example Pegylation variant).In another embodiment, described protein is FGF-18 or osteopontin.
In a preferred specific embodiment, the liquid protein quality sample comprises the protein of one or more treatment usefulness.Preferably, these samples do not comprise non--protein therapeutic material, for example small molecular weight compounds.
Follicle-stimulating hormone (FSH) used herein or FSH refer to the FSH that obtains as the total length mature protein, include but not limited to that reorganization produces or is derived from people's (for example postmenopausal women's urine) and separates the people FSH that obtains i.e. " hFSH ".
Method of the present invention can be used for protein natural and reorganization.In a specific embodiment, described protein formulation is people's RECFSH, LH, CG, TSH, GH or IFN-β.
Follicle-stimulating hormone (FSH) (FSH) is the glycoprotein that belongs to gonadotrophins.FSH is used for the treatment of women and the patient's male sex sterility and reproductive disease, for example induces the oligospermia male sex's sperm to generate.
Metakentrin (LH) is the promoting sexual gland hormone by the anteriorpituitary secretion.LH and FSH unite OI (ovulation induction) and the COH (controlled super ovulation) that is used for female patients, it is extremely low or to the patient of LH tolerance to be particularly useful for endogenous LH levels, for example suffers from hypogonadotropic hypogonadism (HH, WHO organizes I) women or older patient (promptly 35 years old or more than), and the embryo implants the patient that problem or early abortion are arranged.
Human chorionic gonadtropin (CG) is the promoting sexual gland hormone that is produced and obtained from pregnant woman urine by placenta.CG acts on the acceptor identical with LH and causes identical reaction.The circulating half-life of CG is longer than LH, therefore is used as the long-acting source of LH-activity usually.CG is used for intending like natural LH peak and triggers ovulating in OI and the COH therapy.A kind of human chorionic gonadotrophin (hCG) injection is used for share to stimulate at FSH or FSH and LH triggering ovulation when finishing.In order to provide LH-activity to the patient who needs the LH-activity as previously mentioned between stimulation period, CG also can be used for OI and COH with FSH jointly between stimulation period.
FSH, LH and CG are heterodimer, the member of glycoprotein hormones family (also comprising thyrotropic hormone (TSH)).This family member is a heterodimer, comprises α-and β-subunit.These subunits combine by noncovalent interaction.People FSH (hFSH) heterodimer is made up of following: (i) 92 amino acid whose ripe glycoprotein α subunits (also are common in the plain family member's (being human chorionic gonadtropin (" CG "), metakentrin (" LH ") and thyrotropic hormone (" TSH ")) of other human protein kinase; (ii) distinctive 111 amino acid whose ripe β subunits of FSH.People LH heterodimer is made up of following: (i) 92 amino acid whose ripe glycoprotein α subunits; (ii) 112 of a distinctive maturation of LH amino acid whose β subunits.Because interact with anticorrisive agent, surfactant and other excipient, the α of glycoprotein and β subunit may dissociate easily in the preparation.And the defection of separating of subunit causes bioactive forfeiture.
FSH is used for intramuscular (IM) or subcutaneous (SC) injection by preparation.In the specific embodiment, FSH provides with freeze-drying (solid) form, is packaged in the bottle or ampoule bottle of 75IU/ bottle and 150IU/ bottle, and the shelf life is 2-25 ℃ a year and a half to two year.Freeze-drying prods and water for injection (WFI) are mixed, reconstitute the solution that is used to inject.In addition, also have a kind of FSH preparation (Gonal-F pen) of liquid, contain 22 μ g/0.5ml, 33 μ g/0.75ml or 66 μ g/1.5ml FSH and poloxamer 188, sucrose, buffer solution, methionine and metacresol.
Therefore, FSH has been mixed with single dose and the multiple dose liquid form in bottle or the ampoule bottle.Single dose form must keep stable and effective storage period before use.It is stable and effectively that the multiple dose form not only must keep before use storage period, and during the administration of whole multiple dose operational version, behind the unseal of ampoule bottle, also to keep stablizing, effective and relative aseptic.For this reason, the multiple dose form contains bacteriostatic agent usually, for example phenmethylol or metacresol.
LH is used for intramuscular (IM) or subcutaneous (SC) injection by preparation.LH provides with freeze-drying (solid) form, is packaged in the bottle or ampoule bottle of 75IU/ bottle, and the shelf life is 2-25 ℃ a year and a half to two year.To reconstitute the solution that is used to inject after freeze-drying prods and water for injection (WFI) mixing.Except that LH, Luveris
TMAlso contain following excipient: sucrose, buffer solution, Polysorbat 20, methionine.Recently, the LH preparation that contains poloxamer 188 also has description (WO 2004/087213).
The composition of liquid medicine that contains hCG is also arranged on the market.For example, Ovitrelle
TMContain sweet mellow wine, methionine, poloxamer 188 in pH 7 phosphate buffers.
Term " variant " refers to and comprises between amino acid sequence, glycosylation pattern or subunit and to connect different with people FSII, LH, CG, TSH, IFN-β or GH but show corresponding bioactive those molecules of FSH, LH, CG, TSH, IFN-β or GH.Example comprises CTP-FSH, and it is a long-acting modification RECFSH, is made up of wild type α-subunit and heterozygosis β-subunit, and wherein the β of the carboxyl terminal peptide of hCG and FSH-subunit C holds fusion, sees (Endocrinology such as LaPoIt; 1992,131,2514-2520) or (development of long-acting r-hFSH activator and evaluation such as Klein; Human Reprod.2003,18, description 50-56).Also comprise strand CTP-FSH, it is a single chain molecule, is made up of following sequence (holding the end to C-from N-):
| βFSH | βhCG-CTP(113-145) | αFSH |
Wherein, β FSH represents β-subunit of FSH, β hCG CTP (113-145) represents the carboxyl terminal peptide of hCG, α FSH represents α-subunit of FSH, describes (pharmacokinetics and the pharmacodynamics of strand recombined human follicular stimulating hormone in the rhesus macaque body that contains the human chorionic gonadotrophin c-terminal peptides such as seeing Klein; Fertility ﹠amp; Sterility; 2002,77,1248-1255).The example of other FSH variant comprises the FSH molecule that mixes additional glycosylation site on α and/or the β-subunit, referring to the claim 10 of WO 01/58493 (Maxygen), particularly WO 01/58493 and 11 described those; And the FSH molecule with S-S key between subunit, referring to WO 98/58957.
FSH variant as referred to herein also comprises the carboxyl-terminal deletion of β-subunit, and they are shorter than the total length FSH protein of maturation.FSH heterodimer or FSH variant heterodimer all can produce by suitable method, for example recombinant technique, obtain from the natural origin isolated or purified, or by chemical synthesis, or its any combination.
" variant " also comprises the Pegylation form of protein.
The use of term " reorganization " is meant adopts recombinant DNA technology (for example referring to WO 85/01958) to produce FSH, LH, CG, TSH, GH, IFN β or variant.For example, a kind of method of utilizing recombinant technique to express FSH or LH is by using coding FSH or the α of LH and the dna sequence dna transfecting eukaryotic cells of β subunit, and no matter to be contained in a carrier still be on two carriers having separately each subunit of promoter, European patent: EP 0 211 894 and EP 0 487 512 are seen in description.Another example that utilizes recombinant technique to produce FSH or LH is to connect with operability with homologous recombination allos is regulated the endogenous sequence that fragment is inserted coding FSH or LH subunit, and European patent is seen in its description: EP 0 505 500 (Applied Research Systems ARS Holding NV (Applied Research Systems ARS Holding NV)).
FSH that the present invention uses or FSH variant not only can be by recombination method (comprising from mammalian cell) generations, and can for example originate that the urine purifying obtains from other biological.Acceptable method comprises those methods of following document description: Hakola, K.Molecular and Cellular Endocrinology, 127:59-69,1997; Keene etc., J.Biol.Chem., 264:4769-4775,1989; Cerpa-Poljak etc., Endocrinology, 132:351-356,1993; Dias etc., J.Biol.Chem., 269:25289-25294,1994; Flack etc., J.Biol.Chem., 269:14015-14020,1994; With Valove etc., Endocrinology, 135:2657-2661,1994; United States Patent (USP) 3,119,740 and United States Patent (USP) 5,767,067.
Metakentrin used herein or LH refer to the LH that obtains as the total length mature protein, include but not limited to reorganization generation or the people LH that obtains from people source (for example postmenopausal women's urine) separation.The protein sequence of human glucoprotein α subunit is shown in SEQ ID NO:1, and the protein sequence of people LH β subunit is shown in SEQ ID NO:6.LH recombinates in a preferred specific embodiment.
Term " LH variant " refers to and comprises between amino acid sequence, glycosylation pattern or subunit and to connect different with people LH but show those molecules of LH activity.
LH heterodimer or LH variant heterodimer can produce with any suitable method, for example recombinant technique, obtain from the natural origin isolated or purified, or by chemical synthesis, or its any combination.
Liquid protein quality sample of the present invention also comprises the mixture (WO2004/087213) of FSH/LH and variant, and the mixture of FSH and hCG and variant (WO 2004/105788).
Term " aqueous diluent " refers to moisture liquid flux.The aqueous solvent system can only be made up of water, also can be by water and one or more mixable solvent composition, and the solute that can contain dissolving is as sugar, buffer solution, salt or other excipient.Nonaqueous solvents commonly used is the organic alcohol of short chain as methyl alcohol, ethanol, propyl alcohol etc., as the chain ketones of acetone, as the polyalcohol of glycerine.
Term " antibacterial " or " bacteriostatic agent " refer to compound or the composition that adds as antiseptic in preparation.The storage preparation that the present invention contains FSH or FSH variant or FSH and LH preferably meets the guide of legal or regulation of the anticorrosion validity of the multipurpose product of implementing about commerce (particularly supplying the product of human).The example of bacteriostatic agent comprises phenol, metacresol, paracresol, orthoresol, chloreresol, phenmethylol, alkyl paraben class (as methyl esters, ethyl ester, propyl ester, butyl ester etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal.
Term " buffer solution " or " physiologically acceptable buffer solution " refer to that known application on pharmacy or animal doctor is safe and has the compound solution of the pH value of preparation being kept or is controlled at the effect in the ideal pH value scope of said preparation to preparation.Control pH value includes but not limited to following compound at moderate acid pH value to the acceptable buffer solution of medium basic pH value: phosphate, acetate, citrate, arginine, TRIS and histidine." TRIS " refers to 2-amino-2-methylol-1, ammediol and pharmaceutically acceptable salt thereof.Preferred buffer solution is the phosphate buffer that contains salt or acceptable salt.
Term " phosphate buffer " refers to the solution that contains phosphoric acid or its salt and transferred to the ideal pH value.Usually, phosphate buffer is from phosphoric acid or phosphate (including but not limited to sodium and sylvite) preparation.Some phosphate have been known in this area, for example sodium dihydrogen phosphate and potassium dihydrogen phosphate, disodium-hydrogen and potassium phosphate,monobasic and sodium phosphate and potassium phosphate.Known that phosphate is that hydrate forms with salt exists.The pH value scope of phosphate buffer is very wide, and for example about pH 4 is to the scope of about pH 10, and preferably about pH 5 is to the scope of about pH 9, and more preferably from about pH 6.0 is to the scope of about pH 8.0, and most preferably from about pH 7.0.
Term " bottle " general reference is fit to preserve with the sealed, sterile state reservoir of solid or liquid form FSH.For example, the bottle that herein uses comprises that ampoule, injection tube, blister-pack or other are fit to by syringe, pumping (comprise etc. ooze), conduit, percutaneous plaster, lung or carry the reservoir of FSH through the mucous membrane spray regime to the patient.The bottle that is fit to parenteral, lung, uses through mucous membrane and cutaneous penetration packaging product is known to those skilled in the art and generally acknowledged.
Term " multiple dose use " comprises the FSH preparation that uses single bottle, ampoule or injection tube or uses the once described protein formulation of above injection, for example inject 2,3,4,5,6 times or more than.Preferably, be at least or be at least about 12 hours, in 24 hours, 48 hours equal times, preferably injecting mostly being most or being about at most in time of 12 days.Also can inject, for example every injection in 6,12,24,48 or 72 hours by the time interval.
Term " stability " refers to physics, chemistry and the conformational stability (comprising keeping of biological effect) of given protein in the preparation of the present invention.The chemical degradation of protein molecule or the polymer, the heterodimer that assemble to form higher progression are dissociated at least a bioactive other structural modifications of the included polypeptide of single aggressiveness, de-glycosylation, glycosylation modified, oxidation (particularly α-subunit oxidation in the heterodimer) or any minimizing the present invention, all can cause the protein formulation instability.
" stable " solution or preparation are meant such solution or preparation: the degree of aspects such as protein degradation wherein, modification, gathering, loss of bioactivity is to control acceptably, and can unacceptably not increase along with the time.Preferably, preparation is reaching the activity of proteins that keeps in the time in 2 years at least about 80% mark.
The problem to be solved in the present invention provides a kind of method of determining the amount of poloxamer in the protein example easily and quickly.The poloxamer useful as surfactants, it is selected from the block copolymer of oxirane and expoxy propane, preferred Pluronic
F77, Pluronic F87, Pluronic F88 and Pluronic
F68, preferred especially Pluronic F68 (BASF, Pluronic F68 is also referred to as poloxamer 188).
The front is mentioned, and the shelf life of pharmaceutical preparation should be 2-25 ℃ and reaches 2 years.This means that the preparation expection during this period of time keeps stable.Because liquid preparation contains multiple excipient, might be directly or by decomposing the stability of remote-effects protein formulation, therefore, be necessary to develop the analysis tool of the described preparation stability of assessment.
More particularly, the surfactant poloxamer is the block copolymer of oxirane (EO) and expoxy propane (PO).Expoxy propane (PO) is embedded between two oxirane (EO) section, is sandwich shape.
Poloxamer class (poloxamers) is synthetic by two-step method:
Add expoxy propane with control mode in two hydroxyls in propane diols, produce hydrophobe with desired molecular weight; And
Add oxirane, described hydrophobe is clipped between the hydrophilic radical.
Poloxamer surfactants is also referred to as pluoronics (pluronics).
Pluronic
The polyoxyethylene of F77 (hydrophilic) percentage is 70%, and hydrophobe (polyoxypropylene) molecular weight is about 2,306Da.
The polyoxyethylene of Pluronic F87 (hydrophilic) percentage is 70%, and hydrophobe (polyoxypropylene) molecular weight is about 2,644Da.
The polyoxyethylene of Pluronic F88 (hydrophilic) percentage is 80%, and hydrophobe (polyoxypropylene) molecular weight is about 2,644Da.
The polyoxyethylene of Pluronic F68 (hydrophilic) percentage is 80%, and hydrophobe (polyoxypropylene) molecular weight is about 1,967Da.
The representative property of Pluronic F77 is as follows:
Mean molecule quantity: 6600;
Molten/pour point: 48 ℃;
20 ℃ of physical form: solid;
Cps:480[25 ℃ of liquid of viscosity (Brookfield), 60 ℃ of pastes, 77 ℃ of solids];
25 ℃ of surface tension, dynes/cm:
0.1% concentration: 47.0
0.01% concentration: 49.3
0.001% concentration: 52.8
25 ℃ of interfacial tensions, dynes/cm (with respect to Nujol):
0.1% concentration: 17.7
0.01% concentration: 20.8
0.01% concentration: 25.5
25 ℃ of Draves wetting times (second)
1.0% concentration:＞360
0.1% concentration:＞360
Foam height
Ross Miles method, 0.1%, 50 ℃ of mm: 100
Ross Miles method, 0.1%, 26 ℃ of mm: 47
Dynamically, 0.1%, mm, 400ml/min:＞600
Cloud point in the aqueous solution, ℃
1% concentration:＞100
10% concentration:＞100
HLB (hydrophile-lipophile balance): 25
The representative property of Pluronic F87 is as follows:
Mean molecule quantity: 7700;
Molten/pour point: 49 ℃;
20 ℃ of physical form: solid;
25 ℃ of viscosity (Brookfield) cps:700[liquid, 60 ℃ of pastes, 77 ℃ of solids];
25 ℃ of surface tension, dynes/cm:
0.1% concentration: 44.0
0.01% concentration: 47.0
0.001% concentration: 50.2
25 ℃ of interfacial tensions, dynes/cm (with respect to Nujol);
0.1% concentration: 17.4
0.01% concentration: 20.3
0.01% concentration: 23.3
25 ℃ of Draves wetting times (second)
1.0% concentration:＞360
0.1% concentration:＞360
Foam height
Ross Miles method, 0.1%, 50 ℃ of mm: 80
Ross Miles method, 0.1%, 26 ℃ of mm: 37
Dynamically, 0.1%, mm, 400ml/min:＞600
Cloud point in the aqueous solution, ℃
1% concentration:＞100
10% concentration:＞100
HLB (hydrophile-lipophile balance): 24
The representative property of Pluronic F88 is as follows:
Mean molecule quantity: 11400;
Molten/pour point: 54 ℃;
20 ℃ of physical form: solid;
25 ℃ of viscosity (Brookfield) cps:2300[liquid, 60 ℃ of pastes, 77 ℃ of solids];
25 ℃ of surface tension, dynes/cm;
0.1% concentration: 48.5
0.01% concentration: 52.6
0.001% concentration: 55.7
25 ℃ of interfacial tensions, dynes/cm (with respect to Nujol);
0.1% concentration: 20.5
0.01% concentration: 23.3
0.01% concentration: 27.0
25 ℃ of Draves wetting times (second)
1.0% concentration:＞360
0.1% concentration:＞360
Foam height
Ross Miles method, 0.1%, 50 ℃ of mm: 80
Ross Miles method, 0.1%, 26 ℃ of mm: 37
Dynamically, 0.1%, mm, 400ml/min:＞600
Cloud point in the aqueous solution, ℃
1% concentration:＞100
10% concentration:＞100
HLB (hydrophile-lipophile balance): 28
The representative property of Pluronic F68 is as follows:
Mean molecule quantity: 8400;
Molten/pour point: 52 ℃;
20 ℃ of physical form: solid;
25 ℃ of viscosity (Brookfield) cps:1000[liquid, 60 ℃ of pastes, 77 ℃ of solids];
25 ℃ of surface tension, dynes/cm;
0.1% concentration: 50.3
0.01% concentration: 51.2
0.001% concentration: 53.6
25 ℃ of interfacial tensions, dynes/cm (with respect to Nujol);
0.1% concentration: 19.8
0.01% concentration: 24.0
0.01% concentration: 26.0
25 ℃ of Draves wetting times (second)
1.0% concentration:＞360
0.1% concentration:＞360
Foam height
Ross Miles method, 0.1%, 50 ℃ of mm: 35
Ross Miles method, 0.1%, 26 ℃ of mm: 40
Dynamically, 0.1%, mm, 400ml/min:＞600
Cloud point in the aqueous solution, ℃
1% concentration:＞100
10% concentration:＞100
HLB (hydrophile-lipophile balance): 29
Character and top listed similarly other poloxamer polymer also can be used for preparation of the present invention.Preferably, the poloxamer surfactants that is present in the protein formulation with this method analysis is Pluronic F68 (=poloxamer 188).
The Pluronic-preferably about 0.01mg/ml of concentration of Pluronic F68-extremely in about 1mg/ml scope particularly in the liquid protein preparation, more preferably from about 0.05mg/ml is to about 0.5mg/ml scope, more preferably from about 0.2mg/ml is to about 0.4mg/ml scope, most preferably from about 0.1mg/ml.
The pH of the protein formulation of analyzing according to the inventive method is about 6.0 to about 8.0 scopes, more preferably about 6.8 to about 7.8 scopes, comprises about pH7.0, pH7.2 and 7.4.Preferred buffer solution foot phosphate buffer, preferred equilibrium ion is sodium or potassium ion.The phosphate brine buffer solution has been well known in the art, for example Dulbecco ' s phosphate buffered saline (PBS).The concentration of buffer solution can change between about 5mM, 9.5mM, 10mM, 50mM, 100mM, 150mM, 200mM, 250mM and 500mM in total solution.Preferably, the about 10mM of buffer concentration.Particularly preferred situation is the phosphate buffer of 10mMpH7.0.
The pH of the FSH preparation of analyzing according to the inventive method is preferably about 6.0 to about 8.0 scopes, more preferably about 6.8 to about 7.8 scopes, comprises about pH7.0, pH7.2 and 7.4.Preferred buffer is a phosphate buffer, and preferred equilibrium ion is sodium or potassium ion.
Preferably, the FSH that analyzes according to the inventive method and the pH of LH mixture preparation be about 6.0 to about 9.0 scopes, more preferably about 6.8 to about 8.5 scopes, comprises about pH7.0, pH8.0 and 8.2, most preferably from about pH8.0.
The pH of the hCG preparation of analyzing according to the inventive method is preferably about 6.0 to about 8.0 scopes, more preferably about 6.8 to about 7.8 scopes, comprises about pH7.0, pH7.2 and 7.4.
The liquid preparation of preferably single agent of protein example or multi-agent administration.The liquid preparation that the present invention is used for the multi-agent purpose preferably contains bacteriostatic agent, for example phenol, metacresol, paracresol, orthoresol, chloreresol, phenmethylol, alkyl paraben class (as methyl esters, ethyl ester, propyl ester, butyl ester etc.), thymol, benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal.Particularly preferably be phenol, phenmethylol and metacresol, more preferably phenmethylol and metacresol, most preferably metacresol.The consumption of bacteriostatic agent makes the concentration of acquisition can keep preparation essentially no bacterium (being fit to injection) during the multi-agent injection effectively, and the described multi-agent injection phase can be about 12 or 24 hours to about 12 or 14 days, preferred about 6 days to 12 days.Preferably, to about 2.0% scope, more preferably from about 0.2% to about 1.0% scope in about 0.1% (bacteriostatic agent quality/solvent quality) for the concentration of bacteriostatic agent.For phenmethylol, its preferred concentration is 0.9%.For phenol, its preferred concentration about 0.5%.For metacresol, about 0.3% (for example about 3mg/ml in WFI) of its preferred concentration.
The SEC post is well known to those skilled in the art.Protein and poloxamer concrete are per sample selected for use.The gel of chromatographic column should be based on the matrix of polymer.Preferred chromatographic column is that the TosoHaas logo is the SE-HPLC post of TSK G3000PW.The size of matrix pearl is 10 or 17 μ m, and the aperture is 200
This chromatographic column is commercially available.
Detecting step can carry out on any RI detection system known to those skilled in the art.
Have now found that acidity is strong more, the easy more and Separation of Proteins of poloxamer.
Therefore, flowing that the inventive method is used is aqueous solvent mutually, for example water or buffer solution.
In a specific embodiment, the pH that regulates the phase that flows is lower than 7, preferably is lower than 3, more preferably from about between the 1.6-2.0, most preferably between the 1.9-2.0.In a specific embodiment, described protein is heterodimer, for example FSH, CG, LH or TSH.Under the acid condition, heterodimer protein trends towards resolving into more easily mobile subunit, has therefore improved and has separated and detection step (separating degree).The acid reagent that is suitable for regulating pH can be selected by those skilled in the art.Most preferred acid is trifluoroacetic acid (TFA).
Usually, testing sample is ready to, is injected into chromatographic column.Write down the refractive index that described method detects step (acquisition), obtain chromatogram, it contains at least one other peak of zone, poloxamer peak and protein and excipient.Peak area is calculated the poloxamer that exists in quantitative institute's analytic sample.Making the calibration curve of poloxamer class can realize quantitatively (seeing embodiment).
Embodiment
The present invention will be described for mode that below will be by embodiment.
Embodiment 1 (with reference to the accompanying drawings 1)
The purpose of present embodiment is to analyze commercially available liquid hCG preparation Ovitrelle
TMRoom temperature storage concentration of poloxamer 188 in its sample after 18 months, and thus about the stability of poloxamer 188 assessment liquid preparations.The concentration of poloxamer 188 is about 100mcg/ml during preparation.Ovitrelle
TMThe quantitative detection scheme of middle poloxamer 188 is as follows:
Liquid injectable Ovitrelle
TMContain following composition: choriogonadotropin alfa, sweet mellow wine, L-methionine, poloxamer 188, phosphoric acid, NaOH and water.
1. equipment and material
Supplier
HPLC ALLIANCE model 269 water generation (Waters)
RI detector models 2414 water generation
Software Empower water generation
PC IBM (or similar supply
The merchant)
Poloxamer 188-Lutrol F68 lot number 010293-1 BASF
Trifluoroacetic acid (TFA) lot number 9470 (10 * 1mL) JT Bakes (J.T.Baker)
Analytical column TSKgel G3000 PW * 1 lot number, 08021 Tao Song (TOSOH)
D (-) sweet mellow wine lot number 1.05983 Merck (MERCK)
L-methionine lot number 1.05707 Merck
Orthophosphoric acid 85% lot number 1.00573 Merck
NaOH 50% lot number 7067 JT Bakes
Ethanol gradient level lot number 1.11727 Merck
Ovitrelle syringe-hCG liquid preparation 250mcg Sai Ruolong (SERONO)
2. step
2.1 eluent A (H
2O/TFA 0.5%)
In 1 liter of graduated cylinder, 5mL joins among the pure water 950ml with trifluoroacetic acid (TFA), complements to 1000ml under stirring.The pH of eluent is between the 1.7-1.9.
2.2 eluent B (20% ethanol)
In 1 liter of graduated cylinder, ethanol 200ml is joined among the pure water 750ml, complement to 1000ml under stirring.
2.3 automatic sampler cleaning fluid (10% methyl alcohol)
In 1 liter of graduated cylinder, methyl alcohol 100ml is joined among the pure water 850ml, complement to 1000ml under stirring.
2.4 sealing ring cleaning agent (5% methyl alcohol)
In 1 liter of graduated cylinder, methyl alcohol 50ml is joined among the pure water 900ml, complement to 1000ml under stirring.
2.5 calibration curve dilution (liquid preparation that does not contain poloxamer 188)
In 1 liter of graduated cylinder, D (-) sweet mellow wine 54.6g, 85% orthophosphoric acid 0.98g and L-methionine 200mcg are joined among the pure water 850ml.Dropwise 5 0% sodium hydrate regulator solution complements to 1ml to pH 7.0.Solution filters with 0.45 μ m filter.
Perhaps available water is as the calibration curve dilution.
2.6 dense calibration curve solution
In the 100ml graduated cylinder, 188 200mg are dissolved among the pure water 80ml with poloxamer, complement to 100ml.According to the expection prepared at concentrations calibration curve in the sample to be analyzed.In the present embodiment, estimate liquid injectable Ovitrelle
TMThe concentration of middle poloxamer 188 is approximately 100mcg/ml, so the scope of calibration curve is 50-160mcg/ml.
3 sample preparations
Blank
Sample introduction calibration curve dilution 0.05ml.
Sample
All samples are without any preliminary treatment, sample size 0.05ml.
4. operating condition
4.1. instrument setting
Following solution inserts in the HPLC pipeline:
Pipeline A: eluent A (H
2O/TFA 0.5%)
Pipeline B: eluent B (20% ethanol)
Pipeline A and B are filled with,, washed 3 minutes with the flow velocity cleaning systems of 2mL/min.If online degasser is arranged, then open.
Before the detection, the valve opening solenoid of RI flow detector was taken over A at least 30 minutes to cleaning mode.The cleaning flow cell is with the fresh mobile reference side of injecting the pond mutually.
Chromatographic column is connected to instrument, imports following parameter:
RI detector temperature: 30 ℃
Column temperature :+20 ± 5 ℃
Column flow rate: 0.5ml/min
Helium (Elium) flow velocity: 20ml/min (in the absence of online degasser)
Auto injection actuator temperature :+4 ℃
Auto injection ring: 200mcl
Syringe capacity: 250mcl
Analysis time: 40min
Next sampling interval time: 5min
4.2 column equilibration
Wash chromatographic column with the balance chromatographic column with eluent A.Wash to baseline when steady, balance finishes.
4.3 chromatographic column is preserved
After detection is finished,, wash with 20% ethanol 30ml then with pure water 30ml flushing chromatographic column at least.
4.4Ovitrelle the concentration of poloxamer 188 detects in the sample
Following line style regression formula is used for calculating Ovitrelle sample poloxamer concentration as model:
Y＝a+bx
Wherein
Y=poloxamer 188 gross areas
The a=intercept
The b=slope
X=poloxamer 188 concentration, the μ g/ of unit sample introduction ml.
Carry out the sample integration according to accompanying drawing 1.
Intercept of basis of calculation curve (a) and slope (b) adopt software Statgraphics plus to calculate linear regression.
Following formula is used to calculate the concentration of poloxamer 188:
Equation 1 provides the concentration of poloxamer 188 in each Ovitrelle sample, the μ g/ml of unit.
The concentration of poloxamer is approximately 94mcg/ml (seeing accompanying drawing 1) in the present embodiment sample.
Embodiment 2
The purpose of present embodiment is to analyze commercially available liquid hFSH preparation Gonal-F RFF Pen
TMRoom temperature storage is 188 concentration of the poloxamer in its sample after 18 months, and assess the stability of liquid preparations thus about poloxamer 188.The concentration of poloxamer 188 is about 100mcg/ml during preparation.Except column flow rate 0.75ml/min, Gonal-F RFF Pen
TMIn identical about shown in the Ovitrelle among quantitative scheme and the embodiment 1 of poloxamer 188.
Poloxamer can separate with hFSH and be quantitative separately.
Embodiment 3
The purpose of present embodiment is to analyze commercially available liquid hGH formulations Serostim
TMPoloxamer 188 concentration in the sample.Scheme is identical with embodiment 2.
Poloxamer can separate with hGH and be quantitative separately.
Embodiment 4
The purpose of present embodiment is poloxamer 188 concentration of analyzing in the liquid hIFN-beta formulations sample.
Scheme is identical with embodiment 2.
Poloxamer can separate with hlFN-β and be quantitative separately.
Claims (15)
1. the method for poloxamer in the quantitative assay liquid protein quality sample comprises the step of described sample being carried out following processing:
(a) separating step of employing SEC post;
(b) elution step of the mobile phase of employing; With
(c) the detection step of detection poloxamer;
Wherein, the molecular weight of described protein is 5-70kDa, and the pH of the mobile phase of using in the wherein said elution step is adjusted into and is lower than 3.
2. the method for claim 1, wherein described poloxamer is a poloxamer 188.
The method of claim 1, wherein the molecular weight of described protein between 20-70kDa.
4. as the described method of one of claim 1-3, wherein, described protein is heterodimer protein.
5. method as claimed in claim 4, wherein, described protein is the promoting sexual gland hormone that is selected from follicle-stimulating hormone (FSH), metakentrin, human chorionic gonadotrophin, thyrotropic hormone.
6. as the described method of one of claim 1-3, wherein, described protein is interferon beta or growth hormone.
7. as the described method of one of claim 1-3, wherein, described sample is the aqueous pharmaceutical compositions that contains follicle-stimulating hormone (FSH), metakentrin, human chorionic gonadotrophin, thyrotropic hormone, growth hormone or interferon beta.
8. as the described method of one of claim 1-3, wherein, described flowing is aqueous solvent mutually.
9. method as claimed in claim 8, wherein, described flowing is the solvent of buffering mutually.
10. as each described method among the claim 1-3, wherein, the pH of described mobile phase is adjusted to 1.9-2.0.
11. as the described method of one of claim 1-3, wherein, the detection step of described poloxamer comprises the analytical refraction rate.
12. as the described method of one of claim 1-3, wherein, described SEC post is to have filled the SE-HPLC post that contains the polymer substrate pearl.
13. method as claimed in claim 12, wherein, the granular size of described matrix pearl is 10 or 17 μ m.
14. method as claimed in claim 12, wherein, the aperture of described matrix pearl is
15. method as claimed in claim 13, wherein, the aperture of described matrix pearl is
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| US60/717,642 | 2005-09-16 | ||
| PCT/EP2006/066383 WO2007031566A1 (en) | 2005-09-14 | 2006-09-14 | Method for the quantitative determination of poloxamers |
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| WO2014159813A1 (en) | 2013-03-13 | 2014-10-02 | Moderna Therapeutics, Inc. | Long-lived polynucleotide molecules |
| EP3250925B1 (en) * | 2015-01-29 | 2019-04-10 | Ares Trading S.A. | Immunoassays for highly positively charged proteins |
| CN113424057B (en) * | 2019-04-19 | 2024-04-05 | 海正生物制药有限公司 | Detection method of poloxamer 188 in composition |
| CN111289652A (en) * | 2020-03-16 | 2020-06-16 | 康立泰药业有限公司 | Method for detecting poloxamer 188 in injection |
| CN111830168B (en) * | 2020-07-23 | 2022-07-22 | 吉林医药学院 | LC-HR-MS/MS quantitative analysis method of poloxamer |
| JP7367262B1 (en) | 2021-12-01 | 2023-10-23 | 中外製薬株式会社 | Method for preparing antibody-containing preparations |
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| Title |
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| Yun Mao et al.Quantitation of poloxamers inpharmaceuticalformulaitonsusing size exclusionchromatography andcolorimetricmethods.Journal of Pharmaceutical and Biomedical Analysis35.2004,351127-1142. * |
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