CN102380096B - Medicine combination containing fusion protein for suppressing angiogenesis and application - Google Patents
Medicine combination containing fusion protein for suppressing angiogenesis and application Download PDFInfo
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- CN102380096B CN102380096B CN201010267503.7A CN201010267503A CN102380096B CN 102380096 B CN102380096 B CN 102380096B CN 201010267503 A CN201010267503 A CN 201010267503A CN 102380096 B CN102380096 B CN 102380096B
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Abstract
The invention discloses a medicine combination containing fusion protein for suppressing angiogenesis and application, and particularly relates to a medicine combination containing fusion protein of an extracellular protein domain 2 (Flt-2) of a vascular endothelial growth factor (VEGF) receptor 1, extracellular protein domains 3 and 4 (KDR-3 and 4) of a VEGF receptor 2 and human normal immunoglobulin 1(G1) Fc. The medicine combination can keep the fusion protein stable, has the most outstanding advantage of capability of effectively suppressing fusion protein polymer so as to avoid reduction of purity, and accordingly keeps bioactivity of effective components.
Description
Technical field
The present invention relates to field of pharmaceutical preparations, relate to and comprise the pharmaceutical composition that vegf receptor fragment and immunoglobulin Fc merge the albumen forming, with and application in medical treatment.
Background technology
Along with the biological development of modern cellular elements, cytokine and the effect of cell surface correlation molecule in ophthalmic diseases are paid close attention to widely and are studied, VEGF is that vascular endothelial cell mitogenesis element has the biological nature that increases vascular permeability, very important to the generation of blood vessel at fetal period VEGF, after birth, level declines.Under physiological status, VEGF is low expression level state, for the function that maintains blood vessel, is necessary.Up-to-date result of study prompting, for senile retinal vasculopathy (Age-related macular degeneration, referred to as AMD), the relevant disease of angiogenesis such as diabetic renal papillary necrosis (Diabetic retinopathy, DR) all plays a part extensive and important.AMD is multiple to be born in more than 45 years old, and its prevalence increases with the growth at age, is the important diseases of current middle-aged and elderly people blinding.Moist AMD is mainly the destruction of glass-film, choroidal artery is invaded under retina and is formed choroidal neovascularization, there is serosity or hemorrhagic disciform detachment under macular area retinal pigment epithelium or under neuroepithelium, finally become machine cicatrix, effectively suppress to cause the blood vessel endothelial cell growth factor VEGF of moist maculopathy, thereby blocking VEGF or vegf receptor reach angiogenesis inhibiting, there is the important important therapeutical effect that has.In DR, in cell and body fluid, the content of VEGF is higher than normal level.VEGF increases, and causes that capillary permeability changes, and causes that retina oozes out, hemorrhage and macula retinae edema, and induction of vascular generates element (Angiogenin) and generates increase, and the collaborative formation that promotes retinal neovascularization, causes inpairment of vision.
Sat linkage, hydrogen bond, disulfide bond and hydrophobic interaction are Protein requirement conformation Stabilization power.The interaction of metal ion, substrate, cofactor and other low relative molecular weight parts makes protein conformation stable.Protein and other biomacromolecule be the effect of protein and fat especially.In vivo, protein normal with lipid or the polysaccharide formation complex that interacts, shielded the hydrophobic region of protein surface, thereby significantly increased the stability of protein.Albumen is unstable mainly to be caused by following factor: (1) physical action: the polar water molecules being regulated by Brownian movement can cause protein unstable with contacting of protein hydrophobic core.(2) chemical action: the Oxidation of the amino acid residue of active site is one of the most common mechanism of enzyme deactivation.As the indole ring of the sulfydryl of cysteine and tryptophan, responsive especially to oxidation.(3) biological action: proteolytic enzyme effect.Microorganism and foreign protein hydrolytic enzyme effect catalysis peptide bond hydrolysis.During by genetic engineering bacterium purification eukaryotic cell polypeptide, yield is low, because external Proteolytic enzyme causes.
First polymerization makes the hydrophobic amino acid residues of embedding be exposed to aqueous solvent, causes protein reversible degeneration; Secondly, protein molecule associates each other, to reduce the unfavorable exposed of hydrophobic amino acid; Finally, if protein molecule contains cysteine plus cystine residue, can there is intermolecular disulfide bond exchange reaction.Polymerization can, by reducing and reoxidizing the natural disulfide bond of regeneration, make protein reactivation sometimes.Polymerization and simple precipitation are distinguishing, and the latter does not make protein that significant conformation change occurs.
It is that pharmaceutical solutions easily forms soluble polymer and soluble granule after long term storage that the inventor also observes an obvious problem, how to address this problem, find a kind of on physics and chemistry stable pharmaceutical compositions all, can suppress polymer and generate, and can after long term storage, form less soluble polymer and insoluble particles.In addition, because its component is pharmaceutically acceptable component, ocular disease be can be used for the treatment of, intravitreal injection and outside administration comprised.In addition, the inventor finds that the preparation prescription obtaining is more stable in syringe than in bottle.
In the present invention, adopting can angiogenesis inhibiting, by the extracellular domain 2 (Flt-2) of vascular endothelial cell growth factor (VEGF) receptor 1 and extracellular domain 3 and the 4 (KDR-3 of vegf receptor 2,4) albumen (FP3 albumen) forming with the fusion of human normal immunoglobulin 1 (G1) Fc is expressed and after purification, reached medicinal purity by the working cell of recombinant technique, through changing liquid formulation subpackage, makes suitable pharmaceutical preparation.These preparation preferred liquid preparation or lyophilized formulations, be applicable to the treatment of disease of eye, in particular for intravitreal injection.
FP3 albumen is as protein drug, and its stability is more far short of what is expected than common micromolecule chemicals, and than naturally occurring immunoglobulin, its stability is also poorer.Fusion rotein described in Chinese patent is that the prescription of describing in Chinese patent " application of vegf receptor fusion rotein in treatment disease of eye " (patent No. ZL200610066257.2) need to be-20 ℃ of preservations, production to medicine, transportation is preserved and application has all proposed higher requirement.As everyone knows, the stability of the fusion rotein of general restructuring is all poor, can be subject to the impact of multiple environmental factors, as temperature in preservation process, appropriateness, oxygen, ultraviolet etc. can be that multiple physics or chemical change occur fusion rotein, cause the polymerization of protein, decompose, oxidation or degeneration etc.These variations can make the activity decreased of albumen, and therapeutic effect declines and causes serious toxic and side effects.Therefore, developing fusion protein formulations stable and that be easy to transport and preserve is to have very important clinical meaning.
Summary of the invention
One of object of the present invention is to provide the extracellular domain 2 (Flt-2) of a kind of vascular endothelial cell growth factor containing (VEGF) receptor 1 and extracellular domain 3 and the 4 (KDR-3 of vegf receptor 2,4) with the pharmaceutical composition of the fusion rotein of human normal immunoglobulin 1 (G1) Fc, more particularly can be for the liquid preparation of intravitreal injection, this liquid preparation can make fusion rotein keep stable, its the most outstanding feature is can effectively suppress the generation of fusion rotein polymer and the purity that causes declines, thus the biological activity of the component of remaining valid.
One aspect of the present invention provides a kind of pharmaceutical composition that contains the fusion rotein that suppresses blood vessel hyperplasia, it is characterized in that comprising
(a) extracellular domain 2 of the vegf receptor 1 of 0.1-100mg/ml and the extracellular domain 3 of vegf receptor 2 and 4 and the fusion rotein of human normal immunoglobulin Fc, the aminoacid sequence that comprises SEQ ID No:1;
(b) 5-100mM buffer, acid is wherein selected from Tris-HCl, citric acid, dibastic sodium phosphate, sodium dihydrogen phosphate, acetic acid, succinic acid, one or more in hydrochloric acid;
(c) 5-500mM basic amino acid is selected from lysine, arginine, and a kind of or its combination in histidine;
(d) 0.1-30% salt penetration is pressed agent regulator, and sugar is wherein selected from sucrose, trehalose, and mannitol, glycerol, propylene glycol, one or more in Pyrusussuriensis ester alcohol, salt is selected from pharmaceutically a kind of or its combination in acceptable salt of sodium chloride or other;
(e) one or more surfactants or the cosolvent of 0.01-0.1%, be selected from Polyethylene Glycol, polysorbas20, and Tween 80, propylene glycol, dimethyl sulfoxide or other be one or more in acceptable surfactant pharmaceutically;
(f) regulating pH is 7.5～8.3.
Wherein pharmaceutical composition is preferably the pharmaceutical composition that comprises following component:
(a) 10-40mg/ml's as the fusion rotein of SEQ ID No:1;
(b) one or both of the citric acid of 5-100mM or sodium dihydrogen phosphate;
(c) one or both of the arginine of 5-500mM or histidine;
(d) one or both of the sucrose of 8-30% or trehalose;
(e) one or both of the tween 20 of 0.01-0.1% or Polyethylene Glycol;
(f) regulating pH is 7.5～8.3.
Preferred pharmaceutical composition of the present invention can also contain sodium chloride.
The present invention is more excellent provide a kind of by 10-40mg/ml as the citric acid of the fusion rotein of SEQ ID No:1,10mM, 5% sucrose, the arginine of 100mM, 0.05% polysorbas20, the pharmaceutical composition that pH7.5～8.3 form.
The present invention also further provide a kind of 10-40mg/ml as the citric acid of the fusion rotein of SEQ ID No:1,55mM, 12.5% sucrose, the arginine of 250mM, 0.05% polysorbas20, pH7.9 buffer, pH7.5～8.3.
The dosage form that aforementioned pharmaceutical compositions is made can be liquid preparation or lyophilized formulations, and wherein liquid preparation is preferably ophthalmic preparation, especially eye drop; It can also be pre-filled injection.
Aforementioned pharmaceutical compositions can be used for the treatment of the disease that angiogenesis or growth cause, is preferably age-related macular degeneration.
In the present invention, described fusion rotein is the fusion rotein of describing in Chinese patent " application of vegf receptor fusion rotein in treatment disease of eye " (patent No. ZL200610066257.2), the content of specifically FP3 fusion rotein, so ZL200610066257.2 can be used for further setting forth the present invention.
The specific embodiment
Embodiment 1 10mg/ml FP3 fusion rotein original prescription stability study in 3ml glass ampule at 4 ℃
Write out a prescription as follows:
FP3 fusion rotein 10mg/ml
Sodium succinate 10mM
Trehalose 9.0%
Polysorbas20 0.05%
With hydrochloric acid regulating system pH to 6.0～6.5
Albumen stock solution is changed after liquid, aseptic subpackaged obtaining in 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, December working sample, determines stability by SEC-HPLC.Result demonstration, this prescription can not effectively suppress the generation of polymer, causes product purity to decline, and reduces with the affinity of VEGF, enters in body and may bring out immunoreation later.
Table 1.10mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 6.0 | 10.0 | 0.3 | 10.6 |
| 1 | Qualified | 6.0 | 9.4 | 1.9 | 9.5 |
| 2 | Qualified | 6.0 | 10.2 | 2.8 | 9.1 |
| 3 | Qualified | 6.0 | 10.2 | 3.6 | 8.7 |
| 6 | Qualified | 6.0 | 10.0 | 9.1 | 8.4 |
| 9 | Qualified | 6.0 | 10.0 | 18.2 | 7.7 |
| 12 | Qualified | 6.1 | 10.3 | 31.8 | 7.0 |
Embodiment 2 10mg/ml FP3 fusion rotein stability study in 3ml glass ampule at 4 ℃
FP3 fusion rotein 10mg/ml
Sodium hydrogen phosphate 10mM
Sucrose 10%
Sodium chloride 0.5%
Polysorbas20 0.05%
pH 7.5～8.3
Albumen stock solution is changed after liquid, aseptic subpackaged obtaining in 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, December working sample, determines stability by SEC-HPLC.Result demonstration, this prescription can not effectively suppress the generation of polymer, causes product purity to decline, and reduces with the affinity of VEGF, enters in body and may bring out immunoreation later.
Table 2.10mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 7.7 | 10.1 | 0.2 | 10.7 |
| 1 | Qualified | 7.9 | 10.1 | 2.0 | 9.9 |
| 2 | Qualified | 7.7 | 10.7 | 3.2 | 9.3 |
| 3 | Qualified | 7.8 | 10.6 | 5.4 | 8.4 |
| 6 | Qualified | 7.7 | 10.3 | 9.9 | 8.1 |
| 9 | Qualified | 7.5 | 10.1 | 13.1 | 8.5 |
| 12 | Qualified | 7.9 | 10.3 | 23.3 | 7.3 |
Embodiment 3 10mg/ml FP3 fusion rotein stability study in 3ml glass ampule at 4 ℃
FP3 fusion rotein 10mg/ml
Citric acid 5mM
Sucrose 8.0%
Polysorbas20 0.05%
pH 7.5～8.3
Albumen stock solution is changed after liquid, aseptic subpackaged obtaining in 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, December working sample, determines stability by SEC-HPLC.Result demonstration, this prescription can not effectively suppress the generation of polymer, causes product purity to decline, and reduces with the affinity of VEGF, enters in body and may bring out immunoreation later.
Table 3.10mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 7.9 | 10.2 | 0.3 | 10.4 |
| 1 | Qualified | 7.9 | 10.2 | 1.4 | 10.0 |
| 2 | Qualified | 7.8 | 10.6 | 2.6 | 9.7 |
| 3 | Qualified | 7.8 | 10.7 | 3.6 | 8.1 |
| 6 | Qualified | 8.1 | 10.5 | 9.4 | 7.9 |
| 9 | Qualified | 8.3 | 10.1 | 18.5 | 9.7 |
| 12 | Qualified | 8.0 | 10.4 | 19.1 | 9.6 |
Embodiment 4 10mg/ml FP3 fusion rotein stability study in 3ml glass ampule at 4 ℃
FP3 fusion rotein 10mg/ml
Citric acid 10mM
Sucrose 8.0%
Arginine 5mM
Polysorbas20 0.05%
pH 7.5～8.3
Albumen stock solution is changed after liquid, aseptic subpackaged obtaining in 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, December working sample, determines stability by SEC-HPLC.Result demonstration, this prescription can effectively suppress the generation of polymer, and product purity declines very slow, and the affinity of fusion rotein and VEGF is almost constant.
Table 4.10mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 7.7 | 10.1 | 0.4 | 10.5 |
| 1 | Qualified | 7.9 | 10.1 | 0.5 | 10.4 |
| 2 | Qualified | 7.5 | 10.7 | 0.7 | 10.1 |
| 3 | Qualified | 7.8 | 10.6 | 0.8 | 10.0 |
| 6 | Qualified | 7.7 | 10.3 | 0.9 | 9.9 |
| 9 | Qualified | 7.9 | 10.1 | 1.8 | 9.7 |
| 12 | Qualified | 7.9 | 10.3 | 1.9 | 9.6 |
Embodiment 5 10mg/ml FP3 fusion rotein stability study in 3ml glass ampule at 4 ℃
FP3 fusion rotein 10mg/ml
Citric acid 100mM
Sucrose 20.0%
Arginase 12 50mM
Polysorbas20 0.10%
pH 7.5～8.3
Albumen stock solution is changed after liquid, aseptic subpackaged obtaining in 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, December working sample, determines stability by SEC-HPLC.Result demonstration, this prescription can effectively suppress the generation of polymer, and product purity declines very slow, and the affinity of fusion rotein and VEGF is almost constant.
Table 5.10mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 7.9 | 10.0 | 0.3 | 10.4 |
| 1 | Qualified | 7.9 | 10.1 | 0.3 | 10.3 |
| 2 | Qualified | 7.9 | 10.0 | 0.4 | 10.4 |
| 3 | Qualified | 7.9 | 10.1 | 0.5 | 10.2 |
| 6 | Qualified | 7.9 | 10.2 | 0.7 | 10.1 |
| 9 | Qualified | 7.9 | 10.0 | 0.8 | 10.2 |
| 12 | Qualified | 7.9 | 10.1 | 0.9 | 10.0 |
Embodiment 6 10mg/ml FP3 fusion rotein stability study in 3ml glass ampule at 4 ℃
FP3 fusion rotein 10mg/ml
Sodium dihydrogen phosphate 5mM
Trehalose 10.0%
Arginine 100mM
PEG400 0.01％
pH 7.5～8.3
Albumen stock solution is changed after liquid, aseptic subpackaged obtaining in 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, December working sample, determines stability by SEC-HPLC.
Table 6.10mg/ml FP3 fusion rotein is the stability of 4 ℃
Embodiment 7 20mg/ml FP3 fusion rotein stability study in 3ml glass ampule at 4 ℃
FP3 fusion rotein 20mg/ml
Citric acid 5mM
Sucrose 4.0%
Sodium chloride 4.0%
Arginine 100mM
Histidine 100mM
Polysorbas20 0.05%
PEG400 0.05％
pH 7.5～8.3
Albumen stock solution is changed after liquid, aseptic subpackaged obtaining in 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, December working sample, determines stability by SEC-HPLC.
Table 7.20mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 8.0 | 19.8 | 1 | 14.3 |
| 1 | Qualified | 7.8 | 20.7 | 1.5 | 10.2 |
| 2 | Qualified | 7.9 | 19.8 | 1.3 | 12.6 |
| 3 | Qualified | 7.9 | 20.6 | 2 | 10.5 |
| 4 | Qualified | 7.8 | N/D | 1.7 | N/D |
| 5 | Qualified | 7.8 | N/D | 2.2 | N/D |
| 6 | Qualified | 8.0 | N/D | 2.7 | N/D |
| 7 | Qualified | 7.8 | N/D | 2.7 | N/D |
| 8 | Qualified | 7.9 | N/D | 2.7 | N/D |
| 9 | Qualified | 7.9 | 19.5 | 2.7 | 12.5 |
| 10 | Qualified | 7.8 | N/D | 3.1 | N/D |
| 11 | Qualified | 7.8 | N/D | 3.5 | N/D |
| 12 | Qualified | 7.9 | 20.3 | 3.6 | 14.3 |
N/D represents not detect.
Embodiment 8 20mg/ml FP3 fusion rotein stability study in 3ml glass ampule at 4 ℃
FP3 fusion rotein 20mg/ml
Citric acid 10mM
Sucrose 30%
Arginine 500M
Polysorbas20 0.1%
pH 7.5～8.3
Albumen stock solution is changed after liquid, aseptic subpackaged obtaining in 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, December working sample, determines stability by SEC-HPLC.
Table 8.20mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 7.7 | 20.0 | 0.1 | 10.2 |
| 1 | Qualified | 7.9 | 20.0 | 0.1 | 10.2 |
| 2 | Qualified | 7.7 | 20.2 | 0.1 | 10.3 |
| 3 | Qualified | 7.8 | 20.2 | 0.2 | 10.3 |
| 6 | Qualified | 7.7 | 20.1 | 0.2 | 9.9 |
| 9 | Qualified | 7.9 | 20.3 | 0.3 | 10.0 |
| 12 | Qualified | 7.9 | 20.0 | 0.5 | 10.3 |
Embodiment 9 10mg/ml FP3 fusion rotein stability study in 3ml glass ampule at 4 ℃
FP3 fusion rotein 10mg/ml
Citric acid 10mM
Sucrose 5%
Arginine 100M
Polysorbas20 0.05%
pH 7.5～8.3
Albumen stock solution is changed after liquid, aseptic subpackaged obtaining in 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, December working sample, determines stability by SEC-HPLC.
Table 9.10mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 7.7 | 10.1 | 0.4 | 10.5 |
| 1 | Qualified | 7.9 | 10.1 | 0.5 | 10.4 |
| 2 | Qualified | 7.7 | 10.7 | 0.7 | 10.1 |
| 3 | Qualified | 7.8 | 10.6 | 0.8 | 10.0 |
| 6 | Qualified | 7.7 | 10.3 | 0.9 | 9.9 |
| 9 | Qualified | 7.9 | 10.1 | 1.8 | 7.7 |
| 12 | Qualified | 7.9 | 10.3 | 1.9 | 6.3 |
Embodiment 10 10mg/ml FP3 fusion rotein stability study in 3ml glass ampule at 4 ℃
By FP3 fusion rotein stock solution through Vivaflow is concentrated aseptic subpackaged after changing liquid after subpackage obtain semi-finished product, put the citric acid of 55mM, 12.5% sucrose, the arginine of 250mM, 0.05% polysorbas20, pH7.9 buffer, regulates FP3 fusion rotein to 10mg/ml, aseptic subpackaged obtaining in 3ml glass ampule, 4 ℃ keep sample, 0,1, and 2,3,4,5,6,7,8,9,10,11, December working sample, determines stability by SEC-HPLC.
Table 10.10mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 8.3 | 10.0 | 0.2 | 9.7 |
| 1 | Qualified | 8.3 | 10.1 | 0.2 | 9.6 |
| 2 | Qualified | 8.2 | 10.2 | 0.3 | 9.6 |
| 3 | Qualified | 7.9 | 10.0 | 0.4 | 9.3 |
| 6 | Qualified | 8.0 | 10.1 | 0.5 | 9.3 |
| 9 | Qualified | 7.9 | 10.1 | 0.6 | 9.1 |
| 12 | Qualified | 7.9 | 10.3 | 1.0 | 9.4 |
Embodiment 11 20mg/ml FP3 fusion rotein stability study in 3ml glass ampule at 4 ℃
By FP3 fusion rotein stock solution through Vivaflow is concentrated aseptic subpackaged after changing liquid after subpackage obtain semi-finished product, put the citric acid of 55mM, 12.5% sucrose, the arginine of 250mM, 0.05% polysorbas20, pH7.5～8.3 buffer, regulates FP3 fusion rotein to 20mg/ml, aseptic subpackaged obtaining in 3ml glass ampule, 4 ℃ keep sample, 0,1, and 2,3,4,5,6,7,8,9,10,11, December working sample, determines stability by SEC-HPLC.
Table 11.20mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 7.9 | 19.9 | 0.7 | 10.7 |
| 1 | Qualified | 7.9 | 20.1 | 1.2 | 10.2 |
| 2 | Qualified | 7.7 | 20.7 | 1.3 | 10.3 |
| 3 | Qualified | 7.8 | 21.6 | 1.4 | 10.4 |
| 6 | Qualified | 8.0 | 21.6 | 1.4 | 10.4 |
| 9 | Qualified | 7.9 | 21.1 | 2.6 | 9.6 |
| 12 | Qualified | 7.9 | 21.3 | 3.6 | 9.4 |
The stability study of 40mg/ml FP3 fusion rotein lyophilized formulations at 12 4 ℃ of embodiment
FP3 fusion rotein 40mg/ml
Citric acid 250mM
Sucrose 8.0%
Histidine 100mM
Polysorbas20 0.10%
pH 7.5～8.3
FP3 fusion rotein solution is adjusted to 40mg/ml, after the rear subpackage in pH regulator to 7.5～8.3, divide and install in 3ml glass ampule, the condition of the freeze-drying curve of optimizing is :-50 ℃ of rapid pre-freezes 4 hours, be controlled at-20 ℃ of distillations and remove most of moisture, then the temperature of dividing plate of progressively raising is further removed residual moisture, final step application 25 spend and the vacuum that reaches capacity, and makes moisture reduction, make simultaneously sample temperature rise can be too not high.After lyophilizing finishes, Asser, goes out freeze drying box, rolls aluminium lid, carries out the analyses such as moisture and purity, and 4 ℃ keep sample, and 0,1,6, December adds equal-volume water for injection to redissolve, and working sample, determines stability by SEC-HPLC.
Table 12.40mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance after redissolving | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 7.9 | 39.9 | 0.1 | 11.2 |
| 1 | Qualified | 7.9 | 40.1 | 0.2 | 10.6 |
| 6 | Qualified | 7.9 | 40.7 | 0.3 | 10.1 |
| 12 | Qualified | 8.3 | 40.6 | 0.4 | 10.0 |
The stability study of 40mg/ml FP3 fusion rotein lyophilized formulations at 13 4 ℃ of embodiment
FP3 fusion rotein 40mg/ml
Citric acid 10mM
Sucrose 5%
Arginine 100mM
Polysorbas20 0.05%
pH 7.5～8.3
FP3 fusion rotein solution is adjusted to 20mg/ml, after the rear subpackage in pH regulator to 7.5～8.3, divide and install in 3ml glass ampule, the condition of the freeze-drying curve of optimizing is :-50 ℃ of rapid pre-freezes 4 hours, be controlled at-20 ℃ of distillations and remove most of moisture, then the temperature of dividing plate of progressively raising is further removed residual moisture, final step application 25 spend and the vacuum that reaches capacity, and makes moisture reduction, make simultaneously sample temperature rise can be too not high.After lyophilizing finishes, Asser, goes out freeze drying box, rolls aluminium lid, carries out the analyses such as moisture and purity, and 4 ℃ keep sample, and 0,1,6, December adds equal-volume water for injection to redissolve, and working sample, determines stability by SEC-HPLC.
Table 13.40mg/ml FP3 fusion rotein is the stability of 4 ℃
| Time (moon) | Outward appearance after redissolving | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 8.0 | 40 | 0.1 | 10.2 |
| 1 | Qualified | 8.3 | 40 | 0.1 | 10.5 |
| 6 | Qualified | 7.9 | 40 | 0.2 | 10.2 |
| 12 | Qualified | 8.2 | 40.1 | 0.3 | 10.3 |
The stability study of 20mg/ml FP3 fusion rotein in glass pre-filled syringe at 14 4 ℃ of embodiment
FP3 fusion rotein 20mg/ml
Citric acid 55mM
Sucrose 12.5%
Arginase 12 50mM
Polysorbas20 0.05%
pH 7.5～8.3
By FP3 fusion rotein stock solution through concentrated aseptic subpackaged after changing liquid after subpackage obtain semi-finished product, regulate FP3 fusion rotein to 20mg/ml's, aseptic subpackaged to having in the 1ml glass glass pre-filled syringe of FluroTec coating, 4 ℃ keep sample, 0,1,6, December working sample, determines stability by SEC-HPLC.
The stability study of 20mg/ml FP3 fusion rotein in glass pre-filled syringe at table 14.4 ℃
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | Qualified | 7.9 | 20.0 | 0.1 | 11.2 |
| 1 | Qualified | 7.9 | 20.1 | 0.1 | 11.6 |
| 6 | Qualified | 7.9 | 20.1 | 0.1 | 11.1 |
| 12 | Qualified | 8.2 | 20.1 | 0.1 | 11.0 |
The gel exclusion chromatography analysis (SEC-HPLC) of embodiment 15 polymer
Bio-sil column stability is high, heat-resistant pressure-resistant, life-span long, can realize the sharp separation of biomacromolecule.TSK G3000SWxl is Bio-sil post, the scope 10-500kD of particle diameter 5 μ m, aperture 250 dusts, separated globulin, and sample separation time 30min, is to analyze in the world the chromatographic column that biomacromolecule is generally used.In the present invention, we select Bio-sil post TSKG3000SWxl chromatographic column, instrument is 2695 high performance liquid chromatographs of Waters, the phosphate buffer of pH to 7.20 of take is mobile phase, flow velocity is 0.5ml/ml, column temperature is 25 ℃, and detection wavelength is 280nm, and the purity of Recombinant human vascular endothelial growth factor receptor-antibody FP3 fusion rotein is detected.
Embodiment 16 rabbit intravitreal injection repeat administration tests
12 Japan large ear rabbits, are divided into 2 groups at random, are respectively test sample group and solvent control group, and 6 every group, male and female half and half.At the right eye of each treated animal, single intravitreal injection gives test sample (0.5mg/50 μ L/ eye) or equal-volume solvent control product respectively, and opposite side rehearses and injects contrast or blank.After administration, carry out perusal every day, regularly uses indirect ophthalmoscope and slit lamp to check.Within after administration 14 days, put to death animal, get eyeball and carry out histopathological examination.By observed result and histopathological examination result, investigate the local toxicity of test sample.Found that: Japan large ear rabbit vitreum inner injecting and administering, in the eyes that 6 give test sample, there is 1 perusal to see and have slight hyperemia, see a small amount of secretions for 1; Slit lamp observation is shown in that 1 occurs lenticular opacity.Meanwhile, observe same phenomenon giving solvent control side, false injection side and blank branch hole eyeball respectively, and incidence rate and time of origin and to give test sample branch hole eyeball basically identical.Can get rid of thus these phenomenons is the irritant reaction that caused by test sample.Histopathological examination is shown in that all eyeball tissue structures are all normal.Therefore under this experimental condition, lagophthalmos single intravitreal injection gives Recombinant human vascular endothelial growth factor receptor-antibody fusion protein injection, do not cause local irritation or tissue injury's reaction.
Claims (10)
1. a pharmaceutical composition that contains the fusion rotein that suppresses blood vessel hyperplasia, it is characterized in that composed of the following components: the fusion rotein as shown in SEQ ID No:1 of 10-40mg/ml, the citrate buffer solution of 10mM, 5% sucrose, the arginine of 100mM, 0.05% polysorbas20, pH7.5～8.3.
2. a pharmaceutical composition that contains the fusion rotein that suppresses blood vessel hyperplasia, it is characterized in that composed of the following components: the fusion rotein as shown in SEQ ID No:1 of 10-40mg/ml, the citrate buffer solution of 55mM, 12.5% sucrose, the arginine of 250mM, 0.05% polysorbas20, pH7.5～8.3.
3. according to the pharmaceutical composition described in any one in claim 1 or 2, it is characterized in that the described fusion rotein as SEQ ID No:1 is 10mg/ml.
4. in claim 1 or 2, the pharmaceutical composition described in any one is liquid preparation.
5. in claim 1 or 2, the pharmaceutical composition described in any one is lyophilized formulations.
6. pharmaceutical composition according to claim 4, is characterized in that the liquid preparation of making is ophthalmic preparation.
7. pharmaceutical composition according to claim 6, is characterized in that described ophthalmic preparation is eye drop.
8. pharmaceutical composition according to claim 4, is characterized in that dosage form is pre-filled injection.
9. according to the pharmaceutical composition described in any one in claim 1-8, it is characterized in that the purposes in preparing the medicine for the treatment of the disease being caused by angiogenesis or growth.
10. pharmaceutical composition according to claim 9, is characterized in that described disease is age-related macular degeneration.
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| CN201010267503.7A CN102380096B (en) | 2010-08-31 | 2010-08-31 | Medicine combination containing fusion protein for suppressing angiogenesis and application |
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| EP3753548A1 (en) | 2006-06-16 | 2020-12-23 | Regeneron Pharmaceuticals, Inc. | Vegf antagonist formulations suitable for intravitreal administration |
| SG10201509193UA (en) | 2011-01-13 | 2015-12-30 | Regeneron Pharma | Use of a vegf antagonist to treat angiogenic eye disorders |
| CN107115294B (en) * | 2012-01-19 | 2019-10-18 | 北京康弘生物医药有限公司 | A kind of eye drops containing VEGF antagonist |
| CN104940926B (en) * | 2014-09-25 | 2017-09-22 | 信达生物制药(苏州)有限公司 | Recombination fusion protein preparation |
| CN105435222B (en) * | 2014-09-25 | 2018-05-29 | 信达生物制药(苏州)有限公司 | Recombination fusion protein preparation |
| CN105543204A (en) * | 2015-12-30 | 2016-05-04 | 海口奇力制药股份有限公司 | Composition and application thereof, and stabilizer containing composition |
| WO2018195912A1 (en) * | 2017-04-28 | 2018-11-01 | 苏州思坦维生物技术股份有限公司 | Ophthalmic pharmaceutical composition and use thereof |
| WO2021108530A1 (en) * | 2019-11-26 | 2021-06-03 | University Of Massachusetts | Recombinant adeno-associated virus for delivery of kh902 (conbercept) and uses thereof |
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