CN102380096A - 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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- CN102380096A CN102380096A CN2010102675037A CN201010267503A CN102380096A CN 102380096 A CN102380096 A CN 102380096A CN 2010102675037 A CN2010102675037 A CN 2010102675037A CN 201010267503 A CN201010267503 A CN 201010267503A CN 102380096 A CN102380096 A CN 102380096A
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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 vegf receptor fragment and immunoglobulin Fc and merge the proteic pharmaceutical composition that forms, 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 the biological nature that vascular endothelial cell mitogenesis element has increases vascular permeability; Very important to the generation of blood vessel at fetal period VEGF, birth back level descends.VEGF is the low expression level state under the physiological status, is necessary for the function of keeping blood vessel.Up-to-date result of study prompting; For senile retinal vasculopathy (Age-related macular degeneration; Abbreviate AMD as), (Diabetic retinopathy DR) waits the relevant disease of angiogenesis to play a part extensive and important to diabetic renal papillary necrosis.The AMD pilosity was born in more than 45 years old, and its prevalence increases with the growth at age, was the important diseases of current middle-aged and elderly people blinding.Moist AMD is mainly the destruction of glass-film; Choroidal artery is invaded retina and is constituted CNV down; Take place under the macular area retinal pigment epithelium or serosity or hemorrhagic disciform detachment under the neuroepithelium; Finally become the machine cicatrix, effectively suppress to cause the blood vessel endothelial cell growth factor VEGF of moist maculopathy, have the important important therapeutical effect that has thereby blocking VEGF or vegf receptor reach angiogenesis inhibiting.In DR, the content of VEGF is higher than normal level in cell and the body fluid.VEGF increases, and causes that capillary permeability changes, and causes that retina oozes out, hemorrhage and macula retinae edema, and induction of vascular generates plain (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 to keep protein 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 and lipid or polysaccharide interaction formation complex have shielded the hydrophobic region of protein surface, thereby have significantly increased proteinic stability.The albumen instability is mainly caused by following factor: (1) physical action: the polar water molecules of 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.Like the sulfydryl of cysteine and the indole ring of tryptophan, responsive especially to oxidation.(3) biological action: proteolytic enzyme effect.Microorganism and foreign protein hydrolytic enzyme effect catalysis peptide bond hydrolysis.Yield is low during by genetic engineering bacterium purification eukaryotic cell polypeptide, because external Proteolytic enzyme causes.
Polymerization at first makes the hydrophobic amino acid residues of embedding be exposed to aqueous solvent, causes the protein reversible degeneration; Secondly, protein molecule associates each other, to reduce the unfavorable exposed of hydrophobic amino acid; At last, if protein molecule contains the cysteine plus cystine residue, then the intermolecular disulfide bond exchange reaction can take place.Polymerization can and reoxidize the natural disulfide bond of regeneration through reduction sometimes, makes the protein reactivate.Polymerization and simple precipitation are distinguishing, and the latter does not make protein that significant conformation change takes place.
It is that pharmaceutical solutions forms soluble polymer and soluble granule easily after long term storage that the inventor also observes a tangible problem; How to address this problem; Find a kind of at physics and chemically all stable pharmaceutical composition; Can suppress polymer and generate, and can after long term storage, form less soluble polymer and insoluble particles.In addition,, ocular disease be can be used to treat, intravitreal injection and outside administration comprised because its component is pharmaceutically acceptable component.In addition, the inventor finds that the preparation prescription that obtains is more stable in syringe than in bottle.
Adopting among the present invention can angiogenesis inhibiting; By the extracellular domain 2 (Flt-2) of vascular endothelial cell growth factor (VEGF) receptor 1 and the extracellular domain 3 and the 4 (KDR-3 of vegf receptor 2; 4) albumen (FP3 albumen) that forms with the fusion of human normal immunoglobulin 1 (G1) Fc is to express and reach medicinal purity through behind the purification through the working cell of recombinant technique, makes suitable pharmaceutical preparation through changing the liquid formulation packing.These preparation preferred liquid preparation or lyophilized formulations are 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.The described fusion rotein of Chinese patent is that the prescription of describing in the Chinese patent " application of vegf receptor fusion rotein in the treatment disease of eye " (patent No. ZL200610066257.2) need be-20 ℃ of preservations; To production of medicine, transportation is preserved and is used and all proposed higher requirement.As everyone knows, the stability of the fusion rotein of general reorganization is all poor, in the preservation process, can receive multiple Effect of Environmental, like temperature; Appropriateness, oxygen, ultraviolet etc. can be that multiple physics or chemical change take place fusion rotein; Cause proteinic polymerization, decompose oxidation or degeneration etc.These variations can make proteic active the reduction, and therapeutic effect descends and causes serious toxic and side effects.Therefore, develop fusion protein formulations stable and that be easy to transport and preserve and have crucial clinical meaning.
Summary of the invention
One of the object of the invention is to provide the extracellular domain 2 (Flt-2) of a kind of vascular endothelial cell growth factor that contains (VEGF) receptor 1 and the 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; The liquid preparation that more particularly can be used for intravitreal injection; This liquid preparation can make fusion rotein keep stable; Its most outstanding characteristic is can effectively suppress the generation of fusion rotein polymer and the purity that causes descends, 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 3 of the extracellular domain 2 of the vegf receptor 1 of 0.1-100mg/ml and vegf receptor 2 and 4 and the fusion rotein of human normal immunoglobulin Fc comprises the aminoacid sequence of SEQ ID No:1;
(b) 5-100mM buffer, acid wherein is selected from Tris-HCl, citric acid, dibastic sodium phosphate, sodium dihydrogen phosphate, acetic acid, succinic acid, one or more in the hydrochloric acid;
(c) the 5-500mM basic amino acid is selected from lysine, a kind of or its combination in arginine and the histidine;
(d) the 0.1-30% salt penetration is pressed the agent regulator, and sugar wherein is selected from sucrose, and trehalose, mannitol, glycerol, propylene glycol, one or more in the Pyrusussuriensis ester alcohol, salt are selected from pharmaceutically a kind of or its combination in the acceptable salt of sodium chloride or other;
(e) one or more surfactants or the cosolvent of 0.01-0.1% are selected from Polyethylene Glycol, and polysorbas20, Tween 80, propylene glycol, dimethyl sulfoxide or other be one or more in the acceptable surfactant pharmaceutically;
(f) regulating pH is 7.5～8.3.
Wherein pharmaceutical composition is preferably the pharmaceutical composition that comprises following component:
(a) the fusion rotein of 10-40mg/ml like 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 according to the invention can also contain sodium chloride.
The present invention is more excellent provide a kind of by 10-40mg/ml like the fusion rotein of SEQ ID No:1, the citric acid of 10mM, 5% sucrose, the arginine of 100mM, 0.05% polysorbas20, the pharmaceutical composition that pH7.5～8.3 are formed.
The present invention also further provide a kind of 10-40mg/ml like the fusion rotein of SEQ ID No:1, the citric acid of 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 processed can be liquid preparation or lyophilized formulations, and wherein liquid preparation is preferably ophthalmic preparation, especially eye drop; Can also be filling injection agent in advance.
Aforementioned pharmaceutical compositions can be used to treat the disease that angiogenesis or growth cause, is preferably AMD.
In the present invention; Described fusion rotein is the fusion rotein of describing in the Chinese patent " application of vegf receptor fusion rotein in the treatment disease of eye " (patent No. ZL200610066257.2); Particularly be the FP3 fusion rotein, so the content of ZL200610066257.2 can be used for further setting forth the present invention.
The specific embodiment
The stability study of the former prescription of embodiment 1 10mg/ml FP3 fusion rotein in 4 ℃ of following 3ml glass ampules
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
After albumen stock solution changed liquid, aseptic subpackaged obtaining in the 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, the December working sample is confirmed stability through SEC-HPLC.The result shows that this prescription can not effectively suppress the generation of polymer, causes product purity to descend, and with the affinity reduction of VEGF, possibly bring out immunoreation after the entering body is interior.
Table 1.10mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 |
The stability study of embodiment 2 10mg/ml FP3 fusion rotein in 4 ℃ of following 3ml glass ampules
FP3 fusion rotein 10mg/ml
Sodium hydrogen phosphate 10mM
Sucrose 10%
Sodium chloride 0.5%
Polysorbas20 0.05%
pH 7.5～8.3
After albumen stock solution changed liquid, aseptic subpackaged obtaining in the 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, the December working sample is confirmed stability through SEC-HPLC.The result shows that this prescription can not effectively suppress the generation of polymer, causes product purity to descend, and with the affinity reduction of VEGF, possibly bring out immunoreation after the entering body is interior.
Table 2.10mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 |
The stability study of embodiment 3 10mg/ml FP3 fusion rotein in 4 ℃ of following 3ml glass ampules
FP3 fusion rotein 10mg/ml
Citric acid 5mM
Sucrose 8.0%
Polysorbas20 0.05%
pH 7.5～8.3
After albumen stock solution changed liquid, aseptic subpackaged obtaining in the 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, the December working sample is confirmed stability through SEC-HPLC.The result shows that this prescription can not effectively suppress the generation of polymer, causes product purity to descend, and with the affinity reduction of VEGF, possibly bring out immunoreation after the entering body is interior.
Table 3.10mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 |
The stability study of embodiment 4 10mg/ml FP3 fusion rotein in 4 ℃ of following 3ml glass ampules
FP3 fusion rotein 10mg/ml
Citric acid 10mM
Sucrose 8.0%
Arginine 5mM
Polysorbas20 0.05%
pH 7.5～8.3
After albumen stock solution changed liquid, aseptic subpackaged obtaining in the 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, the December working sample is confirmed stability through SEC-HPLC.The result shows that this prescription can effectively suppress the generation of polymer, and product purity descends very slow, and the affinity of fusion rotein and VEGF is almost constant.
Table 4.10mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 |
The stability study of embodiment 5 10mg/ml FP3 fusion rotein in 4 ℃ of following 3ml glass ampules
FP3 fusion rotein 10mg/ml
Citric acid 100mM
Sucrose 20.0%
Arginase 12 50mM
Polysorbas20 0.10%
pH 7.5～8.3
After albumen stock solution changed liquid, aseptic subpackaged obtaining in the 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, the December working sample is confirmed stability through SEC-HPLC.The result shows that this prescription can effectively suppress the generation of polymer, and product purity descends very slow, and the affinity of fusion rotein and VEGF is almost constant.
Table 5.10mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 |
The stability study of embodiment 6 10mg/ml FP3 fusion rotein in 4 ℃ of following 3ml glass ampules
FP3 fusion rotein 10mg/ml
Sodium dihydrogen phosphate 5mM
Trehalose 10.0%
Arginine 100mM
PEG400 0.01％
pH 7.5～8.3
After albumen stock solution changed liquid, aseptic subpackaged obtaining in the 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, the December working sample is confirmed stability through SEC-HPLC.
Table 6.10mg/ml FP3 fusion rotein is 4 ℃ stability
The stability study of embodiment 7 20mg/ml FP3 fusion rotein in 4 ℃ of following 3ml glass ampules
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
After albumen stock solution changed liquid, aseptic subpackaged obtaining in the 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, the December working sample is confirmed stability through SEC-HPLC.
Table 7.20mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 representes not detect.
The stability study of embodiment 8 20mg/ml FP3 fusion rotein in 4 ℃ of following 3ml glass ampules
FP3 fusion rotein 20mg/ml
Citric acid 10mM
Sucrose 30%
Arginine 500M
Polysorbas20 0.1%
pH 7.5～8.3
After albumen stock solution changed liquid, aseptic subpackaged obtaining in the 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, the December working sample is confirmed stability through SEC-HPLC.
Table 8.20mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 |
The stability study of embodiment 9 10mg/ml FP3 fusion rotein in 4 ℃ of following 3ml glass ampules
FP3 fusion rotein 10mg/ml
Citric acid 10mM
Sucrose 5%
Arginine 100M
Polysorbas20 0.05%
pH 7.5～8.3
After albumen stock solution changed liquid, aseptic subpackaged obtaining in the 3ml glass ampule, 4 ℃ keep sample, and 0,1,2,3,4,5,6,7,8,9,10,11, the December working sample is confirmed stability through SEC-HPLC.
Table 9.10mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 |
The stability study of embodiment 10 10mg/ml FP3 fusion rotein in 4 ℃ of following 3ml glass ampules
With FP3 fusion rotein stock solution through Vivaflow concentrate change liquid after aseptic subpackaged back packing obtain semi-finished product, put the citric acid of 55mM, 12.5% sucrose, the arginine of 250mM, 0.05% polysorbas20, pH7.9 buffer; Regulate the FP3 fusion rotein to 10mg/ml, aseptic subpackaged obtaining in the 3ml glass ampule, 4 ℃ keep sample, 0,1,2; 3,4,5,6,7,8; 9,10,11, the December working sample is confirmed stability through SEC-HPLC.
Table 10.10mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 |
The stability study of embodiment 11 20mg/ml FP3 fusion rotein in 4 ℃ of following 3ml glass ampules
With FP3 fusion rotein stock solution through Vivaflow concentrate change liquid after aseptic subpackaged back packing 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; Regulate the FP3 fusion rotein to 20mg/ml, aseptic subpackaged obtaining in the 3ml glass ampule, 4 ℃ keep sample, 0,1,2; 3,4,5,6,7,8; 9,10,11, the December working sample is confirmed stability through SEC-HPLC.
Table 11.20mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 12 4 ℃ of following 40mg/ml FP3 fusion rotein lyophilized formulations 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; Divide after pH regulator to the 7.5～8.3 back packing to install in the 3ml glass ampule, the condition of the freeze-drying curve of optimization is :-50 ℃ of rapid pre-freezes 4 hours are controlled at-20 ℃ of distillations and remove most of moisture; The temperature of dividing plate of progressively raising is then further removed residual moisture; Final step application 25 degree and the vacuum that reach capacity reduce moisture, make simultaneously sample temperature rise can be too not high.After lyophilizing finished, the Asser went out freeze drying box, rolls aluminium lid, carries out analyses such as moisture and purity, and 4 ℃ keep sample, and 0,1,6, December adds equal-volume water for injection and redissolves, and working sample is confirmed stability through SEC-HPLC.
Table 12.40mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 13 4 ℃ of following 40mg/ml FP3 fusion rotein lyophilized formulations 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; Divide after pH regulator to the 7.5～8.3 back packing to install in the 3ml glass ampule, the condition of the freeze-drying curve of optimization is :-50 ℃ of rapid pre-freezes 4 hours are controlled at-20 ℃ of distillations and remove most of moisture; The temperature of dividing plate of progressively raising is then further removed residual moisture; Final step application 25 degree and the vacuum that reach capacity reduce moisture, make simultaneously sample temperature rise can be too not high.After lyophilizing finished, the Asser went out freeze drying box, rolls aluminium lid, carries out analyses such as moisture and purity, and 4 ℃ keep sample, and 0,1,6, December adds equal-volume water for injection and redissolves, and working sample is confirmed stability through SEC-HPLC.
Table 13.40mg/ml FP3 fusion rotein is 4 ℃ stability
| 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 14 4 ℃ of following 20mg/ml FP3 fusion rotein of embodiment in the glass pre-filled syringe
FP3 fusion rotein 20mg/ml
Citric acid 55mM
Sucrose 12.5%
Arginase 12 50mM
Polysorbas20 0.05%
pH 7.5～8.3
With FP3 fusion rotein stock solution through concentrate change liquid after aseptic subpackaged back packing obtain semi-finished product, regulate the FP3 fusion rotein to 20mg/ml, aseptic subpackaged in 1ml glass glass pre-filled syringe with FluroTec coating; 4 ℃ keep sample; 0,1,6; The December working sample is confirmed stability through SEC-HPLC.
The table 14.4 ℃ stability study of following 20mg/ml FP3 fusion rotein in the glass pre-filled syringe
| 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
The Bio-sil column stability high, heat-resisting withstand voltage, the life-span long, can realize the sharp separation of biomacromolecule.TSK G3000SWxl is the Bio-sil post, the scope 10-500kD of particle diameter 5 μ m, aperture 250 dusts, separation globulin, and sample separation time 30min is to analyze the chromatographic column that biomacromolecule generally uses in the world.Among the present invention; We select Bio-sil post TSKG3000SWxl chromatographic column for use, and instrument is 2695 high performance liquid chromatographs of Waters, are mobile phase with the phosphate buffer of pH to 7.20; Flow velocity is 0.5ml/ml; Column temperature is 25 ℃, and the detection wavelength is 280nm, and the purity of reorganization human vascular endothelial growth factor receptor-antibody FP3 fusion rotein is detected.
The test of embodiment 16 rabbit intravitreal injection repeat administrations
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, the single intravitreal injection gives test sample (0.5mg/50 μ L/ eye) or equal-volume solvent control article respectively, and opposite side rehearses and injects contrast or blank.Carry out perusal every day after the administration, regularly uses indirect ophthalmoscope and slit lamp to check.Put to death animal in 14 days after the administration, get eyeball and carry out histopathological examination.Through observed result and histopathological examination result, investigate the local toxicity of test sample.The result finds: Japan large ear rabbit vitreum inner injecting and administering, and in the eyes that 6 give test sample, there is 1 perusal to see slight hyperemia is arranged, see a small amount of secretions for 1; Slit lamp observation sees that 1 lenticular opacity occurs.Simultaneously, observe same phenomenon giving solvent control side, false injection side and blank branch hole eyeball respectively, and incidence rate and time of origin and give test sample branch hole eyeball basically identical.Can get rid of these phenomenons thus is the IRs that caused by test sample.Histopathological examination sees that all eyeball tissue structures are all normal.So under this experimental condition, lagophthalmos single intravitreal injection gives recombined human vascular endothelial growth factor receptor-antibody fusion protein injection, does not cause local irritation or tissue injury responses.
Claims (10)
1. a pharmaceutical composition that contains the fusion rotein that suppresses blood vessel hyperplasia is characterized in that comprising
(a) extracellular domain 3 of the extracellular domain 2 of the vegf receptor 1 of 0.1-100mg/ml and vegf receptor 2 and 4 and the fusion rotein of human normal immunoglobulin Fc comprises the aminoacid sequence of SEQ ID No:1;
(b) 5-100mM buffer, acid wherein is selected from Tris-HCl, citric acid, dibastic sodium phosphate, sodium dihydrogen phosphate, acetic acid, succinic acid, one or more in the hydrochloric acid;
(c) the 5-500mM basic amino acid is selected from lysine, a kind of or its combination in arginine and the histidine;
(d) the 0.1-30% salt penetration is pressed the agent regulator, and sugar wherein is selected from sucrose, and trehalose, mannitol, glycerol, propylene glycol, one or more in the Pyrusussuriensis ester alcohol, salt are selected from pharmaceutically a kind of or its combination in the acceptable salt of sodium chloride or other;
(e) one or more surfactants or the cosolvent of 0.01-0.1% are selected from Polyethylene Glycol, and polysorbas20, Tween 80, propylene glycol, dimethyl sulfoxide or other be one or more in the acceptable surfactant pharmaceutically;
(f) regulating pH is 7.5～8.3.
2. pharmaceutical composition according to claim 1 is characterized in that comprising
(a) the fusion rotein of 10-40mg/ml like 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.
3. pharmaceutical composition according to claim 2 is characterized in that containing sodium chloride.
4. pharmaceutical composition according to claim 2; It is characterized in that composed of the following components: 10-40mg/ml like the fusion rotein of SEQ ID No:1, the citric acid of 10mM, 5% sucrose, the arginine of 100mM; 0.05% polysorbas20, pH7.5～8.3.
5. pharmaceutical composition according to claim 2; It is characterized in that composed of the following components: 10-40mg/ml like the fusion rotein of SEQ ID No:1, the citric acid of 55mM, 12.5% sucrose, the arginine of 250mM; 0.05% polysorbas20, pH7.5～8.3.
6. pharmaceutical composition according to claim 4 is characterized in that described fusion rotein like SEQ ID No:1 is 10mg/ml.
7. according to each described pharmaceutical composition among the claim 1-5, it is characterized in that processing liquid preparation or lyophilized formulations.
8. pharmaceutical composition according to claim 6 is characterized in that the liquid preparation of processing is ophthalmic preparation, especially eye drop.
9. pharmaceutical composition according to claim 6 is characterized in that dosage form is preparatory filling injection agent.
10. according to each described pharmaceutical composition among the claim 1-8, it is characterized in that purposes, especially AMD in the medicine of the disease that the preparation treatment is caused by angiogenesis or growth.
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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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| CN105435222A (en) * | 2014-09-25 | 2016-03-30 | 信达生物制药(苏州)有限公司 | Recombinant 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 |
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| CN109562166A (en) * | 2017-04-28 | 2019-04-02 | 苏州思坦维生物技术股份有限公司 | A kind of medical composite for eye and application 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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