CN103816115B - A kind of pharmaceutical composition containing the fusion protein for suppressing blood vessel hyperplasia and purposes - Google Patents
A kind of pharmaceutical composition containing the fusion protein for suppressing blood vessel hyperplasia and purposes Download PDFInfo
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- CN103816115B CN103816115B CN201410067935.1A CN201410067935A CN103816115B CN 103816115 B CN103816115 B CN 103816115B CN 201410067935 A CN201410067935 A CN 201410067935A CN 103816115 B CN103816115 B CN 103816115B
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Abstract
The invention discloses it is a kind of containing suppress blood vessel hyperplasia fusion protein pharmaceutical composition and purposes, and in particular to it is a kind of containing vascular endothelial growth factor（VEGF）The extracellular domain 2 (Flt 2) of acceptor 1 and the (KDR 3 of extracellular domain 3 and 4 of vegf receptor 2,4) with the pharmaceutical composition of the fusion protein of human immunoglobulin(HIg) 1 (IG1) Fc, the pharmaceutical composition can make fusion protein keep stabilization, generation that fusion protein polymer can effectively be suppressed and the purity that causes declines, so as to keep the bioactivity of active principle.
Description
It is on 08 31st, 2010 the applying date that the application is, Application No. 201010267503.7 is entitled " a kind of
The divisional application of pharmaceutical composition and purposes containing the fusion protein for suppressing blood vessel hyperplasia ".
Technical field
The present invention relates to field of pharmaceutical preparations, it is related to the egg merged comprising vegf receptor fragment and immunoglobulin Fc
White pharmaceutical composition, and it is in application medically.
Background technology
With the development of INVENTIONModern cell molecular biology, cell factor and cell surface correlation molecule are in ophthalmology disease
Effect is widely paid close attention to and is studied, and VEGF is that vascular endothelial cell mitogenesis element has increase vasopermeability
Biological nature, particularly important in generations of the fetal period VEGF to blood vessel, level declines after birth.VEGF is in low under physiological status
Horizontal expression state, the function for maintaining blood vessel is necessary.Newest result of study prompting, for senile retinal blood
Pipe lesion (Age-related macular degeneration, referred to as AMD), diabetic retinopathy (Diabetic
Retinopathy, DR) etc. the related disease of angiogenesis all play extensive and important effect.AMD is multiple be born in 45 years old with
On, its illness rate increases with the growth at age, is the important diseases of current the elderly's blinding.Moist AMD is mainly glass
The destruction of film, choroidal artery constitutes CNV under invading retina, occurs under macular area retinal pigment epithelium
Or serosity or hemorrhagic disciform detachment under neural epithelium, machine scar is eventually become, effectively suppress to cause wet macular disease
The blood vessel endothelial cell growth factor VEGF of change, blocking VEGF or vegf receptor so as to reach angiogenesis inhibiting have it is important
There is important therapeutic action.In DR, the content of VEGF is higher than normal level in cell and body fluid.VEGF increases, and causes hair
Thin vascular permeability is sexually revised, and causes retina to ooze out, bleeding and macular edema, induction of vascular generation element
(Angiogenin) generation increases, and collaboration promotes the formation of retinal neovascularization, causes inpairment of vision.
Sat linkage, hydrogen bond, disulfide bond and hydrophobic effect are Protein requirement conformational stability active forces.It is metal ion, substrate, auxiliary
Helping the factor and interaction of other low phases to molecular weight ligands makes protein conformation stabilization.Protein and other large biological molecules
The especially effect of protein and fat.In vivo, protein often interacts to form compound with lipid or polysaccharide, shielding
The hydrophobic region of protein surface, so as to dramatically increasing the stability of protein.Albumen is unstable mainly to be drawn by following factor
Rise：(1) physical action：The polar water molecules contact with protein hydrophobic core adjusted by Brownian movement can cause protein unstable
It is fixed.(2) chemical action：The oxidation of the amino acid residue of active site is one of most common mechanism of enzyme inactivation.Such as half Guang
The sulfydryl of propylhomoserin and the indole ring of tryptophan, it is especially sensitive to aoxidizing.(3) biological action：Proteolytic enzyme is acted on.Micro- life
Thing and foreign protein hydrolysis enzyme effect catalysis peptide bond hydrolysis.By genetic engineering bacterium purify eukaryotic polypeptide when yield it is low, be by
Caused in external proteolysis.
Polymerization makes the hydrophobic amino acid residues of embedding exposed to aqueous solvent first, causes protein reversible to be denatured；
Secondly, protein molecule associates each other, to reduce the unfavorable exposed of hydrophobic amino acid；Finally, if protein molecule contains half
Cystine and cystine residue, then can occur intermolecular disulfide bond exchange reaction.Polymerization sometimes can be by reduction and oxygen again
Change regeneration natural disulphide bonds, be re-activated protein.Polymerization and simple precipitation are distinguishing, and the latter occurs protein
Significant conformation change.
The inventors have observed that an obvious problem is that pharmaceutical solutions easily forms solvable polymerization after long term storage
How thing and insoluble particle, solve this problem, finds a kind of in physics and chemically all stable pharmaceutical composition, can
Suppress polymer generation, and less soluble polymer and insoluble particles can be formed after long term storage.Further, since its group
It is pharmaceutically acceptable component to divide, and can be used for treating eye disease, including intravitreal injection and topical administration.Additionally,
The inventors discovered that the preparation prescription for obtaining compares in syringe more stablizing in the vial.
In the present invention using can angiogenesis inhibiting, by the cell of vascular endothelial growth factor (VEGF) acceptor 1
The extracellular domain 3 and 4 (KDR-3,4) of extracellular portion 2 (Flt-2) and vegf receptor 2 and human immunoglobulin(HIg) 1 (IG1) Fc
The albumen (FP3 albumen) of fusion be to be expressed by the working cardial cell of recombinant technique and medicinal pure by reaching after purification
Degree, suitable pharmaceutical preparation is obtained by changing liquid formulation packing.These preparation preferred liquid preparations or lyophilized formulations, it is adaptable to eye
The treatment of eyeball disease, in particular for intravitreal injection.
, used as protein drug, its stability is more far short of what is expected than common small-molecule chemical medicine, compared to naturally occurring for FP3 albumen
Immunoglobulin, its stability is also worse.Fusion protein described in Chinese patent is Chinese patent " vegf receptor fusion protein
Prescription described in application in disease of eye is treated " (patent No. ZL200610066257.2) is needed in -20 DEG C of preservations, right
The production of medicine, transport is preserved and application is proposed requirement higher.It is well known that the stabilization of the fusion protein of general restructuring
Property it is all poor, can be influenceed by various environmental factors during preservation, such as temperature, appropriateness, oxygen, ultraviolet etc. can
It is that fusion protein occurs various polymerizations for physically or chemically changing, causing protein, decomposes, oxidation or denaturation etc..These changes
The activity reduction of albumen can be made, therapeutic effect declines and causes serious toxic and side effect.Therefore, develop stabilization and be easy to
The fusion protein formulations of transport and storage are with highly important clinical meaning.
The content of the invention
An object of the present invention be provide it is a kind of containing vascular endothelial growth factor (VEGF) acceptor 1 it is thin
The extracellular domain 3 and 4 (KDR-3,4) of ectodomain 2 (Flt-2) and vegf receptor 2 and human immunoglobulin(HIg) 1 (G1) Fc
Fusion protein pharmaceutical composition, can more particularly be used for the liquid preparation of intravitreal injection, the liquid preparation can make to melt
Hop protein keeps stabilization, its most it is prominent be characterized in the generation and the purity that causes that can effectively suppress fusion protein polymer under
Drop, so as to keep the bioactivity of active principle.
One aspect of the present invention provides a kind of pharmaceutical composition containing the fusion protein for suppressing blood vessel hyperplasia, and its feature exists
In comprising
The extracellular domain 2 of the vegf receptor 1 of (a) 0.1-100mg/ml and the He of extracellular domain 3 of vegf receptor 2
The fusion protein of 4 and human immunoglobulin(HIg) Fc, comprising SEQ ID No:1 amino acid sequence；
B () 5-100mM buffer solutions, acid therein is selected from Tris-HCl, citric acid, dibastic sodium phosphate, sodium dihydrogen phosphate, vinegar
Acid, succinic acid, one or more in hydrochloric acid；
C () 5-500mM basic amino acids are selected from lysine, one kind or its combination in arginine, and histidine；
D () 0.1-30% salt penetrations press agent conditioning agent, sugar therein to be selected from sucrose, trehalose, mannitol, glycerine, the third two
Alcohol, one or more in sorb ester alcohol, salt is selected from sodium chloride or other one kind or its group pharmaceutically in acceptable salt
Close；
One or more surfactant or cosolvent of (e) 0.01-0.1%, selected from polyethylene glycol, polysorbas20, tween
80, propane diols, dimethyl sulfoxide (DMSO) or other pharmaceutically in acceptable surfactant one or more；
F () regulation pH is 7.5~8.3.
Wherein pharmaceutical composition is preferably the pharmaceutical composition comprising following component：
(a) 10-40mg/ml such as SEQ ID No:1 fusion protein；
The citric acid of (b) 5-100mM or sodium dihydrogen phosphate one or two；
The arginine of (c) 5-500mM or histidine one or two；
The sucrose of (d) 8-30% or trehalose one or two；
The Tween-20 of (e) 0.01-0.1% or polyethylene glycol one or two；
F () regulation pH is 7.5~8.3.
Pharmaceutical composition of the present invention, it is more preferably composed of the following components：10mg/ml such as SEQ ID
No：The citric acid of fusion protein, 10mM shown in 1,8% sucrose, the arginine of 5mM, 0.05% polysorbas20, pH7.5~
8.3；
Described pharmaceutical composition of the present invention, it is further preferably composed of the following components：10mg/ml such as SEQ ID
No：The citric acid of fusion protein, 100mM shown in 1,20% sucrose, the arginine of 250mM, 0.1% polysorbas20, pH7.5
~8.3.
Pharmaceutical composition of the present invention, it is more preferably composed of the following components：20mg/ml such as SEQ ID
No：The citric acid of fusion protein, 10mM shown in 1,30% sucrose, the arginine of 500mM, 0.1% polysorbas20, pH7.5
~8.3.
Pharmaceutical composition of the present invention, it is more preferably composed of the following components：40mg/ml such as SEQ ID
No：The citric acid of fusion protein, 250mM shown in 1,8% sucrose, the histidine of 100mM, 0.1% polysorbas20, pH7.5
~8.3.
Sodium chloride can also be contained in above-mentioned pharmaceutical composition.
Pharmaceutical composition of the present invention, it is more preferably composed of the following components：20mg/ml such as SEQ ID
No：The citric acid of fusion protein, 5mM shown in 1,4% sucrose, 4% sodium chloride, the arginine of 100mM, the group of 100mM
Propylhomoserin, 0.05% polysorbas20,0.05% PEG400, pH7.5~8.3.
Pharmaceutical composition of the present invention, it is more preferably composed of the following components：10mg/ml such as SEQ ID
No：The sodium dihydrogen phosphate of fusion protein, 5mM shown in 1,10.0% trehalose, the arginine of 100mM, 0.01%
PEG400, pH7.5~8.3.
The dosage form that aforementioned pharmaceutical compositions are made can be liquid preparation or lyophilized formulations, and wherein liquid preparation is preferably
Eye-drops preparations, especially eye drops；Can also be pre-filled syringes.
Aforementioned pharmaceutical compositions can be used for treating the disease that angiogenesis or growth cause, and preferably age related is yellow
Spot is denatured.
In the present invention, described fusion protein is that " vegf receptor fusion protein is in disease of eye is treated for Chinese patent
Application " fusion protein described in (patent No. ZL200610066257.2), specifically FP3 fusion proteins, therefore
The content of ZL200610066257.2 can be used to the present invention is expanded on further.
Specific embodiment
Stability study of the 10mg/ml FP3 fusion proteins original prescriptions of embodiment 1 in 3ml glass ampules at 4 DEG C
Prescription is as follows：
After albumen stoste is changed into liquid, aseptic subpackaged to obtain 3ml glass ampules, 4 DEG C keep sample, 0,1,2,3,4,5,6,7,
8,9,10,11, December determination sample determines stability by SEC-HPLC.Result shows that the prescription can not effectively suppress polymerization
The generation of thing, causes product purity to decline, the affinity reduction with VEGF, and immune response may be induced later into internal.
Stability of the 10mg/ml FP3 fusion proteins of table 1. at 4 DEG C
Stability study of the 10mg/ml FP3 fusion proteins of embodiment 2 in 3ml glass ampules at 4 DEG C
After albumen stoste is changed into liquid, aseptic subpackaged to obtain 3ml glass ampules, 4 DEG C keep sample, 0,1,2,3,4,5,6,7,
8,9,10,11, December determination sample determines stability by SEC-HPLC.Result shows that the prescription can not effectively suppress polymerization
The generation of thing, causes product purity to decline, the affinity reduction with VEGF, and immune response may be induced later into internal.
Stability of the 10mg/ml FP3 fusion proteins of table 2. at 4 DEG C
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | It is qualified | 7.7 | 10.1 | 0.2 | 10.7 |
| 1 | It is qualified | 7.9 | 10.1 | 2.0 | 9.9 |
| 2 | It is qualified | 7.7 | 10.7 | 3.2 | 9.3 |
| 3 | It is qualified | 7.8 | 10.6 | 5.4 | 8.4 |
| 6 | It is qualified | 7.7 | 10.3 | 9.9 | 8.1 |
| 9 | It is qualified | 7.5 | 10.1 | 13.1 | 8.5 |
| 12 | It is qualified | 7.9 | 10.3 | 23.3 | 7.3 |
Stability study of the 10mg/ml FP3 fusion proteins of embodiment 3 in 3ml glass ampules at 4 DEG C
After albumen stoste is changed into liquid, aseptic subpackaged to obtain 3ml glass ampules, 4 DEG C keep sample, 0,1,2,3,4,5,6,7,
8,9,10,11, December determination sample determines stability by SEC-HPLC.Result shows that the prescription can not effectively suppress polymerization
The generation of thing, causes product purity to decline, the affinity reduction with VEGF, and immune response may be induced later into internal.
Stability of the 10mg/ml FP3 fusion proteins of table 3. at 4 DEG C
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | It is qualified | 7.9 | 10.2 | 0.3 | 10.4 |
| 1 | It is qualified | 7.9 | 10.2 | 1.4 | 10.0 |
| 2 | It is qualified | 7.8 | 10.6 | 2.6 | 9.7 |
| 3 | It is qualified | 7.8 | 10.7 | 3.6 | 8.1 |
| 6 | It is qualified | 8.1 | 10.5 | 9.4 | 7.9 |
| 9 | It is qualified | 8.3 | 10.1 | 18.5 | 9.7 |
| 12 | It is qualified | 8.0 | 10.4 | 19.1 | 9.6 |
Stability study of the 10mg/ml FP3 fusion proteins of embodiment 4 in 3ml glass ampules at 4 DEG C
After albumen stoste is changed into liquid, aseptic subpackaged to obtain 3ml glass ampules, 4 DEG C keep sample, 0,1,2,3,4,5,6,7,
8,9,10,11, December determination sample determines stability by SEC-HPLC.Result shown, polymer can be effectively suppressed at this
Generation, product purity declines very slow, and fusion protein is almost unchanged with the affinity of VEGF.
Stability of the 10mg/ml FP3 fusion proteins of table 4. at 4 DEG C
Stability study of the 10mg/ml FP3 fusion proteins of embodiment 5 in 3ml glass ampules at 4 DEG C
After albumen stoste is changed into liquid, aseptic subpackaged to obtain 3ml glass ampules, 4 DEG C keep sample, 0,1,2,3,4,5,6,7,
8,9,10,11, December determination sample determines stability by SEC-HPLC.Result shown, polymer can be effectively suppressed at this
Generation, product purity declines very slow, and fusion protein is almost unchanged with the affinity of VEGF.
Stability of the 10mg/ml FP3 fusion proteins of table 5. at 4 DEG C
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | It is qualified | 7.9 | 10.0 | 0.3 | 10.4 |
| 1 | It is qualified | 7.9 | 10.1 | 0.3 | 10.3 |
| 2 | It is qualified | 7.9 | 10.0 | 0.4 | 10.4 |
| 3 | It is qualified | 7.9 | 10.1 | 0.5 | 10.2 |
| 6 | It is qualified | 7.9 | 10.2 | 0.7 | 10.1 |
| 9 | It is qualified | 7.9 | 10.0 | 0.8 | 10.2 |
| 12 | It is qualified | 7.9 | 10.1 | 0.9 | 10.0 |
Stability study of the 10mg/ml FP3 fusion proteins of embodiment 6 in 3ml glass ampules at 4 DEG C
After albumen stoste is changed into liquid, aseptic subpackaged to obtain 3ml glass ampules, 4 DEG C keep sample, 0,1,2,3,4,5,6,7,
8,9,10,11, December determination sample determines stability by SEC-HPLC.
Stability of the 10mg/ml FP3 fusion proteins of table 6. at 4 DEG C
Stability study of the 20mg/ml FP3 fusion proteins of embodiment 7 in 3ml glass ampules at 4 DEG C
After albumen stoste is changed into liquid, aseptic subpackaged to obtain 3ml glass ampules, 4 DEG C keep sample, 0,1,2,3,4,5,6,7,
8,9,10,11, December determination sample determines stability by SEC-HPLC.
Stability of the 20mg/ml FP3 fusion proteins of table 7. at 4 DEG C
N/D is represented and not detected.
Stability study of the 20mg/ml FP3 fusion proteins of embodiment 8 in 3ml glass ampules at 4 DEG C
After albumen stoste is changed into liquid, aseptic subpackaged to obtain 3ml glass ampules, 4 DEG C keep sample, 0,1,2,3,4,5,6,7,
8,9,10,11, December determination sample determines stability by SEC-HPLC.
Stability of the 20mg/ml FP3 fusion proteins of table 8. at 4 DEG C
Stability study of the 10mg/ml FP3 fusion proteins of embodiment 9 in 3ml glass ampules at 4 DEG C
After albumen stoste is changed into liquid, aseptic subpackaged to obtain 3ml glass ampules, 4 DEG C keep sample, 0,1,2,3,4,5,6,7,
8,9,10,11, December determination sample determines stability by SEC-HPLC.
Stability of the 10mg/ml FP3 fusion proteins of table 9. at 4 DEG C
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | It is qualified | 7.7 | 10.1 | 0.4 | 10.5 |
| 1 | It is qualified | 7.9 | 10.1 | 0.5 | 10.4 |
| 2 | It is qualified | 7.7 | 10.7 | 0.7 | 10.1 |
| 3 | It is qualified | 7.8 | 10.6 | 0.8 | 10.0 |
| 6 | It is qualified | 7.7 | 10.3 | 0.9 | 9.9 |
| 9 | It is qualified | 7.9 | 10.1 | 1.8 | 7.7 |
| 12 | It is qualified | 7.9 | 10.3 | 1.9 | 6.3 |
Stability study of the 10mg/ml FP3 fusion proteins of embodiment 10 in 3ml glass ampules at 4 DEG C
By FP3 fusion proteins stoste through Vivaflow concentration change liquid after it is aseptic subpackaged after packing obtain semi-finished product, put 55mM
Citric acid, 12.5% sucrose, the arginine of 250mM, 0.05% polysorbas20, pH7.9 buffer solutions, regulation FP3 fusion egg
It is white that to 10mg/ml, aseptic subpackaged to obtain 3ml glass ampules, 4 DEG C keep sample, 0,1,2,3,4,5,6,7,8,9,10,11,
December determination sample, stability is determined by SEC-HPLC.
Stability of the 10mg/ml FP3 fusion proteins of table 10. at 4 DEG C
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | It is qualified | 8.3 | 10.0 | 0.2 | 9.7 |
| 1 | It is qualified | 8.3 | 10.1 | 0.2 | 9.6 |
| 2 | It is qualified | 8.2 | 10.2 | 0.3 | 9.6 |
| 3 | It is qualified | 7.9 | 10.0 | 0.4 | 9.3 |
| 6 | It is qualified | 8.0 | 10.1 | 0.5 | 9.3 |
| 9 | It is qualified | 7.9 | 10.1 | 0.6 | 9.1 |
| 12 | It is qualified | 7.9 | 10.3 | 1.0 | 9.4 |
Stability study of the 20mg/ml FP3 fusion proteins of embodiment 11 in 3ml glass ampules at 4 DEG C
By FP3 fusion proteins stoste through Vivaflow concentration change liquid after it is aseptic subpackaged after packing obtain semi-finished product, put 55mM
Citric acid, 12.5% sucrose, the arginine of 250mM, 0.05% polysorbas20, pH7.5~8.3 buffer solution, regulation FP3 melt
To 20mg/ml, aseptic subpackaged to obtain 3ml glass ampules, 4 DEG C keep sample hop protein, 0,1,2,3,4,5,6,7,8,9,10,
11, December determination sample determines stability by SEC-HPLC.
Stability of the 20mg/ml FP3 fusion proteins of table 11. at 4 DEG C
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | It is qualified | 7.9 | 19.9 | 0.7 | 10.7 |
| 1 | It is qualified | 7.9 | 20.1 | 1.2 | 10.2 |
| 2 | It is qualified | 7.7 | 20.7 | 1.3 | 10.3 |
| 3 | It is qualified | 7.8 | 21.6 | 1.4 | 10.4 |
| 6 | It is qualified | 8.0 | 21.6 | 1.4 | 10.4 |
| 9 | It is qualified | 7.9 | 21.1 | 2.6 | 9.6 |
| 12 | It is qualified | 7.9 | 21.3 | 3.6 | 9.4 |
The stability study of 40mg/ml FP3 fusion protein lyophilized formulations at 12 4 DEG C of embodiment
By FP3 fusion proteins solution regulation to 40mg/ml, 3ml glass is dispensed into after being dispensed after pH regulations to 7.5~8.3
In ampoule, the condition of the freeze-drying curve of optimization is：- 50 DEG C of rapid 4 hours of pre-freeze, control removes most of in -20 DEG C of distillations
Moisture, the temperature for being then stepped up dividing plate further removes the moisture of residual, and 25 degree of final step application simultaneously reaches capacity true
Sky, reduces moisture, at the same rise sample temperature will not be too high.After lyophilized end, Asser goes out freeze drying box, rolls aluminium lid, carries out
Moisture and purity etc. are analyzed, and 4 DEG C keep sample, 0,1,6, and December adds isometric water for injection to redissolve, determination sample, by SEC-
HPLC determines stability.
Stability of the 40mg/ml FP3 fusion proteins of table 12. at 4 DEG C
| Time (moon) | Outward appearance after redissolution | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | It is qualified | 7.9 | 39.9 | 0.1 | 11.2 |
| 1 | It is qualified | 7.9 | 40.1 | 0.2 | 10.6 |
| 6 | It is qualified | 7.9 | 40.7 | 0.3 | 10.1 |
| 12 | It is qualified | 8.3 | 40.6 | 0.4 | 10.0 |
The stability study of 40mg/ml FP3 fusion protein lyophilized formulations at 13 4 DEG C of embodiment
By FP3 fusion proteins solution regulation to 20mg/ml, 3ml glass is dispensed into after being dispensed after pH regulations to 7.5~8.3
In ampoule, the condition of the freeze-drying curve of optimization is：- 50 DEG C of rapid 4 hours of pre-freeze, control removes most of in -20 DEG C of distillations
Moisture, the temperature for being then stepped up dividing plate further removes the moisture of residual, and 25 degree of final step application simultaneously reaches capacity true
Sky, reduces moisture, at the same rise sample temperature will not be too high.After lyophilized end, Asser goes out freeze drying box, rolls aluminium lid, carries out
Moisture and purity etc. are analyzed, and 4 DEG C keep sample, 0,1,6, and December adds isometric water for injection to redissolve, determination sample, by SEC-
HPLC determines stability.
Stability of the 40mg/ml FP3 fusion proteins of table 13. at 4 DEG C
Stability study of the 20mg/ml FP3 fusion proteins in glass pre-filled syringe at 14 4 DEG C of embodiment
By FP3 fusion protein stostes it is concentrated change after liquid it is aseptic subpackaged after packing obtain semi-finished product, adjust FP3 fusion proteins
To 20mg/ml, aseptic subpackaged 4 DEG C keep sample in the 1ml glass glass pre-filled syringes with FluroTec coatings,
0,1,6, December determination sample determines stability by SEC-HPLC.
Stability study of the 20mg/ml FP3 fusion proteins in glass pre-filled syringe at 14. 4 DEG C of table
| Time (moon) | Outward appearance | PH value | Concentration (mg/ml) | Polymer (%) | Affinity (pM) |
| 0 | It is qualified | 7.9 | 20.0 | 0.1 | 11.2 |
| 1 | It is qualified | 7.9 | 20.1 | 0.1 | 11.6 |
| 6 | It is qualified | 7.9 | 20.1 | 0.1 | 11.1 |
| 12 | It is qualified | 8.2 | 20.1 | 0.1 | 11.0 |
Gel exclusion chromatography analysis (SEC-HPLC) of the polymer of embodiment 15
Bio-sil column stability is high, heat-resistant pressure-resistant, long lifespan, quick separating that large biological molecule can be realized.TSK
G3000SWxl is Bio-sil post, and 5 μm of particle diameter, 250 angstroms of aperture, the scope 10-500kD for separating globulin separate sample time
30min, is to analyze the chromatographic column that large biological molecule commonly uses in the world.In the present invention, we select Bio-sil post
TSKG3000SWxl chromatographic columns, instrument is 2695 high performance liquid chromatographs of Waters, with the phosphate buffer of pH to 7.20
It is mobile phase, flow velocity is 0.5ml/ml, and column temperature is 25 DEG C, and Detection wavelength is 280nm, and Recombinant human vascular endothelial growth factor is received
The purity of body-antibody FP3 fusion proteins is detected.
The rabbit intravitreal injection repeat administration of embodiment 16 is tested
12 Japan large ear rabbits, are randomly divided into 2 groups, are respectively test sample group and solvent control group, every group 6, male and female
Half and half.In the right eye of each group animal, single intravitreal injection gives test sample (0.5mg/50 μ L/) or isometric solvent respectively
Reference substance, opposite side rehearses and injects control or blank.Visually observed daily after administration, periodically used indirect ophthalmoscope
And slit-lamp is checked.Put to death animal within 14 days after administration, taking eyeball carries out histopathological examination.By observing result and group
Pathological examination result is knitted, the local toxicity of test sample is investigated.Result finds：It is administered in Japan large ear rabbit vitreum
Medicine, 6 are only given in the eyes of test sample, have 1 to visually observe and see there is slight hyperemia, and 1 is seen a small amount of secretion；Slit lamp observation
See that 1 phacoscotasmus occurs.Meanwhile, it is visually observed giving solvent control side, false injection side and blank side respectively
Same phenomenon, and incidence and time of origin are with to give test sample branch hole eyeball basically identical.Thus can exclude these phenomenons be by
The IR that test sample causes.Histopathological examination is shown in that all eyeball tissue structures are normal.Therefore in this experimental condition
Under, lagophthalmos single intravitreal injection gives Recombinant human vascular endothelial growth factor receptor-antibody fusion protein parenteral solution, does not cause
Local irritation or tissue damage are reacted.
Claims (12)
1. it is a kind of containing suppress blood vessel hyperplasia fusion protein pharmaceutical composition, it is characterised in that it is composed of the following components：
10mg/ml such as SEQ ID No：The citric acid of fusion protein, 10mM shown in 1,8% sucrose, the arginine of 5mM,
0.05% polysorbas20, pH7.5~8.3.
2. it is a kind of containing suppress blood vessel hyperplasia fusion protein pharmaceutical composition, it is characterised in that it is composed of the following components：
10mg/ml such as SEQ ID No：The citric acid of fusion protein, 100mM shown in 1,20% sucrose, the arginine of 250mM,
0.1% polysorbas20, pH7.5~8.3.
3. it is a kind of containing suppress blood vessel hyperplasia fusion protein pharmaceutical composition, it is characterised in that it is composed of the following components：
10mg/ml such as SEQ ID No：The sodium dihydrogen phosphate of fusion protein, 5mM shown in 1,10.0% trehalose, the essence of 100mM
Propylhomoserin, 0.01% PEG400, pH7.5~8.3.
4. it is a kind of containing suppress blood vessel hyperplasia fusion protein pharmaceutical composition, it is characterised in that it is composed of the following components：
20mg/ml such as SEQ ID No：The citric acid of fusion protein, 10mM shown in 1,30% sucrose, the arginine of 500mM,
0.1% polysorbas20, pH7.5~8.3.
5. it is a kind of containing suppress blood vessel hyperplasia fusion protein pharmaceutical composition, it is characterised in that it is composed of the following components：
40mg/ml such as SEQ ID No：The citric acid of fusion protein, 250mM shown in 1,8% sucrose, the histidine of 100mM,
0.1% polysorbas20, pH7.5~8.3.
6. it is a kind of containing suppress blood vessel hyperplasia fusion protein pharmaceutical composition, it is characterised in that it is composed of the following components：
20mg/ml such as SEQ ID No：The citric acid of fusion protein, 5mM shown in 1,4% sucrose, 4% sodium chloride, 100mM
Arginine, the histidine of 100mM, 0.05% polysorbas20,0.05% PEG400, pH7.5~8.3.
7. the pharmaceutical composition according to any one of claim 1-6, it is characterised in that be made liquid preparation or lyophilized system
Agent.
8. pharmaceutical composition according to claim 7, it is characterised in that the liquid preparation is eye-drops preparations.
9. pharmaceutical composition according to claim 7, it is characterised in that the liquid preparation is eye drops.
10. the pharmaceutical composition according to any one of claim 1-6, it is characterised in that be made pre-filled syringes.
11. pharmaceutical composition according to any one of claim 1-10, it is characterised in that treatment is new by blood vessel preparing
Purposes in the medicine of the disease that raw or growth causes.
12. pharmaceutical compositions according to claim 11, it is characterised in that the disease is AMD.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410067935.1A CN103816115B (en) | 2010-08-31 | 2010-08-31 | A kind of pharmaceutical composition containing the fusion protein for suppressing blood vessel hyperplasia and purposes |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201010267503.7A CN102380096B (en) | 2010-08-31 | 2010-08-31 | Medicine combination containing fusion protein for suppressing angiogenesis and application |
| CN201410067935.1A CN103816115B (en) | 2010-08-31 | 2010-08-31 | A kind of pharmaceutical composition containing the fusion protein for suppressing blood vessel hyperplasia and purposes |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201010267503.7A Division CN102380096B (en) | 2010-08-31 | 2010-08-31 | Medicine combination containing fusion protein for suppressing angiogenesis and application |
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| Publication Number | Publication Date |
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| CN103816115A CN103816115A (en) | 2014-05-28 |
| CN103816115B true CN103816115B (en) | 2017-06-27 |
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| CN201410067935.1A Active CN103816115B (en) | 2010-08-31 | 2010-08-31 | A kind of pharmaceutical composition containing the fusion protein for suppressing blood vessel hyperplasia and purposes |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104940926B (en) * | 2014-09-25 | 2017-09-22 | 信达生物制药(苏州)有限公司 | Recombination fusion protein preparation |
| CN111556744B (en) * | 2017-12-22 | 2023-12-29 | 韩商三星Bioepis 股份有限公司 | Liquid compositions comprising VEGF antagonists |
| CN108671229B (en) * | 2018-05-08 | 2022-03-25 | 华博生物医药技术（上海）有限公司 | Pharmaceutical composition preparation of recombinant human vascular endothelial growth factor receptor-antibody fusion protein |
| CN112739323A (en) * | 2018-05-10 | 2021-04-30 | 瑞泽恩制药公司 | Formulations containing high concentrations of VEGF receptor fusion proteins |
| WO2024056058A1 (en) * | 2022-09-16 | 2024-03-21 | 齐鲁制药有限公司 | Stable high-concentration self-buffering pharmaceutical composition |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1993138A (en) * | 2004-06-01 | 2007-07-04 | 阿雷斯贸易股份有限公司 | Method of stabilizing proteins |
| US20090264358A1 (en) * | 2006-03-31 | 2009-10-22 | Chengdu Kanghong Biotechnologies, Co., Ltd | Vegf receptor fusion proteins, their pharmaceutical compositions and therapeutic applications for the eye diseases |
-
2010
- 2010-08-31 CN CN201410067935.1A patent/CN103816115B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1993138A (en) * | 2004-06-01 | 2007-07-04 | 阿雷斯贸易股份有限公司 | Method of stabilizing proteins |
| US20090264358A1 (en) * | 2006-03-31 | 2009-10-22 | Chengdu Kanghong Biotechnologies, Co., Ltd | Vegf receptor fusion proteins, their pharmaceutical compositions and therapeutic applications for the eye diseases |
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| CN103816115A (en) | 2014-05-28 |
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