CN108379561A - A kind of PEGylated uricoxidase freeze dried powder and preparation method thereof - Google Patents
A kind of PEGylated uricoxidase freeze dried powder and preparation method thereof Download PDFInfo
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- CN108379561A CN108379561A CN201810171512.2A CN201810171512A CN108379561A CN 108379561 A CN108379561 A CN 108379561A CN 201810171512 A CN201810171512 A CN 201810171512A CN 108379561 A CN108379561 A CN 108379561A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/44—Oxidoreductases (1)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
- C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
- C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase
Abstract
The present invention relates to the stabilization formulations of PEGylated uricoxidase conjugate more particularly to a kind of PEGylated uricoxidase freeze dried powder and preparation method thereof.The solid constituent of a kind of PEGylated uricoxidase freeze dried powder, the freeze dried powder includes PEGylated uricoxidase, protective agent and freeze-dried excipient, and the protective agent uses sucrose, freeze-dried excipient to use arginine, according to the mass fraction：0.5 5.0 parts of PEGylated uricoxidase；15 35 parts of sucrose；5.0 25 parts of arginine.The freeze dried powder improves the stability of PEGylated uricoxidase freeze dried powder, and forming.The present invention also provides the preparation methods of above-mentioned freeze dried powder.
Description
Technical field
The present invention relates to the stabilization formulations of PEGylated uricoxidase conjugate more particularly to a kind of Pegylations
Urate oxidase freeze dried powder and preparation method thereof.
Background technology
Urate oxidase (Urate oxidase, UOX) is the key enzyme of purine metabolism in organism, catalysis uric acid oxidation
Allantoic acid is formed, is made of four identical subunits, each subunit contains 301 amino acid, and molecular weight is 34kD or so.It is clinical
On, urate oxidase not only can be used for the detection of uric acid in blood concentration as diagnostic reagent, but also can be used as biological therapy medicine
Object is used for the treatment of Patients with Hyperuricemia.The natural limited source of urate oxidase significantly limits its research and answers
With.Currently, the urate oxidase largely used is produced using gene engineering method, that is, recombinate urate oxidase.For
For human body, recombination urate oxidase is foreign body, and human body can generate it prodigious immunological rejection, recombinate uric acid oxygen
After change enzyme enters human body, foreign protein is easily identified as by immune system, to cause serious allergic reaction or even cause
Quick property shock.PEGylated uricoxidase by by water-soluble macromolecule polymer polyethylene glycol with recombination urate oxidase
The modified medicaments of covalent bond Cheng Xin reduce its immunogenicity, and extend the half-life period of recombination urate oxidase in vivo.
So Pegylation human urine acid oxidase is polyethyleneglycol modified to reduce it to recombination urate oxidase progress
Change structure drug for the purpose of immunogenicity and extension Half-life in vivo.However, although PEGylated uricoxidase is in clinic
It is upper effective, but such conjugate in clinical practice in use, during producing and being distributed to health institution, need energy
Enough preparations stored for a long time.
The PEGylated uricoxidase drug pegloticase of Savient companies of U.S. production, using by 8mg/mL
PEGylated uricoxidase, bis- hypophosphite monohydrate disodium hydrogens of 12.2mmol/L, bis- hypophosphite monohydrate sodium dihydrogens of 2.8mmol/L,
The liquid preparation of 150mmol/L sodium chloride composition.The shortcomings that due to this liquid preparation in the presence of that can not vibrate, can not freeze, therefore not
It is appropriate for transporting for long-distance.Freeze-drying is to overcome this deficiency, provides and a kind of preparing stable Pegylation uric acid
The method of oxidase preparation.
Freeze-drying is a kind of method for making water distil from composition after composition freezing.It is water-soluble in this method
Surpass drug relatively unstable after a period of time and biological products in liquid, dosage appearance can be placed in tractable liquid form
In device, it is dried without using damaging heating, and to extend period of storage under drying regime.It is designed to freeze-drying
Preparation generally comprises expanded constituents and protective agent, antifreezing agent and other stabilizers for increasing solid material amount, with protection activity
Ingredient damage during freeze-drying and after freeze-drying.
Invention content
It is an object of the present invention to provide a kind of PEGylated uricoxidase freeze dried powder, which improves
The stability of PEGylated uricoxidase freeze dried powder, and forming.Second object of the present invention is to provide
The preparation method for the freeze dried powder stated.
The present invention provides the PEGylated uricoxidase that can stablize during long term storage at 2 DEG C -8 DEG C and system is newly lyophilized
Agent, and the method for preparing these preparations.The part provide these preparations, their preparation and use detailed description.This is retouched
It states in a manner of gradually detailed and specific, by several exemplary for example, being retouched to the various embodiments of the present invention
It states.These examples are non-limiting, and the obvious associated change of those skilled in the art is included in claims.
In order to which the present invention can be more easily understood, which especially defines certain technical and scientific terms.Herein
The every other technical and scientific term used has that general technical staff of the technical field of the invention is normally understood contains
Justice.
In order to realize first above-mentioned purpose, present invention employs technical solutions below：
The solid constituent of a kind of PEGylated uricoxidase freeze dried powder, the freeze dried powder includes Pegylation urine
Acid oxidase, protective agent and freeze-dried excipient, the protective agent use sucrose, and freeze-dried excipient uses arginine, by quality
Number meter：
0.5-5.0 parts of PEGylated uricoxidase
15-35 parts of sucrose
5.0-25 parts of arginine.
Preferably, according to the mass fraction：
1.5-3.0 parts of PEGylated uricoxidase
20-30 parts of sucrose
10-20 parts of arginine.
As a further improvement, the solid constituent of the freeze dried powder further includes buffer, buffer is by solution before freeze-drying
PH maintains 7.0-9.0, it is preferable that pH maintains 7.5-8.5；Further preferably, pH maintains 7.8-8.2.Preferably, buffer selects
With disodium hydrogen phosphate and sodium dihydrogen phosphate system.
As a further improvement, the freeze dried powder is freezed under conditions of moisture is less than 3% freeze dried powder
It is dry；It is preferred that the moisture of freeze dried powder is 0.5%-2.5%, more preferably 1%-2%.
Terms used herein " PEGylated uricoxidase " refer to one or more polyethylene glycol (PEG) molecule and one
The conjugate of a or multiple recombination urate oxidase molecules.Preferred conjugate for invention formulation each recombinates uric acid oxidation
Enzyme molecule has a 5-13 PEG molecule, more preferable conjugate for 7-11 PEG molecule and single recombination urate oxidase molecule it
Between conjugate.
PEG molecules in conjugate can have different molecular weight.It is preferred that PEG molecules have the mean molecule of 2000-20000
Amount.In particularly preferred embodiments, using PEG5000Prepare conjugate, PEG5000Indicate the PEG molecules tool in conjugate
There is about 5000 average molecular weight.
The urate oxidase part of the PEGylated uricoxidase used in the present invention can be those skilled in the art
Known any recombination urate oxidase.Most preferably by the recombination uric acid oxygen of the aspergillus flavus sequence of escherichia expression system expression
Change enzyme to prepare the active constituent of invention formulation.
PEG and recombination urate oxidase molecule it is conjugated can by any conjugation reaction well known by persons skilled in the art into
Row.It is preferred that poly- by recombinating urate oxidase and mPEG-SC (mPEG-SC), methoxyl group
Ethylene glycol-succinimide acetic acid esters (mPEG-SCM), methoxy poly (ethylene glycol)-succinimidyl propionate (mPEG-SPA) in
PH7.5-9.5 reacts, more preferably by recombinating urate oxidase and methoxy poly (ethylene glycol)-succinimide acetic acid esters (mPEG-
SCM), methoxy poly (ethylene glycol)-succinimidyl propionate (mPEG-SPA) is reacted in pH7.5-9.5, most preferably passes through recombination
Urate oxidase is reacted with methoxy poly (ethylene glycol)-succinimidyl propionate (mPEG-SPA) in pH7.5-9.5, prepares poly- second
Diolation urate oxidase.
The most preferably PEGylated uricoxidase used in invention formulation is PEG5000Recombined Aspergillus flavus uric acid oxygen
Change enzyme.
The PEGylated uricoxidase content used in invention formulation is between 0.5-5mg/ml, preferred real
It applies in scheme, PEGylated uricoxidase content is in 1-4mg/ml, preferred PEGylated uricoxidase content
In 1.5-3mg/ml.
In addition to PEGylated uricoxidase, invention formulation also includes protective agent, which protects polyethylene glycol
Change destruction, absorption and loss of the urate oxidase from vacuum used in freeze-drying.Protective agent is also used at freeze-drying
During reason, and PEGylated uricoxidase is set to stablize in obtained freeze dried powder.It is generally basede on using protectant amount
The total volume of preparation solution.In an experimental program, the existing amount of protective agent is 5mg/ml-25mg/ml.Preferably implementing
In scheme, protectant amount is 10mg/ml-20mg/ml.
Invention formulation also includes excipient, which can make lyophilized preparation appearance obtained loose, full, complete
It is whole, it is very suitable for industrialized production.The amount of excipient is generally basede on the total volume of preparation liquid.In an experimental program, assign
The existing amount of shape agent is 15mg/ml-35mg/ml.In preferred embodiments, the amount of excipient is 20mg/ml-30mg/ml.
Invention formulation liquid includes buffer, the range for the pH of preparation to be maintained to 7.0-9.0.Preferred reducing will
Between pH maintains 7.5-8.5, pH is most preferably maintained into 7.8-8.2.Preferred buffer includes 12 hypophosphite monohydrate hydrogen two
Sodium and a hypophosphite monohydrate sodium dihydrogen, in 20mmol/L-100mmol/L, preferred concentration is the total concentration of these compounds in preparation
30mmol/L-50mmol/L。
In order to realize second above-mentioned purpose, present invention employs technical solutions below：
A kind of preparation method preparing the PEGylated uricoxidase freeze dried powder, this method includes below
Step：
1) prepared by Solutions in Freeze-drying, and PEGylated uricoxidase, protective agent, excipient are dissolved in note with the amount selected
It penetrates in water, the pH of solution is maintained by 7.0-9.0 by buffer；
2) solution is fitted into suitable for being used freeze-dryer and suitable for the glass container that lyophilized preparation is stored；
3) it is freeze-dried under conditions of being suitble to preparation moisture to be less than 3% freeze dried powder.
Preferably, the step 3) is as follows：
1) under 25 DEG C or 25 DEG C or less, environmental pressure, glass container is fitted into freeze-dryer；
2) it can be pre-chilled 0.5-2 hours under the conditions of 2 DEG C -8 DEG C, under -50 DEG C to -40 DEG C, environmental pressure, make loading
Container experience freezing 2-4 hours；Then with 0.05-0.15 millibars, under the vacuum of preferably 0.06-0.10 millibars of pressure, with two
Walk the solution being frozen and dried；
3) initial drying step can carry out 14-20 hours in -40 DEG C to -30 DEG C, and second step drying can be in 20 DEG C to 30
DEG C carry out 5-9 hours；
4) under 25 DEG C or 25 DEG C or less, environmental pressure, the glass container containing freeze dried powder is unloaded.
Preferably, the glass container is 7ml cillin bottles, preferred loading amount is 2ml/ branch.
In order to prepare invention formulation, by PEGylated uricoxidase, protective agent, excipient, buffer to select
Amount be dissolved in water for injection, which can obtain suitable for being freeze-dried the constituent concentration of solid.Terms used herein
" water for injection " refers to sterile purified water, meets particle, dissolved solid, organic matter, inorganic matter, microorganism and endotoxin contaminants
Normative standard.Once all formulations ingredient dissolve in the solution, by solution is fitted into suitable for freeze-dryer use with
Suitable for the glass container of lyophilized preparation storage.Preferred container is 7ml cillin bottles, and preferred loading amount is 2ml/ branch.Usually
Cillin bottle is placed directly on the pallet of freeze-dryer room or the frame of direct heat transfer.
Glass container containing PEGylated uricoxidase preparation of the present invention is less than being suitble to preparation moisture
It is freeze-dried under conditions of 3% freeze dried powder.It is preferred that the moisture of freeze dried powder is 0.5%-2.5%, more preferably
For 1%-2%.
It selects freeze-drying condition to reach the cake defect of acceptable level, while maintaining ideal low moisture content.This
The phrase " cake defect " that text uses refer to such as collapsed with one or more physical imperfections, shrinkage, cake with cover or melt back.Usually by
Cake is collapsed caused by the excessive heating of preparation during freeze-drying, aesthetic appearances is lost with product and stability difference is related.It is low
Shrinkage caused by the lyophilization cycle of efficiency may lead to part or micro the phenomenon that collapsing, and can lead to Pegylation
Urate oxidase is poor in stable upon storage.With cover is a kind of physical imperfection, and wherein the top of cake is formed and cake overall separation
Film or skin.Melt back refers to the common form collapsed, mainly since preparation distillation is incomplete.Melt back and Pegylation uric acid oxygen
The physical form variation for changing enzyme or moisture bag is related, the poly- second that PEGylated uricoxidase can be caused unstable and combined
Glycol falls off.The percentage of this kind of cake defect is preferably smaller than 50% in freeze dried powder, and more preferably less than 10%, even more preferably less than
2%, more preferably less than 1%.
The preferably freeze drying cycle for being used to prepare the PEGylated uricoxidase that the present invention is lyophilized see the table below 1 always
Knot.
Table 1. is used to be freeze-dried the preferably freeze drying cycle of the PEGylated uricoxidase of the present invention
The preferred lyophilization cycle needs to complete not less than 35 hours, including temperature changes between each step when application
The required time.
According to previously mentioned, the PEGylated uricoxidase of preparation is highly stable during 25 DEG C of storage.One
In a embodiment, after 25 DEG C store 60 days, the potency of freeze dried powder is dropped by less than 30%, preferably smaller than 20%, most preferably
Less than 10%.Free polyethylene glycol is less than 3%, preferably smaller than 2%, more preferably less than 1.5%.PEGylated uricoxidase
Purity is more than 95%, preferably greater than 96%, most preferably greater than 97%.
According to previously mentioned, the PEGylated uricoxidase of preparation is highly stable during 2-8 DEG C of storage.One
In a embodiment, after 2-8 DEG C stores 24 months, the potency of freeze dried powder is dropped by less than 30%, preferably smaller than 20%, more excellent
Choosing is less than 15%, more preferably less than 10%.Free polyethylene glycol is less than 3%, preferably smaller than 2%, more preferably less than 1.5%.It is poly-
Glycation urate oxidase purity is more than 95%, preferably greater than 96%, most preferably greater than 97%.
Preferred invention formulation is freeze-dried in 7ml cillin bottles, and mainly by the PEG of 4.8-5.2mg amounts5000Weight
Group Aspergillus flavus uricoxidase, 50mg arginine, 30mg sucrose, 10.4mg disodium hydrogen phosphates, 0.65mg sodium dihydrogen phosphates composition.
It includes designated substance that terms used herein " mainly by ... form ", which refer to freeze dried powder not only, also includes other substances, these
Other substances do not interfere with the bioactivity or stability of preparation substantially, and it is any specific substantially to influence preparation
It is required that characteristic, such as moisture or cake defect level.Particularly preferred preparation in this section by enumerating at being grouped as.
Description of the drawings
Fig. 1 capillary isoelectric focusings (cIEF) detect the isoelectric point of PEGylated uricoxidase.
Appearance detection (being from left to right followed successively by prescription 1- prescriptions 9) after the freeze-drying of Fig. 2 difference preparation prescriptions.
The dsc analysis of Fig. 3 PEGylated uricoxidase preparation prescriptions.
Specific implementation mode
Below will by embodiment, the present invention will be further described, but following embodiments are only present invention examples therein
Son does not represent interest field defined by the present invention.
Embodiment one
By the isoelectric point of PEGylated uricoxidase, suitable pharmaceutical formulation pH value is selected.
The isoelectric point of PEGylated uricoxidase is detected by capillary isoelectric focusing (cIEF), testing result is shown in figure
1, it is analyzed by Fig. 1, the isoelectric point of PEGylated uricoxidase designs preparation between 4.0-6.0, according to the result
PH value, pH value range design between 6.0-10.
8 buffer systems by designing following different pH value are tested, and the poly- second of 2.5mg/ml is added in each formula
Diolation urate oxidase, detection accelerate active situation of change after 1h at 30 DEG C.
Retentive activity after PEGylated uricoxidase Acceleration study under 2. condition of different pH of table
By carrying out 30 DEG C of Acceleration studies to 9 prescriptions, the PEGylated uricoxidase after Acceleration study is carried out
Activity determination is experimentally confirmed PEGylated uricoxidase and is not sufficiently stable under the conditions of meta-acid or parlkaline, therefore
The pH value of preparation prescription is arranged between 7.0-9.0, between preferably 7.5-8.5, most preferred pH is between 7.8-8.2.
Embodiment two
It is screened by different preparation prescriptions, selects the preparation prescription ingredient of suitable PEGylated uricoxidase.
The preparation of freeze-drying generally comprises the expanded constituents for increasing solid material amount and protective agent, antifreezing agent and other stabilizations
Agent, the damage during freeze-drying and after freeze-drying with protection activity ingredient.
Wherein in freeze drying protectant, our preferred carbohydrates are as protective agent, because carbohydrate plays during freezing
The function of cryoprotective agent, and dehydration protection agent can be played the role of during drying and dehydrating.Carbohydrate divide reduced form sugar and it is non-also
Prototype sugar, in freezing dry process, no matter reduced form sugar and non-reduced carbohydrate can have a good protecting effect, but
The presence of reduced form sugar makes dried frozen aquatic products that Maillard reactions occur during the storage of freeze-drying, finally dried frozen aquatic products is made to become
Matter.So preferably sucrose and mannitol are as protective agent in protective agent screening process.Because sucrose and mannitol can be in height
Effective protection biomolecular structure is not destroyed under the mal-conditions such as warm, high and cold, dry dehydration, to keep protein active.
Excipient main function is to prevent PEGylated uricoxidase from distilling loss with vapor, while slow
It can be crystallized when quick-frozen knot, to provide structural support for PEGylated uricoxidase, make PEGylated uricoxidase
Freeze-drying molding.Excipient should select to stablize, be not easy the substance of oxidation by air in sterile solution.So being sieved in protective agent
Preferably glycine, alanine, sucrose, Tween-80 is as protective agent during choosing, because these substances can be in freezing dry process
In for PEGylated uricoxidase provide structural support, and will not react with active component.
The PEGylated uricoxidase solution containing different protective agent and excipient is prepared, is fitted into 7ml cillin bottles
Freeze-drying tests the cake defect of obtained freeze-drying pulvis, the potency of moisture and PEGylated uricoxidase, dissociates and gather
Ethylene glycol content.The following table 3 is described using the freeze-drying condition enumerated in above-mentioned table 1, and be freeze-dried nine kinds these are molten
The composition of liquid.
Table 3.
(use the lyophilization cycle listed in table 1) after freeze-drying, determine due to collapse cake, cake with cover,
The cake and melt back of catastrophic collapse and the number (appearance results are shown in Fig. 2) for being typically considered to underproof cake, and determine moisture and contain
Amount.It the results are shown in Table shown in 4.
Table 4. is prepared the cake defective work percentage of preparation by solution 1,2,3 and 4
| Preparation | Cake defective work (%) | Moisture (%) |
| 1 | ＜ 1 | ＜ 2.5 |
| 2 | 100 | ＜ 3 |
| 3 | ＜ 2 | ＜ 2.5 |
| 4 | ＜ 1 | ＜ 3 |
| 5 | ＜ 35 | ＜ 2 |
| 6 | 100 | ＜ 3 |
| 7 | ＜ 1 | ＜ 3 |
| 8 | ＜ 2 | ＜ 2.5 |
| 9 | ＜ 1 | ＜ 2 |
In table 4 statistics indicate that, can be reduced after the freeze-drying of the liquid composite of prescription 1,3,4,7,8,9 because cake defect (collapse,
Shrinkage, with cover or melt back) caused by disqualification rate, and keep lower lyophilized preparation moisture.
By measuring the potency of freeze dried powder, shadow of each prescription to PEGylated uricoxidase stability is evaluated
Ring, when freeze dried powder in 25 DEG C preserve 2 months, 2 DEG C -8 DEG C storage 24 months when, potency reduction the results are shown in Table shown in 5, table 6.
The potency for the freeze dried powder that table 5. is prepared by solution 1-9 declines percentage and compares (25 DEG C)
The potency for the freeze dried powder that table 6. is prepared by solution 1-9 declines percentage and compares (2-8 DEG C)
Statistics indicate that, after the liquid composite freeze-drying of prescription 2,3,6,7,8,9, poly- second can be preferably kept in 5 table 6 of table
The potency of diolation urate oxidase.
By measuring the free polyethylene glycol percentage of freeze dried powder, each prescription also can be evaluated to Pegylation uric acid
Aoxidize enzyme stability influence, when freeze dried powder in 25 DEG C store 60 days, 2 DEG C -8 DEG C storage 24 months when, occur dissociate poly- second
The change of glycol content (%) the results are shown in Table shown in 7, table 8.
Table 7. by the solution 1-9 freeze dried powders prepared free polyethyleneglycol content (25 DEG C)
Table 8. by the solution 1-9 freeze dried powders prepared free polyethyleneglycol content (2-8 DEG C)
In table 7, table 8 statistics indicate that, the freeze drying protectant in 25 DEG C and 2 DEG C -8 DEG C, prescription 1,2,3,4,5,6,8,9
And excipient all has effect steady in a long-term to free polyethylene glycol amount.
In addition, the purity of the PEGylated uricoxidase by measuring freeze dried powder, also can be evaluated each prescription pair
The influence of PEGylated uricoxidase stability, when freeze dried powder stores 60 days in 25 DEG C, 2 DEG C -8 DEG C store 24 months
When, the change for purity occur the results are shown in Table shown in 9, table 10.
Table 9. by the solution 1-9 freeze dried powders prepared purity (25 DEG C)
Table 10. by the solution 1-9 freeze dried powders prepared purity (2-8 DEG C)
In table 9, table 10 statistics indicate that, freeze drying protectant in 25 DEG C and 2 DEG C -8 DEG C, prescription 3,4,5,6,9 and
Excipient has effect steady in a long-term to PEGylated uricoxidase.
In summary data, preferably sucrose is as protective agent, preparation of the arginine as excipient, as Pegylation
The preparation of urate oxidase forms.
Embodiment three
Select the suitable range of buffer concentration of pharmaceutical formulation.
The different buffer solution formulas of design 8 are tested, and 2.5mg/ml Pegylation uric acid is added in each formula
Oxidizing ferment detects the aggregate and precipitate and activity decline percentage of PEGylated uricoxidase after multigelation, the results are shown in Table
11。
The different buffer concentration PEGylated uricoxidase multigelation experiments of table 11.
| Number | Buffer selects | PH of cushioning fluid | State | Retentive activity |
| 1 | 5mM phosphate buffers | 8.0 | Slight turbid | 33% |
| 2 | 10mM phosphate buffers | 8.0 | Clarification | 51% |
| 3 | 20mM phosphate buffers | 8.0 | Clarification | 76% |
| 4 | 30mM phosphate buffers | 8.0 | Clarification | 82% |
| 5 | 40mM phosphate buffers | 8.0 | Clarification | 98% |
| 6 | 60mM phosphate buffers | 8.0 | Clarification | 89% |
| 7 | 100mM phosphate buffers | 8.0 | Clarification | 77% |
| 8 | 150mM phosphate buffers | 8.0 | Slight turbid | 62% |
By to the comparison of different buffer concentrations can be seen that due to PEGylated uricoxidase product itself
Characteristic, too low buffer concentration can not provide frozen samples and be effectively protected, and excessively high buffer concentration is in refrigerating process
In can influence sample activity, therefore the optional buffer solution range of PEGylated uricoxidase preparation is in 20-100mM phosphate
Buffer solution, preferably 30-60mM phosphate buffers.
Example IV
The appropriate concentration range of protective agent and excipient in selection formula.
The protective agent and excipient that various concentration is selected in preparation, have larger impact, therefore to the quality of product after freeze-drying
Filter out suitable protective agent and excipient concentration.The different buffer solution formulas of design 9 are tested, and are added in each formula
Enter 2.5mg/ml PEGylated uricoxidases, appearance and moisture is investigated after freeze-drying, and testing result is shown in Table 12.
The different protective agents of table 12. and excipient proportioning freeze-drying result
Compared by protective agent to various concentration and excipient as can be seen that since PEGylated uricoxidase produces
The self character of product, too low protective agent can not carry out effective protection to PEGylated uricoxidase, and protect agent concentration
The excessively high moisture that can lead to product after being lyophilized is unqualified；The appearance of product is collapsed after too low excipient freeze-drying.According to reality
The concentration range of the optional protective agent sucrose of result is tested in 5-25mg/ml, preferably 10-20mg/ml；The arginic concentration of excipient
Range is in 15-35mg/ml, preferably 20-30mg/ml.
Embodiment five
Product quality research after the PEGylated uricoxidase freeze-drying of various concentration.
Content 0.5mg/ml, 1mg/ containing PEGylated uricoxidase are respectively configured using prescription 9 in embodiment 2
Ml, 1.5mg/ml, 2.5mg/ml, 3mg/ml, 4mg/ml, 5mg/ml, 10mg/ml are lyophilized using the lyophilized technique of table 1,
Ocular estimate and water content detection are carried out to product after freeze-drying, use 2ml injection waters to redissolve respectively, investigates and redissolves time and clarity,
It the results are shown in Table 13.
The different protective agents of table 13. and excipient proportioning freeze-drying result
By product quality after the PEGylated uricoxidase freeze-drying to various concentration studies have shown that working as albumen concentration
The redissolution time of product increases after being lyophilized when excessively high, and transparency also becomes micro- turbid, therefore optional albumen concentration is in 0.5-5mg/
Ml, while for the ease of using, albumen concentration is unsuitable too low, therefore the preferred 1-4mg/ of PEGylated uricoxidase concentration
Ml, more preferable 1.5-3mg/ml.
Embodiment six
According to being preferably formulated in embodiment, freeze drying process is set.
According to preferred preparation prescription in embodiment：2.5mg/ml PEGylated uricoxidase+40mM phosphate is slow
Fliud flushing+15mg/ml sucrose+25mg/ml arginine (pH8.0) carries out heat analysis by differential scanning calorimetry instrument (DSC), first will
Sample is cooled to -55 DEG C, is then warming up to 25 DEG C with 10 DEG C/min.Heat analysis result is shown in Fig. 3.
According to DSC scanning results, refrigerating process parameter is set, freeze-drying process is shown in Table 14.
Table 14. is used to be freeze-dried the preferably freeze drying cycle of the PEGylated uricoxidase of the present invention
Embodiment seven
According to being preferably formulated in embodiment, compare PEGylated uricoxidase with urate oxidase albumen identical
Stability result in preparation prescription compares.
Urate oxidase is after polyethyleneglycol modified, and due to the shielding action of polyethylene glycol, biological characteristics have occurred
Huge change, therefore to the preparation prescription that PEGylated uricoxidase stability is beneficial to, to urate oxidase egg
White stability has larger impact.Using applying preferred preparation prescription in example：40mM phosphate buffer+15mg/ml sucrose+
25mg/ml arginine (pH8.0) is separately added into the PEGylated uricoxidase and urate oxidase albumen of 2.5mg/ml,
Its stability under the conditions of 2-8 DEG C is investigated, it is main to investigate retentive activity and appearance, it the results are shown in Table 15.
15. same preparation prescription PEGylated uricoxidase of table and urate oxidase stability compare
Be shown experimentally that cook protective agent using sucrose, arginine makees the prescription of excipient to the steady of urate oxidase albumen
It is qualitative to have an impact, but it is directed to PEGylated uricoxidase, which helps to increase PEGylated uricoxidase
Stability.
Claims (14)
1. the solid constituent of a kind of PEGylated uricoxidase freeze dried powder, the freeze dried powder includes Pegylation uric acid
Oxidizing ferment, protective agent and freeze-dried excipient, which is characterized in that the protective agent uses sucrose, and freeze-dried excipient is using smart ammonia
Acid, according to the mass fraction：
0.5-5.0 parts of PEGylated uricoxidase
15-35 parts of sucrose
5.0-25 parts of arginine.
2. a kind of PEGylated uricoxidase freeze dried powder according to claim 1, which is characterized in that press mass parts
Number meter：
1.5-3.0 parts of PEGylated uricoxidase
20-30 parts of sucrose
10-20 parts of arginine.
3. a kind of PEGylated uricoxidase freeze dried powder according to claim 1, which is characterized in that the freeze-dried powder
The solid constituent of agent further includes buffer, and the pH of solution before freeze-drying is maintained 7.0-9.0 by buffer, it is preferable that pH is maintained
7.5-8.5；Further preferably, pH maintains 7.8-8.2.
4. a kind of PEGylated uricoxidase freeze dried powder according to claim 3, which is characterized in that buffer selects
With disodium hydrogen phosphate and sodium dihydrogen phosphate system.
5. a kind of PEGylated uricoxidase freeze dried powder according to claim 1, which is characterized in that the freeze-dried powder
Agent is freeze-dried under conditions of moisture is less than 3% freeze dried powder；It is preferred that the moisture of freeze dried powder is
0.5%-2.5%, more preferably 1%-2%.
6. a kind of PEGylated uricoxidase freeze dried powder according to claim 1, which is characterized in that polyethylene glycol
The conjugate for changing urate oxidase one or more peg molecule and one or more recombination urate oxidase molecules, is conjugated
It is 7-11 polyethylene glycol point that object, which each recombinates urate oxidase molecule to have 5-13 peg molecule, more preferable conjugate,
Conjugate between sub and single recombination urate oxidase molecule.
7. a kind of PEGylated uricoxidase freeze dried powder according to claim 6, which is characterized in that polyethylene glycol
Molecule has the average molecular weight of 2000-20000.
8. a kind of PEGylated uricoxidase freeze dried powder according to claim 6, which is characterized in that recombination uric acid
Oxidizing ferment selects the recombination urate oxidase of the aspergillus flavus sequence of escherichia expression system expression.
9. a kind of PEGylated uricoxidase freeze dried powder according to claim 6, which is characterized in that recombination uric acid
Oxidizing ferment and mPEG-SC, methoxy poly (ethylene glycol)-succinimide acetic acid esters, methoxy
Base polyethylene glycol-succinimidyl propionate is reacted in pH7.5-9.5；It is more preferably poly- with methoxyl group by recombinating urate oxidase
Ethylene glycol-succinimide acetic acid esters, methoxy poly (ethylene glycol)-succinimidyl propionate are reacted in pH7.5-9.5, most preferably
It is reacted in pH7.5-9.5 with methoxy poly (ethylene glycol)-succinimidyl propionate by recombinating urate oxidase, prepares poly- second two
Refine urate oxidase.
10. a kind of PEGylated uricoxidase freeze dried powder prepared described in claim 1~9 any one claim
Preparation method, which is characterized in that this method includes the following steps：
1) prepared by Solutions in Freeze-drying, and PEGylated uricoxidase, protective agent, excipient are dissolved in injection with the amount selected
In water, the pH of solution is maintained by 7.0-9.0 by buffer；
2) solution is fitted into suitable for being used freeze-dryer and suitable for the glass container that lyophilized preparation is stored；
3) it is freeze-dried under conditions of being suitble to preparation moisture to be less than 3% freeze dried powder.
11. preparation method according to claim 10, which is characterized in that step 3) is as follows：
1) under 25 DEG C or 25 DEG C or less, environmental pressure, glass container is fitted into freeze-dryer；
2) it can be pre-chilled 0.5-2 hours under the conditions of 2 DEG C -8 DEG C, under -50 DEG C to -40 DEG C, environmental pressure, make the appearance of loading
Device experience freezing 2-4 hours；Then with 0.05-0.15 millibars, it is dry with two steps under the vacuum of preferably 0.06-0.10 millibars of pressure
The solution of dry freezing；
3) initial drying step can in -40 DEG C to -30 DEG C carry out 14-20 hour, and second step dry can in 20 DEG C to 30 DEG C into
Row 5-9 hours；
4) under 25 DEG C or 25 DEG C or less, environmental pressure, the glass container containing freeze dried powder is unloaded.
12. preparation method according to claim 11, which is characterized in that step 3) is as follows：
1) under 25 DEG C or 25 DEG C or less, environmental pressure, glass container is fitted into freeze-dryer；
2) it can be pre-chilled 1 hour under the conditions of 4 DEG C, under -45 DEG C, environmental pressure, the container of loading made to undergo freezing 3 hours；
Then with the solution that under 0.1 millibar of vacuum, is frozen and dried with two steps；
3) initial drying step can carry out 17 hours in -35 DEG C, and second step drying can carry out 7 hours in 25 DEG C；
4) under 25 DEG C of environmental pressures, the glass container containing freeze dried powder is unloaded.
13. according to the preparation method described in claim 10 or 11 or 12, which is characterized in that glass container is 7ml cillin bottles, excellent
The loading amount of choosing is 2ml/ branch.
14. according to the preparation method described in claim 10 or 11 or 12, which is characterized in that lyophilization cycle needs are not less than
It completes within 35 hours, including temperature changes the required time between each step when application.
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