CN103068366B - The liquid preparation of long-acting human growth hormone conjugate - Google Patents
The liquid preparation of long-acting human growth hormone conjugate Download PDFInfo
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- CN103068366B CN103068366B CN201180039263.8A CN201180039263A CN103068366B CN 103068366 B CN103068366 B CN 103068366B CN 201180039263 A CN201180039263 A CN 201180039263A CN 103068366 B CN103068366 B CN 103068366B
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Abstract
Disclose the liquid preparation without albuminous long-acting human growth hormone (hGH) conjugate, it can ensure that the described long-acting hGH conjugate stability when long-term storage, and wherein said long-acting human growth hormone conjugate comprises and the human growth hormone that immunoglobulin fc region is connected and the internal stability compared with native form with prolongation.The liquid preparation of the hGH conjugate comprising the buffer agent of pH5.0～6.0, sugar alcohol, salt and nonionic surfactant is without human serum albumin and other potential risk factor polluted by virus, and can be provided as the splendid bin stability of the long-acting hGH conjugate customization being made up of hGH polypeptide and immunoglobulin fc region, described conjugate has higher molecular weight and internal persistency compared with described native form.
Description
Technical field
The present invention relates to the liquid preparation without albuminous long-acting human growth hormone conjugate, it can when long-term storage
Guarantee that the stability of described long-acting human growth hormone conjugate, wherein said long-acting human growth hormone conjugate comprise and immunity ball
The human growth hormone that albumen Fc district is connected, and there is compared with native form the internal stability of prolongation.
Background technology
Human growth hormone (hereinafter referred to " hGH ") is by antepituitary gland (anterior pituitarygland) point
Not glycosyafated (aglycosylated) peptide hormone secreted, and with the specific receptor on the cell surface of Various Tissues
Interact, thus stimulate other somatomedin of secretion, with the size of responsible increase health some.Since finding from people
Since the growth hormone of pituitary gland is effective therapeutic agent of growth hormone deficiency dwarfism (pituitary dwarfism), the need to hGH
Ask and occur in that and increase explosively.But, can be extremely limited from the supply of the hGH of human pituitary extraction.It addition, accepting corpse
The child of the hGH in source there occurs sick (the degenerative neurological disorder) Ke-refined of degenerative neurological
After sick (Creutzfeldt-Jacob disease), U.S. FDA has prohibitted the use of the hGH extracted from corpse pituitary gland, this
Be based on the assumption that the infectious prion (prion) causing disease propagate with the hGH of cadaveric origin (Roger, L.,
Science234:22,1986).At present, the biosynthetic human growth hormone that gene recombination technology is produced is used by E.coli
Ratified commercially available by FDA.
Polypeptide (such as hGH) is prone to regression (degenerate) due to its low stability, and easily by serum albumin
Enzymatic degradation and being removed by kidney or liver.Therefore, comprise polypeptide must execute to patient continually as the medicine of active constituents of medicine
In order to maintain its serum levels and titer.But, by frequently using (being in most of the cases with the form of injection) albumen
Matter medicine maintains the high serum levels of active polypeptide to be painful for patient.
In order to solve these problems, the serum stability by improving pharmaceutical grade protein and long term maintenance albumen are attempted
The high serum levels of matter medicine makes drug effect maximize.Accordingly, it would be desirable to there is the stability of raising in patients and maintain
The preparation of the pharmaceutical grade protein of the most high-caliber activity (and not causing immunne response).
In order to stable protein preventing contacts with protease and kidney loss, routinely by highly soluble polymer
(such as Polyethylene Glycol (PEG)) chemistry adds the surface to pharmaceutical grade protein.Non-specific some of target protein of being conjugated to
Or behind multiple position, PEG can improve the dissolubility of target protein, makes protein stabilization and prevents it from degrading, without causing
Significantly side effect (Sada etc., J.Fermentation Bioengineering, 1991,71:137-139).PEG puts together can
Contribute to stablizing of protein, but its activity can be significantly decreased.The PEG of higher molecular weight there is reduction with protein
Reactivity, therefore reduces yield.
It is by using gene recombinaton for improving a kind of alternative strategy of the internal stability of physiologically active protein matter
Target protein and the protein with high serum stability being blended, this is to be connected with each other by each gene of coded protein
The method connecing and cultivating zooblast with fusion gene conversion.For example, it has been reported that fused protein, the most known carry
Albumin or its fragment of high protein stability blend (International Patent Publication by gene recombinaton and target protein
No.WO93/15199 and No.WO93/15200, European Patent Publication No.EP413,622).
United States Patent (USP) No.5,045,312 discloses use cross-linking agent and puts together with bovine serum albumin or rat immune globulin
HGH there is the activity of enhancing compared with the growth hormone of unmodified.The unique cross-linking agent mentioned in that patent is low molecule
Quantify compound (such as, carbodiimide or glutaraldehyde).But, such low-molecular-weight cross-linking agent is due to its non-specific connection
Do not ensure that homogeneous composition and be likely to be of toxicity in vivo.It addition, this patent only discloses improves life by chemical coupling
The activity of long hormone, but do not demonstrate the chemical coupling effect for the activity for other polypeptide drugs, and do not have
The dependency of the stability (such as, persistency and the raising of serum half-life) of understanding and protein.
Recently, introduce the conjugate formed between physiological active polypeptide and immunoglobulin fc region and nonpeptidic polymer to make
For durative action preparation, it makes pharmaceutical grade protein have both the activity of Min. reduction and the stability of raising, such as Korea S
Patent No.10-0567902 (Physiologically Active Polypeptide Conjugate Having
Improved In Vivo Durability) and Korean Patent No.10-0725315 (Protein Complex Using An
Immunoglobulin FragmentAnd Method For The Preparation Thereof) described in.
According to these methods, hGH can be used as physiological active polypeptide thus can prepare long-acting hGH conjugate.These are treated
Long-acting hGH conjugate as medicine, it is necessary to maintain its drug effect in vivo, suppresses it that physicochemical change, example occur simultaneously
The degeneration that causes such as the impurity in light, heat or additive, assemble, adsorb or hydrolyze.Compared with hGH, long-acting hGH conjugate is at chi
Very little bigger with on molecular weight, the most more it is difficult to stabilize it.
Generally, protein has the shortest half-life, and when being exposed to unsuitable temperature, water-air interface, height
Show degeneration after pressure, physics or mechanical stress, organic solvent, microorganism pollution etc., such as monomer aggregation, aggregate and precipitate and
Absorption is on vessel surface.Once degeneration, protein loses its intrinsic physicochemical properties and physiologically active.Due in big portion
In the case of Fen, protein denaturation is irreversible, so for the protein of degeneration, recovering its intrinsic character almost
Impossible.
Absorbed protein is prone to because of its degeneration assemble.The protein assembled may act as when being expelled in health resisting
Immunogenic substance, it is therefore necessary to use sufficiently stable protein.Have studied the multiple method (John preventing protein denaturation
Geigert, J.Parenteral Sci.Tech., 43, No5,220-224,1989, David Wong,
Pharm.Tech.October, 34-48,1997, WeiWang., Int.J.Pharm., 185,129-188,1999, Willem
Norde, Adv.ColloidInterface Sci., 25,267-340,1986, Michelle etc., Int.J.Pharm.120,
179-188,1995).
Some pharmaceutical grade proteins use lyophilizing technique to avoid stability problem.But, freeze-drying prods is inconvenient to be dissolved in note
Penetrate with in solvent.It addition, lyophilization needs extensive freezer dryer, this improves in the production of pharmaceutical grade protein and invests into
This.Also been proposed with spray dryer make protein powder with the stability of Protein requirement, but due to low yield
The most unhelpful.Additionally, the high temperature being exposed to spray drying produces negative side effect to protein itself.
Have studied the stabilizer occurred as the alternative method overcoming these to limit, this is because when being added
During to pharmaceutical grade protein solution, it is possible to suppress the physicochemical change of pharmaceutical grade protein and guarantee internal drug effect, even growing
Also it is such after phase storage.Human serum albumin has been widely used as the stabilizer of multiple proteins medicine, and its performance
Have been obtained for proving (Edward Tarelli etc., Biologicals (1998) 26,331-346).
When using together with human serum albumin, patient Mao Zhe is exposed to biological pollutant or pathogen (such as, Zhi Yuan
Body, Protein virus, antibacterial and virus) risk, this is because while that the technique for purification of albumin includes inactivation, screens or examine
Look into this type of biological pollutant or pathogen, but these possibly cannot be eliminated or inactivate completely.Such as, screening technology includes
For some virus checking donor's serum, but this inspection is the most reliable.Especially, very small amount of some virus (as
If fruit exists) possibly cannot detect.
Due to the chemical differences of different proteins, they can during storing under different conditions with different speed by
Gradually inactivate.It is to say, by stabilizer extend storage period be different for different protein.To this end, according to institute
State the physicochemical properties of target protein, stabilizer to be used and target protein in ratio, concentration and type
Difference,.Unexpectedly, when used in combination, due to the competition between stabilizer and interaction, negative shadow can be caused
Ring.Additionally, due to the character of target protein or concentration can change during storing, thus the stabilizer used can provide with
Expect different effects.Accordingly, it would be desirable to substantial amounts of effort and preventive measure are with the protein in stable solution.
Especially, by be connected physiologically active peptide hGH with immunoglobulin fc region to improve in hGH body durability and
Stability and the long-acting hGH conjugate needs prepared specifically form for stable protein, this is because they are at molecular weight
Different from typical hGH ten points with in size.
International Patent Publication No.W093/19776 disclose comprise the buffer agent of pH6.0～7.0, aminoacid, mannitol and
The liquid preparation of the stable hGH of optional preservative (such as benzyl alcohol).International Patent Publication No.WO94/03198 discloses
Buffer agent, nonionic surfactant, preservative and optional neutral salt containing hGH, pH6.0 or the stable liquid of mannitol
Body preparation.United States Patent (USP) No.6,448,225 discloses the buffer agent containing hGH, pH6.0, nonionic surfactant and optionally
Neutral salt or the stable pharmaceutically acceptable liquid preparation of mannitol (but need not glycine).Korean Patent No.10-0537260
Disclose containing PEG (replacing nonionic surfactant and preservative), buffer agent and isotonic agent stablizing as active component
Changing the liquid preparation of hGH, this is owing to nonionic surfactant and preservative cause hGH deaminizating significantly.
But, although hGH and immunoglobulin fc region both of which are peptide or protein, but they have different physics
Chemical property, and the two needs the most stabilized.As it has been described above, different protein is because its chemical differences is during storing
Gradually inactivate with different speed under different conditions.Against one's expectation, use and be suitable for stabilized peptide in combination
Or the stabilizer of protein can have a negative impact because of the competition between them and interaction.Therefore, long-acting hGH is sewed
Compound, the compositions of its stabilization formulations is different from those of the preparation for individually stablizing hGH.Indeed, it is difficult to find to be used for
Stablize the preparation of both hGH and immunoglobulin fc region.
By the following present invention that creates: by the present inventor, long-term safety is stored long-acting hGH-immunoglobulin
What Fc conjugate was carried out deeply and studies thoroughly, results in a finding that and comprises the buffer agent of pH5.0～6.0, non-ionic surface active
The stabiliser compositions of agent, sugar alcohol and salt can provide the most useful liquid preparation with long-acting hGH conjugate, said preparation
The stability improving long-acting hGH conjugate can be greatly promoted pollute without worry virus at long-term storage period.
Disclosure of the invention content
Technical problem
Therefore, it is an object of the present invention to provide a kind of improve bin stability comprise long-acting hGH conjugate,
The liquid preparation of the long-acting hGH conjugate of the buffer agent of pH5.0～6.0, sugar alcohol, salt and nonionic surfactant.
Technical scheme
An aspect according to it, the invention provides long-acting hGH conjugate, the pH5.0～6.0 comprising medicine effective quantity
The liquid preparation of long-acting hGH conjugate of buffer agent, sugar alcohol, salt and nonionic surfactant.
Term used herein " long-acting hGH conjugate " means such conjugate, wherein by physiologically active peptide life
Long hormone is connected with immunoglobulin fc region and its physiologically active persistent period compared with natural hGH is longer.
Term used herein " long-acting " means that physiologically active has the persistent period more longer than natural hGH.
The hGH that can be used for the present invention has the aminoacid sequence of wild type or has the close phase of activity similar with wild type
Close the aminoacid sequence of analog.The present invention can use any hGH (the most natural or restructuring).It is preferably used
The restructuring hGH that E.coli is prepared as host.This area can use by the replacement of amino acid residue, lack or insert and spread out
It is conigenous any mutant of natural hGH, as long as its biological activity does not significantly change.
As for can be used for the immunoglobulin Fc of the present invention, it can be human normal immunoglobulin Fc or closely-related with it
Analog or animal (such as, cattle, goat, pig, mice, rabbit, hamster, rat, Cavia porcellus etc.) can be derived from.Immunoglobulin Fc
District can be derived from IgG, IgA, IgD, IgE, IgM or a combination thereof or heterozygote.Immunoglobulin fc region can be selected from IgG,
The heterozygosis Fc district in the different structure territory of the immunoglobulin of IgA, IgD, IgE and IgM, or can be the knot with identical source
The dimer of the single-chain immunoglobulins in structure territory or polymer.Spreading out of the serum half-life of the most known raising ligand binding protein
It is conigenous the Fc district of IgG or IgM (it is the abundantest in human blood), and most preferably derived from the Fc district of IgG.Can by with some
Protease Treatment natural IgG adaptive immune globulin Fc or use gene recombination technology are produced immune globulin by inverted cell
White Fc.Preferably, described immunoglobulin Fc is the recombined human immunoglobulin Fc produced in E.coli.
IgG is divided into IgG1, IgG2, IgG3 and IgG4 hypotype, and the present invention can include a combination thereof or heterozygote.Preferably
IgG2 and IgG4 hypotype, and seldom there is effector functions (such as, CDC (CDC
(Complement Dependent Cytotoxicity))) the Fc district of IgG4.It is to say, most suitable as medicine of the present invention
The immunoglobulin fc region of thing carrier is the not glycosyafated Fc district in human IgG 4 source.The Fc district in people source is than the Fc in inhuman source
District it is further preferred that the Fc district in described inhuman source can serve as antigen in human body and cause less desirable immunne response, such as, produces
The raw new antibodies for antigen.
Preparation can be fetched by being connected with the immunoglobulin fc region produced by said method by hGH and be used for the length of the present invention
Effect hGH conjugate.This method of attachment by being cross-linked mutually with immunoglobulin Fc by hGH via non-peptidyl polymer or can be passed through
Produce fused protein (wherein using gene recombinaton to be blended by hGH) with immunoglobulin fc region to realize.Preferably hGH with exempt from
Epidemic disease globulin Fc connects via non-peptidyl polymer between district.
The non-peptidyl polymer that can be used for described crosslinking is selected from Polyethylene Glycol, polypropylene glycol, ethylene glycol and propylene glycol
Copolymer, polyoxyethylated polyols, polyvinyl alcohol, polysaccharide, glucosan, polyvinyl ethyl ether, biodegradable polymerization
Thing (such as, PLA (poly-(lactic acid)) and PLGA (PLGA)), lipid polymer, chitin kind (chitins),
Hyaluronic acid and combinations thereof.Most preferably Polyethylene Glycol.Its derivant being known in the art and side known in the art can be used
The derivant that method is easy to preparation is also within the scope of the invention.
For preparing long-acting hGH conjugate, can refer to Korean Patent No.0725315, the disclosure of which is by quoting entirety
And in herein.Those skilled in the art can refer to described document and produce the long-acting hGH conjugate of the present invention.
The liquid preparation of the long-acting hGH conjugate of the present invention comprises the long-acting hGH conjugate of medicine effective quantity.Generally, hGH
Medicine effective quantity corresponding to about 1～3mg in the bottle that is intended for single use.Concentration for the long-acting hGH conjugate of the present invention
Scope is 1mg/mL to 55mg/mL, preferably 15mg/mL to 25mg/mL.
Term used herein " stabilizer " means the material allowing long-acting hGH conjugate stably to store.Term is " steady
Fixed " mean and lose a certain percentage ratio less than active component, usually less than 10%, preferably shorter than 5%.When storing at 10 ± 3 DEG C
Deposit 2 years, at 25 ± 2 DEG C, store 6 months or store at 40 ± 2 DEG C after one to two week long-acting hGH compared with initial activity sew
The activity of compound keeps 90% or higher level (preferably from about 92～the level of 95%), then think that preparation is stable.Right
In protein (the most long-acting hGH conjugate), its bin stability produces its antigen forms potentially in suppression and guarantees it
It is very important on correct dose.
Although being kept together by the long-acting hGH conjugate of the present invention, physiologically active peptide hGH and immunoglobulin fc region
Physicochemical properties different from each other and must be by Simultaneous Stabilization.Physical chemical differences between them can cause different
Peptide or protein gradually inactivate with respective speed during storing at different conditions.It was unexpectedly determined that make in combination
Can contend with one other because of them or interact with the stabilizer being suitable to bioactive peptide or protein and have a negative impact.
It is designed for both Simultaneous Stabilization physiologically active peptide hGH and immunoglobulin fc region so that long-acting hGH conjugate
Activity can maintain desired level for a long time, and the stabilizer of the present invention comprises specific buffer agent, sugar alcohol, salt and non-ionic surface
Activating agent.
The effect of buffer agent is to maintain the pH of solution in predetermined scope to prevent from may result in long-acting hGH inactivation
Drastically pH change.The present invention can use any buffer agent, as long as pharmaceutically acceptable pH buffer agent as known in the art.Can
Buffer agent for the present invention includes basic salt (alkaline salt) (sodium phosphate or potassium phosphate or its hydrogen salt or dihydro
Salt), sodium citrate/citric acid, acetate/acetic and combinations thereof.Optimization citric acid salt buffer agent and phosphate buffer, more excellent
Select citrate buffer agent.The citrate buffer agent that can be used for the present invention can comprise preferred 5mM to 100mM amount and more excellent
Select the citrate of 10mM to 50mM concentration.
Owing to the reaction in solution can change, so the pH of stabilizer is extremely important according to the pH of buffer agent in solution.
React pH when occurring and they are different because protein is different on the impact of dissolubility.Therefore, it is difficult to maintain high-dissolvability
Accurately three dimensional structure and do not produce the mode stable protein of degeneration impurity.Conventional hGH preparation generally use pH6.0～
7.0 or higher buffer agents are to reduce the dissolubility producing and improving protein of impurity.
The stabilizer of the present invention comprises pH5.0～6.0, preferred pH5.2～6.0 and the buffering of more preferably pH5.2～5.5
Agent.In one embodiment of the invention, (deaminizating of corresponding hGH is miscellaneous for #6 and the #7 impurity measured after storing 3 months
Matter) content reduction (Fig. 1) in particular under low pH (such as, pH5.2).These data show, because of by hGH and immunoglobulin
The long-acting hGH conjugate that Fc district is constituted is different from single hGH in nature, so being designed for long stable effect hGH conjugate
The liquid preparation of the hGH of routine must be different from pH with the liquid preparation of both the dissolubility improving long-acting hGH conjugate.
Additionally, the effect of sugar alcohol is to improve the stability of long-acting hGH conjugate.In the present invention, based on described preparation
Cumulative volume, sugar alcohol preferably with the amount of 1 to 10% (w/v) and more preferably uses with the amount of 5% (w/v).Can be used for the present invention
The example of sugar alcohol include but not limited to mannitol, sorbitol and combinations thereof.Such as understood from the data of table 4, by 0.5%
The stability of long-acting hGH conjugate is not affected by L-Arg-HCl interpolation to mannitol.
When the solution of hGH conjugate is injected in health, salt has stablizes long-acting hGH conjugate in solution further
And the effect as the isotonic agent maintaining suitable osmotic pressure.Generally, salt is water-soluble inorganic salt, and preferably sodium chloride.
In the formulation, salt is preferably with the amount of 5 to 200mM and more preferably with the amount existence of 150mM, and its content can
Type and amount according to composition are adjusted, so that preparation is isotonic.
In one embodiment of the invention, under salt presence or absence, to comprise pH5.0～6.0 buffer agent,
In the preparation of sugar alcohol and nonionic surfactant, the stability of long-acting hGH conjugate is evaluated.As a result, with there is not NaCl
Under compare, in the presence of NaCl (especially 150mMNaCl) in the preparation containing the mannitol of 5% at 25 DEG C store 4
Zhou Shi, the stability maintenance of long-acting hGH conjugate is in significantly higher level (table 2).Compared with conventional hGH, according to the present invention
Long-acting hGH conjugate can containing 1～10% (w/v) sugar alcohol and 5～200mM NaCl preparation in stabilisation and also can
Stabilisation in the preparation of the buffer agent containing pH5.0～6.0, sugar alcohol, nonionic surfactant and salt.
Nonionic surfactant reduces the surface tension of protein solution, to prevent protein on hydrophobic surface
Absorption or gathering.The example of the nonionic surfactant that can be used for the present invention include polysorbate (polysorbate),
Poloxamer (poloxamer) and combinations thereof, preferably polysorbate.Wherein the nonionic surfactant of polysorbate is
Polysorbate20, polysorbate40, polysorbate60 and polysorbate80.Preferably polysorbate80.
According to one embodiment of the invention, it was observed that the stability of long-acting hGH conjugate is at polysorbate80
In the presence of increase (table 8).When substituting polysorbate80 with polysorbate20, until two weeks, long-acting put together
The stability of thing is identical, but after storage 4 weeks, the stability although surfactant is mutually similar, between long-acting conjugate
There is significant difference.
In the liquid preparation of the present invention, preferably with the amount of 0.1% (w/v) or less, more preferably with 0.001 to 0.05%
(w/v) amount and the most more preferably comprise nonionic surfactant with the amount of 0.005% (w/v).Norditropin (purchases
From the hGH liquid preparation of Nordisk) use 3mg/mL PLURONICS F87 as surfactant (table 9).According to the present invention's
One embodiment, it was observed that the long-acting hGH conjugate in the preparation containing 3mg/mL PLURONICS F87 is stored at 25 DEG C
(table 8) is assembled after two weeks.These data show, the type of surfactant (serving as the stabilizer of pharmaceutical grade protein) and dense
Degree must be drug specificity.
In one embodiment of the invention, find to go back in addition to the buffer agent and nonionic surfactant of pH5.2
Containing 1～10% the preparation of (w/v) sugar alcohol and 5～200mM NaCl to considerably improve the storage of long-acting hGH conjugate stable
Property, show that the combination of the buffer agent of pH5.2, nonionic surfactant, sugar alcohol and salt shows long-acting hGH conjugate steady
Synergism qualitatively.
Preferably, the stabilizer of the present invention does not contains albumin.Because it originates from human serum, so always there being following possibility
Property: can be used as polluting of the pathogenic virus that the human serum albumin of protein stabilizing agent can be originated.Some patients
In, gelatin or bovine serum albumin can cause disease or can readily generate allergy.Do not have heterologous protein (such as people or
The serum albumin of animal origin or purified gel), the stabilizer of the present invention does not cause the probability of virus pollution.
In addition to buffer agent, salt, sugar alcohol and the nonionic surfactant of pH5.0～6.0, the liquid preparation of the present invention can
Also comprise composition well known in the art or material (unless they reduce the effect of the present invention).Such as, the preparation of the present invention can be also
Comprise sugar, polyhydric alcohol or neutral amino acid.
Also may be included in preparation with improve long-acting conjugate bin stability sugar preferred embodiment include monosaccharide (example
As, mannose, glucose, fucose and xylose) and polysaccharide (such as, lactose, sucrose, cottonseed sugar (raffinose) and Portugal are poly-
Sugar).Can be additionally used in the polyhydric alcohol of the present invention have propylene glycol, low molecular poly, glycerol, molecular weight polypropylene glycol and
Combination.Cumulative volume based on preparation, sugar and polyhydric alcohol each can be with the amounts of 1 to 10% (w/v) and preferably with 5% (w/v)
Amount use.
According to one of them preferred embodiment, the present invention provide comprise 20mM sodium citrate buffer agent (pH5.2～
6.0), 5～200mM NaCl, 1～10% (w/v) mannitol and 0.001～0.05% liquid preparation of polysorbate80.?
In one embodiment of the invention, sodium citrate buffer agent (pH5.2), 5% (w/v) mannitol, 150mM NaCl will be comprised
With the liquid preparation of long-acting hGH conjugate of 0.005% (w/v) polysorbate80 and the hGH preparation purchased from Nordisk
Norditropin compares.The liquid preparation of the long-acting hGH conjugate of the present invention show high as Norditropin or
Than its higher bin stability (table 10).In the test according to the long term storage stability of another embodiment, find
The liquid preparation of the long-acting hGH conjugate of the present invention makes the activity of long-acting hGH conjugate maintain 6 months (tables on high level
11)。
Due to viral risk of pollution not together and simple and have splendid bin stability, the present invention does not contains
The liquid preparation (being designed to provide for the stability of long-acting hGH conjugate) of albuminous long-acting hGH conjugate is stable with other
Agent or lyophilized preparation are compared has economic benefit.
Additionally, due to the liquid preparation of the present invention comprise have compared with native form higher internal persistent long-acting
HGH conjugate, the liquid preparation of the present invention allows the activity of protein to maintain Gao Shui for a long time compared with typical hGH preparation
Flat, therefore can be used as effective pharmaceutical preparation.
Beneficial effects of the present invention
The hGH comprising the buffer agent of pH5.0～6.0, sugar alcohol, salt and nonionic surfactant according to the present invention puts together
The liquid preparation of thing is without human serum albumin and other potential risk factors polluted by virus, and can be provided as by hGH
What polypeptide was constituted with immunoglobulin fc region has more macromolecule and internal persistent long-acting hGH conjugate compared with natural
The splendid bin stability of customization.
Accompanying drawing is sketched
By below in conjunction with the details of accompanying drawing describe understand with will be apparent from the above and other purpose of the present invention, feature with
And other advantages, wherein:
Fig. 1 is to store three at long-acting hGH conjugate at 4 DEG C in the buffer agent of multiple pH value as described in Example 4
Analyzed the representative IE-HPLC chromatogram obtained after described conjugate stability by IE-HPLC during Yue；With
Fig. 2 is to store six months phases at 4 DEG C in the buffer agent of pH5.2 at long-acting hGH conjugate as described in Example 7
Between use IE-HPLC to analyze the figure obtained after described conjugate stability.
Detailed description of the invention
By following can obtain for embodiment illustrated, the present invention is better understood from, but these embodiments are not
It is interpreted to limit the present invention.
The preparation of [embodiment 1] long-acting hGH conjugate
By ALD-PEG-ALD (IDB) (3.4kDa Polyethylene Glycol, and there is aldehyde radical at each end) and hGH (Mw 22
KDa) puting together mutually, the N end with the not glycosyafated Fc district (Mw 50 kDa) in human IgG 4 source is connected afterwards, and then purification is to carry
For hGH-PEG-Fc conjugate.
[embodiment 2] measures the stability of long-acting hGH conjugate under salt presence or absence
In order to evaluate under salt presence or absence in the preparation comprising buffer agent, sugar alcohol and nonionic surfactant
The stability of long-acting hGH conjugate, long-acting hGH conjugate is stored 4 weeks in the preparation of table 1 below at 25 DEG C, makes afterwards
With ion exchange chromatography (ion exchange chromatography, IEC) and size exclusion chromatography (SEC) (size
Exclusion chromatography, SEC) analyze its stability.In described preparation, citrate buffer agent is used as slow
Electuary, mannitol is used as sugar alcohol, and polysorbate80 is used as nonionic surfactant.In table 2, IE-HPLC (%)
It is expressed as (area %/initial area %) with SE-HPLC (%), shows long-acting hGH conjugate remnants compared with initial purity
Purity (residual purity).
Table 1
[table 1]
Table 2
[table 2]
As understood from data, compared with under there is not NaCl, when long-acting hGH conjugate exists NaCl (especially
It is 150mM NaCl) under the preparation containing 5% mannitol at 25 DEG C, store the stability maintenance of 4 weeks significantly higher
Level.
[embodiment 3] measures the stability of long-acting hGH conjugate for sugar alcohol
Long-acting hGH is comprising the buffer agent as stabilizer, NaCl, nonionic surfactant and sugar as isotonic agent
The preparation of alcohol detects during storage the sugar alcohol impact on the stability of long-acting hGH.
In described preparation, citric acid buffer agent (sodium citrate, pH5.2) is used as buffer agent, mannitol or sorbitol
As sugar alcohol, and polysorbate80 is used as nonionic surfactant.
Long-acting hGH conjugate is stored 4 weeks in the preparation of table 3 below at 25 DEG C, uses ion exchange chromatography afterwards
And size exclusion chromatography (SEC) (SEC) analyzes its stability (IEC).In table 4, IE-HPLC (%) and SE-HPLC (%) is expressed as
(area %/initial area %), shows the long-acting hGH conjugate remaining purity compared with initial purity.
Table 3
[table 3]
Table 4
[table 4]
As above visible in table, sugar alcohol mannitol is similar with the stability of sorbitol.It addition, 0.5%L-Arg-HCl is added
Add to the mannitol stability on long-acting hGH conjugate without impact.
[embodiment 4] is for the stability of the pH long-acting hGH conjugate of mensuration of buffer agent
The stability of long-acting hGH is evaluated in the buffer agent of multiple pH value.In this case, long-acting hGH conjugate is existed
The preparation of table 5 below is stored three months at 4 DEG C, uses ion exchange chromatography (IEC) to analyze its stability afterwards.At table 6
In, IE-HPLC (%) is expressed as (area %/initial area %), show long-acting hGH conjugate and impurity respectively with as main peak
The remaining purity (Fig. 1) compared with the initial purity of impurity peaks.
Table 5
[table 5]
Table 6
[table 6]
After storing three months, the content of #6 with #7 impurity has dropped under pH5.2 compared with under pH5.5 and pH6.0
Low, illustrate that long-acting hGH conjugate stability in the buffer agent of pH5.2 improves to some extent (Fig. 1).By peptide mapping (peptide
Mapping) analysing impurity, then determines molecular weight by LC-MS/MS.Determine that #6 and #7 impurity is deaminizating impurity.
[embodiment 5] measures the stability of long-acting hGH conjugate for nonionic surfactant
Comprise nonionic surfactant polysorbate80 or 20 or PLURONICS F87 as the preparation of stabilizer
In at 25 DEG C, store 4 weeks after, use SEC and IEC measure long-acting hGH conjugate stability.At the preparation as seen in table 7
In, citric acid buffer agent and the mannitol of pH5.2 are used separately as buffer agent and sugar alcohol.In table 8, IE-HPLC (%) and SE-
HPLC (%) discloses the long-acting hGH conjugate remaining purity compared with initial purity.
Table 7
[table 7]
*Corresponding 0.005% polysorbate80 of 0.05mg/mL polysorbate80
Table 8
[table 8]
ADue to aggregate and precipitate, so data are unavailable
As understood from data, it was observed that under there is polysorbate80, the stability of long-acting hGH conjugate is
Improve.In the case of polysorbate20, until not detecting and polysorbate in the stability of long-acting conjugate for two weeks
The difference of 80, but after storage 4 weeks, the stability between them has significant difference (although polysorbate20 is with poly-
PS80 is similar).It addition, long-acting hGH conjugate in the preparation containing 2mg/mL PLURONICS F87 25
Assemble after storing two weeks at DEG C.
The comparison of stability between [embodiment 6] liquid long-acting hGH conjugate and marketed drugs Norditropin
By compared with commercial liquid hGH preparation Norditropin (Novo Nordisk) relatively to by Citrate buffer
Agent (pH5.2), NaCl, mannitol and polysorbate80 (finally being selected by the Stability Determination of embodiment 2 to 5) are constituted
The stabilisation ability of preparation be evaluated.The concentration of Norditropin is 10mg/mL and its composition is given in Table 9 below.
They are stored 4 weeks at 25 DEG C.In order to confirm the multiformity of change, use the capillary electrophoresis as described in European Pharmacopoeia
(capillary electrophoresis, CE) and SEC measure Norditropin.On the other hand, IEC and SEC is used to analyze
The long-acting hGH conjugate (measuring similar with CE in principle) of the present invention.Result is given in Table 10 below.CE (%), or IE-HPLC
(%) the long-acting hGH conjugate remaining purity compared with initial purity is represented with SE-HPLC (%).
Table 9
[table 9]
Table 10
[table 10]
As can be seen, the preparation of the present invention guarantee hGH with level high as commercially available hGH preparation Norditropin or
Stabilisation is carried out than its higher level.These results prove, the liquid preparation of the long-acting hGH of the present invention can be that hGH provides pole
Good bin stability.
[embodiment 7] measures the long term storage stability of liquid long-acting hGH conjugate
In order to evaluate the liquid system being made up of citrate buffer agent (pH5.2), NaCl, mannitol and polysorbate80
The long term storage stability of agent, stores the post analysis sample of six months in described preparation at 4 DEG C.Result is at Fig. 2 and Biao 11
In be given.IE-HPLC (%), SE-HPLC (%) and protein content (%) disclose long-acting hGH conjugate and initial purity phase
The remaining purity of ratio.
Table 11
[table 11]
Observe the long-acting hGH conjugate of the present invention in liquid preparation stably more than 6 months.
Although disclosing the preferred embodiments of the invention for exemplary purposes, but those skilled in the art should managing
Solve, multiple amendment can be carried out, add and replace, without departing from the scope and spirit of claims present invention disclosed
?.
Claims (14)
- The liquid preparation of the most long-acting human growth hormone conjugate, its described long-acting human growth hormone comprising medicine effective quantity puts together Thing and without albuminous stabilizer, in described long-acting human growth hormone conjugate, human growth hormone is via Polyethylene Glycol even Connect is connected with immunoglobulin fc region, described stabilizer substantially consists of: pH 5.2 or higher but less than 5.5 Buffer agent, sugar alcohol, nonionic surfactant and salt, wherein said nonionic surfactant be polysorbate80 and with The amount of 0.001% to 0.05% (w/v) of cumulative volume based on described preparation uses, and wherein said salt is sodium chloride.
- The liquid preparation of long-acting human growth hormone conjugate the most according to claim 1, wherein said sugar alcohol is selected from: manna Alcohol, sorbitol and combinations thereof.
- The liquid preparation of long-acting human growth hormone conjugate the most according to claim 1, wherein said sugar alcohol is with based on institute The amount of 1% to 10% (w/v) stating the cumulative volume of preparation uses.
- The liquid preparation of long-acting human growth hormone conjugate the most according to claim 1, wherein said buffer agent is Fructus Citri Limoniae Hydrochlorate or phosphate buffer.
- The liquid preparation of long-acting human growth hormone conjugate the most according to claim 1, wherein said salt is with 5mM extremely The concentration of 200mM uses.
- The liquid preparation of long-acting human growth hormone conjugate the most according to claim 1, wherein said stabilizer also comprises One or more of selected from sugar, polyhydric alcohol and amino acid whose composition.
- The liquid preparation of long-acting human growth hormone conjugate the most according to claim 1, wherein said long-acting people grows sharp Element conjugate comprises the human growth hormone and immunoglobulin fc region connected via Polyethylene Glycol, and described stabilizer comprises pH 5.2 or citrate buffer agent, 1～10% (w/v) mannitol, 0.001～0.05% polysorbate higher but less than 5.5 80 and 5～200mM NaCl.
- The liquid preparation of long-acting human growth hormone conjugate the most according to claim 1, wherein said human growth hormone has There is the aminoacid sequence identical with Native human growth hormone.
- The liquid preparation of long-acting human growth hormone conjugate the most according to claim 1, wherein said immunoglobulin Fc District is derived from IgG, IgA, IgD, IgE or IgM.
- The liquid preparation of long-acting human growth hormone conjugate the most according to claim 9, wherein said immunoglobulin Fc District is the heterozygosis Fc district in the different structure territory of immunoglobulin, and described immunoglobulin is selected from IgG, IgA, IgD, IgE and IgM.
- The liquid preparation of 11. long-acting human growth hormone conjugates according to claim 9, wherein said immunoglobulin Fc District is dimer or the polymer of the single-chain immunoglobulins of the domain with identical source.
- The liquid preparation of 12. long-acting human growth hormone conjugates according to claim 9, wherein said immunoglobulin Fc District is IgG4Fc district.
- The liquid preparation of 13. long-acting human growth hormone conjugates according to claim 12, wherein said immunoglobulin Fc district is not glycosyafated human IgG 4Fc district.
- 14. for producing the liquid preparation of the long-acting human growth hormone conjugate as according to any one of claim 1 to 13 Method, comprising:A) long-acting human growth hormone conjugate is prepared；WithB) by described long-acting human growth hormone conjugate and combination of stabilizers, described stabilizer substantially consists of: pH 5.2 or higher but less than 5.5 buffer agent, sugar alcohol, nonionic surfactant and salt, wherein said nonionic surfactant It is polysorbate80 and the amount use of 0.001% to 0.05% (w/v) with cumulative volume based on described preparation, Yi Jiqi Described in salt be sodium chloride.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2010-0067796 | 2010-07-14 | ||
| KR1020100067796A KR101337797B1 (en) | 2010-07-14 | 2010-07-14 | A liquid formulation of long acting human growth hormone conjugate |
| PCT/KR2011/005194 WO2012008779A2 (en) | 2010-07-14 | 2011-07-14 | A liquid formulation of long-acting human growth hormone conjugate |
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| CN103068366A CN103068366A (en) | 2013-04-24 |
| CN103068366B true CN103068366B (en) | 2016-11-30 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1723219A (en) * | 2003-11-13 | 2006-01-18 | 韩美药品工业株式会社 | Protein complex using immunoglobulin fragment andmethod for the preparation thereof |
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1723219A (en) * | 2003-11-13 | 2006-01-18 | 韩美药品工业株式会社 | Protein complex using immunoglobulin fragment andmethod for the preparation thereof |
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