CN109929025A - A kind of preparation method of the polyethylene glycol Luo Saina peptide based on kinetics - Google Patents
A kind of preparation method of the polyethylene glycol Luo Saina peptide based on kinetics Download PDFInfo
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- CN109929025A CN109929025A CN201711368308.1A CN201711368308A CN109929025A CN 109929025 A CN109929025 A CN 109929025A CN 201711368308 A CN201711368308 A CN 201711368308A CN 109929025 A CN109929025 A CN 109929025A
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- saina peptide
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Abstract
The preparation method of the present invention relates to a kind of polyethylene glycol Luo Saina peptide based on kinetics.Particularly, the present invention relates to a kind of preparation method of polyethylene glycol Luo Saina peptide, the synthetic method is established on the basis of the process optimization control technology based on kinetics, and maximizing improves Luo Saina peptide and mPEG2The material utilization of-MAL (40kD), reduces side reaction generation and impurity generates.The purification process uses cation-exchange chromatography, can obtain the polyethylene glycol Luo Saina peptide of high-purity and high-recovery.The simple process, efficient, suitable industrialized production.
Description
Technical field
The invention belongs to bioengineering and field of pharmaceutical engineering, it is related to the polyethyleneglycol modified of albumen and polypeptide drug,
More particularly to the preparation method of polyethylene glycol Luo Saina peptide, especially synthesis and purification process.
Background technique
Diabetes are a kind of global prevalence diseases, and annual lethal number is only second to tumour and cardiovascular and cerebrovascular disease.Glycosuria
The body that is mainly characterized by of disease is in hyperglycemia state.According to the generation period of diabetes and the cause of disease, International Diabetes Federation
(IDF) diabetes are divided into type 1 diabetes, diabetes B and gestational diabetes mellitus.In all diabetics made a definite diagnosis, surpass
The diabetic for crossing 90% is type 2 diabetic patient.The characteristics of diabetes B is that insulin inhibits and pancreas Instreptozotocin Induced
Damage, leads to insulin deficit and hyperglycemia.Diabetes B can occur at any age, and opposite type 1 diabetes be not easy by
It was found that usually found by blood or urine detection.Diabetes B is usually accompanied by obesity, hypertension, blood lipid
The complication such as obstacle and cardiovascular disease, these complication and the increase of diabetes lethality have direct relation.Currently, 2 types of control
The method of diabetes has movement, keeps a diet and drug therapy.Most common drug mainly includes its Lei of insulin Ji Si Wu ﹑ sulphur
The bis- Gua Lei ﹑ alpha-glucosidase restrainer of acyl Niao Lei ﹑ and insulin sensitizer.These drugs emphasize that internal blood glucose is promoted to tend to just
Often, the complication as caused by diabetes can not be corrected, and the side effects such as hypoglycemia and weight gain can be generated, wherein weight gain
It may be decreased compliance, thereby increases and it is possible to increase the risk that cardiovascular disease occurs, therefore exploitation is highly-safe, patient's biddability is good, secondary
Low novel therapeutic diabetes B drug is acted on as the focus of attention.
Glucagon-like-peptide-1 (GLP-1) receptor stimulant medicine has blood glucose dependence insulin secretion accelerating, suppression
Glicentin processed secretion, the effects of promoting beta cell hyperplasia, protect Instreptozotocin Induced, sharpest edges be no risk of hypoglycemia,
The effects of can reduce weight, some drugs also show the effect unique that cardiovascular and cerebrovascular benefits, just can overcome the disadvantages that traditional 2 type for the treatment of
The defect of diabetes medicament becomes new research and development focus.Natural GLP-1 is 4 that Proglucagon transcription post-processing is formed
One of a maturation peptide fragment, there is two kinds of forms of 7-37,7-36, in vivo can be rapidly by dipeptidyl peptidase IV (DPP- based on 7-37
IV N-terminal amino acid) is cut off from the C-terminal of the 8th alanine and loses bioactivity, and half-life short (only 1-2min) does not have
Standby druggability, thus long-acting GLP-1 receptor stimulant medicine is developed as Development Trend.Currently, GLP-1 receptor stimulating agent
Class drug includes the Exenatide listed, Liraglutide, lixisenatide, albiglutide, Du Lalu peptide, the Suo Ma that will be listed
Shandong peptide and at least more than 20 in the new drug ground.Used long-actingization technology includes that amino acid sequence is transformed, fatty acid chain is repaired
Decorations, polyethylene glycol (PEG) modification, albumin coupling (non-fused), antibody coupling (non-fused), Fc are coupled (non-fused), ELPs
(elastin laminin fusion), HyFc fusion, Fc fusion, microsphere sustained-release, PEGylated+PLGA microballoon, subdermal osmotic pumps etc..Just because of
The drugs such as Exenatide, Liraglutide are really applied to clinic, and the New-type long-acting technology continuously emerged makes GLP-1 receptor
Stimulant medicine market capacity rapid development, becomes the maximum impetus of diabetes medicament market dilatation.
In the GLP-1 receptor stimulant medicine listed, Exenatide is the GLP-1 receptor agonism of first listing
Agent class drug, administration frequency are twice a day.Exenatide is the polypeptide containing 39 amino acid, is initially the saliva in the huge lizard in America
It is found in liquid.It is different from N-terminal sequence alanine (His-Ala-Glu) of GLP-1, the N-terminal second-to-last amino of Exenatide
Acid is glycine (His-G1y-Glu), is not easy to be degraded by DPP-IV, and half-life period is obviously prolonged, but still needs to be taken twice daily.Together
When due to there was only 53% homology with people GLP-1, immunogenicity is stronger.Polyethylene glycol Luo Saina peptide (PEX168) is by Jiangsu person of outstanding talent
The weekly GLP-1 receptor stimulant medicine that gloomy medicine company Group Co., Ltd is developed based on PEG modification technique, is to end
Fill in and carry out modification transformation in that chemistry of peptides structure basis, by the 2nd Gly, 14 Met and 28 Asn sport respectively D-Ala,
Nle and Gln improves enzyme Jie stability and chemical stability based on polypeptide backbone；It has changed Peptide C end Ser into Cys simultaneously, has made
The modification of specificity is carried out with polyethylene glycol, reduces the probability of drug enzymatic hydrolysis, to extend drug half-life period in vivo and work
With the time, its bioavilability is improved, and then improves the therapeutic effect of drug.Compared with Exenatide, pass through changing for amino acid
It makes and is modified with PEG, significant to extend the half-life period of drug in vivo, administration frequency is weekly；PEG modification simultaneously can also reduce
Immunogenicity overcomes the strong defect of Exenatide immunogenicity to a certain extent.
The quality control of polyethylene glycol Luo Saina peptide is the key that its industrialization.Establish the poly- second two for being suitble to industrialized production
Alcohol Luo Saina peptide preparation process is particularly important.Polyethylene glycol Luo Saina peptide preparation process mainly includes synthesis and purifying work
Skill, wherein amplification production is limited by synthesis technology to a certain extent.Therefore, it is necessary to the synthesis to polyethylene glycol Luo Saina peptide
Technique carries out stringent control.The synthesis of polyethylene glycol Luo Saina peptide is influenced by many factors, such as pH, temperature, mPEG2-MAL
(40kD)/Luo Saina peptide molar ratio and reaction time etc..Wherein, pH not only will affect the rate of reaction, also will affect side reaction
Generation, substrate and product stability, to influence the generation of impurity.mPEG2- MAL (40kD)/Luo Saina peptide molar ratio
It is particularly important key factor, mPEG2- MAL (40kD) excessively will form more modified outcomes, Luo Saina peptide Lip river plug easy to form
That peptide dimer, and mPEG2- MAL excess or Luo Saina peptide excessively can bring difficulty to later separation.Reaction time is to poly-
The generation of ethylene glycol Luo Saina peptide and impurity also has a significant impact.Therefore, it is necessary to these influence factors carry out accurate control and
Optimization.However, since influence factor is more, using conventional experimental method to mPEG2- MAL (40kD) modifies the anti-of Luo Saina peptide
Answering process to optimize control still has very big difficulty.
Summary of the invention
The purpose of the present invention is to provide a kind of systems of polyethylene glycol Luo Saina peptide that is more efficient, being more suitable for industrialized production
Preparation Method.
Synthetic method of the present invention uses the process optimization control technology based on kinetics, can be to mPEG2-MAL
(40kD) modification Luo Saina peptide reaction process accurately controlled and optimized, obtain optimal reaction condition, as pH, temperature,
mPEG2- MAL (40kD)/Luo Saina peptide molar ratio and reaction time etc., maximizing Luo Saina peptide and mPEG2- MAL's (40kD)
Raw material availability, reduces side reaction generation and impurity generates.Purification process uses cation-exchange chromatography, can be effectively removed not
Participate in the mPEG of reaction2- MAL (40kD) and other impurity, can obtain high-purity (being greater than 99.5%) and high-recovery (is greater than
75%) polyethylene glycol Luo Saina peptide.Finally obtain simple, efficient polyethylene glycol Luo Saina peptide industrialized producing technology.
To achieve the purpose of the present invention, technical solution of the present invention is implemented by following scheme:
(1) the polyethylene glycol Luo Saina peptide symthesis method based on kinetics:
(i) mPEG based on kinetics is established2- MAL (40kD) modifies Luo Saina peptide process optimization control technology, really
Determine mPEG2The technological parameter of-MAL (40kD) modification Luo Saina peptide；
(ii) above-mentioned technological parameter is finely adjusted by the stability of analysis process and end product quality, is determined best
MPEG2- MAL (40kD) modifies Luo Saina peptide technological parameter；
(iii) polyethylene glycol Luo Saina peptide crude product solution is obtained after modification reaction；
It further, may include the method for purifying polyethylene glycol Luo Saina peptide crude product: pure using cation-exchange chromatography
Change polyethylene glycol Luo Saina peptide crude product solution obtained by step (1), obtains polyethylene glycol Luo Saina peptide salting liquid；Again through RP-HPLC
Desalination, reduced pressure, freeze-drying obtain polyethylene glycol Luo Saina peptide.
Wherein, the polyethylene glycol Luo Saina peptide is mPEG2The Luo Saina peptide of-MAL (40kD) modification, structure such as formula
(1) shown in:
The wherein mPEG2- MAL (40kD) is branched polyethylene glycol derivative, molecular weight 40kDa, wherein described
Shown in the structure of Luo Saina peptide such as formula (2):
Wherein, the mPEG based on kinetics described in step (i)2- MAL (40kD) modifies the process optimization control of Luo Saina peptide
Technology processed the following steps are included:
(a) building reaction Kinetics Model is as follows:
Above-mentioned 2 reactions can with following differential equation group (ordinary differential equations,
ODEs it) describes:
Wherein, k1For mPEG2The reaction rate constant of-MAL (40kD) modification Luo Saina peptide；De-PEG is inactive forms
mPEG2- MAL (40kD), correspondingly, kdFor mPEG2The inactivation reaction rate constant of-MAL (40kD)；CloxenatideFor Luo Saina
The concentration of peptide；CPEGFor reactive mPEG2The concentration of-MAL (40kD)；CDe-PEGFor the mPEG of inactive forms2- MAL (40kD) is dense
Degree；T is the reaction time；
(b) by the combination experiment under the conditions of differential responses, investigating influences polyethyleneglycol modified key factor, such as different
PH, temperature, polyethyleneglycol derivative/Luo Saina peptide molar ratio, reaction time etc. obtains mPEG under the conditions of differential responses2-
The experimental data that the reaction substrate and production concentration of MAL (40kD) modification Luo Saina peptide change over time is quasi- for kinetic parameter
It closes；
(c) experimental data obtained based on step (b), the reaction power established using MATLAB software fit procedure (a)
Model is learned, to relevant kinetic parameter (k1And kd) solved；
(d) kinetic parameter (k that the reaction Kinetics Model and step (c) established using step (a) are obtained1And kd), it is inverse
MPEG under the conditions of to prediction differential responses2The reaction process of-MAL (40kD) modification Luo Saina peptide；To obtain maximum Luo Saina
Peptide and mPEG2- MAL molar yield and shortening reaction time etc. are target, determine the mPEG of optimization2- MAL (40kD) modification
Luo Saina peptide technological parameter, and carry out corresponding experimental verification.
In above-mentioned steps (1), step (ii) the optimal mPEG2- MAL (40kD) modifies Luo Saina peptide technological parameter
Are as follows: mPEG2- MAL (40kD) modifies the sodium phosphate buffer that the action solvent of Luo Saina peptide is 0.1mol/L；Sodium ascorbyl phosphate is slow
The pH of fliud flushing is 5.5-7.7, preferably 6.3-7.0, most preferably 7.0；Reaction temperature is 15-30 DEG C；mPEG2-MAL(40kD)/
Luo Saina peptide molar ratio is 0.92:1-1.38:1, preferably 1.12:1-1.15:1；Reaction time is 15-90min, preferably
15-60min。
In above-mentioned purification step, the cation-exchange chromatography medium is strong cation exchange material Sepharose
SP-HP；The cation-exchange chromatography uses salt gradient linear elution mode；The ion-exchange chromatography using a certain concentration and
The acetate buffer of pH as mobile phase liquid A, a certain concentration and pH acetate buffer as Mobile phase B, setting is linear
Gradient are as follows: increase to 50%B from 0%B in 150min, be correspondingly reduced to 50%A from 100%A, collect polyethylene glycol Luo Saina
Peptide main peak；Or setting linear gradient are as follows: increase to 30%B from 0%B in 150min, be correspondingly reduced to 70% from 100%A
A collects polyethylene glycol Luo Saina peptide main peak.
The object of the invention is also to provide a kind of preparation method of polyethylene glycol Luo Saina peptide, this method includes following step
It is rapid:
(1) Luo Saina peptide and a certain amount of mPEG are weighed2- MAL is dissolved in buffer；
The pH of buffer is 6.3-7.7, and reaction temperature is 15-30 DEG C, mPEG2The molar ratio of-MAL and Luo Saina peptide is
1.10:1-1.15:1 reaction time 15-90min obtains polyethylene glycol Luo Saina peptide crude product solution after reaction；
Optional, further includes:
(2) purifying of polyethylene glycol Luo Saina peptide
The polyethylene glycol Luo Saina peptide crude product solution obtained by cation-exchange chromatography purification step (1), obtains polyethylene glycol
Luo Saina peptide salting liquid；Polyethylene glycol Luo Saina peptide sterling is obtained through RP-HPLC desalination, reduced pressure, freeze-drying again.
Preferably, the buffer is phosphate buffer, and the pH of buffer is 6.3 or 7.0, and reaction temperature is room temperature
Or 20 DEG C, mPEG2- MAL/ Luo Saina peptide molar ratio is 1.12-1.15:1, reaction time 15-60min.
Preferably, cation-exchange chromatography described in step (2) carries out in AKTA process system, using being equipped with
The BPG200 chromatographic column of 1.25L Sepharose SP-HP filler, flow velocity 125mL/min, Detection wavelength 280nm were operated
Journey is as follows: the PEG Luo Saina peptide crude product that step (1) obtains is used the acetic acid-sodium acetate buffer solution of 0.02mol/L as slow by (i)
Fliud flushing A is diluted to suitable concentration, and adjusting pH is 4.3；(ii) it is loaded to suitable loading volume with buffer solution A pre-balance
In good chromatographic column, then with the buffer solution A cleaning of certain column volume；(iii) linear gradient elution mode is used, elutes and collects
Polyethylene glycol Luo Saina peptide main peak component；It is further preferred that the linear gradient can be interior from 0% buffer solution B for 150min
(acetic acid-sodium acetate buffer solution+the 1mol/L NaCl, pH4.3 of 0.02mol/L) increases to 50%B, correspondingly, collects poly- second two
Alcohol Luo Saina peptide main peak；Alternatively, linear gradient may be to increase to 30%B from 0%B in 150min, correspondingly, poly- second is collected
Glycol Luo Saina peptide main peak.
Preparation method of the invention compared with existing preparation process, existing advantage at least that:
(1) method of the invention modifies Luo Saina peptide process optimization control technology, energy using the PEG based on kinetics
Enough optimize determines Optimal pH, mPEG2- MAL (the 40kD)/synthesis technologic parameters such as Luo Saina peptide molar ratio and reaction time, can be most
Improve mPEG to big degree2The raw material availability of-MAL (40kD) and Luo Saina peptide, to significantly improve mole of Luo Saina peptide
Conversion ratio (up to 97% or more)；Reaction pH7.7 is down to 6.3-7.0 simultaneously, it is possible to reduce the degradation or polymerization of Luo Saina peptide,
Reduce mPEG2- MAL (40kD) modifies the probability of amino, reduces impurity and generates, is conducive to the purifying of subsequent PEG Luo Saina peptide.
(2) the polyethylene glycol Luo Saina that method of the invention is obtained using cation-exchange chromatography purifying synthesis technology step
Peptide crude product solution.Under the conditions of slant acidity pH, polyethylene glycol Luo Saina peptide can be adsorbed on cation exchange medium well, not
The PEG of reaction can be removed in the balance elution process after loading process and loading, overcome the limitation of RP-HPLC chromatography
Property；And during linear elution, PEG Luo Saina peptide can also be separated with Luo Saina peptide and Luo Saina peptide dimer well, from
And obtain the PEG Luo Saina peptide of high-purity (being greater than 99.5%) and high-recovery (being greater than 75%).
(3) total recovery for the PEG Luo Saina peptide sterling that method of the invention is prepared and product quality are than existing system
Standby technique is significantly increased.
Detailed description of the invention
MPEG when Fig. 1 pH is 7.72- MAL (40kD) modifies the kinetics of Luo Saina peptide, when wherein abscissa represents
Between (minute), ordinate represents Pegylation percentage (%).
MPEG when Fig. 2 pH is 7.02- MAL (40kD) modifies the kinetics of Luo Saina peptide, when wherein abscissa represents
Between (minute), ordinate represents Pegylation percentage (%).
MPEG when Fig. 3 pH is 6.32- MAL (40kD) modifies Luo Saina peptide kinetics, and wherein abscissa represents the time
(minute), ordinate represent Pegylation percentage (%).
MPEG when Fig. 4 pH is 5.52- MAL (40kD) modifies the kinetics of Luo Saina peptide, when wherein abscissa represents
Between (minute), ordinate represents Pegylation percentage (%).
MPEG when Fig. 5 temperature is 30 DEG C2- MAL (40kD) modifies the kinetics of Luo Saina peptide, and wherein abscissa represents
Time (minute), ordinate represent Pegylation percentage (%).
MPEG when Fig. 6 temperature is 25 DEG C2- MAL (40kD) modifies the kinetics of Luo Saina peptide, and wherein abscissa represents
Time (minute), ordinate represent Pegylation percentage (%).
MPEG when Fig. 7 temperature is 20 DEG C2- MAL (40kD) modifies the kinetics of Luo Saina peptide, and wherein abscissa represents
Time (minute), ordinate represent Pegylation percentage (%).
MPEG when Fig. 8 temperature is 15 DEG C2- MAL (40kD) modifies the kinetics of Luo Saina peptide, and wherein abscissa represents
Time (minute), ordinate represent Pegylation percentage (%).
Fig. 9 mPEG2MPEG when-MAL/ Luo Saina peptide molar ratio is 0.92:12- MAL (40kD) modifies the anti-of Luo Saina peptide
Dynamics is answered, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
Figure 10 mPEG2MPEG when-MAL/ Luo Saina peptide molar ratio is 1.15:12- MAL (40kD) modifies the anti-of Luo Saina peptide
Dynamics is answered, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
Figure 11 mPEG2MPEG when-MAL/ Luo Saina peptide molar ratio is 1.27:12- MAL (40kD) modifies the anti-of Luo Saina peptide
Dynamics is answered, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
Figure 12 mPEG2MPEG when-MAL/ Luo Saina peptide molar ratio is 1.38:12- MAL (40kD) modifies the anti-of Luo Saina peptide
Dynamics is answered, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
20 DEG C of Figure 13, under the conditions of pH is 6.3, PEG/ Luo Saina peptide molar ratio is 1:1 the reaction time to polyethylene glycol Lip river
The influence of that peptide synthesis is filled in, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
20 DEG C of Figure 14, under the conditions of pH is 6.3, PEG/ Luo Saina peptide molar ratio is 1.05:1 the reaction time to poly- second two
The influence of alcohol Luo Saina peptide synthesis, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
20 DEG C of Figure 15, under the conditions of pH is 6.3, PEG/ Luo Saina peptide molar ratio is 1.1:1 the reaction time to polyethylene glycol
The influence of Luo Saina peptide synthesis, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
20 DEG C of Figure 16, under the conditions of pH is 6.3, PEG/ Luo Saina peptide molar ratio is 1.12:1 the reaction time to poly- second two
The influence of alcohol Luo Saina peptide synthesis, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
20 DEG C of Figure 17, under the conditions of pH is 6.3, PEG/ Luo Saina peptide molar ratio is 1.15:1 the reaction time to poly- second two
The influence of alcohol Luo Saina peptide synthesis, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
20 DEG C of Figure 18, under the conditions of pH is 6.3, PEG/ Luo Saina peptide molar ratio is 1.27:1 the reaction time to poly- second two
The influence of alcohol Luo Saina peptide synthesis, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
20 DEG C of Figure 19, under the conditions of pH is 7.0, PEG/ Luo Saina peptide molar ratio is 1:1 the reaction time to polyethylene glycol Lip river
The influence of that peptide synthesis is filled in, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
20 DEG C of Figure 20, under the conditions of pH is 7.0, PEG/ Luo Saina peptide molar ratio is 1.05:1 the reaction time to poly- second two
The influence of alcohol Luo Saina peptide synthesis, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
20 DEG C of Figure 21, under the conditions of pH is 7.0, PEG/ Luo Saina peptide molar ratio is 1.1:1 the reaction time to polyethylene glycol
The influence of Luo Saina peptide synthesis, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
20 DEG C of Figure 22, under the conditions of pH is 7.0, PEG/ Luo Saina peptide molar ratio is 1.12:1 the reaction time to poly- second two
The influence of alcohol Luo Saina peptide synthesis, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
20 DEG C of Figure 23, under the conditions of pH is 7.0, PEG/ Luo Saina peptide molar ratio is 1.15:1 the reaction time to poly- second two
The influence of alcohol Luo Saina peptide synthesis, wherein abscissa represents time (minute), and ordinate represents Pegylation percentage (%).
Specific embodiment
In order to more clearly show technical solution of the present invention and its technical effect outstanding obtained, will pass through below
Embodiment describes a specific embodiment of the invention in detail, but specific embodiment should not be construed as limiting the invention.Such as
Without specified otherwise, used experimental method is conventional method, and agents useful for same etc. can chemically Reagent Company be bought, used
Chemical reagent is that analysis is pure.
1, material and instrument
Luo Saina peptide raw material (liquid phase purity is greater than 98%) is provided by Jiangsu Hao Sen medicine company Group Co., Ltd；mPEG2-
(molecular weight 40kDa, reactivity are about 95%) to be provided by Jiangsu Hao Sen medicine company Group Co., Ltd to MAL；Polyethylene glycol Lip river plug
That poly saccharide peptide standard product is provided by Jiangsu Hao Sen medicine company Group Co., Ltd, and liquid phase purity is greater than 99%；The pure acetonitrile of liquid chromatogram and
Trifluoroacetic acid is purchased from Sigma-Aldrich；The equipment and separating medium used includes: liquid chromatographic system
(Agilent 1200), protein layer analysis system AKTA process system, reversed-phase liquid chromatography column Waters C18 (4.6 mm
× 100 mm, 2.5 μm), BPG200 chromatographic column and strong cation exchange material Sepharose SP-HP (U.S. GE
Healthcare company).
2, based on the mPEG of kinetics2- MAL (40kD) modifies Luo Saina peptide process optimization control technology
(1) reaction Kinetics Model is constructed
1. forming Luo Saina peptide dimer in reaction process and more PEG modification Luo Saina peptide prod being less than 1%, can neglect
Slightly；2. the mPEG in reaction process, more than 10%2- MAL can inactivate loss, cannot ignore.
Based on the above two o'clock, the reaction Kinetics Model of foundation is as follows:
Above-mentioned 2 reactions can with following differential equation group (ordinary differential equations,
ODEs it) describes:
Wherein, k1For mPEG2The reaction rate constant of-MAL (40kD) modification Luo Saina peptide；De-PEG is inactive forms
mPEG2- MAL, correspondingly, kdFor mPEG2The inactivation reaction rate constant of-MAL；CloxenatideFor the concentration of Luo Saina peptide；CPEG
For reactive mPEG2The concentration of-MAL；CDe-PEGFor the mPEG of inactive forms2- MAL concentration；T is the reaction time；
(2) experimental data under the conditions of acquisition differential responses is used for models fitting
10 mg Luo Saina peptides and a certain amount of mPEG are weighed respectively2It is slow that-MAL is dissolved in 10 mL, 0.1 mol/L sodium ascorbyl phosphate
Fliud flushing is reacted.Reaction condition is as follows: sodium phosphate buffer pH is respectively 5.5-7.7, and temperature is respectively 15-30 DEG C,
mPEG2The molar ratio of-MAL/ Luo Saina peptide is respectively 0.92:1-1.38:1, and the reaction time is 0-120 min.In 120min
Different time intervals sampling, is terminated with excessive DTT solution and is reacted.Sample is detected using RP-HPLC analysis method, is measured
The content of Luo Saina peptide and PEG Luo Saina peptide, and calculate the molar yield of Luo Saina peptide；The Luo Saina peptide of acquisition and the Lip river PEG
The experimental data for filling in the concentration changes with time of that peptide is fitted for kinetic parameter；
(3) experimental data obtained based on step (b), the reaction power established using MATLAB software fit procedure (1)
Model is learned, to relevant kinetic parameter (k1And kd) solved；
(4) kinetic parameter (k that the reaction Kinetics Model and step (3) established using step (1) are obtained1And kd), it is inverse
MPEG under the conditions of to prediction differential responses2The reaction process of-MAL (40kD) modification Luo Saina peptide；To obtain maximum Luo Saina
Peptide and mPEG2- MAL molar yield and shortening reaction time etc. are target, determine the mPEG of optimization2- MAL (40kD) modification
Luo Saina peptide technological parameter, and carry out corresponding experimental verification.
The differential equation is solved with the ode45 (four step Runge-Kutta) in software for calculation MATLAB 7.10 (R2010a)
Group, lsqcurvefit (nonlinear least squares fitting) carry out nonlinear fitting, obtain optimal solution.
3, the preparation of PEG Luo Saina peptide
(1) synthesis of polyethylene glycol Luo Saina peptide
3g Luo Saina peptide and a certain amount of mPEG are weighed respectively2- MAL be dissolved in 1L 0.1mol/L sodium phosphate buffer into
Row reaction.Reaction condition is as follows: sodium phosphate buffer pH is respectively 6.3-7.0, and temperature is respectively 15-30 DEG C, mPEG2-
The molar ratio of MAL/ Luo Saina peptide is respectively 1.10:1-1.15:1, reaction time 15-90min；Poly- second is obtained after reaction
Glycol Luo Saina peptide crude product solution；
(2) purifying of polyethylene glycol Luo Saina peptide
Using polyethylene glycol Luo Saina peptide crude product solution obtained by cation-exchange chromatography purification step (1), poly- second two is obtained
Alcohol Luo Saina peptide salting liquid；Polyethylene glycol Luo Saina peptide sterling is obtained through RP-HPLC desalination, reduced pressure, freeze-drying again.
Wherein, cation-exchange chromatography described in step (2) carries out in AKTA process system, using being equipped with
The BPG200 chromatographic column of 1.25L Sepharose SP-HP filler, flow velocity 125mL/min, Detection wavelength 280nm.Specific behaviour
It is as follows to make process: PEG Luo Saina peptide crude product buffer solution A (acetic acid-sodium acetate of 0.02mol/L that (i) obtains step (1)
Buffer, pH4.3) it is diluted to suitable concentration, adjusting pH is 4.3；(ii) it is then loaded to suitable loading volume with buffering
In the good chromatographic column of liquid A pre-balance, then with the buffer solution A cleaning of certain column volume；(iii) linear gradient elution is finally used
Mode elutes and collects polyethylene glycol Luo Saina peptide main peak component.
Wherein, linear gradient described in the step (iii) in step (2) can be interior from 0% buffer solution B for 150min
(acetic acid-sodium acetate buffer solution+the 1mol/L NaCl, pH4.3 of 0.02mol/L) increases to 50%B, correspondingly, collects poly- second two
Alcohol Luo Saina peptide main peak.
Wherein, linear gradient described in the step (iii) in step (2) may be to increase in 150min from 0%B
30%B correspondingly collects polyethylene glycol Luo Saina peptide main peak.
4, analysis method
The PEG of Luo Saina peptide and polyethylene glycol Luo Saina peptide content, purifying in the polyethylene glycol Luo Saina peptide crude product of synthesis
The purity of Luo Saina peptide salting liquid, PEG Luo Saina peptide sterling after freeze-drying, the Luo Saina peptide and mPEG used2The purity of-MAL
It is all made of RP-HPLC analysis method shown in table 1 and condition detection.
1 RP-HPLC analysis method of table
| Chromatographic column | Waters C18(4.6mm×100mm,2.5μm) |
| Operating parameter | Flow velocity 1.0mL/min；60 DEG C of column temperature；Detection wavelength 214nm；20 μ L of sample volume |
| Mobile phase | Trifluoroacetic acid-water of mpA:0.05%；Trifluoroacetic acid-acetonitrile of mpB:0.05% |
| Gradient | 0-30min:30-68B%；30-31min:68-30B%；31-36min:30-30B% |
| Reference substance | PEX-168:10mg/mL；Luo Saina peptide: 1mg/mL；Using quantified by external standard method |
5, the computational representation of conversion ratio and yield
(1) Luo Saina peptide molar yield calculation formula are as follows:
(2)mPEG2- MAL molar yield calculation formula are as follows:
(3) obtained polyethylene glycol Luo Saina peptide rate of recovery calculation formula is purified:
(4) polyethylene glycol Luo Saina peptide sterling total moles yield calculation formula:
(i) it is calculated by Luo Saina peptide:
Wherein, polyethylene glycol Luo Saina peptide sterling is theoretical must measure 1.=Luo Saina peptide initial charge (mol)；
(ii) mPEG is pressed2- MAL is calculated:
Wherein, polyethylene glycol Luo Saina peptide sterling is theoretical must measure 2.=mPEG2- MAL initial charge (mol).
MPEG of the embodiment 1 based on kinetics2It is (small that-MAL (40kD) modifies Luo Saina peptide process optimization control technology
Examination scale)
The mPEG based on kinetics is established according to the method in above-mentioned 22- MAL (40kD) modifies Luo Saina peptide process
Optimized-control Technique.Wherein, sodium phosphate buffer pH is respectively 5.5,6.3,7.0 and 7.7, and temperature is respectively 15,20,25 and 30
DEG C, mPEG2The molar ratio of-MAL/ Luo Saina peptide is respectively 0.92:1,1.15:1,1.27:1 and 1.38:1, reaction time 0-
120min.With sodium phosphate buffer pH 6.3,20 DEG C of temperature, mPEG2The reaction of the molar ratio 1.15:1 of-MAL/ Luo Saina peptide
Condition is used to analyze the influence of single factor as reference standard.
MPEG under different condition2- MAL (40kD) modifies Luo Saina peptide kinetics as shown in Fig. 1-Figure 12, tests number
The kinetic parameter obtained according to fitting kinetic model is as shown in table 2.
As can be seen that pH influences significantly kinetics from Fig. 1-Figure 12 and table 2.When pH is 7.7, in 6min
Fully reacting, and PEG Luo Saina peptide content is begun to decline after 6min, illustrates have a small amount of two modified outcome to generate；When pH is 7.0,
30min can fully reacting；When pH is 6.3,90min can fully reacting；When pH is 5.5,120min unreacted is complete.Comprehensive point
Analysis selects pH more suitable for 7.0 or 6.3.When temperature range is 15-30 DEG C, the influence to kinetics is less.mPEG2-
When the molar ratio of MAL/ Luo Saina peptide is higher than 1.15:1, the basic fully reacting of Luo Saina peptide.
Reactive kinetics parameters under 2 different condition of table
When further using 20 DEG C of reactive kinetics parameters Dynamics Optimization temperature in reaction Kinetics Model and table 2,
PEG/ Luo Saina peptide molar ratio and reaction time under the conditions of pH6.3 or 7.0, and corresponding experimental verification is carried out, as a result such as Figure 13-
Shown in Figure 23 and table 3.According to Figure 13-Figure 23 and table 3 as a result, determining that the PEG of optimization modifies Luo Saina peptide reaction condition are as follows:
PH7.0, temperature are 20 DEG C, and PEG/ Luo Saina peptide molar ratio is 1.12-1.15:1, reaction time 15-60min.Under this condition,
The molar yield of Luo Saina peptide is 97.0-98.9%.Influence due to temperature to kinetics is less, in actual production
It carries out, temperature can not accurately be controlled at room temperature.
The optimum results of PEG/ Luo Saina peptide molar ratio and reaction time under the conditions of 3 20 DEG C of table, pH6.3 or 7.0
The synthesis (pilot-scale) of 1 polyethylene glycol Luo Saina peptide of embodiment 2 and comparative example
Embodiment 2-1: the synthesis of polyethylene glycol Luo Saina peptide is carried out by above-mentioned 3 method.Wherein, sodium phosphate buffer pH
It is 7.0, temperature is room temperature (between 20-25 DEG C), mPEG2The molar ratio of-MAL/ Luo Saina peptide is 1.15:1, and the reaction time is
30min.Polyethylene glycol Luo Saina peptide crude product solution is obtained after reaction.The crude product solution is measured with RP-HPLC analysis method
The content of Luo Saina peptide and PEG Luo Saina peptide, analyzes the purity of PEG Luo Saina peptide, and calculates Luo Saina peptide and mPEG2- MAL's
Molar yield, the results are shown in Table 4.
Embodiment 2-2: it is carried out according to the method for embodiment 2-1, the difference is that PEG2Mole of-MAL/ Luo Saina peptide
Than replacing with 1.13:1 and 60min respectively with the reaction time.
Comparative example 1: it is carried out according to the method for embodiment 2-1, the difference is that technological parameter are as follows: sodium phosphate buffer pH,
PEG2The molar ratio of-MAL/ Luo Saina peptide and reaction time replace with 7.7,0.97:1 and 120min respectively.
Influence of the 4 differential responses condition of table to polyethylene glycol Luo Saina peptide synthesis
| Number | Embodiment 2-1 | Embodiment 2-2 | Comparative example 1 |
| pH | 7.0 | 7.0 | 7.7 |
| mPEG2The molar ratio of-MAL/ Luo Saina peptide | 1.15:1 | 1.13:1 | 0.97:1 |
| Reaction time (min) | 30 | 60 | 120 |
| Polyethylene glycol Luo Saina peptide crude product purity (%) | 94.1 | 95.3 | 82.6 |
| Luo Saina peptide molar yield (%) | 98.0 | 98.2 | 84.2 |
| mPEG2The molar yield (%) of-MAL | 82.4 | 84.7 | 84.2 |
As can be seen from Table 4, in the polyethylene glycol Luo Saina peptide crude product solution that embodiment 2-1 and embodiment 2-2 are obtained
The purity of polyethylene glycol Luo Saina peptide is significantly improved than comparative example 1, can reduce its subsequent separating difficulty.Meanwhile embodiment 2-1
It is significantly improved compared with comparative example 1 with the Luo Saina peptide molar yield of embodiment 2-2, the raw material for improving Luo Saina peptide utilizes
Rate, and the molar yield of mPEG-MAL does not have notable difference.
The purifying (pilot-scale) of 3 polyethylene glycol Luo Saina peptide of embodiment
Embodiment 3-1: the polyethylene glycol Luo Saina peptide crude product solution that embodiment 2-1 is obtained is divided into two parts of equivalent, is taken
A copy of it is purified by above-mentioned 3 method with cation-exchange chromatography.Wherein, the linear gradient of elution be 150min in from
0%B increases to 50%B, is correspondingly reduced to 50%A from 100%A, collects polyethylene glycol Luo Saina peptide main peak.Obtained poly- second
Glycol Luo Saina peptide salting liquid measures the content of PEG Luo Saina peptide with RP-HPLC analysis method, analyzes the pure of PEG Luo Saina peptide
Degree, and the polyethylene glycol Luo Saina peptide rate of recovery is calculated, the results are shown in Table 5.
Embodiment 3-2: according to embodiment 3-1 method by another sample with elution linear gradient be in 150min from
The mode that 0%B increases to 30%B is purified, and is correspondingly reduced to 70%A from 100%A, collects polyethylene glycol Luo Saina peptide
Main peak the results are shown in Table shown in 5.
The different elutions of table 5 and influence of the collection mode to polyethylene glycol Luo Saina peptide purification
As can be seen from Table 5, using the linear eluent gradient of cation-exchange chromatography of the invention and the poly- second two of collection
Alcohol Luo Saina peptide main peak can obtain high-purity (> 99.5%) and the polyethylene glycol Luo Saina peptide salt of high-recovery (> 75%) is molten
Liquid.
The preparation (pilot-scale) of 2 polyethylene glycol Luo Saina peptide of embodiment 4 and comparative example
Embodiment 4-1:
(1) according to the method for embodiment 2-1, polyethylene glycol Luo Saina peptide crude product solution is obtained；
(2) according to the method for embodiment 3-1, polyethylene glycol Luo Saina peptide salting liquid is obtained；
(3) polyethylene glycol Luo Saina peptide salting liquid obtained by step (2) is concentrated under reduced pressure, is lyophilized through RP-HPLC column desalination
To polyethylene glycol Luo Saina peptide sterling.Weighing polyethylene glycol Luo Saina peptide sterling, and Luo Saina peptide and mPEG are pressed respectively2- MAL meter
Polyethylene glycol Luo Saina peptide sterling total moles yield is calculated, the results are shown in Table 6.In addition, detection polyethylene glycol Luo Saina peptide sterling
Key Quality Indicator, the results are shown in Table 7.
Embodiment 4-2: it is carried out according to the method for embodiment 4-1, the difference is that the embodiment 3-1 in step (2) is replaced
For embodiment 3-2, it is specifically shown in Table 6.
Comparative example 2: it is carried out according to the method for embodiment 4-1, the difference is that the embodiment 2-1 in step (1) is replaced with
Comparative example 1, is specifically shown in Table 6.
As can be seen from Table 6, the mPEG of embodiment 4-12- MAL total moles yield and comparative example 2 are suitable, but Luo Saina
Peptide total moles yield is significantly increased than comparative example 2.The mPEG of embodiment 4-22- MAL total moles yield and Luo Saina peptide always rub
You are significantly increased than comparative example 2 at yield.
The influence that 6 different technology conditions of table prepare polyethylene glycol Luo Saina peptide
| Number | Embodiment 4-1 | Embodiment 4-2 | Comparative example 2 |
| Synthetic method | It is identical as embodiment 2-1 | It is identical as embodiment 2-1 | It is identical as comparative example 1 |
| Purification process | It is identical as embodiment 3-1 | It is identical as embodiment 3-2 | It is identical as embodiment 3-1 |
| Luo Saina peptide total moles yield (%) | 68.7 | 75.5 | 60.1 |
| mPEG2- MAL total moles yield (%) | 57.8 | 63.5 | 57.5 |
7 different technology conditions of table count the related substance of polyethylene glycol Luo Saina peptide
| Number | It is total miscellaneous | Specific impurities A | Specific impurities B | Maximum unknown list is miscellaneous |
| Embodiment 4-1 | 0.75 | 0.13 | 0.12 | 0.28 |
| Embodiment 4-2 | 0.69 | 0.11 | 0.07 | 0.28 |
| Comparative example 2 | 0.80 | 0.15 | 0.11 | 0.25 |
As can be seen from Table 7, the mass ratio for the polyethylene glycol Luo Saina peptide sterling that embodiment 4-1 and 4-2 are obtained compares
Example 2 is significantly increased.
Claims (10)
1. a kind of preparation method of the polyethylene glycol Luo Saina peptide based on kinetics, method includes the following steps:
I) mPEG based on kinetics is established2- MAL (40kD) modifies Luo Saina peptide process optimization control technology, determines
mPEG2The technological parameter of-MAL (40kD) modification Luo Saina peptide；
Ii) above-mentioned technological parameter is finely adjusted by the stability of analysis process and end product quality, is determined optimal
mPEG2- MAL (40kD) modifies Luo Saina peptide technological parameter；
Iii polyethylene glycol Luo Saina peptide crude product solution) is obtained after modification reaction；
Optional, it further include the method to polyethylene glycol Luo Saina peptide purifying crude, comprising:
Using cation-exchange chromatography purification step iii) gained polyethylene glycol Luo Saina peptide crude product solution, obtain polyethylene glycol Lip river
Fill in that peptide salting liquid；Polyethylene glycol Luo Saina peptide is obtained through RP-HPLC desalination, reduced pressure, freeze-drying again.
2. the preparation method of the polyethylene glycol Luo Saina peptide according to claim 1 based on kinetics, feature exist
In the polyethylene glycol Luo Saina peptide is mPEG2The Luo Saina peptide of-MAL (40kD) modification, shown in structure such as formula (1):
The wherein mPEG2- MAL (40kD) is branched polyethylene glycol derivative, molecular weight 40kDa, Qi Zhong Suo Shu Luo Saina
Shown in the structure of peptide such as formula (2):
3. the preparation method of the polyethylene glycol Luo Saina peptide according to claim 1 based on kinetics, feature exist
In the mPEG based on kinetics described in step i)2- MAL (40kD) modifies Luo Saina peptide process optimization control technology
Following steps:
A) building reaction Kinetics Model is as follows:
Above-mentioned 2 reactions can be described with following differential equation group:
Wherein, k1For mPEG2The reaction rate constant of-MAL (40kD) modification Luo Saina peptide；De-PEG is inactive forms
mPEG2- MAL (40kD), correspondingly, kdFor mPEG2The inactivation reaction rate constant of-MAL (40kD)；CloxenatideFor Luo Saina
The concentration of peptide；CPEGFor reactive mPEG2The concentration of-MAL (40kD)；CDe-PEGFor the mPEG of inactive forms2- MAL (40kD) is dense
Degree；T is the reaction time；
B) by the combination experiment under the conditions of differential responses, investigating influences polyethyleneglycol modified key factor, obtains different anti-
MPEG under the conditions of answering2The experimental data that the reaction substrate and production concentration of-MAL (40kD) modification Luo Saina peptide change over time is used
It is fitted in kinetic parameter；
C) experimental data obtained based on step b), the reaction Kinetics Model established using MATLAB software fit procedure a),
To relevant kinetic parameter k1And kdIt is solved；
D) the kinetic parameter k obtained using the step a) reaction Kinetics Model established and step c)1And kd, backward-predicted is not
With mPEG under reaction condition2The reaction process of-MAL (40kD) modification Luo Saina peptide；To obtain maximum Luo Saina peptide and mPEG2-
MAL molar yield and shortening reaction time etc. are target, determine the mPEG of optimization2- MAL (40kD) modifies Luo Saina peptide work
Skill parameter, and carry out corresponding experimental verification.
4. the preparation method of the polyethylene glycol Luo Saina peptide according to claim 3 based on kinetics, feature exist
In investigating influences polyethyleneglycol modified key factor, rubs including different pH, temperature, polyethyleneglycol derivative/Luo Saina peptide
That ratio, reaction time etc..
5. the preparation method of the polyethylene glycol Luo Saina peptide according to claim 1 based on kinetics, feature exist
In step ii) the optimal mPEG2- MAL (40kD) modifies Luo Saina peptide technological parameter are as follows: mPEG2- MAL (40kD) modification
The action solvent of Luo Saina peptide is the sodium phosphate buffer of 0.1mol/L；The pH of sodium phosphate buffer is 5.5-7.7, preferably
It is 7.0；Reaction temperature is 15-30 DEG C；mPEG2- MAL (40kD)/Luo Saina peptide molar ratio is 0.92:1-1.38:1, preferably
1.12:1-1.15:1；Reaction time is 15-90min, preferably 15-60min.
6. the preparation method of the polyethylene glycol Luo Saina peptide according to claim 1 based on kinetics, feature exist
In the cation-exchange chromatography medium is strong cation exchange material Sepharose SP-HP；The cation-exchange chromatography
Using salt gradient linear elution mode；The ion-exchange chromatography is using the acetate buffer of a certain concentration and pH as flowing
Linear gradient is arranged as Mobile phase B in the acetate buffer of phase liquid A, a certain concentration and pH, correspondingly, collects polyethylene glycol
Luo Saina peptide main peak.
7. the preparation method of the polyethylene glycol Luo Saina peptide according to claim 6 based on kinetics, feature exist
In linear gradient setting are as follows: increase to 50%B from 0%B in 150min, be correspondingly reduced to 50%A from 100%A, collect poly-
Ethylene glycol Luo Saina peptide main peak；Or setting linear gradient are as follows: increase to 30%B from 0%B in 150min, correspondingly from 100%
A is reduced to 70%A, collects polyethylene glycol Luo Saina peptide main peak.
8. a kind of preparation method of polyethylene glycol Luo Saina peptide, method includes the following steps:
1) Luo Saina peptide and a certain amount of mPEG are weighed2- MAL is dissolved in buffer；
The pH of buffer is 6.3-7.7, and reaction temperature is 15-30 DEG C, mPEG2The molar ratio of-MAL and Luo Saina peptide is 1.10:
1-1.15:1, reaction time 15-90min obtain polyethylene glycol Luo Saina peptide crude product solution after reaction；
Optional, further includes:
2) purifying of polyethylene glycol Luo Saina peptide
With cation-exchange chromatography purification step 1) gained polyethylene glycol Luo Saina peptide crude product solution, obtain polyethylene glycol Luo Saina
Peptide salting liquid；Polyethylene glycol Luo Saina peptide sterling is obtained through RP-HPLC desalination, reduced pressure, freeze-drying again.
9. the preparation method of polyethylene glycol Luo Saina peptide according to claim 8, which is characterized in that the buffer is phosphorus
Phthalate buffer, the pH of buffer are 6.3 or 7.0, and reaction temperature is room temperature or 20 DEG C, mPEG2- MAL/ Luo Saina peptide mole
Than for 1.12-1.15:1, reaction time 15-60min.
10. the preparation method of polyethylene glycol Luo Saina peptide according to claim 8, which is characterized in that described in step 2)
Cation-exchange chromatography carries out in AKTA process system, using equipped with 1.25L Sepharose SP-HP filler
BPG200 chromatographic column, flow velocity 125mL/min, Detection wavelength 280nm；
Preferred operating process is as follows: the i) acetic acid-acetic acid for the PEG Luo Saina peptide crude product 0.02mol/L for obtaining step 1)
Sodium buffer is diluted to suitable concentration as buffer solution A, and adjusting pH is 4.3；Ii it) is loaded to suitable loading volume with slow
In the good chromatographic column of fliud flushing A pre-balance, then with the buffer solution A cleaning of certain column volume；Iii linear gradient elution side) is used
Formula elutes and collects polyethylene glycol Luo Saina peptide main peak component；It is further preferred that the linear gradient can be in 150min
Increase to 50%B from 0% buffer solution B, be correspondingly reduced to 50%A from 100%A, collects polyethylene glycol Luo Saina peptide main peak；Or
Person, linear gradient may be to increase to 30%B from 0%B in 150min, are correspondingly reduced to 70%A from 100%A, collect poly-
Ethylene glycol Luo Saina peptide main peak, wherein buffer solution B is the acetic acid-sodium acetate buffer solution+1mol/L NaCl of 0.02mol/L,
pH4.3。
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