CN105820232B - The preparation method and its product of mono-modified polyethylene glycol Recombinant Human Erythropoietin and application - Google Patents
The preparation method and its product of mono-modified polyethylene glycol Recombinant Human Erythropoietin and application Download PDFInfo
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Abstract
The invention discloses a kind of preparation method of mono-modified polyethylene glycol Recombinant Human Erythropoietin and its product and applications.The present invention carries out the modification of polyethylene glycol chemistry to Recombinant Human Erythropoietin albumen active in regular, and obtaining has the mono-modified polyethylene glycol Recombinant Human Erythropoietin for promoting hemopoiesis and long-acting activity in vivo.The present invention passes through buffer system pH value, the Recombinant Human Erythropoietin protein concentration to modification reaction, and Recombinant Human Erythropoietin albumen and the raw material ratio of polyethylene glycol etc. optimize, keep the yield of prepared mono-modified polyethylene glycol Recombinant Human Erythropoietin high, and to modify ratio low more, is conducive to purifying.Compared with the Recombinant Human Erythropoietin albumen before modification, mono-modified polyethylene glycol Recombinant Human Erythropoietin vivo biodistribution specific activity prepared by the present invention is high, and half-life period is obviously prolonged, and can be applied to the drug of preparation treatment anemic disorders, especially renal anemia.
Description
Technical field
The present invention relates to the preparation of the carbowax modifier of Recombinant Human Erythropoietin albumen more particularly to mono-modified poly- second two
The preparation method of alcohol Recombinant Human Erythropoietin and prepared mono-modified polyethylene glycol Recombinant Human Erythropoietin, the invention further relates to described
Application of the mono-modified polyethylene glycol Recombinant Human Erythropoietin in the drug of preparation treatment anemic disorders, especially renal anemia,
Belong to the preparation and application field of polyethylene glycol Recombinant Human Erythropoietin.
Background technique
Nephritic disease is more serious chronic disease, is not easy to cure completely.The annual morbidity of Chinese chronic nephritis is about
0.25%, wherein there is considerable part patient eventually to switch to renal failure.Anaemia is the major complications of Renal Failure Patients, is received according to statistics
Anaemia can occur for the Renal Failure Patients 97% of dialysis treatment.
Since renal anemia is a kind of chronic disease, the delay time is longer, is not easy to cure completely, it is therefore desirable to be used for a long time
Treat anaemia drug.Recombinant Human Erythropoietin class drug is the significant active drug for treating renal anemia, and no other drugs can generation
It replaces, market capacity is very big.Recombinant epo is given expression to from Chinese hamster ovary celI and base similar in natural EPO using technique for gene engineering
Because of engineering drug, succeeded in developing by Amgen, the U.S. within 1989.Recombinant epo becomes treatment renal anemia and Surgery During Perioperative Period
The active drug that red blood cell is mobilized, but need to be used for a long time.
Under the premise of not significantly affecting drug activity in vivo, mainly pass through albumen changes structure to exploitation new E PO class drug
Or manually modified mode come improve epo protein matter stability and extend half-life period, thus reduce frequency of injection and patient pain
Hardship improves the competitiveness of product and the level of update.It is protein modified most with the application of PEG class dressing agent.
Polyethylene glycol (PEG) is the linear or branch-like polyethers high score with hydroxyl end being polymerized by ethylene glycol monomers
Sub- compound, the hydrophily with height have biggish hydrodynamics volume in aqueous solution, and do not have immunogenicity, energy
Change the biological distribution behavior and dissolubility of drug in aqueous solution, generate physical barrier around the drug of its modification, reduces
The enzymatic hydrolysis of drug avoids being eliminated quickly in the metabolism of kidney, and enables cell recognition of the drug by immune system.PEG is
Through Food and Drug Adminstration of the US (FDA) ratify only a few can be medicinal as internal injection one of synthetic polymer.PEG is repaired
It adorns protein techniques and has carried out developing Journal of Sex Research first in the 1970s by Davis.1991, the 1st kind was repaired with polyethylene glycol
The protein drug PEG- adenosine deaminase of decorations obtains FDA approval listing, for treating immunization programs for children Defect, has had at present more
Kind PEG modification protein drug listing.
The existing method that Recombinant Human Erythropoietin albumen (EPO) is chemically modified using PEG, different degrees of presence
The defects of monosubstituted rate is low, and polysubstituted rate is high, purification difficult, therefore urgently develop a kind of new mono-modified polyethylene glycol recombined human
The preparation method of erythropoietin reduces polysubstituted rate while improving monosubstituted rate to reach.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of preparation sides of mono-modified polyethylene glycol Recombinant Human Erythropoietin
Method, this method is modified using polyethylene glycol with Recombinant Human Erythropoietin albumen (EPO) active in regular, to obtain
There must be the mono-modified polyethylene glycol Recombinant Human Erythropoietin (PEG-EPO) for promoting hemopoiesis and long-acting activity in vivo,
And the yield of this method PEG-EPO is high, and ratios of modifying low more, is conducive to purifying；
It is red that another technical problem to be solved by this invention is to provide prepared mono-modified polyethylene glycol recombined human rush
Application of the element in the drug of preparation treatment anemic disorders, especially renal anemia.
In order to solve the above technical problems, the technical solution used in the present invention is:
The present invention discloses a kind of preparation method of mono-modified polyethylene glycol Recombinant Human Erythropoietin, including following step first
It is rapid: carbowax modifier being added in the buffer system of Xiang Hanyou Recombinant Human Erythropoietin albumen (EPO), carries out polyethyleneglycol modified
Reaction；Terminate reaction, purifying to get；Wherein, the molar ratio of Recombinant Human Erythropoietin albumen and carbowax modifier is 1:1-5；
Preferably, the molar ratio of the Recombinant Human Erythropoietin albumen and carbowax modifier is 1:2-4, more preferably 1:2-3.
Carbowax modifier of the present invention is methoxy poly (ethylene glycol) butyric acid succinimide ester (mPEG-SBA).Institute
The PEG modification reaction site for stating polyethyleneglycol modified reaction is predominantly located at the N-terminal α amino of Recombinant Human Erythropoietin albumen, and 45
Or any one site in 52 lysine ε amino.
In preparation method of the present invention, the buffer system is 100-150mM K2HPO4, pH value 6.0-9.0；Preferably,
The buffer system is 150mM K2HPO4, pH value 7.5.In the buffer system, the concentration of Recombinant Human Erythropoietin albumen is 1-
5mg/ml, preferably 2-4mg/ml, most preferably 3mg/ml.The condition of the polyethyleneglycol modified reaction includes: 20-25 DEG C and stirs
It mixes 2-4 hours；Preferably, it stirs 2 hours for 20-25 DEG C；Wherein, the revolving speed of stirring is not particularly limited, as a reference, stirring
Revolving speed is 80-130rpm.
Recombinant Human Erythropoietin albumen of the present invention is what people's Erythropoietin was obtained through eukaryotic cell expression system expression；
Preferably, people's Erythropoietin is through CHO-dhfr- cell (that is: dihyrofolate reductase deficiency Chinese hamster ovary celI (Chinese hamster ovum
Nest cell)) expression acquisition.It specifically include that, using recombinant DNA technology, will turn with the recombinant plasmid of someone's Erythropoietin
Contaminate CHO-dhfr- cell line, by cell culture, separation and it is highly purified after be made.The ammonia of Recombinant Human Erythropoietin albumen (EPO)
Base acid sequence is shown in SEQ ID No.1.The preparation method of Recombinant Human Erythropoietin albumen is known in the art, such as be can refer to
" open the progress and clinical application China blood purification for waiting long-acting erythropoietin quietly, the 9th phase of volume 2012,11
.519-521.”。
In the preparation method of the mono-modified polyethylene glycol Recombinant Human Erythropoietin of the present invention, the termination reaction is adjustment reaction solution
PH value be 2.0-4.5, preferably 3.0-4.0, more preferably 3.5-4.0, most preferably 4.0；Wherein, it is adjusted and is reacted with acetic acid
The pH value of liquid.The purifying uses ion-exchange chromatography, the filler of the ion-exchange chromatography be SP Sepharose F.F. or
Any one in MacroCap SP, preferably MacroCap SP；The step of ion-exchange chromatography includes: successively with flat
Weighing apparatus liquid is balanced, loading, and rebalancing after end of the sample is washed with cleaning solution, eluted with eluent, collects elution
Product to get；Wherein, the equilibrium liquid is 30mM NaAc, pH value 2.0-4.5, preferably 3.0-4.0, more preferably 3.5-
4.0, most preferably 4.0；The cleaning solution be 100mM NaCl, 30mM NaAc, pH value 2.0-4.5, preferably 3.0-4.0,
More preferably 3.5-4.0, most preferably 4.0；The eluent is 175mM NaCl, 30mM NaAc, pH value 2.0-4.5, excellent
It is selected as 3.0-4.0, more preferably 3.5-4.0, most preferably 4.0；The regenerated liquid is 750mM NaCl, 30mM NaAc, pH value
For 2.0-4.5, preferably 3.0-4.0, more preferably 3.5-4.0, most preferably 4.0.
The preparation method of the mono-modified polyethylene glycol Recombinant Human Erythropoietin of the present invention, further includes: carry out product after purification
Concentration, commutation；Wherein, the concentration is to be concentrated by ultrafiltration with 10KDa ultrafilter；The commutation liquid of the commutation includes: 10-
20mM sodium phosphate, 40-50mM sodium sulphate, pH value 5.5-7.5, preferably 5.8-7.0, more preferably 6.0-6.5；Preferably,
The commutation liquid is 10mM sodium phosphate, 40mM sodium sulphate, 3% mannitol, pH value 6.2.
The buffer system pH value in mono-modified polyethylene glycol Recombinant Human Erythropoietin preparation method is optimized in the present invention,
The result shows that within the scope of pH6.0-9.0, under pH7.5 and pH9.0 reaction condition, the mono-modified PEG-EPO component ratio of preparation
It is essentially identical, respectively 48.85% and 51.20%, and under pH6.0 reaction condition, mono-modified PEG-EPO component ratio is obvious
It is relatively low, only 40.28%.Although the mono-modified a little higher than pH7.5 of PEG-EPO component ratio under the reaction condition of pH9.0,
PH9.0 is alkaline condition, is unfavorable for protein stabilization.Therefore, the present invention selects pH7.5 for best modification reaction pH value.
The present invention is further optimized the raw material ratio of polyethyleneglycol modified reaction, the results showed that EPO:PEG
(mPEG-SBA) under the reaction condition of molar ratio 1:4, PEG-EPO component ratios of modifying larger more, up to 43%, purpose product ratio
It is relatively low, only 44.38%, and it is unfavorable for later-period purification.And under the reaction condition of EPO:PEG molar ratio 1:2,1:3, purpose product
The ratio of mono-modified PEG-EPO component is maximum, and respectively 47.01% and 51.37%, no significant difference.Therefore, the present invention selects
EPO:PEG (mPEG-SBA) molar ratio 1:2-1:3 (mass ratio 1:3.5-1:5) is best modification reaction raw material ratio.
The present invention is also to Recombinant Human Erythropoietin albumen (EPO) in mono-modified polyethylene glycol Recombinant Human Erythropoietin preparation method
Protein concentration is optimized, the results showed that under the reaction condition of EPO concentration 5.68mg/ml, EPO:PEG molar ratio 1:2, mesh
The mono-modified PEG-EPO component ratio of product be 49.35%；In the reaction item of EPO concentration 3mg/ml, EPO:PEG molar ratio 1:2
Under part, the mono-modified PEG-EPO component ratio of purpose product is 47.89%.The two is compared, the mono-modified PEG- of the former purpose product
EPO component ratio is slightly higher, but difference is unobvious, and the former more modification PEG-EPO component ratios are also relatively high, reach
24.11%.In view of the concentration of EPO stoste production is generally in 3mg/ml or so, and the removal for more modifying PEG-EPO component is difficult
Spend larger, therefore the present invention selects EPO concentration for 2-4mg/ml.
To sum up, the present invention is 2-4mg/ml, EPO:PEG (mPEG-SBA) molar ratio 1:2-1:3, buffer body in EPO concentration
Be pH value 7.5 reaction condition under, the ratio of the mono-modified PEG-EPO component of purpose product obtained is maximum, reaches 42.91%-
51.37%, and the ratio of more modification PEG-EPO components is low, below 24.99%.Reaction product is through SP cation-exchange chromatography
After purification, the purity of the mono-modified PEG-EPO of electroresis appraisal is greater than 95%, carries out Purity using SEC-HPLC method, purity is
98.82%.
The PEG modification reaction mild condition that the present invention uses, easily makes protein properties tend towards stability；Modifier mPEG-SBA
Quality meet pharmaceutical raw material requirement.The purifying of the mono-modified polyethylene glycol Recombinant Human Erythropoietin (PEG-EPO) of the present invention uses one
SP cation-exchange chromatography, simple process are walked, amplification is easy, and the rate of recovery is high；After PEG-EPO is purified, PEG is remained in finished product
Less than or equal to 50 μ g/mg albumen, endotoxin pyrogen is less than or equal to 10EU/mg；Effective control is able to carry out to impurity, heat source
System.PEG-EPO commutation after purification can directly carry out preparation with liquid, packing, no longer need to replacement buffer body to the commutation liquid
System.
The mono-modified polyethylene glycol Recombinant Human Erythropoietin that the method for the present invention is prepared, which has, promotes hemopoiesis,
Compared with the EPO before modification, vivo biodistribution specific activity is high, and specific activity 10105.8U/mg is more suitable than work with standard items, partly declines
Phase is obviously prolonged, and can be applied to the drug of preparation treatment anemic disorders, especially renal anemia.
Technical solution of the present invention compared with prior art, has the advantages that
The present invention is using carbowax modifier (mPEG-SBA) to Recombinant Human Erythropoietin egg active in regular
White (EPO) is modified, and mono-modified polyethylene glycol Recombinant Human Erythropoietin (PEG-EPO) is prepared.The present invention passes through to modification reaction
Buffer system pH value, EPO concentration and PEG and EPO raw material ratio optimize, keep prepared PEG-EPO yield high, it is more
The ratio for modifying PEG-EPO is low, is conducive to purifying.Compared with the EPO before modification, PEG-EPO vivo biodistribution prepared by the present invention
Specific activity is high, and half-life period is obviously prolonged, and can be applied to the drug of preparation treatment anemic disorders, especially renal anemia.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of Recombinant Human Erythropoietin albumen (EPO)；
Fig. 2 is methoxy poly (ethylene glycol) butyric acid succinimide ester (mPEG-SBA) molecular structural formula；
Fig. 3 is that HPLC detects PEG modification reaction proportion of products of the present invention；
Fig. 4 is the structural formula of PEG-EPO；
Fig. 5 is that PEG-EPO stoste reversed-phased high performace liquid chromatographic (RP-HPLC) analyzes result；
Fig. 6 is that PEG-EPO stoste gel high performance liquid chromatography (SEC-HPLC) analyzes result；
Fig. 7 is PEG-EPO stoste electrophoretic analysis result.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.It should be understood that described, examples are merely exemplary, does not constitute any restrictions to the scope of the present invention.This field
Technical staff should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and
Form is modified or is replaced, but these modifications or substitutions each fall within protection scope of the present invention.
1, material
(concentration 2.84mg/ml buffers as 10mM PB, pH7.0, by Shandong A Hua biology medicine company limited public affairs EPO stoste
Department's production)；
MPEG-SBA (methoxy poly (ethylene glycol) butyric acid succinimide ester) (Beijing Jian Kai company, purity > 95%, molecule
Amount is 30kD)；
Acetic acid (analyzes pure, Chinese medicines group)；Dipotassium hydrogen phosphate (analyzes pure, Chinese medicines group).
The preparation of Preparative Example 1 Recombinant Human Erythropoietin albumen (EPO) stoste
EPO stoste is using recombinant DNA technology, by the Transfected Recombinant Plasmid CHO-dhfr- (two with someone's Erythropoietin
Hydrogen folic acid reductase gene defection type cell) cell line, by cell culture, separation and it is highly purified after be made.It is specific as follows:
1, the basic demand of raw material and auxiliary material
Production and calibrating facility, raw material and auxiliary material, water, utensil, animal etc. should meet current edition " Chinese Pharmacopoeia " three
The related requirement of " note on the use ".
2, the preparation of EPO stoste
2.1 engineering cell
2.1.1 title and source
Polyethylene glycol Recombinant Human Erythropoietin engineering cell system is the CHO- of the Transfected Recombinant Plasmid with someone's Erythropoietin
Dhfr- (dihydrofolate reductase gene deficient cell) cell line.
2.1.2 cell bank is established, passes on and is saved
It is passed on, is frozen in liquid nitrogen after amplification, as master cell bank, master cell bank is at most not by the cell of master cell bank
Obtain more than 2 cell generations；It passes on from the cell of master cell bank, is frozen in liquid nitrogen after amplification, as working cardial cell library, work
Cell bank is necessarily limited to 1 cell generation.In liquid nitrogen, assay approval rear can be used for producing cell cryopreservation.
2.1.3 the calibrating of master cell bank and working cardial cell library cell
" Chinese Pharmacopoeia " three " biological products produce the preparation of calibrating zooblast matrix and vertification regulation " rule should be met
It is fixed.
2.1.3.1 cell identification experiment
Application cell method, is identified under an optical microscope, should be typical Chinese hamster ovary celI.
2.1.3.2 sterility test
Cell culture supernatant sample is taken, bacterium and Fungus examination are carried out, should be negative.
2.1.3.3 mycoplasma and interior external source virokine inspection
Cell culture supernatant sample is taken, mycoplasma and interior external source virokine inspection are carried out, should be negative.
2.1.3.4 people's erythropoietin expression quantity detects
In growth medium, the detection of cell bottle culture expression quantity external activity should be not less than 4000IU/ml.
2.1.3.5 target gene nucleotide sequence inspection (exempting to do in working cardial cell library)
Target gene nucleotide sequence should be consistent with the sequence of approval.
The preparation of 2.2EPO stoste
2.2.1 cell culture
2.2.1.1 cell bottle culture
Seed cell is taken out from working cardial cell library, is melted in 39 DEG C of warm water immediately.In sterile immigration culture bottle, it is added
In 37 DEG C, CO after growth medium2It is cultivated in incubator.The cell of recovery is passed in growth medium, is expanded, for thin
The inoculation of born of the same parents' culture tank is used, and inoculum density is not less than 5.0 × 105cells/ml.The cow's serum containing inactivation in growth medium.
2.2.1.2 cell tank culture
2.2.1.2.1 growth period
Growth period uses growth medium.Glucose concentration of medium in tank is measured after inoculation, when sugared concentration is down to 1~
When 2g/L, starts perfusion culture, concentration of glucose is maintained to be not higher than 1.5g/L.
2.2.1.2.2 production period
Production period uses production medium, is free of cow's serum and any antibiotic.When switching to production period by growth period, with thin
Production medium is changed to after the washing of born of the same parents' cleaning solution.Concentration of glucose in tank is measured, when concentration of glucose is down to 1.5~2.5g/L
When, start to be perfused with production medium and cultivate, maintains concentration of glucose to be not higher than 3g/L, collect culture solution.Harvest liquid needs 2~8
It is DEG C stored refrigerated and handle in time, no more than 2 weeks.
2.2.1.2.3 tank culture control parameter
In tank incubation, temperature control is 37 DEG C, and mixing speed control is 150~200rpm, pH value control for 7.0 ±
0.2, dissolved oxygen is not less than 38%.
2.2.2 isolating and purifying
2.2.2.1 affinity chromatography
Using affinity media, medium is balanced with equilibrium liquid, and loading harvest liquid is balanced with equilibrium liquid, with washing for sodium chloride-containing
De- liquid elution, collects eluting peak, and desalination.
2.2.2.2 ion-exchange chromatography
Using anionic exchange medium, medium is balanced with equilibrium liquid.Desalination collection liquid elder generation prepurification, i.e., with balance after loading
Liquid balance, with the elution of sodium chloride-containing.Loading again after collection liquid dilution is eluted, is balanced with equilibrium liquid, uses acetate buffer
Liquid washing, is balanced with rebalancing liquid, with elution, collects eluting peak.
2.2.2.3 reversed phase chromatography
Using inverted medium, medium is balanced with equilibrium liquid.Step elution collection liquid, is balanced with equilibrium liquid in loading.With balance
The elution of the continuous gradient of liquid and dehydrated alcohol, collects main elution peak.Collection liquid is diluted with equilibrium liquid, and loading anion exchange is situated between
Matter, and elute, collect eluting peak.
2.2.2.4 gel chromatography
Using gel separation media, medium is balanced with equilibrium liquid, and step elution collection liquid, is balanced with equilibrium liquid in loading, is received
Collect protein peak.After aseptic filtration, as Recombinant Human Erythropoietin stoste is frozen or for next step modification reaction.
3, the peptide sequence of EPO, EPO structural schematic diagram
The amino acid sequence of EPO peptide chain is shown in SEQ ID No.1:
ALA PRO PRO ARG LEU ILE CYS ASP SER ARG VAL LEU GLU ARG TYR LEU LEU
GLU ALA LYS GLU ALA GLU ASN*ILE THR THR GLY CYS ALA GLU HIS CYS SER LEU ASN
GLU ASN*ILE THR VAL PRO ASP THR LYS VAL ASN PHE TYR ALA TRP LYS ARG MET GLU
VAL GLY GLN GLN ALA VAL GLU VAL TRP GLN GLY LEU ALA LEU LEU SER GLU ALA VAL
LEU ARG GLY GLN ALA LEU LEU VAL ASN*SER SER GLN PRO TRP GLU PRO LEU GLN LEU
HIS VAL ASP LYS ALA VAL SER GLY LEU ARG SER LEU THR THR LEU LEU ARG ALA LEU
GLY ALA GLN LYS GLU ALA ILE SER PRO PRO ASP ALA ALA SER**ALA ALA PRO LEU ARG
THR ILE THR ALA ASP THR PHE ARG LYS LEU PHE ARG VAL TYR SER ASN PHE LEU ARG
GLY LYS LEU LYS LEU TYR THR GLY GLU ALA CYS ARG THR GLY ASP ARG；
The structural schematic diagram of EPO is shown in Fig. 1.
Preparation-PEG modification reaction of the mono-modified polyethylene glycol Recombinant Human Erythropoietin (PEG-EPO) of embodiment 1
The EPO stoste of assay approval is taken, adjustment buffer system is 150mM K2HPO4, pH7.5, EPO concentration is 2.84mg/
ml。
It is the ratio of 1:3 (mass ratio 1:5) in EPO:PEG molar ratio, weighs mPEG-SBA (methoxy poly (ethylene glycol) fourth
Sour succinimide ester, straight chain, 30KDa, molecular structural formula are shown in Fig. 2), it is added in EPO stoste adjusted, slowly stirs molten
Solution carries out average rate stirring (80-130rpm) using electric mixer at (20-25 DEG C) of room temperature, reacts 2 hours.After reaction
5 times of dilution and with glacial acetic acid tune pH 4.0, is placed in stored under refrigeration in 2~8 DEG C of refrigerators, the time was no more than 3 days；Such as more than 3 days,
It needs to carry out stored frozen in -20 DEG C of refrigerators.
Identification-PEG modification reaction of the mono-modified polyethylene glycol Recombinant Human Erythropoietin of embodiment 2
Using the PEG for the mono-modified polyethylene glycol Recombinant Human Erythropoietin that RP HLPC method prepares embodiment 1
Modification ratio is identified.Mobile phase A is trifluoroacetic acid aqueous solution (0.3%TFA)；Mobile phase B is trifluoroacetic acid and acetonitrile water
Solution (0.2%TFA, 84% acetonitrile)；Flow velocity is 1ml/min；Detection wavelength is 220nm；Column temperature is 60 DEG C；Sample volume is 5-20
μg.Chromatographic column uses Agilent Poroshell 300SB C8 (5 μm, 300A, 2.1x75mm).
Gradient:
The qualification result of PEG modification ratio is shown in Fig. 3 and table 1, the mono-modified PEG-EPO's of purpose product (structural formula is shown in Fig. 4)
Ratio is 42.91%.
The chromatographic peak result of 1 PEG of table modification ratio
The identification of the mono-modified polyethylene glycol Recombinant Human Erythropoietin of embodiment 3-PEG-EPO purifying
The mono-modified polyethylene glycol Recombinant Human Erythropoietin prepared to embodiment 1 purifies.Ion exchange column is assembled,
Fillers selection strong cation medium MacroCap SP, pillar height connects online UV detector after 15cm or so, column, 2~8
Chromatographic purifying is carried out in DEG C environment.
With 30mM NaAc (sodium acetate), pH4.0 buffer balance is no less than 5 column volumes.Prepared by loading embodiment 1
Modification reaction solution, applied sample amount are no more than 2mg albumen/ml medium (optimal for 1mg albumen/ml medium or so).After end of the sample
5 column volumes are no less than with the buffer balance of 30mM NaAc, pH4.0.
3~4 column volumes are washed with 100mM NaCl, 30mM NaAc, pH4.0；With 175mM NaCl, 30mM NaAc,
PH4.0 elutes 3 column volumes, collects eluting peak；750mM NaCl, 30mM NaAc, pH4.0 regeneration are used again.
Elution collection liquid is concentrated by ultrafiltration using 10KDa ultrafilter, and commutation, to 10mM sodium phosphate, 40mM sodium sulphate, 3% is sweet
Reveal alcohol, is mono-modified polyethylene glycol Recombinant Human Erythropoietin (PEG-EPO) stoste after aseptic filtration in the solution of pH6.2.
Purity detecting is carried out to PEG-EPO using RP HLPC method, purity is 98.52% (Fig. 5).
Identification-PEG-EPO Purity of the mono-modified polyethylene glycol Recombinant Human Erythropoietin of embodiment 4
It is identified using purity of the SEC-HPLC method to the PEG-EPO that embodiment 3 purifies.Mobile phase: 5mM phosphoric acid hydrogen two
Sodium/500mM sodium chloride/1.5mM potassium dihydrogen phosphate, pH7.3.Chromatographic column: Tosohaas tsk gel G3000SWxl (7.8mm x
30cm, 5 μm).
Chromatographic condition are as follows: flow velocity 0.6ml/ minutes (isocratic), applied sample amount: according to protein content into proper volume (5~20 μ
G), column temperature is room temperature, and sample cell temperature is 8 DEG C ± 1 DEG C, and environment temperature is room temperature, Detection wavelength 220nm.
As a result see Fig. 6, purity 98.82%.
The identification of the mono-modified polyethylene glycol Recombinant Human Erythropoietin of embodiment 5-electrophoresis purity identification
Electrophoresis purity identification is carried out to the PEG-EPO that embodiment 3 purifies.
1, solution is prepared: A liquid: 1.5M Tris-HCl (pH8.8) (100ml): weighing Tris 18.15g, dissolved with water,
Concentrated hydrochloric acid tune pH to 8.8 is settled to 100ml with water.B liquid: 0.5M Tris-HCl (pH6.8) (100ml): Tris is weighed
6.05g is dissolved with water, and concentrated hydrochloric acid tune pH to 6.8 is settled to 100ml with water.C liquid: acrylamide and the double propylene of N.N '-methene
Amide reservoir (100ml): 29.2 grams of acrylamide, 0.8 gram of N.N '-methene acrylamide is dissolved, and be settled to 100ml with water
It filters (setting in brown reagent bottle, be kept in dark place).D liquid: 10%SDS (100ml): 10 grams of SDS are weighed, is dissolved with water, and constant volume
To 100ml.E liquid: 10%APS solution, prepared before use.F liquid: TEMED.Sample treatment liquid: Tris 0.303g, bromine phenol are weighed
Blue 2mg, SDS 0.8g, measurement concentrated hydrochloric acid 0.189ml, glycerol 4ml mixing are dissolved in water and are diluted to 10ml.Electrode buffer
(1000ml): glycine 14.4g, Tris 3.0g, SDS 1.0g is dissolved to 900ml with water, adjusts pH to 8.3, be settled to water
1000ml。
2, it sample treatment: before sample and standard protein loading, is heated 3~5 minutes in 100 DEG C of water-baths.
3, glue: separation gel (10%) composition: 4.1ml water, 2.1ml A liquid, 3.3ml C liquid, 100ul D liquid, 100ul E
Liquid, 10ul F liquid.Glue (4.5%) composition: 2.9ml water, 1.25ml A liquid, 0.75ml C liquid, 50ul D liquid, 50ul E is concentrated
Liquid, 5ul F liquid.Separation sol solution is poured into sustained height in mold, with water seal top, after glue polymerization, incline water, with water
It washes, filter paper wipe dry, pours into concentration glue, be inserted into comb, pay attention to avoiding the occurrence of bubble.Comb is taken out after polymerization, upper and lower slot falls
Enter electrode buffer.
4, electrophoresis: according to quantity of sample handling loading 2ug-5ug, conspicuousness standard, sensitivity standard press sample loading respectively
5%, the 1% of amount carries out loading.150V is concentrated, and into after separation gel, voltage is increased to 200V, when bromophenol blue reaches bottom,
Terminate electrophoresis.
5, dye: fixed: with 50% methanol, 12% acetic acid, 100 μ l formaldehyde are settled to 100ml, room with water for injection
Running gel is fixed in warm shaking table 1 hour.Rinsing: being rinsed with 50% methanol aqueous solution for injection 100ml, altogether rinsing 3 times, and every time 5
Minute.Sensitization: with 0.02% Na2S2O3Aqueous solution for injection 100ml is sensitized 1 minute.Cleaning: being cleaned 3 times with water for injection,
0.5 minute every time.Silver staining: claiming 0.2g silver nitrate, 75 μ l formaldehyde, is dissolved into 100ml with water for injection, dyes 20 minutes.Washing:
It is cleaned 2 times, every time 0.5 minute with water for injection.Colour developing: claim the Na of 12g natrium carbonicum calcinatum, 100 μ l formaldehyde, 20 μ l 2%2S2O3
Solution, being settled to 200ml mixing with water for injection is developing solution, after being developed the color 0.5 minute with 100ml developing solution, outwells developing solution,
100ml developing solution is added, it is clear to protein band to develop the color.Color development stopping: it is terminated with the acetic acid 100ml of 50% methanol, 12%
Colour developing.It saves: should be saved after gel is cleaned with water for injection after color development stopping in aqueous solution or kept dry.
As a result see Fig. 7, purity is greater than 95%.
Preparation-PEG modification reaction of the mono-modified polyethylene glycol Recombinant Human Erythropoietin (PEG-EPO) of embodiment 6
The EPO stoste of assay approval is taken, adjustment buffer system is 150mM K2HPO4, pH7.5, EPO concentration is 3mg/ml.
It is the ratio of 1:3 (mass ratio 1:5) in EPO:PEG molar ratio, weighs mPEG-SBA (methoxy poly (ethylene glycol) butyric acid succinyl
Imines ester, straight chain, 30KDa), it is added in EPO stoste adjusted, slowly agitation dissolution, uses electricity at (20-25 DEG C) of room temperature
Dynamic blender carries out average rate stirring (80-130rpm), reacts 2 hours.5 times are diluted after reaction and with glacial acetic acid tune pH
4.0, it is placed in stored under refrigeration in 2~8 DEG C of refrigerators, the time was no more than 3 days；Such as more than 3 days, need to carry out cold in -20 DEG C of refrigerators
Freeze storage.Ratio is modified using PEG of the RP HLPC method to prepared mono-modified polyethylene glycol Recombinant Human Erythropoietin
Example is identified that the ratio of the mono-modified PEG-EPO of purpose product is 51.30%.
The identification of the mono-modified polyethylene glycol Recombinant Human Erythropoietin of embodiment 7-vivo biodistribution specific activity detection
1, experimental material
1.1 experimental raw
PEG-EPO stoste standard items (reference substance) PE20120101BZ (being produced by Shandong Ahua Biopharmaceuticals Co., Ltd)；
EPO stoste (test sample) E201001 (being produced by Shandong Ahua Biopharmaceuticals Co., Ltd)；
PEG-EPO sample (test sample) 20120101 (PEG-EPO sample used is the PEG-EPO of the embodiment of the present invention 3 after purification).
1.2 laboratory apparatus
Blood analyser
1.3 reagent
Dilution: 0.1% bovine serum albumin(BSA) physiological saline；
EDTAP dipotassium ethylene diamine tetraacetate (EDTA-K2) anti-coagulants: 100mg EDTA-K2 is dissolved in 10ml physiological saline, is mixed
It closes uniformly, 200 μ l is added into every clean tube, it is spare.
1.4 experimental animal
Cleaning grade female BAl BIc/C mice, 6~8 week old.
2, experimental method
2.1 standard items and sample dilution
Reference substance and sample are diluted to equal unit concentrations with dilution.By basic, normal, high 3 dosage groups (example: 12.5,
25.0,50.0ng/ mouse) mouse is subcutaneously injected respectively (ratio of adjacent high low dose group wants equal).Every dosage group injection 4
Mouse.
2.2 test processes
Normal detection is taken a blood sample in the 5th day (96h) eye socket of injection, anticoagulant with EDTA-K2 anticoagulant tube.Above-mentioned anticoagulation is taken,
Ratio of the reticulocyte counts to Erythrocytes of every mouse is counted with full-automatic reticulocyte determination analyzer
(Ret%).
2.3 calculating
The potency (U/ml) of measuring samples is calculated by injection dosage (ng) the parallel line analysis method of Ret% value.It calculates
Method is detailed in version Chinese Pharmacopoeia (three) annex Ⅹ IV in 2010.
2.4 points for attention
Experimental result is determined by statistics determination method.If it is determined that this time experiment is invalid, should reform.
3, experimental result
PEG-EPO standard items PE20120101BZ is diluted to equal unit concentrations 125ng/ml with dilution.Make 5 altogether
Mouse 0.2ml is subcutaneously injected by middle dose group (25.0ng/ mouse) in group, 4 mouse of every group of injection respectively.It is injecting respectively
Take within the 3rd day (48h), the 4th day (72h), the 5th day (96h), the 6th day (120h), the 7th day (144h) blood sampling of one group of eye socket respectively,
It is anticoagulant with EDTA-K2 anticoagulant tube.Above-mentioned anticoagulation is taken, the net of every mouse is counted with full-automatic reticulocyte determination analyzer
Red blood cell number is knitted to the ratio (Ret%) of Erythrocytes, concrete outcome is shown in Table 2,96h time Ret% and reaches peak.
2 PEG-EPO each group Ret% test result of table
EPO stoste is diluted to equal unit concentrations 250ng/ml with dilution.5 groups of work altogether, 4 mouse of every group of injection,
Mouse 0.2ml is subcutaneously injected respectively by middle dose group (50.0ng/ mouse).Respectively at the 3rd day (48h), the 4th day of injection
(72h), 1 group of eye socket blood sampling is taken within the 5th day (96h), the 6th day (120h), the 7th day (144h) respectively, it is anti-with EDTA-K2 anticoagulant tube
It is solidifying.Above-mentioned anticoagulation is taken, counts the reticulocyte counts of every mouse to red thin with full-automatic reticulocyte determination analyzer
As shown in table 3,72h time Ret% reaches peak to the ratio (Ret%) of born of the same parents' sum.
3 EPO each group different time points Ret% test result of table
It can be concluded that the best blood sampling time of PEG-EPO is the 5th day (96h) in table 2.Therefore, with PE20120101BZ
Criticizing PEG-EPO standard items is reference substance, carries out Activity determination by test sample of 20120101 batches of PEG-EPO samples, adopted at the 5th day
Blood.(note: PE20120101BZ batches of PEG-EPO standard items, 20120101 batches of PEG-EPO samples are the PEG- that embodiment 3 purifies
EPO).PEG-EPO reference substance and sample are diluted to equal unit concentrations 125ng/ml with dilution.Each 5 groups of work altogether, every group
4 mouse are injected, mouse 0.2ml is subcutaneously injected respectively by middle dose group (25.0ng/ mouse).Experimental implementation and result calculate
It requires unanimously, according to small raticide generation and Ret% numerical value feature, to select 2-8 with version Chinese Pharmacopoeia (three) annex Ⅹ IV in 2010
All mouse are injected, and are taken a blood sample within the 5th day.Surveyed test sample specific activity is 10105.8U/mg, more suitable than work with standard items, not low
In 8.0 × 103U/mg。
The optimization of 1 PEG modification reaction pH value of test example
1, experimental method
The EPO stoste for taking 3 parts of assay approvals, is respectively added K2HPO4To final concentration of 150mM K2HPO4, EPO concentration be 2~
4mg/ml, adjustment buffer system pH value is respectively pH6.0, pH7.5, pH9.0.By EPO:PEG molar ratio 1:3 (mass ratio 1:5)
Ratio be added mPEG-SBA, dissolution, under room temperature (20-25 DEG C) environment stir (80-130rpm) react 2 hours.Reaction knot
Shu Hou is terminated with acetic acid tune pH4.0 and is reacted, and sampling carries out SEC-HPLC detection, with single maximum pH value of PEG-EPO component ratio
Optimum condition as PEG modification reaction.
2, experimental result
Under condition of different pH, SEC-HPLC testing result is shown in Table 4.
4 condition of different pH SEC-HPLC testing result of table
Under the reaction condition of pH7.5 and pH9.0, after reaction 2 hours, mono-modified PEG-EPO component ratio is basic
It is identical, and after pH6.0 reacts 2 hours, mono-modified PEG-EPO component ratio is obviously relatively low.Therefore, can select pH7.5 and
Two kinds of reaction conditions of pH9.0.Although under the reaction condition of pH9.0, mono-modified PEG-EPO component ratio is slightly higher, pH9.0 is alkali
Property condition, is unfavorable for protein stabilization, so selecting pH7.5 for best modification reaction pH value.
The ratio optimization of test example 2 modification reaction EPO and PEG
1, experimental method
The EPO stoste of 3 parts of assay approvals is taken, adjustment buffer system is 150mM K2HPO4, pH7.5, EPO concentration be 2~
4mg/ml.MPEG-SBA, dissolution, in room temperature (20-25 DEG C) environment is added in the ratio of EPO:PEG molar ratio 1:2,1:3,1:4
Lower stirring (80-130rpm) is reacted 2 hours.After reaction, it is terminated and is reacted with acetic acid tune pH4.0, sampling carries out SEC-HPLC
Detection, using single maximum raw material of PEG-EPO component ratio than the optimum condition as PEG modification reaction.
2, experimental result
Under the usage ratio of different modifying reaction EPO:PEG, SEC-HPLC testing result is shown in Table 5.
5 modification reaction different material ratio SEC-HPLC testing result of table
After reaction 2 hours, the reaction condition of EPO:PEG molar ratio 1:4, PEG-EPO component ratios of modifying larger more,
Purpose product ratio is relatively low, and is unfavorable for later-period purification.The reaction condition of EPO:PEG molar ratio 1:2,1:3, purpose product list are repaired
It is maximum to adorn PEG-EPO component ratio, and no significant difference, therefore selects EPO:PEG molar ratio 1:2~1:3 (mass ratio 1:3.5
~1:5) condition be best modification reaction raw material ratio.
3 modification reaction epo protein concentration optimization of test example
1, experimental method
The EPO stoste for taking assay approval, is suitably concentrated through 10kD ultra-filtration centrifuge tube, and adjustment buffer system is 150mM
K2HPO4, pH7.5, EPO concentration is 2.84mg/ml.A stoste adjusted is taken, with 150mM K2HPO4, pH7.5 buffer
One times of dilution is divided into two parts to 1.42mg/ml；A stoste adjusted is separately taken, is concentrated with 10kD ultra-filtration centrifuge tube
One times, to 5.68mg/ml, is equally divided into two parts；Remaining stoste adjusted is equally divided into two parts.Again by each group sample
Product are divided into identical two groups, and mPEG- is added in the ratio of EPO:PEG molar ratio 1:2 and 1:3 (mass ratio 1:3.5 and 1:5) respectively
SBA, stirring and dissolving.Continue to stir (80-130rpm) reaction 2 hours at room temperature.After reaction respectively with acetic acid tune pH4.0
Reaction is terminated, sampling carries out SEC-HPLC detection, modifies using the maximum epo protein concentration of single PEG-EPO component ratio as PEG
The optimum condition of reaction.
2, experimental result
In the case where different modifying reacts epo protein concentration, SEC-HPLC testing result is shown in Table 6.
6 different modifying of table reacts epo protein concentration SEC-HPLC testing result
As seen from Table 6, at EPO concentration 5.68mg/ml, the reaction condition of molar ratio 1:2, the mono-modified PEG- of purpose product
EPO component ratio is 49.35%.At EPO concentration 2.84mg/ml, the reaction condition of molar ratio 1:2, purpose product is mono-modified
PEG-EPO component ratio is 47.89%.The two is compared, and the mono-modified PEG-EPO component ratio of the former purpose product is slightly higher, but
Difference is unobvious, and the former more modification PEG-EPO component ratios are also relatively high.In view of the concentration of EPO stoste production
Generally in 3mg/ml or so, and the removal difficulty for more modifying PEG-EPO component is larger, therefore the present invention does not use EPO concentration
5.68mg/ml, the reaction condition of molar ratio 1:2.
Comprehensively consider, is 2~4mg/ml, EPO:PEG molar ratio 1:2~1:3 (mass ratio 1:3.5~1:5) in EPO concentration
Reaction condition under, the mono-modified PEG-EPO component ratio of purpose product is maximum, therefore it is most preferably to modify that the present invention, which selects this condition,
Reaction condition.
The optimization of 4 commutation liquid composition of test example
1, influence of the pH to thermal stability
The thermal stability of PEG-EPO in various commutation formula of liquid can be analyzed by differential scanning calorimetry (DSC).Generally
The thermal denaturation transition temperature for thinking differential scanning calorimetry measurement is the efficiency index of heat stability of protein.Transition temperature mentions
Height shows the thermal stability enhancing of protein.
In order to determine Optimal pH, PEG-EPO in 4~9 range of pH (purifying of embodiment 3) thermal denaturation temperature is had studied,
30mM Na2HPO4, 30mM sodium citrate analyzes the thermal stability of protein example in 30mM borate.The result shows that maximum turn
Temperature is in 6~9 range of pH, and pH sharply declines lower than 5.5 transition temperatures.Therefore, Optimal pH should be higher than that 5.5.
2, the selection of buffer
Influence of the buffer substance to albumen thermal stability is had studied by differential scanning calorimetry (DSC).The result shows that high
The most suitable buffer of thermal stability is sulfate, phosphate or citrate.
3, sulfate radical buffer influences protein stability
When pH6.2, in the case where the total mole number of salinity is respectively 50mM and 150mM, the phosphate of part is changed
At the sodium sulphate of corresponding molal quantity, albumen transition temperature is significantly raised, so, sulfate is appropriate buffer when pH6.2.
In addition, albumen transition temperature difference is little, low concentration when the total mole number of salinity is respectively 50mM and 150mM
Salt it is preferably be used as commutation liquid.
4, the protein stability in different preparation prescriptions
Formulated solution carries out PEG-EPO study on the stability respectively.Preparation A (10mM sodium phosphate, 100mM sodium chloride,
pH7.5)；Preparation B (10mM sodium phosphate, 40mM sodium sulphate, 3% mannitol, pH6.2)；Formulation C (10mM sodium phosphate, 100mM chlorine
Change sodium, pH7.0)；Preparation D (10mM sodium phosphate, 120mM sodium sulphate, pH6.2)；Preparation E (40mM arginine, 30mM sodium sulphate,
3% (w/v) mannitol, 1mM CaCl2, pH6.2).
By above-mentioned five kinds of component samples, respectively in 4 DEG C, 25 DEG C, 30 DEG C, 40 DEG C of storages, after 6 months, sampling carries out anti-
Phase high performance liquid chromatography, exclusion chromatography and SDS-PAGE detection, calculate protein recovery, evaluate sample stability.
The result shows that preparation B protein recovery is significantly higher than other groups under identical storage condition, illustrate that preparation B matches
Side is relatively stable.Therefore, the present invention selects preparation B for commutation liquid.
Claims (8)
1. a kind of preparation method of mono-modified polyethylene glycol Recombinant Human Erythropoietin, comprising the following steps: Xiang Hanyou recombined human promotees red
Carbowax modifier is added in the buffer system of fibroin, carries out polyethyleneglycol modified reaction；Terminate reaction, purifying to get；
It is characterized by: the molar ratio of the Recombinant Human Erythropoietin albumen and carbowax modifier is 1:2-3；
The carbowax modifier is methoxy poly (ethylene glycol) butyric acid succinimide ester；
The buffer system is 150mM K2HPO4, pH value 7.5；
In the buffer system, the concentration of Recombinant Human Erythropoietin albumen is 2-4mg/ml；
The termination reaction is that the pH value of adjustment reaction solution is 4.0.
2. preparation method described in accordance with the claim 1, it is characterised in that: in the buffer system, Recombinant Human Erythropoietin albumen
Concentration be 3mg/ml.
3. preparation method described in accordance with the claim 1, which is characterized in that the condition of the polyethyleneglycol modified reaction includes:
20-25 DEG C stirring 2-4 hours；
The Recombinant Human Erythropoietin albumen is what people's Erythropoietin was obtained through eukaryotic cell expression system expression；
The pH value for terminating the reaction solution of reaction is adjusted with acetic acid.
4. preparation method described in accordance with the claim 3, which is characterized in that the condition of the polyethyleneglycol modified reaction includes:
20-25 DEG C is stirred 2 hours；
The Recombinant Human Erythropoietin albumen is that people's Erythropoietin is obtained through dihyrofolate reductase deficiency expressing cho cell
's.
5. preparation method described in accordance with the claim 1, which is characterized in that it is described purifying use ion-exchange chromatography, it is described from
The filler of sub- displacement chromatography is any one in SP Sepharose F.F. or MacroCap SP；The ion-exchange chromatography
The step of include: successively be balanced with equilibrium liquid, loading, rebalancing after end of the sample washed with cleaning solution, with elution
Liquid is eluted, and eluted product is collected；
Wherein, the equilibrium liquid is 30mM NaAc, pH value 3.0-4.0；
The cleaning solution is 100mM NaCl, 30mM NaAc, pH value 3.0-4.0；
The eluent is 175mM NaCl, 30mM NaAc, pH value 3.0-4.0.
6. preparation method according to claim 5, which is characterized in that the filler of the ion-exchange chromatography is MacroCap
SP；
The equilibrium liquid is 30mM NaAc, pH value 3.5-4.0；
The cleaning solution is 100mM NaCl, 30mM NaAc, pH value 3.5-4.0；
The eluent is 175mM NaCl, 30mM NaAc, pH value 3.5-4.0.
7. preparation method according to claim 5, which is characterized in that the equilibrium liquid is 30mM NaAc, pH value 4.0；
The cleaning solution is 100mM NaCl, 30mM NaAc, pH value 4.0；
The eluent is 175mM NaCl, 30mM NaAc, pH value 4.0.
8. preparation method described in accordance with the claim 1, which is characterized in that further include: product after purification is concentrated, is changed
Phase；
Wherein, the concentration is to be concentrated by ultrafiltration with 10KDa ultrafilter；
The commutation liquid of the commutation is 10mM sodium phosphate, 40mM sodium sulphate, 3% mannitol, pH value 6.2.
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