The preparation method of mono-modified Polyethylene Glycol Recombinant Human Erythropoietin and goods thereof and application
Technical field
The present invention relates to the preparation of the carbowax modifier of Recombinant Human Erythropoietin albumen, particularly relate to the preparation method of mono-modified Polyethylene Glycol Recombinant Human Erythropoietin and prepared mono-modified Polyethylene Glycol Recombinant Human Erythropoietin, the invention still further relates to described mono-modified Polyethylene Glycol Recombinant Human Erythropoietin at preparation treatment anemic disorders, the especially application in the medicine of renal anemia, belongs to preparation and the application of Polyethylene Glycol Recombinant Human Erythropoietin.
Background technology
Nephritic disease is the more serious chronic disease of ratio, is difficult to cure completely.The annual morbidity of China's chronic nephritis is about 0.25%, wherein has considerable part patient eventually to transfer renal failure to.Anemia is the major complications of Renal Failure Patients, and the Renal Failure Patients 97% accepting dialysis treatment according to statistics can occur anemia.
Owing to renal anemia is a kind of chronic disease, the delay time is longer, is difficult to cure completely, it is therefore desirable to life-time service treatment anemia medicine.Recombinant Human Erythropoietin class medicine is the notable active drug for the treatment of renal anemia, can substitute for without other drug, and market capacity is the biggest.Recombinant epo is that application technique for gene engineering gives expression to the genetically engineered drug close with natural EPO from Chinese hamster ovary celI, within 1989, is succeeded in developing by Amgen of the U.S..Recombinant epo becomes treatment renal anemia and the active drug of Surgery During Perioperative Period erythrocyte mobilization, but needs life-time service.
On the premise of not appreciable impact medicine activity in vivo, if exploitation new E PO class drug main by albumen change structure or manually modified by the way of improve epo protein matter stability and extend the half-life, thus reduce frequency of injection and patient's misery, improve competitiveness and the level of update of product.Protein modified most with the application of PEG class dressing agent.
Polyethylene Glycol (PEG) be by ethylene glycol monomers be polymerized with the linear of hydroxyl end or branch-like polyether high molecular compound, there is the hydrophilic of height, there is bigger hydrodynamics volume in aqueous, and there is no immunogenicity, medicine biological distribution behavior in aqueous and dissolubility can be changed, physical barrier is produced around its medicine modified, reduce the enzymolysis of medicine, avoid quickly being eliminated in the metabolism of kidney, and make the medicine can be by immune cell recognition.PEG is that the only a few ratified through FDA (Food and Drug Adminstration) (FDA) can be as one of medicinal synthetic polymer of internal injection.First PEG modified protein technology has been carried out pioneering research by Davis at 20 century 70s.1991, the 1st kind obtained FDA approval listing by polyethyleneglycol modified protein drug PEG-ADA Adenosine deaminase, is used for treating immunization programs for children Defect, has had multiple PEG modified protein medicine to list.
The method that Recombinant Human Erythropoietin albumen (EPO) is chemically modified by the existing PEG of utilization, the monosubstituted rate of existence in various degree is low, polysubstituted rate is high, the defects such as purification difficult, the most urgently develop the preparation method of a kind of new mono-modified Polyethylene Glycol Recombinant Human Erythropoietin, to reach to reduce while improving monosubstituted rate polysubstituted rate.
Summary of the invention
The technical problem to be solved is to provide the preparation method of a kind of mono-modified Polyethylene Glycol Recombinant Human Erythropoietin, the method utilizes Polyethylene Glycol to modify the Recombinant Human Erythropoietin albumen (EPO) with normal activity in vivo, thus obtain and there is promotion hemopoietic and the mono-modified Polyethylene Glycol Recombinant Human Erythropoietin (PEG-EPO) of long-acting activity in vivo, and the yield of the method PEG-EPO is high, ratios of modifying low more, beneficially purification;
Another technical problem to be solved by this invention is to provide the application in the medicine of preparation treatment anemic disorders, especially renal anemia of the prepared mono-modified Polyethylene Glycol Recombinant Human Erythropoietin.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
First the present invention discloses the preparation method of a kind of mono-modified Polyethylene Glycol Recombinant Human Erythropoietin, comprises the following steps: adds carbowax modifier in the buffer system containing Recombinant Human Erythropoietin albumen (EPO), carries out polyethyleneglycol modified reaction;Terminate reaction, purification, to obtain final product;Wherein, Recombinant Human Erythropoietin albumen is 1:1-5 with the mol ratio of carbowax modifier;Preferably, described Recombinant Human Erythropoietin albumen is 1:2-4, more preferably 1:2-3 with the mol ratio of carbowax modifier.
Carbowax modifier of the present invention is methoxy poly (ethylene glycol) butanoic acid succinimide ester (mPEG-SBA).The PEG modification reaction site of described polyethyleneglycol modified reaction is predominantly located at the N-terminal α amino of Recombinant Human Erythropoietin albumen, any one site in 45 or 52 lysine ε amino.
In preparation method of the present invention, described buffer system is 100-150mMK2HPO4, pH value is 6.0-9.0;Preferably, described buffer system is 150mMK2HPO4, pH value is 7.5.In described buffer system, the concentration of Recombinant Human Erythropoietin albumen is 1-5mg/ml, preferably 2-4mg/ml, most preferably 3mg/ml.The condition of described polyethyleneglycol modified reaction includes: 20-25 DEG C is stirred 2-4 hour;Being preferably, 20-25 DEG C is stirred 2 hours;Wherein, being not particularly limited the rotating speed of stirring, as reference, speed of agitator is 80-130rpm.
Recombinant Human Erythropoietin albumen behaviour Erythropoietin of the present invention obtains through eukaryotic cell expression system expression;Being preferably, people's Erythropoietin expresses acquisition through CHO-dhfr-cell (that is: dihydrofolate reductase deficiency Chinese hamster ovary celI (Chinese hamster ovary cell)).Concrete includes, uses recombinant DNA technology, by the Transfected Recombinant Plasmid CHO-dhfr-cell line with people's Erythropoietin, cultivate through cell, separate and be highly purified after prepare.The aminoacid sequence of Recombinant Human Erythropoietin albumen (EPO) is shown in SEQIDNo.1.The preparation method of Recombinant Human Erythropoietin albumen is known in the art, such as refer to " open and wait quietly. the progress of long-acting erythropoietin and clinical practice. China's blood purification, volume 2012,11 the 9th phase .519-521. ".
In the preparation method of the present invention mono-modified Polyethylene Glycol Recombinant Human Erythropoietin, described termination reaction is 2.0-4.5 for adjusting the pH value of reactant liquor, preferably 3.0-4.0, more preferably 3.5-4.0, most preferably 4.0;Wherein, the pH value of reactant liquor is adjusted with acetic acid.Described purification uses ion-exchange chromatography, and the filler of described ion-exchange chromatography is any one in SPSepharoseF.F. or MacroCapSP, preferably MacroCapSP;The step of described ion-exchange chromatography includes: successively with balance liquid be balanced, loading, reequilibrate after end of the sample, wash with cleaning mixture, carry out eluting with eluent, collect eluted product, to obtain final product;Wherein, described balance liquid is 30mMNaAc, and pH value is 2.0-4.5, preferably 3.0-4.0, more preferably 3.5-4.0, most preferably 4.0;Described cleaning mixture is 100mMNaCl, 30mMNaAc, and pH value is 2.0-4.5, preferably 3.0-4.0, more preferably 3.5-4.0, most preferably 4.0;Described eluent is 175mMNaCl, 30mMNaAc, and pH value is 2.0-4.5, preferably 3.0-4.0, more preferably 3.5-4.0, most preferably 4.0;Described regenerated liquid is 750mMNaCl, 30mMNaAc, and pH value is 2.0-4.5, preferably 3.0-4.0, more preferably 3.5-4.0, most preferably 4.0.
The preparation method of the present invention mono-modified Polyethylene Glycol Recombinant Human Erythropoietin, also includes: carry out product after purification concentrating, commutation;Wherein, described concentration is for be concentrated by ultrafiltration with 10KDa ultrafilter;The commutation liquid of described commutation includes: 10-20mM sodium phosphate, 40-50mM sodium sulfate, and pH value is 5.5-7.5, preferably 5.8-7.0, more preferably 6.0-6.5;Preferably, described commutation liquid is 10mM sodium phosphate, 40mM sodium sulfate, 3% mannitol, and pH value is 6.2.
Buffer system pH value in mono-modified Polyethylene Glycol Recombinant Human Erythropoietin preparation method is optimized by the present invention, result shows in the range of pH6.0-9.0, under pH7.5 and pH9.0 reaction condition, the mono-modified PEG-EPO component ratio of preparation is essentially identical, it is respectively 48.85% and 51.20%, and under pH6.0 reaction condition, mono-modified PEG-EPO component ratio is the most on the low side, 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, but pH9.0 is alkalescence condition, is unfavorable for protein stabilization.Therefore, the present invention selects pH7.5 to be optimal modification reaction pH value.
The raw material ratio of polyethyleneglycol modified reaction is optimized by the present invention further, result shows under the reaction condition of EPO:PEG (mPEG-SBA) mol ratio 1:4, many modification PEG-EPO component ratios are bigger, reach 43%, purpose proportion of products is on the low side, it is only 44.38%, and is unfavorable for later-period purification.And under the reaction condition of EPO:PEG mol ratio 1:2,1:3, the ratio of purpose product mono-modified PEG-EPO component is maximum, respectively 47.01% and 51.37%, no significant difference.Therefore, the present invention selects EPO:PEG (mPEG-SBA) mol ratio 1:2-1:3 (mass ratio 1:3.5-1:5) to be optimal modification reaction raw material ratio.
The protein concentration of Recombinant Human Erythropoietin albumen (EPO) in mono-modified Polyethylene Glycol Recombinant Human Erythropoietin preparation method is also optimized by the present invention, result shows in EPO concentration 5.68mg/ml, under the reaction condition of EPO:PEG mol ratio 1:2, purpose product mono-modified PEG-EPO component ratio is 49.35%;Under EPO concentration 3mg/ml, the reaction condition of EPO:PEG mol ratio 1:2, purpose product mono-modified PEG-EPO component ratio is 47.89%.Both compare, and the former purpose product mono-modified PEG-EPO component ratio is slightly higher, but difference is inconspicuous, and the former many modification PEG-EPO component ratios are the most of a relatively high, reach 24.11%.The concentration produced in view of EPO stock solution is typically at about 3mg/ml, and the removal difficulty modifying PEG-EPO component relatively big more, and therefore the present invention selects EPO concentration to be 2-4mg/ml.
To sum up, the present invention is 2-4mg/ml in EPO concentration, EPO:PEG (mPEG-SBA) mol ratio 1:2-1:3, under the reaction condition of buffer system pH value 7.5, the ratio of the purpose product mono-modified PEG-EPO component prepared is maximum, reach 42.91%-51.37%, and the ratio of many modification PEG-EPO components is low, below 24.99%.Through SP cation-exchange chromatography after purification, the purity of the mono-modified PEG-EPO of electroresis appraisal is more than 95% to product, uses SEC-HPLC method to carry out Purity, and purity is 98.82%.
The PEG modification reaction mild condition that the present invention uses, easily makes protein properties tend towards stability;The quality of trim mPEG-SBA meets pharmaceutical raw material requirement.The purification of the present invention mono-modified Polyethylene Glycol Recombinant Human Erythropoietin (PEG-EPO) uses a step SP cation-exchange chromatography, and technique is simple, amplifies easily, and the response rate is high;After PEG-EPO is purified, in finished product, PEG residual is less than or equal to 10EU/mg less than or equal to 50 μ g/mg albumen, endotoxin pyrogen;Impurity, thermal source can be control effectively.PEG-EPO commutation after purification, to described commutation liquid, can directly carry out preparation dosing, subpackage, it is not necessary to change buffer system again.
The mono-modified Polyethylene Glycol Recombinant Human Erythropoietin that the inventive method prepares has promotion hemopoietic, compared with the EPO before modification, vivo biodistribution specific activity is high, specific activity is 10105.8U/mg, more suitable than living with standard substance, half-life is obviously prolonged, it is possible to be applied to the medicine 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 utilizes carbowax modifier (mPEG-SBA) to modify the Recombinant Human Erythropoietin albumen (EPO) with normal activity in vivo, prepares mono-modified Polyethylene Glycol Recombinant Human Erythropoietin (PEG-EPO).The present invention, by being optimized the buffer system pH value of modification reaction, EPO concentration, and PEG with EPO raw material ratio, makes prepared PEG-EPO yield high, and the ratios modifying PEG-EPO low more, beneficially purification.Compared with the EPO before modification, the PEG-EPO vivo biodistribution specific activity prepared by the present invention is high, and the half-life is obviously prolonged, it is possible to be applied to the medicine of preparation treatment anemic disorders, especially renal anemia.
Accompanying drawing explanation
Fig. 1 is the structural representation of Recombinant Human Erythropoietin albumen (EPO);
Fig. 2 is methoxy poly (ethylene glycol) butanoic 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 PEG-EPO stock solution reversed phase high-performance liquid chromatography (RP-HPLC) analysis result;
Fig. 6 is PEG-EPO stock solution gel high performance liquid chromatography (SEC-HPLC) analysis result;
Fig. 7 is PEG-EPO stock solution electrophoretic analysis result.
Detailed description of the invention
Below in conjunction with specific embodiment further describe the present invention, advantages of the present invention and feature will be with describe and apparent.It should be understood that described embodiment is only exemplary, the scope of the present invention is not constituted any restriction.It will be understood by those skilled in the art that and the details of technical solution of the present invention and form can be modified or replace lower without departing from the spirit and scope of the present invention, but these amendments or replacement each fall within protection scope of the present invention.
1, material
EPO stock solution (concentration is 2.84mg/ml, buffers as 10mMPB, pH7.0, Shandong Ahua Biopharmaceuticals Co., Ltd produces);
MPEG-SBA (methoxy poly (ethylene glycol) butanoic acid succinimide ester) (Beijing Jian Kai company, purity > 95%, molecular weight is 30kD);
Acetic acid (analytical pure, traditional Chinese medicines group);Dipotassium hydrogen phosphate (analytical pure, traditional Chinese medicines group).
The preparation of Preparative Example 1 Recombinant Human Erythropoietin albumen (EPO) stock solution
EPO stock solution is to use recombinant DNA technology, by Transfected Recombinant Plasmid CHO-dhfr-(dihydrofolate reductase gene deficient cell) cell line with people's Erythropoietin, cultivate through cell, separate and be highly purified after make.Specific as follows:
1, raw material and the basic demand of adjuvant
Production and calibrating facility, raw material and adjuvant, water, utensil, animal etc. should meet the relevant requirement of current edition " Chinese Pharmacopoeia " three " notes on the use ".
2, the preparation of EPO stock solution
2.1 engineering cell
2.1.1 title and source
Polyethylene Glycol Recombinant Human Erythropoietin engineering cell system is CHO-dhfr-(dihydrofolate reductase gene deficient cell) cell line of the Transfected Recombinant Plasmid with people's Erythropoietin.
2.1.2 cell bank is set up, is passed on and preserve
By the passage of master cell bank, frozen in liquid nitrogen after amplification, as master cell bank, master cell bank at most must not exceed 2 cell generations;From the passage of master cell bank, frozen in liquid nitrogen after amplification, as working cell storehouse, working cell storehouse is necessarily limited to 1 cell generation.Cell cryopreservation is in liquid nitrogen, and assay approval rear can be used for producing.
2.1.3 master cell bank and the calibrating of working cell storehouse cell
" Chinese Pharmacopoeia " three " biological product produce calibrating zooblast substrate to be prepared and vertification regulation " regulations should be met.
2.1.3.1 cell identification experiment
Application cell method, differentiates under an optical microscope, should be typical case's Chinese hamster ovary celI.
2.1.3.2 sterility test
Take cell culture supernatant sample, carry out antibacterial and Fungus examination, should be negative.
2.1.3.3 mycoplasma and interior external source virokine check
Take cell culture supernatant sample, carry out mycoplasma and interior external source virokine inspection, should be negative.
2.1.3.4 people's erythropoietin expression detection
In growth medium, the detection of expression external activity cultivated by cell bottle should be not less than 4000IU/ml.
2.1.3.5 genes of interest nucleotide sequence checks (working cell storehouse exempts to do)
Genes of interest nucleotide sequence should be consistent with the sequence of approval.
Prepared by 2.2EPO stock solution
2.2.1 cell is cultivated
2.2.1.1 cell bottle is cultivated
Seed cell is taken out from working cell storehouse, melts in 39 DEG C of warm water immediately.In aseptic immigration culture bottle, at 37 DEG C, CO after addition growth medium2Incubator is cultivated.The cell of recovery carries out passing on, expanding in growth medium, inoculates for bioreactor, and inoculum density is not less than 5.0 × 105cells/ml.Containing inactivation Ox blood serum in growth medium.
2.2.1.2 cell tank is cultivated
2.2.1.2.1 trophophase
Trophophase uses growth medium.Measure glucose concentration of medium in tank after inoculation, when sugar concentration is down to 1~2g/L, start perfusion cultures, maintain concentration of glucose to be not higher than 1.5g/L.
2.2.1.2.2 production period
Production period uses production medium, without Ox blood serum and any antibiotic.When being transferred to production period by trophophase, after washing with cell washing solution, it is replaced by production medium.Measure concentration of glucose in tank, when concentration of glucose is down to 1.5~2.5g/L, start to use production medium perfusion cultures, maintain concentration of glucose to be not higher than 3g/L, collect culture fluid.Harvest liquid needs 2~8 DEG C of stored refrigerated and processes in time, no more than 2 weeks.
2.2.1.2.3 tank is cultivated and is controlled parameter
In tank incubation, it is 37 DEG C that temperature controls, and it is 150~200rpm that mixing speed controls, and it is 7.0 ± 0.2 that pH value controls, and dissolved oxygen is for being not less than 38%.
2.2.2 it is isolated and purified
2.2.2.1 affinity chromatograph
Using affinity media, medium balance liquid balances, and loading harvest liquid balances with balance liquid, with the elution of sodium chloride-containing, collects eluting peak, and desalination.
2.2.2.2 ion-exchange chromatography
Using anionic exchange medium, medium balance liquid balances.Liquid elder generation prepurification is collected in desalination, i.e. balances, with the elution of sodium chloride-containing with balance liquid after loading.Eluting collects loading again after liquid dilution, balances with balance liquid, washs with acetate buffer, balances with reequilibrate liquid, with elution, collects eluting peak.
2.2.2.3 reversed phase chromatography
Using inverted medium, medium balance liquid balances.Walk eluting in loading and collect liquid, balance with balance liquid.With the continuous gradient eluting of balance liquid and dehydrated alcohol, collect main elution peak.Collect liquid balance liquid to dilute, loading anionic exchange medium, and eluting, collect eluting peak.
2.2.2.4 gel chromatography
Using gel separation media, medium balance liquid balances, and loading walks eluting and collects liquid, balance with balance liquid, collect protein peak.After aseptic filtration, it is Recombinant Human Erythropoietin stock solution, frozen or for next step modification reaction.
3, the peptide sequence of EPO, EPO structural representation
The aminoacid sequence of EPO peptide chain is shown in SEQIDNo.1:
ALAPROPROARGLEUILECYSASPSERARGVALLEUGLUARGTYRLEULEUGLUALALYSGLUALAGLUASN*ILETHRTHRGLYCYSALAGLUHISCYSSERLEUASNGLUASN*ILETHRVALPROASPTHRLYSVALASNPHETYRALATRPLYSARGMETGLUVALGLYGLNGLNALAVALGLUVALTRPGLNGLYLEUALALEULEUSERGLUALAVALLEUARGGLYGLNALALEULEUVALASN*SERSERGLNPROTRPGLUPROLEUGLNLEUHISVALASPLYSALAVALSERGLYLEUARGSERLEUTHRTHRLEULEUARGALALEUGLYALAGLNLYSGLUALAILESERPROPROASPALAALASER**ALAALAPROLEUARGTHRILETHRALAASPTHRPHEARGLYSLEUPHEARGVALTYRSERASNPHELEUARGGLYLYSLEULYSLEUTYRTHRGLYGLUALACYSARGTHRGLYASPARG;
The structural representation of EPO is shown in Fig. 1.
The preparation PEG modification reaction of embodiment 1 mono-modified Polyethylene Glycol Recombinant Human Erythropoietin (PEG-EPO)
Taking the EPO stock solution of assay approval, adjustment buffer system is 150mMK2HPO4, pH7.5, EPO concentration is 2.84mg/ml.
In the ratio that EPO:PEG mol ratio is 1:3 (mass ratio is 1:5), weigh mPEG-SBA (methoxy poly (ethylene glycol) butanoic acid succinimide ester, straight chain, 30KDa, molecular structural formula is shown in Fig. 2), join in the EPO stock solution after adjustment, slowly stir dissolving, use motor stirrer to carry out average rate stirring (80-130rpm) in room temperature (20-25 DEG C), react 2 hours.Reaction dilutes 5 times and adjusts pH4.0 with glacial acetic acid after terminating, be placed in stored under refrigeration in 2~8 DEG C of refrigerators, and the time not can exceed that 3 days;As more than 3 days, need to carry out stored frozen in-20 DEG C of refrigerators.
The qualification PEG modification reaction of embodiment 2 mono-modified Polyethylene Glycol Recombinant Human Erythropoietin
The PEG of the mono-modified Polyethylene Glycol Recombinant Human Erythropoietin that embodiment 1 is prepared by employing RP HLPC method modifies ratio and identifies.Mobile phase A is trifluoroacetic acid aqueous solution (0.3%TFA);Mobile phase B is trifluoroacetic acid and acetonitrile solution (0.2%TFA, 84% acetonitrile);Flow velocity is 1ml/min;Detection wavelength is 220nm;Column temperature is 60 DEG C;Sample size is 5-20 μ g.Chromatographic column uses AgilentPoroshell300SBC8 (5 μm, 300A, 2.1x75mm).
Gradient:
PEG modifies the qualification result of ratio and sees that Fig. 3 and Biao 1, the ratio of the mono-modified PEG-EPO of purpose product (structural formula is shown in Fig. 4) are 42.91%.
Table 1PEG modifies the chromatographic peak result of ratio
The qualification PEG-EPO purification of embodiment 3 mono-modified Polyethylene Glycol Recombinant Human Erythropoietin
The mono-modified Polyethylene Glycol Recombinant Human Erythropoietin of embodiment 1 preparation is purified.Assembling ion exchange column, Fillers selection strong cation medium MacroCapSP, post height connects online UV-detector after about 15cm, post, carries out chromatography purification in 2~8 DEG C of environment.
With 30mMNaAc (sodium acetate), pH4.0 buffer balance is no less than 5 column volumes.The modification reaction solution of loading embodiment 1 preparation, applied sample amount is less than 2mg albumen/ml medium (optimum is about 1mg albumen/ml medium).By the buffer balance of 30mMNaAc, pH4.0 no less than 5 column volumes after end of the sample.
With 100mMNaCl, 30mMNaAc, pH4.0 wash 3~4 column volumes;With 175mMNaCl, 3 column volumes of 30mMNaAc, pH4.0 eluting, collect eluting peak;Again with 750mMNaCl, 30mMNaAc, pH4.0 regenerate.
Use 10KDa ultrafilter that eluting is concentrated by ultrafiltration and collect liquid, and commutation is to 10mM sodium phosphate, 40mM sodium sulfate, 3% mannitol, in the solution of pH6.2, is mono-modified Polyethylene Glycol Recombinant Human Erythropoietin (PEG-EPO) stock solution after aseptic filtration.
Using RP HLPC method that PEG-EPO is carried out purity detecting, purity is 98.52% (Fig. 5).
The Purity of the qualification PEG-EPO of embodiment 4 mono-modified Polyethylene Glycol Recombinant Human Erythropoietin
Use SEC-HPLC method that the purity of the PEG-EPO of embodiment 3 purification is identified.Flowing phase: 5mM disodium hydrogen phosphate/500mM sodium chloride/1.5mM potassium dihydrogen phosphate, pH7.3.Chromatographic column: TosohaasTSK gel G3000SWxl (7.8mmx30cm, 5 μm).
Chromatographic condition is: flow velocity 0.6ml/ minute (isocratic), applied sample amount: entering proper volume (5~20 μ g) according to protein content, column temperature is room temperature, and sample cell temperature is 8 DEG C ± 1 DEG C, and ambient temperature is room temperature, and detection wavelength is 220nm.
Result is shown in Fig. 6, and purity is 98.82%.
The qualification electrophoresis purity of embodiment 5 mono-modified Polyethylene Glycol Recombinant Human Erythropoietin is identified
The PEG-EPO of embodiment 3 purification is carried out electrophoresis purity qualification.
1, solution preparation: A liquid: 1.5MTris-HCl (pH8.8) (100ml): weigh Tris18.15g, with water dissolution, concentrated hydrochloric acid is adjusted pH to 8.8, is settled to 100ml with water.B liquid: 0.5MTris-HCl (pH6.8) (100ml): weigh Tris6.05g, with water dissolution, concentrated hydrochloric acid is adjusted pH to 6.8, is settled to 100ml with water.C liquid: acrylamide and N.N '-methylene diacrylamide reservoir (100ml): acrylamide 29.2 grams, N.N '-methene acrylamide 0.8 gram, with water dissolution, and it is settled to 100ml and filters (putting in brown reagent bottle, keep in Dark Place).D liquid: 10%SDS (100ml): weigh SDS10 gram, with water dissolution, and is settled to 100ml.E liquid: 10%APS solution, prepared before use.F liquid: TEMED.Sample treatment liquid: weigh Tris0.303g, bromophenol blue 2mg, SDS0.8g, measure concentrated hydrochloric acid 0.189ml, glycerol 4ml mixing be dissolved in water and be diluted to 10ml.Electrode buffer (1000ml): glycine 14.4g, Tris3.0g, SDS1.0g, with water dissolution to 900ml, adjusts pH to 8.3, is settled to 1000ml with water.
2, sample treatment: before sample and standard protein loading, heats 3~5 minutes in 100 DEG C of water-baths.
3, glue: separation gel (10%) forms: 4.1ml water, 2.1mlA liquid, 3.3mlC liquid, 100ulD liquid, 100ulE liquid, 10ulF liquid.Concentrate glue (4.5%) to form: 2.9ml water, 1.25mlA liquid, 0.75mlC liquid, 50ulD liquid, 50ulE liquid, 5ulF liquid.Separation gel solution is poured in mould to sustained height, use water seal top, after glue is polymerized, inclines and anhydrate, with washing, filter paper wipe dry, pour concentration glue into, insert comb, note avoiding the occurrence of bubble.Taking out comb after polymerization, upper and lower groove pours electrode buffer into.
4, electrophoresis: according to quantity of sample handling loading 2ug-5ug, significance standard, susceptiveness standard carry out loading by the 5% of sample applied sample amount, 1% respectively.150V concentrates, and after entering separation gel, voltage is increased to 200V, when bromophenol blue arrives the end, terminates electrophoresis.
5, dyeing: fixing: to use 50% methanol, the acetic acid of 12%, 100 μ l formaldehyde, be settled to 100ml with water for injection, fix running gel in room temperature shaker 1 hour.Rinsing: rinse with 50% methanol aqueous solution for injection 100ml, altogether rinsing 3 times, each 5 minutes.Sensitization: with the Na of 0.02%2S2O3Aqueous solution for injection 100ml sensitization 1 minute.Clean: with water for injection clean 3 times, each 0.5 minute.Silver staining: claim 0.2g silver nitrate, 75 μ l formaldehyde, be dissolved into 100ml with water for injection, dye 20 minutes.Washing: with water for injection clean 2 times, each 0.5 minute.Colour developing: claim 12g natrium carbonicum calcinatum, 100 μ l formaldehyde, the Na of 20 μ l2%2S2O3Solution, being settled to 200ml mixing with water for injection is nitrite ion, after developing the color 0.5 minute with 100ml nitrite ion, outwells nitrite ion, adds 100ml nitrite ion, develop the color clear to protein band.Color development stopping: by 50% methanol, the acetic acid 100ml color development stopping of 12%.Preserve: after color development stopping, gel water for injection should preserve in aqueous or kept dry after cleaning.
Result is shown in Fig. 7, and purity is more than 95%.
The preparation PEG modification reaction of embodiment 6 mono-modified Polyethylene Glycol Recombinant Human Erythropoietin (PEG-EPO)
Taking the EPO stock solution of assay approval, adjustment buffer system is 150mMK2HPO4, pH7.5, EPO concentration is 3mg/ml.In the ratio that EPO:PEG mol ratio is 1:3 (mass ratio is 1:5), weigh mPEG-SBA (methoxy poly (ethylene glycol) butanoic acid succinimide ester, straight chain, 30KDa), join in the EPO stock solution after adjustment, slowly stir dissolving, use motor stirrer to carry out average rate stirring (80-130rpm) in room temperature (20-25 DEG C), react 2 hours.Reaction dilutes 5 times and adjusts pH4.0 with glacial acetic acid after terminating, be placed in stored under refrigeration in 2~8 DEG C of refrigerators, and the time not can exceed that 3 days;As more than 3 days, need to carry out stored frozen in-20 DEG C of refrigerators.Using RP HLPC method that the PEG of prepared mono-modified Polyethylene Glycol Recombinant Human Erythropoietin is modified ratio to identify, the ratio of the mono-modified PEG-EPO of purpose product is 51.30%.
The qualification vivo biodistribution specific activity detection of embodiment 7 mono-modified Polyethylene Glycol Recombinant Human Erythropoietin
1, experiment material
1.1 experimental raw
PEG-EPO stock solution standard substance (reference substance) PE20120101BZ (being produced by Shandong Ahua Biopharmaceuticals Co., Ltd);
EPO stock solution (test sample) E201001 (being produced by Shandong Ahua Biopharmaceuticals Co., Ltd);
PEG-EPO sample (test sample) 20120101 (PEG-EPO sample used is the embodiment of the present invention 3 PEG-EPO after purification).
1.2 experimental apparatus
Blood analyser
1.3 reagent
Diluent: 0.1% bovine serum albumin normal saline;
Ethylenediamine tetraacetic acid,dipotassium salt (EDTA-K2) anticoagulant: 100mgEDTA-K2 is dissolved in 10ml normal saline, mix homogeneously, adds 200 μ l in every clean tube, standby.
1.4 laboratory animal
Cleaning grade female BAl BIc/C mice, 6~8 week old.
2, experimental technique
2.1 standard substance and diluted sample
With diluent by unit concentration such as reference substance and diluted sample one-tenth.By basic, normal, high 3 dosage groups (example: 12.5,25.0,50.0ng/ mices) subcutaneous injection mice (ratio of adjacent high low dose group is equal) respectively.Every dosage group 4 mices of injection.
2.2 process of the test
Normal detection is in (96h) eye socket blood sampling in the 5th day of injection, with EDTA-K2 anticoagulant tube anticoagulant.Take above-mentioned anticoagulation, count the reticulocyte counts ratio (Ret%) to Erythrocytes of every mice with full-automatic reticulocyte determination analyser.
2.3 calculate
By injection dosage (ng), the parallel line analysis method of Ret% value is calculated the titer (U/ml) of measuring samples.Computational methods refer to version Chinese Pharmacopoeia (three) annex Ⅹ IV in 2010.
2.4 points for attention
By statistics decision method, experimental result is judged.If it is determined that this experiment is false, should reform.
3, experimental result
It is diluted to wait unit concentration 125ng/ml by PEG-EPO standard substance PE20120101BZ with diluent.Make 5 groups, often 4 mices of group injection altogether, by middle dosage group (25.0ng/ mice) subcutaneous injection mice 0.2ml respectively.One group of eye socket blood sampling is taken respectively, with EDTA-K2 anticoagulant tube anticoagulant respectively at the 3rd day (48h), the 4th day (72h), the 5th day (96h) of injection, the 6th day (120h), the 7th day (144h).Taking above-mentioned anticoagulation, count the reticulocyte counts ratio (Ret%) to Erythrocytes of every mice with full-automatic reticulocyte determination analyser, concrete outcome is shown in Table 2, and 96h time Ret% reaches peak.
Table 2PEG-EPO respectively organizes Ret% result of the test
It is diluted to wait unit concentration 250ng/ml by EPO stock solution with diluent.Make 5 groups, often 4 mices of group injection altogether, by middle dosage group (50.0ng/ mice) subcutaneous injection mice 0.2ml respectively.1 group of eye socket blood sampling is taken respectively, with EDTA-K2 anticoagulant tube anticoagulant respectively at the 3rd day (48h), the 4th day (72h), the 5th day (96h) of injection, the 6th day (120h), the 7th day (144h).Taking above-mentioned anticoagulation, count the reticulocyte counts of every mice with full-automatic reticulocyte determination analyser as shown in table 3 to the ratio (Ret%) of Erythrocytes, 72h time Ret% reaches peak.
Table 3EPO respectively organizes different time points Ret% result of the test
The optimal blood sampling time that can draw PEG-EPO in table 2 is the 5th day (96h).Therefore, criticize PEG-EPO standard substance as reference substance with PE20120101BZ, carry out Activity determination with 20120101 batches of PEG-EPO samples for test sample, blood sampling in the 5th day.(note: PE20120101BZ criticizes PEG-EPO standard substance, 20120101 batches of PEG-EPO samples are the PEG-EPO of embodiment 3 purification).The unit concentration 125ng/ml such as with diluent, PEG-EPO reference substance and sample are all diluted to.Each 5 groups of work altogether, often 4 mices of group injection, by middle dosage group (25.0ng/ mice) subcutaneous injection mice 0.2ml respectively.Experimental implementation and result calculate and require consistent with version Chinese Pharmacopoeia (three) annex Ⅹ IV in 2010, according to little raticide generation and Ret% numerical value feature, select 2-8 week mice to inject, blood sampling in the 5th day.Surveyed test sample specific activity is 10105.8U/mg, more suitable than living with standard substance, is not less than 8.0 × 103U/mg。
The optimization of test example 1PEG modification reaction pH value
1, experimental technique
Take the EPO stock solution of 3 parts of assay approvals, respectively add K2HPO4To final concentration of 150mMK2HPO4, EPO concentration is 2~4mg/ml, adjusts buffer system pH value and is respectively pH6.0, pH7.5, pH9.0.Add mPEG-SBA in the ratio of EPO:PEG mol ratio 1:3 (mass ratio 1:5), dissolve, under room temperature (20-25 DEG C) environment, stir (80-130rpm) react 2 hours.After reaction terminates, adjusting pH4.0 to terminate reaction with acetic acid, sampling carries out SEC-HPLC detection, using the maximum pH value of single PEG-EPO component ratio as the optimum condition of PEG modification reaction.
2, experimental result
Under condition of different pH, SEC-HPLC testing result is shown in Table 4.
Table 4 condition of different pH SEC-HPLC testing result
Under the reaction condition of pH7.5 and pH9.0, reacting 2 hours after terminating, mono-modified PEG-EPO component ratio is essentially identical, and after pH6.0 reacts 2 hours, mono-modified PEG-EPO component ratio is the most on the low side.Therefore, it can select two kinds of reaction conditions of pH7.5 and pH9.0.Although under the reaction condition of pH9.0, mono-modified PEG-EPO component ratio is slightly higher, but pH9.0 is alkalescence condition, is unfavorable for protein stabilization, so selecting pH7.5 is optimal modification reaction pH value.
The ratio optimization of test example 2 modification reaction EPO Yu PEG
1, experimental technique
Taking the EPO stock solution of 3 parts of assay approvals, adjustment buffer system is 150mMK2HPO4, pH7.5, EPO concentration is 2~4mg/ml.Add mPEG-SBA in the ratio of EPO:PEG mol ratio 1:2,1:3,1:4, dissolve, under room temperature (20-25 DEG C) environment, stir (80-130rpm) react 2 hours.After reaction terminates, adjusting pH4.0 to terminate reaction with acetic acid, sampling carries out SEC-HPLC detection, is used for the optimum condition of PEG modification reaction with the raw material that single PEG-EPO component ratio is maximum.
2, experimental result
Under the usage ratio of different modifying reaction EPO:PEG, SEC-HPLC testing result is shown in Table 5.
Table 5 modification reaction different material is than SEC-HPLC testing result
Reacting 2 hours after terminating, the reaction condition of EPO:PEG mol ratio 1:4, many modification PEG-EPO component ratios are relatively big, and purpose proportion of products is on the low side, and is unfavorable for later-period purification.EPO:PEG mol ratio 1:2, the reaction condition of 1:3, purpose product mono-modified PEG-EPO component ratio is maximum, and no significant difference, therefore selecting EPO:PEG mol ratio 1:2~1:3 (mass ratio 1:3.5~1:5) condition is optimal modification reaction raw material ratio.
Test example 3 modification reaction epo protein concentration optimization
1, experimental technique
Taking the EPO stock solution of assay approval, suitably concentrate through 10kD ultra-filtration centrifuge tube, adjustment buffer system is 150mMK2HPO4, pH7.5, EPO concentration is 2.84mg/ml.Take the stock solution after a adjustment, use 150mMK2HPO4, pH7.5 buffer dilution one times is to 1.42mg/ml, and is divided into two parts;Separately take the stock solution after a adjustment, with 10kD ultra-filtration centrifuge tube concentration one times to 5.68mg/ml, be divided into two parts equally;Stock solution after residue adjusts is divided into two parts equally.Each group of sample is divided into identical two group again, adds mPEG-SBA, stirring and dissolving in the ratio of EPO:PEG mol ratio 1:2 and 1:3 (mass ratio 1:3.5 and 1:5) respectively.At room temperature continue to stir (80-130rpm) and react 2 hours.Reaction adjusts pH4.0 to terminate reaction with acetic acid after terminating respectively, and sampling carries out SEC-HPLC detection, using the maximum epo protein concentration of single PEG-EPO component ratio as the optimum condition of PEG modification reaction.
2, experimental result
Under different modifying reaction epo protein concentration, SEC-HPLC testing result is shown in Table 6.
Table 6 different modifying reaction epo protein concentration SEC-HPLC testing result
As seen from Table 6, under EPO concentration 5.68mg/ml, the reaction condition of mol ratio 1:2, purpose product mono-modified PEG-EPO component ratio is 49.35%.Under EPO concentration 2.84mg/ml, the reaction condition of mol ratio 1:2, purpose product mono-modified PEG-EPO component ratio is 47.89%.Both compare, and the former purpose product mono-modified PEG-EPO component ratio is slightly higher, but difference is inconspicuous, and the former many modification PEG-EPO component ratios are the most of a relatively high.The concentration produced in view of EPO stock solution is typically at about 3mg/ml, and the removal difficulty modifying PEG-EPO component relatively big more, and therefore the present invention does not use EPO concentration 5.68mg/ml, the reaction condition of mol ratio 1:2.
Consider, it is 2~4mg/ml in EPO concentration, under the reaction condition of EPO:PEG mol ratio 1:2~1:3 (mass ratio 1:3.5~1:5), purpose product mono-modified PEG-EPO component ratio is maximum, and therefore the present invention selects this condition to be optimal modification reaction condition.
The optimization of test example 4 commutation liquid composition
1, the pH impact on heat stability
The heat stability of PEG-EPO in various commutation formula of liquid, can pass through differential scanning calorimetry (DSC) and analyze.It is generally acknowledged that the thermal denaturation transition temperature that differential scanning calorimetry measures is the efficiency index of heat stability of protein.The raising of transition temperature shows that the heat stability of protein strengthens.
In order to determine Optimal pH, have studied PEG-EPO (embodiment 3 purification) thermal denaturation temperature in the range of pH4~9, at 30mMNa2HPO4, 30mM sodium citrate, the heat stability of analysing protein samples in 30mM borate.Result shows, maximum transition temperature is in the range of pH6~9, and pH drastically declines less than 5.5 transition temperatures.Therefore, Optimal pH should be higher than that 5.5.
2, the choosing of buffer agent
The buffer substance impact on albumen heat stability is have studied by differential scanning calorimetry (DSC).Result shows that the most suitable buffer agent of high thermal stability is sulfate, phosphate or citrate.
3, protein stability is affected by sulfate radical buffer agent
During pH6.2, in the case of the total mole number of salinity is respectively 50mM and 150mM, the phosphate of part changing the sodium sulfate of corresponding molal quantity into, albumen transition temperature is significantly raised, so, during pH6.2, sulfate is appropriate buffer.
During it addition, the total mole number of salinity is respectively 50mM and 150mM, albumen transition temperature difference is little, and the salt of low concentration is preferably 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 sulfate, 3% mannitol, pH6.2);Formulation C (10mM sodium phosphate, 100mM sodium chloride, pH7.0);Preparation D (10mM sodium phosphate, 120mM sodium sulfate, pH6.2);Preparation E (40mM arginine, 30mM sodium sulfate, 3% (w/v) mannitol, 1mMCaCl2, pH6.2).
By above-mentioned five kinds of component sample, respectively 4 DEG C, 25 DEG C, 30 DEG C, 40 DEG C of storages, after 6 months, sampling carries out reversed-phase high-performance liquid chromatography, exclusion chromatography and SDS-PAGE detection, calculates protein recovery, evaluates sample stability.
Result shows, under identical storage condition, preparation B protein recovery is significantly higher than other groups, illustrates that preparation B formula is relatively stable.Therefore, the present invention selects preparation B to be commutation liquid.