CN110554121A - Recombinant protein peptide map analysis method - Google Patents
Recombinant protein peptide map analysis method Download PDFInfo
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- CN110554121A CN110554121A CN201910990448.5A CN201910990448A CN110554121A CN 110554121 A CN110554121 A CN 110554121A CN 201910990448 A CN201910990448 A CN 201910990448A CN 110554121 A CN110554121 A CN 110554121A
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
Abstract
The invention discloses a recombinant protein peptide map analysis method which comprises the following steps of S1 sample preparation, including S101, taking a recombinant protein reference substance and a sample to be detected, dissolving or diluting with ultrapure water to enable the final concentration to be 1mg/mL, S102, weighing trypsin solution, dissolving 5mg of the trypsin with the ultrapure water to enable the concentration to be 1mg/mL, subpackaging the trypsin in an EP tube, storing the trypsin in a tube at the temperature of 50 mu L/mL for later use, weighing S103, 100mmol/L NH 4 HCO 3 solution, weighing NH 4 HCO 3 395.3mg, dissolving with ultrapure water and fixing the volume to 50mL, taking S104 and 10% TFA solution, putting 100 mu L of the TFA solution in the EP tube, adding 900 mu L of the ultrapure water, uniformly mixing the solution for later use, aiming at recombinant human erythropoietin fusion protein, establishing a stable and controllable peptide map analysis method for quality control of rhEPO-Fc through optimization of denaturation, disulfide bond breakage, chromatographic conditions and the like, and solving the problems that the key structural effect and the ideal fragment are sufficient, more, the peptide map is difficult to be obtained through enzymolysis and the accurate detection of the recombinant protein map.
Description
Technical Field
The invention belongs to the technical field of peptide map analysis, and particularly relates to a recombinant protein peptide map analysis method.
Background
Peptide Mapping (Peptide Mapping) is one of important means for controlling the quality of protein polypeptide drugs, and is a process of utilizing an enzymatic cracking method or a chemical cracking method to crack protein macromolecules into smaller fragments according to the characteristics of the molecular weight and the amino acid composition of protein, and forming a characteristic map by a certain separation and detection means. It has important significance for the structural research and characteristic identification of protein drugs and becomes one of the conventional indexes for controlling the quality standard of protein polypeptide drugs.
For the inspection of peptide graph of recombinant protein drug, the method adopted in the third part of the 'Chinese pharmacopoeia' of 2015 edition is generally Trypsin cracking-reversed phase high performance liquid chromatography, the peptide graph analysis of the item in early stage is directly carried out according to the method of pharmacopoeia, although the single experimental sample and the peptide graph of a reference substance have better consistency, the separation degree of partial peaks of the peptide graph is obviously insufficient, and the peak graphs obtained by different time experiments have certain difference.
Disclosure of Invention
The invention aims to: in order to solve the technical problems related to the background technology, a recombinant protein peptide map analysis method is provided.
The technical scheme adopted by the invention is as follows:
A method for analyzing a peptide map of a recombinant protein, comprising the following steps:
S1, sample preparation: the method comprises the following steps of S101, taking a recombinant protein reference substance and a sample to be detected, and dissolving or diluting the reference substance and the sample to be detected by using ultrapure water to ensure that the final concentration is 1 mg/ml; s102, trypsin solution: weighing trypsin 5mg, dissolving with ultrapure water to obtain 1mg/mL solutionSubpackaging in EP tube, 50 μ L/tube, storing at-20 deg.C for use; s103, 100mmol/L NH4HCO3Solution, weighing NH4HCO3395.3mg, dissolving the ultra-pure water and fixing the volume to 50 ml; s104, 10% TFA solution: adding 100 mu l of TFA solution into an EP tube, adding 900 mu l of ultrapure water, and uniformly mixing for later use;
S2, preparing a mobile phase group of the equilibrium chromatographic column;
The method comprises the following steps of S201, preparation of a mobile phase A: 100% ultrapure water (containing 0.1% TFA) solution: taking a clean measuring cylinder, measuring 1000ml of ultrapure water in a blue-cap bottle, adding 1.0ml of TFA, uniformly mixing, and carrying out ultrasonic treatment for 30min for later use.
s202, mobile phase B: 90% acetonitrile (containing 0.1% TFA) solution, taking a clean measuring cylinder, measuring acetonitrile 900ml, metering ultrapure water to 1000ml, adding TFA 1.0ml, mixing uniformly, and performing ultrasonic treatment for 30min for later use.
S203, mobile phase C: 100% acetonitrile, taking appropriate amount of acetonitrile (about 500ml), and performing ultrasonic treatment for 30min for later use;
S204, mobile phase D: taking an appropriate amount of ultrapure water (about 500ml) with 100% ultrapure water, and carrying out ultrasonic treatment for 30min for later use;
S3, sample treatment: a clean 1.5ml EP tube was taken and 100mM NH was added4HCO3200 mul of solution and 200 mul of sample to be measured are used as a standard control and a blank control at the same time, and after being uniformly mixed, the solution is heated for 30min in a boiling water bath at 100 ℃; after heating, taking out the mixture to a refrigerator at 4 ℃ for cooling for 5min, adding 4 mu l of trypsin solution according to the enzyme-substrate ratio of 1:50, uniformly mixing, and reacting for 1h at 37 ℃ in a constant-temperature water tank; then adding 20 mul 10% TFA solution to terminate the reaction, mixing evenly and then centrifuging, taking 150 mul supernatant liquid in a liquid phase sample injection bottle, and waiting for HPLC sample injection analysis;
S4, HPLC detection, including S401, HPLC equipment opening: pre-washing the chromatographic column, and then balancing the chromatographic column with an initial elution mobile phase A for 20min until the baseline is stable for later use;
S402, sample detection: firstly, balancing a chromatographic column for 80min according to the lower chromatographic condition, and then carrying out sample injection detection;
S5, result processing: overlapping and comparing the maps of the reference substance and the test substance, and storing the result;
And S6, judging results, wherein the blank control has no obvious absorption peak within 4-60 min, and the sample to be detected is qualified if the peptide map of the sample to be detected is consistent with that of the control product, otherwise, the sample to be detected is unqualified.
as a further technical scheme of the invention: s3, centrifuging in the sample treatment process, wherein a centrifuge is adopted, and the rotation speed is 10000rpm for 10 min.
As a further technical scheme of the invention: and in the step S401, starting an instrument according to Waters e2695 type high performance liquid chromatograph standard operating procedures, completing preparation of a solvent management system, and connecting the required chromatographic column.
As a further technical scheme of the invention: in the step S401, the chromatographic column is washed in advance, and is washed for 10min by 50% ultrapure water-50% ACN at the flow rate of 1 ml/min.
As a further technical scheme of the invention: the chromatographic conditions in S402 are: chromatographic column, Symmetry300TM C18(5 μm, 4.6 × 150 mm); column temperature, 30 +/-5 ℃; flow rate, 1 ml/min; detection wavelength, 214 nm; sample introduction volume, 80 μ l; run time, 76 min.
as a further technical scheme of the invention: the overlap condition in S5: 90 degree overlap with a pitch of 0.
As a further technical scheme of the invention: further comprising S7, detection post-processing: and after the sample detection is finished, washing the chromatographic column by using a lower chromatographic elution method, then washing four HPLC pipelines by using 100% ACN, and turning off the instrument and the power supply after ensuring that all the pipelines are washed clean.
As a further technical scheme of the invention: the chromatographic column is detached when the chromatographic column is not used within a week and is properly stored in a shady and cool place; the two-way connection is used for connecting the equipment pipeline.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. The invention aims at recombinant human erythropoietin (Fc) fusion protein, and establishes a stable and controllable peptide map analysis method for the quality control of rhEPO-Fc through optimization of denaturation, disulfide bond breakage, chromatographic conditions and the like.
2. The invention mainly solves the technical problems that complete enzymolysis is difficult to obtain an ideal cracking peak image due to the structural steric effect and more fragments, and improves the accuracy of peptide image inspection of recombinant protein drugs.
drawings
FIG. 1 is an enlarged peptide diagram of the HPLC analysis of recombinant human erythropoietin (Fc) fusion protein of the present invention.
Detailed Description
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
1. Instrument and reagent consumables
1.1 instrumentation
High performance liquid chromatography (e2695, Waters), check balance (BT2202S/BP221S, Sartorius), ultrapure water machine (Milli-Q Advantage a10, Millipore), electric-heating constant temperature water tank (DK-AAD, shanghai-constant technology), microcomputer induction cooker (LBC-20EH3, LUBY), centrifuge (5415D, eppendorf), and the like.
1.2 reagent consumables
TPCK-Trypsin protease (Sigma), chromatographic grade acetonitrile (Merck), chromatographic grade trifluoroacetic acid (Merck), chromatographic grade methanol (Sigma), Milli-Q ultrapure water, ammonium bicarbonate (Sigma), EP tube (AXYGEN), pipette (eppendorf), Symmetry300TM C18(5 μm, 4.6 × 150mm) chromatography column, etc.
2. solution preparation
2.1 sample: the rhEPO-Fc reference substance and the sample to be tested are taken and dissolved or diluted by ultrapure water so as to lead the final concentration to be 1 mg/ml.
2.2 Trypsin solution: weighing 5mg of trypsin, dissolving with ultrapure water to obtain a solution with a concentration of 1mg/mL, subpackaging in an EP tube, 50 mu L/tube, and storing at-20 ℃ for later use.
2.3 100mmol/L NH4HCO3Solution, weighing NH4HCO3395.3mg, ultra pure water was dissolved and made to volume of 50 ml.
2.410% TFA solution: mu.l of TFA solution was taken out and put into an EP tube, and 900. mu.l of ultrapure water was added thereto and mixed well for use.
2.5 mobile phase A: 100% ultrapure water (containing 0.1% TFA) solution: taking a clean measuring cylinder, measuring 1000ml of ultrapure water in a blue-cap bottle, adding 1.0ml of TFA, uniformly mixing, and carrying out ultrasonic treatment for 30min for later use.
2.6 mobile phase B: 90% acetonitrile (with 0.1% TFA): taking a clean measuring cylinder, measuring acetonitrile 900ml, metering ultrapure water to 1000ml, adding TFA 1.0ml, mixing uniformly, and carrying out ultrasonic treatment for 30min for later use.
2.7 mobile phase C: 100% acetonitrile, taking appropriate amount of acetonitrile (about 500ml), and performing ultrasonic treatment for 30min for later use.
2.8 mobile phase D: taking an appropriate amount of ultrapure water (about 500ml) with 100% ultrapure water, and carrying out ultrasonic treatment for 30min for later use.
3. Experimental procedure
3.1 sample treatment: a clean 1.5ml EP tube was taken and 100mM NH was added4HCO3200 mul of solution and 200 mul of sample to be measured are used as a standard control and a blank control at the same time, and after being uniformly mixed, the solution is heated for 30min in a boiling water bath at 100 ℃; after heating, taking out the mixture to a refrigerator at 4 ℃ for cooling for 5min, adding 4 mu l of trypsin solution according to the enzyme-substrate ratio of 1:50, uniformly mixing, and reacting for 1h at 37 ℃ in a constant-temperature water tank; then 20. mu.l of 10% TFA solution was added to terminate the reaction, after mixing well, the mixture was centrifuged at 10000rpm in a centrifuge for 10min, and the supernatant (about 150. mu.l) was taken in a liquid phase sample bottle for HPLC analysis.
3.2HPLC detection
3.2.1HPLC equipment on: the instrument was started and the solvent management system preparation was completed as per Waters e2695 model HPLC Standard operating procedures, the desired column was attached, the column was rinsed with 50% ultrapure water-50% ACN for about 10min (1ml/min), and then equilibrated to baseline plateau (about 20min) with the initial elution mobile phase (mobile phase A) for a further time.
3.2.2 sample detection: the column was equilibrated (80min) first according to the following chromatographic conditions and then assayed by injection. Chromatographic stripA piece: chromatographic column, Symmetry300TM(5 μm, 4.6 × 150 mm); column temperature, 30 +/-5 ℃; flow rate, 1 ml/min; detection wavelength, 214 nm; sample introduction volume, 80 μ l; run time, 76 min; mobile phase gradient elution is shown in table 1 below.
TABLE 1 gradient elution table for mobile phase of recombinant human erythropoietin (Fc) fusion protein
4. Result processing
and (4) overlapping and comparing the maps of the reference substance and the test substance (overlapping condition: 90 degrees overlapping, interval 0), and storing the result.
5. Determination of results
the blank control has no obvious absorption peak within 4-60 min, the recombinant human erythropoietin (Fc) fusion protein reference substance peptide map is shown as the following reference picture (shown in figure 1), the test result is true, the sample to be tested is consistent with the reference substance peptide map, the sample to be tested is qualified, otherwise, the sample is not qualified.
6. Note that:
after the sample detection is finished, the chromatographic column is washed by a lower chromatographic elution method (see table 2) (if the chromatographic column is not used within one week, the chromatographic column is detached and properly stored in a shady place; a two-way connection device pipeline is used), then the HPLC four pipelines are washed by 100% ACN, and after the pipelines are completely washed, the instrument and the power supply are turned off.
TABLE 2 elution table for chromatographic column cleaning mobile phase
the above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. a method for analyzing a peptide map of a recombinant protein, comprising: the method comprises the following steps:
S1, sample preparation:
S101, taking an SUBS-4 reference substance and a sample to be detected, and dissolving or diluting the SUBS-4 reference substance and the sample to be detected by using ultrapure water to ensure that the final concentration is 1 mg/ml; s102, trypsin solution: weighing 5mg of trypsin, dissolving with ultrapure water to obtain a solution with the concentration of 1mg/mL, subpackaging the solution in an EP tube and a 50 mu L/tube, and storing at-20 ℃ for later use; s103, 100mmol/L NH4HCO3Solution, weighing NH4HCO3395.3mg, dissolving the ultra-pure water and fixing the volume to 50 ml; s104, 10% TFA solution: adding 100 mu l of TFA solution into an EP tube, adding 900 mu l of ultrapure water, and uniformly mixing for later use;
S2, preparing a mobile phase group of the equilibrium chromatographic column;
The method comprises the following steps of S201, preparation of a mobile phase A: 100% ultrapure aqueous solution with 0.1% TFA: taking a clean measuring cylinder, measuring 1000ml of ultrapure water in a blue-cap bottle, adding 1.0ml of TFA, uniformly mixing, and carrying out ultrasonic treatment for 30min for later use;
S202, mobile phase B: taking a clean measuring cylinder, measuring acetonitrile 900ml, metering ultrapure water to 1000ml, adding TFA 1.0ml, uniformly mixing, and carrying out ultrasonic treatment for 30min for later use.
S203, mobile phase C: 100% acetonitrile, taking a proper amount of acetonitrile 500ml, and carrying out ultrasonic treatment for 30min for later use;
S204, mobile phase D: taking a proper amount of ultrapure water 500ml for 100 percent, and carrying out ultrasonic treatment for 30min for later use;
S3, sample treatment: a clean 1.5ml EP tube was added with 100mM, NH4HCO3200 mul of solution and 200 mul of sample to be measured are used as a standard control and a blank control at the same time, and after being uniformly mixed, the solution is heated for 30min in a boiling water bath at 100 ℃; after heating, taking out the mixture to a refrigerator at 4 ℃ for cooling for 5min, adding 4 mu l of trypsin solution according to the enzyme-substrate ratio of 1:50, uniformly mixing, and reacting for 1h at 37 ℃ in a constant-temperature water tank; then adding 20 mul 10% TFA solution to terminate the reaction, mixing uniformly and then centrifuging, taking 150 mul supernatant liquid in a liquid phase sample injection bottle, and waiting for HPLC sample injection analysis;
S4, HPLC detection, including S401, HPLC equipment opening: pre-washing the chromatographic column, and then balancing the chromatographic column with an initial elution mobile phase A for 20min until the baseline is stable for later use;
S402, sample detection: firstly, balancing a chromatographic column for 80min according to the lower chromatographic condition, and then carrying out sample injection detection;
S5, result processing: overlapping and comparing the blank reference, the reference and the test sample maps, and storing the result;
S6, judging the result: and (4) the blank control has no obvious absorption peak within 4-60 min, the to-be-detected sample is consistent with the peptide map of the control, the to-be-detected sample is qualified, and otherwise, the to-be-detected sample is unqualified.
2. the method of analyzing a peptide map of a recombinant protein according to claim 1, wherein: s3, centrifugation in sample treatment: and (4) centrifuging for 10min at 10000rpm by adopting a centrifuge.
3. The method of analyzing a peptide map of a recombinant protein according to claim 1, wherein: and in the step S401, starting an instrument according to Waters e2695 type high performance liquid chromatograph standard operating procedures, completing preparation of a solvent management system, and connecting the required chromatographic column.
4. The method of analyzing a peptide map of a recombinant protein according to claim 1, wherein: in the step S401, the chromatographic column is washed in advance, and is washed for 10min by 50% ultrapure water-50% ACN at the flow rate of 1 ml/min.
5. The method of analyzing a peptide map of a recombinant protein according to claim 1, wherein: the chromatographic conditions in S402 are: chromatographic column, Symmetry300TM，5 μm, 4.6X 150 mm; column temperature, 30 +/-5 ℃; flow rate, 1 ml/min; detection wavelength, 214 nm; sample introduction volume, 80 μ l; run time, 76 min.
6. The method of analyzing a peptide map of a recombinant protein according to claim 1, wherein: the overlap condition in S5: 90 degree overlap with a pitch of 0.
7. The method according to any one of claims 1 to 6, wherein: further comprising S7, detection post-processing: and after the sample detection is finished, washing the chromatographic column by using a lower chromatographic elution method, then washing four HPLC pipelines by using 100% ACN, and turning off the instrument and the power supply after ensuring that all the pipelines are washed clean.
8. The method of claim 7, wherein the peptide mapping comprises: the chromatographic column is detached when the chromatographic column is not used within a week and is properly stored in a shady and cool place; the two-way connection is used for connecting the equipment pipeline.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111220723A (en) * | 2019-12-30 | 2020-06-02 | 北京博康健基因科技有限公司 | Method for detecting peptide pattern of teriparatide |
| CN114965839A (en) * | 2022-05-11 | 2022-08-30 | 朗肽生物制药股份有限公司 | Peptide map analysis method of human basic fibroblast growth factor |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7326837B2 (en) * | 2005-01-07 | 2008-02-05 | Academia Sinica | Clinical applications of crystalline diamond particles |
| WO2009110873A2 (en) * | 2007-12-05 | 2009-09-11 | The Gov. Of The Usa, As Represented By The Secretary Dept. Of Health & Human Services | Improved viral protein quantification and vaccine quality control therewith |
| CN108267534A (en) * | 2016-12-31 | 2018-07-10 | 江苏众红生物工程创药研究院有限公司 | The Peptides figure analysis method of polyethylene glycol modified protein drug |
| CN109725094A (en) * | 2019-01-17 | 2019-05-07 | 中国食品药品检定研究院 | Double enzymatic treatment methods of glycoprotein |
-
2019
- 2019-10-17 CN CN201910990448.5A patent/CN110554121A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7326837B2 (en) * | 2005-01-07 | 2008-02-05 | Academia Sinica | Clinical applications of crystalline diamond particles |
| WO2009110873A2 (en) * | 2007-12-05 | 2009-09-11 | The Gov. Of The Usa, As Represented By The Secretary Dept. Of Health & Human Services | Improved viral protein quantification and vaccine quality control therewith |
| CN108267534A (en) * | 2016-12-31 | 2018-07-10 | 江苏众红生物工程创药研究院有限公司 | The Peptides figure analysis method of polyethylene glycol modified protein drug |
| CN109725094A (en) * | 2019-01-17 | 2019-05-07 | 中国食品药品检定研究院 | Double enzymatic treatment methods of glycoprotein |
Non-Patent Citations (3)
| Title |
|---|
| ANA PAULA DE ARAUJO: "Desenvolvimento de Metodologia para Mapeamento Peptídico da Eritropoetina Humana Recombinante visando o Controle em Processo da Produção em Bio-Manguinhos", 《HTTPS://WWW.ARCA.FIOCRUZ.BR/BITSTREAM/ICICT/5902/2/ANA-PAULA-ARAUJO.PDF》 * |
| 李响 等: "重组人促红素理化对照品的质量评价", 《药物分析杂志》 * |
| 王敏力 等: "重组人促红细胞生成素的HPLC与UPLC肽图谱分析", 《中国预防医学杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111220723A (en) * | 2019-12-30 | 2020-06-02 | 北京博康健基因科技有限公司 | Method for detecting peptide pattern of teriparatide |
| CN114965839A (en) * | 2022-05-11 | 2022-08-30 | 朗肽生物制药股份有限公司 | Peptide map analysis method of human basic fibroblast growth factor |
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Application publication date: 20191210 |