CN115032317B - Method for detecting recombinant human erythropoietin - Google Patents
Method for detecting recombinant human erythropoietin Download PDFInfo
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- 101000987586 Homo sapiens Eosinophil peroxidase Proteins 0.000 title claims abstract description 93
- 101000920686 Homo sapiens Erythropoietin Proteins 0.000 title claims abstract description 93
- 102000044890 human EPO Human genes 0.000 title claims abstract description 93
- 238000000034 method Methods 0.000 title claims description 26
- 238000001514 detection method Methods 0.000 claims abstract description 75
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- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims abstract description 6
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- ADCGAPKUMAQOLJ-UHFFFAOYSA-N azane;formic acid Chemical compound N.OC=O.OC=O ADCGAPKUMAQOLJ-UHFFFAOYSA-N 0.000 description 3
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- ALSPKRWQCLSJLV-UHFFFAOYSA-N azanium;acetic acid;acetate Chemical compound [NH4+].CC(O)=O.CC([O-])=O ALSPKRWQCLSJLV-UHFFFAOYSA-N 0.000 description 2
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- 108010092408 Eosinophil Peroxidase Proteins 0.000 description 1
- 102100031939 Erythropoietin Human genes 0.000 description 1
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- 101150064138 MAP1 gene Proteins 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- OZZOVSQSDIWNIP-UHFFFAOYSA-N acetic acid;azane Chemical compound [NH4+].[NH4+].CC([O-])=O.CC([O-])=O OZZOVSQSDIWNIP-UHFFFAOYSA-N 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
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- 235000019257 ammonium acetate Nutrition 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
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- BSXZJWHTFAEYDT-UHFFFAOYSA-N diazanium diformate Chemical compound [NH4+].[NH4+].[O-]C=O.[O-]C=O BSXZJWHTFAEYDT-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
-
- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
- G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate
Abstract
The invention provides a detection method of recombinant human erythropoietin, which comprises the following steps: obtaining a control of recombinant human erythropoietin; carrying out chromatography on a cell culture supernatant containing recombinant human erythropoietin to prepare a sample to be detected; respectively carrying out anion high performance liquid chromatography detection on the reference substance and the sample to be detected; wherein, the conditions for anion high performance liquid chromatography detection include: the mobile phase A is an ammonium formate solution with the concentration of 20mmol/L to 40mmol/L, the mobile phase B is a formic acid solution with the concentration of 20mmol/L to 40mmol/L, the mobile phase C is a mixed solution of tris (hydroxymethyl) aminomethane and sodium chloride, the concentration of tris (hydroxymethyl) aminomethane in the mobile phase C is 5mmol/L to 15mmol/L, and the concentration of sodium chloride is 0.8mol/L to 1.2mol/L; gradient elution is used.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a detection method of recombinant human erythropoietin.
Background
Erythropoietin in humans is a hormone-like substance secreted by the perirenal cortical tubular interstitial cells and the liver, which promotes erythropoiesis. Endogenous erythropoietin is a polypeptide consisting of 165 amino acids and containing 3N-glycosylation sites and 1O-glycosylation site, and has a molecular weight of about 30kDa, and sugar chains accounting for about 40% of the total molecular weight, and have a large influence on the biological activity in vivo. An important factor in the glycosylation of erythropoietin is the sialic acid content, and galactose with exposed sugar chain ends, which are not subjected to sialic acid modification, is recognized by hepatic galactose receptors and is thus rapidly cleared. Further studies have shown that sialic acid, if modified by O-acetylation, is also enhanced in protection. More specifically, large amounts of sialic acid are associated with increased biological activity and half-life in EPO. The commercial erythropoietin is recombinant human erythropoietin (rhEPO), which has the amino acid sequences identical to those of endogenous erythropoietin with only minor differences in the glycosyl moiety. Recombinant human erythropoietin contains more tri-and tetra-antenna oligosaccharides than endogenous erythropoietin and has a greater difference in the degree of sialylation. The difference in sialylation results in the presence of charge heterogeneity in recombinant human erythropoietin, which forms different charge isoforms.
Conventional methods for detecting charge isomers include isoelectric focusing electrophoresis (IEF), capillary isoelectric focusing electrophoresis (cif), ion exchange Chromatography (CEX), and the like. The standard method described in the three chinese pharmacopoeia 2020 edition relies on the separation of the charge isomers of recombinant human erythropoietin by isoelectric focusing electrophoresis. FIG. 1 shows an isoelectric focusing electrophoresis pattern of a recombinant human erythropoietin standard having mainly 4-7 charge isomers of recombinant human erythropoietin, 4 bands in total, and 3 bands of the isomer being relatively insignificant. Isoelectric focusing electrophoresis can rapidly detect isoelectric points of proteins, has wide popularity, but is a dispersive strip on an IEF map, and each charge isomer cannot be accurately analyzed, so that the isoelectric focusing electrophoresis method is mainly used for qualitative analysis, and the accurate quantification of the recombinant human erythropoietin charge isomer cannot be performed.
Compared with isoelectric focusing electrophoresis, capillary isoelectric focusing can separate the charge isomer of each recombinant human erythropoietin more accurately, and like the principle of isoelectric focusing electrophoresis, the capillary isoelectric focusing electrophoresis separates various variants according to the difference of the surface charge distribution of proteins, but the capillary isoelectric focusing electrophoresis is time-consuming, difficult to standardize, and also needs to be equipped with special electrophoresis equipment, and has higher cost.
Disclosure of Invention
Based on the above, the invention provides a method for detecting recombinant human erythropoietin, which can rapidly complete the detection of the content of the acidic isomer of the recombinant human erythropoietin, and has higher accuracy and lower requirement on the purity of a sample.
The invention is realized by the following technical scheme.
A method for detecting recombinant human erythropoietin, comprising the steps of:
obtaining a control of recombinant human erythropoietin;
carrying out chromatography on a cell culture supernatant containing recombinant human erythropoietin to prepare a sample to be detected;
respectively carrying out anion high performance liquid chromatography detection on the reference substance and the sample to be detected;
wherein, the conditions for anion high performance liquid chromatography detection include: the mobile phase A is an ammonium formate solution with the concentration of 20mmol/L to 40mmol/L, the mobile phase B is a formic acid solution with the concentration of 20mmol/L to 40mmol/L, the mobile phase C is a mixed solution of tris (hydroxymethyl) aminomethane and sodium chloride, the concentration of tris (hydroxymethyl) aminomethane in the mobile phase C is 5mmol/L to 15mmol/L, and the concentration of sodium chloride is 0.8mol/L to 1.2mol/L; gradient elution is adopted; the gradient elution procedure included: 0-10 min, wherein the volume percentage of the mobile phase A is 100%; 10-60 min, wherein the volume percentage of the mobile phase A is changed from 100% to 0%, and the volume percentage of the mobile phase B is changed from 0 to 100%; 60-70 min, wherein the volume percentage of the mobile phase B is kept to be 100%; 70-71 min, wherein the volume percentage of the mobile phase B is changed from 100% to 0%, and the volume percentage of the mobile phase C is changed from 0% to 100%;71 min-85 min, the volume percentage of the mobile phase C is kept to be 100%; 85-86 min, wherein the volume percentage of the mobile phase A is changed from 0% to 100%, and the volume percentage of the mobile phase C is changed from 100% to 0%;86 min-100 min, the volume percentage of the mobile phase A is kept to be 100%.
In one embodiment, the conditions for anion high performance liquid chromatography detection further comprise: the flow rate was 1 mL/min.+ -. 0.1mL/min.
In one embodiment, the conditions for anion high performance liquid chromatography detection further comprise: the detection wavelength is 280nm + -5 nm.
In one embodiment, the conditions for anion high performance liquid chromatography detection further comprise: the column temperature was 30 ℃ + -2 ℃.
In one embodiment, the conditions for anion high performance liquid chromatography detection further comprise: the chromatographic column is a strong anion exchange column.
In one embodiment, subjecting the cell culture supernatant comprising recombinant human erythropoietin to chromatography comprises the steps of:
balancing a first chromatographic column by adopting a first balancing solution, mixing the cell culture supernatant containing the recombinant human erythropoietin with the first balancing solution, loading the sample, eluting by using a mixed solution of a first eluent and the first balancing solution, and collecting target proteins; wherein the first chromatographic column is an anion exchange chromatographic column; the first equilibrium liquid is 5-15 mmol/L of tris (hydroxymethyl) aminomethane; the first eluent is a mixed solution of tris (hydroxymethyl) aminomethane and sodium chloride, wherein the concentration of tris (hydroxymethyl) aminomethane in the first eluent is 5-15 mmol/L, and the concentration of sodium chloride in the first eluent is 450-550 mmol/L; or (b)
Balancing a second chromatographic column by adopting a second balancing solution, loading the cell culture supernatant containing the recombinant human erythropoietin, washing the column by using the second plate Heng Ye, eluting by using a mixed solution of a second eluent and the second balancing solution, and collecting target proteins; wherein the second chromatographic column is a reversed phase chromatographic column; the second equilibrium liquid is 5 mmol/L-15 mmol/L of tris (hydroxymethyl) aminomethane; the second eluent is a mixed solution of the tris and acetonitrile or ethanol, wherein the concentration of the tris in the second eluent is 5-15 mmol/L, and the volume fraction of the acetonitrile or ethanol is 75-85%; or (b)
Balancing a third chromatographic column by using a third balancing solution, loading the cell culture supernatant containing the recombinant human erythropoietin, washing the column by using the third balancing solution, eluting by using a third eluent, and collecting target proteins; wherein the third chromatographic column is a blue agarose gel chromatographic column; the third equilibrium liquid is a mixed solution of the tris and the sodium chloride, wherein the concentration of the tris in the third equilibrium liquid is 15-25 mmol/L, and the concentration of the sodium chloride is 0.13-0.18 mol/L; the third eluent is a mixed solution of the tris and the sodium chloride, wherein the concentration of the tris in the third eluent is 15-25 mmol/L, and the concentration of the sodium chloride is 1-1.4 mol/L.
In one embodiment, chromatography is performed at a flow rate of 2mL/min to 4mL/min.
In one embodiment, the method further comprises the following steps:
establishing a standard curve between the total peak area mean value of the acid isomer of the recombinant human erythropoietin control and the concentration of the recombinant human erythropoietin control.
In one embodiment, the total peak area mean of the acid isoforms of the recombinant human erythropoietin control is y, the concentration of the recombinant human erythropoietin control is x, and the function equation of the standard curve is y= 1763787.6x-77217.1.
In one embodiment, the preparation of the recombinant human erythropoietin-containing cell culture supernatant comprises the steps of:
culturing Chinese Hamster Ovary (CHO) cells which efficiently express human erythropoietin, and preparing a fermentation harvest liquid;
centrifuging the fermentation harvest liquid, filtering and taking supernatant.
Compared with the prior art, the detection method of the recombinant human erythropoietin has the following beneficial effects:
the invention adopts anion high performance liquid chromatography to screen out proper mobile phase and limits gradient elution program, and finally successfully establishes a detection method for chromatographic capturing recombinant human erythropoietin sample. The detection method disclosed by the invention requires few sample processing steps, can be used for rapidly completing the detection of the content of the acid isomer of the recombinant human erythropoietin in the cell culture supernatant, has higher accuracy, and can also more truly reflect the quality of the recombinant human erythropoietin in the cell culture supernatant. Meanwhile, the detection method has lower requirement on the purity of the sample, and the purity of the sample is 40-70%, so that the sample processing time is further saved, and the cost is reduced.
Drawings
FIG. 1 is an isoelectric focusing electrophoretogram of a recombinant human erythropoietin standard;
FIG. 2 is a chart of a blank solution sample injection of 50ul anion detection;
FIG. 3 shows an anion detection pattern-1 of 50 μl sample injection of a 0.6mg/ml control provided in the example of the present invention;
FIG. 4 shows an anion detection pattern-2 of 50 μl sample injection of a 0.6mg/ml control provided in the example of the present invention;
FIG. 5 shows an anion detection pattern-1 of 50 μl sample injection of a 0.9mg/ml control provided in the example of the present invention;
FIG. 6 is a graph of anion detection profile-2 of 50. Mu.l sample injection of 0.9mg/ml control provided in the example of the present invention;
FIG. 7 shows an anion detection pattern-1 of 50 μl sample injection of 1.2mg/ml control provided in the example of the present invention;
FIG. 8 is a graph of anion detection profile-2 of 50. Mu.l sample injection of 1.2mg/ml control provided in the example of the present invention;
FIG. 9 is a sample injection of 50. Mu.l of anion detection map-1 for a 1.5mg/ml control provided in the example of the present invention;
FIG. 10 is a graph of anion detection profile-2 of 50. Mu.l sample injection of 1.5mg/ml control provided in the example of the present invention;
FIG. 11 is a graph of an anion detection pattern-1 of 50. Mu.l sample injection of a 1.8mg/ml control provided in an embodiment of the present invention;
FIG. 12 is a graph of anion detection profile-2 of 50. Mu.l sample injection of 1.8mg/ml control provided in the example of the present invention;
FIG. 13 is a chromatography map of QHP one-step capture recombinant human erythropoietin provided by an embodiment of the present invention;
FIG. 14 is a graph of the detection of anions of a sample of QHP-captured recombinant human erythropoietin provided by an embodiment of the present invention;
FIG. 15 is a one-step capture chromatogram of recombinant human erythropoietin on a Resource RPC 3ml chromatographic column provided by an embodiment of the invention;
FIG. 16 is a one-step detection spectrum of a recombinant RPC 3ml chromatographic column for capturing a recombinant human erythropoietin sample provided by an embodiment of the present invention;
FIG. 17 is a chromatography chart of one-step capture of recombinant human erythropoietin by blue gel provided by an embodiment of the invention;
FIG. 18 is a graph of anion detection of a sample of recombinant human erythropoietin captured in one step with blue gel provided by an embodiment of the present invention;
FIG. 19 is an anion detection profile of a recombinant human erythropoietin standard provided in comparative example 1 of the present invention using an ammonium acetate-acetate buffer system;
FIG. 20 is an anion detection profile of recombinant human erythropoietin standard provided in comparative example 1 using an ammonium formate-formate buffer system;
FIG. 21 is a graph of the anion detection profile of a linear elution volume of 10CV for a recombinant human erythropoietin standard provided in comparative example 1 of the present invention;
FIG. 22 is a graph of the anion detection profile of a linear elution volume of 20CV for a recombinant human erythropoietin standard provided in comparative example 1 of the present invention;
FIG. 23 is a graph of the anion detection profile of a linear elution volume of 30CV for a recombinant human erythropoietin standard provided in comparative example 1 of the present invention.
Detailed Description
In order that the invention may be readily understood, a more complete description of the invention will be rendered by reference to the appended drawings. The drawings illustrate preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include at least one such feature. In the description of the invention, the meaning of "plurality" means at least two, for example, two, three, etc., unless specifically defined otherwise. In the description of the present invention, the meaning of "several" means at least one, such as one, two, etc., unless specifically defined otherwise.
The words "preferably," "more preferably," and the like in the present invention refer to embodiments of the invention that may provide certain benefits in some instances. However, other embodiments may be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.
When a range of values is disclosed herein, the range is considered to be continuous and includes both the minimum and maximum values for the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values of the range is included. Further, when multiple range description features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, all ranges disclosed herein are to be understood to include any and all subranges subsumed therein.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The invention provides a detection method of recombinant human erythropoietin, which comprises the following steps:
obtaining a control of recombinant human erythropoietin;
carrying out chromatography on a cell culture supernatant containing recombinant human erythropoietin to prepare a sample to be detected;
respectively carrying out anion high performance liquid chromatography detection on the reference substance and the sample to be detected;
wherein, the conditions for anion high performance liquid chromatography detection include: the mobile phase A is an ammonium formate solution with the concentration of 20mmol/L to 40mmol/L, the mobile phase B is a formic acid solution with the concentration of 20mmol/L to 40mmol/L, the mobile phase C is a mixed solution of tris (hydroxymethyl) aminomethane and sodium chloride, the concentration of tris (hydroxymethyl) aminomethane in the mobile phase C is 5mmol/L to 15mmol/L, and the concentration of sodium chloride is 0.8mol/L to 1.2mol/L; gradient elution is adopted; the gradient elution procedure included: 0-10 min, wherein the volume percentage of the mobile phase A is 100%; 10-60 min, wherein the volume percentage of the mobile phase A is changed from 100% to 0%, and the volume percentage of the mobile phase B is changed from 0 to 100%; 60-70 min, wherein the volume percentage of the mobile phase B is kept to be 100%; 70-71 min, wherein the volume percentage of the mobile phase B is changed from 100% to 0%, and the volume percentage of the mobile phase C is changed from 0% to 100%;71 min-85 min, the volume percentage of the mobile phase C is kept to be 100%; 85-86 min, wherein the volume percentage of the mobile phase A is changed from 0% to 100%, and the volume percentage of the mobile phase C is changed from 100% to 0%;86 min-100 min, the volume percentage of the mobile phase A is kept to be 100%.
In a specific example, the invention provides a method for detecting the acid isomer content of recombinant human erythropoietin.
More specifically, mobile phase A was a 30mmol/L ammonium formate solution at a pH of 5.5.+ -. 0.1.
More specifically, mobile phase B was a 30mmol/L formic acid solution at a pH of 2.6.+ -. 0.1.
More specifically, the mobile phase C is a mixed solution of 10mmol/L of tris and 1mol/L of sodium chloride, and the pH value is 7.0 + -0.2.
In a specific example, the conditions for anion high performance liquid chromatography detection further include: the flow rate was 1 mL/min.+ -. 0.1mL/min.
In a specific example, the conditions for anion high performance liquid chromatography detection further include: the sample injection volume is 5 mu L-100 mu L.
In a specific example, the conditions for anion high performance liquid chromatography detection further include: the detection wavelength is 280nm + -5 nm.
In a specific example, the conditions for anion high performance liquid chromatography detection further include: the column temperature was 30 ℃ + -2 ℃.
In a specific example, the conditions for anion high performance liquid chromatography detection further include: the chromatographic column is a strong anion exchange column.
More specifically, the chromatographic column used for anion high performance liquid chromatography detection is a nano-spectrum BioCore SAX column, and the specification comprises: the column length was 150mm, the inner diameter was 4.6mm, and the particle diameter was 10. Mu.m.
In a specific example, subjecting the cell culture supernatant comprising recombinant human erythropoietin to chromatography comprises the steps of:
balancing the first chromatographic column by adopting a first balancing solution, mixing a cell culture supernatant containing recombinant human erythropoietin with the first balancing solution, loading the sample, eluting by using a mixed solution of a first eluent and the first balancing solution, and collecting target proteins; wherein the first chromatographic column is an anion exchange chromatographic column; the first equilibrium liquid is 5 mmol/L-15 mmol/L of tris (hydroxymethyl) aminomethane; the first eluent is a mixed solution of the tris and the sodium chloride, wherein the concentration of the tris in the first eluent is 5-15 mmol/L, and the concentration of the sodium chloride is 450-550 mmol/L.
Specifically, the anion exchange chromatography column is selected from Q Sepharose High Performance, qff, DEAE, source 15Q or Source 30Q. Preferably, the anion exchange chromatography column is a Q Sepharose High Performance column. More specifically, the packed column of Q Sepharose High Performance column had a packed Column Volume (CV) of 13ml, a bed height of 16.5cm and a column inner diameter of 1cm.
More specifically, the first equilibrium liquid was 10mmol/L of tris (hydroxymethyl) aminomethane, and the pH was 7.
More specifically, in the first eluent, the concentration of tris was 10mmol/L, the concentration of sodium chloride was 500mmol/L, and the pH of the first eluent was 7.
More specifically, the chromatography of the cell culture supernatant containing recombinant human erythropoietin comprises the steps of:
filtering cell culture supernatant containing recombinant human erythropoietin with 0.22 μm filter membrane, adding 3 times volume of first balance liquid for dilution, balancing 3CV with first balance liquid by a first chromatographic column, loading 180ml (the actual sample volume of cell culture supernatant is 45 ml), eluting 3CV with 90% volume of first balance liquid and 10% volume of first eluent at equal degree, eluting 4CV with 74% volume of first balance liquid and 26% volume of first eluent at equal degree, and collecting eluted protein as sample to be detected.
In a specific example, a second chromatographic column is equilibrated with a second equilibration solution, a cell culture supernatant containing recombinant human erythropoietin is loaded, then the column is washed with a second plate Heng Ye, and then the target protein is collected by eluting with a mixture of a second elution solution and the second equilibration solution; wherein the second chromatographic column is a reversed phase chromatographic column; the second equilibrium liquid is 5 mmol/L-15 mmol/L of tris (hydroxymethyl) aminomethane; the second eluent is a mixed solution of the tris and acetonitrile or ethanol, wherein the concentration of the tris is 5-15 mmol/L, and the volume fraction of the acetonitrile or ethanol is 75-85%.
Preferably, the volume fraction of acetonitrile is 80%.
In particular, the reverse phase chromatography column is selected from a Resource RPC, C4, C8 or C18 column. More preferably, the reverse phase chromatography column is a Resource RPC 3ml polymer matrix reverse phase chromatography column. More specifically, resource RPC 3ml polymer matrix reverse phase chromatography column size was 0.6X10 cm, bed volume (CV) 3ml.
More specifically, the second equilibrium liquid was 10mmol/L of tris (hydroxymethyl) aminomethane, and the pH was 7.
More specifically, in the second eluent, the concentration of tris was 10mmol/L, the volume fraction of acetonitrile was 80%, and the pH of the second eluent was 7.
More specifically, the chromatography of the cell culture supernatant containing recombinant human erythropoietin comprises the steps of:
filtering cell culture supernatant containing recombinant human erythropoietin with 0.22 μm filter membrane, balancing the second chromatographic column with a second balancing solution for 10CV, loading 50mL, flowing at 2mL/min, washing with the second balancing solution for 10CV, isocratically eluting with 95% of the second balancing solution and 5% of the second eluent for 10CV, linearly eluting with 95% -0% of the second balancing solution and 5% -100% of the second eluent (the volume ratio of the two solutions changes in a straight line) for 19CV, and collecting the eluted protein as a sample to be detected.
In a specific example, a third equilibrium liquid is adopted to balance a third chromatographic column, a cell culture supernatant containing recombinant human erythropoietin is sampled, then the column is washed by the third equilibrium liquid, and then the target protein is collected by eluting by a third eluent; wherein the third chromatographic column is a blue agarose gel chromatographic column; the third equilibrium liquid is a mixed solution of the tris (hydroxymethyl) aminomethane and sodium chloride, wherein the concentration of the tris (hydroxymethyl) aminomethane in the third equilibrium liquid is 15-25 mmol/L, and the concentration of the sodium chloride in the third equilibrium liquid is 0.13-0.18 mol/L; the third eluent is a mixed solution of the tris and the sodium chloride, wherein the concentration of the tris in the third eluent is 15 mmol/L-25 mmol/L, and the concentration of the sodium chloride is 1 mol/L-1.4 mol/L.
Specifically, the Blue Sepharose chromatography column is selected from Blue Sepharose 6FF, capto Blue or Capto Blue (HS). More preferably, the Blue Sepharose column is a Blue Sepharose 6FF Blue column. More specifically, the Blue Sepharose 6FF Blue gel column had a specification of 1.6X4 cm and a bed volume (CV) of 8ml.
More specifically, in the third equilibrium liquid, the concentration of tris was 20mmol/L, the concentration of sodium chloride was 0.15mol/L, and the pH of the third equilibrium liquid was 7.
More specifically, in the third eluent, the concentration of tris was 20mmol/L, the concentration of sodium chloride was 1.2mol/L, and the pH of the third eluent was 7.
More specifically, the chromatography of the cell culture supernatant containing recombinant human erythropoietin comprises the steps of:
the cell culture supernatant containing recombinant human erythropoietin was first filtered through a 0.22 μm filter, and the third chromatography column equilibrated with a third equilibration solution at 5CV, 80mL, and flow rate at 2mL/min. Then washing 10CV with a third equilibrium solution at a flow rate of 4ml/min, isocratically eluting 5CV with a third eluent, and collecting the eluted protein as a sample to be tested.
It will be appreciated that in the present invention, the same purification effect can be achieved by using different manufacturers, different particle sizes or different types of anion exchange chromatography packing, affinity chromatography packing and reverse phase chromatography packing in the chromatography of the cell culture supernatant containing recombinant human erythropoietin.
In a specific example, the solvents used to formulate mobile phase a, mobile phase B, mobile phase C, first equilibrium liquid, second equilibrium liquid, third equilibrium liquid, first eluent, second eluent, and third eluent at the respective concentrations are water.
In a specific example, the method further comprises the steps of:
establishing a standard curve between the average value of the total peak area of the acid isomer of the recombinant human erythropoietin control and the concentration of the recombinant human erythropoietin control.
In one specific example, the total peak area mean of the acid isoforms of the recombinant human erythropoietin control is y, the concentration of the recombinant human erythropoietin control is x, and the function equation of the standard curve is y= 1763787.6x-77217.1.
It will be appreciated that in the present invention, x is in mg/mL and y is in microvolts seconds.
In a specific example, the preparation of a cell culture supernatant comprising recombinant human erythropoietin comprises the steps of:
culturing Chinese Hamster Ovary (CHO) cells which efficiently express human erythropoietin, and preparing a fermentation harvest liquid;
centrifuging the fermentation harvest liquid, filtering, and taking supernatant.
It will be appreciated that in the present invention, the CHO cells are cultured to secrete recombinant human erythropoietin and the fermentation broth is centrifuged to remove cells and cell debris to obtain a cell culture supernatant containing recombinant human erythropoietin.
The method for detecting recombinant human erythropoietin of the present invention is described in further detail below with reference to specific examples. The raw materials used in the following examples are all commercially available products unless otherwise specified.
Example 1
The embodiment provides a detection method of recombinant human erythropoietin, which specifically comprises the following steps:
1. chromatographic capture of rhEPO in cell culture supernatants
And (3) secreting the rhEPO after culturing the CHO cells, centrifuging a fermentation harvest liquid to remove cells and cell fragments, and obtaining a cell culture supernatant containing the rhEPO.
(1) Q Sepharose High Performance (Q HP) chromatographic column one-step chromatographic capturing rhEPO
Q HP packed Column Volume (CV) 13ml, bed height 16.5cm, column inner diameter 1cm. The equilibration solution was 10mM Tris, pH7.0, and the elution solution was 10mM Tris+0.5M NaCl, pH7.0. The cell culture supernatant was filtered with a 0.22 μm filter membrane, then diluted with 3 volumes of equilibration solution, equilibrated with 3CV of chromatography column, loaded with 180ml (the actual sample volume of cell culture supernatant was 45 ml), flow rate was 2ml/min, then isocratically washed with 10% eluent (50 mM NaCl) for 3CV, isocratically eluted with 26% eluent (130 mM NaCl) for 4CV, and the eluted protein was collected, and 2.87mg of protein was collected as Q HP test sample.
(2) One-step capture of rhEPO by Resource RPC 3ml polymer matrix reverse phase chromatography column
Column size 0.6 x 10cm, bed volume (CV) 3ml. Equilibration solution 10mM Tris, pH7.0, eluate 10mM Tris+80% acetonitrile, pH7.0. The cell culture supernatant was filtered through a 0.22 μm filter, the column equilibrated with equilibration solution at 10CV, and 50ml of the supernatant was loaded at a flow rate of 2ml/min. Then, the sample was washed with a equilibration solution for 10CV, then with a 5% eluent for 10CV, then with a 5-100% eluent for 19CV, and the target protein was collected for 1.32mg as a Resource RPC sample to be measured.
(3) One-step chromatographic capture of rhEPO by Blue Sepharose 6FF Blue gel
Chromatographic column specification 1.6x4cm, bed volume (CV) 8ml. Equilibrium solution 20mM Tris+0.15M NaCl, pH7.0, eluent 20mM Tris+1.2M NaCl, pH7.0. The cell culture supernatant was filtered through a 0.22 μm filter, the column equilibrated with equilibration solution at 5CV, 80ml of sample was loaded, and the flow rate was 2ml/min. Then washing 10CV with balancing solution at a flow rate of 4ml/min, isocratically eluting 5CV with 100% eluent, and collecting target protein, wherein the total amount of target protein is 5.52mg, to obtain blue gel sample.
2. Establishing a standard curve of a reference substance
(1) Solution preparation
A. Balancing solution: 30mM ammonium formate, pH 5.5.+ -. 0.1, conductivity 1.6.+ -. 0.4mS/cm,0.22 μm membrane.
B. Eluent: 30mM formic acid, pH 2.6.+ -. 0.1, conductivity 0.9.+ -. 0.2mS/cm,0.22 μm membrane.
C. Regeneration solution: 10mM Tris+1M NaCl,pH7.0.+ -. 0.2, conductivity 85.+ -. 3mS/cm,0.22 μm.
D. Blank solution: 10mM Tris, pH 7.0.+ -. 0.2, pH conductivity 0.9.+ -. 0.2mS/cm,0.22 μm.
(2) Anion high performance liquid chromatography detection conditions
Sample detection was performed using an Agilent HP 1100 high performance liquid chromatography system. The chromatographic column adopted is a nano-spectrum BioCore SAX column, and the specification comprises: the column length was 150mm, the inner diameter was 4.6mm, and the particle diameter was 10. Mu.m. The flow rate was 1.0ml/min, the temperature was 30℃and the UV detector wavelength was 280nm, the sample volume was 50. Mu.l, and the gradient of the elution solution in the detection method was as shown in Table 1 below:
TABLE 1
| Time (min) | A(％) | B(％) | C(％) |
| 0 | 100 | 0 | 0 |
| 10 | 100 | 0 | 0 |
| 60 | 0 | 100 | 0 |
| 70 | 0 | 100 | 0 |
| 71 | 0 | 0 | 100 |
| 85 | 0 | 0 | 100 |
| 86 | 100 | 0 | 0 |
| 100 | 100 | 0 | 0 |
(3) Integration method
The liquid phase ultraviolet spectrum integration condition is that the peak width is 400, the threshold value is 0.5, the integration interval is 10-70min, and the retention time of each isomer of rhepo is 15-65 min.
(4) Establishing a standard curve between the average value of the total peak area of the acid isomer of the recombinant human erythropoietin control and the concentration of the recombinant human erythropoietin control
The purified intermediate of rhEPO (purchased from kexing biopharmaceutical Co., ltd.) was used as a control, with a purity of 90%. The rhEPO reference substances are respectively prepared according to the proportion of 0.6mg/ml, 0.9mg/ml,1.2mg/ml, 1.5mg/ml and 1.8mg/ml of the sample are prepared, 1 needle is sampled from a blank solution, 2 needles are sampled from each concentration reference substance, and the sample volume is 50 mu l. The detection patterns are shown in fig. 2 to 12. The total peak area mean and corresponding acid isomer concentration data of each concentration control rhEPO acid isomer (charge isomer 4-7) were calculated and the results are shown in Table 2 below, wherein the standard curve is made from the total peak area mean of rhEPO acid isomers to the acid isomer concentration, the standard curve equation is y= 1763787.6x-77217.1, R is the ratio of 2 =0.995, standard curve meets the requirements.
TABLE 2
| Sample name | Acid isomer total peak area mean (microvolts second) | Acid isomer concentration (mg/ml) |
| 0.6mg/ml control | 423957 | 0.28 |
| 0.9mg/ml control | 632383 | 0.42 |
| 1.2mg/ml control | 946396 | 0.57 |
| 1.5mg/ml control | 1215852 | 0.71 |
| 1.8mg/ml control | 1393022 | 0.85 |
3. Sample processing and detection to be detected
Concentrating the sample to be detected by using an ultrafiltration centrifuge tube with the molecular weight cutoff of 10kDa, and replacing the sample with a blank solution D, wherein the concentration of the sample after the concentration and the replacement is more than or equal to 0.6mg/ml, and the conductivity is less than or equal to 3mS/cm. Detecting by adopting the detection method in the second point step (2), injecting 1 needle to obtain a liquid phase map, integrating by adopting the method in the second point step (3) to generate a detection report, calculating the total peak area of the rhEPO acid isomer, substituting the total peak area into a standard curve, and calculating the concentration of the rhEPO acid isomer in the sample to be detected.
Specifically, as shown in fig. 13, the Q HP chromatographic chromatogram is obtained by concentrating the Q HP sample to be tested, changing the solution, detecting the protein concentration by using anion high performance liquid chromatography, loading 50 μl, and substituting the result in fig. 14, wherein the total peak area of the rhEPO acid isomer in the sample to be tested is 375786 microvolts for a second, into a standard curve to calculate the concentration of the rhEPO acid isomer in the sample to be tested to be 0.257mg/ml, and finally calculating the content of the rhEPO acid isomer in the cell culture supernatant to be 17.0ug/ml.
Specifically, as shown in fig. 15, the Resource RPC chromatographic chromatogram is shown, the Resource RPC sample to be detected is concentrated and changed, the protein concentration is 0.89mg/ml, the anion high performance liquid chromatography is used for detection, 50 μl is loaded, the result is shown in fig. 16, the total peak area of the rhEPO acid isomer in the sample to be detected is 824308 microvolts for a second, the concentration of the rhEPO acid isomer in the sample to be detected is calculated to be 0.511mg/ml by substituting into a standard curve, and finally the content of the rhEPO acid isomer in the cell culture supernatant is calculated to be 16.8ug/ml.
Specifically, as shown in fig. 17, the blue gel chromatographic analysis is performed, the blue gel sample to be detected is concentrated and changed, the protein concentration is 0.81mg/ml, the detection is performed by anion high performance liquid chromatography, 50 μl is loaded, the result is shown in fig. 18, the total peak area of the rhEPO acid isomer in the sample to be detected is 309137 microvolts for a second, the concentration of the rhEPO acid isomer in the sample to be detected is calculated to be 0.219mg/ml by substituting into a standard curve, and finally the content of the rhEPO acid isomer in the cell culture supernatant is calculated to be 18.6ug/ml.
4. Results and analysis
The cell culture supernatants used for capturing the rhEPO by the first 3 fillers are in the same batch, and the content of the rhEPO acid isomer in the samples obtained by the 3 capturing modes is obtained after the detection of the anion high performance liquid chromatography, and the results are shown in Table 3. The content data of the rhEPO acid isomer in the cell culture supernatant obtained after conversion are relatively close, the average value is 17.47ug/ml, the relative standard deviation RSD is 5.6%, and the data are relatively stable, which is enough to indicate that the detection of the rhEPO sample after the cell supernatant is captured in one step by using the anion high performance liquid chromatography is stable and reliable.
TABLE 3 Table 3
Comparative example 1
The detection method in the comparative example adopts an acetic acid-ammonium acetate buffer system, and the specific detection method is as follows:
(1) Flow matching
A. Balancing solution: 30mM ammonium acetate, pH 5.5.+ -. 0.1, conductivity 1.6.+ -. 0.4mS/cm,0.22 μm membrane.
B. Eluent: 60mM acetic acid, pH was adjusted to 2.5.+ -. 0.1 with hydrochloric acid, conductivity 1.8.+ -. 0.2mS/cm, and filtration was carried out with a 0.22 μm filter.
C. Regeneration solution: 10mM Tris+1M NaCl,pH7.0.+ -. 0.2, conductivity 85.+ -. 3mS/cm,0.22 μm.
D. Blank solution: 10mM Tris, pH 7.0.+ -. 0.2, pH conductivity 0.9.+ -. 0.2mS/cm,0.22 μm.
(2) Anion high performance liquid chromatography detection conditions
Sample detection was performed using an Agilent HP 1100 high performance liquid chromatography system. The chromatographic column adopted is a nano-spectrum BioCore SAX column, and the specification comprises: the column length was 150mm, the inner diameter was 4.6mm, and the particle diameter was 10. Mu.m. The flow rate was 1.0ml/min, the temperature was 30℃and the UV detector wavelength was 280nm, the sample volume was 50. Mu.l, and the gradient of the elution solution according to the detection method was as shown in Table 4 below:
TABLE 4 Table 4
(3) Integration method
The liquid phase ultraviolet spectrum integration condition is that the peak width is 400, the threshold value is 0.5, the integration interval is 10-70min, and the retention time of each isomer of rhepo is 15-65 min.
(4) Sample detection
Taking rhEPO standard, adopting the detection method and the detection method described in the example 1 to respectively sample 1 needle, and adopting a sample volume of 50 mu l, wherein the results are shown in fig. 19 and 20, when the acetic acid-ammonium acetate system is used for detection, the retention time of each charge isomer of rhEPO is delayed, each charge isomer is overlapped, and the formic acid-ammonium formate system can obviously separate 4 charge isomer peaks, and isomer 3 is relatively unobvious and corresponds to the isoelectric focusing result in fig. 1. Therefore, the formic acid-ammonium formate system has better separation effect.
Comparative example 2
This comparative example examined the separation effect of rhEPO at different linear elution volumes, as shown in table 1 of example 1, 10min to 60min was a linear elution interval, i.e., a linear elution volume of 50ml, about 20CV (CV is the column volume of the analytical column), and this comparative example compared the separation effect of linear elution of 10CV, 20CV and 30CV, respectively. Taking a rhEPO standard substance, adopting a formic acid-ammonium formate buffer system in example 1, setting linear elution volumes of 10CV, 20CV and 30CV, and respectively injecting one needle, wherein the result is shown in figures 21, 22 and 23, the larger the linear elution volume is, the better the separation degree of charge isomers of rhEPO is, but the baseline separation of each charge isomer cannot be achieved. Considering that the larger the linear elution volume, the longer the single sample detection time, we selected the linear elution 20CV as the preferred condition, reducing the sample detection time under conditions where the degree of separation is acceptable.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples merely represent a few embodiments of the present invention, which facilitate a specific and detailed understanding of the technical solutions of the present invention, but are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. It should be understood that, based on the technical solutions provided by the present invention, those skilled in the art may obtain technical solutions through logical analysis, reasoning or limited experiments, which are all within the scope of protection of the appended claims. The scope of the patent is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted as illustrative of the contents of the claims.
Claims (10)
1. A method for detecting recombinant human erythropoietin, comprising the steps of:
obtaining a recombinant human erythropoietin control;
carrying out chromatography on a cell culture supernatant containing recombinant human erythropoietin to prepare a sample to be detected;
respectively carrying out anion high performance liquid chromatography detection on the reference substance and the sample to be detected;
wherein, the conditions for anion high performance liquid chromatography detection include: the mobile phase A is an ammonium formate solution with the concentration of 20mmol/L to 40mmol/L, the mobile phase B is a formic acid solution with the concentration of 20mmol/L to 40mmol/L, the mobile phase C is a mixed solution of tris (hydroxymethyl) aminomethane and sodium chloride, the concentration of tris (hydroxymethyl) aminomethane in the mobile phase C is 5mmol/L to 15mmol/L, and the concentration of sodium chloride is 0.8mol/L to 1.2mol/L; gradient elution is used, and the gradient elution procedure comprises: 0-10 min, wherein the volume percentage of the mobile phase A is 100%; 10-60 min, wherein the volume percentage of the mobile phase A is changed from 100% to 0%, and the volume percentage of the mobile phase B is changed from 0 to 100%; 60-70 min, wherein the volume percentage of the mobile phase B is kept to be 100%; 70-71 min, wherein the volume percentage of the mobile phase B is changed from 100% to 0%, and the volume percentage of the mobile phase C is changed from 0% to 100%;71 min-85 min, the volume percentage of the mobile phase C is kept to be 100%; 85-86 min, wherein the volume percentage of the mobile phase A is changed from 0% to 100%, and the volume percentage of the mobile phase C is changed from 100% to 0%;86 min-100 min, the volume percentage of the mobile phase A is kept to be 100%.
2. The method for detecting recombinant human erythropoietin according to claim 1, wherein the conditions for anion high performance liquid chromatography detection further comprise: the flow rate was 1 mL/min.+ -. 0.1mL/min.
3. The method for detecting recombinant human erythropoietin according to claim 1, wherein the conditions for anion high performance liquid chromatography detection further comprise: the detection wavelength is 280nm + -5 nm.
4. The method for detecting recombinant human erythropoietin according to claim 1, wherein the conditions for anion high performance liquid chromatography detection further comprise: the column temperature was 30 ℃ + -2 ℃.
5. The method for detecting recombinant human erythropoietin according to claim 1, wherein the conditions for anion high performance liquid chromatography detection further comprise: the chromatographic column is a strong anion exchange column.
6. The method for detecting recombinant human erythropoietin according to claim 1, wherein the step of subjecting the cell culture supernatant containing recombinant human erythropoietin to chromatography comprises the steps of:
balancing a first chromatographic column by adopting a first balancing solution, mixing the cell culture supernatant containing the recombinant human erythropoietin with the first balancing solution, loading the sample, eluting by using a mixed solution of a first eluent and the first balancing solution, and collecting target proteins; wherein the first chromatographic column is an anion exchange chromatographic column; the first equilibrium liquid is 5-15 mmol/L of tris (hydroxymethyl) aminomethane; the first eluent is a mixed solution of tris (hydroxymethyl) aminomethane and sodium chloride, wherein the concentration of tris (hydroxymethyl) aminomethane in the first eluent is 5-15 mmol/L, and the concentration of sodium chloride in the first eluent is 450-550 mmol/L; or (b)
Balancing a second chromatographic column by adopting a second balancing solution, loading the cell culture supernatant containing the recombinant human erythropoietin, washing the column by using the second plate Heng Ye, eluting by using a mixed solution of a second eluent and the second balancing solution, and collecting target proteins; wherein the second chromatographic column is a reversed phase chromatographic column; the second equilibrium liquid is 5 mmol/L-15 mmol/L of tris (hydroxymethyl) aminomethane; the second eluent is a mixed solution of the tris and acetonitrile or ethanol, wherein the concentration of the tris in the second eluent is 5-15 mmol/L, and the volume fraction of the acetonitrile or ethanol is 75-85%; or (b)
Balancing a third chromatographic column by using a third balancing solution, loading the cell culture supernatant containing the recombinant human erythropoietin, washing the column by using the third balancing solution, eluting by using a third eluent, and collecting target proteins; wherein the third chromatographic column is a blue agarose gel chromatographic column; the third equilibrium liquid is a mixed solution of the tris and the sodium chloride, wherein the concentration of the tris in the third equilibrium liquid is 15-25 mmol/L, and the concentration of the sodium chloride is 0.13-0.18 mol/L; the third eluent is a mixed solution of the tris and the sodium chloride, wherein the concentration of the tris in the third eluent is 15-25 mmol/L, and the concentration of the sodium chloride is 1-1.4 mol/L.
7. The method for detecting recombinant human erythropoietin according to claim 6, wherein the flow rate used for chromatography is 2mL/min to 4mL/min.
8. The method for detecting recombinant human erythropoietin according to claim 1, further comprising the steps of:
establishing a standard curve between the total peak area mean value of the acid isomer of the recombinant human erythropoietin control and the concentration of the recombinant human erythropoietin control.
9. The method according to claim 8, wherein the total peak area mean of the acid isoforms of the recombinant human erythropoietin control is y, the concentration of the recombinant human erythropoietin control is x, and the function equation of the standard curve is y= 1763787.6x-77217.1.
10. The method for detecting recombinant human erythropoietin according to any one of claims 1 to 9, wherein the preparation of the cell culture supernatant containing recombinant human erythropoietin comprises the steps of:
culturing Chinese hamster ovary cells which efficiently express human erythropoietin, and preparing fermentation harvest liquid;
centrifuging the fermentation harvest liquid, filtering and taking supernatant.
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| EP1428878A1 (en) * | 2002-12-13 | 2004-06-16 | Siegfried Ltd. | Process for the production and purification of erythropoietin |
| CN102040659A (en) * | 2009-10-19 | 2011-05-04 | 深圳新鹏生物工程有限公司 | Method for separating and purifying recombinant human erythropoietin |
| CN102816227A (en) * | 2012-08-30 | 2012-12-12 | 深圳赛保尔生物药业有限公司 | Erythropoietin recovery method |
| CN103278574A (en) * | 2013-04-26 | 2013-09-04 | 首都医科大学附属北京朝阳医院 | Method for detecting hemoglobin by liquid chromatogram-triple tandem quadrupole mass spectrometer |
| CN112940101A (en) * | 2019-12-11 | 2021-06-11 | 深圳赛保尔生物药业有限公司 | Purification method of recombinant human erythropoietin |
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| WO2008068879A1 (en) * | 2006-12-06 | 2008-06-12 | Jcr Pharmaceuticals Co., Ltd. | Method for production of human erythropoietin |
| AR067536A1 (en) * | 2007-07-17 | 2009-10-14 | Hoffmann La Roche | METHOD FOR OBTAINING A MONO-PEGILATED ERYTHROPOYETIN IN A SUBSTANTIALLY HOMOGENOUS FORM |
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| EP1428878A1 (en) * | 2002-12-13 | 2004-06-16 | Siegfried Ltd. | Process for the production and purification of erythropoietin |
| CN102040659A (en) * | 2009-10-19 | 2011-05-04 | 深圳新鹏生物工程有限公司 | Method for separating and purifying recombinant human erythropoietin |
| CN102816227A (en) * | 2012-08-30 | 2012-12-12 | 深圳赛保尔生物药业有限公司 | Erythropoietin recovery method |
| CN103278574A (en) * | 2013-04-26 | 2013-09-04 | 首都医科大学附属北京朝阳医院 | Method for detecting hemoglobin by liquid chromatogram-triple tandem quadrupole mass spectrometer |
| CN112940101A (en) * | 2019-12-11 | 2021-06-11 | 深圳赛保尔生物药业有限公司 | Purification method of recombinant human erythropoietin |
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