CN114773427A - Method for purifying polypeptide containing cysteine - Google Patents
Method for purifying polypeptide containing cysteine Download PDFInfo
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- CN114773427A CN114773427A CN202210632523.2A CN202210632523A CN114773427A CN 114773427 A CN114773427 A CN 114773427A CN 202210632523 A CN202210632523 A CN 202210632523A CN 114773427 A CN114773427 A CN 114773427A
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- mobile phase
- polypeptide
- cysteine
- purifying
- reducing agent
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- 229920001184 polypeptide Polymers 0.000 title claims abstract description 53
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 53
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
- 235000018417 cysteine Nutrition 0.000 title claims abstract description 25
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 25
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000003638 chemical reducing agent Substances 0.000 claims abstract description 15
- 239000007864 aqueous solution Substances 0.000 claims abstract description 8
- 238000001914 filtration Methods 0.000 claims abstract description 8
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 15
- 238000001514 detection method Methods 0.000 claims description 14
- 238000010828 elution Methods 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 claims description 7
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 5
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 claims description 3
- 239000005695 Ammonium acetate Substances 0.000 claims description 3
- 229940043376 ammonium acetate Drugs 0.000 claims description 3
- 235000019257 ammonium acetate Nutrition 0.000 claims description 3
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 claims description 3
- MHQVIGJMLNEBCF-UHFFFAOYSA-N C(=O)=CCOP(OCC=C=O)(OCC=C=O)=O Chemical compound C(=O)=CCOP(OCC=C=O)(OCC=C=O)=O MHQVIGJMLNEBCF-UHFFFAOYSA-N 0.000 claims description 2
- ZRSCWSKNSQLFFI-UHFFFAOYSA-N Cl.C(=O)=CCOP(OCC=C=O)(OCC=C=O)=O Chemical compound Cl.C(=O)=CCOP(OCC=C=O)(OCC=C=O)=O ZRSCWSKNSQLFFI-UHFFFAOYSA-N 0.000 claims 1
- 239000007853 buffer solution Substances 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 13
- 230000003647 oxidation Effects 0.000 abstract description 6
- 238000007254 oxidation reaction Methods 0.000 abstract description 6
- 239000000539 dimer Substances 0.000 abstract description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 10
- 239000012535 impurity Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 239000008213 purified water Substances 0.000 description 5
- 238000002390 rotary evaporation Methods 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 230000009286 beneficial effect Effects 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000011031 large-scale manufacturing process Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 2
- 238000013341 scale-up Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- RZEQTVHJZCIUBT-WDSKDSINSA-N Ser-Arg Chemical compound OC[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N RZEQTVHJZCIUBT-WDSKDSINSA-N 0.000 description 1
- GFIHKCBJSYGAPP-UHFFFAOYSA-N [Cl-].C(=O)=CC[PH+](CC=C=O)CC=C=O Chemical compound [Cl-].C(=O)=CC[PH+](CC=C=O)CC=C=O GFIHKCBJSYGAPP-UHFFFAOYSA-N 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- -1 firstly Substances 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical class [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 125000006239 protecting group Chemical group 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
Abstract
The invention relates to a method for purifying polypeptide containing cysteine, which solves the technical problems of easy oxidation to form disulfide bonds or dimers and the like in the purification process of the polypeptide, and the technical scheme of the invention is as follows: a method of purifying a cysteine-containing polypeptide comprising the steps of: dissolving and filtering a polypeptide product to be purified, and then separating the polypeptide product on a chromatographic column, wherein the polypeptide product is eluted by a mobile phase containing a reducing agent, and the polypeptide product is eluted and separated by a mobile phase A and a mobile phase B, wherein the mobile phase A is a trifluoroacetic acid aqueous solution, and the mobile phase A also comprises the reducing agent. The invention can realize the scale purification of the polypeptide containing cysteine.
Description
Technical Field
The invention relates to a method for purifying polypeptide, in particular to a method for purifying polypeptide containing cysteine.
Background
By chemical synthesis, one can not only insert unnatural amino acids other than the 20 natural amino acids into any position of the sequence, but also achieve posttranslational modification of the protein at a specific site. In the disulfide bond modification of polypeptide, the synthesis of a pair of disulfide bonds in molecules or between molecules is generally easier, the reaction conditions have various choices, such as air oxidation, DMSO oxidation and other mild oxidation processes, and H can also be adopted2O2,I2Mercury salts, etc. The most classical method in polypeptide synthesis is the formation of disulfide bonds by an air oxidation method, which generally comprises the steps of dissolving a polypeptide with a reduced sulfhydryl group in water, and reacting for more than 24 hours under a near-neutral or weakly alkaline condition (pH value of 6.5-10) to form a target disulfide bond. However, during the synthesis and preparation of cysteine-containing polypeptides, non-target disulfide bonds, i.e., impurities, may form within or between molecules, thus increasing the difficulty of subsequent purification. In the prior art, three methods are generally adopted for preparation and purification to control or remove impurities, firstly, impurities are controlled by controlling the liquid concentration of a crude product (or purified fraction), and when the liquid concentration is low, effective components in a solution can fully contact with air and are easily oxidized to generate new impurities; when the concentration is higher, the effective components in the solution can generate molecular bonds to form rings and can also generate new impurities; secondly, removing the generated non-target disulfide bond or dimer impurities by adopting multiple purification, namely removing the impurities by multiple purification of the crude product; thirdly, cysteine with protective group is selected and used in the synthesis to protect the reactive group, such as cysteine with TRT protection, namely Cys (TRT), cysteine with Acm protection, namely Cys (Acm), or cysteine with CAM protection, namely Cys (CAM) and the like.
With the above three approaches, there are the following disadvantages: 1. the distillate liquid has no fixed concentration, is easy to generate new impurities, is not beneficial to production and cannot control the quality; 2. the purification is carried out for many times, so that the sample loss is large, the yield is low, the time consumption is long, and the large-scale production is not facilitated; 3. the cysteine with protection is adopted, the purity of the synthesized polypeptide crude product is low, and due to the protection group, the solubility is poor, and the later purification and the scale-up production are not facilitated.
The invention provides a method for purifying polypeptide containing cysteine, which can overcome the defects of low purity, time and labor waste of the three prior art, is convenient to operate, greatly improves the yield and the product purity, and is easy to amplify production.
Disclosure of Invention
The present invention provides a method for purifying a cysteine-containing polypeptide. The method can effectively solve the technical problems of easy oxidation to form disulfide bonds or dimers and the like in the process of purifying the polypeptide, and can ensure the yield and the product quality.
The technical scheme of the invention is as follows: a method of purifying a cysteine-containing polypeptide comprising the steps of: the polypeptide product to be purified is subjected to a separation on a chromatography column after solubilization filtration, wherein the polypeptide product is eluted with a mobile phase comprising a reducing agent.
Preferably, the mobile phase elutes: and eluting and separating the polypeptide product by using a mobile phase A and a mobile phase B, wherein the mobile phase A is trifluoroacetic acid aqueous solution, and the mobile phase A also comprises a reducing agent.
More preferred is: the reducing agent in the mobile phase A is selected from Dithiothreitol (DTT), tris (2-carbonylethyl) phosphate (TCEP) or beta-mercaptoethanol;
more preferred is: the reducing agent in the mobile phase A is Dithiothreitol (DTT) or beta-mercaptoethanol, and the pH of the phase A is 6.5-9.0, preferably 6.5.
The more preferable scheme is as follows: the reducing agent in mobile phase A is tris (2-carbonylethyl) phosphonium hydrochloride (TCEP) and phase A has a pH of 1.5-8.5, preferably a pH of 1.5-2.5.
The more preferable scheme is as follows: the molar concentration of the reducing agent is 1-100 mM.
The mobile phase A is one of trifluoroacetic acid, ammonium formate and ammonium acetate.
The mobile phase B is one of methanol, acetonitrile or ethanol.
Elution gradient, flow rate and detection wavelength: gradient: 15-50% of mobile phase B; flow rate: 50-250 mL/min; detection wavelength: 220 nm.
The invention has the beneficial effects that: the invention provides a purification method of polypeptide containing cysteine, which solves the problems that 1, distillate liquid has no fixed concentration, is easy to generate new impurities, is not beneficial to production and cannot control quality; 2. the purification is carried out for many times, so that the sample loss is large, the yield is low, the time consumption is long, and the large-scale production is not facilitated; 3. the adoption of protected cysteine can solve the problems of low purity of the synthesized polypeptide crude product and poor solubility which are not beneficial to later purification and scale-up production. The reducing agent is added into the mobile phase, so that the possibility of forming disulfide bonds or dimers can be avoided in the purification process, and the problem that the concentration is difficult to control or impurities are removed through multiple times of purification is effectively solved.
Drawings
FIG. 1 is a high performance liquid chromatography assay of the purified fraction of example 1.
FIG. 2 is a HPLC analysis chart of the purified fraction of example 2.
FIG. 3 is a HPLC analysis chart of the purified fraction of example 3.
FIG. 4 is a HPLC analysis chart of the purified fraction of example 4.
FIG. 5 is a high performance liquid chromatography image of a purified fraction according to a conventional method.
Detailed Description
Example 1
The polypeptide sequence: Tyr-Thr-Ser-His-Lys-Leu-Val-Met-Asn-Arg-Trp-Phe-Cys-Gln-His-Asp
(1) Adding purified water into the polypeptide sample, stirring, ultrasonically dissolving until the polypeptide sample is clear, and filtering by using a 0.45 mu m microporous filter membrane for later use.
(2) Adding the sample into a reverse phase chromatographic system for elution, wherein a mobile phase A is a 0.1% (mass percentage concentration) trifluoroacetic acid aqueous solution of 20mM DTT, and the pH value is 6.5; and the mobile phase B is acetonitrile.
(3) And setting elution gradient, flow rate and detection wavelength. Gradient: the mobile phase B is 15-45%; flow rate: 50 mL/min; detection wavelength: 220 nm.
(4) Collecting the target fraction, and performing reduced pressure rotary evaporation and freeze drying. Calculated recovery after weighing was 23%. The HPLC chromatogram of the product is shown in FIG. 1.
Example 2
Polypeptide sequence: Thr-Ser-Val-Phe-Glu-Cys-Tyr-Pro-Lys-Arg-Ala-Met-Trp-Gln-Cys-His-Tyr-
Ser-Arg
(1) Adding purified water into the polypeptide sample, stirring, ultrasonically dissolving until the polypeptide sample is clear, and filtering by using a 0.45 mu m microporous filter membrane for later use.
(2) Adding the sample into a reverse phase chromatographic system for elution, wherein a mobile phase A is a 0.1% (mass percentage concentration) ammonium formate aqueous solution of 30mM TCEP, and the pH value is 2.5; the mobile phase B is methanol.
(3) And setting elution gradient, flow rate and detection wavelength. Gradient: the mobile phase B is 20-45%; flow rate: 250 mL/min; detection wavelength: 220 nm.
(4) Collecting the target fraction, and performing reduced pressure rotary evaporation and freeze drying. The calculated recovery after weighing was 22.6%. The HPLC chromatogram of the product is shown in FIG. 2.
Example 3
The polypeptide sequence: Ala-Ser-Asp-Phe-Cys-Val-Asn-Met-Cys-Glu-Arg-Tyr-Cys-Pro-His-Gly-Gln-
Phe-Cys-Met-Lys-Leu
(1) Adding purified water into the polypeptide sample, stirring, ultrasonically dissolving until the polypeptide sample is clear, and filtering by using a 0.45 mu m microporous filter membrane for later use.
(2) Adding the sample into a reverse phase chromatographic system for elution, wherein a mobile phase A is a 0.1% (mass percentage concentration) trifluoroacetic acid aqueous solution of 50mM TCEP, and the pH value is 1.5; and the mobile phase B is acetonitrile.
(3) And setting elution gradient, flow rate and detection wavelength. Gradient: the mobile phase B is 25-50%; flow rate: 50 mL/min; detection wavelength: 220 nm.
(4) Collecting the target fraction, and performing reduced pressure rotary evaporation and freeze drying. The calculated recovery after weighing was 24.8%. The HPLC chromatogram of the product is shown in FIG. 3.
Example 4
The polypeptide sequence: Met-Pro-His-Gly-Gln-Phe-Asp-Ala-Ser-Asp-Phe-Cys-Val-Asn-Met-Lys-Leu
(1) Adding purified water into the polypeptide sample, stirring, ultrasonically dissolving until the polypeptide sample is clear, and filtering with a 0.45 mu m microporous filter membrane for later use.
(2) Adding the sample into a reverse phase chromatography system for elution, wherein a mobile phase A phase is a 3% (mass percentage concentration) ammonium acetate aqueous solution of 10mM beta-mercaptoethanol, and the pH value is 6.5; the mobile phase B is ethanol.
(3) And setting elution gradient, flow rate and detection wavelength. Gradient: the mobile phase B is 18-45%; flow rate: 50 mL/min; detection wavelength: 220 nm.
(4) Collecting the target fraction, and performing reduced pressure rotary evaporation and freeze drying. The calculated recovery after weighing was 21.4%. The HPLC chromatogram of the product is shown in FIG. 4.
Example 5
Polypeptide sequence: Tyr-Thr-Ser-His-Lys-Leu-Val-Met-Asn-Arg-Trp-Phe-Cys-Gln-His-Asp
(1) Adding purified water into the polypeptide sample, stirring, ultrasonically dissolving until the polypeptide sample is clear, and filtering with a 0.45 mu m microporous filter membrane for later use.
(2) Adding the sample into a reversed phase chromatography system for elution, wherein a mobile phase A is a trifluoroacetic acid aqueous solution with the concentration of 0.1 percent (mass percentage); the mobile phase B is acetonitrile.
(3) And setting elution gradient, flow rate and detection wavelength. Gradient: the mobile phase B is 15-45%; flow rate: 50 mL/min; detection wavelength: 220 nm.
(4) Collecting the target fraction, and performing reduced pressure rotary evaporation and freeze drying. The HPLC chromatogram of the product is shown in FIG. 5.
Claims (8)
1. A method of purifying a cysteine-containing polypeptide, comprising: the method comprises the following steps: dissolving and filtering a polypeptide product to be purified, and then separating the polypeptide product on a chromatographic column, wherein the polypeptide product is eluted by a mobile phase containing a reducing agent, and the polypeptide product is eluted and separated by a mobile phase A and a mobile phase B, wherein the mobile phase A is a trifluoroacetic acid aqueous solution, and the mobile phase A also comprises the reducing agent.
2. A method of purifying a cysteine-containing polypeptide according to claim 1 wherein: the reducing agent in the mobile phase A is one of dithiothreitol, tris (2-carbonyl ethyl) phosphate and beta-mercaptoethanol.
3. A method of purifying a cysteine-containing polypeptide according to claim 2 wherein: the reducing agent in the mobile phase A is dithiothreitol or beta-mercaptoethanol, and the pH of the mobile phase A is 6.5.
4. A method of purifying a cysteine-containing polypeptide according to claim 2 wherein: the reducing agent in the mobile phase A is tris (2-carbonyl ethyl) phosphate hydrochloride, and the pH value of the mobile phase A is 1.5-2.5.
5. A method according to claim 1, wherein the step of purifying the cysteine-containing polypeptide comprises: the molar concentration of the reducing agent is 1-100 mM.
6. A method according to claim 1, wherein the step of purifying the cysteine-containing polypeptide comprises: the mobile phase A phase buffer solution is one of trifluoroacetic acid, ammonium formate or ammonium acetate.
7. A method according to claim 1, wherein the step of purifying the cysteine-containing polypeptide comprises: the mobile phase B is as follows: methanol, acetonitrile or ethanol.
8. A method of purifying a cysteine-containing polypeptide according to claim 1 wherein: and (3) eluting: elution gradient, flow rate and detection wavelength: gradient: 15-50% of mobile phase B; flow rate: 50-250 mL/min; detection wavelength: 220 nm.
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004101805A1 (en) * | 2003-05-14 | 2004-11-25 | Axxima Pharmaceuticals Ag | Method for recombinant production of mammalian proteins and proteins obtainable by such method |
WO2006102722A1 (en) * | 2005-03-31 | 2006-10-05 | Newsouth Innovations Pty Limited | Process for the production of ϝ-glutamylcysteine |
CN110658289A (en) * | 2019-10-25 | 2020-01-07 | 苏州强耀生物科技有限公司 | Purification method of RGD peptide containing multiple cysteines |
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- 2022-06-07 CN CN202210632523.2A patent/CN114773427A/en active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2004101805A1 (en) * | 2003-05-14 | 2004-11-25 | Axxima Pharmaceuticals Ag | Method for recombinant production of mammalian proteins and proteins obtainable by such method |
WO2006102722A1 (en) * | 2005-03-31 | 2006-10-05 | Newsouth Innovations Pty Limited | Process for the production of ϝ-glutamylcysteine |
CN110658289A (en) * | 2019-10-25 | 2020-01-07 | 苏州强耀生物科技有限公司 | Purification method of RGD peptide containing multiple cysteines |
Non-Patent Citations (1)
Title |
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佚名: "三(2-羰基乙基)磷盐酸盐", pages 1 * |
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Application publication date: 20220722 |