CN116589599A - Purification method of recombinant human growth hormone-Fc fusion protein - Google Patents
Purification method of recombinant human growth hormone-Fc fusion protein Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 53
- 238000000746 purification Methods 0.000 title abstract description 30
- 238000001042 affinity chromatography Methods 0.000 claims abstract description 74
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- 229910000403 monosodium phosphate Inorganic materials 0.000 description 1
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- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
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- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
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- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Endocrinology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a purification method of recombinant human growth hormone-Fc fusion protein, which comprises the following steps: filtering cell sap containing recombinant human growth hormone-Fc fusion protein, and collecting filtrate; subjecting the filtrate to affinity chromatography; wherein, the eluent adopted by the affinity chromatography contains poloxamer 188. The method can effectively improve the yield of the rhGH-Fc fusion protein, and the obtained purified product has the characteristics of high purity of the rhGH-Fc fusion protein, less impurities such as HCP protein, and the like, and the quality meets the requirements, and has the characteristics of simple process steps, high efficiency, large-scale amplification and the like, and has high application value.
Description
Technical Field
The present invention relates to the field of biology. In particular, the invention relates to a method for purifying recombinant human growth hormone-Fc fusion protein.
Background
Human growth hormone (human growth hormone, hGH) is a non-glycosylated protein hormone secreted by the anterior She Shi acid cells of the pituitary gland, is the most important hormone for promoting growth after birth, and has multiple functions of regulating growth metabolism of human body. Recombinant human growth hormone-Fc fusion protein (rhGH-Fc) is formed by 2 molecules with structure and amino acid sequence identical to natural hGH, plus 2 Fc fragments of immunoglobulin, using recombinant DNA technology. The rhGH-Fc fusion protein is widely used for growth hormone deficiency of adults and children, and the recombinant human growth hormone can act in vivo for a longer time due to the increased Fc segment, so that the injection times are reduced.
In the rhGH-Fc fusion protein purification process, due to the related characteristics of natural human growth hormone molecular sequences, the phenomenon of continuous protein precipitation occurs in the purification process, and the continuous protein precipitation still occurs even after the protein is removed by filtration, so that the purification process cannot be continued, and the continuous and stable production of products meeting the quality requirements is difficult. The precipitation phenomenon of the rhGH-Fc fusion protein is particularly obvious when the purification process is amplified, and greatly restricts the large-scale amplification production of the purification process.
Thus, current methods for purification of recombinant human growth hormone-Fc fusion proteins remain to be investigated.
Disclosure of Invention
The present invention aims to solve, at least to some extent, the technical problems existing in the prior art. Therefore, the invention provides a purification method of recombinant human growth hormone-Fc fusion protein, by using the method, the yield of the rhGH-Fc fusion protein can be effectively improved, and the obtained purified product has the characteristics of high purity of the rhGH-Fc fusion protein, less impurities such as HCP protein, and the like, and the quality meets the requirements, and has the characteristics of simple process steps, high efficiency, large-scale amplification and the like, and has high application value.
The present invention has been made based on the findings and knowledge of the inventors regarding the following facts and problems:
The Fc in the rhGH-Fc fusion protein can be used as a label, and the separation purpose can be achieved through affinity chromatography. In order to solve the problem of protein precipitation, the inventors of the present invention have tried to add a pro-solvent to an eluate of affinity chromatography, and studied different pro-solvents, and found that some pro-solvents were not capable of inhibiting protein precipitation or, although having an inhibitory effect, did not show remarkable effect. Through intensive research and screening, poloxamer 188 can effectively inhibit protein precipitation, and eluted protein effluent is clear, so that the purification process is facilitated to be smoothly carried out, the protein yield is ensured, and large-scale production can be realized. In addition, if poloxamer 188 is needed to be added in the subsequent preparation process of the rhGH-Fc fusion protein, the poloxamer 188 is remained in the purified product, so that the poloxamer 188 can be reduced or omitted later, and the production cost and the production efficiency are reduced as a whole.
Further, the inventors tried to subject the effluent containing rhGH-Fc fusion protein isolated by affinity chromatography to ion exchange chromatography. The document reports that the isoelectric point of the host cell protein HCP ranges from 4.5 to 7.0, and the isoelectric point of the target protein rhGH is about 6.0 and is close to the isoelectric point of the HCP, so that the HCP is difficult to be sufficiently separated and removed by ion exchange chromatography, and the HCP content in the purified product is too high. Furthermore, the inventors tried to remove HCP as much as possible in affinity chromatography in order to relieve the pressure of the next stage ion exchange chromatography.
In view of the above, the inventors have found through a large number of experiments that HCP can be effectively removed by adding sodium octoate to the wash buffer of affinity chromatography. Further, the inventor finds that sodium octoate is easy to precipitate under the acidic condition, the pH value of the eluent is acidic, and if the buffer solution containing sodium octoate is washed by the affinity chromatography column and then is directly eluted by the eluent, sodium octoate precipitation can occur in effluent liquid, so that the subsequent process is influenced. Furthermore, the inventor adopts a slightly alkaline flushing buffer solution to flush the chromatographic column before and after flushing the chromatographic column with the buffer solution containing sodium octoate, thereby effectively avoiding the precipitation of sodium octoate, ensuring the smooth operation of the purification process and reducing the impurity content in the purified product.
Therefore, the invention provides a purification method of recombinant human growth hormone-Fc fusion protein. According to an embodiment of the invention, the method comprises: filtering cell sap containing recombinant human growth hormone-Fc fusion protein, and collecting filtrate; subjecting the filtrate to affinity chromatography; wherein, the eluent adopted by the affinity chromatography contains poloxamer 188.
According to the method provided by the embodiment of the invention, poloxamer 188 is added into the eluent, so that precipitation of rhGH-Fc fusion protein can be effectively avoided, the purification process can be smoothly carried out, the protein yield is ensured, and large-scale production can be realized.
According to an embodiment of the present invention, the purification method of the recombinant human growth hormone-Fc fusion protein may further have at least one of the following additional technical features:
according to an embodiment of the invention, the filler used for affinity chromatography is selected from the group consisting of Protein A.
According to an embodiment of the invention, the poloxamer 188 content in the eluent is more than 0.03% by volume, preferably between 0.04 and 0.50% by volume.
According to an embodiment of the invention, the pH of the eluent is 3.0-4.5, preferably 3.2-3.8.
According to an embodiment of the invention, the eluent contains acid and alkali, wherein the acid is at least one selected from acetic acid, a citric acid system, glycine, hydrochloric acid, phosphoric acid and sulfuric acid, preferably acetic acid; the alkali is at least one selected from sodium hydroxide, tris, disodium hydrogen phosphate, arginine, calcium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate and ammonium sulfate, preferably sodium hydroxide.
According to an embodiment of the invention, the affinity chromatography comprises: washing the affinity chromatographic column by using a first buffer solution and an eluent in sequence; the first buffer solution contains sodium octoate.
According to an embodiment of the invention, the pH of the first buffer is 7.0-9.0, preferably 7.0-7.5.
According to an embodiment of the invention, the concentration of sodium octoate in the first buffer is 50-150 mM, preferably 80-120 mM.
According to an embodiment of the present invention, the first buffer contains phosphate.
According to an embodiment of the invention, the affinity chromatography comprises: a) Washing the affinity chromatography column with at least one of a second buffer and a third buffer; b) Washing the affinity chromatography column with the first buffer; c) Washing the affinity chromatography column with a second buffer or a third buffer; d) Washing the affinity chromatography column with a fourth buffer; e) Eluting the affinity chromatographic column by using the eluent, and collecting effluent; the pH value of the second buffer solution is 7.0-8.0, preferably 7.0-7.5; the pH value of the third buffer solution is 7.0-8.0, preferably 7.0-7.5; the pH value of the fourth buffer solution is 4.0-6.0, preferably 5.2-5.8.
According to an embodiment of the present invention, the second buffer is selected from phosphate buffers, the third buffer is selected from phosphate buffers containing sodium chloride, and the fourth buffer contains an acid and a base.
According to an embodiment of the invention, the acid is selected from at least one of acetic acid, citric acid system, glycine, hydrochloric acid, phosphoric acid, sulfuric acid, preferably acetic acid; the alkali is at least one selected from sodium hydroxide, tris, disodium hydrogen phosphate, arginine, calcium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate and ammonium sulfate, preferably sodium hydroxide.
According to an embodiment of the present invention, in step a), the total amount of washing of at least one of the second buffer and the third buffer is not less than 3 column volumes, and the linear flow rate is 60 to 120cm/h.
According to an embodiment of the present invention, in step b), the washing amount of the first buffer solution is not less than 6 times of the column volume, and the linear flow rate is 120 to 180cm/h.
According to an embodiment of the present invention, in step c), the second buffer solution or the third buffer solution has a flushing amount of not less than 2 column volumes, and a linear flow rate of 120 to 180cm/h.
According to an embodiment of the present invention, in step d), the fourth buffer solution has a flushing amount of not less than 6 column volumes and a linear flow rate of 120 to 180cm/h.
According to an embodiment of the present invention, in step e), the eluting amount of the eluent is not less than 5 times of the column volume, and the linear flow rate is 120-180 cm/h.
According to an embodiment of the invention, between steps a) and b), further comprising: f) And flushing the affinity chromatography column by using the second buffer solution or the third buffer solution, wherein the flushing amount is not less than 3 times of column volume, and the linear flow rate is 120-180 cm/h.
According to an embodiment of the invention, the method further comprises: subjecting the effluent collected by eluting the affinity chromatographic column with the eluent to anion exchange chromatography and cation hydrophobic composite chromatography, and collecting the effluent containing recombinant human growth hormone-Fc fusion protein; nanofiltration is carried out on the effluent liquid containing the recombinant human growth hormone-Fc fusion protein, and nanofiltration effluent liquid is collected; ultrafiltering the nanofiltration effluent, and collecting the retentate to obtain the purified recombinant human growth hormone-Fc fusion protein.
According to the embodiment of the invention, the eluting buffer used for anion exchange chromatography contains poloxamer 188; the content is more than 0.03% by volume, preferably 0.04 to 0.50% by volume.
According to an embodiment of the present invention, the anion exchange chromatography is performed by gradient elution using a first elution buffer and a second elution buffer, where the first elution buffer includes: l-histidine and L-histidine hydrochloride, the pH value is 6.6-7.0; the second elution buffer comprises: l-histidine, L-histidine hydrochloride, sodium chloride and poloxamer 188, and the pH value is 6.6-7.0; the gradient elution mode is as follows: and 0-100% of the second eluting buffer solution is linearly eluted with 30 column volumes.
According to an embodiment of the present invention, the washing solution used in the cationic hydrophobic complex chromatography includes: l-histidine, L-histidine hydrochloride and sodium chloride, and the pH value is 6.6-7.0.
According to the embodiment of the invention, the aperture of the nanofiltration membrane adopted by the nanofiltration is 15-40 nm.
According to the embodiment of the invention, the aperture of the ultrafiltration membrane used for ultrafiltration is 5-30 KD.
According to an embodiment of the invention, the effluent collected by eluting the affinity column with an eluent is virus inactivated prior to performing the anion exchange chromatography and the cation-hydrophobic complex chromatography.
According to an embodiment of the invention, the viral inactivation comprises: adding acetic acid into the effluent liquid collected by eluting the affinity chromatographic column by using the eluent until the pH value is 3.0-4.0, standing at the dark room temperature for 0.5-1.5 hours, and then regulating the pH value of the solution to 7.0-7.5 by using Tris-HCl buffer solution.
In addition, the invention provides another purification method of the recombinant human growth hormone-Fc fusion protein. According to an embodiment of the invention, the method comprises:
(1) Affinity chromatography:
loading: loading a sample to be treated into a Protein A affinity chromatography column;
flushing 1: washing the affinity chromatography column 3CV with 80% buffer A and 20% buffer B, wherein the linear flow rate is 90cm/h, the buffer A is selected from disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate, the pH value is 7.2 + -0.2, and the buffer B is selected from disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate and sodium chloride, and the pH value is 7.2 + -0.2;
flushing 2: washing the affinity chromatography column 3CV with 100% buffer B at a linear flow rate of 180cm/h;
flushing 3: washing the affinity chromatography column 6CV with a buffer solution C1, wherein the linear flow rate is 180cm/h, the buffer solution C1 is selected from disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate and sodium octoate, and the pH value is 7.2+/-0.2;
Flushing 4: washing the affinity chromatographic column 2CV with buffer A at linear flow rate of 180cm/h;
flushing 5: washing the affinity chromatographic column 6CV with a buffer solution C2, wherein the linear flow rate is 180cm/h, the buffer solution C2 is selected from acetic acid and sodium hydroxide, and the pH value is 5.5+/-0.2;
eluting: eluting the affinity chromatographic column 5CV with a buffer solution D, wherein the elution is started when the UV280 rises to 100mAU/2mm in the elution process, the UV280 falls to 200mAU/2mm after passing through the peak top, and the collection is stopped, and the buffer solution D is selected from acetic acid, 0.04 volume percent poloxamer 188 and sodium hydroxide, and the pH value is 3.5+/-0.2;
(2) Low pH virus inactivation:
adding acetic acid under the condition that the finally collected sample solution is continuously and uniformly mixed, and regulating the pH of the sample to 3.6+/-0.2;
stopping uniformly mixing, and standing the sample at the dark room temperature for 60+/-15 min;
adding a buffer solution E under the condition that the sample solution after standing is continuously and uniformly mixed, and regulating the pH value of the sample to 7.2+/-0.2, wherein the buffer solution E is selected from the group consisting of tris (hydroxymethyl) aminomethane and tris (hydroxymethyl) aminomethane hydrochloride, and the pH value of the buffer solution E is 9.0+/-0.2;
(3) Anion exchange chromatography:
loading: loading the solution obtained in the previous step into an anion exchange chromatographic column;
flushing: washing the anion exchange chromatographic column 2CV with a buffer F, wherein the linear flow rate is 90cm/h, the buffer F is selected from L-histidine and L-histidine hydrochloride, and the pH value is 6.8+/-0.2;
Eluting: performing 0-100% buffer solution G30CV linear elution by using a buffer solution F and a buffer solution G; after UV280 reaches the peak, 2CV starts to collect protein liquid, the UV280 is reduced to 300mAU/2mm, and then collection is stopped, wherein the buffer solution G is selected from L-histidine, L-histidine hydrochloride, 0.04 volume percent poloxamer 188 and sodium chloride, and the pH value is 6.8+/-0.2;
(4) Cation hydrophobic composite chromatography:
loading: loading the finally collected solution to a cation hydrophobic composite chromatographic column, and collecting the flow-through liquid when the UV280nm of the flow-through liquid reaches more than 50mAU/2 mm; the flow-through liquid collected in the step is used as a first section collecting liquid;
flushing: flushing the cationic hydrophobic complex chromatographic column 2CV by using a buffer solution H, wherein the buffer solution H is selected from L-histidine, L-histidine hydrochloride and sodium chloride, and the pH value is 6.8+/-0.2, and stopping collecting until the UV280nm is reduced to 100mAu/2 mm;
(5) Nanofiltration and ultrafiltration:
and combining the first-stage collecting liquid and the second-stage collecting liquid, and carrying out nanofiltration and ultrafiltration to obtain a purified product.
The method can effectively improve the yield of the rhGH-Fc fusion protein, and the obtained purified product has the characteristics of high purity of the rhGH-Fc fusion protein, less impurities such as HCP protein, and the like, and the quality meets the requirements, and has the characteristics of simple process steps, high efficiency, large-scale amplification and the like, and has high application value.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic flow chart showing a method for purifying a recombinant human growth hormone-Fc fusion protein according to an embodiment of the present invention;
FIG. 2 shows a schematic representation of the turbidity of the effluent after elution of affinity chromatography under different pro-solvent conditions according to one embodiment of the present invention;
FIG. 3 shows a schematic representation of the turbidity of the effluent after elution of anion exchange chromatography under different pro-solvent conditions in accordance with one embodiment of the present invention;
FIG. 4 shows a schematic representation of HCP protein content in an eluted effluent of affinity chromatography with different compositions of buffer C1 and wash mode in affinity chromatography according to an embodiment of the invention;
FIG. 5 shows a schematic representation of HCP protein content in an effluent of affinity chromatography after elution under different composition conditions of buffer C2 in affinity chromatography according to one embodiment of the invention;
FIG. 6 shows a schematic representation of HCP content in an effluent containing rhGH-Fc fusion protein collected by affinity chromatography, anion exchange chromatography and cation-hydrophobic complex chromatography after purification of different-scale cell fluids according to one embodiment of the invention.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," second, "" third, "and fourth" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first", "a second", "a third" and a fourth "may explicitly or implicitly include one or more such feature.
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
In the early-stage research of the purification process, the inventor of the invention comprehensively considers factors influencing the whole purification process, such as the selection of a buffer system, the coping measure influencing quality standard factors, the connection of front and back links and the like, so as to relieve the precision pressure and obtain the recombinant human growth hormone-Fc fusion protein with better quality. Specifically, the invention provides a purification method of recombinant human growth hormone-Fc fusion protein. According to an embodiment of the invention, referring to fig. 1, the method comprises: s100 filtration and S200 affinity chromatography, each of which will be described in detail below.
S100 filtration
In this step, the cell fluid containing the recombinant human growth hormone-Fc fusion protein is filtered, and the filtrate is collected.
The method for obtaining the cell sap containing the recombinant human growth hormone-Fc fusion protein is not strictly limited, and the target gene can be expressed in cells by adopting the conventional technical means in the field so as to obtain the rhGH-Fc fusion protein. Since rhGH-Fc fusion proteins are secreted extracellularly, they can be obtained by filtration of the cell fluid.
S200 affinity chromatography
In this step, the filtrate is subjected to affinity chromatography. Thereby, to isolate rhGH-Fc fusion protein, impurities were removed.
According to an embodiment of the invention, the filler used for affinity chromatography is selected from the group consisting of Protein A. The Protein A can be specifically combined with an immunoglobulin Fc segment of the rhGH-Fc fusion Protein, so that the purposes of separation and impurity removal are realized.
According to an embodiment of the invention, the eluent used for affinity chromatography contains poloxamer 188. The poloxamer 188 can effectively inhibit protein precipitation, and the eluted protein effluent is clear, so that the purification process is facilitated to be smoothly carried out, the protein yield is ensured, and the large-scale production can be realized.
According to embodiments of the invention, the poloxamer 188 content in the eluent is greater than 0.03 vol%, for example, may be 0.04 vol%, 0.05 vol%, 0.08 vol%, 0.1 vol%, 0.12 vol%, 0.15 vol%, 0.2 vol%, 0.25 vol%, 0.3 vol%, 0.35 vol%, 0.4 vol%, 0.45 vol%, etc., preferably 0.04-0.50 vol%, 0.04-0.2 vol%, more preferably 0.04-0.12 vol%. Thus, protein precipitation can be suppressed more effectively.
According to an embodiment of the present invention, the pH of the eluent is 3.0 to 4.5, for example, 3.2, 3.5, 3.8, 4.0, 4.2, etc., preferably 3.2 to 3.8. Therefore, the binding bond between the rhGH-Fc fusion Protein and the Protein A can be broken, and the rhGH-Fc fusion Protein flows out along with the eluent, so that the separation purpose is realized.
According to an embodiment of the invention, the eluent contains acid and alkali, wherein the acid is at least one selected from acetic acid, a citric acid system, glycine, hydrochloric acid, phosphoric acid and sulfuric acid, preferably acetic acid; the alkali is at least one selected from sodium hydroxide, tris, disodium hydrogen phosphate, arginine, calcium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate and ammonium sulfate, preferably sodium hydroxide.
According to an embodiment of the invention, the affinity chromatography comprises: washing the affinity chromatographic column by using a first buffer solution and an eluent in sequence; the first buffer solution contains sodium octoate. The affinity chromatography column is rinsed with a first buffer to remove impurities. Since HCPs have isoelectric points close to those of the target protein, it is difficult to sufficiently separate and remove HCPs in ion exchange chromatography. In the process of affinity chromatography, a first buffer solution containing sodium octoate is washed by an affinity chromatography column, so that HCP can be effectively removed, the removal pressure of subsequent ion exchange chromatography is reduced, and the content of HCP in the finally obtained purified product is low. Meanwhile, the interference to the target protein can be reduced by adopting sodium octoate for washing, and the denaturation and loss caused by washing from the chromatographic column are avoided.
According to an embodiment of the present invention, the pH of the first buffer is 7.0 to 9.0, for example, 7.1, 7.3, 7.5, 7.7, 7.8, 8.0, 8.2, 8.5, 8.7, 8.9, etc., preferably 7.0 to 7.5. Therefore, the first buffer solution can play a good buffering role, effectively remove impurities such as HCP and the like, maintain the stability of sodium octoate and avoid precipitation.
According to an embodiment of the present invention, the first buffer contains phosphate, and the concentration of sodium octoate is 50 to 150mM, for example, 60mM, 80mM, 100mM, 120mM, 140mM, etc., preferably 80 to 120mM. HCP can be better removed at this sodium octoate concentration.
According to an embodiment of the present invention, step S200 of affinity chromatography comprises:
a) Washing the affinity chromatography column with at least one of a second buffer selected from phosphate buffer having a pH of 7.0 to 8.0, for example, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, etc., preferably 7.0 to 7.5; the third buffer is selected from phosphate buffers containing sodium chloride, and has a pH of 7.0 to 8.0, for example, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, etc., preferably 7.0 to 7.5; the total amount of washing of at least one of the second buffer and the third buffer is not less than 3 column volumes (also referred to herein as "CV"), and may be, for example, 4CV, 5CV, 6CV, etc., and the linear flow rate may be 60 to 120cm/h, for example, 60cm/h, 90cm/h, 120cm/h, etc. Thereby, it can act as a buffer system and remove impurities.
In the present invention, the kind of the term "phosphate" is not strictly limited, and may be a component of a phosphate buffer commonly known in the art, for example, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the like.
b) The affinity chromatography column is washed with a first buffer. The first buffer solution may be washed in an amount of not less than 6 column volumes, for example, 4CV, 5CV, 6CV, etc., and the linear flow rate may be 120 to 180cm/h, for example, 120cm/h, 150cm/h, 180cm/h, etc. Thus, HCP can be effectively removed.
According to an embodiment of the invention, between steps a) and b), further comprising: f) The affinity chromatography column is washed with the second buffer solution or the third buffer solution in an amount of not less than 3 times the column volume, for example, 4CV, 5CV, 6CV, etc., and the linear flow rate is 120 to 180cm/h, for example, 120cm/h, 150cm/h, 180cm/h, etc. Thereby, impurities can be removed better.
c) Washing the affinity chromatography column with a second buffer or a third buffer; the second buffer or the third buffer may be washed in an amount of not less than 2 column volumes, for example, 3CV, 4CV, 5CV, 6CV, etc., and the linear flow rate may be 120 to 180cm/h, for example, 120cm/h, 150cm/h, 180cm/h, etc. Thereby, impurities can be removed better.
d) Washing the affinity chromatography column with a fourth buffer solution containing an acid and a base, the pH value of the fourth buffer solution being 4.0 to 6.0, for example, 4.2, 4.4, 4.5, 4.8, 5.0, 5.2, 5.4, 5.6, 5.8, etc., preferably 5.2 to 5.8; the acid is at least one selected from acetic acid, citric acid system, glycine, hydrochloric acid, phosphoric acid and sulfuric acid, preferably acetic acid; the alkali is at least one selected from sodium hydroxide, tris, disodium hydrogen phosphate, arginine, calcium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate and ammonium sulfate, preferably sodium hydroxide; the fourth buffer solution may have a washing amount of not less than 6 column volumes, for example, 7CV, 8CV, 9CV, 10CV, etc., and a linear flow rate of 120 to 180cm/h, for example, 120cm/h, 150cm/h, 180cm/h, etc.
Because the eluent is strong in acidity, if the chromatographic column is directly eluted by the strong-acid eluent after being washed by the slightly alkaline washing buffer solution, the chromatography filler ligand is easy to fall off, the service life of the chromatography filler is reduced, the elution and collection of a sample are not facilitated, and the target protein is easy to pass through the isoelectric point, so that the target protein is separated out in the chromatographic column. Therefore, the weak acid fourth buffer solution is adopted to wash the chromatographic column, so that the equilibrium bridging effect is achieved, the subsequent eluent is facilitated to better elute the target protein, and the service life of the chromatographic column is prolonged.
e) Eluting the affinity chromatographic column by using eluent, and collecting effluent; the eluting amount of the eluent is not less than 5 column volumes, and may be, for example, 7CV, 8CV, 9CV, 10CV, etc., and the linear flow rate may be 120 to 180cm/h, for example, 120cm/h, 150cm/h, 180cm/h, etc.
The inventor finds that sodium octoate is easy to precipitate under the acidic condition, the pH value of the eluent is acidic, and if the buffer solution containing sodium octoate is washed by the affinity chromatography column and then is directly eluted by the eluent, sodium octoate precipitation can occur in effluent liquid, thereby influencing the subsequent process. Furthermore, the inventor adopts a slightly alkaline flushing buffer solution to flush the chromatographic column before and after flushing the chromatographic column with the first buffer solution containing sodium octoate, thereby effectively avoiding the precipitation of sodium octoate and ensuring the smooth proceeding of the purification process.
The steps S100 and S200 belong to the prior coarse purification, and impurities can be removed as much as possible on the premise of ensuring the yield, so that the subsequent fine purification pressure is reduced, the purification efficiency is improved as a whole, the cost is reduced, and the method is suitable for large-scale amplification and is suitable for large-scale production. Specifically, according to an embodiment of the present invention, the purification method of the recombinant human growth hormone-Fc fusion protein further comprises the following purification steps: s400 anion exchange chromatography, S500 cation hydrophobic complex chromatography, S600 nanofiltration, S700 ultrafiltration, each of which will be described in detail below.
S400 anion exchange chromatography and S500 cation hydrophobic composite chromatography
In this step, the effluent collected by eluting the affinity column with the eluent is subjected to anion exchange chromatography and cation-hydrophobic composite chromatography, and the effluent containing the recombinant human growth hormone-Fc fusion protein is collected. Thereby, in order to remove impurities.
The invention does not strictly limit the sequence of anion exchange chromatography and cation hydrophobic composite chromatography, and can firstly carry out anion exchange chromatography and then cation hydrophobic composite chromatography, or can firstly carry out cation hydrophobic composite chromatography and then anion exchange chromatography, and preferably selects to carry out anion exchange chromatography and then cation hydrophobic composite chromatography, so that the impurity removal effect is better.
According to an embodiment of the invention, the elution buffer used for anion exchange chromatography contains poloxamer 188. Thus, precipitation of the rhGH-Fc fusion protein can be effectively avoided. According to an embodiment of the invention, the poloxamer 188 content in the elution buffer is greater than 0.03 vol%, for example, may be 0.03 vol%, 0.04 vol%, 0.05 vol%, 0.08 vol%, 0.1 vol%, 0.12 vol%, 0.15 vol%, 0.2 vol%, 0.25 vol%, 0.3 vol%, 0.35 vol%, 0.4 vol%, 0.45 vol%, etc., preferably 0.02-0.5 vol%, more preferably 0.04-0.12 vol%. Thus, protein precipitation can be suppressed more effectively.
According to an embodiment of the present invention, the elution process of anion exchange chromatography uses a first elution buffer and a second elution buffer for gradient elution, the first elution buffer comprising: l-histidine and L-histidine hydrochloride, the pH value is 6.6-7.0; the second elution buffer comprises: l-histidine, L-histidine hydrochloride, sodium chloride and poloxamer 188, and the pH value is 6.6-7.0; the gradient elution mode is as follows: and 0-100% of the second eluting buffer solution is linearly eluted with 30 column volumes. Therefore, the impurity removing effect can be better.
According to an embodiment of the present invention, a washing solution used in cationic hydrophobic complex chromatography includes: l-histidine, L-histidine hydrochloride and sodium chloride, and the pH value is 6.6-7.0. Thereby, the residual protein on the lower column was washed.
The packing material used in the anion exchange chromatography of the present invention may be any packing material commonly used in the art and suitable for separating rhGH proteins, for example, unigel30 DEAEE, capto sphere, capto Q, etc. Wherein, the Unigel30DEAE has smaller particle size and better purification and separation effect, and can be used for chromatographic packing with large-scale amplification.
The filler used in the cationic hydrophobic composite chromatography can be a filler which is commonly used in the field and is suitable for separating rhGH protein, and can be, for example, diamond MMC, capto S impact, SP HP and the like. Wherein, diamond MMC can adsorb impurity such as HCP effectively, and the edulcoration effect is better.
30DEAE according to an embodiment of the present invention, the effluent collected from the elution affinity chromatography column is subjected to virus inactivation S300 prior to performing the step S400 of anion exchange chromatography and cation-hydrophobic complex chromatography. Thereby, the safety of the final purified product is satisfied.
According to an embodiment of the invention, the viral inactivation comprises: adding acetic acid into the effluent liquid collected by eluting the affinity chromatographic column by using eluent until the pH value is 3.0-4.0, standing for 0.5-1.5 hours in a dark place, and then regulating the pH value of the solution to 7.0-7.5 by using Tris-HCl buffer solution.
The virus can be effectively inactivated under the virus inactivation condition, especially under the low pH value condition, and the effluent collected by eluting the affinity chromatographic column is acidic, so that the use of acid can be reduced, and the operation is convenient.
S600 nanofiltration
In this step, the effluent containing the recombinant human growth hormone-Fc fusion protein is subjected to nanofiltration, and the nanofiltration effluent is collected. Thereby, impurities can be effectively removed.
According to an embodiment of the present invention, nanofiltration membranes used for nanofiltration have a pore size of 15 to 40nm, for example, 18nm, 20nm, 22nm, 25nm, 28nm, 30nm, 32nm, 35nm, 38nm, etc. Thus, the rhGH-Fc fusion protein can be effectively separated, and impurities can be removed.
S700 ultrafiltration
In this step, the nanofiltration effluent is subjected to ultrafiltration, and the retentate is collected to obtain a purified recombinant human growth hormone-Fc fusion protein. Therefore, the rhGH-Fc fusion protein can be effectively separated, impurities are removed, and the rhGH-Fc fusion protein stock solution with high purity and high yield is obtained.
According to the embodiment of the invention, the ultrafiltration membrane used for ultrafiltration has a pore diameter of 5-30 KD, such as 8KD, 10KD, 12KD, 15KD, 18KD, 20KD, 22KD, 25KD, 28KD, etc. Thus, the rhGH-Fc fusion protein can be effectively separated, impurities are removed, and the purpose of concentration is achieved.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
In this example, the rhGH-Fc fusion protein was purified as follows:
1. reagent(s)
The reagents used in affinity chromatography, anion exchange chromatography and cationic hydrophobic complex chromatography are shown in tables 1-3.
TABLE 1 affinity chromatography reagents
TABLE 2 anion exchange chromatography reagents
TABLE 3 cationic hydrophobic composite chromatographic reagents
2. Step (a)
Dividing the sample to be treated into 4 parts, respectively carrying out affinity chromatography and low pH virus inactivation on each part of sample, then merging the obtained treatment solutions, respectively carrying out anion exchange chromatography on the two parts of the treatment solutions, merging the obtained treatment solutions, and carrying out cation-hydrophobic composite chromatography, nanofiltration and ultrafiltration to obtain the final purified product. The method comprises the following specific steps:
2.1 affinity chromatography
2.1.1 loading: the sample to be treated is applied to an affinity chromatography column (pre-packedPro Protein A).
2.1.2 flushing 1: the affinity column was washed with 80% buffer A and 20% buffer B at a linear flow rate of 90cm/h at 3 CV.
2.1.3 flushing 2: the affinity column was washed with 100% buffer B at a linear flow rate of 180cm/h at 3 CV.
2.1.4 rinse 3: the affinity column was washed with buffer C1 at a linear flow rate of 180cm/h at 6 CV.
2.1.5 rinse 4: the affinity column was washed with buffer A at 2CV and a linear flow rate of 180cm/h.
2.1.6 rinse 5: the affinity column was washed with buffer C2 at a linear flow rate of 180cm/h at 6 CV.
2.1.7 elution: and (3) eluting the affinity chromatography column 5CV by using a buffer solution D, starting to collect the eluent when the UV280 rises to 100mAU/2mm in the eluting process, and stopping collecting after the UV280 falls to 200mAU/2mm after passing through the peak top.
2.2 Low pH Virus inactivation
2.2.1 under the condition that the finally collected sample solution in the last step is continuously and evenly mixed, acetic acid is slowly and evenly added, the pH value of the sample is regulated to 3.6+/-0.1, and the regulating time is controlled to be not more than 20 minutes.
2.2.2 stopping the mixing operation, and standing the sample at the dark room temperature (18-26 ℃) for 60+/-15 min.
2.2.3 under the condition that the sample solution is continuously and evenly mixed, the buffer solution E is slowly and evenly added, and the pH of the sample is regulated to 7.2+/-0.1. And (5) adjusting the pH value to be within 20 minutes.
2.3 anion exchange chromatography
2.3.1 loading: the solution obtained in the above step was applied to an anion exchange chromatography column (pre-packed with UniGel 30 DEAE).
2.3.2 flushing: the anion exchange chromatography column was washed with buffer A at a linear flow rate of 90cm/h at 2 CV.
2.3.3 elution: the buffer B30CV linear elution was performed with buffer A and buffer B at 0-100%. When the UV280 reaches the peak (the following two conditions are preferable condition 1, the peak judgment reference condition 1 is that the absorbance value of the UV280 exceeds 700mAU/2mm, the peak judgment reference condition 2 is that the numerical value of the UV280 changes by plus or minus 50mAU and fluctuation starts to enter a gentle state), the collection of protein liquid is started by 2CV, and the collection is stopped after the UV280 is reduced to 300mAU/2 mm.
2.4 cationic hydrophobic Complex chromatography
2.4.1 loading: loading the finally collected solution to a cation hydrophobic composite chromatographic column (pre-filled with Diamond MMC) which is subjected to balancing in advance, and collecting the flow-through liquid when the UV280nm of the flow-through liquid reaches more than 50mAU/2 mm; the total sample injection amount of the sample is controlled not to exceed the sample loading amount specified by the chromatographic column. The flow through collected in this step was used as the first stage collection.
2.4.2 flushing: the cationic hydrophobic interaction chromatography column was washed with buffer solution at 2CV until the UV280nm was reduced to 100mAu/2mm and collection was stopped. The collected flow-through from this step served as the second stage collection.
2.5 nanofiltration
And filtering the finally collected solution by adopting a virus filtering membrane, and collecting filtrate.
2.6 Ultrafiltration
Ultrafiltering the filtrate obtained in the last step with ultrafilter membrane (30 KD), and collecting the trapped fluid to obtain purified product.
2.7 determination
The compositions of the purified intermediates of the affinity chromatography (abbreviated as "chromatography one"), the anion exchange chromatography (abbreviated as "chromatography two") and the cation hydrophobic composite chromatography (abbreviated as "chromatography three") are respectively determined by the following determination methods:
determination of rhGH-Fc fusion protein content: the protein content of the purified products of each step is determined by adopting the general rule 0731Lowry method of three parts (2015 edition) of Chinese pharmacopoeia.
HCP protein assay: HCP protein assay was performed using the CHO HCP ELISA Kit kit.
SEC-HPLC purity analysis: the chromatographic column adopts TSK3000SW, the mobile phase is phosphate buffer solution (pH 6.8) containing 0.3mol/L NaCl, the loading amount is 100 mu L, the flow rate is 0.6mL/min, the detection wavelength is 280nm, and the detection time is set to 22min.
Related protein (RP-HPLC) analysis: chromatographic column C18, mobile phase A is 0.1% TFA aqueous solution, mobile phase B is 0.1% TFA acetonitrile solution, gradient elution condition is 0-5 min,45% mobile phase A and 55% mobile phase B; 5-45 min,45% -31% of mobile phase A,55% -69% of mobile phase B, the flow rate is 0.6mL/min, the detection wavelength is 280nm, the sample loading amount is 50 mu L, and the detection time is set to be 45min.
The results are shown in Table 4. It can be seen that a part of impurities such as HCP can be removed after the first chromatography treatment, and that impurities such as HCP can be further efficiently removed after the second chromatography treatment. Therefore, the purification method provided by the application can effectively remove impurities, and the yield of the target protein is high.
Table 4 200l clinical batch stock preparation purification intermediate quality data
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Note that: "N/A" means undetected.
Example 2
In this example, the problem of protein precipitation was investigated using different treatments, as follows:
1. the inventors added glycine, arginine, mgCl to buffer D of affinity chromatography (without poloxamer 188) and buffer B of anion exchange chromatography (without poloxamer 188) of example 1, respectively 2 Histidine and collecting the effluent after eluting in two chromatographic modes, wherein each of the effluent is divided into three parts, one part is used for directly measuring turbidity, and the other part is used for measuring after filteringTurbidity of the filtrate, after another portion was allowed to stand overnight, turbidity was measured.
As shown in FIG. 2, the addition of glycine improves protein precipitation, but protein precipitation in unfiltered solution is still serious, protein precipitation is obviously improved after standing overnight, but protein precipitation still exists, and protein precipitation is continued even if protein is removed by filtration.
For histidine, arginine and MgCl 2 In other words, the addition of the modified starch significantly promotes protein precipitation, and even though the modified starch is left standing overnight and filtered, the modified starch still has obvious protein precipitation.
For poloxamer 188, the addition of both 0.05% poloxamer 188 and 0.1% poloxamer 188 significantly improved protein precipitation with little apparent precipitation compared to the addition of 0.01% poloxamer 188.
The results of anion exchange chromatography are shown in FIG. 3, and the addition of glycine and histidine improves protein precipitation, but the protein precipitation in unfiltered solution is serious, the protein precipitation is obviously improved after standing overnight, but protein precipitation still exists, and protein precipitation is continued even if the protein is removed by filtration.
For arginine and MgCl 2 In other words, protein precipitation is promoted at low concentrations, and improved after high concentrations, standing overnight and filtration, but still significant protein precipitation occurs.
For poloxamer 188, the addition of both 0.05% poloxamer 188 and 0.1% poloxamer 188 significantly improved protein precipitation with little apparent precipitation compared to the addition of 0.01% poloxamer 188.
2. The inventors added 64mg/ml sucrose, 64mg/ml trehalose, 10mM EDTA, 0.01% Tween-80, 2% DMSO, 0.05-0.5% poloxamer 188 and 2% ethanol to buffer D of affinity chromatography (without poloxamer 188) and buffer B of anion exchange chromatography (without poloxamer 188) of example 1, respectively, and collected the effluent after elution in two chromatography modes, put them into a penicillin bottle, and under irradiation of a formulation searchlight, it was observed whether floc was generated or not, and the more "+" indicates the more flocs and the higher the turbidity degree.
The results are shown below, with "+" indicating a turbidity level, and with more "+" indicating a higher turbidity level. It can be seen that poloxamer 188 has a better dissolution-promoting effect than other dissolution-promoting agents, and can effectively reduce protein precipitation.
(1) Sucrose 64 mg-ml (+ ++)
(2) Trehalose 64mg per ml (+ ++)
(3)10mM EDTA(++++)
(4) Tween-80,0.01% (++)
(5)DMSO 2％(+++)
(6) Poloxamer 188 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5% (+)
(7) Ethanol 2% ++ + (+)
3. The components in the elution buffer of chromatography II (buffer B) were studied and the effluent obtained from the different components was compared for the presence of flocs in the same manner as in step 2 above.
The results are shown below, and it can be seen that the addition of histidine, sodium chloride and more than 0.03% poloxamer 188 is effective in preventing protein precipitation.
1.20mM PB,250mM sodium chloride, pH7.2 (ineffective, protein precipitated)
2.25mM Tris,250mM sodium chloride, pH7.2 (ineffective, protein precipitated)
3.40mM PB,250mM sodium chloride, pH7.2 (ineffective, protein precipitated)
4.20mM PB,250mM sodium chloride pH6.5 (null, protein precipitation)
5.32.5mM L-histidine, 7.5mM L-histidine hydrochloride, 250mM sodium chloride, pH6.8 (null, protein precipitation)
6.32.5mM L-histidine, 7.5mM L-histidine hydrochloride, 0.3M arginine, 0.15M sodium chloride, pH6.8 (null, protein precipitation)
7.200mM Tris,250mM sodium chloride, pH6.8 (ineffective, protein precipitated)
8.32.5mM L-histidine, 7.5mM L-histidine hydrochloride, 0.8M arginine, 250mM sodium chloride, pH6.8 (null, protein precipitation)
9.30mM citric acid/sodium citrate, 30mg/ml sucrose, 250mM sodium chloride, 35mM Tris, pH6.8 (ineffective, protein precipitated, conditions unsuitable)
10.20 mM-histidine, 30mg/ml sucrose, 250mM sodium chloride, 35mM Tris, pH6.8 (conditions not applicable)
11.6.554mg/ml Na 2 HPO 4 0.377mg/ml anhydrous citric acid, 20mg/ml sucrose, 40mg/ml mannitol, 250mM sodium chloride, pH6.8 (ineffective, protein precipitated, conditions unsuitable)
12.32.5mM L-histidine, 7.5mM L-histidine hydrochloride, 250mM sodium chloride, 0.4% poloxamer 188, pH6.8 (conditions applicable, no protein precipitation)
13.32.5mM L-histidine, 7.5mM L-histidine hydrochloride, 250mM sodium chloride, 0.05% poloxamer 188, pH6.8 (conditions applicable, no protein precipitation)
14.32.5mM L-histidine, 7.5mM L-histidine hydrochloride, 250mM sodium chloride, 0.04% poloxamer 188, pH6.8 (conditions applicable, no protein precipitation)
15.32.5mM L-histidine, 7.5mM L-histidine hydrochloride, 250mM sodium chloride, 0.01% poloxamer 188, pH6.8 (conditions unsuitable, protein precipitated)
16.32.5mM L-histidine, 7.5mM L-histidine hydrochloride, 250mM sodium chloride, 0.03% poloxamer 188, pH6.8 (conditions unsuitable, protein precipitated)
Example 3
In this example, the removal of HCP proteins using different treatments was studied, as follows:
the inventors designed the composition of buffer C1 and the washing procedure of example 1 as follows, and performed affinity chromatography in accordance with the method of example 1 to determine the HCP protein content of the eluted effluent.
(1) 30mM acetic acid, buffer pH 5.5, rinse 6CV;
(2) 30mM acetic acid, 0.8M arginine, buffer pH 5.5, rinse 6CV;
(3) 600mM acetic acid, 0.8M arginine, buffer pH 5.5, rinse 6CV;
(4) 100mM sodium octoate, 20mM phosphate buffer, pH 7.2, rinse 6CV;
(5) 100mM sodium octoate, 20mM phosphate buffer, pH 7.2, rinse 3CV;
(6) 1% Triton 100, 20mM PB, buffer pH 7.2, 6CV.
As a result, as shown in FIG. 4, HCP protein was efficiently removed by using sodium octoate, and the removal effect of HCP protein was better when the washing volume was at least 6CV.
To further confirm the HCP protein content of the eluted effluent, the invention proceeds with affinity chromatography as in example 1 and further studies on the composition of buffer C2.
(1) 50mM sodium octoate, 0.1M Tris, buffer pH 9.0, 3CV wash;
(2) 100mM sodium octoate, 30mM acetic acid, buffer pH 7.2, rinse 6CV;
(3) 500mM acetic acid, 445.5mmol/L sodium hydroxide, buffer pH 5.5, rinse 6CV;
(4) 200mM sodium octoate, 30mM acetic acid, buffer pH 7.2, rinse 4CV;
(5) 600mM acetic acid, 0.8M arginine, buffer pH 5.5, rinse 3CV;
(6) 20mM phosphate, 1.5M sodium chloride, buffer pH 7.2, rinse 4CV.
As shown in FIG. 5, the washing of 6CV with 500mM acetic acid at pH 5.5 effectively removes HCP protein, and the washing of at least 6CV results in better removal of HCP protein.
Example 4
Using the method of example 1, 2L, 15L, 30L and 200L of the cell sap containing rhGH-Fc fusion protein were purified, and the HCP content in the effluent containing rhGH-Fc fusion protein collected by affinity chromatography, anion exchange chromatography and cation-hydrophobic complex chromatography was measured, respectively.
The results are shown in FIG. 6. It can be seen that HCP protein can be effectively removed by adopting the purification method of the invention, and the residual quantity is controlled within a reasonable range, thereby meeting the requirement of below 100 ppm.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.
Claims (10)
1. A method for purifying a recombinant human growth hormone-Fc fusion protein, comprising:
filtering cell sap containing recombinant human growth hormone-Fc fusion protein, and collecting filtrate;
subjecting the filtrate to affinity chromatography;
wherein, the eluent adopted by the affinity chromatography contains poloxamer 188.
2. The method of claim 1, wherein the filler used for affinity chromatography is selected from the group consisting of Protein a.
3. The method according to claim 1, wherein the poloxamer 188 content in the eluent is more than 0.03% by volume, preferably between 0.04 and 0.50% by volume;
optionally, the pH of the eluate is 3.0 to 4.5, preferably 3.2 to 3.8;
optionally, the eluent contains acid and alkali, wherein the acid is at least one selected from acetic acid, a citric acid system, glycine, hydrochloric acid, phosphoric acid and sulfuric acid, preferably acetic acid; the alkali is at least one selected from sodium hydroxide, tris, disodium hydrogen phosphate, arginine, calcium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate and ammonium sulfate, preferably sodium hydroxide.
4. The method of claim 1, wherein the affinity chromatography comprises: washing the affinity chromatographic column by using a first buffer solution and an eluent in sequence;
the first buffer solution contains sodium octoate;
optionally, the pH of the first buffer is 7.0 to 9.0, preferably 7.0 to 7.5;
optionally, the concentration of sodium octoate in the first buffer is 50 to 150mM, preferably 80 to 120mM;
optionally, the first buffer comprises phosphate.
5. The method of claim 4, wherein the affinity chromatography comprises:
a) Washing the affinity chromatography column with at least one of a second buffer and a third buffer;
b) Washing the affinity chromatography column with the first buffer;
c) Washing the affinity chromatography column with a second buffer or a third buffer;
d) Washing the affinity chromatography column with a fourth buffer;
e) Eluting the affinity chromatographic column by using the eluent, and collecting effluent;
the pH value of the second buffer solution is 7.0-8.0, preferably 7.0-7.5; the pH value of the third buffer solution is 7.0-8.0, preferably 7.0-7.5; the pH value of the fourth buffer solution is 4.0-6.0, preferably 5.2-5.8;
Optionally, the second buffer is selected from phosphate buffer, the third buffer is selected from phosphate buffer containing sodium chloride, and the fourth buffer contains acid and alkali;
optionally, the acid is selected from at least one of acetic acid, citric acid system, glycine, hydrochloric acid, phosphoric acid, sulfuric acid, preferably acetic acid; the alkali is at least one selected from sodium hydroxide, tris, disodium hydrogen phosphate, arginine, calcium hydroxide, sodium carbonate, sodium bicarbonate, sodium acetate and ammonium sulfate, preferably sodium hydroxide.
6. The method according to claim 5, wherein in step a), the total amount of flushing of at least one of the second buffer and the third buffer is not less than 3 column volumes, and the linear flow rate is 60 to 120cm/h;
optionally, in step b), the flushing amount of the first buffer solution is not less than 6 times of the column volume, and the linear flow rate is 120-180 cm/h;
optionally, in step c), the second buffer solution or the third buffer solution has a flushing amount of not less than 2 times of column volume and a linear flow rate of 120-180 cm/h;
optionally, in step d), the fourth buffer solution has a flushing amount of not less than 6 times of the column volume and a linear flow rate of 120-180 cm/h;
Optionally, in step e), the eluting amount of the eluent is not less than 5 times of the column volume, and the flow rate is linear flow rate of 120-180 cm/h;
optionally, between steps a) and b), further comprising:
f) And flushing the affinity chromatography column by using the second buffer solution or the third buffer solution, wherein the flushing amount is not less than 3 times of column volume, and the linear flow rate is 120-180 cm/h.
7. The method as recited in claim 1, further comprising:
subjecting the effluent collected by eluting the affinity chromatographic column with the eluent to anion exchange chromatography and cation hydrophobic composite chromatography, and collecting the effluent containing recombinant human growth hormone-Fc fusion protein;
nanofiltration is carried out on the effluent liquid containing the recombinant human growth hormone-Fc fusion protein, and nanofiltration effluent liquid is collected;
ultrafiltering the nanofiltration effluent, and collecting the retentate to obtain the purified recombinant human growth hormone-Fc fusion protein.
8. The method according to claim 7, wherein the elution buffer used for the anion exchange chromatography contains poloxamer 188 in an amount of more than 0.03 vol%, preferably between 0.04 and 0.50 vol%;
optionally, the anion exchange chromatography adopts a first eluting buffer solution and a second eluting buffer solution to carry out gradient elution during the eluting process,
The first elution buffer comprises: l-histidine and L-histidine hydrochloride, the pH value is 6.6-7.0;
the second elution buffer comprises: l-histidine, L-histidine hydrochloride, sodium chloride and poloxamer 188, and the pH value is 6.6-7.0;
the gradient elution mode is as follows: 0-100% of the second elution buffer 30 column volumes is eluted linearly;
optionally, the washing solution employed in the cationic hydrophobic complex chromatography comprises: l-histidine, L-histidine hydrochloride and sodium chloride, and the pH value is 6.6-7.0.
9. The method of claim 7, wherein the effluent collected by eluting the affinity chromatography column with an eluent is virus inactivated prior to performing the anion exchange chromatography and the cation hydrophobic complex chromatography;
optionally, the viral inactivation comprises:
adding acetic acid into the effluent liquid collected by eluting the affinity chromatographic column by using the eluent until the pH value is 3.0-4.0, standing at the dark room temperature for 0.5-1.5 hours, and then regulating the pH value of the solution to 7.0-7.5 by using Tris-HCl buffer solution.
10. A method for purifying a recombinant human growth hormone-Fc fusion protein, comprising:
(1) Affinity chromatography:
loading: loading a sample to be treated into a Protein A affinity chromatography column;
Flushing 1: washing the affinity chromatography column 3CV with 80% buffer A and 20% buffer B, wherein the linear flow rate is 90cm/h, the buffer A is selected from disodium hydrogen phosphate dodecahydrate and sodium dihydrogen phosphate monohydrate, the pH value is 7.2 + -0.2, and the buffer B is selected from disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate and sodium chloride, and the pH value is 7.2 + -0.2;
flushing 2: washing the affinity chromatography column 3CV with 100% buffer B at a linear flow rate of 180cm/h;
flushing 3: washing the affinity chromatography column 6CV with a buffer solution C1, wherein the linear flow rate is 180cm/h, the buffer solution C1 is selected from disodium hydrogen phosphate dodecahydrate, sodium dihydrogen phosphate monohydrate and sodium octoate, and the pH value is 7.2+/-0.2;
flushing 4: washing the affinity chromatographic column 2CV with buffer A at linear flow rate of 180cm/h;
flushing 5: washing the affinity chromatographic column 6CV with a buffer solution C2, wherein the linear flow rate is 180cm/h, the buffer solution C2 is selected from acetic acid and sodium hydroxide, and the pH value is 5.5+/-0.2;
eluting: eluting the affinity chromatographic column 5CV with a buffer solution D, wherein the elution is started when the UV280 rises to 100mAU/2mm in the elution process, the UV280 falls to 200mAU/2mm after passing through the peak top, and the collection is stopped, and the buffer solution D is selected from acetic acid, 0.04 volume percent poloxamer 188 and sodium hydroxide, and the pH value is 3.5+/-0.2;
(2) Low pH virus inactivation:
adding acetic acid under the condition that the finally collected sample solution is continuously and uniformly mixed, and regulating the pH of the sample to 3.6+/-0.2;
stopping uniformly mixing, and standing the sample at the dark room temperature for 60+/-15 min;
adding a buffer solution E under the condition that the sample solution after standing is continuously and uniformly mixed, and regulating the pH value of the sample to 7.2+/-0.2, wherein the buffer solution E is selected from the group consisting of tris (hydroxymethyl) aminomethane and tris (hydroxymethyl) aminomethane hydrochloride, and the pH value of the buffer solution E is 9.0+/-0.2;
(3) Anion exchange chromatography:
loading: loading the solution obtained in the previous step into an anion exchange chromatographic column;
flushing: washing the anion exchange chromatographic column 2CV with a buffer F, wherein the linear flow rate is 90cm/h, the buffer F is selected from L-histidine and L-histidine hydrochloride, and the pH value is 6.8+/-0.2;
eluting: performing 0-100% buffer solution G30CV linear elution by using a buffer solution F and a buffer solution G; starting to collect protein liquid when UV280 reaches the peak and 2CV, stopping collecting after the UV280 is reduced to 300mAU/2mm, wherein the buffer solution G is selected from L-histidine, L-histidine hydrochloride, sodium chloride and 0.04 volume percent poloxamer 188, and the pH value is 6.8+/-0.2;
(4) Cation hydrophobic composite chromatography:
loading: loading the finally collected solution to a cation hydrophobic composite chromatographic column, and collecting the flow-through liquid when the UV280nm of the flow-through liquid reaches more than 50mAU/2 mm; the flow-through liquid collected in the step is used as a first section collecting liquid;
Flushing: flushing the cationic hydrophobic complex chromatographic column 2CV by using a buffer solution H, wherein the buffer solution H is selected from L-histidine, L-histidine hydrochloride and sodium chloride, and the pH value is 6.8+/-0.2, and stopping collecting until the UV280nm is reduced to 100mAu/2 mm;
(5) Nanofiltration and ultrafiltration:
and combining the first-stage collecting liquid and the second-stage collecting liquid, and carrying out nanofiltration and ultrafiltration to obtain a purified product.
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