CN114316072B - Purification method of recombinant human GLP-1-Fc fusion protein - Google Patents
Purification method of recombinant human GLP-1-Fc fusion protein Download PDFInfo
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Abstract
The invention discloses a purification method of recombinant human GLP-1-Fc fusion protein, which comprises the following steps: (1) Pretreating a cell fermentation broth of the recombinant human GLP-1-Fc fusion protein to obtain a crude recombinant human GLP-1-Fc fusion protein; (2) preparing an affinity chromatography column; (3) Loading the crude recombinant human GLP-1-Fc fusion protein sample onto an affinity chromatographic column, eluting with an eluent after leaching treatment, and collecting the eluent containing a sample peak to obtain a pure recombinant human GLP-1-Fc fusion protein product. According to the invention, through optimizing the affinity filler and optimizing the pH value of the eluting buffer solution and the pH value of the eluting buffer solution, the optimal collection range and the optimal loading dynamic load of the eluting solution are screened, the HCP can be removed to the maximum extent by the finally established purification method, the eluting volume is small, and the purity and the yield of the prepared recombinant human GLP-1-Fc fusion protein are high.
Description
Technical Field
The invention relates to a purification method of fusion protein, in particular to a purification method of recombinant human GLP-1-Fc fusion protein, belonging to the purification field of recombinant human GLP-1-Fc fusion protein.
Background
In recent years, the prevalence of Chinese diabetes is increasing year by year, and the prevalence of type 2 diabetes complications is urgent by constantly developing excellent medicines, stably and effectively controlling the blood sugar level of patients, and reducing the prevalence of the patients.
The recombinant human GLP-1-Fc fusion protein is formed by connecting the C terminal ends of two recombinant human glucagon-like peptide-1 analogues with IgG4 Fc (CH 2) and Fc (CH 3) through short peptides respectively, the half life is prolonged to nearly 4 days, 90% of the amino acid sequence of the recombinant human GLP-1-Fc fusion protein has homology with endogenous human GLP-1, and the recombinant human GLP-1-Fc fusion protein promotes insulin secretion and reduces fasting blood glucose of a patient by activating GLP-1 receptors; inhibiting postprandial glucagon secretion and reducing postprandial blood glucose in a patient; prolonging gastric discharge time, reducing appetite, improving islet beta cell function, and reducing blood sugar.
Affinity chromatography is a key step in the purification of Fc fusion proteins, capturing the protein of interest by specific adsorption, but requires an optimized process to maintain a balance of protein purity, yield and host protein HCP removal capacity, as well as multimeric content. Along with the improvement of the upstream cell culture process and the improvement of the expression level, the impurities are increased, and the effect between the impurities and the target proteins and the existence of nonspecific adsorption cause great difficulty to the affinity purification process.
Disclosure of Invention
The main object of the present invention is to provide a method for purifying recombinant human GLP-1-Fc fusion protein, which can remove host cell residual protein (HCP) to the maximum extent under the premise of ensuring purity and yield.
The above object of the present invention is achieved by the following technical solutions:
a method of purifying a recombinant human GLP-1-Fc fusion protein comprising:
(1) Pretreating a cell fermentation broth of the recombinant human GLP-1-Fc fusion protein to obtain a crude recombinant human GLP-1-Fc fusion protein; (2) preparing an affinity chromatography column; (3) Loading the recombinant human GLP-1-Fc fusion protein crude sample on an affinity chromatographic column, eluting by using an eluent after leaching treatment, and collecting the eluent containing a sample peak to obtain a recombinant human GLP-1-Fc fusion protein pure product.
As a preferred embodiment, the method for pretreating the cell fermentation broth of the recombinant human GLP-1-Fc fusion protein to obtain a crude recombinant human GLP-1-Fc fusion protein comprises the following steps: adding a detergent into a deep filtration collection liquid of a cell fermentation liquid of the recombinant human GLP-1-Fc fusion protein, stirring, incubating and filtering to obtain a crude sample of the recombinant human GLP-1-Fc fusion protein.
As a preferred embodiment, the preparation of the affinity chromatography column according to the invention comprises: filling the column with affinity chromatography packing, and rinsing the column with a balancing solution; sterilizing the chromatographic column with NaOH solution; the column is then equilibrated with an equilibration solution, wherein the affinity chromatography packing used includes, but is not limited to, any of MabSelect Sure, eshmuno A, or Mab Capture A. According to the invention, the purification effect of different affinity chromatography fillers on purifying recombinant human GLP-1-Fc fusion proteins is examined, and the result shows that the purification result by adopting MabSelect Sure as the affinity chromatography filler is optimal, the purity and the yield of the recombinant human GLP-1-Fc fusion proteins are high, the elution volume is small, and the HCP removal effect is good, so that the MabSelect Sure is most preferably adopted as the affinity chromatography filler of the affinity chromatography column; the balancing solution is preferably 50mM Tris-HAc,150mM NaCl,pH 7.4.
As a preferred embodiment, the crude recombinant human GLP-1-Fc fusion protein is loaded on an affinity chromatography column in the step (3) and the dynamic loading of the recombinant human GLP-1-Fc fusion protein is required to be analyzed, and the loading is influenced by the content of HCP in the sample. According to the invention, through an affinity filler dynamic load optimization test, the retention time of a sample in affinity chromatography is preferably 5-6min, and the load range of a recombinant human GLP-1-Fc fusion protein sample is preferably 10-20g/L, and most preferably 8-16g/L.
As a preferred specific embodiment, the leaching treatment is sequentially performed by three leaching solutions, namely leaching 1 buffer solution, leaching 2 buffer solution and leaching 3 buffer solution, wherein the leaching 1 buffer solution is 50mM Tris-HAc,150mM NaCl,pH 7.4, and the leaching 2 buffer solution is any one of acetic acid-sodium acetate buffer solution, tris-hydrochloric acid buffer solution, citric acid buffer solution or phosphate buffer solution; most preferably, the wash 2 buffer is 50mM Tris-HCl,1M NaCl,pH 9.0.
The invention discovers through optimization experiments that the pH value of the leaching 2 buffer solution has great influence on removing some impurities which are not specifically adsorbed. The invention detects the monomer content and the host cell protein impurity content of the recombinant human GLP-1-Fc fusion protein of the eluent to screen the pH of the eluting 2 buffer solution through calculating the recovery rate, finally discovers that the pH range of the eluting 2 buffer solution is preferably 5.0-9.0, especially 8.5-9.0, can remove HCP to the maximum extent, simultaneously ensures the purity and the yield of the recombinant human GLP-1-Fc fusion protein in the eluent,
experiments show that when the eluting solution is used for eluting, the eluting condition greatly affects the collection of the recombinant human GLP-1-Fc fusion protein sample, the invention determines the proper eluting pH range by detecting the monomer content and HCP content of the recombinant human GLP-1-Fc fusion protein of the eluting solution through the screening test of the pH of the eluting solution and calculating the recovery rate, and finally, the purity and the yield of the recombinant human GLP-1-Fc fusion protein in the eluting solution can be effectively improved when the pH of the eluting solution is 3.0-4.0, especially 3.5-3.7; the most preferred eluents of the present invention are: 50mM NaAc-HAc, pH 3.5.
According to the invention, experiments show that the target protein recombinant human GLP-1-Fc fusion protein and impurities are distributed differently on a column bed, so that the eluting sample quality can be better controlled by optimizing the eluting solution collecting range when the eluting solution is collected, and the preferable eluting peak collecting range is finally determined to be 50 mAU/mm-peak top-150 mAU/mm, and most preferably 100 mAU/mm-peak top-200 mAU/mm by detecting the content of the recombinant human GLP-1-Fc fusion protein monomer and host protein.
To achieve a better elution effect, tris-HCl solution with pH of 9.0 is added into the collected eluent, and the pH is adjusted to 6.5-7.0.
In order to save the cost, the invention can recycle the used affinity chromatographic column after being regenerated, rinsed and disinfected; wherein, the regeneration treatment can be carried out by adopting 1M acetic acid or 0.1M glycine solution with 3-5 times of column volume.
According to the invention, through optimizing the affinity filler, optimizing the pH of the eluting buffer solution 2 and the eluting buffer solution, optimizing the collecting range of the eluting solution and determining the optimal dynamic loading, the purification method can remove HCP to the maximum extent, and the purity and the yield of the recombinant human GLP-1-Fc fusion protein are high, so that technical support is provided for the affinity purification process of Fc fusion protein medicines.
Terms and definitions relating to the present invention
The term "recombinant human GLP-1-Fc fusion protein" is a fusion protein consisting of two recombinant human glucagon-like peptide-1 analogs, C-terminal linked to IgG4 Fc (CH 2) and Fc (CH 3), respectively, via a short peptide.
The term "HCP" is a host cell residual protein.
Drawings
FIG. 1 is a graph showing the liquid chromatography of the test example 2 when the pH of the buffer solution for elution 2 is 9.0.
FIG. 2 is a plot of affinity chromatography loading versus percent flow-through in test example 5.
FIG. 3 is a graph of retention time and dynamic binding capacity for test example 5.
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
Test instrument
The chromatographic system used in the invention is AKTA Purifier100, and the analytical high performance liquid instrument is Agilent 1260.
The detection items involved in affinity chromatography are:
a) Protein concentration, detected by an ultraviolet visible spectrophotometer UV 280;
b) Purity, detected by size exclusion high performance liquid chromatography (SEC-HPLC);
c) Host cell residual protein (HCP) was detected by enzyme-linked immunosorbent assay (ELISA).
Purity detection method of recombinant human GLP-1-Fc fusion protein
The instrument is Agilent 1260, the analytical column is TSKgel G3000SWXL molecular exclusion chromatographic column, the column temperature is 25 ℃, the mobile phase is 50mM phosphate, 500nM arginine hydrochloride, 150nM sodium chloride, the flow rate is 0.5ml/min, isocratic elution is carried out for 35min, detection is carried out at the wavelength of 280nM, the contents of high polymer, monomer and low molecular weight impurities are calculated by adopting a peak area normalization method, and the content of the monomer is the purity of the recombinant human GLP-1-Fc fusion protein.
Test example 1 Effect of affinity Filler on purification Effect of recombinant human GLP-1-Fc fusion protein
Adding 0.5% detergent into the deep filtration collection liquid of the cell fermentation liquid of the recombinant human GLP-1-Fc fusion protein, stirring at 25 ℃ for 300r/min, incubating for 6h, and filtering by using a 0.22um capsule filter to obtain an upper sample (step 1).
The affinity filler is selected from protein A affinity chromatography fillers of three manufacturers of MabSelect SuRe of GE company, eshmuno A of Miliipore company and Mab Capture A of Thermo Fisher company, and the affinity filler suitable for the invention is selected. The three experimental parameters are kept consistent, and the loading capacity is 15g/L of filler. The optimal affinity chromatography packing was selected by calculating the recovery, elution volume, and detecting the SEC-LC monomer content and HCP content of the eluate.
The equilibrium solution is 50mM Tris-HAc,150mM NaCl,pH 7.4, the buffer solution 1 is the equilibrium solution, and the buffer solution 2 is: 50mM NaAc-HAc, pH 5.6, eluent A:50mM NaAc-HAc, pH 5.5, eluent B:50mM HAc, pH 3.13; the rinsing liquid is water for injection; the disinfectant is 0.1M NaOH solution; the preservation solution is 20% ethanol.
Loading the affinity packing column, and rinsing the chromatographic column with a balancing solution with the volume of 4 times of the column (step 2 a); sterilizing the chromatographic column with 3 column volumes of NaOH solution (step 2 b); equilibrate the chromatographic column with 3 column volumes of equilibration liquid (step 2 c); slowly loading the sample so that the sample remains on the column for 6min (step 2 d); eluting the chromatographic column with 5 column volumes of elution 1 buffer (step 3 a); eluting the chromatographic column with 5 column volumes of elution 2 buffer (step 3 b); eluting the chromatographic column with eluent A and eluent B, eluting with 0-100% B and 10CV, collecting eluting peak, adding Tris-HCl with pH of 9.0, adjusting pH to 6.6 (step 4), and detecting; then regenerating with 3 column volumes of 1M acetic acid (step 5); the water for injection, the disinfectant and the water for injection are used for rinsing, disinfecting and rinsing successively, and then the water for injection is preserved by 20% ethanol (step 6).
The eluted samples were collected for detecting purity and HCP content, the concentration was measured for calculating yield, the elution volume was counted, and the final results of the different fillers are shown in table 1.
TABLE 1 purification results of recombinant human GLP-1-Fc fusion protein by affinity chromatography packing
| Affinity filler | MabSelect SuRe | Eshmuno A | Mab Capture A |
| Purity (%) | 94.6 | 94.8 | 93.1 |
| Elution volume (CV) | 3.8 | 7.7 | 4.4 |
| HCP content (ppm) | 7960 | 16125 | 8717 |
| Yield (%) | 88.5 | 90.7 | 35.7 |
As can be seen from Table 1, the purity levels of the three fillers have insignificant differences, the yield of MabCapture A is lowest, the HCP removal capacity of Eshmuno A is poor, and the maximum elution volume is unfavorable for the large scale production, so that the MabSelect SuRe is selected as an affinity chromatography filler of the recombinant human GLP-1-Fc fusion protein in comprehensive consideration.
Test example 2 test of influence of pH value of elution 2 buffer on purification effect of recombinant human GLP-1-Fc fusion protein
Adding 0.6% detergent into the deep filtration collection liquid of the cell fermentation liquid of the recombinant human GLP-1-Fc fusion protein, stirring at 23 ℃ for 300r/min, incubating for 8 hours, and filtering by using a 0.22um capsule filter to obtain an upper sample (step 1).
The affinity filler is MabSelect SuRe of GE company, and the size of the chromatographic column is 0.5cm x 10.2cm. Four experiments were performed with 50mM Tris-HCl,1M NaCl, pH 5.6,7.0,8.0,9.0 for each buffer, and other parameters kept consistent with a loading of 18g/L of filler. The optimum pH of the elution 2 buffer was selected by calculating the yield, detecting the SEC-LC monomer content and HCP content of the eluate.
The equilibrium solution is 50mM Tris-HAc,150mM NaCl,pH 7.4, the buffer solution 1 is the equilibrium solution, and the buffer solution 3 is: 50mM NaAc-HAc, pH 5.0, eluent: 50mM NaAc-HAc, pH 3.5, eluent collection range of 50-capping-50 mAU/mM; the rinsing liquid is water for injection; the disinfectant is 0.1M NaOH solution; the preservation solution is 20% ethanol.
Loading the affinity packing column, and rinsing the chromatographic column with a balancing solution with the volume of 5 times of the column volume (step 2 a); sterilizing the chromatographic column with 4 column volumes of NaOH solution (step 2 b); equilibrate the column with 5 column volumes of equilibration liquid (step 2 c); slowly loading the sample so that the sample remains on the column for 6min (step 2 d); eluting the chromatographic column with 5 column volumes of eluent 1 (step 3 a); eluting the chromatographic column with 5 column volumes of eluent 2 (step 3 b); eluting the chromatographic column with 3 times the column volume of eluent 3 (step 3 c); eluting the chromatographic column with 5 times of column volume eluent, collecting eluting peak, adding Tris-HCl with pH of 9.0, adjusting pH to 6.8 (step 4), and detecting; then regenerated with 4 column volumes of 1M acetic acid (step 5); the water for injection, the disinfectant and the water for injection are used for rinsing, disinfecting and rinsing successively, and then the water for injection is preserved by 20% ethanol (step 6).
The eluted samples were collected for detecting purity and HCP content, the concentration was measured, the yield was calculated, and the final results of purification of recombinant human GLP-1-Fc fusion protein using different pH eluting 2 buffers are shown in Table 2.
TABLE 2 affinity purification results with different pH of the buffer for elution 2
| Elution 2 buffer pH | pH 5.6 | pH 7.0 | pH 8.0 | pH 9.0 |
| Purity (%) | 96.3 | 95.3 | 94.8 | 95.4 |
| HCP content (ppm) | 7075 | >5442 | >5734 | 2781 |
| Yield (%) | 95.8 | 96.1 | 92.4 | 92.7 |
As can be seen from Table 2, the pH of the elution 2 buffer was changed without significant changes in monomer purity and yield in the eluate, but with the best HCP removal capacity when the pH of the solution was increased to 9.0, 50mM Tris-HCl,1M NaCl,pH 9.0 was chosen as the elution 2 buffer for affinity chromatography of the recombinant human GLP-1-Fc fusion protein.
Test example 3 Effect of pH value of eluent on purification Effect of recombinant human GLP-1-Fc fusion protein
Adding 0.4% detergent into the deep filtration collection liquid of the cell fermentation liquid of the recombinant human GLP-1-Fc fusion protein, stirring at 26 ℃ for 300r/min, incubating for 7h, and filtering by using a 0.22um capsule filter to obtain an upper sample (step 1).
The affinity filler is MabSelect SuRe of GE company, the size of a chromatographic column is 0.5cm, 10.2cm, and the loading capacity is 18g/L of filler.
The equilibrium solution is 50mM Tris-HAc,150mM NaCl,pH 7.4, the buffer solution 1 is the equilibrium solution, and the buffer solution 2 is: 50mM NaAc-HAc,1M NaCl,pH 5.0, elution 3 buffer was: 50mM NaAc-Hac, pH 5.0; the eluent is 50mM NaAc-HAc, and the pH is selected to be 3.4, 3.5 and 3.6; the rinsing liquid is water for injection; the disinfectant is 0.1M NaOH solution; the preservation solution is 20% ethanol.
Loading the affinity packing column, and rinsing the chromatographic column with a balancing solution with the volume of 5 times of the column volume (step 2 a); sterilizing the chromatographic column with 3 column volumes of NaOH solution (step 2 b); equilibrate the column with 5 column volumes of equilibration liquid (step 2 c); slowly loading the sample so that the sample remains on the column for 7min (step 2 d); eluting the chromatographic column with 4 column volumes of eluent 1 (step 3 a); eluting the chromatographic column with 5 column volumes of eluent 2 (step 3 b); eluting the chromatographic column with 4 column volumes of eluent 3 (step 3 c); eluting the chromatographic column by using eluent with different pH values and 5 times of column volume, collecting the eluent, adding Tris-HCl with pH value of 9.0 into the eluate, regulating the pH value to 7.0 (step 4), and detecting; then regenerating with 5 column volumes of 1M acetic acid (step 5); the water for injection, the disinfectant and the water for injection are used for rinsing, disinfecting and rinsing successively, and then the water for injection is preserved by 20% ethanol (step 6).
The eluted samples were collected for detecting purity and HCP content, concentration was measured, yield was calculated, and the final results of purification of recombinant human GLP-1-Fc fusion protein using different pH eluents are shown in Table 3.
TABLE 3 influence of different pH of the eluate on the affinity purification results
| Eluent pH | pH 3.4 | pH 3.5 | pH 3.6 |
| Purity content (%) | 95.8 | 96.3 | 97.0 |
| HCP content (ppm) | 6781 | 6357 | 4171 |
| Yield (%) | 100.5 | 99.2 | 90.6 |
As is clear from Table 3, the host cell protein removal was best when eluted at pH 3.6, but the yield was lower. There is no significant difference in the purity and yield of the elution peak under both conditions when eluting at pH 3.4 and pH 3.5, but the target protein may not be stable when eluting at lower pH. In summary, 50mM NaAc-HAc, pH 3.5 was chosen as the affinity chromatography eluent for recombinant human GLP-1-Fc fusion protein.
Experimental example 4 influence of elution peak collection range on purification effect of recombinant human GLP-1-Fc fusion protein experimental test 0.7% detergent was added to a depth filtration collection solution of a cell fermentation broth of recombinant human GLP-1-Fc fusion protein, stirred at 26 ℃ for 300r/min, incubated for 8 hours, and then filtered with a 0.22um capsule filter to obtain a sample (step 1).
The affinity filler is MabSelect SuRe of GE company, the size of a chromatographic column is 0.5cm, the size of the chromatographic column is 10.2cm, and the loading capacity is 20g/L of filler.
The equilibrium solution is 50mM Tris-HAc,150mM NaCl,pH 7.4, the buffer solution 1 is the equilibrium solution, and the buffer solution 2 is: 50mM NaAc-HAc,1M NaCl,pH 9.0, elution 3 buffer was: 50mM NaAc-Hac, pH 5.0; the eluent is 50mM NaAc-HAc, pH 3.5; the rinsing liquid is water for injection; the disinfectant is 0.1M NaOH solution; the preservation solution is 20% ethanol.
Loading the affinity packing column, and rinsing the chromatographic column with a balancing solution with the volume of 4 times of the column (step 2 a); sterilizing the chromatographic column with 4 column volumes of NaOH solution (step 2 b); equilibrate the column with 5 column volumes of equilibration liquid (step 2 c); slowly loading the sample so that the sample remains on the column for 7min (step 2 d); eluting the chromatographic column with 3 column volumes of eluent 1 (step 3 a); eluting the chromatographic column with 5 column volumes of eluent 2 (step 3 b); eluting the chromatographic column with 4 column volumes of eluent 3 (step 3 c); eluting the chromatographic column with 5 times of column volume eluent, collecting the eluting peak in a segmented manner, adding Tris-HCl with pH of 9.0 into the eluting collecting liquid, regulating the pH to 6.7 (step 4), and detecting; then regenerated with 4 column volumes of 1M acetic acid (step 5); the water for injection, the disinfectant and the water for injection are used for rinsing, disinfecting and rinsing successively, and then the water for injection is preserved by 20% ethanol (step 6).
Samples collected from each elution stage were collected to determine the monomer, polymer and low molecular weight and HCP content, and the specific results are shown in Table 4.
TABLE 4 influence of the collection ranges of elution peaks of affinity chromatography on the affinity purification results
Note that: "-" indicates no detection.
As is clear from Table 4, the high polymer and HCP are mainly concentrated at the tail of the peak, the impurity can be removed from the peak at the tail of the peak, the yield and the impurity distribution condition are comprehensively considered, and finally the collection range of the elution peak of the protein A affinity chromatography is 50 mAU/mm-the main peak-150 mAU/mm.
Test example 5 test of the Effect of affinity Filler dynamic Loading on the purification Effect of recombinant human GLP-1-Fc fusion protein
Adding 0.7% detergent into the deep filtration collection liquid of the cell fermentation liquid of the recombinant human GLP-1-Fc fusion protein, stirring at 25 ℃ for 300r/min, incubating for 6h, and filtering by using a 0.22um capsule filter to obtain an upper sample (step 1).
The affinity filler is MabSelect SuRe of GE company, and the size of a chromatographic column is 0.5cm x 10.2cm; the equilibration solution, i.e., the leacheate, was 50mM Tris-HAc,150mM NaCl,pH 7.4.
Loading the affinity packing column, and rinsing the chromatographic column with a balancing solution with the volume of 4 times of the column (step 2 a); sterilizing the chromatographic column with 4 column volumes of NaOH solution (step 2 b); equilibrate the column with 5 column volumes of equilibration liquid (step 2 c); slowly loading, wherein the loading concentration is 3.52g/L, the loading volumes are different, and the retention time is 3min,5min and 7min respectively.
Collecting the flow-through liquid, detecting the content of target protein, and determining the residual percentage (C/C 0 ) The maximum dynamic loading is determined. C (C) 0 For the target protein concentration in the sample, C is the target protein concentration in the flow-through. According to dynamicsLoad experimental parameters and test results, the load and flow through percentage (C/C 0 ) See fig. 1.
Equations (1) - (3) are empirical equations for calculating filler loading at different retention times, and based on the results of table 5, the filler loading at different retention times for 5% and 10% sample flow-through is calculated, and based on the loadings, the DBCmax and K values are calculated. The results of the calculations are shown in Table 5 and the retention time versus dynamic binding capacity is plotted in FIG. 2.
TABLE 5 affinity chromatography dynamic loading analysis
According to the result, the retention time of the recombinant human GLP-1-Fc fusion protein is 6min, the carrying capacity of a 5% sample when flowing through is 25g/L, the safety factor is 0.8, and the carrying capacity is initially set to be less than or equal to 20g/L.
Considering that the affinity chromatography loading will have an effect on the removal of HCPs, the present invention investigated the relationship between HCP content and loading. The load of the recombinant human GLP-1-Fc fusion protein affinity chromatography is determined by calculating the yield, detecting the purity of SEC-LC and the content of HCP. The experimental parameters and results are shown in Table 6.
TABLE 6 determination of affinity chromatography load Range of recombinant human GLP-1-Fc fusion proteins
| Loading capacity (g/L) | 20 | 10 |
| Purity (%) | 96.3 | 96.8 |
| HCP content (ppm) | 6357 | 8046 |
| Yield (%) | 99.2 | 90.1 |
As can be seen from table 6, there is no significant change in the purity of the eluting peak monomer at both loadings, but the yield at lower loadings is significantly reduced and host protein removal is poor, so in order to ensure yield and sample quality, the lower limit of the affinity chromatography loading of the recombinant human GLP-1-Fc fusion protein needs to be set. The final recombinant human GLP-1-Fc fusion protein has the affinity chromatography load range of 10-20g/L by comprehensively considering the filler utilization rate and the sample quality.
Claims (9)
1. A method for purifying a recombinant human GLP-1-Fc fusion protein, comprising:
(1) Pretreating a cell fermentation broth of the recombinant human GLP-1-Fc fusion protein to obtain a crude recombinant human GLP-1-Fc fusion protein; (2) preparing an affinity chromatography column; (3) Loading the recombinant human GLP-1-Fc fusion protein crude sample on an affinity chromatographic column, eluting by using an eluent after leaching treatment, and collecting the eluent containing a sample peak to obtain a recombinant human GLP-1-Fc fusion protein pure product;
wherein, the preparation of the affinity chromatography column comprises the following steps: filling the column with affinity chromatography packing, and rinsing the column with a balancing solution; disinfecting the chromatographic column with a disinfecting solution; balancing the chromatographic column with balancing solution; wherein the affinity chromatography filler is MabSelect Sure or MabCapture A;
in the step (3), when the recombinant human GLP-1-Fc fusion protein crude sample is loaded on an affinity chromatography column, the loading range of the recombinant human GLP-1-Fc fusion protein crude sample is 10-20 g/L;
the leaching treatment in the step (3) is to sequentially adopt leaching 1 buffer solution, leaching 2 buffer solution and leaching 3 buffer solution for leaching treatment; wherein, the leaching 2 buffer solution is: 50mM Tris-HCl,1M NaCl,pH 9.0;
the pH value of the eluent in the step (3) is 3.4-3.6;
the collection range of the elution peak in the step (3) is 50 mAU/mm-peak top-150 mAU/mm.
2. The method of purifying according to claim 1, wherein the method of pretreating a cell fermentation broth of a recombinant human GLP-1-Fc fusion protein in step (1) to obtain a crude recombinant human GLP-1-Fc fusion protein comprises: adding a detergent into a deep filtration collection liquid of a cell fermentation liquid of the recombinant human GLP-1-Fc fusion protein, stirring, incubating and filtering to obtain a crude sample of the recombinant human GLP-1-Fc fusion protein.
3. The method according to claim 1, wherein the equilibration liquid is 50mM Tris-HAc,150mM NaCl, pH 7.4.
4. The method according to claim 1, wherein the retention time of the crude recombinant human GLP-1-Fc fusion protein on the affinity chromatography is 2-10 min after the crude recombinant human GLP-1-Fc fusion protein is loaded on the affinity chromatography column in the step (3).
5. The method according to claim 4, wherein the retention time of the crude recombinant human GLP-1-Fc fusion protein in the affinity chromatography is 5-6min after the crude recombinant human GLP-1-Fc fusion protein is loaded on the affinity chromatography column in the step (3).
6. The purification method according to claim 1, wherein the loading of the crude recombinant human GLP-1-Fc fusion protein in step (3) is in the range of 8-16g/L when the crude recombinant human GLP-1-Fc fusion protein is loaded onto an affinity chromatography column.
7. The purification method according to claim 1, wherein the pH of the eluent in step (3) is 3.5.
8. The purification method according to claim 1, wherein the eluent in step (3) is: 50mM NaAc-HAc, pH 3.5.
9. The purification method according to claim 1, wherein the step (3) is to add Tris-HCl solution having pH9.0 to the collected eluate and adjust the pH to 6.5 to 7.0.
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| CN104327187A (en) * | 2014-10-11 | 2015-02-04 | 上海兴迪金生物技术有限公司 | Recombinant human GLP-1-Fc fusion protein |
| CN107793469A (en) * | 2017-10-16 | 2018-03-13 | 上海药明生物技术有限公司 | A kind of affinity purification technique for removing host cell proteins content |
| CN109336969A (en) * | 2018-11-09 | 2019-02-15 | 杭州奕安济世生物药业有限公司 | A kind of purification process of antibody |
| CN110172102A (en) * | 2019-05-16 | 2019-08-27 | 北京军科华仞生物工程技术研究有限公司 | A kind of purification process purifying Fc fusion protein |
| WO2020132452A1 (en) * | 2018-12-20 | 2020-06-25 | Shire Human Genetic Therapies, Inc. | Purification of iduronate-2-sulfatase immunoglobulin fusion protein |
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| CN104327187A (en) * | 2014-10-11 | 2015-02-04 | 上海兴迪金生物技术有限公司 | Recombinant human GLP-1-Fc fusion protein |
| CN107793469A (en) * | 2017-10-16 | 2018-03-13 | 上海药明生物技术有限公司 | A kind of affinity purification technique for removing host cell proteins content |
| CN109336969A (en) * | 2018-11-09 | 2019-02-15 | 杭州奕安济世生物药业有限公司 | A kind of purification process of antibody |
| WO2020132452A1 (en) * | 2018-12-20 | 2020-06-25 | Shire Human Genetic Therapies, Inc. | Purification of iduronate-2-sulfatase immunoglobulin fusion protein |
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