CN112876567A - Fc fusion protein and purification method thereof - Google Patents
Fc fusion protein and purification method thereof Download PDFInfo
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- 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
Abstract
The invention relates to the field of protein purification, and particularly provides an Fc fusion protein and a purification method thereof. According to the method for purifying the Fc fusion protein, a sample to be purified is subjected to ion exchange purification to obtain the Fc fusion protein, wherein an additive is contained in an ion exchange purification buffer solution, and the additive comprises at least one of proline, histidine or aspartic acid. The inventor researches and discovers that the additive inhibits the generation of aggregates in the purification process, effectively solves the aggregation problem of Fc fusion protein, and obviously improves the recovery rate of products.
Description
Technical Field
The invention relates to the field of protein purification, and particularly relates to an Fc fusion protein and a purification method thereof.
Background
The Fc fusion protein is a bifunctional fusion protein based on antibody structure modification, and is formed by connecting a segment of functional protein molecules at the C end of a traditional antibody, namely the CH3 end of a heavy chain, through a proper linker. The constructed bifunctional fusion protein can play double or multiple biological functions through Fab of an antibody and the fused functional protein, and has wide application prospects in the fields of pharmacy and diagnosis.
At present, the platform process of monoclonal antibodies is mature, and because antibodies and Fc fusion proteins have similar molecular structures, the platform process of antibodies is often referred to in the research and development of Fc fusion proteins and in the design of production processes. Wherein, in the purification of the antibody, the target antibody in the fermentation liquid is captured by Protein A, and then the Protein purity is further improved by using 1-2 steps of ion exchange chromatography, so as to meet the purity and quality requirements of different products. Under the current technical conditions, the purification platform is most beneficial to the purification and preparation of antibody proteins. The various Fc fusion proteins produced by antibody engineering with the Fc fragment retained are also generally purified with direct reference thereto. However, there is a problem that the modified Fc fusion proteins are often not stable to antibodies, including bifunctional fusion proteins, and different fusion forms may have different purification preparation problems, such as: the low expression level results in low yield and high production and preparation cost; the product is sensitive to acid, so that a sample is easy to precipitate, aggregate and degrade after being washed and removed by acid, and further the development of a purification process is difficult; the product structure is unstable, and the development is difficult compared with an antibody preparation formula, and the like. These problems restrict the development of bifunctional fusion proteins in terms of technology and cost. In the purification process, the instability of the property of the Fc fusion protein often causes the Fc fusion protein to be unevenly adsorbed on a chromatographic filler to generate a local high-concentration area, the local high concentration can cause the increase of the viscosity of the protein along with the increase of the loading capacity, and when the protein is unstable, the problem of aggregate formation is more likely to occur.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a method for purifying Fc fusion protein, so as to relieve the problems of easy protein aggregation, low product recovery rate and cost increase in the prior art for Fc fusion protein purification.
The second object of the present invention is to provide a purified Fc fusion protein obtained by the above purification method.
In order to achieve the above purpose of the present invention, the following technical solutions are adopted:
a purification method of Fc fusion protein comprises the steps of purifying a sample to be purified through ion exchange to obtain the Fc fusion protein;
the ion exchange purification buffer contains an additive, and the additive comprises at least one of proline, histidine or aspartic acid.
Further, the additives include proline, histidine and aspartic acid.
Further, the concentration of proline, histidine or aspartic acid is 0.05-0.4 mol/L.
Further, the purified Fc fusion protein is preserved in a preservation solution, wherein the preservation solution contains a protective agent, and the protective agent comprises proline and/or histidine.
Further, the concentration of the histidine is 0.05-0.3 mol/L;
preferably, the concentration of the proline is 0.1-0.3 mol/L.
Further, the ion exchange purification comprises cation exchange chromatography or anion exchange chromatography, preferably cation exchange chromatography, further preferably Capo MMC.
Further, the sample to be purified is subjected to ion exchange purification after being captured and purified;
preferably, the method of capture purification comprises affinity chromatography, preferably Protein a affinity chromatography.
Further, after the sample to be purified is subjected to ion exchange purification, fine purification is carried out to obtain Fc fusion protein;
preferably, the method of fine purification comprises gel filtration chromatography.
Further, the sample to be purified comprises cell fermentation broth, preferably fusion protein cell fermentation broth of anti-CD 40 antibody and CTLA-4.
The purified Fc fusion protein obtained by the above purification method.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a method for purifying Fc fusion protein, which is characterized in that a sample to be purified is subjected to ion exchange purification to obtain the Fc fusion protein, wherein an ion exchange purification buffer solution contains an additive, and the additive comprises at least one of proline, histidine or aspartic acid. The inventor finds that protein macromolecules easily form a solution with higher viscosity in a high-concentration state due to the characteristics and intermolecular interaction force of the protein macromolecules in the research and development of a purification process of Fc fusion protein, protein aggregates are increased after the Fc fusion protein is subjected to ion exchange purification operation, the recovery rate is obviously reduced, and the step is a key step for limiting the purification process of the Fc fusion protein. Therefore, the inventor proposes a technical scheme for solving the problems, in the process of ion exchange purification, at least one of proline, histidine or aspartic acid is added into a purification buffer solution to reduce the viscosity of the solution, so that the high viscosity condition of the Fc fusion protein during enrichment on a chromatographic column is relieved, the generation of aggregates in the purification process is inhibited, the aggregation problem of the Fc fusion protein is effectively solved, and the recovery rate of the product is remarkably improved.
The Fc fusion protein obtained by the purification method provided by the invention has the advantages that the aggregate content is obviously reduced, the monomer content is obviously increased, and the activity and the purity of the Fc fusion protein are good.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph of the R0362 MabE purity analysis SEC after affinity chromatography in example 1 of the present invention;
FIG. 2 is a graph of the purity analysis SEC of R0362MMC E2 after complex mode chromatography in example 1 of the present invention;
FIG. 3 is a graph of the purity analysis SEC of R0362MMC E3 after complex mode chromatography in example 1 of the present invention;
FIG. 4 is a graph of purity analysis SEC for SEC E5 after gel filtration chromatography in example 1 of the present invention;
FIG. 5 shows the content of the dimer of R0362 purified in example 3 of the present invention after storage at 4 ℃ for different days in different protective solutions;
FIG. 6 shows the monomer content of R0362 purified in example 3 of the present invention after storage at 4 ℃ for different days in different protective solutions;
FIG. 7 shows the content of the dimer of R0362 purified in example 3 of the present invention after storage at 37 ℃ for different days in different protective solutions;
FIG. 8 shows the monomer content of R0362 purified in example 3 of the present invention after storage at 37 ℃ for different days in different protective solutions;
FIG. 9 is a SEC plot of 1.213mg/ml R0362 samples after 12 hours of standing in example 3 of the invention;
FIG. 10 is a SEC plot after 12 hours of standing of a 19.03mg/ml R0362 sample in example 3 of the present invention;
FIG. 11 is a SEC plot of a 38.9mg/ml R0362 sample after 12 hours of standing in example 3 of the invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
A method for purifying Fc fusion protein comprises the step of carrying out ion exchange purification on a sample to be purified to obtain the Fc fusion protein, wherein an additive is contained in an ion exchange purification buffer solution, and the additive comprises at least one of proline, histidine or aspartic acid.
The inventor finds that protein macromolecules easily form a solution with higher viscosity under a high concentration state due to the characteristics and intermolecular interaction force of the protein macromolecules in the research and development of a purification process of Fc fusion protein, the recovery rate of the Fc fusion protein is obviously reduced after the Fc fusion protein is subjected to intermediate purification operation, the step is a key step for restricting the purification process of the Fc fusion protein, and further finds that the increase of protein aggregates possibly causes the reduction of the product recovery rate. Therefore, the inventor proposes a technical scheme for solving the problems, and adds at least one of proline, histidine or aspartic acid in the ion exchange purification process to reduce the viscosity of the solution, so as to relieve the high viscosity condition of the Fc fusion protein during enrichment on a chromatographic column, inhibit the generation of aggregates in the purification process, effectively solve the aggregation problem of the Fc fusion protein and remarkably improve the recovery rate of the product. Additives may be, for example, but are not limited to, proline and histidine, histidine and aspartic acid, proline and histidine and aspartic acid, and the like.
In a preferred embodiment, the additive comprises proline (Pro), histidine (His) and aspartic acid (Arg). The inventor finds that the Fc fusion protein has better purification effect when the additive is a plurality of components.
In a preferred embodiment, the concentration of proline, histidine or aspartic acid is each independently 0.05-0.4 mol/L. It is understood that the concentrations of proline, histidine and aspartic acid may each independently be 0.05-0.4mol/L when the additive is a single component or multiple components, and preferably 0.05-0.2mol/L when the additive is a multiple component. The concentration of proline is typically, but not limited to, 0.05mol/L, 0.1mol/L, 0.15mol/L, 0.2mol/L, 0.25mol/L, 0.3mol/L, 0.35mol/L, or 0.4 mol/L; histidine is typically, but not limited to, at a concentration of 0.05mol/L, 0.1mol/L, 0.15mol/L, 0.2mol/L, 0.25mol/L, 0.3mol/L, 0.35mol/L, or 0.4 mol/L; the concentration of aspartic acid is typically, but not limited to, 0.05mol/L, 0.1mol/L, 0.15mol/L, 0.2mol/L, 0.25mol/L, 0.3mol/L, 0.35mol/L, or 0.4 mol/L.
In a preferred embodiment, the purified Fc-fusion protein is stored in a preservation solution comprising a protecting agent comprising proline and/or histidine. The purified Fc fusion protein is also unstable and easy to aggregate, and particularly, more aggregates are formed along with the increase of the concentration of the protein after concentration, so that the inventor finds that the problem of instability of the Fc fusion protein can be obviously improved, the overall performance of the product is improved, and the effective period is prolonged because proline and/or histidine are contained in the storage solution of the Fc fusion protein after purification.
In a preferred embodiment, the concentration of histidine in the preservation solution is between 0.05 and 0.3 mol/L. The concentration of histidine is typically, but not limited to, 0.05mol/L, 0.1mol/L, 0.15mol/L, 0.2mol/L, 0.25mol/L, or 0.3 mol/L. In the case of histidine or histidine and proline in the preservation solution, the concentration of histidine is 0.05 to 0.3 mol/L.
In a preferred embodiment, the concentration of proline in the preservation solution is 0.1-0.3 mol/L. The concentration of proline is typically, but not limited to, 0.1mol/L, 0.15mol/L, 0.2mol/L, 0.25mol/L, or 0.3 mol/L. In the case of proline, or histidine and proline in the preservation solution, the concentration of proline is 0.1 to 0.3 mol/L.
In a preferred embodiment, the ion exchange purification comprises cation exchange chromatography or anion exchange chromatography, preferably cation exchange chromatography, further preferably Capo MMC. Capo MMC is a weak cation exchange chromatography medium of a complex mode ligand, uses weak cation exchange as a main part and simultaneously has the functions of hydrophobic and hydrogen bonds. The Capo MMC can remove impurities such as aggregates, Protein A, HCP and the like in the purification of the Fc fusion Protein. Due to the instability of Fc fusion proteins, non-uniform adsorption on chromatographic packing during Capo MMC purification, high local concentrations can lead to increased protein viscosity, easy formation of aggregates, and reduced recovery, however, the addition of the additives of the present invention can effectively solve the above problems.
In a preferred embodiment, the sample to be purified is subjected to ion exchange purification after capture purification by means of affinity chromatography, preferably Protein a affinity chromatography. The capture purification refers to the preliminary purification of a sample to be purified, and the impurities are preliminarily removed through separation and concentration.
In a preferred embodiment, the sample to be purified is subjected to ion exchange purification and then to fine purification to obtain the Fc fusion protein. Methods for fine purification include gel filtration chromatography. By fine purification is meant achieving a final high level of purity, which may be, for example, ion exchange chromatography or gel filtration chromatography, etc., to give the final high purity Fc fusion protein.
In a preferred embodiment, the sample to be purified comprises a cell fermentation broth, preferably a fusion protein of an anti-CD 40 antibody and CTLA-4. The fusion protein of the anti-CD 40 antibody and CTLA-4 is abbreviated as R0362 in the embodiment.
The purified Fc fusion protein obtained by the purification method has the advantages of obviously reduced aggregate content, obviously increased monomer content and good activity and purity of the Fc fusion protein.
The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.
Taking R0362 as an example, the technical scheme of the invention is introduced:
the purification technological process adopts affinity chromatography-cation exchange-gel filtration chromatography.
Materials and methods
The feed liquid (batch number: 190808, expression form: hybridoma) harvested by cell fermentation department, AKTA Avant 150/AKTA pure 150(GE medical treatment), Protein A affinity chromatography medium (the chromatography medium is from GE Mab Select SuRe LX), cation exchange chromatography medium (from GE Capto S Impact), gel filtration chromatography medium (from BestChrom Chromdex 200prep grade), Epoch (BioTeK, with Take3 micropore plate), ultrafiltration concentration tube (Millipore), portable pH meter, portable conductivity meter, etc.
Example 1
First, affinity chromatography
A chromatographic column: mab Select SURE LX, column volume 53 ml;
Buffer:A:1×PBS;B:20mM NaAc,pH3.4;
CIP buffer:0.1M NaOH;
preservation solution: 20% EtOH (ethanol).
1. Sample treatment: the feed liquid samples harvested by the cell fermentation department were sterile filtered using a 0.22 μm flat top filter.
2. Balancing: and (3) balancing the system and the chromatographic column by using Buffer A, wherein the balance volume is as follows: 5CV, Flow rate: 15 ml/min.
3, Loading: sample loading was performed using a flow rate of 15ml/min, in a bubble-induced fashion, with flow-through collection setting of 20mAU-20mAU (AKTA instruments UV reading units; for the purpose of collecting penetrating proteins during loading).
4, Wash: buffer A wash, 265ml in volume, Flow rate 15 ml/min.
5. And (3) elution: eluting with 100% Buffer B5 CV at constant rate, wherein the Flow rate is 15ml/min, and collecting 20mAU-20 mAU;
CIP:CIP Buffer upflow 3CV,Hold 3min;
Buffer A down-flow 5CV,Hold 0min。
the collected sample was designated as R0362 MabE and the purity of the sample after affinity chromatography was approximately 60%, as shown in FIG. 1.
Second, compound mode chromatography (Capto MMC)
A chromatographic column: capto MMC, column volume 17 ml;
sample source: r0362 MabE;
Buffer A:20mM Citrate pH5.5;
Buffer B:20mM Citrate+2M Nacl pH5.5;
CIP buffer:0.5M NaOH;
preservation solution: 20% EtOH.
1. Sample treatment before loading: r0362 MabE pH was adjusted to 5.5 and diluted to a conductance of 6.1 mS/cm; the sample was filtered using a 0.22 μm bottle top filter. Taking a small amount of samples for concentration measurement; sample loading concentration: 1.816mg/ml, total amount of loaded protein: 627.446 mg.
2. Balancing: and (3) balancing the system and the chromatographic column by using Buffer A, wherein the balance volume is as follows: 5CV, Flow rate: 7 ml/min.
3. Loading: sample loading was performed using a flow rate of 5ml/min, bubble induction, flow through collection setting of 20mAU-20 mAU.
4, Wash: collecting 20mAU-20 mAU; buffer A, wash volume 170 ml; flow rate ═ 7 ml/min.
5. And (3) elution: collecting: 20mAU-20 mAU;
20% Buffer B5 CV isocratic elution, Flow rate 7ml/min, marked as MMC E2;
50% Buffer B5 CV isocratic elution, Flow rate 7ml/min, marked as MMC E3;
100% Buffer B5 CV isocratic elution, Flow rate 7ml/min, marked as MMC E4;
washing with water: 5CV isocratic elution, Flow rate ═ 7 ml/min;
CIP:CIP Buffer down-flow;3CV;Hold 3min;
Buffer A down-flow;3CV;Hold 0min。
the sample elution information is shown in table 1 below:
TABLE 1
Total protein recovery rate of loaded protein: 90.43 percent.
The purity of each component is shown in table 2 below, and the SEC results for MMC E2 are shown in fig. 2, and the SEC results for MMC E3 are shown in fig. 3:
TABLE 2
Sample name | Polymers% | Monomer% | Fraction% |
R0362 MMC E2 | 17.96 | 81.31 | 0.74 |
R0362 MMC E3 | 35.46 | 63.84 | 0.70 |
R0362 MMC E4 | 54.28 | 44.22 | 1.50 |
R0362 MMC Loading | 34.79 | 64.13 | 1.08 |
The total amount of monomer recovered after Capo MMC chromatography: 323.03mg, monomer recovery: 80.27%, total protein recovered: 56.93 percent.
Total amount of polymer recovered: 237.92mg, Polymer recovery: 109%, total protein recovered: 41.93 percent.
The results show that the purity of MMC E2 samples can be improved (from 60% to 81%) through the Capto MMC chromatography of the current round, but the purity improvement is not obvious relative to all proteins, and the content of the polymer is obviously increased (the recovery rate is more than 100%), and the supposedly possible reason that the protein is too much, the protein is adsorbed on the chromatographic column in a large amount during the chromatography, the viscosity is very high, and the polymer is increased.
Third, gel filtration chromatography
A chromatographic column: chromodex PG200, column volume 188.91 ml;
sample source: capto MMC elution collect samples (i.e. R0362MMC E2);
Buffer A:PBS;
Buffer B:PBS;
CIP buffer:0.1M NaOH;
preservation solution: 0.1M NaOH.
1. A portion of the Capto MMC E2 sample was concentrated by ultrafiltration and then sterile filtered using a 0.22 μm needle filter.
2. Balancing: buffer a, equilibrium volume 1.5CV (Flow rate ═ 5 ml/min).
3. Loading: (20 mAU-20mAU collected) Flow rate 5 ml/min.
4. And (3) elution: (20 mAU-20mAU collected) Buffer A wash volume 285ml, Flow rate 5 ml/min;
CIP:CIP Buffer down-flow 2CV,Hold 0min。
the results are shown in Table 3:
TABLE 3
The SEC results of the collected sample SEC E5 are shown in FIG. 4, and the purity of the target protein reaches more than 95%. The chromatography can effectively remove target protein and improve the purity of the sample. The result calculation shows that the monomer recovery protein amount in the gel filtration chromatography is as follows: 78.34mg, loading protein monomer content: 117.23 × 81.31% ═ 95.32mg, monomer recovery rates were: 82.19 percent.
Example 2
This example optimizes Capto MMC in the purification process.
Adding additive Pro
A chromatography system: AKTA pure 150, device number: FAPON-1-203;
a chromatographic column: capto MMC, column number: CEX001, column volume: 1 ml;
Buffer A:20mM Citrate pH5.5;
Buffer B:20mM Citrate+2M NaCl pH5.5;
CIP buffer:0.5M NaOH;
preservation solution: 20% EtOH.
1. Sample loading and processing: the protein sample (SEC E4 sample obtained after gel filtration chromatography in example 1) was pH adjusted to 5.5 and the conductance value was diluted to 5.7ms/cm using a buffer of 0.1M proline; loading protein concentration: 0.581mg/ml, total loading protein: 30.17 × 0.581 ═ 17.528 mg.
2. Balancing: buffer A5CV (Flow rate ═ 1 ml/min).
3. Loading: (10 mAU. about. top. about. 10 mAU. about. were collected) Flow rate of 1 ml/min.
4, Wash: (10 mAu. about. top. about. 10 mAu. about. were collected) Buffer A wash volume 10ml, Flow rate 1 ml/min.
5. And (3) elution: collecting: 20 mAU-top-20 mAU;
20% Buffer B20 CV Linear elution, Flow rate 1 ml/min;
30% Buffer B5 CV isocratic elution, Flow rate ═ 1 ml/min;
100% Buffer B5 CV isocratic elution, Flow rate ═ 1 ml/min;
washing with water: 5CV Linear isocratic elution, Flow rate ═ 1 ml/min;
CIP Buffer down-flow 3CV,Hold 3min;
Buffer A down-flow 3CV,Hold 0min。
the information collected is as follows in table 4:
TABLE 4
The sample was sent for SEC testing with the results shown in table 5 below:
TABLE 5
Sample name | Polymers% | Monomer% | Fraction% |
R0362 MMC Loading | 40.89 | 58.43 | 0.68 |
R0362 MMC FT | 38.36 | 60.93 | 0.71 |
R0362 MMC E2 | 17.34 | 81.61 | 1.05 |
R0362 MMC E5 | 22.18 | 77.00 | 0.82 |
R0362 MMC E15 | 30.04 | 69.05 | 0.90 |
R0362 MMC E17 | 64.79 | 34.13 | 1.08 |
Total amount of loading monomeric protein: 58.43% × 17.528 ═ 10.24 mg. Total amount of loading aggregate protein: 40.89% × 17.528 ═ 7.17mg.
The total amount of the loaded protein is 17.528mg, the total amount of the recovered protein is 13.677mg,
the total amount of recovered monomer after MMC chromatography is 9.827mg, the monomer recovery rate is 95.96%, and accounts for the total recovered protein: 71.85 percent.
Total polymer recovery of 3.811mg, 53.15% polymer recovery, total protein recovered: 27.86 percent.
II, adding no additive Pro
Referring to the first step, the step of adding the additive Pro, the different operation processes are as follows:
1. sample loading and processing: the protein sample (SEC E4 sample obtained after gel filtration chromatography in example 1) was pH-adjusted to 5.5 and the conductance value was diluted to 5.7ms/cm using equilibration buffer (20mM Citrate pH 5.5); loading protein concentration: 1.2mg/ml, total amount of loaded protein: 36.6 × 1.2 ═ 43.92 mg.
5. And (3) elution: collecting: 20 mAU-top-20 mAU;
20% Buffer B6 CV isocratic elution, Flow rate ═ 1 ml/min;
30% Buffer B5 CV isocratic elution, Flow rate ═ 1 ml/min;
100% Buffer B5 CV isocratic elution, Flow rate ═ 1 ml/min;
washing with water: 5CV Linear isocratic elution, Flow rate ═ 1 ml/min.
The elution information is shown in table 6 below:
TABLE 6
The SEC results are shown in table 7 below:
TABLE 7
Sample name | Polymers% | Monomer% | Fraction% |
R0362 MMC Loading | 40.89 | 58.43 | 0.68 |
R0362 MMC E1 | 34.28 | 65.17 | 0.55 |
R0362 MMC E2 | 76.07 | 21.47 | 2.46 |
Total amount of loading monomeric protein: 58.43% × 43.92 ═ 25.66mg, total amount of recovered monomer 20.559 mg;
total amount of loading aggregate protein: 40.89% × 43.92 ═ 17.96mg, total amount of recovered polymer: 13.282 mg.
After MMC chromatography, the total protein recovery was: 34mg, monomer recovery ratio: 60.47%, polymer recovery ratio: 39.06 percent.
Three, different additives
The inventors also examined the purification effect of other additives, and the specific components of each additive and the results in Capo MMC chromatography are shown in table 8 below:
TABLE 8
According to the results, when no stabilizing protective agent is added in the Capo MMC experiment, target protein aggregation is caused due to high protein concentration in a local area, so that the purification effect is poor; the results show an increase in monomer recovery and a decrease in aggregates after addition of the screening adjuvant. The protein stability can be effectively protected and the purification effect can be improved by adding the additive in the protein composite mode chromatography process.
Example 3 stability study
Due to the problem of the stability of the R0362 fusion protein, precipitates are easily generated in the preservation process, and the screening experiment of the Buffer and the additive is carried out on the sample preservation conditions.
The experimental operation flow comprises the following steps: 1ml of a sample (protein obtained after gel filtration chromatography in example 1) was added to a storage solution (specific components are shown in table 9 below) shown in table 9 below, and mixed; filtering with 13mm sterile needle filter into 1.5ml sterile centrifuge tube, collecting 1ml filtered sample to 3ml penicillin bottle, sealing with sealing membrane;
and (5) the residual samples are subjected to A280, SEC and electrophoresis according to requirements, and the detection results are used as evaluation initial data.
And (3) stability assessment: the samples are placed at 4 ℃ and 37 ℃ for examination, and the Day3, the Day7 and the Day14 are respectively aseptically sampled and sent to the SEC for examination; the precipitation was visually observed by photographing and recording.
The experimental groups are shown in table 9 below:
TABLE 9
Sample number | Preserving fluid | Sample | Preserving fluid | |
1 | |
2 | 100mM Tris, |
|
3 | 100mM PB, |
4 | 100mM PBS, |
|
5 | 100mM Citrate, |
6 | 100mM His, |
|
7 | 100mM Arg, |
8 | 100mM Gly, |
|
9 | 10 |
10 | 0.2M sucrose, |
|
11 | 0.02% Tween-80 | 12 | 0.2M Pro pH 6.5 |
Specific sample stability SEC results are shown in fig. 5-8.
From the results, it can be seen that:
general stability examination shows that R0362 is easy to form high polymer, and after the sample is placed for 7 days, the polymer content of the sample at 4 ℃ and 37 ℃ is obviously increased, and is particularly obvious at 37 ℃;
the addition of His and Pro can better inhibit the aggregation of R0362, so that the sample keeps high purity.
To verify the effect of high concentrations on protein aggregation, we performed the following experiments: r0362 protein (1.213mg/ml) was concentrated to different high concentrations using an ultrafiltration concentration tube, left for about 12 hours, and subjected to SEC detection simultaneously with a control sample (which was not concentrated).
The censored protein concentrations and SEC purities are shown in Table 10 below:
watch 10
SEC results are shown in FIGS. 9-11.
From the results, it is known that a high concentration in a local region during chromatography causes aggregation of the sample (83% purity is reduced to 57%).
While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.
Claims (10)
1. A purification method of Fc fusion protein is characterized in that a sample to be purified is purified by ion exchange to obtain the Fc fusion protein;
the ion exchange purification buffer contains an additive, and the additive comprises at least one of proline, histidine or aspartic acid.
2. The purification method according to claim 1, wherein the additive comprises proline, histidine and aspartic acid.
3. The purification method according to claim 1, wherein the concentration of proline, histidine or aspartic acid is 0.05-0.4 mol/L.
4. The purification method according to claim 1, wherein the purified Fc fusion protein is stored in a storage solution containing a protecting agent comprising proline and/or histidine.
5. The purification method according to claim 4, wherein the concentration of histidine is 0.05-0.3 mol/L;
preferably, the concentration of the proline is 0.1-0.3 mol/L.
6. Purification method according to claim 1, characterized in that the ion exchange purification comprises cation exchange chromatography or anion exchange chromatography, preferably cation exchange chromatography, further preferably Capo MMC.
7. The purification method according to claim 1, wherein the sample to be purified is subjected to ion exchange purification after being captured and purified;
preferably, the method of capture purification comprises affinity chromatography, preferably Protein a affinity chromatography.
8. The purification method according to claim 1, wherein the sample to be purified is subjected to ion exchange purification and then to fine purification to obtain the Fc fusion protein;
preferably, the method of fine purification comprises gel filtration chromatography.
9. The purification method according to any one of claims 1 to 8, wherein the sample to be purified comprises a cell fermentation broth, preferably a cell fermentation broth of a fusion protein of an anti-CD 40 antibody and CTLA-4.
10. A purified Fc fusion protein obtained by the purification method of any one of claims 1-9.
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