CN110724204B - Method for purifying Fc fusion protein - Google Patents
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- C—CHEMISTRY; METALLURGY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- C07K14/5434—IL-12
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- C—CHEMISTRY; METALLURGY
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- C12P21/00—Preparation of peptides or proteins
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention discloses a purification method of Fc fusion protein, and relates to the technical field of biology. The disclosed method for purifying an Fc fusion protein comprises adjusting the pH of an eluate containing the Fc fusion protein to 4.9-5.1. The purification method provided by the invention can obviously reduce the high-aggregate Fc fusion protein in the product and improve the purity of the Fc fusion protein.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a purification method of Fc fusion protein.
Background
Interleukin-10 (IL-10) cytokine, first reported by Mosmannand et al in 1989, is a pleiotropic cytokine with bi-directional regulation of the immune system that regulates a variety of immune responses by T cells, B cells, macrophages and antigen presenting cells. In one aspect, IL-10 can inhibit an immune response primarily associated with inflammation by inhibiting the expression of IL-1 α, IL-1 β, IL-6, IL-8, TNF- α, GM-CSF and G-CSF in activated monocytes and activated macrophages; on the other hand, John B, Mumm et al found that IL-10 induced and stimulated tumor-infiltrating CD8+T secretes cytotoxic cytokine gamma interferon (IFN-gamma) and further mediates the production of cytotoxic substance granzyme B (granzyme B), perforin (perfolin) and the like, thereby inhibiting and killing tumor cells and showing corresponding immune stimulation. The dose dependence of pegylated IL-10(PEG-IL-10, AM0010) was reported in ARMO BIOSCIENCES, INCAnti-tumor activity.
In view of the prevalence and severity of IL-10-associated diseases, disorders and conditions, IL-10-related drugs that modulate aspects of IL-10 function are of great therapeutic value.
The molecular weight of pure IL-10 is only about 37KDa, and the pure IL-10 is easily metabolized by organs such as kidney and the like in a human body, so that the extremely short half-life period (2-6 hours) in vivo is caused, and the curative effect of the pure IL-10 is greatly influenced. It is therefore necessary to extend its half-life in vivo by some means.
Fc fusion proteins (Fc-fusion proteins) are an effective way to extend the half-life of functional proteins in vivo.
Immunoglobulin G (IgG) molecules are mainly based on the fact that their Crystallizable fragment (Fc) binds to neonatal Fc receptor (FcRn) in a pH-dependent manner to avoid degradation by nuclear endosomes, extending the half-life of the mab, which is important for improving dosing frequency, patient compliance, and cost.
The Fc fusion Protein recombines IgG Fc gene and target Protein gene by DNA recombination technology, and the obtained expression product has obviously prolonged half-life compared with the target Protein before recombination, and simultaneously, due to the introduction of Fc fragment, the purification process of the target Protein can be greatly simplified based on affinity chromatography technologies with strong specificity such as Protein A, G and the like.
The Fc fusion protein harvested by adopting the high specificity affinity chromatography technology of the ProteinA and G types is easy to form aggregates, and can cause more serious immunogenicity in clinic if being directly applied to clinic, which is not allowed for biological recombinant protein drugs.
Other chromatographic purification methods, such as anion and cation exchange chromatography, gel filtration chromatography, and hydrophobic interaction chromatography, can remove aggregates to some extent. However, the use of such chromatographic purification methods often involves expensive chromatography equipment, chromatography columns and chromatography packing, and the hardware configuration requirements are high; at the same time, part of the target is inevitably lost, resulting in a yield lower than the expected yield of the target, which brings difficulty to the production of the target. Secondly, the process development based on chromatography is also extremely complex, cumbersome and time consuming.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The present invention aims to provide a method for purifying an Fc fusion protein to overcome the above problems.
The invention is realized by the following steps:
in a first aspect, embodiments of the present invention provide a method for purifying an Fc fusion protein, comprising: the pH of the eluate containing the Fc fusion protein was adjusted to 4.9-5.1.
The inventor of the present invention found that in the purification of Fc fusion protein by affinity chromatography, some extreme elution conditions such as low pH (generally pH 3.0-3.5, even lower such as pH 2.0-2.7) are inevitably involved in the elution process, and thus, for Fc fusion protein with poor stability such as pH, it is often more prone to form aggregates.
In response, the inventors of the present invention creatively adjusted the pH of the eluate containing the Fc fusion protein to a range of 4.9-5.1, and as a result, surprisingly found that high aggregates (aggregation > 2) in the product are significantly reduced and the purity of the Fc fusion protein in the desired dimeric form is greatly improved. Compared with the existing affinity chromatography method, the purification method provided by the invention can remove most of Fc fusion protein in a high polymer (aggregation degree > 2) form in a product without losing the target form (dimer) of the Fc fusion protein. Therefore, the purification method provided by the invention can be used as a replacement scheme of a chromatography purification technology which is complex, tedious and time-consuming to operate and relates to expensive chromatography equipment, a chromatography column and chromatography fillers, so that the preparation or purification process cost of the Fc fusion protein is greatly reduced, and the purification method has the characteristics of simple operation and time saving.
In an alternative embodiment, the purification method further comprises:
eluting the Fc fusion protein from an affinity chromatography column to obtain the eluate containing Fc fusion, obtaining the eluate containing the Fc fusion protein.
The type of the affinity column is not particularly limited in the present invention, and the above-mentioned affinity column may be one that specifically binds to Fc fusion protein by adsorption such as protein a or G, or one that binds to Fc fusion protein by other adsorption means (for example, his6 tag). So long as it is capable of specifically binding to the Fc fusion protein, it may be applicable to the present invention.
In alternative embodiments, the affinity chromatography column is selected from Protein a or Protein g or Protein L.
In an alternative embodiment, the Fc fusion protein is eluted from the affinity chromatography column using an initial eluent, and the eluted initial eluent is collected to obtain the Fc fusion-containing eluent, wherein the initial eluent has a pH of 2 to 3.5.
In an alternative embodiment, the initial eluent has a pH of 3.4.
The term "initial eluate" refers to an eluate which is used to elute the Fc fusion protein from the affinity chromatography column but which does not contain the Fc fusion protein.
In order to facilitate elution of the Fc fusion protein from the affinity chromatography column, the pH of the initial eluent is usually 2-3.5, but this does not mean that the pH of the initial eluent of the present invention is limited to 2-3.5, and in some embodiments, it is easy for one skilled in the art to appropriately adjust the pH of the initial eluent to facilitate smooth elution of the Fc fusion protein according to the characteristics of the Fc fusion protein.
In an alternative embodiment, when collecting the initial eluate after elution, since the initial eluate containing the target protein after elution is at a relatively low pH, for the non-stable Fc fusion protein, the Fc protein in the target form is easily aggregated, resulting in loss of its biological activity, so that a neutralization buffer is added in advance to the collection container to make the pH of the solution after mixing the initial eluate after elution and the neutralization buffer 6-7, that is, to obtain the eluate containing the Fc fusion protein;
in an alternative embodiment, the neutralization buffer is a Tris solution, pH 7.5-8.5, at a concentration of 0.8-1.2M.
In an alternative embodiment, the initial eluent is selected from any one of a sodium acetate solution (NaAc), sodium citrate and glycine solution.
It should be noted that the initial eluent used in the present invention is not limited to the above-listed solutions, and those skilled in the art can easily think of using other types of buffers for elution, and it is within the scope of the present invention to use any type of buffer as the initial eluent for elution.
In an alternative embodiment, the initial eluent is a sodium acetate solution.
In alternative embodiments, the concentration of the sodium acetate solution is 18-22 mM.
It should be noted that the concentration of the sodium acetate solution can be adjusted according to actual conditions, and the eluent with a suitable concentration is favorable for eluting the Fc fusion protein, and those skilled in the art can easily think that the sodium acetate solution with a suitable concentration is selected for elution according to the characteristics of the Fc fusion protein, and it is within the scope of the present invention to use any sodium acetate solution with any concentration for elution.
In an alternative embodiment, the purification method further comprises: washing the affinity chromatography column with PBS buffer to wash away some non-specific adsorbed proteins with weak affinity before eluting with the initial eluent;
the concentration of the PBS buffer contained: 9-11mM PB, 140-160mM NaCl, pH 7.2-7.4.
In an alternative embodiment, the purification method further comprises: applying the mixture containing the Fc fusion protein to the affinity chromatography column.
In an alternative embodiment, the mixture is a fermentation supernatant.
It is noted that the mixture containing the Fc fusion protein may be a fermentation supernatant, but in other embodiments it may be a pure protein mixture of the Fc fusion protein with other non-target proteins. The present invention is not particularly limited with respect to the type of the mixture. Any system may be used as long as the Fc fusion protein is purified from the system, and the system may be purified as the mixture.
In alternative embodiments, the fermentation supernatant is fermented from a host cell that expresses the Fc fusion protein.
In alternative embodiments, the host cell is a mammalian cell.
In alternative embodiments, the mammalian cell is selected from HEK293 or CHO.
In an alternative embodiment, the pH of the eluent is adjusted using a citric acid solution or a glycine solution.
In an alternative embodiment, the pH of the eluent is adjusted using a citric acid solution.
The solution for adjusting the pH of the eluent is not limited to the citric acid solution, and other solutions may be selected for adjustment, and any solution that can be used to adjust the pH of the eluent is within the scope of the present invention.
In an alternative embodiment, the citric acid solution has a concentration of 0.4 to 0.6M.
It should be noted that the concentration of the citric acid solution is not limited to 0.4-0.6M, and the concentration of the citric acid solution can be selected according to actual needs, and whatever concentration of the citric acid solution is selected, the citric acid solution is within the protection scope of the present invention.
In an alternative embodiment, the purification method further comprises: after adjusting the pH of the eluent, the mixed solution of the citric acid solution and the eluent is allowed to stand.
In an alternative embodiment, it is allowed to stand for more than 1 hour to allow the non-target dimer form of the target to be sufficiently precipitated.
In an alternative embodiment, the purification method further comprises: and after standing, centrifuging and filtering the mixed solution to obtain a crude Fc fusion protein solution.
In alternative embodiments, the crude Fc fusion protein solution has a SE-HPLC (molecular sieve-high performance liquid chromatography) purity of >80%, further > 90%.
In an alternative embodiment, the conditions of centrifugation are: the rotating speed is 3000-4000rpm, and the time is 5-15 min.
In an alternative embodiment, filtration is carried out using a filter membrane having a pore size of 0.2 to 0.24. mu.m.
In alternative embodiments, the Fc fusion protein comprises an Fc fragment domain and a functional protein domain.
In alternative embodiments, the functional protein domain is selected from human or mouse IL-10.
It should be noted that the functional protein of the functional protein domain can be selected according to the needs, and the invention is not limited to any specific type, and any type of functional protein is within the scope of the invention.
In alternative embodiments, the Fc fragment domain is an Fc fragment of a mammalian IgG protein.
In alternative embodiments, the mammal is selected from any one of a human and a mouse.
It should be noted that the IgG protein can be derived from not only human and mouse, but also other mammals such as rabbit, cow, horse, pig, etc., and the Fc fragment of the IgG protein of any mammal is considered to be within the scope of the present invention.
In alternative embodiments, the IgG protein is selected from any one of IgG1, IgG2, IgG3, and IgG 4.
The subtype of the IgG protein may be selected according to actual needs, and may be, for example, IgG1, IgG2, IgG3 or IgG4, or a mutant form of these subtypes, and the like, and all fall within the scope of the present invention.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the statistics of the total peak area and the target peak area after the eluents of examples 1-5 are adjusted to pH5 and 8 and detected by SE-HPLC.
FIG. 2 shows the statistics of the superpolymers and the target peaks detected by SE-HPLC after adjusting the pH of the eluents of examples 1 to 5 and 8.
FIG. 3 shows the results of isoelectric point measurements of the eluate of example 3 after pH8.0 adjustment.
FIG. 4 shows the results of isoelectric point measurements of the eluate of example 4 after pH8.0 adjustment.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The Fc fusion protein provided in this example was purified as follows:
(1) obtaining a fermentation supernatant containing Fc fusion protein
(a) Seed liquid preparation
Recovering the monoclonal antibody (human embryonic kidney cell (HEK 293) containing the plasmid vector for expressing the Fc fusion protein), and culturing at 37 ℃ for 3-4 days until the cell density is 0.8-2.5 multiplied by 106cells/mL, then cultured for 3 days until the cell density is 0.8-2.5X 106cells/mL are continuously passed, and the cells are cultured for 3 days until the cell density is 1-2 multiplied by 106cells/mL to obtain seed solution.
Wherein, the structure of the Fc fusion protein comprises: murine IgG1Fc-Linker 1-human IL-10.
The structure of the plasmid vector contains a coding gene of mouse IgG1Fc, a coding gene of Linker1 and a coding gene of human IL-10.
(b) Supplemented fermentation
Mixing the obtained seed liquid at 6 × 105cells/mL are inoculated into 50mL/250mL shake flasks and cultured for 3 days until the cell density is 1.5-2 × 106At cells/mL, the fed-batch culture of the cells was started, and the Feed ratio was maintained at 2%, glucose 4g/L, glutamic acid 4mM, and cysteine 0.2%. The culture was continued until day 7 to obtain a fermentation supernatant containing Fc fusion protein.
It should be noted that the fermentation step is an optional step.
(2) Separating and purifying
Materials and equipment:
chromatography packing/column: MabSelectSuRe LX, Column Volume (CV) 5 or 26ml, GE Healthcare;
a chromatography system: AKTA pure150, GE Healthcare;
portable pH meter, Horiba;
an Epoch microplate reader, BioTek;
buffer solution:
Buffer A：1×PBS；
Buffer B：20mM NaAc，pH3.4；
CIP buffer solution: 0.1M NaOH;
neutralization buffer: 1M Tris, pH8.0.
(a) The purification process is as follows:
regenerating the chromatographic column, then using Buffer A balance 10 CV-Reset UV Monitor-loading Sample (Sample Application, loading all samples in a bubble induction mode, in this example, the Sample is the fermentation supernatant of the step (1) — Buffer A colony wash 20 CV-100% Buffer B step elution (step with fill) 7CV, collecting 20 milli absorption units (mAU) -20 mAU, 280 nanometer (nm) ultraviolet absorption components, obtaining the eluent containing Fc fusion protein, and adding 5% neutralization Buffer in advance into a collection tube to make the pH of the final elution mixture in the range of 6.0-7.0-cleaning Clean In Place (CIP), washing 3CV with CIP Buffer upwards (Hold) for 3min, maintaining (Hold) for 3min, then washing 5CV with Buffer A Buffer downwards (down-flow), and ending.
(b) Adjusting the pH
The pH of the eluate containing the Fc fusion protein obtained in the above step was adjusted to 5 using a 0.5M citric acid solution as an experimental treatment group; the eluate containing the Fc fusion protein obtained in the above step was adjusted to pH8 using 1M Tris-HCl as a control, and the actual pH was measured by a pH meter, and the results are shown in Table 1.
(c) After adjustment, standing for 1-2 hours, centrifuging each solution, 3500rpm, 10min, filtering with a filter membrane with the aperture of 0.22um, performing molecular sieve-high performance liquid chromatography (SE-HPLC) on the filtrate by molecular exclusion chromatography (according to pharmacopoeia of people's republic of China, 2015 edition, three parts, general rules 0514), wherein the mobile phase adopts 50 mM phosphate, 300 mM sodium chloride, pH 7.0 +/-0.1, and the chromatographic column adopts TSKgel G3000SWXL7.8 × 300 mm, 5 μm, and carrying out quantitative analysis on the result by adopting a peak area normalization method. And respectively calculating the peak area percentages of the dimer, the high polymer and the low molecular weight impurity. The peak area percentage of the dimer is taken as the sample purity, and the peak area percentage of the high polymer and the low molecular weight impurity is taken as the content of the impurity in the sample. ) Purity detection is carried out, meanwhile, the eluent sample before pH adjustment is detected, and the results are shown in tables 1-4.
Examples 2 to 5
Examples 2-5 were prepared in substantially the same manner as the Fc fusion protein of example 1, except that the Fc fusion protein was slightly different in structure as follows:
the Fc fusion protein structure of example 2 is as follows:
human IL-10-Linker 1-human IgG4 Fc;
the Fc fusion protein structure of example 3 is as follows:
human IL-10-Linker 2-human IgG4 Fc;
the Fc fusion protein structure of example 4 is as follows:
human IgG4Fc-Linker 2-human IL-10;
the Fc fusion protein structure of example 5 is as follows:
murine IgG1Fc-Linker 1-human IL-10.
Wherein Linker1 and Linker2 correspond to (GSGSGSGS) and [ (GGGGS)3]Meanwhile, capillary isoelectric focusing electrophoresis (CIEF, referred to according to the third method of pharmacopoeia of the people's republic of china, 2015 edition, three, general rule 0542) was performed on each of the pH8.0 samples of the eluates containing the Fc fusion proteins obtained after pH adjustment in example 3 and example 4, and the isoelectric point pI test was performed on the samples.
The corresponding test results are shown in tables 1-6 and FIGS. 1-4.
TABLE 1 actual pH values of the pH-adjusted eluents of examples 1 to 5
Table 1 shows the actual pH of the eluents of the examples after pH adjustment, the pH of the eluents of the experimental group of examples 1 to 5 being 4.92 to 5.01, and the pH of the eluents of the control group being 7.93 to 8.04.
TABLE 2 results of SE-HPLC purity measurement of eluate containing Fc fusion protein before pH adjustment obtained in examples 1 to 5
Table 2 shows the content of Fc fusion proteins in each aggregate form in the eluates of the examples before pH adjustment, wherein the content of Fc fusion proteins (non-target components) in the high-polymer form (degree of polymerization > 2) is about 18% to 39%, and the content of Fc fusion proteins in the dimer form, i.e., the target form (i.e., the purity of the sample at this time) is about 54% to 78%.
TABLE 3 Peak area results for SE-HPLC purity measurements of pH adjusted eluents of examples 1-5
Table 3 and figure 1 show the peak area results of SE-HPLC purity measurements of eluents adjusted to pH5 and pH8, although the total peak area of the eluents of each example at pH8 (representing the total protein content in the mixture) is clearly greater than pH5 (with the exception of R0301), but the target peak areas (i.e. representing the target protein content) are nearly identical (figure 1), but for the sample at pH5, the target peak purity (target component percentage content) is significantly higher than that of the sample at pH8 (80% -90% VS 50% -70%) (figure 2), the impurities are mainly derived from the high polymer and almost precipitated at pH5 as a high polymer component.
TABLE 4 comparison of the results of SE-HPLC purity measurements of the pH adjusted eluents of examples 1-5 before and after pH adjustment
Wherein the target component percentage (%) represents the purity of the target protein in the mixed sample, the polymer percentage (%) represents the content of the Fc fusion protein (non-target component) in a polymer form (degree of polymerization > 2) in the mixed sample, and the debris peak percentage (%) represents the content of the target protein degradation fragment (non-target component). The total peak area represents the total protein content in the mixed sample, and the target peak area represents the absolute content of the target form protein, i.e., the dimer form protein, in the mixed sample.
TABLE 5 results of isoelectric point measurement of eluate of example 3 after pH adjustment to 8.0
TABLE 6 results of isoelectric point measurement of eluate of example 4 after pH adjustment to 8.0
Table 4 and fig. 2 show the content of Fc fusion proteins in different aggregates after pH adjustment in each example, and in combination with table 2, it can be clearly seen that the content of Fc fusion proteins in the form of multimers is significantly reduced after pH adjustment to 5, especially to 0.98% in example 5, with almost complete removal of the multimers and, at the same time, the content and purity of Fc fusion proteins in the form of dimers are improved. As a control, it was found that when the pH of the eluate was adjusted to 8, the change in the content of the Fc fusion protein in the form of a high polymer was not significant from that when it was not adjusted, the content and purity of the Fc fusion protein in the form of a dimer were not improved, and even the content of the Fc fusion protein in the form of a dimer of example 5 was reduced. In addition, the isoelectric point tests of example 3 and example 4 after adjusting pH8.0 also showed that the isoelectric points of the components in the sample containing the Fc fusion protein in the target form after affinity elution ranged from 5.5 to 6.3 and 5.7 to 6.7, respectively, indicating that the precipitation caused by the pH adjustment of 5.0 was not due to the sample being at the isoelectric point of impurities. Therefore, after the Fc fusion protein is eluted by the affinity chromatography column, the pH of the eluent containing the Fc fusion protein is adjusted to about 5, so that the high-polymer Fc fusion protein which can obviously reduce or even completely remove the final product is obtained, and the purity of the dimer Fc fusion protein is improved.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method for purifying an Fc fusion protein, comprising: eluting the Fc fusion protein from the affinity chromatography column by using an initial eluent, collecting the eluted initial eluent to obtain an eluent containing the Fc fusion protein, and adjusting the pH value of the eluent containing the Fc fusion protein to 4.9-5.1;
wherein the pH value of the initial eluent is 2-3.5; the affinity chromatography column is selected from ProteinA or ProteinG;
when the initial eluent after elution is collected, adding a neutralization buffer solution into a collection container to ensure that the pH of the mixed solution of the initial eluent after elution and the neutralization buffer solution is 6-7, thus obtaining the eluent containing the Fc fusion protein;
the Fc fusion protein consists of an Fc segment structural domain and a functional protein structural domain, and the Fc segment structural domain is connected with the functional protein structural domain through a Linker; the functional protein domain is selected from human IL-10;
the Fc fragment domain is selected from human IgG4 or murine IgG 1.
2. The purification process according to claim 1, wherein the initial eluent has a pH of 3.4.
3. The purification process according to claim 2, wherein the neutralization buffer is a Tris solution, having a pH of 7.5 to 8.5 and a concentration of 0.8 to 1.2M.
4. A purification process according to claim 3, wherein the initial eluent is selected from any one of a sodium acetate solution, a sodium citrate solution and a glycine solution.
5. The purification process according to claim 4, characterized in that the initial eluent is a sodium acetate solution.
6. The purification process according to claim 5, wherein the concentration of the sodium acetate solution is 18 to 22 mM.
7. The purification method of claim 3, further comprising: washing the affinity chromatography column with PBS buffer prior to elution with the initial eluent;
the PBS buffer solution is: 9-11mM PB, 140-160mM NaCl, pH 7.2-7.4.
8. The purification method of claim 7, further comprising: applying the fermentation supernatant containing the Fc fusion protein to the affinity chromatography column.
9. The purification process of claim 8, wherein the fermentation supernatant is fermented by a host cell that expresses the Fc fusion protein.
10. The purification method according to claim 9, wherein the host cell is a mammalian cell; the mammalian cell is selected from HEK293 or CHO.
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