CN116948039A - Purification method of anti-B7H 3 antibody-cytokine fusion protein - Google Patents
Purification method of anti-B7H 3 antibody-cytokine fusion protein Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
- C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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Abstract
The invention provides a purification method of an anti-B7H 3 antibody-cytokine fusion protein, which comprises the following steps: affinity chromatography; low pH virus inactivation; anion exchange chromatography; cation exchange chromatography. The purification method has good impurity removal effect and high protein recovery rate, and finally obtains the fusion protein with high purity and no virus, thereby being beneficial to improving the quality control level and the drug stability of the product.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a purification process for effectively purifying fusion protein and reducing impurities.
Background
A fusion protein is a protein comprising one, two or more polypeptides derived from different naturally occurring proteins or engineered proteins that are artificially combined to form one protein, including but not limited to the following exemplified forms: 1. for a fusion protein consisting of one polypeptide chain, which are fused to each other by the same or different polypeptides to form one polypeptide chain comprising the different polypeptides; 2. for fusion proteins composed of two or more polypeptide chains, wherein optionally one or more polypeptide chains are fused to each other by the same or different polypeptides to form a polypeptide chain comprising said same or different polypeptides, these polypeptide chains are combined with each other in a covalent or non-covalent form to form the protein.
Multimers refer to aggregates that exist in the form of dimers or multimers. Multimers are produced mainly during the cell culture, protein purification, formulation prescription and drug storage processes, and are formed by the interaction of the exposed relevant regions to induce aggregation due to partial decomposition and folding or misfolding of proteins caused by the influence of culture process, purification conditions and certain factors during formulation storage. In protein samples, some antibody fragments, HCP, HCD, endotoxin, virus, etc. may also be present.
These impurities may not only reduce or even lose bioactivity, thereby affecting the efficacy, but may also cause allergic immunogenic reactions in the recipient, thereby affecting the recipient's health. Thus, these impurities are required to be monitored and removed during the preparation of the antibody protein drug.
In the process of antibody fusion protein drug production, although the production of impurities can be controlled by changing the operating conditions, there is no way to achieve the control effect of completely eliminating impurities in the product, so that effective impurity removal means is crucial for impurity control.
The current purification process for removing impurities of fusion protein drugs mainly comprises gel filtration chromatography, affinity chromatography, ion chromatography and hydrophobic chromatography. However, these methods are not thorough in removing impurities, cannot achieve a good purification effect on various impurities at the same time, and do not have a general combined purification scheme suitable for purification of fusion proteins.
Disclosure of Invention
In order to overcome the technical problem, the purification steps and parameters of affinity chromatography, low pH virus inactivation, anion chromatography, cation chromatography and the like, and the virus inactivation steps and parameters are reasonably arranged and selected, so that the fusion protein can be purified rapidly in a pipelining manner, the effect of removing impurities in the fusion protein is better, the efficiency is high, the protein recovery rate is higher, and finally the fusion protein with high purity and no virus is obtained, thereby being beneficial to improving the quality control level and the drug stability of the product.
Specifically, the invention relates to a purification method of an anti-B7H 3 antibody-cytokine fusion protein, which comprises the following steps: a) Affinity chromatography; b) Low pH virus inactivation; c) Anion exchange chromatography; d) Cation exchange chromatography.
In one embodiment, the first heavy chain amino acid sequence of the fusion protein is selected from SEQ ID NO. 1, the second heavy chain amino acid sequence is selected from SEQ ID NO. 2, and the amino acid sequences of the first light chain and the second light chain are selected from SEQ ID NO. 3.
In one embodiment, step (a) comprises loading, wherein loading should be controlled to within 20mg/mL of filler, preferably within 15mg/mL of filler.
In one embodiment, the step (a) comprises the steps of: step (1), treating chromatographic packing and loading; step (2), leaching by using leaching buffer solution; step (3), eluting and collecting the fusion protein by using an elution buffer; the loading should be controlled to within 20mg/mL of filler, preferably within 15mg/mL of filler.
In one embodiment, step (1) is to flush the equilibrated chromatography column with 2-4 column volumes of equilibration buffer at a flow rate of 1.0-1.4mL/min, loading is performed at a flow rate of 1.0-1.4mL/min, wherein the equilibration buffer is 10-30mm pb,120-160mm nacl, ph 7.2-7.6.
In one embodiment, step (2) is a wash with 2-4 column volumes of re-equilibration buffer at a flow rate of 1.0-1.4mL/min, then with 2-4 column volumes of elution buffer 1 at a flow rate of 1.0-1.4mL/min, and finally with 2-4 column volumes of elution buffer 2 at a flow rate of 1.0-1.4mL/min, wherein the equilibration buffer is 10-30mm pb,120-160mm nacl, ph 7.2-7.6; the leaching buffer solution 1 is 15-25mM NaAc-HAc,450-550mM NaCl, and the pH is 5.8-6.2; the leaching buffer solution 2 is 40-60mM NaAc-HAc, and the pH is 4.7-5.3.
In one embodiment, step (3) uses an isocratic elution method, eluting with 3-5 column volumes of elution buffer at a flow rate of 1.0-1.4mL/min, the elution buffer being 50mM NaAc-HAc, pH 3.5.
In one embodiment, the step (b) is adding 18% glacial acetic acid, adjusting the pH to 3.6.+ -. 0.2, standing at room temperature for 1-2 hours, and adding 2M Tris to adjust the pH to 6.5.+ -. 0.2.
In one embodiment, the conditions of step (c) are flow rate: less than 205cm/h; column height: 15+/-2 cm; equilibration/re-equilibration buffer: 20mM PB+30mM NaCl,pH6.0 + -0.4; the pH value of the sample is 6.5+/-0.2, and the electric conductivity is 5.0-8.0mS/cm; the loading is less than or equal to 80mg/mL.
In one embodiment, the conditions of step (d) are flow rate: less than 100cm/h; column height: 15+/-2 cm; equilibration/re-equilibration buffer: 50mM NaAc-HAc, pH 5.0.+ -. 0.4; eluting buffer: 50mM NaAc-HAc,200mM NaCl,pH5.0; eluent: 50mM NaAc-HAc,350mM NaCl,pH5.0; the pH value of the sample is 5.0+/-0.2, and the electric conductivity is 5.0-10.0mS/cm; the loading is less than or equal to 40mg/mL.
Detailed Description
The experimental methods of the present invention, in which specific conditions are not specified, are generally carried out under conventional conditions, and the experimental materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores.
Example 1 nucleotide sequence acquisition and optimization
The amino acid sequence of the fusion protein of the invention is as follows: the first heavy chain is SEQ ID NO. 1, the corresponding light chain is SEQ ID NO. 3, and the second heavy chain is SEQ ID NO. 2. Converting the target amino acid sequences into nucleotide sequences, designing heavy chain genes and light chain genes respectively encoding the antibodies, and designing nucleotide sequences encoding signal peptides obtained by optimizing the amino acid sequences at the 5' ends of the heavy chain and the light chain respectively; in addition, stop codons were added to the 3' ends of the light and heavy chain nucleotide sequences, respectively. The nucleotide sequence of the finally obtained optimized coded antibody is as follows: the nucleotide sequence encoding the first heavy chain is SEQ ID NO. 4, the nucleotide sequence encoding the second heavy chain is SEQ ID NO. 5, and the nucleotide sequence encoding the light chain is SEQ ID NO. 6.
EXAMPLE 2 Gene synthesis and construction of expression vectors
The pcDNA3.1-G418 vector is adopted as a special vector for expressing the light chain and the heavy chain of the multifunctional antibody. The pcDNA3.1-G418 vector contains the Promoter CMV Promoter used for the heavy chain, the eukaryotic selectable marker G418 tag and the prokaryotic selectable marker Ampicillin. The nucleotide sequences of the first heavy chain, the second heavy chain and the light chain coding genes expressed by the antibody (namely target genes) are respectively obtained through gene synthesis, hindIII and XhoI are used for carrying out double enzyme digestion on the vector and the target fragment, the vector and the target fragment are recovered and then are subjected to enzyme ligation through DNA ligase, and E.coli competent cells DH5 alpha are transformed, positive clones are selected, plasmid extraction and enzyme digestion verification are carried out, and the recombinant plasmid containing the first heavy chain, the second heavy chain and the light chain coding genes of the antibody is obtained.
EXAMPLE 3 plasmid extraction
According to the method described in the molecular cloning laboratory guidelines (2002, scientific Press), recombinant plasmids containing the above-mentioned genes of interest were transformed into E.coli competent cells DH 5. Alpha. And the transformed bacteria were spread on LB plates containing 100. Mu.g/mL ampicillin, cultured, plasmid clones were selected and cultured in liquid LB medium, shaking at 260rpm for 14h, plasmids were extracted by endotoxin-free plasmid megapump kit, dissolved in sterile water and concentration was measured using a nucleic acid protein quantitative analyzer.
EXAMPLE 4 plasmid transfection, transient expression and antibody purification
At 37℃C, 8% CO 2 ExpiCHO was cultured at 100rpm to a cell density of 6X 10 6 And each mL. The constructed vector plasmids were transfected into the above cells using liposomes at a mass concentration of 1:1:1, respectively, with a transfected plasmid concentration of 1mg/mL, and with reference to the liposome concentration of ExpiCHO TM Expression System kit, 5% CO at 32 ℃C 2 Culturing at 100rpm for 7-10 days. The feed was fed once after 18-22h and between day 5 of transfection. The above culture product was placed in a centrifuge, centrifuged at 4000g, filtered through a 0.22 μm filter membrane and the culture supernatant was collected, and the resulting antibody protein was purified using protein A, ion column and the eluate was collected.
The specific operation steps of ProteinA and ion column purification are as follows: the cell culture fluid is centrifuged at high speed, and the supernatant is subjected to affinity chromatography by using a GE protein A chromatography column. Chromatography uses equilibration buffer 1 XPBS (pH 7.4), cell supernatants were combined, washed with PBS to UV light back to baseline, then eluted with elution buffer 0.1M glycine (pH 3.0), and stored with Tris to adjust pH to neutral. The pH of the product obtained by affinity chromatography is adjusted to 1-2 pH units below or above isoelectric point pI, and the product is diluted appropriately to control the sample conductivity below 5 ms/cm. The collection tube in which the fusion protein of the present invention is located is selected for subsequent purification experiments according to SDS-PAGE using appropriate conditions corresponding to pH buffers such as phosphate buffer, acetate buffer, etc., and NaCl gradient elution under corresponding pH conditions using ion exchange chromatography methods conventional in the art such as anion exchange or cation exchange.
EXAMPLE 5 affinity chromatography
Sample: antibody sample 1 was prepared according to the method of examples 1-4, concentration: 4.65mg/mL; antibody sample 2, concentration: 3.74mg/mL; antibody sample 3, concentration: 4.02mg/mL
Chromatography column: omnifit 6.6mm/330mm column, packing: bogera Long Naijian ProteinADiamond, column volume: 6.158mL
Equilibration buffer: 20mM PB+140mM NaCl,pH7.4
Elution buffer 1:20mM PB+500mM NaCl,pH6.0
Elution buffer 2:50mM NaAc-HAc, pH5.0
Elution buffer: 50mM NaAc-HAc, pH3.5
The pipeline is rinsed with balance buffer solution in advance. The column was equilibrated with equilibration buffer at 2 column volumes to baseline plateau at a flow rate of 1.2mL/min. The sample was applied at a volume of 45mL and a flow rate of 1.2mL/min. After loading was complete, the column volume was re-equilibrated 2 times with equilibration buffer to baseline plateau at a flow rate of 1.2mL/min. Eluting with eluting buffer 1, eluting 2 times column volume, and flowing speed 1.2mL/min. Eluting with eluting buffer 2, eluting 2 times column volume, and flowing speed 1.2mL/min. Elution was performed with elution buffer at a flow rate of 1.2mL/min and the elution peak was collected. The purification results of each sample are shown in Table 1. The recovery rate in the step is high, the purity reaches about 83%, and the purification purity reaches about 79%.
TABLE 1
EXAMPLE 6 Low pH Virus inactivation
The steps are as follows: the pH was adjusted to 3.6 with 18% glacial acetic acid, allowed to stand at room temperature for 60min, and then adjusted back to 6.5 with 2M Tris. Then deep filtration is carried out. The results of each sample are shown in Table 2.
TABLE 2
| Sample of | Recovery% | Purity% |
| Antibody sample 1 | 91.99％ | 78.43％ |
| Antibody sample 2 | 91.74％ | 79.15％ |
| Antibody sample 3 | 93.01％ | 80.96％ |
EXAMPLE 7 anion chromatography
Sample: antibody samples 1, 2, 3 inactivated by low pH virus
Chromatography column: omnifit 6.6mm/330mm column, packing: capto Q, column volume: 5.8mL
Equilibration buffer: 20mM PB,30mM NaCl,pH6.5
Eluting buffer: 20mM PB,1.0M NaCl,pH6.5
The pipeline is rinsed with balance buffer solution in advance. The column was equilibrated with 5 column volumes of equilibration buffer to baseline plateau at a flow rate of 1.16mL/min. Samples were loaded and breakthrough peaks were collected at a loading of 80mg/mL with a flow rate of 1.16mL/min. After loading was complete, the column volume was re-equilibrated 2 times with equilibration buffer to baseline plateau at a flow rate of 1.16mL/min. Eluting with eluting buffer solution, wherein the eluting rate is 1.16mL/min and the column volume is 1.5 times. Washing with regeneration buffer, eluting 2 times of column volume, and flowing speed is 0.5mL/min. The purification results of each sample are shown in Table 3. The recovery rate of the step is high, the purity reaches about 94%, the purification purity reaches about 80%, and further purification is needed.
TABLE 3 Table 3
EXAMPLE 8 cationic chromatography
Sample: antibody samples 1, 2, 3 via anion chromatography
Chromatography column: omnifit 6.6mm/330mm column, packing: GE Capto S ImpAct, column volume: 6mL of
Equilibration buffer: 50mM NaAc-HAc, pH5.0
Eluting buffer: 50mM NaAc-HAc,200mM NaCl,pH5.0
Elution buffer: 50mM NaAc-HAc,350mM NaCl,pH5.0
The pH value of the sample is regulated to 5.0, the sample is diluted to the electric conduction of 5-10mS/cm by water injection, the pipeline is pre-rinsed by a balancing buffer solution, the chromatographic column is flushed by a balancing buffer solution with the volume of 2 times of the column volume at the flow rate of less than 100cm/h, and the sample is loaded at the flow rate of less than 100 cm/h. Washing with 2 times of column volume balancing buffer solution at a flow rate of less than 100cm/h, and then washing with 4 times of column volume washing buffer solution at a flow rate of less than 100cm/h; eluting with eluting buffer at a rate of less than 100cm/h, and collecting eluting peaks in segments. The purification results of each sample are shown in Table 4. The recovery rate of the step is higher, the recovery rate reaches about 68%, and the purification purity reaches about 97%.
TABLE 4 Table 4
In conclusion, each sample can obtain a sample with higher purity through one-step affinity chromatography, low-pH virus inactivation, one-step anion chromatography and one-step cation chromatography, the purity is more than 96%, various indexes meet the requirements, and the total yield is between 45 and 52%.
It should be understood that the foregoing description is only of the preferred embodiments of the present invention and is not intended to limit the scope of the invention, but is intended to cover any and all modifications, equivalents, and alternatives falling within the spirit and principles of the invention.
Claims (10)
1. A method for purifying an anti-B7H 3 antibody-cytokine fusion protein, comprising the steps of: (a) affinity chromatography; (b) low pH viral inactivation; (c) anion exchange chromatography; (d) cation exchange chromatography.
2. The method of claim 1, wherein the first heavy chain amino acid sequence of the fusion protein is selected from the group consisting of SEQ ID NO. 1, the second heavy chain amino acid sequence is selected from the group consisting of SEQ ID NO. 2, and the amino acid sequences of the first light chain and the second light chain are selected from the group consisting of SEQ ID NO. 3.
3. The method according to claim 1, wherein step (a) comprises loading, wherein loading should be controlled to within 20mg/mL of filler, preferably within 15mg/mL of filler.
4. The method of claim 1, wherein step (a) comprises the steps of: step (1), treating chromatographic packing and loading; step (2), leaching by using leaching buffer solution; step (3), eluting and collecting the fusion protein by using an elution buffer; the loading should be controlled to within 20mg/mL of filler, preferably within 15mg/mL of filler.
5. The method of claim 4, wherein step (1) is performed by flushing the equilibrated chromatography column with 2-4 volumes of equilibration buffer at a flow rate of 1.0-1.4mL/min, and loading at a flow rate of 1.0-1.4mL/min, wherein the equilibration buffer is 10-30mm pb,120-160mm nacl, and ph 7.2-7.6.
6. The method of claim 4, wherein step (2) is performed with 2-4 column volumes of rebalancing buffer at a flow rate of 1.0-1.4mL/min, then with 2-4 column volumes of rinsing buffer 1 at a flow rate of 1.0-1.4mL/min, and finally with 2-4 column volumes of rinsing buffer 2 at a flow rate of 1.0-1.4mL/min, wherein the equilibration buffer is 10-30mm pb,120-160mm nacl, ph 7.2-7.6; the leaching buffer solution 1 is 15-25mM NaAc-HAc,450-550mM NaCl, and the pH is 5.8-6.2; the leaching buffer solution 2 is 40-60mM NaAc-HAc, and the pH is 4.7-5.3.
7. The method according to claim 4, wherein the step (3) uses an isocratic elution method, eluting with 3-5 column volumes of elution buffer at a flow rate of 1.0-1.4mL/min, wherein the elution buffer is 50mM NaAc-HAc, pH 3.5.
8. The purification method according to claim 1, wherein the step (b) is to add 18% glacial acetic acid, adjust the pH to 3.6.+ -. 0.2, leave it at room temperature for 1-2 hours, and add 2M Tris to adjust the pH to 6.5.+ -. 0.2.
9. The purification process according to claim 1, wherein the conditions of step (c) are flow rate: less than 205cm/h; column height: 15+/-2 cm; equilibration/re-equilibration buffer: 20mM PB+30mM NaCl,pH6.0 + -0.4; the pH value of the sample is 6.5+/-0.2, and the electric conductivity is 5.0-8.0mS/cm; the loading is less than or equal to 80mg/mL.
10. The purification process according to claim 1, wherein the conditions of step (d) are flow rate: less than 100cm/h; column height: 15+/-2 cm; equilibration/re-equilibration buffer: 50mM NaAc-HAc, pH 5.0.+ -. 0.4; eluting buffer: 50mM NaAc-HAc,200mM NaCl,pH5.0; eluent: 50mM NaAc-HAc,350mM NaCl,pH5.0; the pH value of the sample is 5.0+/-0.2, and the electric conductivity is 5.0-10.0mS/cm; the loading is less than or equal to 40mg/mL.
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