CN118344486A - Purification method for producing recombinant anti-CD 19-CD3 bispecific antibody Fab fragment - Google Patents
Purification method for producing recombinant anti-CD 19-CD3 bispecific antibody Fab fragment Download PDFInfo
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Abstract
The invention discloses a purification method for producing recombinant anti-CD 19-CD3 bispecific antibody Fab fragments, which comprises the following steps: in the affinity chromatography process, the supernatant of the CHO culture is loaded into an affinity chromatography filler after being sterilized and filtered, and a first balance solution, a first eluent, a second eluent and a first eluent are sequentially loaded on the affinity chromatography filler, and an affinity chromatography elution product is obtained by collecting, so that the affinity chromatography is realized; and in the cation exchange chromatography process, the elution product of the affinity chromatography is loaded into a cationic chromatography filler, a second equilibrium solution is loaded on the cationic chromatography filler, and after the linear elution of the second equilibrium solution, the recombinant anti-CD 19-CD3 bispecific antibody Fab fragment is collected. The method for purifying the recombinant anti-CD 19-CD3 bispecific antibody Fab fragment has few steps, solves the problems of low recovery rate, low purity and difficult amplification in the prior art, and simultaneously uses domestic filler with higher economic benefit.
Description
Technical Field
The invention relates to the field of biological medicine purification, in particular to a purification method for producing recombinant anti-CD 19-CD3 bispecific antibody Fab fragments.
Background
CD19 belongs to the immunoglobulin superfamily and is a receptor expressed on the surface of B cells and plays an important role in regulating the growth, activation and differentiation of B lymphocytes as an important signaling molecule. CD3 belongs to the immunoglobulin superfamily and is an important component of the T lymphocyte receptor complex involved in T cell expression, antigen recognition and signaling.
Bispecific antibodies have two antigen binding targets, which can bind to target cells and effector cells respectively, and guide the effector cells to target and kill tumors, so that bispecific antibodies for treating tumors are attracting more and more attention. Prior studies have shown that bispecific molecules comprising anti-CD 19-CD3 have a relatively definite clinical efficacy. At present, the main expression mode is to express recombinant anti-CD 19-CD3 bispecific antibody Fab fragments in Chinese hamster ovary Cells (CHO) by genetic engineering technology, but the CHO expression supernatant contains a plurality of impurities, such as HCP, HCD, polymers and the like, and the impurities must be strictly purified to meet the quality requirements.
The method for purifying the Fab fragment of the CHO-expressed recombinant anti-CD 19-CD3 bispecific antibody is mainly disclosed as follows:
The WO99/54440 patent and CN200480014513.2 disclose a method for purifying a pharmaceutical composition of a dual-specificity anti-CD 3 and anti-CD 19 antibody construct, which is characterized in that 6 histidines are carried at the C end of protein, so that the protein is captured by metal chelating chromatography and separated by a molecular sieve, and the final sample purity is more than 95 percent (SDS-PAGE electrophoresis method), but the patent does not mention the recovery rate of the product, the loading amount of the molecular sieve chromatography is small (the loading amount is less than or equal to 4 percent of the volume of the column), the time consumption is long, and the amplified production is not facilitated.
2. Chinese patent CN116769044a discloses an anti-CD 3 and CD19 bispecific antibody protein and a chromatographic purification method thereof, which adopts hydroxyapatite chromatography to separate impurities, and retains one or more steps of chromatography means such as ion exchange chromatography, hydrophobic chromatography, etc. before the hydroxyapatite chromatography, the recovery rate of the final sample is 77.5%, the process steps are increased, the recovery rate of the product is lower, and the production period is prolonged.
3. Chinese patent CN107903324a discloses a method for separating bispecific antibody of human CD19 and CD3 from CHO cell culture, which adopts two-step chromatography method of cation exchange and anion exchange to prepare bispecific antibody with purity greater than 95%, but the ultrafiltration step is required to replace buffer before anion, the process is more complex, the two-step ion exchange purification principle is single, and the purity of the obtained product can not meet the production requirement.
Based on the above, the existing anti-CD 19-CD3 bispecific antibody purification technology has the disadvantages of high cost, low yield, poor selectivity, long period and the like, and is not suitable for large-scale production. Therefore, it is important to develop a purification process which is suitable for expressing anti-CD 19-CD3 bispecific antibody by CHO cell system, has high purity and recovery rate, short period and low cost.
Disclosure of Invention
The present invention is directed to a purification method for producing recombinant anti-CD 19-CD3 bispecific antibody Fab fragments, which solves the above-mentioned problems in the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions: a purification method for the production of recombinant anti-CD 19-CD3 bispecific antibody Fab fragments, comprising:
S1: affinity chromatography process: loading the supernatant of the CHO culture medium into an affinity chromatography packing after sterilization and filtration, sequentially loading a first balance liquid, a first eluent and a second eluent on the affinity chromatography packing, and finally loading the first eluent and collecting to obtain an affinity chromatography eluting product to realize affinity chromatography;
s2: cation exchange chromatography process: and (3) loading the affinity chromatography elution product into a cationic chromatography packing, sequentially loading a second equilibrium solution on the cationic chromatography packing, and collecting the recombinant anti-CD 19-CD3 bispecific antibody Fab fragment after linear elution by the second elution solution.
Preferably, the CHO culture medium in S1 is a CHO cell culture medium expressing recombinant anti-CD 19-CD3 bispecific antibody Fab fragment, and the cell culture medium is centrifuged at 3000rpm for 20min to remove cells and cell debris, and the supernatant is collected.
Preferably, the pore size of the sterilization filtration membrane used in the sterilization filtration in the step S1 is 0.22 μm, and the sterilization filtration membrane is made of PES.
Preferably, the loading amount of the recombinant anti-CD 19-CD3 bispecific antibody Fab fragment loaded into the affinity chromatography filler in S1 is 30-40mg/ml, and the affinity chromatography filler in S1 is NMab Pro L; the chromatographic system for affinity chromatography was Ä KTA Avant150 chromatographic system (Cytiva).
Preferably, the first equilibrium liquid in the S1 is 20mM PB, the NaCl concentration is 0-0.15mol/L, the pH is 7.0, and the loading amount of the first equilibrium liquid after the sample is loaded is more than or equal to 3 column volumes.
Preferably, the first eluent in the S1 is 20mM PB, the NaCl concentration is 0.5-1.0mol/L, the pH is 7.0, and the loading amount of the first eluent is more than or equal to 3 column volumes.
Preferably, the second eluent in the S1 is a 20-50mM sodium acetate system, the pH is 5.0-5.5, and the loading amount of the second eluent is more than or equal to 3 column volumes.
Preferably, the first eluent in S1 is a 20-50mM sodium acetate system, the pH is 3.3-3.7, and the loading amount of the first eluent is greater than or equal to 4 column volumes.
Preferably, the feed liquid loaded to the cationic filler in the S2 is an affinity elution collection liquid, the pH value of which is 4.5-5.0 after 2M Tris sample adjustment, and the conductivity is less than 5.000mS/cm.
Preferably, the pore size of the sterilization filtration membrane used in the sterilization filtration in the step S2 is 0.22 μm, and the sterilization filtration membrane is made of PES.
Preferably, the loading of recombinant anti-CD 19-CD3 bispecific antibody Fab fragments loaded into the cationic chromatographic packing in S2 is 30-40mg/ml, cationic chromatographic packing NanoGel-50SP HP in S2.
Preferably, the second balancing solution in the step S2 is 20mM PB, the pH is 4.5-5.0, and the loading amount of the second balancing solution before the completion of loading is greater than or equal to 4 column volumes.
Preferably, the second balancing solution in the step S2 is 20mM PB, the pH is 4.5-5.0, and the loading amount of the second balancing solution after the loading is more than or equal to 3 column volumes.
Preferably, the second eluent in S2 is 20mM PB, the pH is 4.5-5.0, the NaCl concentration is 1.0mol/L, the linear condition is an ascending NaCl concentration gradient, wherein the NaCl concentration is 0.0-1.0mol/L, and the elution volume is 10-30CV.
Compared with the prior art, the invention has the beneficial effects that:
1. The invention has the advantages that the invention provides a purification method, which solves the problems of low recovery rate, low purity and difficult amplification of production in the prior art by combining two process steps, and simultaneously uses domestic filler, thereby having higher economic benefit.
2. The invention can obtain the product with purity more than 99% and recovery rate more than 90% after two-step chromatography.
Drawings
FIG. 1 is an affinity chromatography chromatogram of example 1;
FIG. 2 is a cationic chromatography map of example 2;
FIG. 3 is a graph showing the results of SEC-HPLC analysis of the cationic analytical product in example 2;
FIG. 4 is a graph showing the analysis results of CE-NR-SDS of the cationic chromatography product of example 2;
Detailed Description
The following describes the technical solution in the embodiment of the present invention in detail with reference to the drawings in the embodiment of the present invention. The embodiment is not limited to the present invention, and all other examples obtained by a person of ordinary skill in the art without making any inventive effort based on the examples in the present invention are within the scope of the present invention.
Example 1: affinity chromatography
The CHO cell culture expressing the recombinant anti-CD 19-CD3 bispecific antibody Fab fragment was centrifuged at 3000rpm for 20min to remove cells and cell debris and the supernatant was collected. Then, the supernatant was sterilized and filtered by using a sterilizing filter having a pore size of 0.22. Mu.m, and the filtrate was collected.
Affinity chromatography was performed using Ä KTA Avant150 chromatography system (Cytiva) with chromatography packing NMab Pro L (5 ml, nano-micro). The instrument operation was performed according to the instructions, with the A1 pump being Buffer A (20 mM PB, pH 7.0).
The specific chromatography steps are as follows:
(1) Cleaning: washing NMab Pro L resin columns with 0.1M NaOH at a flow rate of 1ml/min, washing NMab Pro L resin columns with 2 column volumes, suspending for 15min, and washing NMab Pro L resin columns with 0.1M NaOH at a flow rate of 1 ml/min;
(2) Balance: washing NMab Pro L column volumes of resin column to UV baseline with 20mM PB, pH 7.0 buffer, flow rate 1 ml/min;
(3) Loading: loading at a flow rate of 1 ml/min;
(4) Balance: 3 Column Volumes (CV) of the NMab Pro L resin columns were washed with 20mM PB, pH 7.0 buffer, flow rate 1 ml/min;
(5) Leaching 1: 3 Column Volumes (CV) of NMab Pro L resin columns were washed with 20mM PB+0.5M NaCl,pH 7.0, a flow rate of 1 ml/min;
(6) Leaching 2: the NMab Pro L resin column was washed with 50mM Na-Ac, pH 5.5, flow rate 1ml/min, 3 Column Volumes (CV);
(7) Eluting: washing NMab Pro L resin column with 50mM Na-Ac, pH 3.5, flow rate 1ml/min, 4 Column Volumes (CV), and collecting eluate with UV collection range of 50mAu-50 mAu;
(8) Regeneration: 3 Column Volumes (CV) of the NMab Pro L resin columns were washed with 1M HAc, flow rate 1 ml/min;
(9) Balance: 3 Column Volumes (CV) of the NMab Pro L resin columns were washed with 20mM PB, pH 7.0 buffer, flow rate 1 ml/min;
(10) Cleaning: washing NMab Pro L resin columns for 2 column volumes with 0.1M NaOH at a flow rate of 1ml/min, suspending for 15min, and washing NMab Pro L resin columns for 1 column volume with 0.1M NaOH at a flow rate of 1ml/min to remove impurity residues on the resin;
(11) Balance: the resin column was washed NMab Pro L with 20mM PB, pH 7.0 buffer, flow 1ml/min, 4 Column Volumes (CV);
(12) And (3) preserving: the NMab Pro L resin columns were washed 3 column volumes with 20% ethanol at a flow rate of 1 ml/min.
The affinity chromatography is shown in figure 1.
Example 2: cationic chromatography
And (3) sampling an affinity chromatography elution product, filtering, loading the elution product into a cationic chromatography filler, sequentially loading a second balancing solution on the cationic chromatography filler, linearly eluting the elution product by the second eluting solution, and collecting the elution product to obtain the recombinant anti-CD 19-CD3 bispecific antibody Fab fragment.
Cationic chromatography was performed using Ä KTA Avant150 chromatography system (Cytiva) with chromatography packing of NanoGel-50SP HP (5 ml, nano-micro). The instrument was operated according to the instructions, with the A1 pump being Buffer A (20 mM PB, pH 4.5) and the B1 pump being Buffer B (20mM PB+1M NaCl,pH. Sup.4.5).
The specific chromatography steps are as follows:
(1) Cleaning: washing NanoGel-50SP HP resin column with 1M NaOH at a flow rate of 1ml/min, pausing for 15min, and washing NanoGel-50SP HP resin column with 1M NaOH at a flow rate of 1 ml/min;
(2) Pre-balancing: washing NanoGel-50SP HP resin column 3 column volumes with 1M NaCl at a flow rate of 1ml/min to UV baseline;
(3) Balance: washing the NanoGel-50SP HP resin column with 20mM PB, pH 4.5 buffer, flow rate 1ml/min, 4 column volumes to UV baseline;
(4) Loading: loading at a flow rate of 1 ml/min;
(5) Balance: 3 Column Volumes (CV) of NanoGel-50SP HP resin columns were washed with 20mM PB, pH 4.5 buffer, flow rate 1 ml/min;
(6) Eluting: performing linear elution with Buffer A (20 mM PB, pH 4.5), buffer B (20mM PB+1M NaCl,pH4.5) at a flow rate of 1.00ml/min, wherein the linear elution gradient is 100% -0% of Buffer A, 0% -100% of Buffer B, the elution volume is 20CV, and collecting the eluent with the UV collection range of 50mAu-50 mAu;
(7) Regeneration: 3 Column Volumes (CV) of NanoGel-50SP HP resin columns were washed with 1M NaCl, flow rate 1 ml/min;
(8) Cleaning: washing NanoGel-50SP HP resin column with 1M NaOH at a flow rate of 1ml/min, suspending for 15min, and washing NanoGel-50SP HP resin column with 1M NaOH at a flow rate of 1ml/min to remove impurity residues on the resin;
(9) And (3) preserving: the NanoGel-50SP HP resin column was flushed with 0.1M NaOH at a flow rate of 1ml/min for 3 column volumes.
The cationic chromatography is shown in FIG. 2.
Purity detection
The purity of the recombinant anti-CD 19-CD3 bispecific antibody Fab fragments obtained after one-step sterilization and two-step chromatography was analyzed by SEC-HPLC and CE-NR-SDS methods, in combination with example 1, example 2. The detection results are shown in table 1, fig. 3 and fig. 4:
TABLE 1
From the results of the tests shown in Table 1, FIG. 3 and FIG. 4, it is clear that the purification process according to the present invention can obtain a product with a purity of more than 99% and a recovery rate of more than 90%.
Examples 1-2 are the full flow process of the present invention, with cationic chromatography set up after affinity chromatography capture, which can further remove host cell proteins, further enhancing product purity. In conclusion, the method process of the invention prepares the high-purity recombinant anti-CD 19-CD3 bispecific antibody Fab fragment by a two-step chromatography mode, has high product recovery rate, less purification steps and short production period, can realize industrial production, and has good market application prospect.
Although embodiments of the present invention have been shown and described in detail, it will be apparent to those skilled in the art that modifications, improvements or variations can be made to these embodiments without departing from the spirit and principles of the invention, and such modifications, improvements or variations are intended to be within the scope of the invention as claimed.
Claims (10)
1. A purification method for the production of a recombinant anti-CD 19-CD3 bispecific antibody Fab fragment comprising the steps of:
S1: affinity chromatography process: the CHO cell culture expressing recombinant anti-CD 19-CD3 bispecific antibody Fab fragment is loaded into an affinity chromatography packing after centrifugation, sterilization and filtration, a first equilibrium solution and a first and a second eluent are sequentially loaded on the affinity chromatography packing, and finally, the first eluent is loaded and collected to obtain an affinity chromatography eluting product, so that affinity chromatography is realized;
s2: cation exchange chromatography process: and (3) sampling an affinity chromatography elution product, filtering, loading the elution product into a cationic chromatography filler, sequentially loading a second balancing solution on the cationic chromatography filler, linearly eluting the elution product by the second eluting solution, and collecting the elution product to obtain the recombinant anti-CD 19-CD3 bispecific antibody Fab fragment.
2. The purification method for producing a recombinant anti-CD 19-CD3 bispecific antibody Fab fragment according to claim 1, wherein: the loading amount of the recombinant anti-CD 19-CD3 bispecific antibody Fab fragment loaded in the S1 into the affinity chromatography filler is 30-40mg/ml, and the affinity chromatography filler in the S1 is NMab Pro L.
3. The purification method for producing a recombinant anti-CD 19-CD3 bispecific antibody Fab fragment according to claim 1, wherein: in the S1, the first equilibrium liquid is 20mM PB, the NaCl concentration is 0-0.15mol/L, the pH is 7.0, and the loading amount of the first equilibrium liquid is more than or equal to 3 column volumes.
4. The purification method for producing a recombinant anti-CD 19-CD3 bispecific antibody Fab fragment according to claim 1, wherein: the first eluent in the S1 is 20mM PB, the NaCl concentration is 0.5-1mol/L, the pH is 7.0, and the loading amount of the first eluent is more than or equal to 3 column volumes; the second eluent in the S1 is a 20-50mM sodium acetate system, the pH is 5.0-5.5, and the loading amount of the second eluent is more than or equal to 3 column volumes.
5. The purification method for producing a recombinant anti-CD 19-CD3 bispecific antibody Fab fragment according to claim 1, wherein: the first eluent in the S1 is a 20-50mM sodium acetate system, the pH is 3.3-3.7, the loading amount of the first eluent is greater than or equal to 4 column volumes, and the UV collection range is 50mAu-50mAu.
6. The purification method for producing a recombinant anti-CD 19-CD3 bispecific antibody Fab fragment according to claim 1, wherein: and (2) the pH value of the affinity elution collection liquid loaded to the cationic filler in the S2 is 4.5-5.0 after the affinity elution collection liquid is subjected to 2M Tris sample adjustment, and the electric conductivity is less than 5.000mS/cm.
7. The purification method for producing a recombinant anti-CD 19-CD3 bispecific antibody Fab fragment according to claim 1, wherein: the loading amount of the affinity chromatography elution product loaded into the cationic chromatography packing in S2 is 30-40mg/ml, and the cationic chromatography packing NanoGel-50SP HP in S2.
8. The purification method for producing a recombinant anti-CD 19-CD3 bispecific antibody Fab fragment according to claim 1, wherein: and the second balancing liquid in the S2 is 20mM PB, the pH is 4.5-5.0, and the loading amount of the second balancing liquid before the completion of the loading is greater than or equal to 4 column volumes.
9. The purification method for producing a recombinant anti-CD 19-CD3 bispecific antibody Fab fragment according to claim 1, wherein: and the second balancing liquid in the S2 is 20mM PB, the pH is 4.5-5.0, and the loading amount of the second balancing liquid is more than or equal to 3 column volumes after the sample loading is finished.
10. The purification method for producing a recombinant anti-CD 19-CD3 bispecific antibody Fab fragment according to claim 1, wherein: the elution condition of the cation chromatography in S2 is linear elution, the second eluent is 20mM PB, the pH is 4.5-5.0, the NaCl concentration is 1.0mol/L, the linear condition is an increasing NaCl concentration gradient, the NaCl concentration is 0.0-1.0mol/L, the elution volume is 10-30CV, and the UV collection range is 50mAu-50mAu.
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| CN118344486A true CN118344486A (en) | 2024-07-16 |
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