TW202241924A - Method for purifying bispecific antibody - Google Patents

Abstract

A method for purifying a bispecific antibody, in particular relating to a method for purifying a protein by using two-step chromatography, and the method comprises affinity chromatography and composite mode anion exchange chromatography; the protein is the bispecific antibody, and comprises a first protein domain of a Fab structure and a second protein domain comprising two heavy chain variable domain VH structures. The purity of a sample obtained by using the method is improved from about 87% to at least 95% through size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) detection. The packing used can operate at a higher flow rate and a higher column bed height and maintain a high resolution, and large-scale production can be easily implemented.

Description

A kind of purification method of bispecific antibody

This application claims the priority of Chinese patent application 202110442397X with a filing date of April 23, 2021, and Chinese patent application 2022103882003 with a filing date of April 13, 2022. This application cites the full text of the above-mentioned Chinese patent application.

The invention relates to the technical field of antibody purification, in particular to a method for purifying a bispecific antibody, especially a BCMA×CD3 bispecific antibody.

Bispecific antibodies (referred to as double antibodies) refer to antibodies that can bind to two different epitopes or antigens at the same time, and can exert the synergistic effect of two monoclonal antibodies. Resistance is more advantageous. Due to its unique biological mechanism, bispecific antibodies have become an important branch in the field of antibody drug research and development, and are also one of the most popular research and development and investment directions in the pharmaceutical industry.

However, with its rapid development, it also faces a series of difficulties, including the preparation process. The BCMA×CD3 double antibody involved in the present invention is an asymmetric structure composed of three polypeptide chains. In order to minimize the formation of by-products of heavy chains with mismatches (such as two heavy chain mismatches of anti-CD3 antibodies), a Mutated heterodimeric Fc regions carrying "knob-hole" mutations and engineered disulfide bonds as described in WO2009080251 and WO2009080252. However, all molecular design strategies can only minimize the production of certain types of byproducts, but cannot completely eliminate all byproducts. Not only that, in the process of recombinant expression of double antibodies, incomplete fragment antibodies and aggregates will inevitably be produced. This conclusion has also been confirmed during the process development process. When the proteins captured by affinity chromatography were detected by polyacrylamide gel electrophoresis (SDS-PAGE), more than ten by-product bands were found. Moreover, the biochemical properties of these by-products are very similar to the target double antibody, so it is extremely difficult to separate the target double antibody from them.

Therefore, it is of great significance to develop a purification method suitable for process scale-up with high purity and high recovery rate of the target double antibody.

In order to solve the problem that there are too many by-product bands in the purification process of bispecific antibodies, the biochemical characteristics of the by-products are very similar to the target bi-antibody, which makes it extremely difficult to separate the target bi-antibody from it, the loss of the target bi-antibody is serious during the purification process, and the recovery rate is low To solve the problem, the present invention provides a method for separating and purifying bispecific antibodies with high recovery rate and high purity of the target protein, which can not only obtain high-purity target bispecific antibodies, but also greatly reduce the loss of target bispecific antibodies during the purification process.

In order to solve the above technical problems, one of the technical solutions of the present invention is to provide a method for purifying proteins using two-step chromatography, the method comprising affinity chromatography and multiple mode anion exchange chromatography; the protein is bispecific An antibody comprising a first protein functional region of a Fab structure and a second protein functional region comprising two heavy chain variable region VH structures; preferably, the two heavy chain variable regions are separated by (G ₄ S) _n Connected, the n is a non-zero natural number, preferably 1 to 20, more preferably 3 or 4; more preferably, the bispecific antibody also includes an Fc structure; further more preferably, the first protein functional region label Targeting CD3, the second protein domain targets BCMA.

In a specific embodiment, the bispecific antibody includes polypeptide chain 1, polypeptide chain 2, and polypeptide chain 3, wherein, the polypeptide chain 1 includes VL1-CL sequentially from the N-terminus to the C-terminus, and the polypeptide chain 2 From the N-terminal to the C-terminal, it includes VH1-CH1-CH2-CH3, and the polypeptide chain 3 includes VH2-VH2-CH2-CH3 from the N-terminal to the C-terminal.

In a specific embodiment, the VH1 comprises HCDR1, HCDR2 and HCDR3, the amino acid sequences of which are shown in SEQ ID NO: 5, 6 and 7 respectively; the VL1 comprises LCDR1, LCDR2 and LCDR3, the amino acid sequences of which are The sequences are respectively shown in SEQ ID NO: 10, 11 and 12; the VH2 comprises HCDR1, HCDR2 and HCDR3, and its amino acid sequence is shown in SEQ ID NO: 15, 16 and 17 respectively, or as shown in SEQ ID NO: 20, 21 and 22.

In a specific embodiment, the amino acid sequences of VH1, VL1 and VH2 are shown in SEQ ID NO: 2, SEQ ID NO: 9 and SEQ ID NO: 14, respectively; Alternatively, the amino acid sequences of VH1, VL1 and VH2 are shown in SEQ ID NO: 4, SEQ ID NO: 9 and SEQ ID NO: 19, respectively.

In a specific embodiment, the amino acid sequences of polypeptide chain 1, polypeptide chain 2 and polypeptide chain 3 are shown in SEQ ID NO: 8, SEQ ID NO: 1 and SEQ ID NO: 13, respectively; Alternatively, the amino acid sequences of polypeptide chain 1, polypeptide chain 2 and polypeptide chain 3 are shown in SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 18, respectively.

In a specific embodiment, the affinity chromatography includes the following steps: pre-equilibration, sample loading, equilibrium and elution; the pre-equilibration uses PBS as a pre-equilibration buffer, and the equilibrium uses PBS as an equilibrium buffer, The elution uses glycine-hydrochloric acid as the elution buffer.

Preferably, in the affinity chromatography, the filler is Mabselect sure LX, and/or the chromatography column is XK16/20.

In a specific embodiment, the loading of the culture supernatant of the bispecific antibody is 0.1-50 mg/ml, for example, 40 mg/ml when the sample is loaded, and the flow rate is, for example, 4.5 ml/min; and/or, the pre- The equilibration buffer uses 2 to 10 column volumes, such as 5 column volumes, and the flow rate is, for example, 10ml/min; and/or, the equilibration buffer uses 2 to 10 column volumes, such as 5 column volumes, The flow rate is, for example, 10 ml/min; and/or, the elution buffer is 50-200 mM, such as 100 mM glycine-hydrochloric acid, pH 3.5, and the flow rate is, for example, 5 ml/min.

Preferably, it also includes the step of using acetic acid to inactivate the virus; more preferably after the inactivation, the pH of the neutralized sample is to 6.5~7.5 such as 7.0; further more preferably, the neutralized sample uses 1M Tris-HCl buffer, pH8.0 conduct.

In a specific embodiment, the complex mode anion exchange chromatography comprises the following steps: pre-equilibration, sample loading, equilibration, washing and eluting; wherein, the pre-equilibration buffer used in the pre-equilibration is PBS or Tris -HCl; and/or, the equilibration buffer used in the equilibration is PBS or Tris-HCl; and/or, the rinsing buffer used in the rinsing includes liquid A containing PBS or Tris-HCl, containing spermine Solution B of acid-hydrochloric acid; and/or, the elution buffer used in the elution includes histidine and aspartic acid.

Preferably, in the anion exchange chromatography, the filler used is Capto adhere ImpRes; the chromatographic empty column is Tricorn 5/100; and/or, the bispecific antibody sample captured by the affinity chromatography is loaded into the layer Analysis column, protein sample loading capacity 7mg/ml; and/or, the A solution includes 20mM PBS, pH7.0, or 20mM Tris-HCl, pH7.2; the B solution includes 20mM PBS, pH7.0 , 1M arginine-hydrochloric acid, or 20mM Tris-HCl, pH7.2, 1M arginine-hydrochloric acid; and/or, the flow rate of the washing and/or elution is 0.4ml/min.

In a specific embodiment, the pre-equilibration uses a pre-equilibration buffer of 2 to 10 column volumes, such as 5 column volumes, and the pre-equilibration buffer is 20mM PBS, pH7.0; and/or, The equilibrium uses 2 to 10 column volumes, such as 10 column volumes of equilibrium buffer, the equilibrium buffer is 20mM PBS, pH7.0; and/or, the washing uses A solution and 0 to 100 %B liquid is carried out gradient elution, 20 column volumes, and described A liquid comprises 20mM PBS, pH7.0, and described B liquid comprises 20mM PBS, pH7.0, 1M arginine-hydrochloric acid; And/or, all The elution buffer used in the above elution is 10 mM histidine and 10 mM aspartic acid, pH 5.0.

In a specific embodiment, the pre-equilibration uses a pre-equilibration buffer of 2 to 10 column volumes, for example, 5 column volumes, and the pre-equilibration buffer is 20mM Tris-HCl, pH7.2; and/ Or, the equilibrium uses 2 to 10 column volumes, such as 10 column volumes of equilibration buffer, the equilibration buffer is 20mM Tris-HCl, pH7.2; 30% to 40% Liquid B was subjected to isocratic rinsing, rinsing 20 column volumes, wherein said A liquid included 20mM Tris-HCl, pH7.2, said B liquid included 20mM Tris-HCl, pH7.2, 1M arginine- hydrochloric acid; and/or, the elution buffer used in the elution is 10 mM histidine and 10 mM aspartic acid, pH 5.0.

In a specific embodiment, the purification method also includes the process of analyzing the purity of the product obtained by the affinity chromatography and/or the multiple mode anion exchange chromatography, and the analysis process includes particle size screening chromatography - High performance liquid chromatography (SEC-HPLC), wherein the mobile phase is phosphate buffer containing 15% acetonitrile; preferably, the pH is 7.4.

On the basis of conforming to common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progress effect of the present invention is: 1. The compound mode anion chromatography elution step can remove about 10 kinds of impurities in one step, and the target protein will not be eluted, and the target protein is almost not lost. In the preferred technical scheme, after 0.4M arginine (40% B liquid) or 0.3M arginine (30% B liquid) eluting, the purity of the sample detected by particle size sieve chromatography-high performance liquid chromatography (SEC-HPLC) is about 87% (complex mode cation Chromatography) is improved to more than 95%; in a preferred scheme, the purity detected can reach more than 97%. 2. The Capto adhere ImpRes filler is used. The matrix has both the high flow and high rigidity characteristics of Capto media and the small particle size characteristics of ImpRes. It can operate at higher flow rates and higher column bed heights and maintain high resolution, easy to scale up production . 3. The method of SEC-HPLC was optimized by adding 15% acetonitrile in the mobile phase, which solved the problem that SEC-HPLC could not truly reflect the purity of the product before optimization. The peak shape of the optimized method was more symmetrical and the resolution was also obvious. improve.

detailed description

The present invention will be further described below through examples, but the present invention is not limited to the scope of the examples. For the experimental methods that do not specify specific conditions in the following examples, select according to conventional methods and conditions, or according to the product instructions. Embodiment 1 purification object

The purified object of the present invention is BCMA×CD3 bispecific antibody, which can bind to two targets at the same time, one end of which can recognize BCMA specifically expressed on the surface of tumor cells, and the other end can bind to CD3 on T cells molecular. When the BCMA×CD3 double antibody molecule binds to the surface of tumor cells, it can recruit and activate T cells near the tumor cells, thereby killing tumor cells.

The structure of the BCMA×CD3 bispecific antibody is shown in FIG. 16 .

For each bispecific antibody, three protein chains are involved, which respectively comprise the heavy chain of the corresponding anti-BCMA antibody, and the heavy and light chains of the aforementioned anti-CD3 antibody. In order to minimize by-product formation of heavy chains with mismatches (such as two heavy chain mismatches of anti-CD3 antibodies), mutated heterodimeric Fc regions were used, which carried "knob-hole" mutations and engineered disulfide bonds, as described in WO2009080251 and WO2009080252. The BCMA×CD3 bispecific antibody is IgG1 with Fc mutations L234A and L235A (numbering according to EU index).

Each bispecific antibody was produced by simultaneous co-transfection of three different mammalian expression vectors, each encoding: 1) the heavy chain of the corresponding BCMA antibody, which carries a "Hole" mutation in the Fc region to generate a heterodimer Antibody, CH3 of Fc carries L234A, L235A mutations. 2) The heavy chain of the corresponding CD3 antibody carries a "knob" mutation in the Fc region to produce a heterodimeric antibody, and the CH3 of the Fc carries the L234A and L235A mutations. 3) The light chain of the corresponding CD3 antibody. The "knob" mutations of the human IgG1 Fc region consisted of the following: T366W, and the "Hole" mutations consisted of the following: T366S, L368A, Y407V. In addition, S354C and "Hole" Y349C of the "knob" Fc region can be included to form a pair of disulfide bonds to increase stability and heterodimeric antibody production.

Tables 1 to 3 are the sequence information of the bispecific antibodies PR003178 and PR002299 of the present invention, respectively.

The heavy chain and heavy chain variable region of the CD3 antibody are respectively referred to as HC1 and VH1 in the bispecific antibody, and the heavy chain and heavy chain variable region of the BCMA antibody are respectively referred to as HC2 and VH2 in the bispecific antibody. Table 1 CD3 antibody sequence information for PR003178 PR003178 HC1 sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO: 1） PR003178 VH1 sequence EVQLVESGGGLVQPGGSLKLSCAASGFTFSTYAMNWVRQASGKGLEWVGRIRSKYNNYATYYADSVKDRFTISRDDSKNTAYLQMNSLKTEDTAVYYCTRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 2) PR002299 HC1 sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKDRFTISRDDSKSTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO: 3） PR002299 VH1 sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYAMNWVRQAPGKGLEWVSRIRSKYNNYATYYADSVKDRFTISRDDSKSTLYLQMNSLRAEDTAVYYCVRHGNFGNSYVSWFAYWGQGTLVTVSS (SEQ ID NO: 4) PR003178/ PR002299 VH1 CDR1 (Combined) GFTFSTYAMN (SEQ ID NO: 5) PR003178/ PR002299 VH1 CDR2 (Combined) RIRSKYNNYATYYADSVKD (SEQ ID NO: 6) PR003178/ PR002299 VH1 CDR3 (Combined) HGNFGNSYVSWFAY (SEQ ID NO: 7) PR003178/ PR002299LC serial QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGDKAALTLLGAQPEDEAEYFCALWYSNLWVFGGGTKLTVLGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYEGTHVEPEQWKSHRSYSCKQTV PR003178/ PR002299 VL sequence QAVVTQEPSLTVSPGGTVTLTCRSSTGAVTTSNYANWVQQKPGQAPRGLIGGTNKRAPWTPARFSGSLLGDKAALTLLGAQPEDEAEYFCALWYSNLWVFGGGTKLTVL (SEQ ID NO: 9) PR003178/ PR002299 LCDR1 (Combined) RSSTGAVTTSNYAN (SEQ ID NO: 10) PR003178/ PR002299 LCDR2 (Combined) GTNKRAP (SEQ ID NO: 11) PR003178/ PR002299 LCDR3 (Combined) ALWYSNLWV (SEQ ID NO: 12) Table 2 PR003178 HC2 sequence EVQLVETGGGLIQPGGSLRLSCAASGFTVSDNYMTWVRQAPGKGLEWVSVIFSGGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTALYYCARRNYDDTRGTDVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSEVQLVETGGGLIQPGGSLRLSCAASGFTVSDNYMTWVRQAPGKGLEWVSVIFSGGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTALYYCARRNYDDTRGTDVFDIWGQGTMVTVSSASEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO: 13） PR003178 VH2 sequence EVQLVETGGGLIQPGGSLRLSCAASGFTVSDNYMTWVRQAPGKGLEWVSVIFSGGNTYYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTALYYCARRNYDDTRGTDVFDIWGQGTMVTVSS (SEQ ID NO: 14) PR003178 VH2 CDR1 (Combined) GFTVSDNYMT (SEQ ID NO: 15) PR003178 VH2 CDR2 (Combined) VIFSGGNTYYADSVKG (SEQ ID NO: 16) PR003178 VH2 CDR3 (Combined) RNYDDTRGTDVFDI (SEQ ID NO: 17) table 3 PR002299 HC2 sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMIWVRQAPGKGLEWVSGISESGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCVKDLDDILTGYYKDYWGQGTLVTVSSRTGGGGSKLEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMIWVRQAPGKGLEWVSGISESGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCVKDLDDILTGYYKDYWGQGTLVTVSSASEPKSSDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK（SEQ ID NO: 18） PR002299 VH2 sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMIWVRQAPGKGLEWVSGISESGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYFCVKDLDDILTGYYKDYWGQGTLVTVSS (SEQ ID NO: 19) PR002299 VH2 CDR1 (Combined) GFTFSSYAMI (SEQ ID NO: 20) PR002299 VH2 CDR2 (Combined) GISESGGSTYYADSVKG (SEQ ID NO: 21) PR002299 VH2 CDR3 (Combined) DLDDILTGYYKDY (SEQ ID NO: 22) Embodiment 2 Chromatography method and selection optimization of filler 2.1 Screening of chromatography filler

The screening of filler is shown in Table 4. Table 4 Affinity filler Mabselect SuRe Mabselect PrimsA LambdaFabSelect Composite Mode Filler Capto MMC ImpRes Capto adhere ion exchange packing anion exchange chromatography QFF Capto Q ImpRes Cation Exchange Chromatography Capto SP ImpRes Mono-S Hydrophobic filler Buthl-S 6FF Capto Buthl ImpRes gel filtration Superdex 200

During the purification process of PR003178 culture fluid, different fillers were screened (as shown in Table 4), and the single chromatography purification method did not obtain the ideal recovery rate and purity, and the recovery rate was lower than 40%. 2.2 Affinity chromatography, cation chromatography process 2.2.1 Affinity chromatography

(1) Sample: PR003178 double-antibody culture medium expressed by CHO cells (expressed at 1.73 mg/ml).

(2) Main reagents and instruments: 10× PBS buffer solution, 1M Tris-HCl solution (pH 8.0) and glycine were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; hydrochloric acid was purchased from Shanghai Titan Technology Co., Ltd.; acetic acid Purchased from Avantuo, USA; protein A filler Mabselect SuRe, chromatography column XK16/20 (diameter 1.6 cm, length 20 cm), protein chromatography purification system AKTA pure 150 and protein chromatography purification system AKTA avant 150 were purchased from Stofan Corporation of the United States; Nanodrop One, an ultra-micro-volume spectrophotometer, was purchased from Thermo Fisher Scientific; SevenExcellence™ multi-parameter tester was purchased from Mettler Toledo.

(3) Protein A affinity chromatography method A chromatographic empty column XK16/20 (diameter 1.6 cm) was packed with protein A filler Mabselect SuRe with a packing height of 9 cm and a column volume of 18 ml. Equilibrium buffer (PBS) equilibrates 5 column volumes of the chromatography column, the flow rate is 10ml/min; the culture supernatant sample is loaded onto the chromatography column, the flow rate is 4.5ml/min, and the protein sample load is 40mg/ml resin; Buffer (PBS) equilibrates the chromatography column with 5 column volumes, the flow rate is 10ml/min; the elution buffer (100mM glycine-hydrochloric acid, pH3.5) elutes the chromatography column, the flow rate is 5ml/min, UV When the 280 rises to 50mAU, the peak is collected, and the collection is stopped when the UV drops to 50mAU. The purity of the samples was detected by non-reduced polyacrylamide gel electrophoresis (non-reduced SDS-PAGE) and particle size screening chromatography-high performance liquid chromatography (SEC-HPLC). 2.2.2 Cation exchange chromatography process development

(1) Sample: double antibody protein captured by protein A affinity chromatography.

(2) Main reagents and instruments: disodium hydrogen phosphate and sodium chloride were purchased from Merck & Co., USA; sodium dihydrogen phosphate dihydrate was purchased from Sinopharm Chemical Reagent Co., Ltd.; L-arginine American Avanto Company; Mode cationic filler Capto SP ImpRes, chromatography column Tricorn 5/100 (diameter 0.5cm, length 10cm), protein chromatography purification system AKTA pure 150 and protein chromatography purification system AKTA avant 150 were purchased from Stofan Corporation of the United States; Spectrophotometer Nanodrop One was purchased from Thermo Fisher Scientific; SevenExcellence™ multi-parameter tester was purchased from Mettler Toledo.

(3) Compound mode cation chromatography experiment

A chromatographic empty column Tricorn 5/100 (diameter 1.6 cm) was packed with a composite mode cationic filler Capto SP ImpRes, with a packing height of 10 cm and a column volume of 2 ml. Equilibrium buffer (20mM PBS, pH7.0) equilibrates 5 column volumes of the chromatography column, and the flow rate is 0.4ml/min; the double antibody protein sample captured by protein A affinity chromatography is loaded onto the chromatography column, and the flow rate is 0.4ml/min. min, protein sample loading capacity 7mg/ml resin; equilibration buffer (20mM PBS, pH 7.0) equilibrates the chromatography column with 10 column volumes, and the flow rate is 0.4ml/min.

Sodium chloride linear gradient elution: Equilibrium buffer (20mM PBS, pH7.0) is liquid A, elution buffer (20mM PBS, 1M sodium chloride, pH7.0) is liquid B, use "0-100% B, 20 column volumes, 0.4ml/min" for elution.

Start collecting peaks when UV 280 rises to 50mAU, and stop collecting when UV drops to 50mAU. The collected elution peaks were tested for purity using non-reduced polyacrylamide gel electrophoresis (non-reduced SDS-PAGE) and particle size screening chromatography-high performance liquid chromatography (SEC-HPLC).

(4) Results Affinity chromatography elution chromatography is as shown in Figure 1, A of Figure 2 shows the chromatogram of composite mode cation chromatography, and B of Figure 2 is an enlarged view of the elution peak of cation chromatography, to the elution peak Samples were collected in 6 stages; SDS-PAGE detection was performed on the cation-loaded samples and collected elution peak samples, the results are shown in Figure 3, and the sample elution was concentrated in stages 3-5; using NR-CE-SDS and SEC - HPLC detection of the sample purity of the main peaks eluted by affinity chromatography and cation chromatography, the detection results are shown in Figures 4-5 and Table 5. After the target protein was captured by affinity chromatography, the product-related impurities were removed by cationic chromatography, and the CE-SDS purity was only about 87%. N-SDS-PAGE detects that the highest purity is only within 85%, and the highest recovery is only within 86%. Table 5 Summary of purification data and results step sample name Volume (mL) Concentration (mg/mL) Protein amount (mg) Recovery rate (%) SEC-HPLC(%) NR-CE-SDS(%) HMWS Monomer LMWS Monomer Others fermentation broth RDHL1720121208HF 1.77 400 708 - - - - - - affinity Affinity Elution Peak 10.08 62 624.96 88.27 - - - - - cation Cationic sample loading sample (affinity elution peak callback pH7.0) 8.43 2 16.86 / 16.54 80.79 2.67 23.69 76.31 Cationic chromatography elution peak collection 1 0.11 2 0.22 1.30 - - - - - Cationic chromatography elution peak collection 2 0.39 4 1.56 9.25 - - - - - Cationic chromatography elution peak collection 3~5 (elution main peak) 2.91 5 1.03 86.30 14.93 83.82 1.25 12.71 87.29 Cationic chromatography elution peak collection 6 0.28 2.2 0.616 3.65 - - - - - Example 3 Optimization of analytical methods for reducing polyacrylamide gel electrophoresis (non-reduced SDS-PAGE) and particle size screening chromatography - high performance liquid chromatography (SEC-HPLC) 3.1 Reducing polyacrylamide gel Electrophoresis (non-reduced SDS-PAGE)

(1) Main reagents and instruments: standard molecular weight marker PageRuler Unstained Protein Ladder was purchased from Thermo Fisher Scientific; protein precast gel SurePAGE (Bis-Tris, 10×8, 4-20% BT), sample loading buffer LDS Sample buffer (4×), electrophoresis buffer Tris-MOPS-SDS, protein gel staining solution eStain L1 Stain solution, protein gel destaining solution eStain L1 Destain solution and protein staining instrument eStain L1 were purchased from Nanjing KingScript Biotechnology Co., Ltd.; electrophoresis The basic power supply of the instrument, Powerpac Basic, and the glue tank, Mini-protean Tetra system, were purchased from Bio-Rad Corporation of the United States.

(2) Method: Concentrate or dilute the sample to be analyzed to 0.33mg/ml, and then prepare the sample with the sample loading buffer (4×LDS Sample buffer) in a ratio of 3:1. GenScript's protein precast gel SurePAGE (Bis-Tris, 10×8, 4-20% BT) and electrophoresis buffer Tris-MOPS-SDS were used for electrophoresis experiments. Electrophoresis conditions: 180V constant voltage for 30min. After the electrophoresis, use the protein staining instrument eStain L1 to stain and decolorize the protein glue; the staining solution and decolorization solution are eStain L1 Stain solution and eStain L1 Destain solution respectively. 3.2 Optimization of particle size sieve chromatography-high performance liquid chromatography (SEC-HPLC) analysis method

In the process of purification and analysis, it was found that the original SEC-HPLC method could not truly reflect the purity of the product. The reason for the analysis may be that the target protein has strong hydrophobicity, which produces non-specific adsorption with the SEC column. By adding 15 % acetonitrile for method optimization, it was unexpectedly found that the peak shape of the optimized method was more symmetrical and the resolution was significantly improved (Figure 15).

(1) Main reagents and instruments: 10×PBS buffer was purchased from (Shanghai) Co., Ltd.; acetonitrile was purchased from Merck, USA; liquid chromatography column Welch Xtimate SEC-300 (7.8×300mm) was purchased from Yuexu Technology (Shanghai) Co., Ltd.; high performance liquid chromatography system 1260 Infinity II was purchased from Agilent.

(2) Method before optimization: particle size screening chromatography-high performance liquid chromatography using Agilent high performance liquid chromatography system 1260 Infinity II and liquid chromatography column Welch Xtimate SEC-300 (7.8×300mm). Phosphate buffered saline (PBS, pH 7.4) was used as the mobile phase for isocratic elution, the flow rate was 1.0 ml/min, and the running time was 20 min. Using UV detection at 280nm, the percentages of each species were calculated based on the peak areas corresponding to aggregates, monomers and low molecular weight fragments.

(3) Optimized method: particle size screening chromatography-high performance liquid chromatography using Agilent high performance liquid chromatography system 1260 Infinity II and liquid chromatography column Welch Xtimate SEC-300 (7.8×300mm). Phosphate buffered saline (PBS, pH 7.4, 15% acetonitrile) was used as the mobile phase for isocratic elution, the flow rate was 1.0 ml/min, and the running time was 20 min. Using UV detection at 280nm, the percentages of each species were calculated based on the peak areas corresponding to aggregates, monomers and low molecular weight fragments. Embodiment 4 Affinity chromatography, anion chromatography process development 4.1 Affinity chromatography

(1) Sample: PR003178 double-antibody culture medium expressed by CHO cells (expressed at 1.73 mg/ml).

(2) Main reagents and instruments: 10× PBS buffer, “1M Tris-HCl solution, pH 8.0” and glycine were purchased from Sangon Bioengineering (Shanghai) Co., Ltd.; hydrochloric acid was purchased from Shanghai Titan Technology Co., Ltd.; Acetic acid was purchased from Avantuo, USA; protein A filler Mabselect sure LX, chromatography column XK16/20 (diameter 1.6 cm, length 20 cm), protein chromatography purification system AKTA pure 150 and protein chromatography purification system AKTA avant 150 Purchased from Stofan Corporation of the United States; Nanodrop One, an ultra-micro-volume spectrophotometer, was purchased from Thermo Fisher Scientific; SevenExcellence™ multi-parameter tester was purchased from Mettler Toledo.

(3) Protein A affinity chromatography method The protein A filler Mabselect sure LX was used to fill the chromatography empty column XK16/20 (diameter 1.6cm), the filling height was 9cm, and the column volume was 18ml. Equilibrium buffer (PBS) equilibrates 5 column volumes of the chromatography column, and the flow rate is 10ml/min; the culture supernatant sample is loaded onto the chromatography column, the flow rate is 4.5ml/min, and the loading capacity of the protein sample is 40mg/ml resin; Equilibrium buffer (PBS) equilibrates the chromatography column with 5 column volumes, the flow rate is 10ml/min; the elution buffer (100mM glycine-hydrochloric acid, pH3.5) elutes the chromatography column, the flow rate is 5ml/min, Start collecting peaks when UV 280 rises to 50mAU, and stop collecting when UV drops to 50mAU. The collected eluted peaks were adjusted to pH 3.6 with 5M acetic acid, incubated at room temperature for 1 hour, and then the pH of the sample was neutralized to 7.0 with neutralization buffer (1M Tris-HCl, pH 8.0). The purity of the samples was detected by non-reduced polyacrylamide gel electrophoresis (non-reduced SDS-PAGE) and particle size screening chromatography-high performance liquid chromatography (SEC-HPLC). 4.2 Anion exchange chromatography process development

(1) Sample: double antibody protein captured by protein A affinity chromatography.

(2) Main reagents and instruments: disodium hydrogen phosphate, trishydroxyaminomethane (Tris) and L-histidine were purchased from Merck & Co., USA; sodium dihydrogen phosphate dihydrate was purchased from Sinopharm Chemical Reagent Co., Ltd.; L -Arginine was purchased from Avanto, USA; aspartic acid was purchased from PanReac AppliChem. Composite mode anionic packing Capto adhere ImpRes, chromatography column Tricorn 5/100 (diameter 0.5cm, length 10cm), protein chromatography purification system AKTA pure 150 and protein chromatography purification system AKTA avant 150 were purchased from Stofan Corporation of the United States; Micro-volume spectrophotometer Nanodrop One was purchased from Thermo Fisher Scientific; SevenExcellence™ multi-parameter tester was purchased from Mettler Toledo.

(3) Composite mode anion chromatography pre-experiment: arginine linear gradient elution

A chromatographic empty column Tricorn 5/100 (diameter 1.6cm) was packed with a composite anionic packing Capto adhere ImpRes, the filling height was 10cm, and the column volume was 2ml. Pre-equilibration buffer (20mM PBS, pH7.0) equilibrates 5 column volumes of the chromatography column, and the flow rate is 0.4ml/min; the double antibody protein sample captured by protein A affinity chromatography is loaded onto the chromatography column, and the flow rate is 0.4ml /min, protein sample loading capacity 7mg/ml resin; equilibration buffer (20mM PBS, pH7.0) to equilibrate the chromatography column with 10 column volumes, flow rate 0.4ml/min.

Arginine linear gradient elution: Equilibrium buffer (20mM PBS, pH7.0) is liquid A, washing buffer (20mM PBS, 1M arginine-hydrochloric acid, pH7.0) is liquid B, use "0- 100% B, 20 column volumes, 0.4ml/min" linear gradient for eluting. To find the concentration of arginine suitable for washing impurities to remove impurities and improve purity.

Use elution buffer (10mM histidine, 10mM aspartic acid, pH5.0) to elute the chromatography column at a flow rate of 0.4ml/min, start collecting peaks when the UV 280 rises to 50mAU, and stop when the UV drops to 50mAU collect. The collected elution peaks were tested for purity using non-reduced polyacrylamide gel electrophoresis (non-reduced SDS-PAGE) and particle size screening chromatography-high performance liquid chromatography (SEC-HPLC).

(4) Optimization of multi-mode anion chromatography: arginine isocratic elution

According to the pre-experimental results, the linear gradient elution of arginine is optimized as isocratic elution (0.3M arginine-hydrochloric acid, 0.4M arginine-hydrochloric acid), which is convenient for process amplification and production; PBS buffer was replaced with Tris-HCl buffer: Equilibrium buffer (20mM Tris-HCl, pH7.2) was liquid A, and washing buffer (20mM Tris-HCl, 1M arginine-hydrochloric acid, pH7.2) was Solution B; respectively use 30%B (ie 20mM Tris-HCl, 0.3M arginine-hydrochloric acid, pH7.2) and 40%B (ie 20mM Tris-HCl, 0.4M arginine-hydrochloric acid, pH7.2) Perform a rinse.

The experimental method is as follows: the composite mode anionic packing Capto adhere ImpRes is used to fill the chromatography empty column Tricorn 5/100 (diameter 1.6cm), the filling height is 10cm, and the column volume is 2ml. Pre-equilibration buffer (20mM Tris-HCl, pH7.2) equilibrates 5 column volumes of the chromatography column at a flow rate of 0.4ml/min; the double antibody protein sample captured by protein A affinity chromatography is loaded onto the chromatography column at a flow rate of 0.4ml/min, protein sample loading capacity 7mg/ml resin; equilibration buffer (20mM Tris-HCl, pH7.2) to equilibrate the chromatography column with 10 column volumes, flow rate 0.4ml/min. Isocratic elution with arginine: use 30%B (ie 20mM Tris-HCl, 0.3M arginine-hydrochloric acid, pH7.2) and 40%B (ie 20mM Tris-HCl, 0.4M arginine-hydrochloric acid , pH7.2) for rinsing at a flow rate of 0.4ml/min for 20 column volumes.

Use elution buffer (10mM histidine, 10mM aspartic acid, pH5.0) to elute the chromatography column at a flow rate of 0.4ml/min, start collecting peaks when the UV 280 rises to 50mAU, and stop when the UV drops to 50mAU collect. The collected elution peaks were tested for purity using the method of Example 3.

(5) Results

The results of the multi-mode anion chromatography pre-experiment (arginine linear gradient elution) are shown in Figure 7: SDS-PAGE detection, the linear gradient elution peaks are all impurity proteins, and there is no target protein band; the purity of the target protein in the elution peak higher. It shows that the use of Capto adhere ImpRes composite mode anion chromatography and arginine elution can remove impurities without losing the target protein.

From the chromatogram of the pre-experiment (Figure 6), it can be seen that in the arginine linear gradient elution experiment, the arginine concentration corresponding to the top of the impurity peak is 0.34M, so when optimizing the experiment, the isocratic elution was performed separately 0.3M and 0.4M arginine were chosen.

The optimized experimental chromatograms are shown in Figures 8 and 9. It can be seen from the chromatograms that both 0.3M and 0.4M arginine can elute out protein peaks. The protein peaks eluted by SDS-PAGE (Fig. 10) are all miscellaneous proteins and do not contain the target protein. After elution, the eluted target protein has a high purity. As shown in Table 6, after SEC-HPLC detection and elution with 0.4M arginine and 0.3M arginine, the purity of the eluted samples increased from 83.17% to 97.68% and 97.57%.

Fig. 11 is the SEC-HPLC figure before anion chromatography, and the B of Fig. 11 is its enlarged part of A; Fig. 12 is the SEC-HPLC figure (0.4M arginine washing) after the anion chromatography, and the B of Fig. 12 is its The enlarged part of A; Figure 13 is the SEC-HPLC figure after anion chromatography (0.3M arginine elution), and B in Figure 13 is the enlarged part of A. Figure 14 is the SDS-PAGE figure before and after anion chromatography. Table 6 SEC-HPLC results of anion chromatography optimization experiments sample name SEC-HPLC(%) Polymer monomer fragments Affinity low pH incubated sample after callback (anion sample loaded sample) 2.51 83.17 14.33 Anion elution peak (0.4M arginine elution) 0.53 97.68 1.79 Anion elution peak (0.3M arginine elution) 0.93 97.57 1.50

The PR002299 double antibody culture medium was purified using the same purification method as above, and the purity of the eluted sample also reached more than 95%.

Figure 1 is the chromatogram of the affinity chromatography experiment.

Figure 2 is a chromatogram of cationic chromatography.

Fig. 3 is the non-reducing SDS-PAGE result figure of cationic chromatography experiment; Among them, M: protein marker; L: the sample (cation loaded sample) after affinity low pH incubation callback, 1: cationic chromatography elution peak collection 1,2 : cation chromatography elution peak collection 2, 3: cation chromatography elution peak collection 3, 4: cation chromatography elution peak collection 4, 5: cation chromatography elution peak collection 5, 6: cation chromatography elution peak collection Peak collection 6.

Fig. 4 is the SEC-HPLC diagram of the affinity elution peak and the cation chromatography elution main peak.

Figure 5 is the CE-SDS (NR) diagram of the affinity elution peak and the cation chromatography elution main peak.

Fig. 6 is an anion chromatography pre-experiment chromatogram; (0-1M arginine linear gradient elution).

Figure 7 is the non-reducing SDS-PAGE results of the anion chromatography pre-experiment; among them, M: protein marker, 1: the sample (anion loaded sample) after affinity low pH incubation and callback, 2: the linear gradient elution peak of anion chromatography, 3: Anion chromatography elution peak.

Fig. 8 is the chromatogram of anion chromatography optimization experiment - 0.4M arginine elution.

Fig. 9 is the chromatogram of anion chromatography optimization experiment - 0.3M arginine elution.

Figure 10 is the non-reducing SDS-PAGE results of the anion chromatography optimization experiment; among them, M: protein marker, 1: the sample (anion loaded sample) after affinity low pH incubation and callback, 2: 0.4M arginine elution peak, 3: Anion chromatography elution peak (0.4M arginine elution), 4: 0.3M arginine elution peak, 5: Anion elution peak (0.3M arginine elution).

Figure 11 is the SEC-HPLC figure before anion chromatography.

Figure 12 is the SEC-HPLC chart after anion chromatography (0.4M arginine elution).

Figure 13 is the SEC-HPLC chart after anion chromatography (0.3M arginine elution).

Figure 14 is the SDS-PAGE figure before and after anion chromatography.

Figure 15 is a comparison chart before and after the optimization of the SEC-HPLC method.

Figure 16 is the structure of CD3-BCMA bispecific antibody.

Claims (11)

A method for purifying proteins using two-step chromatography, characterized in that the method includes affinity chromatography and complex mode anion exchange chromatography; the protein is a bispecific antibody, which includes the first protein functional region of the Fab structure and the second protein functional region comprising the VH structure of two heavy chain variable regions; preferably, the two heavy chain variable regions are connected by (G ₄ S) _n , and the n is a non-zero natural number, preferably 1 to 20, more preferably 3 or 4; more preferably, the bispecific antibody also includes an Fc structure; further more preferably, the first protein functional region targets CD3, and the second protein functional region targets BCMA. The method according to claim 1, wherein the bispecific antibody comprises polypeptide chain 1, polypeptide chain 2 and polypeptide chain 3, wherein the polypeptide chain 1 comprises VL1-CL in sequence from the N-terminus to the C-terminus , the polypeptide chain 2 includes VH1-CH1-CH2-CH3 from the N-terminus to the C-terminus, and the polypeptide chain 3 includes VH2-VH2-CH2-CH3 from the N-terminus to the C-terminus in sequence. The method according to claim 2, wherein said VH1 comprises HCDR1, HCDR2 and HCDR3, and its amino acid sequences are shown in SEQ ID NO: 5, 6 and 7 respectively; said VL1 comprises LCDR1, LCDR2 and LCDR3, its amino acid sequence is shown in SEQ ID NO: 10, 11 and 12 respectively; The VH2 comprises HCDR1, HCDR2 and HCDR3, its amino acid sequence is shown in SEQ ID NO: 15, 16 and 17 respectively, Or as shown in SEQ ID NO: 20, 21 and 22. The method as claimed in claim 2, wherein the amino acid sequences of VH1, VL1 and VH2 are respectively shown in SEQ ID NO: 2, SEQ ID NO: 9 and SEQ ID NO: 14; Alternatively, the amino acid sequences of VH1, VL1 and VH2 are shown in SEQ ID NO: 4, SEQ ID NO: 9 and SEQ ID NO: 19, respectively. The method according to any one of claim items 2 to 4, wherein the amino acid sequences of the polypeptide chain 1, the polypeptide chain 2 and the polypeptide chain 3 are respectively as SEQ ID NO: 8, SEQ ID NO: 1 and Shown in SEQ ID NO: 13; Alternatively, the amino acid sequences of polypeptide chain 1, polypeptide chain 2 and polypeptide chain 3 are shown in SEQ ID NO: 8, SEQ ID NO: 3 and SEQ ID NO: 18, respectively. The method according to any one of claim items 1 to 5, wherein the affinity chromatography comprises the following steps: pre-equilibration, sample loading, equilibrium and elution; the pre-equilibration uses PBS as a pre-equilibration buffer , the equilibrium uses PBS as the equilibrium buffer, and the elution uses glycine-hydrochloric acid as the elution buffer; Preferably, in the affinity chromatography, the filler is Mabselect sure LX, and/or the chromatography column is XK16/20. The method according to claim 6, wherein the culture supernatant of the bispecific antibody has a loading capacity of 0.1-50 mg/ml, such as 40 mg/ml, and a flow rate of, for example, 4.5 ml/min when the sample is loaded; and /or, the pre-equilibration buffer uses 2 to 10 column volumes, such as 5 column volumes, and the flow rate is, for example, 10ml/min; and/or, the balance buffer uses 2 to 10 column volumes, such as 5 column volumes, the flow rate is, for example, 10ml/min; and/or, the elution buffer is 50-200mM, such as 100mM glycine-hydrochloric acid, pH3.5, and the flow rate is, for example, 5ml/min; Preferably, it also includes the step of using acetic acid to inactivate the virus; more preferably after the inactivation, the pH of the neutralized sample is to 6.5~7.5 such as 7.0; further more preferably, the neutralized sample uses 1M Tris-HCl buffer, pH8.0 conduct. The method according to any one of claim items 1 to 7, wherein the complex mode anion exchange chromatography comprises the following steps: pre-balance, sample loading, balance, rinsing and elution; wherein, the pre-balance The pre-equilibration buffer used for equilibration is PBS or Tris-HCl; and/or, the equilibration buffer used for the equilibration is PBS or Tris-HCl; and/or, the elution buffer used for the washing includes PBS Or Tris-HCl liquid A, liquid B containing arginine-hydrochloric acid; and/or, the elution buffer used in the elution includes histidine and aspartic acid; Preferably, in the anion exchange chromatography, the filler used is Capto adhere ImpRes; the chromatographic empty column is Tricorn 5/100; and/or, the bispecific antibody sample captured by the affinity chromatography is loaded into the layer Analysis column, protein sample loading capacity 7mg/ml; and/or, the A solution includes 20mM PBS, pH7.0, or 20mM Tris-HCl, pH7.2; the B solution includes 20mM PBS, pH7.0 , 1M arginine-hydrochloric acid, or 20mM Tris-HCl, pH7.2, 1M arginine-hydrochloric acid; and/or, the flow rate of the washing and/or elution is 0.4ml/min. The method according to claim 8, wherein the pre-equilibration uses a pre-equilibration buffer of 2 to 10 column volumes, such as 5 column volumes, and the pre-equilibration buffer is 20mM PBS, pH7. 0; and/or, the equilibrium uses 2 to 10 column volumes, such as 10 column volumes, of the equilibrium buffer, the equilibrium buffer is 20mM PBS, pH7.0; and/or, the washing is used Solution A and 0~100% solution B are subjected to gradient elution, 20 column volumes, the solution A includes 20mM PBS, pH7.0, the solution B includes 20mM PBS, pH7.0, 1M arginine-hydrochloric acid and/or, the elution buffer used in the elution is 10mM histidine and 10mM aspartic acid, pH5.0. The method as described in claim item 8, wherein the pre-equilibration uses 2 to 10 column volumes such as 5 column volumes of pre-equilibration buffer, and the pre-equilibration buffer is 20mM Tris-HCl, pH7.2; and/or, the equilibrium uses 2 to 10 column volumes, such as 10 column volumes, of an equilibrium buffer, the equilibrium buffer being 20mM Tris-HCl, pH7.2; the washing Perform isocratic rinsing with 30%~40% B solution, rinsing 20 column volumes, wherein the A solution includes 20mM Tris-HCl, pH7.2, and the B solution includes 20mM Tris-HCl, pH7.2 , 1M arginine-hydrochloric acid; and/or, the elution buffer used in the elution is 10mM histidine and 10mM aspartic acid, pH5.0. The method according to any one of claims 1 to 10, further comprising analyzing the product obtained by the affinity chromatography and/or the complex mode anion exchange chromatography using SEC-HPLC, wherein the The mobile phase of SEC-HPLC was phosphate buffer containing 15% acetonitrile.

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