CN114324646A - Liquid chromatography analysis method for multi-specific antibody mismatching - Google Patents
Liquid chromatography analysis method for multi-specific antibody mismatching Download PDFInfo
- Publication number
- CN114324646A CN114324646A CN202111607082.2A CN202111607082A CN114324646A CN 114324646 A CN114324646 A CN 114324646A CN 202111607082 A CN202111607082 A CN 202111607082A CN 114324646 A CN114324646 A CN 114324646A
- Authority
- CN
- China
- Prior art keywords
- multispecific antibody
- chromatographic column
- column
- sec
- antibody
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000004458 analytical method Methods 0.000 title claims abstract description 21
- 238000004811 liquid chromatography Methods 0.000 title claims abstract description 14
- 238000000034 method Methods 0.000 claims abstract description 20
- 230000007717 exclusion Effects 0.000 claims abstract description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000000945 filler Substances 0.000 claims abstract description 5
- CHRJZRDFSQHIFI-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;styrene Chemical compound C=CC1=CC=CC=C1.C=CC1=CC=CC=C1C=C CHRJZRDFSQHIFI-UHFFFAOYSA-N 0.000 claims abstract description 3
- 229920000936 Agarose Polymers 0.000 claims abstract description 3
- 125000001165 hydrophobic group Chemical group 0.000 claims abstract description 3
- 229920000193 polymethacrylate Polymers 0.000 claims abstract description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 24
- 150000003839 salts Chemical class 0.000 claims description 17
- 238000004587 chromatography analysis Methods 0.000 claims description 12
- 230000002209 hydrophobic effect Effects 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 239000000337 buffer salt Substances 0.000 claims description 7
- 239000004696 Poly ether ether ketone Substances 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- 229920002530 polyetherether ketone Polymers 0.000 claims description 6
- 229910001220 stainless steel Inorganic materials 0.000 claims description 5
- 239000010935 stainless steel Substances 0.000 claims description 5
- 238000001819 mass spectrum Methods 0.000 claims description 4
- 229920001903 high density polyethylene Polymers 0.000 claims description 3
- 239000004700 high-density polyethylene Substances 0.000 claims description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 claims description 2
- 229910002651 NO3 Inorganic materials 0.000 claims description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 2
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 2
- 239000007983 Tris buffer Substances 0.000 claims description 2
- 239000007979 citrate buffer Substances 0.000 claims description 2
- 238000009826 distribution Methods 0.000 claims description 2
- 150000004820 halides Chemical class 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 239000011148 porous material Substances 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 2
- 230000009977 dual effect Effects 0.000 abstract description 5
- 238000012856 packing Methods 0.000 abstract description 4
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 238000000926 separation method Methods 0.000 description 16
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 10
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 239000001103 potassium chloride Substances 0.000 description 5
- 235000011164 potassium chloride Nutrition 0.000 description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 4
- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000012512 characterization method Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 2
- 102000009109 Fc receptors Human genes 0.000 description 2
- 108010087819 Fc receptors Proteins 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 229910000160 potassium phosphate Inorganic materials 0.000 description 2
- 235000011009 potassium phosphates Nutrition 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 239000012064 sodium phosphate buffer Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000004881 tumor cell Anatomy 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
- 239000005695 Ammonium acetate Substances 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 208000029462 Immunodeficiency disease Diseases 0.000 description 1
- 230000004988 N-glycosylation Effects 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
- 241000580858 Simian-Human immunodeficiency virus Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 229940043376 ammonium acetate Drugs 0.000 description 1
- 235000019257 ammonium acetate Nutrition 0.000 description 1
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical group [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229960003008 blinatumomab Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229960000419 catumaxomab Drugs 0.000 description 1
- 238000001311 chemical methods and process Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940088679 drug related substance Drugs 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000009437 off-target effect Effects 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000009097 single-agent therapy Methods 0.000 description 1
- 238000001542 size-exclusion chromatography Methods 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
Images
Landscapes
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
The invention relates to a liquid chromatography analysis method for multi-specific antibody mismatching. A Sepax BioMix SEC chromatographic column is adopted, the chromatographic column is a mixed-mode size exclusion chromatographic column, the filler is porous spherical silica particles, polymethacrylate, styrene/divinylbenzene (PS/DVB) or agarose, and the surfaces of the porous spherical silica particles are modified with hydrophobic groups. The chromatographic packing of the Sepax BioMix SEC chromatographic column has the dual functions of hydrophobicity and size exclusion, shows strong capability in separating the mismatch of multispecific antibodies with hydrophobicity difference and molecular weight difference, and has significantly higher peak resolution than other types of size exclusion chromatographic columns. The liquid chromatography analysis method is simple and convenient to use, mobile phase is environment-friendly, organic reagents are not used, and the method is suitable for analysis of multi-specific antibody mismatching.
Description
Technical Field
The invention relates to biomacromolecule characterization analysis, in particular to a liquid chromatography analysis method for multi-specific antibody mismatching.
Background
The multispecific antibody is a single antibody molecule which can be simultaneously and specifically combined with a plurality of different antigen epitopes, and the common tumor therapeutic antibody can only be combined with a single antigen, so that the combination specificity is relatively low, and off-target effect is easy to occur. The multispecific antibody can respectively recognize and combine a plurality of different antigens, so that the multispecific antibody can connect immune cells, virus molecules and the like to tumor cells to further enhance the killing effect on target cells, and can also combine different antigens on the same tumor cell to enhance the combination specificity, thereby reducing the side effects such as off-target toxicity and the like. The multifunctional recombinant antibody has higher curative effect than that of a monoclonal antibody as a tumor treatment drug, and the research and analysis of the prior multispecific antibody mainly focuses on two types of antibodies, namely a bispecific antibody and a trispecific antibody.
Bispecific antibodies (BsAb), referred to as diabodies, refer to a single antibody molecule that can specifically bind to 2 different epitopes simultaneously, and BsAb exhibits unique advantages in disease treatment due to its unique dual-target and versatility. Since the concept of bispecific antibody was first proposed by Nisonoff and its collaborators in 1960, along with the rapid progress in related fields such as genetic engineering antibody and immunology, the construction concept, technical platform and product development of bispecific antibody have been continuously innovated.
Currently, at least 57 BsAbs are in clinical trials and are mainly used for the treatment of tumors and autoimmune diseases, and BsAbs approved for marketing include Catumaxomab, emilizumab and Blinatumomab. The existing BsAb construction technology developed exceeds 60, such as Triomab, Crossmab, DAF, IgG-scFv and the like, can realize the dual specificity of a single antibody molecule, wherein the Triomab, Crossmab and the like are adopted to simultaneously express structurally-modified different Heavy Chains (HC) and/or Light Chains (LC) in a production cell strain, and HC-LC is correctly paired by virtue of charge force, hydrophobic effect, steric hindrance and the like to form a heterologous BsAb tetramer, so that the requirement on expression and purification in the BsAb production process is high, and the product structure can be greatly changed by slight change of production parameters, and the safety and effectiveness of the product are influenced. Therefore, such BsAb key characterization needs to be analyzed in detail, and based on this, the potential impact of process change on product quality is determined. The key characteristics to be defined include relative molecular mass, peptide pattern, disulfide bond, N-glycosylation site, N-glycoform and content, affinity with antigen, affinity with Fc receptors (FcRs), and the like, which are the basis for ensuring the correct structural function of the product.
Trispecific antibodies (hereinafter referred to as "tertiary antibodies") have been developed by researchers to kill HIV virus. However, HIV mutates very quickly and escape mutations occur very quickly, limiting the clinical utility of monotherapy. Studies have shown that cocktail therapy of mabs could theoretically be used to cope with the emergence of resistance, but there are huge obstacles in actual development-toxicological and pharmacokinetic assessments of each antibody would greatly increase the preclinical workload. Regulatory agencies often require that each antibody be detected individually or in combination, which results in the production of expensive assays that are impractical. Production and quality control requirements present challenges to them. In contrast, trispecific antibodies provide a simplified strategy. In a new study, american researchers reported that a trispecific antibody made in the laboratory better protected monkeys from infection by two human monkey chimeric immunodeficiency virus (SHIV) strains than the single natural antibody used to make the trispecific antibody.
Such trispecific antibodies were developed by researchers from the National Institutes of Health (NIH) and the pharmaceutical company cenofil, a french press, and also in the laboratory are more potent than single natural antibodies in preventing infection of cells by more HIV strains. However, it should be noted that the triple antibody minimizes off-target and maximizes specificity. Since three antibodies are widely expressed in many cell types, this increases the risk of tri-specific T cell engagers attacking healthy cells. It is therefore more important to confirm the effectiveness and safety of the three antibodies. The mismatch of the multispecific antibody has great influence on the properties of products, so that the characterization of the mismatch is particularly important, and the liquid chromatography for the mismatch analysis of the multispecific antibody is established in order to meet the requirement of the mismatch analysis of the specific antibody.
Disclosure of Invention
The purpose of the invention is as follows: the invention provides a sensitive liquid chromatography analysis method for multi-specific antibody mismatching.
The technical scheme is as follows: the liquid chromatographic analysis method for the multi-specific antibody mismatch adopts a Sepax BioMix SEC chromatographic column which is a mixed-mode size exclusion chromatographic column, the filler is porous spherical silica particles, polymethacrylate, styrene/divinylbenzene (PS/DVB) or agarose, and the surfaces of the porous spherical silica particles are modified with hydrophobic groups.
Further, the mobile phase used is a buffered salt system. The selection range of the buffer salt is as follows: phosphate, sulfate, halide, Tris buffer, citrate buffer, formate, acetate, ammonium or nitrate; the pH of the mobile phase is in the range of 2-8. The salt species can be selected from: NaCl, KCl or a mixed salt of NaCl/KCl; the salt concentration ranges from 0 mol/L to 2 mol/L; selecting the flow rate: 20cm/h to 150 cm/h.
Further, analytical columns of conventional specifications may be used, and semi-preparative columns or preparative columns may also be used. The detector adopts an ultraviolet detector or a mass spectrum detector. Pore size of the spherical silica particles
Particle sizeThe size is 1.7-30 μm. The chromatographic column tube can be made of stainless steel, PEEK (polyetheretherketone) and high-density polyethylene. The inner diameter of the analytical chromatographic column can be selected from 2.0-10mm, and the column length can be selected from 50-600 mm; the inner diameter of the semi-preparative or preparative chromatographic column can be 10-100 mm; the length of the column can be 50-600 mm. The sample suitable for the analysis method is suitable for the sample with molecular weight or hydrophobic difference besides the multispecific antibody with mismatch; the particle size distribution of the filler may be monodisperse, polydisperse.
The main driving force for the different molecular separations in the mixed mode size exclusion chromatography column Sepax Biomix SEC described above is the size exclusion effect of the surface hydrophobic clusters and the chromatographic packing. The Sepax Biomix SEC has strong capacity in separating the mismatched substances of the multispecific antibody, has obviously higher peak resolution than other types of chromatographic columns, and can better separate the mismatched antibodies with molecular weight difference and hydrophobic difference. The method can quantitatively detect even the antibody with low abundance, and provides a new tool for screening different expressions of cell culture clones, monitoring the purification of the cell culture clones and evaluating the purity of drug substances.
One preferred liquid chromatography method for multispecific antibody mismatch analysis is as follows:
(1) sample pretreatment: dissolving a multispecific antibody sample in a buffer;
(2) a chromatographic column: the Sepax BioMix SEC analytical column (PN: 214300-7830) is adopted, the Sepax BioMix SEC analytical column packing takes spherical silica particles with the particle size of 3 mu m, high purity and high mechanical stability as a matrix, a layer of uniform, hydrophilic and neutral nano thick film is bonded on the surface of the Sepax BioMix SEC analytical column packing, and the chemical process of film formation can be well controlled by a special surface technology, so that the separation of multi-specificity antibodies, hydrophobic samples and the like with difference in hydrophobicity and molecular size can be realized with high resolution and high recovery rate. The material of the chromatographic column tube can be stainless steel, PEEK material, high density polyethylene or other high molecular materials, preferably stainless steel and PEEK material, most preferably stainless steel material. a. The chromatographic column size specification range for the analytical method is as follows: the inner diameter of the analytical chromatographic column can be selected from 2.0-10mm, and the inner diameter of the chromatographic column is preferably 4.6-10 mm; the column length of the chromatographic column can be selected from 50-600mm, and the column length of the chromatographic column is preferably 100-300 mm. b. In addition, in the experiment, if a large amount of mismatched peaks need to be collected, particularly, target objects with low content of mismatched peaks are collected, a semi-preparative or preparative chromatographic column can be considered for collecting the target peaks, the size specification of the semi-preparative or preparative chromatographic column can be selected according to actual requirements, the inner diameter of the chromatographic column can be 10-100mm, and the inner diameter of the chromatographic column is preferably 10-30 mm; the column length of the chromatographic column can be selected from 50-600mm, and the column length of the chromatographic column is preferably 100-300 mm.
c. Selecting the column temperature: an external column oven is used, the temperature is 10-80 ℃, preferably 15-40 ℃, and most preferably 20-30 ℃.
d. Selecting a mobile phase: the most commonly used mobile phases are: phosphate buffer salt + salts with different concentrations can change the concentration of the salts in the mobile phase to optimize the separation degree of mismatched peaks, and the types of the salts in the mobile phase of the liquid chromatographic analysis method can be selected as follows: NaCl, KCl or a mixed NaCl/KCl salt, with a salt concentration in the range of 0-2mol/L, preferably 0.3-0.8mol/L, most preferably 0.4-0.6 mol/L.
e. Selecting the flow rate: from 20cm/h to 150cm/h, preferably from 50cm/h to 100cm/h, most preferably from 60cm/h to 80 cm/h.
f. A detector: UV detector, 280nm or the ultraviolet wavelength with the highest response value to the sample.
g. In addition, the ultraviolet detector can only be used for preliminarily judging whether the multispecific antibody has mismatch, if more accurate qualitative analysis is needed to be carried out on each mismatch peak, the mass spectrum detector can be used for analyzing the mass-to-charge ratio of each mismatch peak of the multispecific antibody so as to carry out more accurate qualitative analysis on each mismatch peak, when the mass spectrum detector is used, the mobile phase buffer salt which is not easy to volatilize needs to be changed into volatile buffer salt, and the most commonly used volatile buffer salt is ammonium formate or ammonium acetate buffer salt.
Has the advantages that: the liquid chromatographic analysis method for the multi-specific antibody mismatching improves the separation effect of the mismatching sample, has simple integral method and environment-friendly mobile phase, does not use organic reagent, and is suitable for the analysis of the multi-specific antibody mismatching.
Drawings
FIG. 1 comparison of flow versus mismatch separation results for bispecific antibodies for different sodium chloride salt concentrations;
FIG. 2 comparison of results of BioMix SEC-300 and Zenix-C300 on bispecific antibody isolation;
FIG. 3 comparison of results of BioMix SEC-300, SRT-C SEC-300 and TSKgel G3000SW for bispecific antibody isolation;
FIG. 4 comparison of BioMix SEC-300 and SRT SEC-300 results for trispecific antibody isolation.
Detailed Description
Example 1
Liquid chromatography for the mismatch analysis of multispecific antibodies of this example
1. Mobile phase a (PBS buffered salt): 100mM sodium phosphate buffer salt (pH 7.4)
Mobile phase B: mobile phase A +1M NaCl (pH 7.4)
Experiment the mobile phase A and the mobile phase B are mixed according to the volume ratio of 7: 3, 4: 6 and 3: 7 respectively to obtain 3 sodium phosphate buffer salts with different sodium chloride concentrations, namely PBS +0.3MNaCl, PBS +0.6MNaCl and PBS +0.7 MNaCl.
2. Sample preparation: bispecific antibody-1 (mobile phase A diluted to 1mg/mL)
3. A chromatographic column 1: BioMix SEC-300(3 μm,
7.8×300mm)
and (3) chromatographic column 2: Zenix-C SEC-300(3 μm,
7.8×300mm)
4. flow rate: 62cm/h
5. A detector: UV: 280nm
6. Column temperature: 25 deg.C
The separation of the mismatch peaks can be optimized by adjusting the salt concentration of sodium chloride in the mobile phase when using a BioMix SEC-300 dedicated column. Generally, the higher the concentration of sodium chloride salt in the mobile phase is, the greater the mismatch differentiation is, and the best degree of separation is obtained, and usually the salt concentration is controlled within 1M, this experiment compares the separation comparison results of PBS, PBS +300mMNaCl, PBS +600mMNaCl, PBS +700mMNaCl, and PBS +1 mnacal, and the optimized separation results of different concentrations of sodium chloride salt are shown in fig. 1.
Meanwhile, in the mobile phase condition of PBS +700mM NaCl, the two types of chromatographic columns of BioMix SEC-300 and Zenix-C300 are compared in the laboratory, the sample used is bispecific antibody-1, and the Zenix-C300 is found to have no capability of separating double anti-mismatch, and the comparison result of the BioMix SEC-300 and the Zenix-C300 is shown in FIG. 2. Since the biomex SEC-300 has the dual function of size exclusion and hydrophobic effect, while the Zenix-C SEC-300 has only the function of size exclusion, the biomex SEC-300 has better separation performance when separating mismatched multispecific antibodies with hydrophobic differences.
Example 2
Liquid chromatography for the mismatch analysis of multispecific antibodies of this example
1. Mobile phase: 100mM potassium phosphate +500mM potassium chloride (pH 7.0)
2. Sample preparation: bispecific antibody-2 (mobile phase diluted to 1mg/mL)
3. A chromatographic column 1: BioMix SEC-300(3 μm,
7.8×300mm)
and (3) chromatographic column 2: SRT-C SEC-300(5 μm,
7.8×300mm)
a chromatographic column 3: TSKgel G3000SW (5 μm,
7.8×300mm)
4. flow rate: 62cm/h
5. A detector: UV: 280nm
6. Column temperature: 25 deg.C
Under the same test conditions, the laboratory compares the separating ability of 3 kinds of columns of BioMix SEC-300, SRT-C SEC-300 and TSKgel G3000SW to double-antibody mismatch, and the results show that the separating ability of SRT-C SEC-300 and TSKgel G3000SW to double-antibody mismatch is not the same as that of BioMix SEC-300, and the comparison results of 3 columns are shown in FIG. 3. Since the Biomix SEC-300 has the dual function of size exclusion and hydrophobic effect, while the SRT-C SEC-300 and the TSKgel G3000SW have the function of size exclusion only, the Biomix SEC-300 has better separation performance when mismatched multispecific antibodies with hydrophobic differences are separated.
Example 3
Liquid chromatography for the mismatch analysis of multispecific antibodies of this example
1. Mobile phase: 50mM sodium phosphate +50mM potassium phosphate +250mM sodium chloride +250mM potassium chloride (pH 7.4)
2. Sample preparation: trispecific antibody (mobile phase diluted to 1mg/mL)
3. A chromatographic column 1: BioMix SEC-300(3 μm,
7.8×300mm)
and (3) chromatographic column 2: SRT SEC-300(5 μm,
7.8×300mm)
4. flow rate: 62cm/h
5. A detector: UV: 280nm
6. Column temperature: 25 deg.C
Under the same test conditions, the laboratory compares the separation ability of 2 chromatographic columns of BioMix SEC-300 and SRT SEC-300 to the mismatch of the trispecific antibody, and the separation ability of SRT SEC-300 to the mismatch of the trispecific antibody is found, which is not the same as the comparison result of BioMix SEC-300 and 2 chromatographic columns shown in figure 4. Since the biomex SEC-300 has a dual role of size exclusion and hydrophobic effect, while the SRT SEC-300 has only a size exclusion effect, the biomex SEC-300 has a better separation performance when separating mismatched multispecific antibodies with hydrophobic differences.
Example 4
Multispecific antibody mismatch analysis of this exampleThe liquid chromatography of (1). The procedure is essentially the same as in example 1, using only the chromatographic column BioMix SEC-300(3 μm,
7.8 × 300mm) except that the flow rate: 62cm/h was replaced by 100cm/h and 124cm/h, respectively, and the comparison results showed that the separation of the mismatched peaks of bispecific antibody gradually decreased with the increase of the flow rate of the mobile phase.
Example 5
Liquid chromatography for multispecific antibody mismatch analysis of this example. The procedure is essentially the same as in example 3, using only the chromatographic column BioMix SEC-300(3 μm,
7.8 x 300mm) except that the pH of the mobile phase was adjusted to 5.5 and 6.5, respectively, and the comparison results showed that the separation of the mismatch peaks of the trispecific antibodies was the best at pH 7.4 of the mobile phase.
Claims (10)
1. A method for liquid chromatography of multispecific antibody mismatches, characterised in that: a Sepax BioMix SEC chromatographic column is adopted, the chromatographic column is a mixed-mode size exclusion chromatographic column, the filler is porous spherical silica particles, polymethacrylate, styrene/divinylbenzene (PS/DVB) or agarose, and hydrophobic groups are modified on the surfaces of the porous spherical silica particles.
2. The method for the liquid chromatographic analysis of multispecific antibody mismatches according to claim 1, characterized in that: the mobile phase adopted is a buffer salt system.
3. The method for the liquid chromatographic analysis of multispecific antibody mismatches according to claim 2, characterized in that: the selection range of the buffer salt is as follows: phosphate, sulfate, halide, Tris buffer, citrate buffer, formate, acetate, ammonium or nitrate; the pH of the mobile phase is in the range of 2-8.
4. The method for the liquid chromatographic analysis of multispecific antibody mismatches according to claim 3, characterized in that: the salt species can be selected from: NaCl, KCl or a mixed salt of NaCl/KCl; the salt concentration ranges from 0 mol/L to 2 mol/L; selecting the flow rate: 20cm/h to 150 cm/h.
5. The method for the liquid chromatographic analysis of multispecific antibody mismatches according to claim 1, characterized in that: analytical columns of conventional specifications may be used, as may semi-preparative or preparative columns.
6. The method for the liquid chromatographic analysis of multispecific antibody mismatches according to claim 1, characterized in that: the detector adopts an ultraviolet detector or a mass spectrum detector.
7. The method for the liquid chromatographic analysis of multispecific antibody mismatches according to claim 1, characterized in that: the spherical silica particles have a pore size of
The particle size is 1.7-30 μm.
8. The method for the liquid chromatographic analysis of multispecific antibody mismatches according to claim 1, characterized in that: the chromatographic column tube can be made of stainless steel, PEEK (polyetheretherketone) and high-density polyethylene.
9. The method for the liquid chromatographic analysis of multispecific antibody mismatches according to claim 1, characterized in that: the inner diameter of the analytical chromatographic column can be selected from 2.0-10mm, and the column length can be selected from 50-600 mm; the inner diameter of the semi-preparative or preparative chromatographic column can be 10-100 mm; the length of the column can be 50-600 mm.
10. The method for the liquid chromatographic analysis of multispecific antibody mismatches according to claim 1, characterized in that: the sample suitable for the analysis method is suitable for the sample with molecular weight or hydrophobic difference besides the multispecific antibody with mismatch; the particle size distribution of the filler may be monodisperse, polydisperse.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111607082.2A CN114324646A (en) | 2021-12-24 | 2021-12-24 | Liquid chromatography analysis method for multi-specific antibody mismatching |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111607082.2A CN114324646A (en) | 2021-12-24 | 2021-12-24 | Liquid chromatography analysis method for multi-specific antibody mismatching |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114324646A true CN114324646A (en) | 2022-04-12 |
Family
ID=81012455
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111607082.2A Pending CN114324646A (en) | 2021-12-24 | 2021-12-24 | Liquid chromatography analysis method for multi-specific antibody mismatching |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114324646A (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111491951A (en) * | 2017-12-22 | 2020-08-04 | 豪夫迈·罗氏有限公司 | Antibody variants depleted of light chain mismatches by hydrophobic interaction chromatography |
| CN113281430A (en) * | 2021-05-11 | 2021-08-20 | 苏州君盟生物医药科技有限公司 | Method for separating and identifying bispecific antibody |
-
2021
- 2021-12-24 CN CN202111607082.2A patent/CN114324646A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111491951A (en) * | 2017-12-22 | 2020-08-04 | 豪夫迈·罗氏有限公司 | Antibody variants depleted of light chain mismatches by hydrophobic interaction chromatography |
| CN113281430A (en) * | 2021-05-11 | 2021-08-20 | 苏州君盟生物医药科技有限公司 | Method for separating and identifying bispecific antibody |
Non-Patent Citations (1)
| Title |
|---|
| HAITAO JIANG等: "Characterization of Bispecific Antibody Production in Cell Cultures by Unique Mixed Mode Size Exclusion Chromatography", ANALYTICAL CHEMISTRY * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2601062C (en) | A method of weak partitioning chromatography | |
| KR101484659B1 (en) | Methods of reducing level of one of more impurities in a sample during protein purification | |
| US8853371B2 (en) | Process for preparing unaggregated antibody Fc domains | |
| Azevedo et al. | Integrated process for the purification of antibodies combining aqueous two-phase extraction, hydrophobic interaction chromatography and size-exclusion chromatography | |
| CN114249831A (en) | Methods of purifying bispecific antibodies | |
| CN108686634B (en) | Solid phase for mixed mode chromatographic purification of proteins | |
| JP2010517942A (en) | Methods for enhancing and purifying antibody performance by mixed-mode chromatography in the presence of water-soluble nonionic organic polymers | |
| US10682640B2 (en) | Anionic exchange-hydrophobic mixed mode | |
| US20220119500A1 (en) | Affinity chromatography purification with low conductivity wash buffer | |
| CN105315369A (en) | Protein purification using cation exchange chromatography | |
| CN105837687B (en) | A kind of chromatography method of anti-tnf-alpha class monoclonal antibody | |
| CN114324646A (en) | Liquid chromatography analysis method for multi-specific antibody mismatching | |
| Kimerer et al. | Chromatographic and adsorptive behavior of a bivalent bispecific antibody and associated fragments | |
| US20180327447A1 (en) | Improved protein separation in ion exchange chromatography | |
| WO2010043700A1 (en) | Clarification process at higher cell density | |
| US20230049176A1 (en) | Methods to decrease impurities from recombinant protein manufacturing processes | |
| Marc et al. | 13 Adsorption Chromatography | |
| US20210017223A1 (en) | Separation Method | |
| US20240026432A1 (en) | Polyvinyl sulfonate detection and removal from biomolecule compositions | |
| CN113150123A (en) | Method for preparing and separating protein charge isomers | |
| Chen | A Mini Summary of Affinity-based Separation Methods for Sample Preparation | |
| Ulmer | Continuous Reaction and Separation of IgG | |
| CN116769044A (en) | anti-CD 3 and CD19 bispecific antibody protein and chromatographic purification method thereof | |
| Marc et al. | Adsorption Chromatography in Monolithic Columns | |
| JPH03137565A (en) | Cancer marker and monochronal antibody peculiar to siad marker |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220412 |
|
| RJ01 | Rejection of invention patent application after publication |