CN114014906B - Method for purifying hydrophobic protein by cation exchange chromatography - Google Patents
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a method for purifying hydrophobic protein by cation exchange chromatography. The chromatography steps of the method comprise balancing, loading, washing and eluting, wherein the cation eluent used in the chromatography process does not contain strong electrolyte components, and the conductivity of the cation washing liquid used is higher than that of the loaded sample; salts used in the cationic equilibrium, rinse and eluent include salts of weak acids and/or phosphates. The method can effectively remove HCP and polymer, avoid the phenomenon of cracking peak or multimodal of cation chromatography elution peak, and the yield and purity of the collected bispecific antibody are high and can reach more than 90 percent (the purity can reach more than 99 percent).
Description
Technical Field
The invention relates to the technical field of antibody purification, in particular to a method for purifying hydrophobic protein by cation exchange chromatography.
Background
Hydrophobicity is an important physicochemical property of antibody molecules, and the strength of the hydrophobicity of antibody molecules has an important influence on the selection of fillers for cation exchange chromatography and process development. Generally, antibody molecules with stronger hydrophobicity are easier to elute and multimodal in the cation exchange chromatography purification process, and the problems of poor polymer removal effect, poor stability, low yield and the like are solved. The hydrophobic antibody will be specifically described below.
Through researches and developments for over 30 years, monoclonal antibody (monoclonal antibody, mAb) medicines have made great progress in the field of treatment of tumors and autoimmune diseases, and meanwhile, the medicines become the most rapid and promising development direction in the field of medicines, and bring new hopes for patients who are ineffective in conventional treatment. At present, through genetic engineering improvement and effective quality control, the affinity, stability, biological activity and treatment effect of the medicine are greatly improved.
Bispecific antibodies (Bispecific monoclonal antibody, bsAb) are capable of achieving simultaneous binding of a single molecule to two unique targets, and are observed to have better therapeutic effects than monoclonal antibody combinations for a variety of indications, including tumors and infectious diseases. Combining both targets simultaneously can achieve a unique mechanism of action that is not achieved by monoclonal antibodies, and interest in bispecific antibodies as therapeutic agents is increasing. Chinese patent (application publication nos. CN110305210a and CN 101896504B) discloses the design and preparation of bispecific antibodies. Chinese patent (application publication number CN107922476 a) discloses a method of isolating these bispecific antibodies from a mixture comprising a monospecific antibody having two kappa light chains or parts thereof and a monospecific antibody having two lambda light chains or parts thereof.
Downstream purification is considered to be one of the most challenging stages in the commercial production of monoclonal antibody drugs, and currently a three-step purification strategy is widely adopted: sample capture, moderate purification and fine purification, the strategy is complex in process and strict in operation requirements, resulting in purification costs generally accounting for 50% -80% of the total production cost. Sample capture is typically performed in the first step by protein A affinity chromatography, and intermediate purification and fine purification may be performed by ion exchange chromatography, hydrophobic chromatography, and the like. Downstream processing of diabodies is likely to utilize the same processing strategy as monoclonal antibodies. Although the various platform processes for monoclonal antibody purification all employ several chromatographic methods, the specific parameters and filler selection are different and produce completely different results.
Cation exchange Chromatography (CEX) is widely used in the purification process of platform monoclonal antibodies due to its high loading, selectivity to impurities, scalability and robustness, mainly for removing high polymers, and simultaneously removing host proteins and DNA. The isoelectric point of a mAb is generally neutral or slightly basic, so cation exchange packing is generally suitable for operation in a binding elution mode in the purification process of antibodies, mAb binds to the resin under low conductivity conditions and has a pH below the isoelectric point of the target molecule. Elution of the mAb by increasing conductivity or pH can be achieved by linear gradient or stepwise elution to predetermined conditions. Impurities, particularly high polymers, are typically more tightly bound to the CEX filler than the mAb product and can be separated from the desired components by adjusting the elution conditions and collection ranges.
CEX is generally considered a gentle procedure and is unlikely to cause conformational changes in proteins because it is primarily based on electrostatic interactions. In most cases, the binding protein eluted from the CEX column in a unimodal form, with the eluted salt concentration depending on pH and gradient slope. However, the use of cation exchange chromatography is sometimes affected by the chemical nature of the protein, producing unexpected results. Gillespy et al found that loading a high purity glycosylated antibody onto a cation exchange column and eluting it with a salt gradient resulted in two elution peaks (gillespeie R, nguyen T, macneil S et al Cation exchange surface-mediated denaturation of an aglycosylated immunoglobulin (IgG 1). J chromator a,2012,1251: 101-110), one containing almost entirely a monomeric form of the antibody and the other containing an increased percentage of aggregated species formed within the column. The molecular mechanism of the phenomenon of multiple elution peaks occurring during elution by both cation and anion exchange chromatography is generally believed to result in localized changes in the dynamic conformation of the surface of the protein molecule due to adsorption to the surface of the packing (Kimerer L K, pabst T M, hunter A K et al Chromatographic behavior of bivalent bispecific antibodies on cation exchange columns.II. Biological persevers.J. chromatogrA, 2019,1601: 133-144.). The solvent exposure area of monoclonal antibodies bound to CEX filler increased over time, increasing gradually the aggregation of unstable intermediates upon elution, resulting in a bimodal elution phenomenon (Guo J, carta G.Unfolding and aggregation of a glycosylated monoclonal antibody on a cation exchange column.part II.protein structure effects by hydrogen deuterium exchange mass spline.J chromator A,2014, 1356:129-137). Reversible protonation of the histidine residues in the monoclonal antibody molecule also produces a different bimodal elution event on different cationic fillers (Luo H, cao M, new well K et al Double-peak elution profile of a monoclonal antibody in cation exchange chromatography is caused by histidine-process-based charge derivatives. J chromatogrA, 2015, 1424:92-101). Reversible changes in conformation and irreversible aggregation of certain mab molecules on CEX columns also result in elution of three peaks (Guo J, creasy a D, barker G et al Surface induced three-peak elution behavior of a monoclonal antibody during cation exchange chromatogrj chromatogra, 2016, 1474:85-94). Luo et al report that NaCl in the elution buffer induced a reversible self-association that bound strongly to the CEX column during linear salt gradient elution, and co-eluted with the aggregates, resulting in lower purity of the eluted product and significant peak splitting of the elution (Luo H, macapagal N, newell K et al. Effects of salt-induced reversible self-association on the elution behavior of a monoclonal antibody in cation exchange chromatogrJ chromatogrA, 2014, 1362:186-193).
The appearance of multiple elution peaks for mAbs on CEX columns is typically seen in a linear salt gradient elution mode, and similar phenomena occur for certain mAb molecules when eluted at salt isocratic (Guo J, carta G.Unfolding and aggregation of a glycosylated monoclonal antibody on a cation exchange column.part II.protein structure effects by hydrogen deuterium exchange mass spline.J chromator A,2014, 1356:129-137). In addition to the nature of the mAb molecule itself, the chemical and structural Impact of the cation exchange packing material on the CEX column is also evident by the bimodal elution profile of polymer graft resins such as Capto S image and Eshmuno CPX (Farys M, gibson D, lewis AP et al Isotype dependent on-column non-reversible aggregation of monoclonal anti-ibodies.Biotechnol Bioeng,2018,115 (5): 1279-1287).
Although the capture behavior of bispecific antibody molecules on protein a fillers is similar to that of conventional mabs, due to the multi-domain structure of bispecific antibody molecules and the flexibility of single chain variable fragment (single chain antibody fragment, scFv) ligation, as well as complex impurity composition, cation exchange chromatography may be significantly different, different conformations may bind to the chromatographic surface in different ways, resulting in more complex chromatographic phenomena including the possibility of multimodal elution (Kimerer L K, pabst T M, hunter a K et al Chromatographic behavior of bivalent bispecific antibodies on cation exchange columns.i. experimental observations and phenomenological model j Chromatogr a,2019,1601 121-132). At present, a CEX chromatography purification method of a bispecific antibody mostly adopts a mAb platform purification method, which is easy to cause cation elution peak abnormality, and has the defects of complex process, low loading capacity, low product yield, low purity and the like.
Disclosure of Invention
The invention aims to solve the technical problems of aggregation, low yield, low purity and the like in the process of purifying hydrophobic antibody molecules in the prior art, and provides a method for purifying hydrophobic protein by utilizing cation exchange chromatography, which can effectively remove HCP and polymers, avoid cracking or multimodal phenomenon of cation chromatography elution peaks, and ensure that the yield and purity of the collected hydrophobic protein (such as bispecific antibody) are higher than 90 percent (the purity can even reach more than 99 percent).
The invention mainly solves the technical problems through the following technical scheme.
One of the technical schemes of the invention is as follows: a method for purifying hydrophobic protein by cation exchange chromatography, wherein the chromatography steps comprise balancing, loading, washing and eluting, wherein the cation eluent used in the chromatography process does not contain strong electrolyte components, and the conductivity of the cation washing liquid used is higher than that of the loaded sample;
salts used in the cationic equilibrium, rinse and eluent include salts of weak acids and/or phosphates.
Preferably, the filler used for cation exchange chromatography is a filler containing a catalyst selected from the group consisting of sulfonic acid groups (SO 3 2- ) Strong cation exchangers of groups selected from the group consisting of sulfomethyl (S), sulfopropyl (SP) and phosphate (P) or weak cation exchangers containing ion exchange groups selected from the group consisting of Carboxymethyl (CM), carboxyl (COO-) Optionally containing sulfonic acid groups - Or sulfopropyl groups, etc. The fillers for cation exchange chromatography used in the present invention preferably include, but are not limited to Capto S image and Capto MMC from GE company, POROS 50XS and Fractogel EMD SO3 from POROS 50HS,Merck Millipore from Thermofiser company - (M) and Eshmuno CPX, nanogel-50SP, suzhou Nami, etc.; capto S Impact or Poros 50HS fillers are preferred.
The cation exchange chromatography described in the present invention may be conventional in the art, preferably using a bind-elute mode.
The cation balancing solution, flushing solution and eluent in the invention can be non-strong acid salt, preferably acetic acid-acetate buffer, citric acid-citrate buffer, phosphate buffer or histidine salt buffer, more preferably acetic acid-acetate buffer; preferably, none of the cationic equilibrium liquid, the rinse liquid and the eluent comprises a strong electrolyte component.
In the present invention, the elution may be conventional in the art, preferably salt gradient elution or salt isocratic elution. Wherein, the salt gradient elution is preferably salt linear gradient elution.
Preferably, the conductivity of the eluent for isocratic elution of the salt is 12.55-17.6 mS/cm, and the pH value is 4.8-5.5.
Preferably, the conductivity of the eluent of the salt linear gradient elution is 19.0-25.0 mS/cm, and the pH value is 5.0-5.4.
Wherein: when the packing is Capto S Impact, the conductivity range of the eluent eluted by the salt isocratic is preferably 13.5-15.5 mS/cm; when the packing is Poros 50HS, the range of conductivity of the salt isocratic eluting eluent is preferably 12.55-14.75 mS/cm.
In the present invention, the pH of the cationic equilibrium liquid is preferably 4.9 to 5.5, more preferably 5.0; the conductivity is preferably 3.0 to 10.5mS/cm, more preferably 5.7 to 10.5mS/cm, still more preferably 8.0mS/cm.
In the present invention, the pH of the rinse liquid is preferably 4.9 to 5.5, more preferably 5.0; the conductivity is preferably 3.0 to 10.5mS/cm, more preferably 5.7 to 10.5mS/cm, still more preferably 8.0mS/cm.
The conductivity of the sample to be loaded in the present invention is preferably 3.74 to 10.5mS/cm, more preferably 3.74 to 7.55mS/cm.
The hydrophobic protein is an antibody, such as a bispecific antibody or a monoclonal antibody; the bispecific antibody is preferably an OX40/PD-L1 bispecific antibody; more preferably, the Fab fragment comprises a single domain antigen binding site that specifically binds to PD-L1 and specifically binds to OX 40.
Preferably, the bispecific antibody comprises polypeptide chain 1: VH-CH1-CH2-CH 3-linker-VHH and polypeptide chain 2: VL-CL; preferably, CDR 1-3 of the VHH are respectively shown as SEQ ID NO 1-3 in a sequence table, amino acid sequences of CDR 1-CDR 3 of the VH are respectively shown as SEQ ID NO 4-6, and CDR 1-CDR 3 of the VL are respectively shown as SEQ ID NO 7-9.
Wherein the linker preferably comprises the amino acid sequence (Gly 4 Ser) n, wherein n is a positive integer equal to or greater than 1, e.g., n is a positive integer from 1 to 7, e.g., n is 1, 2, 3, 4, 5, or 6.
The amino acid sequence of the VH is preferably shown as SEQ ID NO 10 in the sequence table.
The amino acid sequence of the VL is preferably shown as SEQ ID NO. 11 in the sequence table.
The amino acid sequence of the VHH is preferably shown as SEQ ID NO. 12 in the sequence table.
Preferably, said polypeptide chain 1 comprises a sequence as shown in SEQ ID NO. 13 or a variant retaining the function of the sequence; more preferably, the variant has a sequence identity of 90% or more, preferably 95% or more, more preferably 99% or more to the sequence shown in SEQ ID NO. 13.
Preferably, said polypeptide chain 2 comprises a sequence as shown in SEQ ID NO. 14 or a variant retaining the function of the sequence; more preferably, the variant has a sequence identity of 90% or more, preferably 95% or more, more preferably 99% or more to the sequence shown in SEQ ID NO. 14.
In a preferred embodiment of the invention, the OX40/PD-L1 bispecific antibody is a fully human bispecific antibody obtained from CHO cell culture; preferably, the OX40/PD-L1 bispecific antibody is more hydrophobic than ipilimumab.
The numbers in the present invention are approximations, by use of the antecedent "about" or "about" herein. The values of the numbers expressed by the salt concentration and the protein concentration may differ by + -10%, the values of the numbers expressed by the pH may differ by + -0.1, and the values of the numbers expressed by the conductivity may differ by + -0.5. Whenever any one of the salt concentration and the protein concentration is disclosed as having N 1 Any number of values, having N 1 The number of +/-10% values will be explicitly disclosed, where +/-means either plus or minus, and N 1 About 10% to N 1 A range between +10% is also disclosed. For example, if the cleaning solution for the cationic filler is 0.5mol/L NaOH, then a value of 0.5 moI/L+/-10% is disclosed simultaneously, and a concentration range of 0.5mol/L to 10% to 0.5mol/L+10% is also within the disclosed range, i.e., a value of 0.45 to 0.55mol/L and between, all within the inclusion range of the cationic CIP solution. Also whenever the numerical values of the numbers shown for pH and conductivity are disclosed as N respectively 2 And N 3 At the same time disclose N 2 Range of + -0.1 and N 3 A range of + -0.5.
Definition:
the "hydrophobic protein" referred to in the present invention is not particularly limited and includes any hydrophobic protein which can be purified from cells by using the ion exchange chromatography method of the present invention. In addition, the "hydrophobic protein" does not refer to a specific hydrophobicity number or range, but refers to any hydrophobicity that imparts insolubility of a target protein in an aqueous solution by binding to a cellular structure, or self-association, and allows purification of the protein by the ion exchange chromatography method of the present invention.
The protein with strong hydrophobicity referred to in the present invention is a protein having a peak time not earlier than the peak time of Ipilimumab under the same conditions, preferably not earlier than the peak time of OX40/PD-L1 bispecific antibody under the same conditions, under the chromatographic conditions of HIC-HPLC described in section 1.3 of the test materials and test methods.
The definition "antibody" of the present invention refers to any immunoglobulin, complex or fragment form thereof. The term includes, but is not limited to, monoclonal or polyclonal antibodies to IgA, igD, igE, igG and IgM, including forms modified naturally or by genetic engineering of human origin, chimeric, synthetic, recombinant, hybrid, mutant, and the like. In embodiments of the invention, the antibody may be a monoclonal antibody IgG of fully human origin.
The definition "monoclonal antibody" or "monoclonal antibody" in the present invention refers to an antibody synthesized by a single effector B cell against a particular epitope. Depending on the stage of development, it may be murine, chimeric, humanized and fully human monoclonal antibodies.
The definition of "bispecific antibody" or "diabody" in the present invention refers to an engineered antibody that can bind two specific epitopes or proteins of interest simultaneously, has the ability to bind two different epitopes simultaneously, and can perform some specific biological functions. In embodiments of the invention, the OX40/PD-L1 bispecific antibody can be a fully human monoclonal antibody having both the variable and constant regions of the antibody are human. OX40 (also known as CD134, TNFRSF4 and ACT 35) is a cell surface glycoprotein and a Tumor Necrosis Factor (TNF) receptor superfamily member that is expressed on T lymphocytes and provides a co-stimulatory signal for proliferation and survival of activated T cells. PD-L1 (also known as differentiation cluster 274 (CD 274) or B7 homolog 1 (B7-H1)) is a type I transmembrane protein of 40 kDa. PD-L1 binds to its receptor PD-1 present on activated T cells, down regulating T cell activation. In an embodiment of the invention, the OX40/PD-L1 bispecific antibody comprises a single domain antigen binding site that specifically binds PD-L1 and a Fab fragment that specifically binds OX 40.
The definition "CHO cell" as used herein refers to mammalian chinese hamster ovary cells, which are the most successful type of cell used to express foreign proteins, and are commonly used mammalian host cells. When a recombinant expression vector encoding an antibody gene is introduced into CHO cells, the host cells may be cultured under appropriate conditions to express or secrete the antibody into the culture medium, resulting in a mixture containing the antibody of interest. In embodiments of the invention, the "CHO cell" may be a chinese hamster ovary cell used to express OX40/PD-L1 bispecific antibody.
The term "impurity" refers to a substance that is different from the desired antibody product. Contaminants include, but are not limited to: host cell material such as Host Cell Proteins (HCPs) and DNA; variants, fragments, aggregates or derivatives of the desired antibodies; cell culture medium composition.
The term "wash buffer" is used herein to refer to a buffer that flows through the chromatography material after loading the composition and before eluting the bispecific antibody of interest. The wash buffer may be used to remove one or more impurities from the chromatographic packing without substantially eluting the desired antibody product.
The term "elution buffer" is used to elute the antibody of interest from the solid phase. Herein, the elution buffer has a higher conductivity or a higher pH relative to the wash buffer such that the desired antibody product elutes from the chromatography medium.
The term "ion exchange" or "ion exchange chromatography" refers to a chromatography method that uses an ion exchanger as a stationary phase to separate solutes according to the difference in electrostatic interaction forces between a dotted solute and the ion exchanger. The ion exchangers can be classified into anion exchangers and cation exchangers according to their properties. The former has exchange capacity to anions, and the active group has positive charge; the latter has an exchange effect on cations and the active groups are negatively charged. Ion exchangers, in turn, have a strong and weak classification, depending on the size of the pH range in which they have ion exchange capacity: the strong ion exchanger has a large pH value range with ion exchange effect, and the ionization rate is less influenced by pH; the weak ion exchanger has a small pH value range for ion exchange, and the ionization rate is greatly influenced by pH. In embodiments of the invention, "cation chromatography" or "cation exchange chromatography" both refer to chromatography using a cation exchanger. In the above step, the target product can be separated by the cation exchanger by binding with different cations in the solution, due to the positive charge in the buffer solution with pH below the isoelectric point, which is higher than the isoelectric point of the antibody. The cation exchangers according to the invention typically contain sulfonic acid groups (SO 3 2- ) Strong cation exchangers containing groups such as sulfomethyl (S), sulfopropyl (SP), and phosphate (P), and carboxyl groups (COO) - ) Weak cation exchangers of plasma exchange groups; preferably SO-containing 3 2- Or SP, more preferably Capto S Impact or Poros 50HS.
The term "polymer" is understood to mean a molecule that is a non-covalent association of the same antibody molecule, and is formed by the association of two or more antibodies. The antibodies may be composed of homogeneous or heterogeneous polypeptides to which single chain antibodies are covalently bound (e.g., disulfide bonds). Dimers are nonspecific binding of two IgG molecules. The formation of multimers is closely related to the deformation-affecting factors of the antibody structure. For example, factors such as high salt, extreme pH induction, and interactions of antibody molecules with filler surface groups may all cause the antibodies to denature to form multimers. The fraction of the polymer that flows out of the SEC analytical column is typically observed as one or more peaks preceding the main peak in the SEC-HPLC chromatogram.
The definition "loading" of the present invention refers to an operation of adding a sample to be separated into a chromatographic column by a sample pump or manually, and bringing it into contact with a chromatographic packing. In embodiments of the invention, loading may be the operation of adding an appropriately treated OX40/PD-L1 bispecific antibody sample to a CEX chromatography column.
The definition of "gradient elution" used in the present invention is to control the composition of mobile phase, such as polarity, ionic strength, pH value, etc. of solvent in one analysis period, and to analyze complex samples with large number of components and great difference in properties. The use of gradient elution can shorten the analysis time, improve the degree of separation, improve the peak shape, and increase the detection sensitivity, but often causes baseline drift and decreases reproducibility. The gradient elution of the two solvent compositions can be mixed to any degree, i.e. there are various elution curves: linear gradients, concave gradients, convex gradients, and stepped gradients, with linear gradients being most commonly used. In embodiments of the invention, the elution step of cation exchange chromatography may employ a linear gradient based on salt concentration and a linear gradient of pH.
The definition "isocratic elution" used in the present invention refers to an elution mode in which the composition ratio and flow rate of an elution buffer are constant during elution by cation exchange chromatography.
On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the invention.
The reagents and materials used in the present invention are commercially available.
The invention has the positive progress effects that:
The method can effectively remove HCP and polymer, avoid the phenomenon of cracking peak or multimodal of cation chromatography elution peak, and the yield and purity of the collected bispecific antibody are high and can reach more than 90 percent (the purity can reach more than 99 percent).
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic representation of the structure of an anti-OX 40/PD-L1 bispecific antibody.
FIG. 2A shows the salt linear gradient elution peaks of the purified bispecific antibody using the cation exchange chromatography packing POROS 50HS.
FIG. 2B is a graph showing the purity of the components of a collection solution for cation exchange chromatography and HCP as a function of collection volume; the cation exchange chromatography packing is POROS 50HS.
FIG. 3 is a chromatographic chart of the purification of bispecific antibodies using the cation exchange chromatography packing POROS 50 XS.
FIG. 4 is a graph showing the monomer and polymer content of bispecific antibodies in each component of a cationic collection.
FIG. 5 is a salt linear gradient elution purification bispecific antibody chromatographic profile of POROS 50HS packing.
FIG. 6 is a salt linear gradient elution purification bispecific antibody chromatography profile for Capto MMC packing.
FIG. 7 is a graph showing the change in elution volume of bispecific antibody from the step of collecting the monomer content and the polymer content of each component in sections during elution on four different cation exchange chromatography packing materials.
FIG. 8 is a graph of cationic chromatography in pH linear gradient elution mode.
FIG. 9 is a graph of cation chromatography in pH isocratic gradient elution mode; the elution buffer was 50mM tris-HCl+50mM NaCl, pH7.2, conductivity 8.52mS/cm.
FIG. 10 is a graph of cation chromatography in pH isocratic gradient elution mode; the elution buffer was 25mM tris-HCl+25mM NaCl, pH7.2, conductivity 4.74mS/cm.
FIG. 11 is a cationic chromatography map. And (3) filling: POROS 50HS, linear elution, elution buffer 500mmol/L acetic acid-sodium acetate, pH5.0, 22.3mS/cm.
FIG. 12 is a cationic chromatography map. And (3) filling: capto S Impact, linear elution, elution buffer 500mmol/L acetic acid-sodium acetate, pH5.0, 22.3mS/cm.
FIG. 13 is a cationic chromatography map. And (3) filling: capto S Impact, isocratic elution, equilibration buffer and wash buffer were both 50mmol/L acetic acid-sodium acetate (pH 5.0), and elution buffer was 325mmol/L acetic acid-sodium acetate (pH 5.0).
FIG. 14 is a cationic chromatography map. And (3) filling: capto S Impact, isocratic elution, equilibration buffer and wash buffer were both 150mmol/L acetic acid-sodium acetate (pH 5.0), and elution buffer was 325mmol/L acetic acid-sodium acetate (pH 5.0).
FIG. 15 is a cationic chromatography map. And (3) filling: capto S Impact, linear gradient elution, washing buffer of 50mmol/L acetic acid-sodium acetate (pH 5.0) and elution buffer of 500mmol/L acetic acid-sodium acetate (pH 5.0).
Detailed Description
The specific embodiment of the invention comprises the following steps:
1) Clarifying and filtering the CHO cell culture solution expressing the bispecific antibody by the cation sample loading sample or centrifugally separating to remove cells and cell fragments to obtain clarified liquid;
2) Purifying the clarified liquid by Protein A affinity chromatography packing, and collecting affinity eluent;
3) Adjusting the pH of the affinity eluent to 4.9-5.5, or adjusting the pH of the affinity eluent to 3.5-3.6 for low pH inactivation, standing at room temperature for 60-180 min, adjusting the pH to 4.9-5.5, and filtering the sample obtained in the two modes by using a 0.22 mu m filter after adjusting the conductivity of the sample to serve as a cation sample;
In addition, the sample with the pH adjusted by the affinity eluent and the sample treated by the low pH inactivation collecting liquid through purification steps such as deep adsorption filtration, anion exchange chromatography, hydrophobic chromatography and the like can be used as a cation sample after pH adjustment and conductivity adjustment.
4) And (3) cation exchange chromatography, wherein the cation balance liquid balances 3-5 column volumes of the chromatographic column, then loading is started, and after loading is finished, elution is directly started or is started after 2-5 column volumes are washed by using a washing buffer solution, and elution components are collected.
In some embodiments, the bispecific antibody of step 1) is characterized by: a Fab fragment comprising a single domain antigen binding site that specifically binds to PD-L1 and specifically binds to OX 40.
In some embodiments, the clarification filtration described in step 1) may be performed using commercial membrane packages from 3M, sartorius, merck Millipore, PALL, etc.
In some embodiments, the centrifugation described in step 1) is performed by subjecting the CHO cell culture broth to a centrifugation of 4000 to 9000 g. In the embodiment of the invention, the centrifugation is performed on a Beckman Avanti J-E centrifuge, and the centrifugation is performed at 4 ℃ for 10-15 min.
In some embodiments, the affinity chromatography packing material of step 2) may be MabSelect SuRe LX (GE), mabSelect prism A (GE), uniMab Protein A (Suzhou Nami), AF-rProtein A HC-650F(Tosoh)、Amsphere A3(JSR)、KanCapAOne of 3G (Kaneka), POROS MabCapture A Select (Thermofisher), eshmuno A (EMD Merck), praest AP (Purolite) fillers.
In some embodiments, the reagent used to adjust the pH from low to high in step 3) is 1-2 mol/L Tris, 0.1-1.0 mol/L NaOH, 0.5mol/L dipotassium hydrogen phosphate solution (pH 9.5), 0.2-0.5 mol/L glycine-sodium hydroxide solution (pH 10.5), 1-2 mol/L arginine solution (pH 9.0), etc.; the reagent used for regulating the pH from high to low is acetic acid solution with the concentration of 1-3 mol/L, citric acid solution with the concentration of 1-3M, HCl with the concentration of 0.1-1.0 mol/L, glycine-hydrochloric acid solution with the concentration of 0.2-0.5 mol/L (pH of 2.3) and the like.
In some embodiments, the sample applied as cation in step 3) is adjusted for electrical conductivity with ultrapure water, a cationic equilibrium liquid or a mixture of both, the electrical conductivity ranging from 4.0 to 10.0mS/cm, preferably from 4.0 to 7.5mS/cm.
In some embodiments, the packing for cation exchange chromatography in step 4) includes, but is not limited to Capto S image and Capto MMC from GE company, POROS 50XS from Thermofiser company, and Fractogel EMD SO3 from POROS 50HS,Merck Millipore company - (M) and Eshmuno CPX, nanogel-50SP, suzhou Nami, etc.
Preferably, the cation exchange chromatography medium is equilibrated with an equilibration buffer prior to loading. More preferably, the cation exchange chromatography column is equilibrated with 3 to 5CV of equilibration buffer.
Preferably, the pH of the equilibration buffer is from 4.9 to 5.5, preferably 5.0. The equilibration buffer includes, but is not limited to, acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, phosphate buffer, preferably acetic acid-sodium acetate buffer.
In some embodiments, the pH of the wash buffer of the cation exchange chromatography in step 4) is 4.9 to 5.5, preferably 5.0, and the conductivity is 3 to 10mS/cm, preferably 6 to 10mS/cm. The washing buffer includes, but is not limited to, acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, phosphate buffer, preferably acetic acid-sodium acetate buffer.
In some embodiments, the pH of the elution buffer of the cation exchange chromatography in step 4) is between 4.8 and 7.2, preferably between 5.0 and 5.5. Elution buffers include, but are not limited to, acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, phosphate buffer, preferably acetic acid-sodium acetate buffer.
In some embodiments, the elution of the bispecific antibody in step 4) may employ salt linear gradient elution, salt isocratic elution, pH linear gradient elution, pH isocratic elution, and elution of both pH and salt gradients, preferably salt linear gradient elution and salt isocratic elution, more preferably salt linear gradient elution.
In some embodiments, the collection conditions for the eluted sample in step 4) are: the collection is started when the ultraviolet absorption value of 280nm is increased to 100mAU/mm during elution, and the collection is stopped when the ultraviolet absorption value of 280nm is reduced to 200-650 mAU/mm.
The water used in the examples of the present invention was ultrapure water having a resistivity of 18.0mΩ×cm (25 ℃).
The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings and detailed description, but it will be understood by those skilled in the art that the examples described below are some, but not all, examples of the present invention, and are intended to be illustrative of the present invention only and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
1.1 bispecific antibody (hereinafter referred to as "diabody")
The diabodies are bispecific antibodies disclosed in PCT/CN2020/073959 that bind both PD-L1 and OX 40. For the purposes of this application, the entire contents of this PCT application are hereby incorporated by reference.
The subtype of the diabody is IgG2, the isoelectric point of the diabody is 8.0-8.6, and the molecular weight of the diabody is 174.3kDa.
The diabodies are anti-OX 40/PD-L1 bispecific antibodies having the structure shown in FIG. 1, wherein antigen A is OX40 and antigen B is PD-L1.
Wherein, the specific sequence in the diabody is as follows:
the amino acid sequence of the VH-CH1-CH2-CH 3-linker-VHH (i.e., IGN-LP peptide chain # 1) in FIG. 1 is shown in SEQ ID NO. 13; the amino acid sequence of the VH of anti-OX 40 antibody ADI-20057 shown in FIG. 1 is shown as SEQ ID NO. 10; the amino acid sequence of CH1 in FIG. 1 is shown as SEQ ID NO. 16; the amino acid sequence of Fc (namely CH2-CH 3) in FIG. 1 is shown as SEQ ID NO: 17; the amino acid sequence of the linker in FIG. 1 is shown as SEQ ID NO. 15; the amino acid sequence of the VHH of the anti-PD-L1 single domain antibody in FIG. 1 is shown as SEQ ID NO. 12; the amino acid sequence of VL-CL (i.e., IGN-LP peptide chain # 2) of FIG. 1 is shown in SEQ ID NO. 14; the amino acid sequence of VL of anti-OX 40 antibody ADI-20057 in FIG. 1 is shown as SEQ ID NO. 11; the amino acid sequence of CL in FIG. 1 is shown as SEQ ID NO. 18.
Furthermore, the amino acid sequences of CDR 1-CDR 3 of the anti-PD-L1 single domain antibody contained in the double antibody are respectively shown as SEQ ID NO. 1-3; the amino acid sequences of HCDR 1-HCDR 3 of the anti-OX 40 antibody ADI-20057 contained in the double antibody are respectively shown as SEQ ID NO. 4-6; the amino acid sequences of LCDR 1-LCDR 3 of anti-OX 40 antibody ADI-20057 contained in the diabody are shown as SEQ ID NO 7-9 respectively.
In one embodiment, the diabody is an anti-OX 40/PD-L1 bispecific antibody recombinantly expressed in 293 cells or CHO cells.
1.2 hydrophobicity validation of diabodies
The following chromatographic conditions were set:
the hydrophobicity of the OX40/PD-L1 bispecific antibody was detected using HIC-HPLC. Analysis was performed using Waters e 2695 or agilent 1260 high performance liquid chromatograph. And (3) selecting the Iilimiumab antibody to carry out retention time comparison with the hydrophobicity of the sample to be detected, wherein the longer the retention time is, the stronger the protein hydrophobicity is.
Chromatographic conditions: a MabPacTM HIC-10LC column (manufacturer: thermo, cat. Number 088480) was used; mobile phase a:1.8mol/L ammonium sulfate, 100mmol/L NaH2PO4.2H2O, pH 6.5, mobile phase B:90% volume fraction of a mixed solution of 100mmol/L NaH2PO4.2H2O (pH 6.5) solution and 10% isopropanol; flow rate: 1.0ml/min; elution procedure: within 0 to 20min, 100% A to 0% A; within 20 to 25min, 100% b; within 25 to 30min, 100% a; acquisition time: 30min; sample injection amount: 10-20 mul; column temperature: 25 ℃; detection wavelength: 280nm.
As a result, under the above chromatographic conditions, the peak time of the Iplilimumab was about 28 minutes and the peak time of the OX40/PD-L1 bispecific antibody was about 31 minutes.
Example 2
(1) Preparation of cation sample
The culture solution of CHO cells expressing OX40/PD-L1 bispecific antibody was centrifuged at 9000g for 15min by Beckman Avanti J-E centrifuge, and the supernatant was filtered with 0.22 μm filter to give a supernatant, which was purified by affinity chromatography with MabSelect SuRe LX (GE) packing, pH of the affinity collection was adjusted to 4.9, conductivity was adjusted to 4.0mS/cm with ultrapure water, and filtered with 0.22 μm filter to give a cation sample. The protein content of the cationic sample was 10.9g/L and the purity was 96.0% (SEC-HPLC method, all purity data below were determined by this method) and the HCP content was 3232.9ppm.
(2) Cation exchange chromatography
High resolution POROS 50HS filler packing Merck Millipore Inc. with Thermofisher IncL Laboratory Column VL 11X 250 column, column height 21.3cm, column volume 22.6mL. After 5 Column Volumes (CV) of the chromatographic column are balanced by a balancing buffer solution (50 mmol/L acetic acid-sodium acetate, pH 4.9), an AKTA pure M purification system (the optical path of an ultraviolet detector is 2 mm) is adopted to load 50g/L filler, the retention time of the sample during loading is 6min, namely 3.77ml/min, a flushing buffer solution (50 mmol/L acetic acid-sodium acetate, pH 5.5) for 3CV is used for finishing loading, and a flushing buffer solution and an elution buffer solution (B solution: 50mmol/L acetic acid-sodium acetate, 0.5mol/L NaCl, pH 5.5) are used for carrying out salt linear gradient elution (0-100% B solution, 20 CV) and ultraviolet absorption value of 280nm wavelength The collection was started when the thickness was increased to 100mAU/mm, and was stopped when the ultraviolet absorbance at 280nm was decreased to 100mAU/mm by 5.0ml per tube. After the elution is finished, the buffer solution containing 1mol/L sodium chloride is used for regenerating 3CV, 0.5mol/L NaOH solution is used for cleaning 15-30 min, 10mmol/L NaOH or 20% ethanol is used for preserving, and the regeneration, cleaning and preserving methods in the follow-up implementation are the same as the embodiment and are not repeated.
(3) Detection and analysis
The protein content, purity and HCP content of each tube collection liquid were detected, the protein content was detected by NanoDrop2000, and the HCP content was detected by using an ELISA kit purchased commercially, and the detection method used in this example was applicable to other examples and will not be described in detail.
As shown in FIG. 2A, the chromatographic chart of cation elution shows a phenomenon of double peaks in the elution peak, and if the elution peak falls to the vicinity of 400mAU to stop collecting, namely, when collecting about 45.2ml (2 CV), the yield is 82.4%, and the purity is 95.2%. If the stop collection point is set to about 1.33CV (the corresponding ultraviolet absorption value at 280nm is about 1300 mAU), the yield is 77.1%, the purity is 98.5%, and both the yield and the purity are low. The total polymer content of the collection fluid was calculated to have exceeded the content of the sample on the sample from the beginning of collection to about 400mAU, indicating that new polymer was produced during the cation elution. Purity and HCP changes with elution column volume as shown in fig. 2B, the purity of bispecific antibody underwent a decrease followed by an increase and then a decrease with increasing salt concentration, resulting in lower purity of the collection solution. The HCP content of the fraction of the collected liquid in each section of the eluent was gradually increased with the increase of the salt concentration, and most of the HCP was removed when the collection was stopped at about 45.2ml (2 CV) of the collected liquid.
Example 3
(1) Preparation of cation sample
The CHO cell culture liquid expressing OX40/PD-L1 bispecific antibody was filtered using Merck milipore Millistak D0HC and X0HC deep adsorption membrane package, and then subjected to affinity chromatography purification by using Protein A filler, pH of the affinity collection liquid was adjusted to 5.0 by using 2mol/L Tris solution, conductivity was adjusted to 3.0mS/cm by using ultrapure water, and the mixture was filtered by using a 0.22 μm filter to obtain cation loading sample. The protein content of the cationic sample is 17.0g/L, the purity is 96.9%, and the HCP content is 60.7ppm.
(2) Cation exchange chromatography
Filling Merck Millipore company with high-load POROS 50XS filler from Thermofisher companyL Laboratory Column VL 11X 250 column, 9.5cm high and 10.1mL column volume. After 5CV of chromatography column is equilibrated by an equilibration buffer (50 mmol/L acetic acid-sodium acetate, pH 5.0), a purification system (with an ultraviolet detector optical path of 2 mm) of AKTA pure 150L is adopted for loading, the loading capacity is 37g/L of filling, the loading flow rate is 1.68ml/min, the loading is finished, 3CV of washing buffer (50 mmol/L acetic acid-sodium acetate, pH 5.0) is used for loading, the washing buffer and elution buffer (B solution: 50mmol/L acetic acid-sodium acetate, 0.5mol/L NaCl, pH 5.0) are used for carrying out salt linear gradient elution (0-100% B solution, 10 CV), the collection is started when the ultraviolet absorption value of 280nm wavelength is increased to 100mAU/mm, the collection is started by adopting a branch pipe, 3.4ml (1/3 CV) is collected by each pipe, and the collection is stopped when the ultraviolet absorption value of 280nm wavelength is reduced to 100 mAU/mm.
(3) Detection and analysis
The chromatographic patterns of cation elution are shown in FIG. 3, and the elution curves of the bispecific antibody are similar to those of example 1, wherein elution collection is started between 20 and 30 percent of eluent components, and the elution peak double peaks (the peak height of the 2 nd elution peak is lower) are all generated, and the maximum elution protein concentration is reached at 0.6 to 0.7 CV. The distribution of the monomer and polymer content of the bispecific antibody in each component of the cationic collection liquid is shown in fig. 4, and the peaks of the fluctuation of the monomer content and the peaks of the fluctuation of the polymer content overlap, so that the polymer in the cationic collection liquid is difficult to remove, the yield and purity of the elution peak intercepted at 1CV are 78.2% and 98.1%, respectively, and the yield and purity of the elution peak intercepted at 1.33CV are 88.4% and 96.9%, respectively. The total polymer content of all the fractions exceeded the content of the sample loaded, indicating that new polymer was produced during the cation elution as in example 1.
Example 4
(1) Preparation of cation sample
The CHO cell culture liquid expressing OX40/PD-L1 bispecific antibody was filtered using a deep adsorption membrane package of SUPRAcap HP PDE8 and PDE2 from PALL company, and then subjected to affinity chromatography purification by using MabSelect prism A filler, the affinity collection liquid was filtered with a 0.22 μm filter after pH was adjusted to 6.0 with 2mol/L Tris solution, and then stored in a 4℃refrigerator, the pH was adjusted to 5.0 with 2mol/L acetic acid, the conductivity was adjusted to 3.76mS/cm with ultrapure water, and the sample was loaded with cations after filtration with a 0.22 μm filter. The protein content of the cationic sample is 4.4g/L, the purity is 96.8%, and the HCP content is 896.8ppm.
(2) Cation exchange chromatography
Selecting 5 kinds of CEX fillers (POROS 50HS, capto S image, capto MMC, factogel EMD SO) 3- (M) and Nanogel-50SP were subjected to cation exchange chromatography, and the buffer solution, the running procedure and the collection conditions of cation chromatography were the same as those of example 2, and were all collected by a separate tube, 1 tube was collected every 1/3CV, and the retention time of elution of the loaded sample was 6min;
the columns were all Omnifit 006EZ-06-25-FF columns (inner diameter 6.6mm, column length 250 mm) from Diba company, wherein column 1 was packed with POROS 50HS packing, column height=19.2 cm, CV=6.57 mL; wherein the chromatographic column 2 is filled with Capto S Impact packing, column height = 18cm, cv = 6.16mL; wherein the chromatographic column 3 is packed with Factogel EMD SO 3- (M) packing, column height = 19cm, cv = 6.5mL; wherein column 4 was packed with Nanogel-50SP packing, column height = 20.5cm, cv = 6.97mL; chromatography column 5 was packed with Capto MMC packing, column height = 19.5cm, cv = 6.67mL.
(3) Detection and analysis
As shown in FIG. 5, the cationic analytical spectrum of POROS 50HS filler shows that the elution peak does not show the double peaks in example 1 and example 2, the purity and the yield are higher, and when the peak is cut off at the 2CV collection volume (the ultraviolet absorption value is about 600 mAU), the yield is 88.9%, and the purity is 98.7%. The chromatographic pattern of Capto MMC filler is shown in FIG. 6, the bispecific antibody molecule has strong hydrophobicity, firm combination with filler, and no collection during elution The samples were hardly eluted in the regeneration liquid containing 1mol/L NaCl (pH 7.2). Capto S Impact and Factogel EMD SO 3- The chromatographic patterns of (M) fillers are similar to those of POROS 50HS filler, while the chromatographic patterns of Nanogel-50SP fillers are similar to those of example 1, and the results of several fillers are shown in Table 1.
TABLE 1 detection results of equal volume mixtures of the first 2 column volumes of the collected fluids for different cationic fillers
The change in polymer content and monomer content of the segmented collection fluid with collection volume for salt linear gradient purification of bispecific antibodies using different fillers is shown in fig. 7, where the polymer content begins to increase rapidly at 1CV collection volume with POROS 50XS filler, resulting in a lower purity of the first 2 CV collection fluid mixture than the loaded sample. The purity of the first 2 CV collection mixtures was higher when POROS 50HS packing was used, which was more suitable for linear gradient elution of bispecific antibodies with cationic salts.
Example 5
(1) Preparation of cation sample
The cell culture broth was clarified and filtered using Zeta Plus 90ZB05A from 3M company and Emphize AEX membrane pack, purified by affinity chromatography using MabSelect prism A packing, and anion exchange chromatography was performed after pH of the affinity collection broth was adjusted to 7.2 with 2mol/L Tris solution, pH of the anion collection broth was adjusted to 5.17 with 1mol/L dilute hydrochloric acid, conductivity was adjusted to 3.56mS/cm with ultrapure water, and the mixture was filtered with 0.22 μm filter to obtain a cation sample. The protein content of the cationic sample is 4.4g/L, the purity is 97.1%, the HCP content is 9.4ppm, and the conductivity is 3.56mS/cm.
(2) Cation exchange chromatography
The effect of cation exchange chromatography on bispecific antibody purification was examined by means of pH linear gradient elution. The pH gradient elution adopts filler Factogel EMD SO 3- (M) packing Omnifit column, wherein column height = 19cm, cv = 6.5mL, chromatographyThe column was washed with 0.5mol/L NaOH before use, the specific chromatographic conditions are shown in Table 2 below, and a tube was collected every 1/3CV using staged collection.
TABLE 2 chromatographic conditions of cations with linear gradient elution mode of pH
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N/A indicates inapplicability
(3) Detection analysis
As shown in FIG. 8, when the eluent component ratio reaches 100%, the eluting peak does not drop to 200mAU yet, and the elution condition that the eluent component ratio is 100% is continuously maintained until the preset collection stop condition is reached, the total collection volume is 8.3CV, and the eluting force is weak. The conductivities of the equilibration and elution solutions were 3.0mS/cm and 6.0mS/cm, respectively, and the conductivities at the initial collection of the salt gradient elution according to examples 1, 2 and 3 were typically between 10 and 15mS/cm, increasing the conductivity of the elution solution to this interval possibly increasing the elution force. The detection result shows that the polymer is distributed on the front side and the rear side of the elution peak, part of the polymer is eluted from the column at low pH and low conductivity, the collecting solutions of the 3 rd tube to the 20 th tube are mixed in equal proportion, and the yield and the purity are respectively 79.6% and 98.6% and are relatively low in yield.
Example 6
(1) Cationic sample preparation
The CHO cell culture liquid is filtered by a deep adsorption membrane package of Millistak C0HC and X0HC of Merck Millipore company, and then is subjected to affinity chromatography purification by using MabSelect prism A filler, the pH is adjusted back to about 5.0 by using 1M Tris, the diluted electric conduction is conducted to about 3.9mS/cm by using ultrapure water, and the diluted electric conduction is filtered by a filter of 0.22 mu M to be used as a cation loading sample. Sample 1 concentration 12.8g/L, pH 5.03, conductivity 3.94mS/cm; sample 2 had a concentration of 14.4g/L, pH 5.02 and conductivity 3.85mS/cm. Both samples had a purity of 97.4% and HCP content of 1323.9ppm.
(2) Cation exchange chromatography
The effect of cation exchange chromatography on bispecific antibody purification was examined by pH isocratic elution. Using a cationic pre-packed column POROS TM GoPure TM HS (POROS 50HS packing, 12mm inside diameter, 5cm height, 5.7mL column volume), sample 1 and sample 2 were loaded with 28mL and 25mL, respectively. The equilibration solutions of sample 1 and sample 2 were 50mmol/L acetic acid-sodium acetate buffer (pH 5.0), the washing solutions were 20mmol/L acetic acid-sodium acetate buffer (pH 5.0), the eluents were 50mmol/L Tris-HCl,50mmol/L NaCl (pH 7.2, conductivity 8.52 mS/cm) and 25mmol/L LTris-HCl,25mmol/L NaCl (pH 7.2, conductivity 4.74 mS/cm), respectively, elution 10CV, and the collection conditions and other steps were the same as in example 4.
(3) Detection analysis
As shown in FIGS. 9 and 10, the cation chromatography is carried out with a relatively high pH fluctuation after the start of elution, the bispecific antibody molecules bound on the cation column are not eluted by 10CV of eluent with low conductivity (FIG. 10), the bispecific antibody molecules are eluted by 10CV of eluent with high conductivity, the elution volume is smaller than 3CV, the yield is relatively high, but the purity of the antibody molecules is relatively low, the removal effect of HCP is poor, the total volume of the 1 st to 6 th tubes is 2CV, the yield is 93.8%, the purity is 97.1%, and the HCP content is 371ppm. Therefore, the conductivity of the eluent is between 4.74 and 8.52mS/cm, and the effect of purifying the bispecific antibody by adopting a pH isocratic elution mode in the purification of the POROS 50HS packing is poor.
Example 7
(1) Preparation of cation sample
The cell culture broth was clarified and filtered using the same conditions as in example 2, and purified by affinity chromatography using the same conditions as in example 3, and the affinity collection broth was subjected to pH inactivation treatment (pH adjustment to 3.5, treatment at room temperature for 65min, pH adjustment to 6.0), further to deep adsorption filtration (ADF), pH adjustment to 5.0, conductivity adjustment with ultrapure water, and filtration through a 0.22 μm filter to give a cation-loaded sample.
(2) Cation exchange chromatography
The purification effect of the cationic filler POROS 50HS under several different conditions was tested using an isocratic elution mode. Test condition 1: the sample concentration was 7.6g/L, the purity was 97.8%, the HCP content was 32.3ppm, the conductivity was 5.92mS/cm, and the loading was 50g/L. The chromatographic column was a GE XK 26X 40 column (CV=132.5 ml), the linear flow rate at loading and elution was 300cm/h, the equilibrium eluent and the rinse eluent were both 50mmol/L acetic acid-sodium acetate buffer (pH 5.0, conductivity about 3.0 mS/cm), the eluent was 50mmol/L acetic acid-sodium acetate+150 mmol/L NaCl (pH 5.0, conductivity about 17.39 mS/cm), elution was 5CV, the ultraviolet absorption value increased to 200mAU to start collection, and the elution was dropped to 400mAU to stop collection.
Test condition 2: the sample concentration was 7.44g/L, pH 5.06, conductivity 3.91mS/cm, purity 97.2%, GE XK16X40 column (CV=40 ml), eluent 50mmol/L acetic acid-sodium acetate+120 mmol/L NaCl (pH 5.0, conductivity about 14.75 mS/cm), other parameters were the same as those of test condition 1.
Test condition 3: the sample concentration on the sample was 13.8g/L, pH 5.04, conductivity 3.17mS/cm, purity 97.2%, HCP content 8.5ppm. The chromatographic column is Merck Millipore VL column, the linear flow rate during loading and elution is 200cm/h, the eluent is 50mmol/L acetic acid-sodium acetate+110 mmol/L NaCl (pH 5.0, the conductivity is about 12.55 mS/cm), the elution is 6CV, and the other conditions are the same as the test condition 1.
(3) Detection analysis
The detection results of the three isocratic elution test conditions are shown in table 3, when the conductivity of the eluent is 17.39mS/cm, the yield is higher, but the effect of removing the polymer is poorer, the purity of the collected liquid is poorer than that of the sample, and the elution conditions are relatively stronger, so that the bispecific antibody generates a new polymer in the elution process; the conductivity is 14.75mS/cm, the yield is reduced, but the purity is better; the conductivity is 12.55mS/cm, the yield is higher, but the yield is lower, the collection volume is larger, and samples are not eluted from the column after 6 CVs are finished, if the elution volume is increased, the collection volume is larger, and the subsequent technological process operation is not facilitated. In combination with the above analysis, when the filler POROS 50HS is used for purifying bispecific antibody by isocratic elution, the conductivity needs to be controlled between 12.55 and 14.75 mS/cm.
Table 3 comparison of the detection results of three isocratic elution test conditions
Example 8
(1) Preparation of cation sample
Cationic loading samples were prepared as in example 1, with a loading sample concentration of 8.4g/L, pH 5.07,4.16mS/cm, purity 97.2% and HCP content 1846.5ppm.
(2) Cation exchange chromatography
In the case of the elution buffer used for downstream cation purification, the effect of the elution buffer containing strong electrolyte NaCl is not ideal in most cases, whether it is linear gradient elution or isocratic elution, and the purification effect of bispecific antibody is examined by using weak acid salt acetic acid-sodium acetate buffer as the only component of the elution buffer.
Test condition 1: capto S Impact packing, VL11 column (cv=23.0 ml), loading 70g/L; test condition 2: POROS 50HS packing, VL11 column (cv=17.4 ml), loading 50g/L. The equilibration and elution of the two test conditions were the same as in example 6, the elution buffer was 500mmol/L acetic acid-sodium acetate (pH 5.0), the elution was 10CV in a linear gradient (0-100%), and the collection conditions and other chromatographic procedures were the same as in example 6.
(3) Detection analysis
The cationic analytical patterns of both test conditions are shown in FIGS. 11 and 12, and the peaks of the projections as in examples 1 and 2 do not appear after the main peak of the elution peak of both cations, and the elution volumes are larger than those of the elution buffer containing NaCl. The elution peak was broad with POROS 50HS packing and the collection volume was large, amounting to about 6 CV, whereas with the same collection conditions with Capto S imact packing, the collection volume was about 3.7 CV. The results of the two salt linear gradient elution test conditions are shown in table 4, and the yield and purity of the two fillers are higher than those of the salt linear elution with NaCl (examples 1, 2 and 3), and the yield of the POROS 50HS filler reaches 92.4% and the purity reaches 99% when the stop collection point is set between 700 and 800 mAU; when the stop collection point is set between 500 and 600mAU, the yield of the Capto S imact filler reaches 89.2 percent and the purity reaches 99.5 percent.
Table 4 comparison of the test results of the two salt linear gradient elution test conditions
Example 9
(1) Preparation of cation sample
Preparation of cationic sample the sample information obtained from the various batch processes of example 6 is shown in table 5.
TABLE 5 cationic sample loading information Table
(2) Cation exchange chromatography
The effect of purifying OX40/PD-L1 diabody under various chromatographic conditions was tested by using Capto S imact packing, the eluent conductivity varied in the range of 12.7-17.6 mS/cm, pH varied in the range of 4.8-5.5, and an isocratic elution mode was used, and the partial cation chromatography buffers and partial test conditions are shown in Table 6 and Table 7. Except 13# test conditions in table 7, GE XK26 column (cv=114.1 mL) was used, VL11 column was used, CV was between 17 and 23.0mL, and the number of eluted volumes was 5 to 6CV.
TABLE 6 cationic assay buffer information
TABLE 7 partial cationic analytical test condition information
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(3) Detection and analysis
The detection results of part of the test conditions are shown in Table 8, and the comparison of test conditions 1# to 3# shows that the conductivity of the eluent is in the range of 13.5-15.5 mS/cm, the yield and purity of the eluent collection liquid are higher, and the purity of the eluent collection liquid can reach more than 99.0% under the condition that the collection is stopped between 400 mAU and 800 mAU. The eluent conductivities of test condition 1# and 3# were relatively close, and the ph of test condition 1# was lower than 3# resulting in lower yields of test condition 3 #. The comparison of test conditions No. 4-No. 7 shows that the conductivity of the eluent is in the range of 13.8-17.3 mS/cm, the purity of the eluent collection liquid is reduced along with the increase of the conductivity, wherein the conductivity is higher in the range of 13.8-14.4, and the effect of removing the polymer is better; comparing test conditions 5# and 8# finds that the conductivity of the loaded sample affects elution, and when the conductivity is 10.0mS/cm, the elution peak does not completely elute in the set elution process of 5CV, and when the conductivity of the same batch of samples is 7.55mS/cm, the elution peak is normal.
Comparison of test conditions 9# and 10# revealed that the low conductivity loading samples (3.74 mS/cm) used different equilibrium and elution conditions, the collection liquid had no difference in yield, purity and HCP content, but the peak-to-peak differences in elution peak were large, and the chromatographic profiles of test conditions 9# and 10# were as shown in FIGS. 13 and 14. At lower equilibrium and wash conductivities, e.g., 3.0mS/cm, elution peak "split" occurs (FIG. 13), and when the fractions collected in stages are re-run with the same procedure, the same elution peak "split" occurs, possibly with histidine protonation or other reversible changes in bispecific antibody molecule conformation during elution. After increasing the conductivity of the equilibration buffer and the wash buffer, the elution peak profile was similar to that of the other test conditions.
Comparison of test conditions 11# and 12# found that a lower pH of the eluate resulted in no elution of the antibody molecules bound to the filler, resulting in no sample collection, a pH of the eluate greater than 5.2, a conductivity in the range of 16.6-17.6 mS/cm, and a higher yield, but little removal of the polymer.
Comparing test conditions 13# to 15# shows that when the conductivities of the equilibration buffer and the rinse buffer are within 5.7-10.5 mS/cm, there is no effect on the purity of the eluted sample, but the yield is slightly higher when the conductivities are around 8.0.
In combination with the above analysis, the conductivity of the sample to be loaded, the conductivities of the equilibration buffer and the rinse buffer, the conductivities of the eluent and the pH have a great influence on the yield and purity of the sample, and should be controlled in a reasonable interval.
TABLE 8 comparison of test results for partial test conditions
| Test conditions | Collection Volume (CV) | Yield (%) | Purity (%) | HCP content (ppm) |
| 1# | 2.3 | 99.9 | 98.5 | 2.3 |
| 2# | 3.3 | 99.3 | 98.9 | 2.4 |
| 3# | 2.7 | 91.2 | 99.1 | 3.6 |
| 4# | 3.0 | 80.9 | 98.8 | 0.9 |
| 5# | 3.0 | 80.1 | 99.5 | 1.0 |
| 6# | 2.7 | 82.2 | 98.5 | 1.5 |
| 7# | 2.0 | 81.4 | 98.2 | 1.1 |
| 8# | 3.6 | 60.0 | 99.6 | 1.0 |
| 9# | 2.7 | 93.3 | 99.2 | Less than 0.2 |
| 10# | 2.7 | 93.3 | 99.2 | Less than 0.2 |
| 11# | 0 | N/A | N/A | N/A |
| 12# | 2.2 | 99.5 | 92.4 | 3.1 |
| 13# | 4.1 | 90.6 | 98.6 | 9.7 |
| 14# | 4.1 | 93.8 | 98.7 | 8.9 |
| 15# | 4.2 | 92.5 | 98.7 | 7.4 |
Note that: N/A indicates inapplicability, and no sample was collected during the elution phase under the 11# test condition.
Example 10
(1) Preparation of cation sample
Preparation of cationic loading samples the cationic loading sample information is the same as in example 6: the concentration is 14g/L, the pH is 5.01, the concentration is 6.88mS/cm, and the purity is 97.6%.
(2) Cation exchange chromatography
With Capto S Impact packing, VL11 column, cv=21.52 mL; the pH value of the eluting buffer solution is 5.0-5.4, the conductivity of the eluting solution is 22.0-27.0 mS/cm, the flushing fluid is 150mmol/L acetic acid-sodium acetate (pH value is 5.0), and the four eluting solutions respectively correspond to test conditions 1# to 4#: (1) 500mmol/L acetic acid-sodium acetate (pH 5.0); (2) 475mmol/L sodium acetate-acetate (pH 5.0); (3) 500mmol/L acetic acid-sodium acetate (pH 5.2); (4) 525mmol/L acetic acid-sodium acetate (pH 5.4). Loading capacity is 80g/L, and the washing liquid and the eluent are eluted by adopting a linear gradient of 20-80% after loading is finished, and the eluting volume is 10CV. Test condition 1 collection conditions were the same as in example 6, and test conditions 2 to 4 were such that the initial collection point was an elution peak ultraviolet absorption value equal to 200mAU and the final collection point was an elution peak ultraviolet absorption value equal to 1200mAU.
(3) Detection analysis
The cationic analytical profile of test condition 1 is shown in FIG. 15, and the morphology of the falling portion of the elution peak differs from that of the cationic analytical profile 12 of example 7 in that the collected volume is large. The yields and purities of the cationic collection under the four test conditions are shown in Table 9, with the UV280 value at termination of collection set between 888-1340mAU being greater than 91.0% and the purity greater than 99.1%. When the UV280 collection was terminated at 1200mAU, comparison of the yields and purities of the four test conditions found that test condition # 2 was the best. The UV280 that terminated the collection stopped at 1300mAU with a yield of greater than 90.0% and a purity of greater than 99.0% for the four tests. By adopting the method of the embodiment, the parameters of the eluent are in a wider operation range (the conductivity range is 19.0-27.0 mS/cm, the pH value is 5.0-5.4), the yield and the purity of the collected liquid are higher, and the conductivity of the collected liquid is in the range of 13.5-15.5 mS/cm.
TABLE 9 yields and purity of cationic collection under four test conditions
Example 11
(1) Preparation of cation sample
Preparation of cationic loading samples the cationic loading sample information is the same as in example 6: the concentration was 10.1g/L, pH 5.05,3.58mS/cm, and the SEC-HPLC purity was 95.5%.
(2) Cation exchange chromatography
With Capto S Impact packing, VL11 column, cv=21.52 mL; equilibration buffer, wash buffer and eluent a:50mM acetic acid-sodium acetate, pH 4.93,2.57mS/cm; eluent B: (B1) 50mM acetic acid-sodium acetate, 500mM KCl, pH5.0, 60.0mS/cm; (B2) 50mM acetic acid-sodium acetate, 250mM Na 2 SO 4 pH5.0, 63.0mS/cm; cation exchange chromatography procedure: balance 5CV, load 80g/L, wash 3CV,0-100% B linear gradient elution 20CV, collect 200mAU-1200mAU fractions, regenerate 3CV, CIP 3CV.
(3) Detection analysis
The yields of the cationic collections using eluents B1 and B2 were 95.7% and 99.6%, respectively, and the SEC-HPLC purities were 96.8% and 96.0%, respectively. The above results show that the cation eluent component contains strong electrolyte such as KCl and Na 2 SO 4 When the salt linear gradient elution is adopted, the yield of the cation collecting liquid is higher, but the removal effect on the polymer is poor, similar to the result that the cation eluent component contains NaCl in the previous example.
Example 12
(1) Preparation of cation sample
Preparation of cationic loading samples the cationic loading sample information is the same as in example 6: the concentration was 8.2g/L, pH 4.95,6.61mS/cm, and the SEC-HPLC purity was 98.0%.
(2) Cation exchange chromatography
With Capto S Impact packing, VL11 column, cv=21.52 mL; experiment 1: equilibration buffer, wash buffer and eluent A1:60mM histidine hydrochloride, pH 4.96,4.77mS/cm; eluent B1:400mM histidine hydrochloride, pH 5.41, 20.5mS/cm; experiment 2: equilibration buffer, wash buffer and eluent A2:50mM citric acid-sodium citrate, pH5.0,6.83mS/cm; eluent B2:400mM citric acid-sodium citrate, pH5.0,6.83mS/cm; cation exchange chromatography procedure: balance 5CV, load 80g/L, wash 3CV,0-100% B linear gradient elution 20CV, collect 200mAU-1200mAU fractions, regenerate 3CV, CIP 3CV.
(3) Detection analysis
The yields of the cationic collections using eluents B1 and B2 were 93.5% and 91.8%, respectively, and the SEC-HPLC purities were 98.1% and 98.4%, respectively, with collection volumes of 2.2CV and 2.1CV, respectively. The results show that the cation eluent comprises weak acid salts such as 400mM histidine hydrochloride and sodium citrate, and the yield of the cation collecting liquid is higher when the salt is adopted for linear gradient elution, but the removal effect on the polymer is not obvious, and the effect can be related to higher purity of the sample. The elution volume was greater than that of the acetic acid-sodium acetate system (about 3 CV), indicating that the two linear gradient elution buffer systems used in this case had a stronger elution force, and that reducing the concentration of histidine hydrochloride and sodium citrate in the eluate or changing the collection conditions could increase the polymer removal capacity.
Claims (11)
1. A method for purifying an OX40/PD-L1 bispecific antibody using cation exchange chromatography, wherein the chromatography steps comprise equilibration, loading, washing and elution, characterized in that the cation eluent used in the chromatography process does not contain strong electrolyte components, and the cation washing liquid used has higher conductivity than the loaded sample;
salts used in the cation balance liquid, flushing liquid and eluent comprise weak acid salts and/or phosphate salts;
wherein the bispecific antibody comprises polypeptide chain 1: VH-CH1-CH2-CH 3-linker-VHH and polypeptide chain 2: VL-CL; and the bispecific antibody is a bispecific antibody that binds both PD-L1 and OX40 as disclosed in PCT/CN 2020/073959.
2. The method according to claim 1, wherein the filler for cation exchange chromatography is a strong cation exchanger containing a group selected from the group consisting of a sulfonic acid group, a sulfomethyl group, a sulfopropyl group and a phosphoric acid group or a weak cation exchanger containing an ion exchange group selected from the group consisting of a carboxymethyl group and a carboxyl group;
and/or, the cation exchange chromatography adopts a combination-elution mode;
and/or the cation balance liquid, the flushing liquid and the eluent are acetic acid-acetate buffer liquid, citric acid-citrate buffer liquid, phosphate buffer liquid or histidine salt buffer liquid;
And/or the elution is salt gradient elution or salt isocratic elution.
3. The method of claim 2, wherein the strong cation exchanger comprises sulfonic acid groups or sulfopropyl groups;
the cation balance liquid, the flushing liquid and the eluent are acetic acid-acetate buffer solution;
the salt gradient elution is salt linear gradient elution.
4. The method of claim 3, wherein none of the cationic equilibrium liquid, the rinse liquid, and the eluent liquid comprises a strong electrolyte component.
5. The method of claim 2, wherein the salt isocratic elution has an eluent conductivity of 12.55-17.6 mS/cm and a pH of 4.8-5.5;
the conductivity of the eluent of the salt linear gradient elution is 19.0-25.0 mS/cm, and the pH value is 5.0-5.4.
6. A method as defined in claim 5, wherein when said filler is Capto S Impact, said salt isocratic eluent has a conductivity in the range of 13.5 to 15.5mS/cm; when the filler is Poros 50HS, the conductivity of the salt isocratic elution eluent ranges from 12.55 mS/cm to 14.75mS/cm.
7. The method according to claim 1, wherein the pH of the rinse solution is 4.9 to 5.5; the conductivity is 3.0-10.5 mS/cm;
And/or the pH value of the balance buffer solution is 4.9-5.5; the conductivity is 3.0-10.5 mS/cm;
and/or the conductivity of the sample is 3.74-10.5 mS/cm.
8. The method according to claim 7, wherein the rinse solution has a pH of 5.0 and a conductivity of 5.7 to 10.5mS/cm;
the pH value of the balance buffer solution is 5.0, and the conductivity is 5.7-10.5 mS/cm;
the conductivity of the sample is 3.74-7.55 mS/cm.
9. The method of claim 8, wherein the rinse solution has a conductivity of 8.0mS/cm;
the conductivity of the equilibration buffer was 8.0mS/cm.
10. The method of claim 1, wherein the OX40/PD-L1 bispecific antibody is a fully human bispecific antibody obtained from CHO cell culture.
11. The method of claim 10, wherein the OX40/PD-L1 bispecific antibody is more hydrophobic than ipilimumab.
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