Method for detecting content of antibody protein in antibody coupling medicine

Abstract

The invention provides a method for detecting the content of antibody protein in an antibody coupling medicine. The detection method comprises the following steps: S1) decomposing a to-be-detected antibody-coupled drug to obtain a first fragment containing no toxin connexon and a second fragment connected with the toxin connexon; s2, the first fragment is subjected to chromatographic quantitative analysis, and the protein content is obtained.By means of the method, the problem that in the prior art, when the antibody protein content in the antibody coupling medicine is measured, the detection effect is poor can be solved, and the method is suitable for the field of antibody coupling medicine detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody drug conjugate detection, in particular, to a method for detecting the content of antibody protein in antibody drug conjugate. BACKGROUND

[0002] As a core index for evaluating the quality of antibody drug conjugate (ADC), the protein content is crucial for ensuring the potency and safety of ADC, and its determination is throughout the whole process of ADC from research and development to production, to product release detection and long-term stability research. The commonly used detection methods mainly include dual-wavelength ultraviolet absorption spectrophotometry (UV dual-wavelength method) and BCA method (Bicinchoninic Acid), although they are popular in the field of biopharmaceuticals, but these two methods have obvious defects when dealing with the complex structure and components of ADC.

[0003] The BCA method is based on the principle that the protein peptide bond reduces the divalent copper ion to monovalent copper ion, and the latter further reacts with the BCA reagent to generate a purple complex. The absorbance of the complex is positively correlated with the protein concentration, and the protein in the sample can be quantified by colorimetric analysis. However, when the toxin linker is coupled with the antibody, it may cause changes in the surface charge, hydrophobicity, and even the overall conformation of the antibody, which may affect the efficiency of the redox reaction of the antibody and the divalent copper. In addition, the metal coordination or reduction ability of the toxin molecule itself may interfere with the BCA color reaction, and even the potential interference of components such as excipients in the sample may cause significant measurement errors in the BCA method in ADC analysis. In addition, due to the complexity of the BCA reaction kinetics, it has considerable variability, which further increases the random error level of the method.

[0004] The UV dual-wavelength method has become a common tool for protein content detection due to its simple sample processing, low method variability, and high precision. ADC is composed of antibody protein and toxin linker, which are connected by covalent bond to form a complete molecule. Antibody and toxin linker exhibit different absorbance characteristics at specific ultraviolet wavelengths, following the Lambert-Beer Law, the absorbance is linearly related to the concentration of the substance. In theory, by selecting two specific wavelengths, the absorbance of ADC at the two wavelengths can be determined by UV absorption spectrum, and the concentration of antibody, i.e. the protein content of ADC, can be accurately calculated by combining linear equations.

[0005] However, the key to the effective implementation of the UV dual-wavelength method is to accurately obtain the extinction coefficients of the antibody and the toxin linker at two wavelengths. The extinction coefficient of the antibody can be obtained by theoretical prediction or actual measurement of a pure antibody standard, but the determination of the extinction coefficient of the toxin linker is difficult. First, if the purity of the toxin linker sample is not high, it will seriously affect the accuracy of the determination, which is particularly prominent in the early stages of product development. Second, the toxin linker may undergo changes in the ultraviolet absorption spectrum after conjugation, making the extinction coefficient of the unconjugated state no longer applicable. Finally, some ADC designs result in a high similarity between the UV absorption spectrum of the toxin linker and the antibody. Even if the extinction coefficient of the toxin linker can be measured, the dual-wavelength method may fail due to the close extinction coefficient ratio, thereby amplifying any small absorbance measurement error and severely distorting the determination results of the protein content.

[0006] In view of the above limitations of the BCA method and the UV dual-wavelength method, in actual operation, especially in the early stages of toxin linker development, or when the toxin linker has similar UV absorption characteristics to the antibody, these two methods often cannot provide accurate ADC protein quantification, prompting researchers to seek more complex, time-consuming, and less accurate alternatives, such as enzyme-linked immunosorbent assay (ELISA), biuret method, Folin phenol method, Coomassie brilliant blue method, and fluorescence method for indirect measurement. Therefore, there are many limitations in the existing technology for detecting the antibody protein content in antibody conjugate drugs, resulting in poor detection effect. SUMMARY

[0007] The main purpose of the present application is to provide a method for detecting the antibody protein content in antibody conjugate drugs, in order to solve the problem of poor detection effect in measuring the antibody protein content in antibody conjugate drugs in the prior art.

[0008] In order to achieve the above-mentioned purpose, according to the first aspect of the present application, a method for detecting the antibody protein content in antibody conjugate drugs is provided, which comprises: S1) decomposing the antibody conjugate drug to be measured to obtain a first fragment without a toxin linker and a second fragment with a toxin linker; S2) performing chromatographic quantitative analysis on the first fragment to obtain the protein content.

[0009] Further, the decomposition treatment comprises decomposing the antibody conjugate drug to be measured by using a protease and / or a reducing agent.

[0010] Further, the protease comprises one or more of IdeS protease, Lys-C protease, Trypsin protease, IgdE protease, or Papain protease.

[0011] Further, the reducing agent comprises one or more of DTT (dithiothreitol), TCEP (tris(2-carboxyethyl)phosphine hydrochloride) or beta-mercaptoethanol.

[0012] Further, the conjugation mode of the antibody conjugated drug comprises site-directed conjugation.

[0013] Further, the site-directed conjugation comprises cysteine conjugation, glycosylation site-directed conjugation or ThioMab site-directed conjugation.

[0014] Further, in the antibody conjugated drug, the toxin linker is connected to F(ab)2 or Fab, and the antibody conjugated drug is subjected to enzymatic cleavage by using a protease, and the first fragment obtained is Fc, and the second fragment is F(ab)2 or Fab connected to the toxin linker.

[0015] Further, in the antibody conjugated drug, the toxin linker is connected to Fc, and the antibody conjugated drug is subjected to enzymatic cleavage by using a protease, and the first fragment is F(ab)2 or Fab, and the second fragment is Fc connected to the toxin linker.

[0016] Further, in the antibody conjugated drug, the toxin linker is connected to the heavy chain, and the antibody conjugated drug is mixed with a reducing agent to react, so that the disulfide bond between the light chain and the heavy chain is reduced, so that the light chain and the heavy chain are dissociated, the first fragment is the light chain, and the second fragment is the heavy chain.

[0017] Further, in the antibody conjugated drug, the toxin linker is connected to the light chain, and the antibody conjugated drug is mixed with a reducing agent to react, so that the disulfide bond between the light chain and the heavy chain is reduced, so that the light chain and the heavy chain are dissociated, the first fragment is the heavy chain, and the second fragment is the light chain.

[0018] Further, the chromatographic quantitative analysis comprises reverse phase chromatography, hydrophobic chromatography, hydrophilic chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography or capillary electrophoresis.

[0019] By applying the technical scheme of the present application, the antibody conjugated drug to be detected is subjected to decomposition treatment, the first fragment without the toxin linker is separated from the second fragment connected to the toxin linker, and the chromatographic quantitative analysis is performed on the first fragment, so that the content of the antibody protein in the antibody conjugated drug to be detected can be obtained. Compared with the detection of the prior art, the detection method of the present application is simple in steps and relatively high in accuracy, which is conducive to obtaining the accurate protein content in the antibody conjugated drug and further evaluating the quality of the antibody conjugated drug, and promoting the pharmaceutical process of ADC. BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an exemplary embodiment of the application and, together with the description, serve to explain the application without imposing undue limitation thereon. In the drawings:

[0021] Figure 1 A schematic diagram showing the IdeS protease hydrolysis of the antibody and cysteine conjugated ADC molecule in the embodiments of the present application to form the F(ab)2 fragment containing the toxin linker and the Fc fragment not containing the toxin linker, wherein, Figure 1 A is the antibody enzymolysis structure schematic diagram in the embodiment of the present application, Figure 1 B is the ADC enzymolysis structure schematic diagram of the ADC molecule containing the toxin linker in the embodiment of the present application (the red pentagram represents the toxin linker).

[0022] Figure 2 A schematic diagram showing the IdeS protease hydrolysis of the glycosylation site conjugated or ThioMab site-specific conjugated ADC molecule in the embodiments of the present application to form the F(ab)2 fragment not containing the toxin linker and the Fc fragment containing the toxin linker (the red pentagram represents the toxin linker).

[0023] Figure 3 A schematic diagram showing the Papain protease hydrolysis of the antibody and glycosylation site conjugated or ThioMab site-specific conjugated ADC molecule in the embodiments of the present application to form the Fab fragment not containing the toxin linker and the Fc fragment containing the toxin linker; wherein, Figure 3 A is the antibody enzymolysis structure schematic diagram in the embodiment of the present application, Figure 3 B is the ADC enzymolysis structure schematic diagram of the ADC molecule containing the toxin linker in the embodiment of the present application (the red pentagram represents the toxin linker).

[0024] Figure 4 A schematic diagram showing the DTT (dithiothreitol) reduction of the antibody and glycosylation site conjugated or ThioMab site-specific conjugated ADC molecule in the embodiments of the present application to form the light chain (LC) not containing the toxin linker and the heavy chain (HC) containing the toxin linker, wherein, Figure 4 A is the antibody enzymolysis structure schematic diagram in the embodiment of the present application, Figure 4 B is the ADC enzymolysis structure schematic diagram of the ADC molecule containing the toxin linker in the embodiment of the present application (the red pentagram represents the toxin linker).

[0025] Figure 5 A Protein A affinity chromatogram of the enzymatic antibody standard working solution in Embodiment 1 of the present application is shown, wherein, Figure 5 A is the global graph of the affinity chromatogram, Figure 5 B is the local magnification graph of the affinity chromatogram.

[0026] Figure 6 The enzyme-digested antibody standard working curve graph in the embodiment 1 of the present application is shown.

[0027] Figure 7 The Protein A affinity chromatogram of the three parallel samples of the enzyme-digested ADC solution in the embodiment 1 of the present application is shown, wherein, Figure 7 A is the global graph of the affinity chromatogram, Figure 7 B is the local enlarged graph of the affinity chromatogram.

[0028] Figure 8 The Protein A affinity chromatogram of the enzyme-digested antibody standard working solution in the embodiment 2 of the present application is shown, wherein, Figure 8 A is the global graph of the affinity chromatogram, Figure 8 B is the local enlarged graph of the affinity chromatogram.

[0029] Figure 9 The enzyme-digested antibody standard working curve graph in the embodiment 2 of the present application is shown.

[0030] Figure 10 The Protein A affinity chromatogram of the three parallel samples of the enzyme-digested ADC solution in the embodiment 2 of the present application is shown, wherein, Figure 10 A is the global graph of the affinity chromatogram, Figure 10 B is the local enlarged graph of the affinity chromatogram. DETAILED DESCRIPTION

[0031] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the embodiments.

[0032] Explanation of terms:

[0033] Fc (Fragment crystallizable): refers to the antibody crystallizable fragment, which is located in the constant region of the heavy chain and mediates the effector function and half-life.

[0034] Fab (Fragment antigen-binding): refers to the antigen-binding fragment, which is composed of the light chain and the heavy chain variable region and part of the constant region.

[0035] Light chain (LC): antibody small molecular peptide chain.

[0036] Heavy chain (HC): antibody large molecular peptide chain.

[0037] As mentioned in the background, the antibody protein content detection effect in the prior art antibody conjugated drugs is poor, based on which, the inventors try to develop a new antibody protein content detection method in the antibody conjugated drugs in the present application, and thus a series of protection schemes of the present application are proposed.

[0038] In the first typical embodiment of the present application, a method for detecting the antibody protein content in an antibody conjugated drug is provided, which comprises: S1) decomposing the antibody conjugated drug to be detected to obtain a first fragment without a toxin linker and a second part connected to the toxin linker; S2) performing chromatographic quantitative analysis on the first fragment to obtain the protein content.

[0039] The ADC product has two main components, namely the antibody protein and the toxin linker, which are connected (i.e. conjugated) by a covalent bond. The most common methods for measuring the protein content of ADCs are UV double wavelength method and BCA method. The traditional UV double wavelength detection method needs to accurately determine the extinction coefficient of the toxin linker, and the BCA method needs to quantify the protein by BCA reaction, which has many factors affecting the accuracy and reliability of the method, increasing the systematic error and / or random error of the analysis method.

[0040] The difficulty of the UV double wavelength method for determining the protein content of ADC is how to accurately obtain the extinction coefficient of the toxin linker conjugated to the antibody. The traditional method is to use a standard solution of high-purity free toxin linker (i.e. unconjugated toxin linker) to determine the extinction coefficient. This method is often affected by the following factors, resulting in failure to obtain the protein content of ADC:

[0041] 1) The purity of the toxin linker sample is low, containing other impurities that can produce ultraviolet absorption, and high-purity standard cannot be obtained. Since the traditional UV double wavelength method does not have the ability to distinguish impurities, part of the absorbance value measured is contributed by impurities with different absorption behavior, thus leading to inaccurate determination of the extinction coefficient of the toxin linker. This situation often occurs during the early research and development stage of the toxin linker, when the synthesis path and purification process are relatively immature. The occurrence of this situation leads to low accuracy of the protein content measured by the UV double wavelength method, directly affecting the progress of subsequent product and process development (such as calculation of target product yield, design of sample purification process, optimization of conjugation process parameters, etc.), and greatly affecting the results of other analysis and detection (such as activity detection) that rely on the key parameter of protein content.

[0042] 2) Some toxin linker contains specific functional groups. After conjugation with antibody, the molecular structure changes, which may cause significant changes in the UV absorption spectrum compared with the un-conjugated (free) molecule spectrum. In this case, the extinction coefficient of the un-conjugated toxin linker standard solution cannot accurately reflect the UV absorption characteristics of the conjugated toxin linker. Therefore, the extinction coefficient calculated using the free molecule in the early stage will produce large errors when applied to the measurement of ADC protein content.

[0043] 3) The design of some ADC molecules causes the UV spectrum of the toxin linker to be very similar to the UV spectrum of the antibody. In this case, even if the extinction coefficient of the toxin linker at the detection wavelength can be accurately determined, the UV dual-wavelength method will introduce large detection errors or even be unsuitable. The calculation formula of the UV dual-wavelength method for measuring protein content is derived as follows:

[0044] (Formula 1);

[0045] ;

[0046] (Formula 2).

[0047] Wherein:

[0048] C mAb : protein content of ADC sample;

[0049] ε: extinction coefficient;

[0050] w1: wavelength 1;

[0051] w2: wavelength 2;

[0052] L: optical path;

[0053] A: absorbance;

[0054] ADC: antibody-drug conjugate;

[0055] PL: toxin linker;

[0056] DF: dilution factor;

[0057] k1: the ratio of the extinction coefficient of the toxin linker at wavelength 2 to the extinction coefficient at wavelength 1;

[0058] k2: the ratio of the extinction coefficient of the antibody at wavelength 2 to the extinction coefficient at wavelength 1.

[0059] From the above converted formula, if the UV spectrum of the toxin linker is similar to that of the antibody, the extinction coefficient ratio of the toxin linker and the antibody at two wavelengths (k1 and k2) will be similar, the difference between the two ratios (k2-k1) will be very small or even close to 0, and the denominator (L x (k2-k1) x w1 mAb ) in formula 2 will also be close to 0, which will cause the deviation of the absorbance of the ADC measured at two wavelengths to be amplified, thereby affecting the final determination of the protein content.

[0060] Due to the above three situations, in actual work, the UV dual-wavelength method cannot be used to accurately determine the protein content of ADC drugs in many cases, and is abandoned and replaced by other methods such as BCA. These methods have complex sample pretreatment, long detection time, large method variability, and poor accuracy and precision.

[0061] When the protein content of ADC is determined by BCA method, a standard curve is generally prepared using the corresponding antibody reference substance, and the protein content is determined by external standard method. After the toxin linker is coupled to the antibody molecule, it may affect the surface charge and its distribution of the antibody, the hydrophobicity of the antibody surface, or cause changes in the spatial structure of the antibody, which may affect the redox reaction of the antibody with divalent copper ions; in addition, if the toxin molecule itself has metal coordination ability or reducing ability, it may interfere with the color reaction of monovalent copper ions with BCA reagent. If the test solution contains substances that can coordinate with copper ions or have reducing properties (such as excipients), it will also interfere with the BCA reaction. These factors may cause large measurement errors. In addition, since the BCA method involves complex redox and color reactions, such chemical reactions are essentially a kinetic analysis method, and the characteristics of kinetic methods are large variability, which makes it difficult for the BCA method to reach a steady state, thereby causing large random detection errors.

[0062] The present application can eliminate the influence of the toxin linker on the determination of the protein content of ADC by decomposing the ADC into different fragments (subunit units) such as F(ab)2, Fab, Fc, heavy chain or light chain fragments, and further selecting fragments without toxin linkers for chromatographic quantitative analysis, thereby obtaining accurate and reliable ADC protein content results through chromatographic analysis.

[0063] The detection method of the present application eliminates the reliance on the extinction coefficient of the toxin linker in the prior art in principle, is not affected by the toxin linker, and therefore completely eliminates the adverse effects of the toxin linker on the traditional UV double-wavelength method and the BCA method. The method creatively integrates ADC decomposition processing and chromatographic quantitative analysis, realizes the determination of the protein content of ADC directly through the standard working curve of the antibody, simplifies the detection process, saves the detection time, and can be applied to ADC products with different DAR values. In addition, the detection method of the present application can also eliminate the interference of other substances in the sample (such as excipients, free toxin impurities, etc.), improve the accuracy and reliability of the detection results, and has wide applicability. Compared with the detection method of the prior art, it is more suitable for the promotion of clinical samples.

[0064] In a preferred embodiment, the decomposition processing includes decomposing the antibody-conjugated drug to be tested by using a protease and / or a reducing agent.

[0065] In a preferred embodiment, the protease includes one or more of IdeS protease, Lys-C protease, Trypsin protease, IgdE protease, or Papain protease.

[0066] The protease in the present application includes a protease that hydrolytically cleaves the peptide bond in the hinge region of the antibody, which includes but is not limited to one or more of IdeS protease, Lys-C protease, Trypsin protease, IgdE protease, or Papain protease.

[0067] IdeS protease can cleave two heavy chains at a single site below the disulfide bond in the hinge region of ADC (or IgG), and the product is F(ab)2 (containing an intact light chain and a partial heavy chain) and an Fc fragment. Further, a first fragment connected without a toxin linker can be obtained, and the protein content of the ADC can be obtained by chromatographic quantitative analysis of the fragment.

[0068] The action sites of IgdE protease and Papain protease are slightly different from IdeS. Papain enzyme cleaves the peptide bond at a position near the Fab region in the hinge region. For some types of ADC, especially ADCs with toxins concentrated in the Fab region or the Fc region, the Fc or Fab fragment (first fragment) without a toxin linker can be separated and analyzed after enzymolysis with Papain, and the protein content of the ADC can also be detected.

[0069] Lys-C protease and Trypsin protease can cleave ADC into smaller peptides, and a suitable peptide segment without a toxin linker can be selected as a first fragment for protein content detection.

[0070] The skilled person can select a suitable protease to hydrolyze the peptide bond in the ADC region according to the structure of the actual ADC molecule and actual needs, as long as the ADC molecule can be decomposed to obtain the first fragment without the toxin linker, which can be subjected to chromatographic quantitative analysis and further obtain the protein content.

[0071] In a preferred embodiment, the reducing agent includes DTT (dithiothreitol), TCEP (tris (2-carboxyethyl) phosphine hydrochloride), or β-mercaptoethanol.

[0072] In addition to the enzymatic method, the ADC is decomposed by a reducing agent, including but not limited to DTT (dithiothreitol), TCEP (tris (2-carboxyethyl) phosphine hydrochloride), or β-mercaptoethanol. The function of the reducing agent is to reduce the disulfide bond between the light chain and the heavy chain and the disulfide bond between the heavy chains to free thiol groups, so as to completely separate the light chain and the heavy chain of the antibody, and then the first fragment without the toxin linker and the second fragment with the toxin linker can be obtained, and the first fragment is selected to quantitatively detect the protein content of the ADC.

[0073] The skilled person can flexibly select IdeS, Papain or other proteases that can effectively decompose the ADC molecule according to the specific structure and coupling mode of the ADC, or select DTT (dithiothreitol), TCEP (tris (2-carboxyethyl) phosphine hydrochloride), β-mercaptoethanol and other reducing agents to separate the heavy chain and the light chain, so as to achieve the most suitable protein fragment separation effect. When the antibody fragment without the toxin linker is successfully separated, a standard working curve can be established by chromatographic quantitative analysis means, and then the antibody protein content of the ADC can be more accurately determined.

[0074] In a preferred embodiment, the coupling mode of the antibody-drug conjugate includes site-specific coupling.

[0075] In a preferred embodiment, the site-specific coupling includes cysteine coupling, glycosylation site-specific coupling, or ThioMab site-specific coupling. The present application can select a suitable method to "decompose" the ADC according to the coupling mode of the ADC to obtain the first fragment without the toxin linker, and then the protein content is subjected to chromatographic quantitative analysis.

[0076] In the site-specific conjugation of ADC, the most common site-specific conjugation is cysteine conjugation, i.e. the toxin linker is conjugated to the free thiol group of the reduced antibody molecule. Cysteine conjugation refers to the reduction of the inter-chain disulfide bond in the antibody molecule into free thiol groups, which are conjugated to the toxin linker containing active groups to achieve site-specific loading of the toxin. This process usually occurs at the disulfide bond site between the light chain and the heavy chain and the disulfide bond site between the hinge region of the heavy chain and the heavy chain of the antibody, so that the toxin linker is only combined with the Fab fragment of the antibody, and the Fc fragment has no toxin linker.

[0077] Site-specific conjugation of glycosylation sites refers to specific site-specific conjugation of drugs through glycosylation modification sites of antibodies, using glycosylation sites in the Fc region of the antibody as the conjugation site for the binding of the toxin linker. The glycosylation on the antibody is removed or modified by glycoengineering, and then the toxin linker is chemically conjugated to the exposed glycosylation site. In this case, the toxin linker is only combined with the Fc fragment of the antibody, and the Fab fragment has no toxin linker.

[0078] ThioMab site-specific conjugation refers to a method for synthesizing ADC by introducing an engineered cysteine site in the antibody to achieve specific conjugation at the introduced site. This technology usually involves genetic engineering of the antibody to ensure site-specific and specific binding of the toxin linker, while maintaining the functional integrity of the Fc end of the antibody. Generally, the artificially introduced cysteine is located on the Fc fragment, and in this case, the toxin linker is only combined with the Fc fragment of the antibody, and the Fab fragment has no toxin linker.

[0079] In the detection method of the present application, as long as the antibody fragment (i.e. the first fragment as described above, such as the Fc fragment in cysteine conjugation ADC, the F(ab)2 fragment, Fab fragment or light chain in glycosylation site conjugation or ThioMab site-specific conjugation ADC) without toxin linker can be separated by enzymatic digestion or reduction, the ADC protein content can be accurately determined using these fragments. By using protease or reducing agent, ADC can be specifically decomposed to produce antibody fragments without toxin linker, which provides the possibility for subsequent chromatographic separation and quantitative analysis. The detection method of the present application not only overcomes the limitations of traditional UV dual-wavelength method and BCA method, but also shows wide applicability and flexibility, which is suitable for various types of ADC molecular structures, and provides technical support for early research and quality control of ADC drugs.

[0080] In a preferred embodiment, in the antibody-drug conjugate, the toxin linker is connected to F(ab)2 or Fab, and the peptide bond of the antibody-drug conjugate is digested by protease to obtain a first fragment Fc and a second fragment F(ab)2 or Fab.

[0081] In a preferred embodiment, in the antibody drug conjugate, the toxin linker is connected to the Fc, and the peptide bond of the antibody drug conjugate is cleaved by the protease, the first fragment is F(ab)2or Fab, and the second fragment is the Fc connected with the toxin linker.

[0082] In a preferred embodiment, in the antibody drug conjugate, the toxin linker is connected to the heavy chain, and the disulfide bond between the light chain and the heavy chain is reduced by mixing the antibody drug conjugate with a reducing agent, so that the light chain and the heavy chain are dissociated, the first fragment is the light chain, and the second fragment is the heavy chain.

[0083] In a preferred embodiment, in the antibody drug conjugate, the toxin linker is connected to the light chain, and the disulfide bond between the light chain and the heavy chain is reduced by mixing the antibody drug conjugate with a reducing agent, so that the light chain and the heavy chain are dissociated, the first fragment is the heavy chain, and the second fragment is the light chain.

[0084] For the ADCs with the Fc region as the site for conjugation, when the toxin linker is site-specifically conjugated to the Fc region of the antibody, the ADC is decomposed into the first fragment, i.e. the F(ab)2or Fab fragment without the toxin linker, and the second fragment, i.e. the Fc fragment with the toxin linker, by hydrolysis treatment with the above-mentioned protease. This separation strategy can ensure that the obtained F(ab)2or Fab fragment only contains the antibody protein fragment, which enters the subsequent chromatographic analysis, so as to accurately determine the protein content in the ADC. Similar to the cysteine conjugation method, the ADCs with the Fc region as the site for conjugation can also use the method of the present application, and the measurement error caused by the toxin linker can also be effectively avoided.

[0085] When the ADC is treated with a reducing agent, the interchain disulfide bond of the ADC is reduced by the reducing agent (such as DTT, TCEP or β-mercaptoethanol, etc.), and the antibody molecule can be completely decomposed into its constituent light chain and heavy chain. After this treatment, the first fragment without the toxin linker can be obtained.

[0086] Taking the IdeS enzyme-cleaved ADC conjugated by cysteine as an example, as shown in Figure 1 , wherein, Figure 1 , A is the schematic diagram of the IdeS enzyme-cleaved antibody, Figure 1Figure 8 shows a schematic diagram of IdeS enzyme cutting cysteine-conjugated ADCs, where the toxin linker is mainly connected to the Fab or F(ab)2 region of the antibody. Using IdeS to hydrolyze the hinge region of the ADC can accurately decompose the intact antibody molecule into two main parts. The first fragment is the Fc fragment without the toxin linker, which completely retains the Fc region of the antibody and is not affected by the toxin linker, and can be used for subsequent protein content quantification. The second structure is the F(ab)2 or Fab fragment carrying the toxin linker, which contains the antigen binding site of the antibody and the conjugated toxin. Due to the presence of the toxin linker, this part cannot be used for protein content analysis.

[0087] For example, as shown in Figure 9, IdeS protease is used to decompose glycosylation site-conjugated or ThioMab site-specific conjugated ADCs into F(ab)2 and Fc fragments connected by a toxin linker. At this time, the F(ab)2 fragment can be quantified to determine the protein content of the ADC. Figure 2 For example, as shown in Figure 9, IdeS protease is used to decompose glycosylation site-conjugated or ThioMab site-specific conjugated ADCs into F(ab)2 and Fc fragments connected by a toxin linker. At this time, the F(ab)2 fragment can be quantified to determine the protein content of the ADC.

[0088] For example, as shown in Figure 10, Papain protease is used to decompose glycosylation site-conjugated or ThioMab site-specific conjugated ADCs into Fab and Fc fragments connected by a toxin linker. At this time, the Fab fragment without the toxin linker can be quantified to determine the protein content of the ADC. Figure 3 For example, as shown in Figure 10, Papain protease is used to decompose glycosylation site-conjugated or ThioMab site-specific conjugated ADCs into Fab and Fc fragments connected by a toxin linker. At this time, the Fab fragment without the toxin linker can be quantified to determine the protein content of the ADC. Figure 3 For example, as shown in Figure 10, Papain protease is used to decompose glycosylation site-conjugated or ThioMab site-specific conjugated ADCs into Fab and Fc fragments connected by a toxin linker. At this time, the Fab fragment without the toxin linker can be quantified to determine the protein content of the ADC. Figure 3 For example, as shown in Figure 10, Papain protease is used to decompose glycosylation site-conjugated or ThioMab site-specific conjugated ADCs into Fab and Fc fragments connected by a toxin linker. At this time, the Fab fragment without the toxin linker can be quantified to determine the protein content of the ADC.

[0089] For example, as shown in Figure 11, a reducing agent is used to reduce the interchain disulfide bonds of glycosylation site-conjugated or ThioMab site-specific conjugated ADCs, decomposing them into free light chains and heavy chains. After reduction, the light chain without the toxin linker can be quantified by chromatography to determine the protein content of the ADC. Figure 4 For example, as shown in Figure 11, a reducing agent is used to reduce the interchain disulfide bonds of glycosylation site-conjugated or ThioMab site-specific conjugated ADCs, decomposing them into free light chains and heavy chains. After reduction, the light chain without the toxin linker can be quantified by chromatography to determine the protein content of the ADC. Figure 4 For example, as shown in Figure 11, a reducing agent is used to reduce the interchain disulfide bonds of glycosylation site-conjugated or ThioMab site-specific conjugated ADCs, decomposing them into free light chains and heavy chains. After reduction, the light chain without the toxin linker can be quantified by chromatography to determine the protein content of the ADC. Figure 4 For example, as shown in Figure 11, a reducing agent is used to reduce the interchain disulfide bonds of glycosylation site-conjugated or ThioMab site-specific conjugated ADCs, decomposing them into free light chains and heavy chains. After reduction, the light chain without the toxin linker can be quantified by chromatography to determine the protein content of the ADC.

[0090] The skilled in the art can combine the enzymatic treatment with the application of reducing agent according to the different structures of ADC to adapt to the more complex and variable characteristics of ADC structure. For example, the ADC can be first cut into F(ab)2 and Fc fragments using IdeS protease, and then further treated by reducing agent to ensure that the antibody fragment (first fragment) without toxin linker is obtained. Such a combination treatment strategy can further expand the application range of the detection method of ADC antibody protein of the present application, and can further improve the accuracy and reliability of detection.

[0091] The skilled in the art can flexibly select one or more of the above processing steps according to the actual structure and coupling mode of ADC to achieve the best antibody fragment separation effect and provide a solid foundation for the protein content determination of ADC. Whether it is a site-specific coupling or a non-site-specific coupling ADC, the detection method of the present application shows its wide application prospect and adaptability, and provides strong support for the early research and development, process development and quality control of ADC drugs.

[0092] In the detection method of the present application, regardless of the coupling mode of ADC, if the antibody fragment without toxin linker (i.e. the first fragment) can be cleaved by the preferred enzymatic or reduction step, further chromatographic separation and quantitative analysis can be carried out, and the standard working curve established by the antibody reference can be used to calculate the protein content of ADC. The detection method of the present application not only overcomes the limitations of traditional UV double-wavelength method and BCA method, but also provides a more universal, efficient and accurate detection method for the early research and development, process development and quality control of ADC.

[0093] In a preferred embodiment, the reaction ratio of the antibody conjugate drug to be tested and the protease is preferably 100 μg of antibody conjugate drug to 100 units of protease, or a suitable reaction ratio according to the instructions for use of the protease, and the skilled in the art can flexibly adjust the reaction ratio according to the actual situation.

[0094] In a preferred embodiment, the reaction ratio of the antibody conjugate drug to be tested and the reducing agent is preferably 100 μg of antibody conjugate drug to 20 mmol of reducing agent, and the skilled in the art can adjust the amount of reducing agent used according to the actual situation of the reaction system.

[0095] According to the structure of the ADC molecule and the coupling method, a suitable enzyme (such as IdeS protease, Lys-C protease, Trypsin protease, IgdE protease, papain protease, etc.) is selected to perform enzymatic digestion on the ADC sample in a buffer system to obtain different antibody fragments (i.e., the first fragment and the second fragment mentioned above), such as F(ab)2, Fab, Fc, etc.; or the ADC is reduced by a reducing agent to obtain free light chains (LC) and heavy chains (HC), obtain an antibody fragment without a toxin linker, and further perform chromatographic quantitative analysis, so as to obtain the protein content in the ADC sample. The process of enzymatic digestion or reduction of the ADC sample requires precise control of the volume removed and accurate calculation of the dilution factor in the pre-treatment process of the ADC sample to avoid errors in subsequent detection.

[0096] In the above manner, the antibody reference sample corresponding to the ADC sample to be tested is subjected to complete sample pre-treatment. The antibody reference sample pre-treatment process also requires precise control of the volume removed and accurate calculation of the dilution factor in the pre-treatment process of the antibody reference sample and the accurate concentration after pre-treatment. The concentration of the antibody reference sample itself can be determined by the traditional UV280 method.

[0097] In a preferred embodiment, the chromatographic quantitative analysis includes reverse phase chromatography, hydrophobic chromatography, hydrophilic chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, or capillary electrophoresis.

[0098] After obtaining the antibody fragment without a toxin linker (the first fragment) by the above-mentioned enzymatic digestion or reduction method, the antibody fragment can be separated and quantified by chromatography, including but not limited to reverse phase chromatography (RP), hydrophobic chromatography (HIC), hydrophilic chromatography (HILIC), size exclusion chromatography (SEC), ion exchange chromatography (IEC), affinity chromatography (such as Protein A, Protein L chromatography, etc.), and capillary electrophoresis (such as CE-SDS, icIEF, CZE, etc.). In the chromatographic analysis, the antibody fragment without a toxin linker is separated from other fragments, and then the chromatographic quantitative analysis is performed by the external standard curve of the antibody reference sample corresponding to the ADC, so as to obtain accurate protein content results.

[0099] A person skilled in the art can select a suitable chromatographic analysis method according to actual needs, integrate the chromatographic peak of the antibody fragment without a toxin linker, take the protein concentration of the antibody reference sample as the abscissa and the chromatographic peak area of the antibody fragment without a toxin linker (the first fragment) as the ordinate, draw a standard working curve of the antibody reference sample, and fit a linear equation by the least squares method.

[0100] The enzyme-digested (or reduced) ADC sample solution is injected and analyzed in the same chromatographic analysis sequence. The chromatographic peak of the antibody fragment (first fragment) without toxin linker is integrated, and the peak area is substituted into the linear equation of the antibody standard working curve to calculate the protein concentration. After multiplying by the dilution factor of the ADC sample pretreatment, the protein content of the ADC sample is obtained.

[0101] The fragments obtained after enzyme digestion or reduction of the ADC sample to be tested are subjected to chromatographic analysis. The peak areas before and after sample treatment are compared to determine the degree of reaction completion. If the ADC chromatogram after the reaction has no obvious observable peak at the retention time of the main peak of the ADC chromatogram before the reaction, or the peak area does not exceed 5% of the main peak area of ​​the ADC before the reaction, it indicates that the ADC enzyme digestion or reduction reaction is close to complete. If the antibody chromatogram after the reaction has no obvious peak at the retention time of the main peak of the antibody chromatogram before the reaction, or the peak area does not exceed 5% of the main peak area of ​​the antibody before the reaction, it indicates that the antibody enzyme digestion or reduction reaction is close to complete. If the degree of enzyme digestion or reduction reaction is insufficient, such as less than 95% completion, it may lead to a large detection error, and the enzyme digestion or reduction reaction conditions need to be optimized.

[0102] Optimization of enzyme digestion or reduction reaction conditions includes, but is not limited to, optimizing the amount of enzyme or reducing agent, the temperature and time of the enzyme digestion or reduction reaction, the pH value of the reaction system, etc. Those skilled in the art can optimize the system according to conventional reaction principles to improve the efficiency of the reaction, and this application does not impose any restrictions.

[0103] Those skilled in the art can also use the method of this application to obtain the ADC protein content according to actual needs, and based on this content value, further determine the extinction coefficient of the ADC at a selected wavelength, and then subsequently determine the ADC protein content rapidly by UV single-wavelength method. For example, the absorbance of the ADC sample at the detection wavelength (e.g., 280 nm) can be measured, and the ADC protein content can be calculated by the extinction coefficient and Lambert-Beer law, which can also shorten the overall detection process and time compared with the prior art.

[0104] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0105] In this application embodiment, two ADC platform molecules from Shanghai Kailaiying Biotechnology Co., Ltd. were selected for experimental confirmation. The results show that the method of this application can accurately detect the protein content of these two ADCs.

[0106] The two ADC platform molecules are anti-CD19-vc-MMAE ADC and anti-Her2-DxD ADC. Both molecules are typical cysteine-coupled, but their target DAR values ​​differ: anti-CD19-vc-MMAE ADC is a DAR4 product, and anti-Her2-DxD ADC is a DAR8 product. Since the toxin linkers are all coupled to the antibody's Fab fragment, the ADCs were cleaved into F(ab)2 and Fc fragments using the IdeS enzyme. All toxin linkers were distributed in the F(ab)2 fragment, while the Fc fragment contained no toxin linkers. Therefore, the ADC protein content was determined using the Fc fragment.

[0107] Example 1: Detection of protein content of anti-CD19-vc-MMAEADC

[0108] Based on the concentration of anti-CD19 antibody, take an appropriate amount of antibody solution (containing approximately 100 µg of antibody), add 100 units of IdeS protease (manufacturer: Promega; catalog number: V751A) and an appropriate amount of 50 mM Tris (pH 7.0) buffer to a total volume of 100 µL, 37 o Incubate at C temperature for 30 minutes.

[0109] Based on the concentration of anti-CD19 antibody, take the same amount of antibody solution (containing approximately 100 µg of antibody), add an appropriate amount of 50 mM Tris (pH 7.0) buffer to a total volume of 100 µL, mix well, and use as undigested antibody control-1.

[0110] The undigested antibody control-1 solution was diluted 100 times with 50 mM Tris (pH 7.0) to serve as undigested antibody control-2. The extent of the enzyme digestion reaction can be determined by comparing the antibody chromatographic peak areas of the digested antibody and the undigested antibody control-2.

[0111] Based on the estimated concentration of anti-CD19-vc-MMAE ADC, take an appropriate amount of ADC solution (containing approximately 100 µg of ADC), add 100 units of IdeS protease and an appropriate amount of 50 mM Tris (pH 7.0) buffer to a total volume of 100 µL, and 37 o Incubate at C temperature for 30 minutes.

[0112] Based on the estimated concentration of anti-CD19-vc-MMAE ADC, take the same amount of ADC solution (containing approximately 100 µg of ADC) and add an appropriate amount of 50 mM Tris (pH 7.0) buffer to a total volume of 100 µL as the undigested ADC control-1.

[0113] The undigested ADC control-1 solution was diluted 100 times with 50 mM Tris (pH 7.0) to serve as undigested ADC control-2. The extent of the enzyme digestion reaction can be determined by comparing the ADC chromatographic peak areas of the enzyme-digested ADC and the undigested ADC control-2.

[0114] Size exclusion chromatography (SEC) was used to analyze undigested antibody control-1, undigested antibody control-2, digested antibody, undigested ADC control-1, undigested ADC control-2, and digested ADC solution under the following mobile phase conditions (100 mM PB-100 mM NaCl, pH 6.8, 7% IPA). The SEC chromatograms of antibodies and ADCs were integrated. The antibody peak of undigested antibody control-2 was detectable. The peak area of ​​the digested antibody was similar to that of undigested antibody control-2, indicating that the antibody digestion reaction was essentially complete (approximately 99%). The ADC peak of undigested ADC control-2 was also detectable. The peak area of ​​the digested ADC sample was comparable to that of undigested ADC control-2, indicating that the ADC digestion reaction was essentially complete (approximately 99%).

[0115] Protein A affinity columns can specifically bind to the Fc terminus of antibodies. In this experiment, a Protein A affinity column (2.1 × 30 mm, 20 μm) was used to quantitatively analyze the Fc fragment (first fragment) without toxin linkers. The main chromatographic conditions (e.g., mobile phase composition, detection wavelength and elution gradient) are shown in Table 1.

[0116] Table 1: Affinity Chromatography Conditions for Protein A

[0117]

[0118] Protein A affinity chromatography was performed using the enzyme-digested antibody solution as an external standard. The chromatogram is shown below. Figure 5 As shown, where, Figure 5 In the middle, A is the global map of the affinity chromatogram. Figure 5 B is a magnified view of a portion of the affinity chromatogram. Figure 5 The horizontal axis "Minutes" refers to minutes; the other figures are explained similarly here. The chromatographic peaks of the Fc fragment without the toxin linker were integrated, with the concentration of the enzyme-digested antibody on the horizontal axis and the peak area of ​​the Fc fragment on the vertical axis. A least-squares linear fit was used to obtain the linear equation for the standard working curve, as shown below. Figure 6 As shown. The correlation coefficient R of the linear fit. 2 A value of 1.0000 indicates good linearity.

[0119] The enzyme-digested ADC solution (three parallel samples were prepared) was used as the test sample for Protein A affinity chromatography analysis. The chromatogram is shown below. Figure 7 As shown, where, Figure 7 In the middle, A is the global map of the affinity chromatogram. Figure 7 Image B is a magnified view of the affinity chromatogram, showing good repeatability. The Fc fragment peak was integrated, and the peak area was substituted into the linear equation of the standard working curve to calculate the concentration, which is the protein concentration of the ADC injection solution. Multiplying this concentration by the dilution factor of the ADC sample pretreatment yields the protein content of the ADC sample. Using this invention, the protein content of the anti-CD19-vc-MMAE ADC sample was determined to be 17.4 mg / mL, while the protein content determined using the traditional UV dual-wavelength method was 16.5 mg / mL. The difference between the two results is approximately 5%, indicating good consistency.

[0120] Example 2: Detection of protein content in anti-Her2-DxD ADC

[0121] Based on the concentration of anti-Her2 antibody, take an appropriate amount of antibody solution (containing approximately 100 µg of antibody), add 100 units of IdeS protease (manufacturer: Promega; catalog number: V751A) and an appropriate amount of 50 mM Tris (pH 7.0) buffer to a total volume of 100 µL, 37 o Incubate at C temperature for 30 minutes.

[0122] Based on the concentration of anti-Her2 antibody, take the same amount of antibody solution (containing approximately 100 µg of antibody), add an appropriate amount of 50 mM Tris (pH 7.0) buffer to a total volume of 100 µL, mix well, and use as undigested antibody control-1.

[0123] The undigested antibody control-1 solution was diluted 100 times with 50 mM Tris (pH 7.0) to serve as undigested antibody control-2. The extent of the enzyme digestion reaction can be determined by comparing the antibody chromatographic peak areas of the digested antibody and the undigested antibody control-2.

[0124] Based on the estimated concentration of anti-Her2-DxD ADC, take an appropriate amount of ADC solution (containing approximately 100 µg of ADC), add 100 units of IdeS protease and an appropriate amount of 50 mM Tris (pH 7.0) buffer to a total volume of 100 µL, and 37 o Incubate at C temperature for 30 minutes.

[0125] Based on the estimated concentration of anti-Her2-DxD ADC, take the same amount of ADC solution (containing approximately 100 µg of ADC) and add an appropriate amount of 50 mM Tris (pH 7.0) buffer to a total volume of 100 µL as the undigested ADC control-1.

[0126] The undigested ADC control-1 solution was diluted 100 times with 50 mM Tris (pH 7.0) to serve as undigested ADC control-2. The extent of the enzyme digestion reaction can be determined by comparing the ADC chromatographic peak areas of the enzyme-digested ADC and the undigested ADC control-2.

[0127] SEC (size exclusion chromatography) was used to analyze undigested antibody control-1, undigested antibody control-2, digested antibody, undigested ADC control-1, undigested ADC control-2, and digested ADC solution under the following mobile phase conditions (100 mM PB-100 mM NaCl, pH 6.8, 7% IPA). After peak integration, the antibody peak of undigested antibody control-2 was detectable, and the antibody peak of the digested antibody was smaller than that of undigested antibody control-2, indicating that the antibody digestion reaction was essentially complete (reaction degree >99%). The ADC peak of undigested ADC control-2 was detectable, while the antibody peak of the digested ADC was not detected, indicating that the ADC digestion reaction was nearly complete (reaction degree approximately 100%).

[0128] Protein A affinity chromatography columns can specifically bind to the Fc terminus of antibodies. In this experiment, a Protein A affinity chromatography column (2.1×30 mm, 20 μm) was used to quantitatively analyze the Fc fragment (first fragment) without toxin linkers. The main chromatographic conditions (e.g., mobile phase composition, detection wavelength and elution gradient) are shown in Table 2.

[0129] Table 2: Affinity Chromatography Conditions for Protein A

[0130]

[0131] The enzyme-digested antibody solution was used as an external standard solution for Protein A affinity chromatography analysis. The chromatogram is shown below. Figure 8 As shown, where, Figure 8 In the middle, A is the global map of the affinity chromatogram. Figure 8 Image B is a magnified view of the affinity chromatogram. The chromatographic peaks of the Fc fragment without the toxin linker were integrated, with the concentration of the enzyme-digested antibody on the x-axis and the peak area of ​​the Fc fragment on the y-axis. A least-squares linear fit was then performed to obtain the linear equation for the standard working curve, as shown below. Figure 9 As shown. The correlation coefficient R of the linear fit.2 A value of 1.0000 indicates good linearity.

[0132] The enzyme-digested ADC solution (three parallel samples were prepared) was used as the test sample for Protein A affinity chromatography analysis. The chromatogram is shown below. Figure 10 As shown, where, Figure 10 In the middle, A is the global map of the affinity chromatogram. Figure 10 Image B is a magnified view of the affinity chromatogram, showing good repeatability. The Fc fragment peak was integrated, and the peak area was substituted into the linear equation of the standard working curve to calculate the concentration, which is the protein concentration of the ADC injection solution. Multiplying this concentration by the dilution factor of the ADC sample pretreatment yields the protein content of the ADC sample. Using this invention, the protein content of the anti-Her2-DxD ADC sample was measured to be 8.6 mg / mL, while the protein content measured using the traditional UV dual-wavelength method was 8.0 mg / mL. The difference between the two results is approximately 7%, indicating good consistency.

[0133] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: The present application decomposes the ADC to be tested to obtain a first fragment without toxin linkers and a second fragment connected with toxin linkers. Subsequent quantitative analysis of the antibody fragment without toxin linkers (the first fragment) is sufficient to further determine the antibody protein content in the ADC. The detection method of the present application does not require information such as the extinction coefficient of the toxin linkers, is not affected by toxin linkers, and is not affected by other matrices in the sample (such as excipients, free toxin impurities, etc.). It is applicable to ADC products with different DAR values, meets the needs of protein content analysis methods in the early research and development stage of ADC products, and has higher detection accuracy and a simpler detection method compared to existing technologies.

[0134] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting the content of an antibody protein in an antibody conjugate drug, characterized by, The detection method comprises: S1) decomposing the antibody conjugated drug to be detected to obtain a first fragment without a toxin linker and a second fragment with the toxin linker; S2) performing chromatographic quantitative analysis on the first fragment to obtain the protein content.

2. The detection method according to claim 1, characterized in that, The decomposition process comprises using a protease and / or a reducing agent to decompose the antibody conjugated drug to be detected.

3. The detection method according to claim 2, characterized in that, The protease comprises one or more of IdeS protease, Lys-C protease, Trypsin protease, IgdE protease, or Papain protease.

4. The detection method according to claim 2, characterized in that, The reducing agent comprises one or more of DTT, TCEP, or β-mercaptoethanol.

5. The method of claim 1, wherein, The conjugation mode of the antibody conjugated drug comprises site-specific conjugation.

6. The detection method according to claim 5, characterized in that, The site-specific conjugation comprises cysteine conjugation, site-specific conjugation of glycosylation sites, or site-specific conjugation of ThioMab.

7. The detection method according to claim 6, characterized in that, In the antibody conjugated drug, the toxin linker is connected to F(ab)2 or Fab, and the antibody conjugated drug is subjected to enzymatic digestion using a protease, and the first fragment obtained is Fc, and the second fragment is F(ab)2 or Fab connected to the toxin linker.

8. The detection method according to claim 6, characterized in that, In the antibody conjugated drug, the toxin linker is connected to Fc, and the antibody conjugated drug is subjected to enzymatic digestion using a protease, and the first fragment is F(ab)2 or Fab, and the second fragment is Fc connected to the toxin linker.

9. The detection method of claim 6, wherein, In the antibody conjugated drug, the toxin linker is connected to the heavy chain, and the reducing agent is mixed with the antibody conjugated drug to react, so that the disulfide bond between the light chain and the heavy chain is reduced, thereby dissociating the light chain and the heavy chain, the first fragment is the light chain, and the second fragment is the heavy chain.

10. The method of claim 6, wherein, In the antibody conjugated drug, the toxin linker is connected to the light chain, and the reducing agent is mixed with the antibody conjugated drug to react, so that the disulfide bond between the light chain and the heavy chain is reduced, thereby dissociating the light chain and the heavy chain, the first fragment is the heavy chain, and the second fragment is the light chain.

11. The method of claim 1, wherein, The chromatographic quantitative analysis comprises reverse phase chromatography, hydrophobic chromatography, hydrophilic chromatography, size exclusion chromatography, ion exchange chromatography, affinity chromatography, or capillary electrophoresis.

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