A method for detecting low-abundance amphoteric compounds using fluorescence imaging capillary isoelectric focusing.

Abstract

This paper provides a method for detecting low concentrations of amphoteric compounds in a sample, comprising: (a) preparing an analytical sample mixture for capillary isoelectric focusing (CIEF) detection using the sample; and (b) detecting the analytical sample mixture using a fluorescence detector via a CIEF system; wherein the concentration of the low concentration amphoteric compound in the analytical sample mixture is less than 0.5 μg / μL. This method is more resistant to background noise interference and can sensitively detect and accurately quantify low concentrations of amphoteric compounds present in the sample.

Description

Technical Field

[0001] This invention relates to the field of bioanalysis; specifically, it relates to a method for accurately determining low-content amphoteric compounds by fluorescence imaging capillary isoelectric focusing. Background Technology

[0002] Monoclonal antibody drugs (MAVs) possess unique advantages such as high specificity, high sensitivity, and high safety, and have become one of the mainstream developments in the global pharmaceutical industry. While MAVs are flourishing, next-generation antibodies, namely bispecific antibody drugs (BIVs), are also rapidly developing. The FDA has approved four BIVs for marketing. Currently, more than 100 BIVs are in clinical trials worldwide. Simultaneously, many pharmaceutical companies are actively developing and deploying fusion protein drugs (e.g., Fc fusion proteins).

[0003] For some asymmetric bispecific antibody drugs, the presence of homologous mismatch impurities can significantly affect drug activity. For some fusion protein drugs, especially protoprotein drugs that are inactive (or have low activity) before activation but only become active after activation or upon reaching a specific site, even very low levels of related homologous mismatch (active) impurities can cause significant changes in activity. Therefore, the detection and monitoring of low-level homologous mismatch impurities are crucial for the production of such protein drugs.

[0004] Existing techniques for detecting homologous mismatch impurities in asymmetric bispecific antibody or Fc fusion proteins include size exclusion chromatography, reversed-phase chromatography, hydrophobic interaction chromatography, capillary electrophoresis, and imaging capillary isoelectric focusing. When there is a significant difference in the isoelectric point between the target protein and its homologous mismatch impurities, UV imaging capillary isoelectric focusing can be used to separate and detect the impurity levels. However, commonly used amphoteric electrolytes suitable for protein separation (such as Servalyt) are less effective. TM The ultraviolet imaging capillary isoelectric focusing method usually produces significant baseline interference. Therefore, the detection of related homologous mismatch impurities using the ultraviolet imaging capillary isoelectric focusing method is limited by factors such as large baseline interference and high quantification limit. It can only detect homologous mismatch impurities with high concentration levels and is difficult to accurately quantify homologous mismatch impurities with low concentration levels.

[0005] Therefore, a method is still needed to detect and quantify low levels of amphoteric compounds (e.g., homologous mismatch impurities) in samples. Summary of the Invention

[0006] After long-term research and exploration, the inventors have developed a method for detecting low levels of amphoteric compounds in samples. This method is better able to resist background noise interference and can sensitively detect and accurately quantify low levels of amphoteric compounds present in samples.

[0007] On the one hand, this article provides a method for detecting low levels of amphoteric compounds in a sample, which includes:

[0008] (a) Preparation of analytical sample mixtures for capillary isoelectric focusing (CIEF) detection using sample preparation; and

[0009] (b) The analytical sample mixture was detected using a fluorescence detector via a CIEF system;

[0010] The concentration of the low-content amphoteric compound in the analytical sample mixture is less than 0.5 μg / μL. Attached Figure Description

[0011] The present invention will be further described below with reference to the accompanying drawings, which are shown only for illustrating embodiments of the present invention and are not intended to limit the scope of the present invention.

[0012] Figure 1 , 2 Exemplary examples of asymmetric bispecific antibodies with different structural features are shown.

[0013] Figure 3 , 4 Exemplary examples of asymmetric Fc fusion proteins with different structural features are shown.

[0014] Figure 5 The results spectrum of the blank sample detected by ultraviolet capillary isoelectric focusing in the exemplary embodiments of this article is shown.

[0015] Figure 6 The results spectrum of the blank sample detected by fluorescent capillary isoelectric focusing in the exemplary embodiments of this article is shown.

[0016] Figure 7 The results of detecting different concentrations of protein X using ultraviolet capillary isoelectric focusing in the exemplary embodiments described herein are shown.

[0017] Figure 8 The results of detecting different concentrations of protein X using fluorescence capillary isoelectric focusing in the exemplary embodiments described herein are shown.

[0018] Figure 9 A linear graph of protein X concentration and peak area is shown in the exemplary embodiments described herein. Detailed Implementation

[0019] The technical terms used in this application have the same meaning as commonly understood by those skilled in the art, unless otherwise stated. In this application, the word "a" or its combination with various quantifiers includes both singular and plural meanings, unless specifically stated. In this application, when multiple numerical values, numerical ranges, or combinations thereof are given for the same parameter or variable, it is equivalent to specifically disclosing these numerical values, range endpoints, and numerical ranges formed by any combination thereof. In this application, any numerical value, whether or not it is modified by words such as "about," encompasses an approximate range that can be understood by those skilled in the art, such as plus or minus 10%, 5%, etc. In this document, each "implementation" equally refers to and covers the implementation methods and systems of this application. In this application, one or more technical features in any implementation can be freely combined with one or more technical features in any one or more other implementations, and the resulting implementation is also part of the disclosure of this application.

[0020] Isoelectric focusing (IEF) is a technique used to separate molecules based on differences in their isoelectric points (pI) (i.e., the pH value where the molecule has zero net charge). This technique separates and analyzes amphoteric compounds along a pH gradient that gradually increases from the anode to the cathode. Capillary isoelectric focusing (CIEF) uses a capillary or microchannel as the focusing column and a carrier amphoteric electrolyte as the separation medium. The carrier amphoteric electrolyte is added to the sample, and then a voltage is applied to establish the desired pH gradient, allowing the amphoteric analytes to be separated according to their different isoelectric points. CIEF enables automated, reproducible detection and quantification of recombinant proteins, and the analytical results can be easily recorded and archived. However, in practice, CIEF requires a thorough understanding of the analyte movement during electrophoresis and a wealth of practical experience.

[0021] The inventors discovered in practice that commonly used amphoteric electrolytes suitable for separating proteins (such as Servalyt) TM The ultraviolet imaging capillary isoelectric focusing method usually produces significant baseline interference. Therefore, the detection of related homologous mismatch impurities using the ultraviolet imaging capillary isoelectric focusing method is limited by factors such as large baseline interference and high quantification limit. It can only detect homologous mismatch impurities with high concentration levels and is difficult to accurately quantify homologous mismatch impurities with low concentration levels.

[0022] After long-term research and exploration, the inventors have developed a method for detecting low levels of amphoteric compounds in samples. This method is better able to resist background noise interference and can sensitively detect and accurately quantify low levels of amphoteric compounds present in samples.

[0023] On the one hand, this article provides a method for detecting low levels of amphoteric compounds in a sample, which includes:

[0024] (a) Preparation of analytical sample mixtures for capillary isoelectric focusing (CIEF) detection using sample preparation; and

[0025] (b) The analytical sample mixture was detected using a fluorescence detector via a CIEF system;

[0026] The concentration of the low-content amphoteric compound in the analytical sample mixture is less than 0.5 μg / μL.

[0027] I. Low content of amphoteric compounds

[0028] The sample may contain low levels of amphoteric compounds that are typically difficult to detect or accurately quantify using a CIEF system with UV or visible light detectors.

[0029] In this article, the term "amphoteric compound" refers to a compound having both basic and acidic functional groups.

[0030] "Low concentration of amphoteric compounds" refers to amphoteric compounds with a concentration of less than 0.5 μg / μL in the analytical sample mixture prepared for CIEF detection.

[0031] In some embodiments, the concentration of the low-content amphoteric compound in the analytical sample mixture is less than 0.50 μg / μL, for example, less than 0.45 μg / μL, less than 0.40 μg / μL, less than 0.35 μg / μL, less than 0.30 μg / μL, less than 0.25 μg / μL, less than 0.20 μg / μL, less than 0.17 μg / μL, less than 0.15 μg / μL, less than 0.12 μg / μL, less than 0.10 μg / μL, less than 0.08 μg / μL, less than 0.05 μg / μL, or less than 0.03 μg / μL.

[0032] In some embodiments, the concentration of the low-content amphoteric compound in the analytical sample mixture is in the range of 0.03 μg / μL to 0.5 μg / μL, for example, 0.03 μg / μL to 0.45 μg / μL, 0.03 μg / μL to 0.40 μg / μL, 0.03 μg / μL to 0.40 μg / μL, 0.03 μg / μL to 0.35 μg / μL, 0.03 μg / μL, 0.03 μg / μL to 0.35 μg / μL, 0.03 μg / μL to 0.45 μg / μL, 0.03 μg / μL to 0.35 μg / μL, 0.03 μg / μL to 0.40 μg / μL, 0.03 μg / μL to 0.35 μg / μL, 0.03 μg / μL to 0.4 ...5 μg / μL, 0.03 μg / μL to 0.45 μg / μL, 0.03 μg / μL to 0.45 μg / μL, 0.03 μg / μL to 0.45 μg / μL, 0.03 μg / μL μg / μL to 0.30μg / μL, 0.03μg / μL to 0.25μg / μL, 0.03μg / μL to 0.20μg / μL, 0.03μg / μL to 0.17 μg / μL, 0.03μg / μL to 0.15μg / μL, 0.03μg / μL to 0.12μg / μL, or 0.03μg / μL to 0.10μg / μL.

[0033] In some embodiments, the low-content amphoteric compound may include peptides, proteins, or combinations thereof.

[0034] In this article, the term "peptide" refers to a compound consisting of 20 to 50 amino acid residues linked by peptide bonds. The term "protein" refers to a substance with a specific spatial structure, consisting of one or more peptide chains of more than 50 amino acid residues linked together and / or coiled and folded in a specific manner.

[0035] In some embodiments, the low-content amphoteric compound may include impurities in the sample. In some embodiments, the low-content amphoteric compound is an impurity in the sample. In some embodiments, the impurity may be a polypeptide, a protein, or a combination thereof. In some embodiments where the sample is an antibody-like protein sample, the impurity may be a homologous mismatch impurity.

[0036] In this article, the term "impurity" refers to a substance that is undesirable to be included or retained in a sample because it does not have the desired effect or affects the stability, efficacy, or even human health of one or more active substances (e.g., antibody drugs). When referring to antibody protein samples, the term "homogeneous mismatch impurity" refers to an impurity formed when two identical polypeptides or proteins are mistakenly bound together by chemical or physical interactions. For antibody protein drugs (e.g., asymmetric bispecific antibodies or fusion protein drugs), the presence of homologous mismatch impurities (even at low levels) can significantly affect drug activity. Therefore, the detection and monitoring of low-level homologous mismatch impurities are crucial for the production of such antibody protein drugs.

[0037] II. Sample

[0038] The sample that can be tested using the method described in this paper can be any liquid sample.

[0039] In some embodiments, the sample may contain one or more dominant components or target components (e.g., target proteins). The dominant component is generally present in a significantly higher concentration than other components (excluding the carrier) in the sample. For example, the dominant component may be present in the sample at a concentration 40%, 50%, 60%, 70%, 80%, or 90% higher than the concentration of its derivatives, analogs, or other components that are undesirable in the sample. The target component generally refers to one or more components that are desired to be present in the sample. In some cases, the target component is the dominant component in the sample.

[0040] In some embodiments, the sample may be a protein sample, i.e., containing one or more proteins as its dominant component. In some embodiments, the sample may be an antibody-based protein sample, i.e., containing one or more antibody-based proteins as its dominant or target component. In some embodiments, the sample may be an asymmetric bispecific antibody or an Fc fusion protein sample. In some embodiments, the sample may be an asymmetric bispecific antibody sample, i.e., containing one or more asymmetric bispecific antibodies as its dominant or target component. In some embodiments, the sample may be a fusion protein (e.g., an asymmetric Fc fusion protein) sample, i.e., containing one or more fusion proteins (e.g., an asymmetric Fc fusion protein) as its dominant or target component.

[0041] In this article, the term "bispecific antibody" usually refers to an artificial protein that can simultaneously bind to two different types of antigens or two different epitopes on the same antigen. The term "asymmetric bispecific antibody" refers to an artificial antibody (e.g., obtained by chemical coupling, recombinant DNA, or cell fusion) that combines two different H chains and two different L chains, and is capable of simultaneously and specifically binding to two different antigenic epitopes with an asymmetrical left-right structure. Figure 1-2 Exemplary examples of asymmetric bispecific antibodies with different structural features are shown.

[0042] In this article, the term "fusion protein" refers to a protein created by linking two or more genes that originally independently encode proteins. The term "Fc fusion protein" refers to a fusion protein in which a biologically active functional protein molecule is fused to the Fc segment of an immunoglobulin (IgG, IgA, etc.). The term "asymmetric Fc fusion protein" refers to a fusion protein in which a biologically active functional protein molecule is fused to the Fc segment of an immunoglobulin (IgG, IgA, etc.) and the left-right structure is asymmetrical. Figure 3-4 Exemplary examples of asymmetric Fc fusion proteins with different structural features are shown.

[0043] In some implementations, the sample is pretreated before being used to prepare the analytical sample mixture.

[0044] In some embodiments, sample pretreatment includes adjusting the concentration of the dominant component in the sample. Advantageously, the concentration of the dominant component (e.g., the target protein in a protein sample) in the sample is adjusted to 1.0 to 2.0 mg / mL by concentration or dilution.

[0045] In an exemplary embodiment where the target protein in a protein sample needs to be concentrated to 1.0 to 2.0 mg / mL, the concentration adjustment can be performed as follows:

[0046] When the concentration of the target protein in the protein sample is <1.0 mg / mL, the sample should be concentrated. Add at least >2 times the required amount of target protein to a 10 kD ultrafiltration tube and centrifuge at 10000-15000 rcf for at least 8 minutes at 10-15°C. Invert the filter element into a new ultrafiltration tube and centrifuge at 3000-5000 rcf for 2 minutes at 10-15°C. Collect the concentrate and mix well. Measure the absorbance of the concentrate at 280 nm using a UV spectrophotometer (e.g., NanoDrop 2000) and calculate the target protein concentration according to Formula 1.

[0047]

[0048] in,

[0049] c: Concentration of the target protein in the sample;

[0050] A 280 : The absorbance of the sample at 280 nm;

[0051] ε': Extinction coefficient of the target protein.

[0052] In some embodiments, sample pretreatment includes desialylation of the sample. In some embodiments, the desialylation treatment includes adding sialidase to the sample.

[0053] In an exemplary embodiment where the protein sample requires desialylation treatment, the desialylation treatment can be performed as follows:

[0054] Take 40-60 μg of protein, add ultrapure water to a final volume of 10-14 μL, add 4 μL of 5× reaction buffer B (e.g., provided in the sialidase A kit) and 2-6 μL of sialidase A, and mix well. After brief centrifugation, incubate at 30-40°C for 40 to 600 minutes.

[0055] It should be understood that samples may undergo any pretreatment to make them suitable for detection or quantification via CIEF.

[0056] III. Analysis of Sample Mixture

[0057] In this article, the term "analytical sample mixture" refers to a mixture prepared using samples for CIEF testing.

[0058] In some implementations, the analytical sample mixture contains both a sample and a carrier amphoteric electrolyte.

[0059] In this article, the term "carrier amphoteric electrolyte" refers to aliphatic polyamino and polycarboxyl molecules with varying chain lengths and branching. These are amphoteric molecules containing ionizable acidic and basic functional groups, and possess a specific pH. Carrier amphoteric electrolytes dissociate into negatively charged anions at pH values ​​above their isoelectric point, migrating towards the positive electrode of an electric field; and dissociate into positively charged cations at pH values ​​below their isoelectric point, migrating towards the negative electrode of an electric field. This migration ceases at a pH equal to their pI (i.e., when the net charge of the protein is zero). Generally, carrier amphoteric electrolytes have an average molecular weight of approximately 200-1000 Da.

[0060] In some implementations, the analytical sample mixture comprises a mixture of two or more carrier ampholytes covering a specific pH range to create a pH gradient during detection. Suitable commercially available carrier ampholyte products include: Ampholine, Pharmalyte (both from Amersham Pharmacia Biotech), Servalyte (Fluka), Bio-Lyte (BioRad), etc. These products consist of various quantities and types of isomers to cover specific pH ranges.

[0061] In some embodiments, the analytical sample mixture contains a carrier amphoteric electrolyte with pH 2-11. In some exemplary embodiments, the analytical sample mixture contains one or more carrier amphoteric electrolytes selected from the following: Servalyt pH2-11, Servalyt pH 6-9, Servalyt pH 5-9, Servalyt pH 9-11, Pharmalyte pH 5-8, Pharmalyte pH 3-10, and Pharmalyte pH 8-10.5, each in a concentration not exceeding 4.45% by volume, based on a total volume of 100% of the analytical sample mixture.

[0062] In some implementations, the analytical sample mixture also contains an electroosmotic inhibitor.

[0063] In this article, the term "electroosmotic flow inhibitor" refers to a substance used to reduce electroosmotic flow in order to prevent the pI-based separation of proteins from being disrupted and to limit diffusion within separation channels (e.g., capillaries).

[0064] In some embodiments, the electroosmotic flow inhibitor comprises a neutral high molecular weight polymer capable of increasing the viscosity of the separation medium. Examples of suitable electroosmotic flow inhibitors include methylcellulose (e.g., hydroxypropyl methylcellulose), polyacrylamide, dextran, and combinations thereof. In some embodiments, the electroosmotic flow inhibitor comprises methylcellulose. In some embodiments, the concentration of the electroosmotic flow inhibitor in the analytical sample mixture is 0.3 vol% to 0.4 vol%, based on a total volume of 100 vol% of the analytical sample mixture.

[0065] In embodiments where no electroosmotic flow inhibitor is present in the analytical sample mixture, electroosmotic flow inhibition can also be achieved by coating the separation channels (e.g., capillaries) with a neutral and hydrophilic polymer coating.

[0066] In some implementations, the analytical sample mixture also contains a co-solvent.

[0067] In this article, the term "solvent" refers to a substance added to allow proteins to dissolve better.

[0068] Examples of suitable co-solvents include urea, formamide, glycerol, sorbitol, propylene glycol, zwitterionic surfactants, neutral surfactants, and combinations thereof. In some embodiments, the co-solvent includes urea. In some embodiments, the concentration of the co-solvent in the analytical sample mixture is 0-8 M.

[0069] In embodiments where urea is used as a co-solvent, a fresh (within 7 days) urea solution is used. In one exemplary embodiment, the urea solution is prepared as follows: Weigh 4.8 ± 0.1 g of solid urea into a 15 mL centrifuge tube, add approximately 5 mL of ultrapure water to completely dissolve the urea, bring the volume to 10 mL with ultrapure water and mix well. Store at 2-8°C for up to 7 days. The volume of the prepared solution can be scaled up as needed.

[0070] In some implementations, the analytical sample mixture also contains an isoelectric point marker.

[0071] In this article, the term "isoelectric point marker" refers to a chemical or polypeptide small molecule isoelectric point marker with a known pI.

[0072] The selection of isoelectric point markers in the analytical sample mixture is based on the theoretical pI of the desired target substance in the sample. For example, when the theoretical pI of the desired target substance in the sample is in the range of 6.00 to 8.00, multiple isoelectric point markers with pI covering the range of 4.50 to 8.20 are selected; when the theoretical pI of the desired target substance in the sample is in the range of 8.00 to 9.20, multiple isoelectric point markers with pI covering the range of 7.00 to 10.50 are selected. In some embodiments, the concentration of the isoelectric point marker in the analytical sample mixture is 0.0 vol% to 1 vol%, based on a total volume of 100 vol% of the analytical sample mixture.

[0073] In some embodiments, the preparation of the analytical sample mixture includes mixing the sample with a carrier amphoteric electrolyte, and optionally, an electroosmotic inhibitor, a co-solvent, and an isoelectric point marker.

[0074] In some embodiments, the preparation of the analytical sample mixture includes: first mixing a carrier amphoteric electrolyte, and optionally, an electroosmotic inhibitor, a solubilizer, and an isoelectric point marker to form a premix, and then mixing the sample with the premix. In some embodiments, the volume of the premix is ​​in the range of 70 to 100 μL, for example, 70 to 95 μL, 70 to 90 μL, 75 to 95 μL, or 80 to 90 μL. In some embodiments, the volume of the sample is in the range of 1 to 30 μL, for example, 2 to 30 μL, 3 to 30 μL, 5 to 30 μL, 8 to 30 μL, 10 to 30 μL, 5 to 25 μL, 8 to 25 μL, 10 to 25 μL, 8 to 20 μL, or 10 to 20 μL. In some embodiments, the volume ratio of the sample to the premixed liquid is in the range of 1:50 to 1:3, for example, 1:50 to 1:4, 1:40 to 1:4, 1:30 to 1:4, 1:20 to 1:4, 1:10 to 1:4, or 1:9 to 1:4.

[0075] In an exemplary embodiment where the theoretical isoelectric point of the target protein is greater than 6.0 and less than or equal to 8.0, the premix can be prepared according to Table 1 below:

[0076] Table 1. Premixed liquid component ratio (6.0 < theoretical isoelectric point ≤ 8.0)

[0077]

[0078]

[0079] (Note: The volume of the mixture can be adjusted according to experimental requirements. Mix thoroughly.)

[0080] In an exemplary embodiment where the theoretical isoelectric point of the target protein is greater than 8.0 and less than or equal to 9.2, the premix can be prepared according to Table 2:

[0081] Table 2. Premixed liquid component ratio (8.0 < theoretical isoelectric point ≤ 9.2)

[0082]

[0083]

[0084] (Note: The volume of the mixture can be adjusted according to experimental requirements. Mix thoroughly.)

[0085] In some exemplary embodiments, the sample and premixed solution can be mixed and processed as follows for CIEF detection:

[0086] Take 10-20 μL of pretreated sample into a centrifuge tube, add 80-90 μL of premixed solution, mix thoroughly, centrifuge at 12000-15000 rcf for 1-2 minutes, transfer 90-95 μL of supernatant into the inner liner tube, and then put the inner liner tube back into the original centrifuge tube or 96-well plate. Centrifuge at 12000-15000 rcf at 10-15℃ for 3-30 minutes. After ensuring that there are no air bubbles in the inner liner tube, transfer the inner liner tube into a glass sample vial, or place it into the sample tray after ensuring that there are no air bubbles in the sample wells of the 96-well plate, for CIEF analysis.

[0087] In some implementations, the volume of the analytical sample mixture is in the range of 80 to 120 μL, for example, 80 to 115 μL, 80 to 110 μL, 85 to 115 μL, 85 to 110 μL, 90 to 115 μL, or 90 to 110 μL.

[0088] In some implementations, the analytical sample mixture is treated (e.g., centrifuged) to remove air bubbles before being used for CIEF detection.

[0089] In some implementations, no other reagents (including water) are added to the analytical sample mixture before it is used for CIEF testing.

[0090] IV. CIEF detection of the sample mixture

[0091] The prepared analytical sample mixture was detected by the CIEF system using a fluorescence detector.

[0092] In one specific implementation, the detection includes:

[0093] (i) The analytical sample mixture is injected into a capillary for CIEF detection, one end of which is connected to a low pH electrode (anode) and the other end is connected to a high pH electrode (cathode).

[0094] (ii) Apply an electric field;

[0095] (iii) After separation and focusing, an isoelectric focusing spectrum is obtained by using a fluorescence detector.

[0096] In one specific embodiment, the detection further includes:

[0097] (iv) Calculate the content and pI of the low-content amphoteric compound in the sample based on the isoelectric focusing spectrum obtained in step (iii).

[0098] In some embodiments, the CIEF is an iCIEF (in-cylinder imaging capillary isoelectric focusing).

[0099] In some implementations, the temperature of the autosampler used for CIEF detection is 10-30°C.

[0100] In some implementations, the focusing voltage and time used for CIEF detection are:

[0101] Phase 1: 1500 volts, lasting 1-2 minutes;

[0102] Second stage: 3000 volts, lasting 5-18 minutes.

[0103] In some implementations, the injection duration for CIEF detection is 50-60 seconds.

[0104] In some implementations, the instrument used for CIEF testing is a Maurice™ instrument (purchased from ProteinSimple).

[0105] In one exemplary embodiment, the analytical parameters of the Maurice instrument (ProteinSimple) are set as shown in Table 3 below, and a fluorescence detector is used for detection.

[0106] Table 3. Parameter settings for the capillary isoelectric focusing electrophoresis analyzer

[0107]

[0108] Example

[0109] The technical solutions of the present invention will be clearly and completely described below by way of exemplary embodiments. Obviously, the described embodiments are exemplary partial (but not all) implementations of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0110] The reagents, consumables, and instruments used in this embodiment are shown in Tables 4, 5, and 6 below.

[0111] Table 4. Reagents used in the method

[0112]

[0113]

[0114]

[0115] Table 5. Consumables used in the method

[0116]

[0117] Table 6. Instruments and Equipment Used in the Method

[0118]

[0119]

[0120] Take 2.2 μg / μL of protein X (homogeneous mismatch impurity) and the corresponding volume of prescription buffer (e.g., L-glutamic acid-NaOH buffer, pH 4.8) as shown in Table 7, mix them thoroughly to obtain protein X solutions with concentrations of 0.005 μg / μL, 0.01 μg / μL, 0.03 μg / μL, 0.06 μg / μL, 0.16 μg / μL, 0.24 μg / μL, and 0.32 μg / μL, respectively.

[0121] Table 7. Protein X and Diluent Volume Table

[0122]

[0123] Take 14 μL of protein X solutions with concentrations of 0.005 μg / μL, 0.01 μg / μL, 0.03 μg / μL, 0.06 μg / μL, 0.16 μg / μL, 0.24 μg / μL, and 0.32 μg / μL, respectively, add 4 μL of 5× reaction buffer B and 2 μL of sialidase A, and mix well. After brief centrifugation, incubate at 37°C for 4 hours ± 5 minutes. Prepare three replicates for each concentration.

[0124] Blank sample preparation: Take 14 μL of ultrapure water, add 4 μL of 5× reaction buffer B and 2 μL of sialidase A, and mix well. After brief centrifugation, incubate at 37°C for 4 hours ± 5 minutes.

[0125] Preparation of corresponding premixed solutions: The premixed solution preparation for a single sample is shown in Table 8 below. The preparation volume can be adjusted according to experimental requirements, and the solution should be thoroughly mixed.

[0126] Table 8. Premixed liquid component ratio

[0127]

[0128]

[0129] Take 10 μL of sialyl-treated enzyme-digested sample into a centrifuge tube, add 90 μL of premixed solution, mix thoroughly, centrifuge at 13000 rcf for 1 minute, transfer 90 μL of supernatant to a 96-well plate, centrifuge at 4000 rcf, 10℃ for 20 minutes, ensuring no air bubbles are present in the sample wells, and then place them in a Maurice C sample tray for iCIEF analysis. The samples should be stored at 10℃±2℃.

[0130] The Maurice C analysis parameters are set as shown in Table 9 below, and the detection was performed using fluorescence and ultraviolet detectors, respectively.

[0131] Table 9. Parameter Settings for Capillary Isoelectric Focusing Electrophoresis Analyzer

[0132]

[0133] Figure 5 , Figure 6 These are the spectra of blank samples detected by UV and fluorescence capillary isoelectric focusing, respectively. From the spectra of blank samples detected by UV and fluorescence capillary isoelectric focusing, it can be seen that the baseline fluctuation of the UV spectrum is large, while the baseline fluctuation of the fluorescence spectrum is small. Fluorescence can better reduce the interference of the baseline on the protein X elution peak.

[0134] Figure 7 These are the spectral results of different concentrations of protein X detected by ultraviolet capillary isoelectric focusing. Figure 8 These are the spectral results of different concentrations of protein X detected by fluorescent capillary isoelectric focusing. From Figure 7 The spectra of different concentrations of protein X detected by UV capillary isoelectric focusing show that a clear distinction between the peak and baseline of protein X can only be observed when the concentration of protein X is 0.16 μg / μL. However, from... Figure 8 The spectra of different concentrations of protein X detected by fluorescence capillary isoelectric focusing show that a significant difference between the peak and the baseline can be observed when the concentration of protein X is 0.03 μg / μL. The limit of quantitation (LOQ) for protein X detected using a fluorescence detector is 81% lower than that detected using a UV detector, significantly reducing the LQ of protein X. A linear relationship was obtained by performing a linearization test on the peak areas of protein X at concentrations of 0.03 μg / μL, 0.06 μg / μL, 0.16 μg / μL, 0.24 μg / μL, and 0.32 μg / μL detected by fluorescence capillary isoelectric focusing, as shown in the figure. Figure 9 R 2The value >0.99 demonstrates a good linearity in peak area corresponding to protein X concentrations from 0.03 μg / μL to 0.32 μg / μL. This indicates that using fluorescent capillary isoelectric focusing not only reduces background noise interference when Severlyt is used as an amphoteric electrolyte, but also allows for accurate quantification of protein X at concentrations as low as 0.03 μg / μL.

[0135] Based on the results of the above embodiments, this method has a lower limit of quantification for low concentrations of homologous mismatch impurities in asymmetric bispecific antibodies and Fc fusion proteins, and can more sensitively detect and accurately quantify low levels of (e.g., unwanted) amphoteric compounds in samples.

[0136] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. 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 low levels of amphoteric compounds in a sample, comprising: (a) Use samples to prepare analytical sample mixtures for capillary isoelectric focusing (CIEF) detection; and (b) The analytical sample mixture was detected using a fluorescence detector via a CIEF system; Wherein, the concentration of the low-content amphoteric compound in the analytical sample mixture is less than 0.5 µg / µL; wherein, the low-content amphoteric compound includes peptides, proteins, or combinations thereof; wherein, the sample is pretreated before preparing the analytical sample mixture for CIEF detection; the pretreatment includes: adjusting the concentration of the target protein in the sample to 1.0 to 2.0 mg / mL; and / or treating the sample with sialidase.

2. The method as described in claim 1, wherein, The analytical sample mixture contains the sample and a carrier amphoteric electrolyte.

3. The method as described in claim 2, wherein, The analytical sample mixture also contains an electroosmotic inhibitor.

4. The method of claim 3, wherein, The electroosmotic inhibitor is selected from: methylcellulose, polyacrylamide, dextran, and combinations thereof.

5. The method of claim 4, wherein, Methylcellulose is hydroxypropyl methylcellulose.

6. The method of claim 2, wherein, The analytical sample mixture also contains a co-solvent.

7. The method of claim 6, wherein, The cosolvent is selected from: urea, formamide, glycerin, sorbitol, propylene glycol, zwitterionic surfactants, neutral surfactants, and combinations thereof.

8. The method of claim 2, wherein, The analytical sample mixture also contains isoelectric point markers.

9. The method of claim 1, wherein, The volume of the sample mixture for analysis is in the range of 80 to 120 μL.

10. The method of claim 1, wherein, The CIEF is iCIEF (in-cylinder imaging capillary isoelectric focusing).

11. The method of claim 1, wherein, In step (b), the autosampler temperature is 10-30°C; specifically, the focusing voltage and time are: first stage: 1500 volts for 1-2 minutes; second stage: 3000 volts for 5-18 minutes; specifically, the injection duration is 50-60 seconds.

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