Method for non-covalent migration of fluorescent labels in SDS-proteins by counter-current electrophoresis in SDS-CGE

Abstract

The invention relates to a method for counter-current electrophoretic migration mediated non-covalent fluorescent labeling of SDS proteins in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE), wherein the gel-buffer system contains a fluorescent labeling agent, preferably propidium iodide.

Description

Technical Field

[0001] This invention relates to a method for non-covalent fluorescent labeling of SDS proteins mediated by countercurrent electromigration in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE), wherein the gel-buffer system contains a fluorescent labeling agent, preferably propidium iodide. Background Technology

[0002] The past decade has witnessed significant progress in the development of therapeutic proteins, with numerous biopharmaceutical products successfully entering the pharmaceutical market and generating substantial impact {Wang, 2022 #26}. Accessibility to substances such as cytokines, enzymes, monoclonal antibodies, and growth factors is continuously improving, significantly altering the way diseases are treated and controlled. As the demand for therapeutic proteins continues to grow, the requirements for analytical methods that can accurately and reliably assess the quality and purity of biopharmaceutical products are also increasing.

[0003] In recent years, various bioanalytical techniques such as high-performance liquid chromatography (HPLC) and capillary electrophoresis have been widely used to characterize therapeutic proteins. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) combined with laser-induced fluorescence (LIF) detection is one of the most widely used methods for achieving rapid and highly sensitive protein characterization {Lacroix, 2005 #4}. Although the intrinsic fluorescence of various amino acid structural units such as tyrosine and tryptophan can be used for fluorescence detection, the detection limits achieved are not always satisfactory {Koutny, 1993 #22}. Therefore, techniques for covalently labeling proteins using fluorescent dyes that can react with amino or thiol groups, as well as methods based on non-covalent interactions between fluorophores and protein molecules, have received increasing attention in recent years {Colyer, 2000 #29}. Furthermore, the process of fluorescent labeling through covalent or non-covalent interactions can be further divided into three different subtypes: i) pre-column labeling, ii) on-column labeling, and iii) post-column labeling. Pre-column labeling refers to labeling analyte molecules with fluorophores before they enter the separation capillary. The characteristic of column labeling lies in the direct interaction between the labeling reagent and the analyte molecules within the separating capillary. In post-column labeling schemes, the labeling of the solute molecules occurs after separation, i.e., at the capillary outlet {Lee, 1998 #10}.

[0004] Some of the most commonly used covalent labeling dyes are 2,3-naphthalenedicarboxyl (NDA) {Kaneta, 2009 #5}, 3-(2-(2-furanoyl)quinoline-2-carboxaldehyde (FQ) {Michels, 2007 #1}, fluorescein isothiocyanate (FITC) {Nguyen, 2016 #24} {Emonts, 2021 #25}, and Chromeo dyes {Duhamel, 1983 #23} {Ramsay, 2009 #27}. Michels et al. proposed a highly efficient sample preparation protocol for the analysis of therapeutic proteins, employing sodium dodecyl sulfate capillary gel electrophoresis with LIF detection, primarily for assessing the purity of recombinant monoclonal antibodies. This method labels therapeutic proteins with the fluorophoretic dye FQ in the presence of a nucleophile, eliminating the need for purification to remove excess dye and achieving a low detection limit {Michels, 2007}. #1}. The Dovichi team developed an electrophoresis-mediated on-column derivatization labeling method that uses FQ to label proteins for native gel electrophoresis analysis {Lee, 1998#6}. The challenge of covalent labeling lies in the fact that a single protein molecule can contain numerous reactive sites, all of which may interact with the fluorescent labeling agent. In other words, due to the differences in reactivity among different sites, it is difficult to uniformly label all available sites. Therefore, covalent labeling methods may produce mixtures containing unlabeled, fully labeled, and partially labeled proteins. When separating this mixture, peak broadening is common, and even multiple peaks may appear for the same sample component {E. Oldenburg, 1997#28}. To address this problem, the Kaneta team proposed a post-column derivatization technique for analyzing proteins using NDA via capillary gel electrophoresis (CGE) in the presence of 2-mercaptoethanol as a reducing agent during fluorophore derivatization {Kaneta, 2009#9}.

[0005] In non-covalent labeling, negatively charged Sypro Red and Sypro Orange offer significant advantages over covalent amine- or thiol-reactive counterparts by simply complexing with SDS-proteins without chemical reaction {Steinberg, 1996 #10} {Harvey, 1998 #2} {Moody, 1999 #3}. This method has also been applied to protein analysis based on two-dimensional electrophoresis using ultrathin gels combined with real-time imaging of the separated samples, utilizing on-column fluorescence staining with Sypro Red, which rapidly co-migrates with and transcends sample molecules {Guttman, 2002 #9}. A microchip electrophoresis platform using non-covalently fluorescently labeled materials has also been developed for protein analysis, capable of separating proteins with molecular weights ranging from 5 kDa to 250 kDa and offering a wide protein loading capacity {Bousse, 2001 #7}. In the field of DNA analysis, non-covalent fluorescent labeling of biopolymers using positively charged ethidium or propidium ions via an intercalation mechanism has been reported for a long time. However, only a few examples have been found of protein imaging by staining with ethidium bromide after separation by SDS-polyacrylamide plate gel electrophoresis {Vincent, 1979 #8}.

[0006] Discovery of the present invention To overcome the problems caused by covalent labeling of proteins, the inventors of this invention conducted extensive experimental research, leading to the present invention. This invention is based on an unexpected discovery that propidium iodide ions are suitable as fluorescent labeling agents in capillary gel electrophoresis (CGE), and can be used in the separation matrix. In this invention, when separating proteins using SDS-CGE technology, the proteins undergo electrophoretic migration in a separation matrix containing propidium iodide; propidium ions, as a general biomass staining agent, bind to the negatively charged SDS-protein complex in a non-covalent manner during the separation process through an equilibrium reaction, thereby obtaining a strongly fluorescent SDS-protein-propidium adduct. Summary of the Invention

[0007] The present invention relates to the technical solutions and preferred embodiments defined in the following numbered paragraphs.

[0008] 1. A method for separating proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using fluorescence detection, wherein the separation matrix contains a positively charged fluorescent labeling agent that forms a non-covalently fluorescently labeled SDS-protein adduct.

[0009] In an embodiment of the method according to the invention, the fluorescent labeling agent has a double positive charge.

[0010] In a preferred embodiment of the method according to the invention, the fluorescent labeling agent is propidium iodide.

[0011] In an embodiment of the method according to the invention, the fluorescence detection is laser-induced fluorescence (LIF) detection.

[0012] 2. Use of a positively charged fluorescent protein labeler, said labeler binding non-covalently to proteins in a capillary during sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE).

[0013] In an embodiment of the use of the fluorescent protein labeling agent according to the present invention, the fluorescent labeling agent has a double positive charge.

[0014] In a preferred embodiment of the use of the fluorescent protein labeling agent according to the present invention, the fluorescent labeling agent is propidium iodide.

[0015] In an embodiment of the use of the fluorescent protein labeler according to the present invention, the fluorescence detection is laser-induced fluorescence (LIF) detection.

[0016] 3. A sodium dodecyl sulfate (SDS) capillary gel separation matrix (used in SDS capillary gel electrophoresis (SDS-CGE)) comprising a positively charged fluorescent labeling agent, wherein the labeling agent binds to proteins in the capillary in a non-covalent manner.

[0017] In an embodiment of the separation matrix according to the present invention, the fluorescent labeling agent has a double positive charge.

[0018] In a preferred embodiment of the separation matrix according to the present invention, the fluorescent labeling agent is propidium iodide.

[0019] In an embodiment of the separation matrix according to the present invention, the fluorescence detection is laser-induced fluorescence (LIF) detection.

[0020] 4. A kit for separating proteins by capillary gel electrophoresis (CGE), the kit comprising... - Separation matrix, preferably sodium dodecyl sulfate (SDS) capillary gel separation matrix, and - Fluorescent labeling agents, The separation matrix contains a fluorescent labeling agent, wherein the fluorescent labeling agent has a positive charge and binds to proteins in the capillary via non-covalent binding.

[0021] In an embodiment of the kit according to the present invention, the fluorescent labeling agent has a double positive charge.

[0022] In a preferred embodiment of the kit according to the present invention, the fluorescent labeling agent is propidium iodide.

[0023] In an embodiment of the kit according to the present invention, the fluorescence detection is laser-induced fluorescence (LIF) detection. Attached Figure Description

[0024] Figure 1 Schematic electrophoresis diagram of the complete (mAb) and its subunit fragments (LC, ngHC, and HC) of daratumumab obtained through embodiments of the present invention. The formula represents the structure of propidium iodide used as a non-covalently bound fluorescent labeling agent. IS is an internal standard.

[0025] Figure 2 : Sodium dodecyl sulfate capillary gel electrophoresis of intact daratumumab (mAb) and its subunit fragments (LC, ngHC, and HC). Lower trace: UV detection (220 nm), separation matrix without propidium iodide. Upper trace: Laser-induced fluorescence (LIF) detection, using a gel-buffer system containing 100 μg / mL propidium iodide. Peaks: 1-lysozyme (internal standard), 2-light chain (LC), 3-non-glycosylated heavy chain (ngHC), 4-heavy chain (HC), and 5-intact daratumumab (mAb); Heavy-heavy-light chain fragment impurities of intact mAbs. Conditions: 10% dextran (2M) / 4% borate-based sieve matrix, BFS capillary with an effective length of 20 cm (total length 30 cm, 50 μm ID), applied potential: 15 kV reverse polarity mode (cathode at the injection end); separation temperature: 25°C; sample tray temperature: 20°C; electric injection: 5 kV / 20 s for denaturing intact mAbs, followed by 5 kV / 20 s for lysozyme, LC of daratumumab, ngHC, and reductive denaturing mixture of HC fragments.

[0026] Figure 3: Effect of increased propidium iodide concentration in the gel-buffer system on the migration time and peak area of ​​sample components. The numbers on the traces represent the concentration of propidium iodide (µg / mL) in the gel-buffer system. (A) UV detection at 220 nm, (B) Laser-induced fluorescence detection (excitation wavelength 488 nm / emission wavelength 600 nm). Conditions: Same as above. Figure 2 The concentration of propidium iodide is as specified on each trace.

[0027] Figure 4 Figure 3 shows the relationship between peak area and propidium iodide concentration in the gel-buffer system. Dashed line: UV 220nm detection (initial propidium iodide concentration is zero); solid line: LIF detection (initial propidium iodide concentration is 10 µg / mL).

[0028] Figure 5 The effect of capillary temperature on sample component separation. Peaks and experimental conditions are the same as above. Figure 2The gel-buffer system contains 100 μg / mL PI, and the temperature is given on the trace.

[0029] Figure 6 The relationship between separation temperature and the separation degree between continuously migrating sample components when using a gel-buffer system containing 100 μg / mL propidium iodide.

[0030] Figure 7 Based on origin Figure 5 data on lysozyme internal standards ( ), and light chains ( ), non-glycosylated heavy chain (□), heavy chain ( Arrhenius diagram (Fig. A) and activation energy diagram (Fig. B) of the SDS-protein-propidium iodide complex of the subunit and the intact mAb (○).

[0031] abbreviation: SDS = Sodium dodecyl sulfate CGE = Capillary Gel Electrophoresis EOF = Electroosmotic flow LIF = Laser-induced fluorescence PI = propidium iodide BFS = Bare Fused Quartz BGE = Background Electrolyte mAb = monoclonal antibody. LC = Light chain ngHC = non-glycosylated heavy chain. HC = heavy chain definition As used herein, the term "sample" refers to a substance comprising a mixture of compounds prepared for separation, for example, for analysis, such as by capillary electrophoresis. The sample may be derived from substances, for example, from environmental sources, such as bodily fluids or tissues of an object, and optionally processed for analysis. The sample used herein preferably comprises proteins, and more preferably proteins of biological or biotechnological value as defined, described, or exemplified herein.

[0032] As used herein, "fluorescent" or "fluorescently labeled" compounds refer to compounds that can be detected by UV or VIS irradiation, wherein the compound absorbs the irradiation light and emits light of a different wavelength (emitted light), preferably with a wavelength longer than that of the irradiation light. Preferably, the fluorescent labeling agent is positively charged, and in a particularly preferred embodiment, it is doubly positively charged. The fluorescent labeling agent present in the gel-buffer system non-covalently binds to the SDS-protein complex during the separation process.

[0033] The term "detection" as used in this article, in a broad sense, refers to obtaining observational results about a target substance or compound (preferably an analyte) by performing separation methods on a sample.

[0034] A "detector" is a device used for detection, typically located at a specific position in the CE capillary, to detect target compounds migrating within it. Preferably, the detector operates based on fluorescence detection, more preferably on laser-induced fluorescence (LIF) detection.

[0035] The terms “comprises,” “comprising,” or “including” are to be construed herein as having a non-exhaustive meaning and allowing for the addition or introduction of further features, method steps, or components into anything that includes the listed features, method steps, or components. “Comprising” may be replaced with “including” if required by a particular language variation; “consisting essentially of” may be used if other components are not essential to carrying out the invention; and “consisting of” may be used if no other elements or components are present.

[0036] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the,” or at least “a” and “an,” include plural references. Detailed Implementation

[0037] This invention relates to a novel method for separating proteins by adding propidium iodide to the separation matrix in SDS-CGE. Migration in progress Staining concept. Positively charged propidium ions, as components of the gel-buffer system, migrate counter-currently with negatively charged SDS-protein in the capillary, and form non-covalent fluorescent adducts in situ during separation.

[0038] The inventors have introduced a countercurrent electromigration-mediated non-covalent fluorescent staining method ( Migration in progress The method involves analyzing electromigrable proteins in a separation matrix containing propidium iodide. Propidium ions, carrying two positive charges, serve as a general biomass staining agent and bind nonvalently to the negatively charged SDS-protein complex during separation via an equilibrium reaction, providing strong fluorescence to the resulting SDS-protein-propidium adduct.

[0039] Due to the doubly positive charge of the propidium ion, the surface charge density of the resulting SDS-protein-propidium adduct is reduced, consequently decreasing the electrophoretic mobility of the complex. Furthermore, the propidium ion neutralizes the negatively charged silanol groups on the exposed inner surface of the molten silica capillary, thereby reducing the countercurrent electroosmotic flow (EOF) to almost zero, which is beneficial for shortening the separation time. According to the detailed results below, in the case of a gel-buffer system containing propidium iodide, the electrophoretic mobility of the intact biotherapeutic protein and all its subunits used as an example, as well as the non-glycosylated lysozyme used as an internal standard, was lower than that of the sample components separated without fluorescent staining, but they were detected with high sensitivity. To achieve the highest possible separation capability, the propidium iodide concentration and separation temperature can be optimized for specific sample components.

[0040] Propidium iodide is one of the commonly used non-covalent fluorescent labels for DNA analysis. Originally known primarily for its staining ability on DNA and RNA, propidium iodide has now expanded beyond its original applications to provide profound insights into protein research.

[0041] To support this invention, the inventors describe a novel based on... Migration in progress An SDS-protein staining method was developed, which successfully utilized propidium iodide as a non-covalent fluorescent agent in the SDS-CGE separation of the intact and subunit forms of the therapeutic anticancer monoclonal antibody daratumumab. Propidium ions carrying two positive charges (P... 2+ As a general biomass staining agent {Rocha-Santos, 2014 #20}, it significantly binds negatively charged SDS-protein complexes (S...) in an equilibrium reaction and non-covalent manner during the separation process. n- The resulting SDS-protein-propidium adduct provides strong fluorescence. Due to the doubly positive charge of the propidium ion, the surface charge density of the resulting SDS-protein-propidium adduct is reduced to SP. （n-2m）- Consequently, the electrophoretic mobility of the complex was reduced.

[0042] S n- +mP 2+ SP （n-2m）- (Equation 1) K=[SP （n-2m）- ] / [S n- ][P 2+ ] m (Equation 2) Where K is the complex formation constant, n is the number of negatively charged SDS molecules that bind to the protein (approximately one SDS for every two amino acids {Shirahama, 1974 #19}), and m is the number of propidium ions in the SDS-protein-propidium adduct.

[0043] Considering the extremely low countercurrent electroosmotic flow (-1.8 × 10⁻⁶) in capillary-filled sieve matrix based on high-viscosity borate ester (salt) crosslinked 2M molecular weight dextran polymers (without propidium iodide), the electroosmotic flow is also low. -10 m 2 / Vs) {Guttman, 2021 #21}, SDS-protein molecule efficient migration (μ S Its apparent mobility (μ) can be calculated. app ) and electroosmotic flow (μ EOF The algebraic sum of ) μ S =μ app +μ EOF (Equation 3) At the pH of the SDS-CGE buffer, electroosmotic flow is cathodic; therefore, the electromigration (EOF) of the negatively charged SDS-protein complex is in the opposite direction to that of the anodic migration. Thus, because the two mobilities have opposite signs, the electroosmotic mobility (μ) needs to be expressed as... EOF The value of ) and apparent mobility (μ) app The values ​​are added together to determine the effective SDS-protein migration rate. Without EOF, all solute molecules migrate faster. The migration of the charged gel matrix under an applied electric field is negligible and therefore not considered in Equation 1.

[0044] However, due to the presence of positively charged propidium ions in the gel-buffer system, these positively charged fluorophores neutralize some of the negatively charged silanol groups on the exposed inner surface of the molten silica capillary, further reducing the EOF. Furthermore, due to the recombination of positively charged propidium ions, the actual electrophoretic mobility (μ) of the solute molecule is also lower than that of its propidium-free counterpart. µ=µ S R S (Equation 4) Among them, R S This refers to the molar ratio of SDS-protein without propidine. By introducing c... S As the total SDS-protein concentration, R S It can be represented as: R S =[S n- ] / c S =1 / (1+K[P 2+ ] m (Equation 5) Therefore, the resulting mobilities of the negatively charged SDS-protein-propidine complex (propidine molecules with double positive charges) are as follows: µ=µ S / (1+K[P 2+ ] m (Equation 6) Equation 6 shows that increasing the complex formation constant and / or propidium concentration leads to a decrease in the electrophoretic mobility of the SDS-protein-propidium complex. When K[P 2+ ] m When the value is much greater than 1, equation 6 can be simplified to: µ=µ S / K[P 2+ ] m (Equation 7) Another noteworthy feature in LIF spectra is the increased peak area bias in certain sample components, particularly noticeable in lysozyme (peak 1) and intact monoclonal antibody (peak 5) components compared to UV spectra (see Example 2). Lysozyme is a highly basic protein with a pI of 11.35, therefore its binding to SDS molecules may occur not only through hydrophobic interactions but also through ionic bonds. In the latter case, the hydrophobic tail of the SDS molecule can attract larger planar propidine molecules, forming tight van der Waals contacts, thus increasing the number of bound fluorophores and generating a stronger fluorescence signal. For intact non-reduced mAbs, denaturation results in the loss of secondary structure, thus possessing additional hydrophobic regions and binding more fluorophores compared to the reduced subunit.

[0045] In sieving media, the electromigration of multi-ion biopolymers is an activation process {Guttman, 2015 #12; Cottet, 2001 #13; Lu, 1994 #14}, in which each solute molecule requires a certain activation energy (E). a Only then can it pass through the network structure. Based on the modified Arrhenius equation {Arrhenius, 1889 #15; Eyring, 1935 #16}, the inventors have previously developed a migration equation {Filep, 2020 #17} for separating SDS-proteins in borate (salt) crosslinked dextran gels within narrow-pore capillaries. This equation can be used to assess the activation energy required for sample components.

[0046] µ=QMw -1 / 6 e -Ea / RT (Equation 8) Where Q and Mw are the total charge and molecular weight of the solute, respectively, and E aHere, R is the activation energy, R is the universal gas constant, and T is the absolute temperature. Combining equations 7 and 8, we can explain the role of ligand concentration and molecular properties of the analyte, including molecular weight, charge, and complexation constant, from the perspective of the activation energy concept.

[0047] QMw -1 / 6 e -Ea / RT =µ S / K[P 2+ ] m (Equation 9) ln(QMw -1 / 6 )-E a / RT=ln(µ) S / K[P 2+ ] m (Equation 10) E a =RTln(K[P 2+ mQ / µ S Mw1 / 6)(Equation 11) Equation 11 shows that, under isothermal separation conditions, the activation energy is a direct logarithmic function of the complex formation constant, the number of propidine molecules bound, and the total charge of the analyte, but it is a reciprocal logarithmic function of the electrophoretic mobility of the SDS-protein complex and the molecular weight of the solute to the power of 1 / 6.

[0048] Based on the above, one aspect of the present invention is a method for separating proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using fluorescence detection, wherein the separation matrix contains a positively charged fluorescent labeling agent that forms a non-covalently fluorescently labeled SDS-protein adduct.

[0049] In one implementation of this method, fluorescence detection is laser-induced fluorescence (LIF) detection.

[0050] In one embodiment of the method, the fluorescent labeling agent has a double positive charge.

[0051] In a preferred embodiment of the method, the fluorescent labeling agent is propidium iodide.

[0052] The invention is further described below by way of non-limiting embodiments. Those skilled in the art will understand that other embodiments or variations thereof may also achieve the objectives of the invention based on these embodiments.

[0053] Example Materials used in the examples: Boric acid, Tris, EDTA·Na2, glycerol, sodium dodecyl sulfate (SDS), 2-mercaptoethanol, hydrochloric acid, and sodium hydroxide were all purchased from VWR (Radino, Pennsylvania, USA). Dextran (2MDa), iodoacetamide, and lysozyme (non-glycosylated) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Propidium iodide was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). The therapeutic monoclonal antibody daratumumab (Darzalex®) was purchased from Janssen Biotech (Horsham, Pennsylvania, USA), and PNGase F enzyme was purchased from the University of Pannonia (Westprém, Hungary).

[0054] Example 1: General Method Gel-buffer and sample preparation For all capillary electrophoresis separation experiments, a 10% dextran (2 MDa) / 4.0% (w / v) borate gel buffer system as described in {Guttman, 2021 #21} was used. For fluorescence detection, 10–200 µg / mL propidium iodide (PI) was added to the separation matrix by adjusting the dilution series with PI-free gel buffer and stirring overnight in the dark at room temperature. The sample buffer contained 100 mM Tris-HCl and 1% SDS (pH 9). Complete mAb samples were prepared by mixing 5 µL of 20 mg / mL therapeutic monoclonal antibody with 5 µL of 250 mM iodoacetamide and 85 µL of sample buffer. For the analysis of reduced proteins, 5 µL of a 20 mg / mL therapeutic monoclonal antibody and 5 µL of a 10 mg / mL lysozyme were denatured separately by adding 2 µL of denaturing mixture (Bio-Science Kft, Budapest, Hungary) and incubating at 70°C for 15 min. N-glycans were released from the 5 µL denatured mAb solution by adding 2 µL of 200 mU / mL PNGase F enzyme and incubating at 37°C for 1 h. The final reduced sample mixture contained 5 µL of reduced mAb, 5 µL of deglycosylated mAb, and 5 µL of 10 mg / mL reduced lysozyme internal standard protein. Both unreduced and reduced samples were denatured at 70°C for 15 min.

[0055] Capillary SDS-gel electrophoresis All separations were performed on a P / ACE MDQ capillary electrophoresis system (Beckman Coulter, Brea, CA, USA), controlled by 32 Karat software (version 10.1), equipped with a UV (220 nm) detector for analyzing gel-buffer systems without fluorophores, and a LIF detector (488 nm excitation, 600 nm emission) for analyzing gel-buffer systems containing 10–200 µg / mL propidium iodide. The effective length of the naked fused silica (BFS) separation capillary was 20 cm (total length 30 cm, 50 μm ID) / 375 μm OD). Prior to separation, the capillary was rinsed sequentially for 2 min each with 0.5 M NaOH, 0.5 M HCl, and HPLC-grade pure water. Injection parameters were as follows: 5 kV / 20 s for intact denatured mAbs, followed by 5 kV / 20 s for reductive denatured sample mixtures containing lysozyme, daratumumab light chains, non-glycosylated heavy chains, and heavy chain fragments. All measurements were repeated three times, and the RSDs for mean migration time and peak area were 0.18% and 0.78%, respectively.

[0056] Example 2 - Comparison of SDS-CGE analysis of intact (mAb) and subunit fragments (LC, ngHC, and HC) of daratumumab using UV detection (without propidium iodide) and LIF detection (with propidium iodide). The results of this embodiment are as follows: Figure 2 As shown in Table 1.

[0057] Figure 2 The separation of intact (mAb) and subunits of light chain (LC), non-glycosylated heavy chain (ngHC), and heavy chain (HC) fragments of therapeutic anticancer monoclonal antibodies was compared using UV detection (lower trace) with a propidium-free gel-buffer (ELB) and laser-induced fluorescence detection (upper trace) with a background electrolyte containing 100 μg / mL propidium iodide. Non-glycosylated lysozyme (14 kDa) was used as an internal standard. It was observed that the migration time (upper trace) of all solute molecules in the propidium-containing gel-buffer system was slower than that of their fluorophore-free counterparts (lower trace). Preliminary analysis suggests that this phenomenon is caused by the non-covalently bound positively charged label leading to a decrease in surface charge density (Equation 7, reducing electrophoretic mobility) and a reduction in countercurrent electroosmosis (Equation 3, increasing electrophoretic mobility). The interaction of these two opposing effects resulted in a surprisingly small increase in migration time in the presence of propidium iodide in the separation medium. It is also important to note the difference in the height / area of ​​the corresponding peaks in the two curves. This difference may be due to the difference in the number of fluorophores bound to the solute molecules (LIF detection) and the number of UV-active groups (UV detection). In other words, one cannot expect to obtain the same spectrum using these two fundamentally different detection systems.

[0058] Table 1 describes the resolution (Rs) and selectivity (α) values ​​of gel-buffer systems containing zero (UV detection) and 100 µg / mL (LIF detection), respectively. With LIF detection, the resolution between consecutive migrating peak pairs is higher, which is clearly due to the higher selectivity. It should be noted that even small differences in α values ​​close to 1.00 can have a significant impact on resolution.

[0059] Table 1: Resolution and selectivity values ​​in gel-buffer systems without (UV detection) and with 100 µg / mL propidium iodide (LIF detection).

[0060]

[0061] Example 3 – Study on the effect of propidium iodide concentration The effects of propidium iodide concentrations in the gel-buffer system ranging from 0 (for UV detection) and 10 µg / mL (for LIF detection) to 200 µg / mL on solute molecule migration time and peak area were investigated. Figure 3 compares the electrophoresis results obtained using UV (Figure A) and LIF (Figure B) detection. Both figures show that with increasing propidium iodide concentration, the migration time increases slightly but continuously (i.e., electrophoretic mobility decreases), consistent with the prediction in Equation 7 and the decrease in EOF in Equation 3. On the other hand, with increasing propidium iodide concentration in the background electrolyte, the peak area for UV detection only increases slightly (Figure A), while the peak area for LIF detection increases significantly (Figure B). It should be noted that when the propidium iodide concentration in the gel-buffer system exceeds 200 µg / mL, the fluorescence background becomes higher and the noise increases.

[0062] Figure 4The graphs in Figure 3 compare the peak area with propidium iodide concentration using UV detection (dashed line) and LIF detection (solid line), respectively. As mentioned above, the peak area shows only a slight increase in UV detection, for example, a 1.23-fold increase for the intact mAb component between 0 and 200 µg / mL PI, while for the same molecule, the peak area shows a significant increase in LIF detection, for example, a 17-fold increase in PI concentration from 10 µg / mL to 200 µg / mL in the gel-buffer system. With LIF detection, the slope is steepest for the intact mAb, while the curve corresponding to the heavy chain fragment shows the gentlest rise. The other three sample components have roughly similar slopes between the two sample components. It should be noted that the heavy chain fragment is glycosylated, and since the sugar moiety does not bind SDS, this leads to reduced propidium binding. LC, ngHC, and lysozyme are all non-glycosylated, and therefore bind to the fluorophore based on their very similar SDS-mediated surface charge density. On the other hand, although intact mAbs are glycosylated, the sugar structure faces inward, thus not affecting their SDS binding {Scanlan, 2008 #11}. However, due to the loss of secondary structure in denatured but unreduced mAb molecules, the additional hydrophobic regions readily bind additional propidium ions, thereby enhancing the fluorescence signal. The limit of detection (LOD) for the lysozyme internal standard was 6 ng / µL, similar to the detection limits provided by column-labeled microchip electrophoresis systems {Bousse, 2001 #7}.

[0063] Example 4 - Study on the Influence of Separation Temperature like Figure 5 As shown, the effect of capillary temperature on sample component separation and peak height / peak area was evaluated every 5°C between 20°C and 40°C. As the temperature increased, the migration rates of all sample components increased as expected, which was due to the decrease in viscosity of the sieving matrix. To assess the possible changes in the PI / SDS-protein complex with temperature, Table 2 lists the resulting percentage values ​​of relative peak area. The trend indicates that the relative peak area of ​​lysozyme (3.5%) and fragments of LC (1.9%), ngHC (1.2%), and HC (1.2%) decreased to varying degrees, but the relative peak area of ​​intact mAb (7.7%) increased, which may be due to changes in the complex formation constant (K in Equations 2, 5, and 6) caused by temperature variations.

[0064] Table 2: Effect of separation temperature on the peak area % distribution of sample mixture components in CGE-LIF in a gel-buffer system containing 100 µg / mL PI.

[0065] The relationship between the resolution of sample components and separation temperature was evaluated using a sieving matrix containing 100 µg / mL propidium iodide. Figure 6 As shown, the separation degree between HC and ngHC improved slightly (3.81%) with increasing temperature. On the other hand, the separation degree between all other continuously migrating sample components decreased with increasing temperature. The largest decrease was observed between LC and lysozyme (17.86%). This indicates that, in addition to considering the concentration dependence of propidium iodide, the separation temperature should be optimized to achieve the best separation effect of the target solute component.

[0066] Example 5 - Study on activation energy requirements An Arrhenius diagram was drawn to obtain the activation energy requirements of the analyte molecules. Figure 5 The data obtained from the process is plotted on Figure 7 In A, as shown in Equation 8, the relationship between logarithmic electrophoretic mobility (corrected by 4% for every 5°C increase in temperature {Demorest, 1991 #18}) and the reciprocal of absolute temperature is revealed.

[0067] use Figure 7 The slope calculation of the curve in section A includes the activation energy values ​​of the lysozyme internal standard and the sample components in both full-form and subunit forms of mAb. For example... Figure 7 As shown in B, the obtained E a Plot the values ​​relative to the molecular weight of the sample components. The dashed line is used to aid observation and highlight the E values ​​of mAb-related components (i.e., sample components with similar amino acid sequences). a The strong correlation with Mw also highlights the outlier position of lysozyme as an irrelevant protein. The distribution of data points clearly shows that high-Mw intact mAbs and lysozyme molecules with the highest electrophoretic mobility require lower activation energies, as they both increase the denominator in Equation 10. It is noteworthy that the lysozyme-LC pair showed the greatest decrease in resolution with increasing temperature, again emphasizing the importance of capillary temperature optimization in separating SDS-protein-propidine complexes.

[0068] Industrial applicability Based on the above findings, a method for non-covalent fluorescent labeling of SDS-protein mediated by countercurrent electromigration in SDS-CGE is proposed, wherein the gel-buffer system contains a fluorescent labeling agent, preferably propidium iodide.

[0069] The method of this invention can eliminate the problem of protein covalent labeling, which can result from the fact that proteins may have many reactive sites, and therefore their labeling may lead to a mixture containing unlabeled, fully labeled, and partially labeled proteins. Separating such a mixture can cause peak broadening, and even the appearance of multiple peaks for the same sample component.

[0070] Furthermore, the method of the present invention provides better separation and allows for reduced backflow electroosmotic flow (EOF), which is beneficial for shortening the separation time.

[0071] Therefore, the method of the present invention is applicable to the efficient separation of various protein mixtures and can provide a promising method for the high-sensitivity detection of biotherapeutic agents.

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Claims

1. A method for separating proteins by sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) and using fluorescence detection, wherein, The separation matrix contains a positively charged fluorescent labeling agent that binds to proteins in the capillary via non-covalent binding.

2. The method according to claim 1, wherein, The fluorescent labeling agent has a double positive charge, and preferably, the fluorescent labeling agent is propidium iodide.

3. The method according to claim 1 or 2, wherein, The fluorescence detection is laser-induced fluorescence (LIF) detection.

4. Use of a positively charged fluorescent protein labeler, wherein the fluorescent protein labeler binds non-covalently to proteins in capillaries during sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE).

5. The use according to claim 4, wherein, The fluorescent labeling agent has a double positive charge, and preferably, the fluorescent labeling agent is propidium iodide.

6. A sodium dodecyl sulfate (SDS) capillary gel separation matrix (used in SDS capillary gel electrophoresis (SDS-CGE)) comprising a positively charged fluorescent labeling agent, wherein the labeling agent binds to proteins in the capillary in a non-covalent manner.

7. The separation matrix according to claim 6, wherein, The fluorescent labeling agent has a double positive charge, and preferably, the fluorescent labeling agent is propidium iodide.

8. A kit for separating proteins by capillary gel electrophoresis (CGE), the kit comprising: a separation matrix, preferably sodium dodecyl sulfate (SDS) capillary gel separation matrix; and a fluorescent labeling agent. in, The separation matrix contains the fluorescent labeling agent, wherein the fluorescent labeling agent has a positive charge and binds to proteins in the capillary in a non-covalent manner.

9. The kit according to claim 8, wherein, The fluorescent labeling agent has a double positive charge, and preferably, the fluorescent labeling agent is propidium iodide.

10. The use according to claim 4 or 5, the separation matrix according to claim 6 or 7, or the kit according to claim 8 or 9, wherein, The fluorescence detection is laser-induced fluorescence (LIF) detection.

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