WO2022227363A1 - Tampon d'échantillon d'électrophorèse sur gel capillaire contenant de la cystéine - Google Patents

Tampon d'échantillon d'électrophorèse sur gel capillaire contenant de la cystéine Download PDF

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Publication number
WO2022227363A1
WO2022227363A1 PCT/CN2021/115987 CN2021115987W WO2022227363A1 WO 2022227363 A1 WO2022227363 A1 WO 2022227363A1 CN 2021115987 W CN2021115987 W CN 2021115987W WO 2022227363 A1 WO2022227363 A1 WO 2022227363A1
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Prior art keywords
buffer
cysteine
sample buffer
sample
sds
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PCT/CN2021/115987
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English (en)
Chinese (zh)
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方伟杰
沈斌彬
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浙江大学
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Publication of WO2022227363A1 publication Critical patent/WO2022227363A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis

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  • the invention belongs to the field of biotechnology, and relates to the analysis of proteins by reducing capillary gel electrophoresis, in particular to a cysteine-containing capillary gel electrophoresis sample buffer that can accurately detect the purity of biopharmaceuticals such as antibodies, and the use of the sample buffer An assay method for the accurate and safe determination of protein samples in buffers.
  • CGE capillary gel electrophoresis
  • m/z mass-to-charge ratio
  • mercaptoethanol has been widely used in the reduction of protein samples in the field of biopharmaceuticals, and has become the default protein reducing agent for reduced CE-SDS analysis due to its strong reducing ability (2020 Pharmacopoeia of the People's Republic of China ⁇ 3127 > Monoclonal antibody size variant assay (CE-SDS method)).
  • DTT dithiothreitol
  • CTT is also used in rare cases (Cherkaoui, S et al. Tracking of antibody reduction fragments by capillary gel electrophoresis during the coupling to microparticles surface. J Pharm Biomed Anal. 2010, 53 (2): 172-8; Santarino, IB et al.
  • the experimenters are advised to wear a lab coat, wear a gas mask and operate in a fume hood, and dispose of the experimental waste properly. Even so, the samples containing mercaptoethanol will be added to the CE-SDS special analysis bottle together with the sample and subjected to capillary electrophoresis analysis. This process has an impact on air pollution and the health of the experimenter. Therefore, it is necessary to develop a protein reducing reagent that has the same reducing effect as mercaptoethanol, but is safer for the environment and laboratory personnel, so that the reduced CE-SDS analysis of biopharmaceuticals can be performed more safely.
  • Cysteine is a common amino acid in living organisms, the molecular formula is C 3 H 7 NO 2 S, the molecular weight is 121.16, and its structure is shown in Figure 1. Cysteine is a colorless crystal, soluble in water, ethanol, acetic acid and ammonia, insoluble in ether, acetone, ethyl acetate, benzene, carbon disulfide and carbon tetrachloride, it can be dissolved in neutral and weak alkaline solutions. Air is oxidized to cystine. L-cysteine has the effect of anti-radiation and treatment of radiation sickness.
  • cysteine as a reducing agent has been reported (Nairn, NW et al. Cysteine as a Monothiol Reducing Agent to Prevent Copper-Mediated Oxidation of Interferon Beta During PEGylation by CuAAC. Bioconjug Chem. 2015, 21; 26 (10 ): 2070-5; Sharma, B et al. Biologically active L-cysteine as a reducing/capping agent for controlled tuning of gold nanoparticles. J. Alloys Compd. 2015, 649: 11-18; Borghei, YS et al. Novel Fluorometric Assay for Detection of Cysteine as a Reducing Agent and Template in Formation of Copper Nanoclusters. J Fluoresc. 2017, 27(2):529-536.).
  • the actual technical problem to be solved by the present invention is to provide a cysteine-containing capillary gel electrophoresis sample buffer solution for the problem that the existing reduced CE-SDS detection method is more harmful.
  • the solution can improve the safety of reducing CE-SDS detection of protein biopharmaceuticals and ensure the accuracy of the results.
  • One aspect of the present invention discloses the application of cysteine as a reducing agent in preparing a sample buffer for capillary gel electrophoresis.
  • Another aspect of the present invention discloses a capillary gel electrophoresis sample buffer containing cysteine, and the cysteine in the sample buffer is used as a reducing agent, and its concentration is 7-70 mM.
  • the buffer is selected from Tris buffer or phosphate buffer, and the pH value of the sample buffer is between 3.0-10.0; the buffer is preferably selected from Tris-HCl buffer or phosphate-citric acid Buffer, pH between 6.5-10.0.
  • the buffer is selected from Tris-HCl buffer at pH 9.0 or phosphate-citrate buffer at pH 6.5.
  • sample buffer further contains sodium dodecyl sulfate, and its content is between 0.1-5% (w/w), preferably 0.5-2% (w/w), more preferably 1% (w/w) w).
  • sample buffer contains the following components:
  • the pH is 9.0.
  • the preparation method is as follows: the Tris buffer salt in the recipe, the sodium lauryl sulfate in the recipe and the cysteine in the recipe are dissolved in deionized water, the pH is adjusted to 9.0 with hydrochloric acid, and then determined with deionized water. Volume to 50mL to obtain sample buffer.
  • sample buffer contains the following components:
  • the pH is 6.5.
  • the preparation method is as follows: dissolving the phosphate in the recipe amount, the sodium lauryl sulfate in the recipe amount and the cysteine in the recipe amount in deionized water, adjusting the pH to 6.5 with citric acid, and then adjusting the pH with deionized water. Volume to 50mL to obtain sample buffer.
  • the present invention also discloses the application of the above-mentioned sample buffer for reducing CE-SDS detection of protein samples, wherein the sample is selected from recombinant anti-ricin humanized monoclonal antibody, recombinant anti-HER2 humanized monoclonal antibody Antibody, T-DM1 and recombinant human tumor necrosis factor receptor type II-antibody fusion protein for injection.
  • cysteine-added capillary gel electrophoresis sample buffer of the present invention according to the sample processing method recommended by CE manufacturer Beckman (heating at 70°C for 15 minutes), the accuracy of the purity of biopharmaceuticals such as antibodies can be accurately characterized, and at the same time Significantly reduce the safety problems caused by traditional reduction methods, reduce environmental pollution and harm to the human body.
  • Figure 8 The effect of 70 mM cysteine in pH 9.0 buffer (without SDS) on the hydrophilicity/hydrophobicity of glucagon in Example 7, the reducing agent was heated at 70°C for 15 minutes in pH 9.0 buffer ( a); Glucagon in pH 9.0 buffer at 70°C for 15 minutes (b); reducing agent and glucagon in pH 9.0 buffer at 70°C for 15 minutes (c).
  • Example 1 The effect of different concentrations of cysteine on the purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) in reduced CE-SDS purity in pH 9.0 buffer
  • Tris 0.606g, SDS 0.5g, cysteine (MW: 121.16) 0.424g dissolve with 42mL deionized water until clear, then adjust pH to 9.0 with 3M hydrochloric acid, and then dilute to 50mL with deionized water, A sample buffer containing 70 mM cysteine was obtained. Due to solubility limitations, a buffer concentration of 700 mM was not available. Then, the sample buffer containing no cysteine and the sample buffer containing 70 mM cysteine were sequentially diluted to obtain the sample buffer containing 7 mM, 0.7 mM and 0.07 mM cysteine. The recombinant anti-ricin humanized monoclonal antibody (mAb1) was diluted to 1 mg/mL with the above-prepared sample buffer, then denatured in a water bath, and analyzed by capillary gel electrophoresis.
  • mAb1 anti-ricin humanized monoclonal antibody
  • Example 2 The effect of different concentrations of cysteine on the purity of recombinant anti-HER2 humanized monoclonal antibody (mAb2, batch in 2013) by reduced CE-SDS in pH 9.0 buffer
  • Example 3 The effect of different concentrations of cysteine on the purity of recombinant anti-HER2 humanized monoclonal antibody (mAb2, batch in 2016) by reduced CE-SDS in pH 9.0 buffer
  • Example 4 The effect of different concentrations of cysteine on the purity of reduced CE-SDS of T-DM1 (ADC) in the buffer of pH 9.0
  • Example 5 Effect of different concentrations of cysteine on the purity of reduced CE-SDS of recombinant human type II tumor necrosis factor receptor-antibody fusion protein (Fusion protein) for injection in pH 9.0 buffer
  • Example 6 The effect of different concentrations of cysteine on the purity of recombinant anti-ricin humanized monoclonal antibody (mAb1) in reduced CE-SDS purity in pH 6.5 buffer
  • Example 7 Effect of 70 mM cysteine in buffer pH 9.0 (no SDS) on the hydrophilicity/hydrophobicity of glucagon
  • Tris-HCl buffer without SDS, pH 9.0 was configured as blank buffer. Dissolve glucagon with blank buffer at a final concentration of 0.5 mg/mL, and perform reverse-phase chromatography analysis after heat incubation to determine whether the hydrophilic/hydrophobicity of glucagon changes with heat treatment. At the same time, a blank buffer containing different reducing agents was prepared, and the final concentration of free sulfhydryl groups was 70 mM. After passing through a water bath, reversed-phase chromatography was performed to serve as a blank control. Finally, glucagon was dissolved in buffers containing different reducing agents, and then reversed-phase chromatography was performed after thermal incubation to determine whether the hydrophilicity/hydrophobicity of glucagon was changed. The results are shown in Figure 8.

Abstract

La présente invention concerne un tampon d'échantillon d'électrophorèse sur gel capillaire contenant de la cystéine. La cystéine agit en tant qu'agent réducteur et sa concentration est de 7 à 70 mM. Le tampon est choisi parmi des tampons Tris et des tampons phosphate, le pH est compris entre 3,0 et 10,0 et le tampon d'échantillon contient en outre entre 0,1 et 5 % en poids de dodécylsulfate de sodium. L'invention concerne également un procédé destiné à la préparation d'un tampon d'échantillon et l'utilisation du tampon d'échantillon pour une détection par CE-SDS réduite d'un échantillon de protéine.
PCT/CN2021/115987 2021-04-28 2021-09-01 Tampon d'échantillon d'électrophorèse sur gel capillaire contenant de la cystéine WO2022227363A1 (fr)

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CN202110469258.6 2021-04-28
CN202110469258.6A CN113189184B (zh) 2021-04-28 2021-04-28 含有半胱氨酸的毛细管凝胶电泳样品缓冲液

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