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Method for separating and enriching peptide impurities in peptide drugs by pulse incubation immune reaction
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An immune reaction, separation and enrichment technology, applied in the field of biochemical analysis, can solve the problems of affinity reduction, capture, and drug peptide impurity analysis, which are rarely reported, and achieve high separation efficiency and reduce the possible effect of simultaneous extraction
Active Publication Date: 2021-05-07
UNIV OF SCI & TECH BEIJING
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[0006] Immunomagnetic bead affinity extraction technology has been widely used in the purification, analysis and detection of protein or peptide drugs, but it is rarely reported in the analysis of drug peptide impurities.
The reason is mainly caused by the following two defects: 1) The antibody binds to the antigenic determinant of the protein or polypeptide, that is, it binds to a local region of the protein or antigen. If they are the same, they will also be captured by antibodies; 2) Compared with the main component, some impurities have slight changes in the conformation of epitopes, although the affinity with antibodies will be reduced, but they will still bind to antibodies
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Embodiment 1
[0034] Example 1 Separation, enrichment and detection of peptide impurities in C-peptide standard
[0035] Preparation of immunomagnetic beads for binding C-peptide
[0036] 1. Washing of Magnetic Beads
[0037] 1) Vortex to resuspend carboxylated magnetic beads. Pipette 100 μL of magnetic beads into two 2 mL centrifuge tubes, magnetically separate and discard the supernatant.
[0038] 2) Add 500 μL of coupling buffer (50 mM MES, pH 6.0, 0.01% Triton X-100) to the two tubes, vortex for 20 s to wash the magnetic beads, place the centrifuge tube on the magnetic stand for 60 s, magnetically separate and discard clear. Repeat the washing step three times.
[0039] 2. Activation of Magnetic Beads
[0040] 1) Prepare EDC (50mg / mL) and NHS (50mg / mL) with coupling buffer (ready-to-use), add 60 μL of coupling buffer, 20 μL of newly prepared EDC solution and 20 μL of newly prepared NHS solution, vortexed to mix.
[0041] 2) After incubating at room temperature for 15 minutes, pla...
Embodiment 2
[0068] Example 2 Comparison of traditional immunoaffinity extraction and the method of the present invention for the separation effect of C peptide impurities
[0069] Prepare 9 standards of C-peptide structurally similar impurities with ultrapure water, so that the concentration is 100ng / mL. Respectively using a single immunomagnetic bead I, immunomagnetic bead II combined with two kinds of immunomagnetic beads in the method of the present invention to separate the impurities in the sample solution, and use high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS ) to verify whether the impurities can be detected, so as to compare the separation efficiency of peptide impurities under the two methods, the results are shown in Table 1.
[0070] The amino acid sequence of the C peptide is: EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ
[0071] The impurity standards used and their sequences are as follows,
[0072] dea6hCP:EAEDLEVGQVELGGGPGAGSLQPLALEGSLQ
[0073] dea9hCP...
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Abstract
The invention relates to a method for separating and enriching peptide impurities in polypeptide drugs by pulse incubation immune reaction, which belongs to the field of biochemical analysis. Using two kinds of immunoaffinity magnetic beads coupled with antibodies against different antigenic determinants of polypeptides, under certain pulse incubation conditions, the polypeptide impurities in the polypeptide solution containing impurities are sequentially combined and captured to separate them from impurities , the peptide impurities in the supernatant after magnetic separation are enriched with a solid-phase extraction column, desalted at the same time, and used for chromatography-mass spectrometry analysis after elution. The invention greatly reduces the possibility of simultaneous extraction of impurities with similar structures; pulse incubation is adopted in the extraction process, and the impurities with relatively weak binding force on the antibody are replaced by the main component with strong binding force through the change of reaction temperature, further reducing The possibility of impurities being extracted at the same time is eliminated, making the separation efficiency higher. The separated impurities are concentrated and desalted by a solid-phase extraction column, freeze-dried and reconstituted, and the peptide impurities can be qualitatively and quantitatively analyzed by liquid chromatography-mass spectrometry.
Description
technical field [0001] The invention belongs to the field of biochemical analysis, and specifically relates to a method for enriching peptide impurities in polypeptide drugs by using immunomagnetic beads and a solid-phase extraction agent under certain pulse incubation conditions and its application. Background technique [0002] In recent years, therapeutic peptides and proteins have gradually become a hot spot in drug research and development, and are widely used because of their advantages such as relatively low cost, strong biological activity, low toxicity and side effects, and specific action targets. Most peptide drugs are obtained by solid-phase peptide synthesis techniques. During the synthesis and storage of polypeptide drugs, related structural impurities are easily formed, such as amino acid loss, amino acid insertion, protective group residue, oxidation / reduction, diastereoisomer impurities produced by racemization of some amino acids, etc. Classical biochemica...
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