CN114216971A - Ultra-high performance liquid chromatography detection of dithiothreitol residue in protein product - Google Patents
Ultra-high performance liquid chromatography detection of dithiothreitol residue in protein product Download PDFInfo
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Abstract
Discloses a method for determining Dithiothreitol (D) in protein biological medicine by using ultra-performance liquid chromatography UPLCTT) residual amount, comprising the steps of: preparing a DTT standard substance aqueous solution; obtaining a filtrate of the protein sample; obtaining ultrapure water as a blank solution; adding excessive Cu (NO) into DTT standard, filtrate of protein sample and ultrapure water3)2Uniformly mixing to obtain a mixture, and carrying out UPLC analysis on the mixture to obtain a DTT peak; and drawing a standard curve of the relative DTT concentration of the DTT peak area obtained by the standard substance aqueous solution, and quantifying the concentration of the residual DTT in the sample by using an external standard method according to the measured DTT peak area.
Description
(1) Field of the invention
The disclosure relates to the field of biological analysis, in particular to an Ultra Performance Liquid Chromatography (UPLC) detection method for determining DTT residual quantity of dithiothreitol in a protein sample.
(2) Background of the invention
Recombinant proteins often undergo polymer formation during their production, and reducing agents are added during protein purification to prevent polymer formation and to ensure molecular biological activity. Dithiothreitol (Dithiothreitol DTT) is a small molecule reducing agent that can be used to prevent disulfide bond formation at cysteine residues of proteins.
DTT belongs to sulfhydryl compounds, and the entering of DTT into the body can destroy the protein structure, thereby causing phenomena such as enzyme inactivation and the like, so that the residual quantity of the reducing agent in samples and final products in the process needs to be strictly monitored. According to the report, the DTT content can be measured by using a cobalt ion back titration method, but the DTT is easy to be oxidized, and the sensitivity of the method is not high. In addition, researchers also research a method for detecting dithiothreitol by using a graphene quantum dot electrode, and the method is simple to operate and high in sensitivity, but is limited to high price of a preparation process and less application in industrial production.
The high performance liquid chromatography is widely applied in industrial production and can be used for detecting the DTT residual quantity. One of the liquid chromatography-based test methods is to use monobromodiamine (mBBr) fluorescence probe to perform derivatization on DTT, and to use reversed-phase liquid chromatography and a fluorescence detector to perform quantitative analysis on DTT after derivatization, and the method has high sensitivity, but the sample pretreatment process is complex, the requirements on reaction environment are strict, and the method is not suitable for detecting the DTT residue in the industrial production process. In addition, liquid chromatography is adopted to simultaneously carry out quantitative analysis on two coexisting forms of the reduction state and the oxidation state of the dithiothreitol, but the method needs to simultaneously generate standard curves of the two substances, and the detection workload is increased.
Further, it has been reported that DTT is entirely converted to an oxidized state by copper ions and then analyzed by liquid chromatography, and LOQ is 0.3. mu.g/mL by a UV detection method in the literature. However, for recombinant protein products with large clinical administration volume, the content of DTT in the preparation needs to be controlled in a lower concentration range, and for the situation, the existing method cannot ensure that the DTT content in the product is in a safe range by the limit of quantitation, so that the method is reduced in the limit of quantitation, the sensitivity is improved, and the method has important significance for ensuring the safety of the protein products. Based on the above needs, there is still a need in the art for better methods for optimizing the liquid phase analysis conditions of DTT and increasing the sensitivity.
(3) Summary of the invention
Therefore, it is an object of the present invention to provide a UPLC method for detecting the residual amount of dithiothreitol in a protein biological product with higher sensitivity.
Therefore, one aspect of the present invention provides a method for determining the residual amount of dithiothreitol DTT in a protein biological medicine by using ultra performance liquid chromatography UPLC, which comprises the following steps:
(1) preparing DTT standard substance water solution with concentration range of 0.001-10 mug/mL, preferably 0.025-1.0 mug/mL;
(2) centrifuging the purified protein sample to be detected without cell debris and culture medium components to remove proteins, preferably at 14000rcf, more preferably in a 10KD ultrafiltration centrifuge tube at 4 ℃, and filtering to obtain a sample filtrate;
(3) respectively transferring ultrapure water with the same volume, the DTT standard substance aqueous solution and the sample filtrate obtained in the step (2), and respectively adding excessive Cu (NO) into the ultrapure water, the DTT standard substance aqueous solution and the sample filtrate3)2Preferably, the concentration is 10mg/mL, and the mixture is uniformly mixed to obtain a mixture for further UPLC analysis;
(4) loading the mixture obtained in the step (3) onto a UPLC column for chromatographic analysis, and taking ultrapure water as a mobile phase A and acetonitrile containing 10% of ultrapure water as a mobile phase B to obtain a DTT peak;
(5) and drawing a standard curve of the relative DTT concentration of the DTT peak area obtained by the standard substance aqueous solution, and quantifying the concentration of the residual DTT in the sample by using an external standard method according to the measured DTT peak area.
In a particular embodiment of this aspect, the proteinaceous biopharmaceutical includes natural or synthetic proteins, such as antibodies, enzymes, fusion proteins; and/or the protein comprises modifications, such as glycosylation, methylation, phosphorylation, acetylation, hydroxylation modifications; and/or the protein comprises unnatural amino acids, e.g., D-amino acids, pyrrolysine.
In a preferred embodiment of the aspect, the preparation method of the DTT standard aqueous solution in step (1) is to weigh 20.0 ± 0.2mg of DTT powder into a 100mL volumetric flask, add ultrapure water to the volumetric flask to fix the volume, and prepare a standard stock solution of 200 μ g/mL; transferring 1.0mL of 200 mu g/mL DTT standard stock solution into a 100mL volumetric flask, adding ultrapure water to a constant volume, and uniformly mixing for later use to prepare a standard substance with the concentration of 2.0 mu g/mL; then, 2.0. mu.g/mL of the standard was diluted in a gradient to give a series of standard solutions having concentrations of 1.0. mu.g/mL, 0.50. mu.g/mL, 0.2. mu.g/mL, 0.1. mu.g/mL, 0.05. mu.g/mL, and 0.025. mu.g/mL.
In another preferred embodiment of this aspect, the UPLC column is a Waters access UPLC HSS T31.8 μm,2.1 x 100 mm.
In another preferred embodiment of this aspect, the column temperature is 30 ℃.
In yet another preferred embodiment of this aspect, step (4) comprises detecting the DTT peak at 285 nm.
In a preferred embodiment of this aspect, the flow rate of the UPLC column is 0.3 mL/min; the amount of sample was 80. mu.L.
In another preferred embodiment of this aspect, the specific course of gradient elution in the chromatographic assay is:
in still another preferred embodiment of this aspect, further comprising using the DTT solution of the specified concentration as a process control in step (3), preferably 0.025 to 1.0. mu.g/mL DTT solution; more preferably 0.025. mu.g/mL DTT in water.
The DTT detection method has the advantages that: by further optimizing liquid phase analysis conditions, including chromatographic column, elution gradient, flow rate and other conditions, and by making a standard curve of a standard sample, the concentration of DTT can be conveniently determined by linear fitting of the peak area of DTT detected at 285nm wavelength, the detection range reaches 0.025 mu g/mL-1.0 mu g/mL, and the linear relation is good; for recombinant protein products with large clinical administration volume, the DTT residual quantity in a lower concentration range can be detected, and the DTT content in the product is ensured to be in a safe range.
Additional features and advantages of various embodiments will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of various embodiments. The objectives and other advantages of the various embodiments will be realized and attained by means of the elements and combinations particularly pointed out in the description and appended claims.
Unless otherwise indicated, reagents, cells, and instrumentation used in the present invention are commonly commercially and publicly available.
(4) Description of the drawings
The present disclosure is further described with reference to the accompanying drawings, wherein the showings are for the purpose of illustrating embodiments of the disclosure only and not for the purpose of limiting the scope of the disclosure.
FIG. 1 shows liquid phase spectra of DTT standard solution and blank solution at a concentration of 0.5. mu.g/mL.
Figure 2 shows a DTT standard curve.
(5) Detailed description of the preferred embodiments
Reference will now be made in detail to some embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents as may be included within the invention as defined by the appended claims.
The Ultra-high Performance Liquid Chromatography (UPLC) used in the invention covers brand-new technologies such as small particle packing, very low system volume, rapid detection means and the like by means of the theory and principle of HPLC (high Performance Liquid Chromatography), and increases the flux, sensitivity and chromatographic peak capacity of analysis. The ultra-high pressure infusion pump is used for realizing the sample loading of the chromatographic column, and the smaller chromatographic column particles are used for realizing the substance separation.
The HSS High Strength Silica gel (High Strength Silica) used in the embodiments of the present invention is designed to improve mechanical Strength and have a suitable morphology to withstand High pressure conditions with good column life and UPLC efficiency, which is capable of retaining water-soluble, highly polar small molecule organic compounds. The HSS T3 stationary phase is a C18 stationary phase compatible with a 100% aqueous mobile phase designed to retain and separate polar organics, much like the chromatographic behavior of an Atlantis T3 HPLC column.
In embodiments of the invention, the protein sample to be detected may be any kind of protein sample purified free of cell debris or culture medium components, including but not limited to natural or synthetic proteins, such as antibodies, enzymes, fusion proteins; and/or the protein comprises modifications, such as glycosylation, methylation, phosphorylation, acetylation, hydroxylation modifications; and/or the protein comprises unnatural amino acids, e.g., D-amino acids, pyrrolysine. The molecular weight of the protein is preferably not less than 10 KD.
Example UPLC detection of Total dithiothreitol residual amount of protein-based biological products
1. Instruments and reagents
1.1 reagents
1.2 instruments
2. Preparation of solutions
2.1 preparation of Standard solutions
Weighing 20mg of dithiothreitol standard substance into a 100mL volumetric flask, adding ultrapure water to a constant volume, and preparing into 0.2mg/mL of standard solution mother liquor; then 0.2mg/mL of the stock solution was diluted to 2.0. mu.g/mL in a 100mL volumetric flask, and then the stock solution of the standard solution was subjected to gradient dilution as shown in the following table. After dilution, 200. mu.L of each standard solution was put into a centrifuge tube, and 4.0. mu.L of Cu (NO3) was added2The solution was mixed and loaded into the liner and ready for UPLC analysis.
2.2 preparation of test solutions
Taking 400 μ L of the recombinant protein product purified by column chromatography, keeping the concentration in the solution at 4.0mg/mL, placing into an ultrafiltration centrifuge tube with 10KD, centrifuging at 14000rcf rate for 30mins at 4 deg.C, taking 200 μ L of the filtrate into the centrifuge tube, adding 4.0 μ L of Cu (NO3)2The solution was mixed and loaded into the liner and ready for UPLC analysis.
3. Methodology validation
3.1 chromatographic conditions: agilent Technologies 1290 ultra high performance liquid chromatograph; a chromatographic column: waters acquisition UPLC HSS T3 column (1.8 μm,2.1 × 100 mm); mobile phase: mobile phase A ultrapure water, and mobile phase B is acetonitrile solution containing 10% ultrapure water; column temperature: 30 ℃; flow rate: 0.3 mL/min; detection wavelength: 285 nm; sample introduction volume: 80 μ L.
3.2 specificity
In order to examine whether an interference peak exists in a blank sample, the specificity of the experimental method is detected. And taking a standard solution with the DTT concentration of 0.5 mu g/mL and a blank solution to obtain a chromatogram shown in figure 1, wherein the result shows that the blank solution without DTT has no interference peak at the position where the DTT appears, and the method has good specificity.
3.3 Linearity and Range
Taking the standard solution prepared in the step 2.1, and detecting according to the method of 3.1. Taking the concentration of dithiothreitol as the abscissa X and the peak area of dithiothreitol as the ordinate Y, performing linear fitting to obtain a linear equation, namely a standard curve (figure 2). The method has good linear relation in the concentration range of dithiothreitol (0.025 mu g/mL-1.0 mu g/mL).
3.5 degree of accuracy
The recovery of the process at different concentrations was investigated. Taking a recombinant protein product which does not contain dithiothreitol and is purified by column chromatography, keeping the concentration of the recombinant protein product in the solution to be 4.0mg/mL, adding a dithiothreitol standard solution into a sample to ensure that the theoretical final concentration of the dithiothreitol is 0.025 mu g/mL, and measuring according to a method of 3.1, wherein the measuring results are shown in the following table. The test results show that the recovery rate can be between 80 and 120 percent at 0.025 mu g/mL, the method has good accuracy and the LOQ is 0.025 mu g/mL.
By combining the results, the method can conveniently and accurately determine the concentration of the residual dithiothreitol in the protein sample, can effectively monitor the residual dithiothreitol in the protein biological products, and provides excellent detection limit and sensitivity.
It will be apparent to those of ordinary skill in the art that various modifications and variations can be made to the various embodiments described herein without departing from the spirit or scope of the teachings herein. Thus, it is intended that the various embodiments cover other modifications and variations of the various embodiments within the scope of the present teachings.
Claims (9)
1. A method for determining the DTT residual quantity in protein biological medicine products by using ultra-high performance liquid chromatography UPLC is characterized by comprising the following steps:
(1) preparing DTT standard substance water solution with concentration range of 0.001-10 mug/mL, preferably 0.025-1.0 mug/mL;
(2) centrifuging the purified protein sample to be detected without cell debris and culture medium components to remove proteins, preferably at 14000rcf, more preferably in a 10KD ultrafiltration centrifuge tube at 4 ℃, and filtering to obtain a sample filtrate;
(3) respectively transferring ultrapure water with the same volume, the DTT standard substance aqueous solution and the sample filtrate obtained in the step (2), and respectively adding excessive Cu (NO) into the ultrapure water, the DTT standard substance aqueous solution and the sample filtrate3)2Preferably, the concentration is 10mg/mL, and the mixture is uniformly mixed to obtain a mixture for further UPLC analysis;
(4) loading the mixture obtained in the step (3) onto a UPLC column for chromatographic analysis, and taking ultrapure water as a mobile phase A and acetonitrile containing 10% of ultrapure water as a mobile phase B to obtain a DTT peak;
(5) and drawing a standard curve of the relative DTT concentration of the DTT peak area obtained by the standard substance aqueous solution, and quantifying the concentration of the residual DTT in the sample by using an external standard method according to the measured DTT peak area.
2. The method of claim 1, wherein the proteinaceous biopharmaceutical comprises a natural or synthetic protein, such as an antibody, an enzyme, a fusion protein; and/or the protein comprises modifications, such as glycosylation, methylation, phosphorylation, acetylation, hydroxylation, and PEG-modification, chemical small molecule modification; and/or the protein comprises unnatural amino acids, e.g., D-amino acids, pyrrolysine.
3. The method of claim 1, wherein the DTT standard aqueous solution in step (1) is prepared by weighing 20.0 ± 0.2mg of DTT powder into a 100mL volumetric flask, adding ultrapure water to a constant volume to prepare a 200 μ g/mL standard stock solution; transferring 1.0mL of 200 mu g/mL DTT standard stock solution into a 100mL volumetric flask, adding ultrapure water to a constant volume, and uniformly mixing for later use to prepare a standard substance with the concentration of 2.0 mu g/mL; then, 2.0. mu.g/mL of the standard was diluted in a gradient to give a series of standard solutions having concentrations of 1.0. mu.g/mL, 0.50. mu.g/mL, 0.2. mu.g/mL, 0.1. mu.g/mL, 0.05. mu.g/mL, and 0.025. mu.g/mL.
4. The method of claim 1, wherein the UPLC column is Waters acquisition UPLC HSS T31.8 μm,2.1 x 100 mm.
5. The method of claim 1, wherein the column temperature is 30 ℃.
6. The method of claim 1, wherein step (4) comprises detecting the DTT peak at 285 nm.
7. The method of claim 1, wherein the flow rate of the UPLC column is 0.3 mL/min; the amount of sample was 80. mu.L.
8. The method of claim 1, wherein the specific course of gradient elution in the assay is:
9. the method of claim 1, further comprising using the DTT solution of the specified concentration as a process control in step (3), preferably 0.025-1.0 μ g/mL DTT solution.
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