WO2024037398A1 - Method for measuring content of pentafluorophenol - Google Patents

Method for measuring content of pentafluorophenol Download PDF

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Publication number
WO2024037398A1
WO2024037398A1 PCT/CN2023/111922 CN2023111922W WO2024037398A1 WO 2024037398 A1 WO2024037398 A1 WO 2024037398A1 CN 2023111922 W CN2023111922 W CN 2023111922W WO 2024037398 A1 WO2024037398 A1 WO 2024037398A1
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mobile phase
pentafluorophenol
column
solution
trifluoroacetic acid
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PCT/CN2023/111922
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French (fr)
Chinese (zh)
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李小华
葛均友
唐懿挺
闵聆
雷富彬
李森武
陈红
谷伟瑶
熊优
张润
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四川科伦博泰生物医药股份有限公司
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Publication of WO2024037398A1 publication Critical patent/WO2024037398A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography

Definitions

  • the invention belongs to the field of analytical chemistry, and specifically relates to a method for determining the content of pentafluorophenol, and in particular to a method for determining the content of pentafluorophenol in drugs using high-performance liquid chromatography.
  • ADCs antibody-drug conjugates
  • lysine conjugation is achieved by binding a lysine residue to an activated ester such as N-hydroxysuccinyl. This is achieved by forming amide bonds between imines (NHS).
  • Pentafluorophenol ester is a potential active ester for protein coupling due to its high reactivity, stability, and high coupling efficiency. It has been widely used in peptide synthesis. Due to its stability in body circulation, it is often used as a connector for ADC drugs. It is a non-cleavable type and relies on intracellular lysosomal degradation to release effective small molecules in ADC. load.
  • Pentafluorophenol (PFP) is a commonly used raw material for the preparation of pentafluorophenyl active esters in peptide synthesis.
  • the quality control of the final ADC product is very critical to the efficacy of the drug in vivo.
  • reactions using pentafluorophenyl ester linkers will produce free PFP impurities.
  • PFP impurities produced during the process have teratogenic toxicity, and premature release of the cytotoxic payload can also lead to off-target toxicity, which poses certain safety risks and requires strict control.
  • the detection of PFP residues in ADC drugs is affected by the small amount of residues and large molecular proteins. The existing reported methods are not suitable for the detection of PFP residues in ADC drugs.
  • the purpose of this application is to establish a reversed-phase high-performance liquid phase detection method for determining the residual amount of PFP.
  • this method is easy to operate, the optimized chromatographic conditions are easy to operate and control, has good durability, has very high sensitivity, can accurately and quantitatively determine extremely low levels of PFP impurities, and can be widely used in small molecules and
  • the quality analysis and control of macromolecular drugs can achieve the goals of efficient, platform-based and environmentally friendly quality control, which is conducive to industrial use.
  • This application provides a method for determining the content of pentafluorophenol using HPLC.
  • chromatographic conditions such as chromatographic columns, mobile phases, and elution procedures
  • the content of pentafluorophenol can be determined accurately and effectively.
  • the method provided in this application has very high detection sensitivity and can accurately measure it quantitatively.
  • This application provides a method for determining the content of pentafluorophenol, which is characterized in that HPLC is used for detection, and the chromatographic conditions of the HPLC include:
  • the chromatographic column is C8 column or C18 column;
  • Mobile phases A and B are respectively selected from trifluoroacetic acid-aqueous solution or trifluoroacetic acid-acetonitrile solution;
  • 0 ⁇ 5min The volume percentages of mobile phase A and mobile phase B are 70% ⁇ 80% and 30% ⁇ 20% respectively;
  • the volume percentages of mobile phase A and mobile phase B are 50% ⁇ 45% and 50% ⁇ 55% respectively;
  • 26 ⁇ 33min The volume percentages of mobile phase A and mobile phase B are 0% and 100% respectively;
  • the volume percentages of mobile phase A and mobile phase B are 70% ⁇ 80% and 30% ⁇ 20% respectively.
  • This application provides a method for determining the content of pentafluorophenol, which is characterized in that HPLC is used for detection, and the chromatographic conditions of the HPLC include:
  • the chromatographic column is C8 column or C18 column;
  • Mobile phases A and B are respectively selected from trifluoroacetic acid-aqueous solution or trifluoroacetic acid-acetonitrile solution;
  • 0 ⁇ 5min The volume percentages of mobile phase A and mobile phase B are 70% ⁇ 80% and 30% ⁇ 20% respectively;
  • the volume percentages of mobile phase A and mobile phase B are 50% ⁇ 45% and 50% ⁇ 55% respectively;
  • the volume percentages of mobile phase A and mobile phase B are 0% and 100% respectively;
  • the volume percentages of mobile phase A and mobile phase B are 70% ⁇ 80% and 30% ⁇ 20% respectively.
  • the mobile phase A is a trifluoroacetic acid-aqueous solution
  • the mobile phase B is a trifluoroacetic acid-acetonitrile solution
  • the mobile phase A is a trifluoroacetic acid-aqueous solution with a volume percentage of 0.08% to 0.12%
  • Phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.08%-0.12%; more preferably, the mobile phase A is a trifluoroacetic acid-water solution with a volume percentage of 0.1%, and the mobile phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.1%.
  • Fluoroacetic acid-acetonitrile solution is a trifluoroacetic acid-aqueous solution
  • the mobile phase B is a trifluoroacetic acid-acetonitrile solution
  • HPLC is used for detection.
  • the chromatographic conditions include: the chromatographic column is a C8 chromatographic column, the mobile phase A is a trifluoroacetic acid-water solution with a volume percentage of 0.1%, and the mobile phase B is a trifluoroacetic acid-water solution with a volume percentage of 0.1%.
  • Acetic acid-acetonitrile solution, gradient elution program selected from any of the following:
  • the chromatographic column is an SB-C8 chromatographic column or a CSH C18 chromatographic column; preferably, the chromatographic column is SB-C8, 5 ⁇ m 4.6 ⁇ 250 mm chromatographic column column or CSH C18, 3.5 ⁇ m 4.6 ⁇ 150mm chromatographic column; preferably, the chromatographic column is Agilent Zorbax 300 SB-C8, 5 ⁇ m 4.6 ⁇ 250mm chromatographic column or Xselect CSH C18, 3.5 ⁇ m 4.6 ⁇ 150mm chromatographic column; more preferably , the column is Agilent Zorbax 300 SB-C8, 5 ⁇ m 4.6 ⁇ 250mm column.
  • a chromatographic column with similar performance to the Agilent Zorbax300SB-C8 column can also be selected.
  • the sample injection volume is 50-100 ⁇ l; preferably, the sample injection volume is 100 ⁇ l.
  • the limit of quantification of pentafluorophenol is 0.1 ⁇ g/ml.
  • the chromatographic conditions also include: the detection wavelength is 254-265nm, the flow rate is 1-1.5ml/min, and the column temperature is 55-65°C; preferably, the chromatography The conditions also include: the detection wavelength is 264nm, the flow rate is 1-1.5ml/min, and the column temperature is 57-63°C.
  • the chromatographic conditions also include: the detection wavelength is 264 nm, the flow rate is 1 ml/min, and the column temperature is 60°C.
  • the preparation method of mobile phase A is: measure 1000 ml of ultrapure water, add 1 ml of trifluoroacetic acid, mix evenly, and filter through a 0.22 ⁇ m filter membrane to obtain ;
  • the preparation method of mobile phase B is: measure 1000ml of acetonitrile, add 1ml of trifluoroacetic acid, mix evenly, and filter through a 0.22 ⁇ m filter membrane to obtain.
  • the method for determining pentafluorophenol content includes the following steps:
  • HPLC is used for detection, and the chromatographic conditions of HPLC include:
  • the chromatographic column is Agilent Zorbax 300 SB-C8, 5 ⁇ m 4.6 ⁇ 250mm column;
  • Mobile phase A is a trifluoroacetic acid-water solution with a volume percentage of 0.1%
  • mobile phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.1%;
  • the injection volume is 100 ⁇ l
  • the detection wavelength is 264nm
  • the flow rate is 1ml/min
  • the gradient elution program is
  • the method for determining pentafluorophenol content includes the following steps:
  • step (3) Inject each solution in step (1) into the liquid chromatograph according to the above injection volume, and use a UV detector to record the chromatogram.
  • the concentration of the pentafluorophenol standard solution is selected from the group consisting of 0.1 ⁇ g/ml, 0.2 ⁇ g/ml, 0.5 ⁇ g/ml, 1.0 ⁇ g/ml, 2.0 ⁇ g/ml and 5.0 ⁇ g/ml.
  • a diluent is used to dissolve the pentafluorophenol reference substance to obtain the pentafluorophenol standard solution; preferably, the diluent is an acetonitrile-water solution with a volume concentration of 30%.
  • the test solution is a pharmaceutical solution.
  • the drug is an ADC or a small molecule compound; preferably, the molecular weight of the ADC is 150 ⁇ 50kD, and the ADC may produce pentafluorophenol during preparation; preferably, the toxin in the ADC drug is Auristatin compounds, or the small molecule compound is a small molecule compound that easily produces pentafluorophenol during the production process; preferably, the small molecule compound is an auristatin compound.
  • the determination method provided by this application can be used to test the pentafluorophenol content in drugs; preferably, the drug is an ADC coupled drug or a small molecule drug; preferably, the molecular weight of the ADC coupled drug is 150 ⁇ 50KD; preferably, the ADC conjugated drug is a recombinant antibody conjugated drug that produces free molecules of pentafluorophenol in the conjugated product.
  • This application provides a high-performance liquid chromatography method for determining the content of pentafluorophenol in drugs, especially ADC macromolecule drugs.
  • the method has simple chromatographic conditions, convenient operation, high sensitivity, good durability, and can effectively separate the components and Quantitative calculations quickly and accurately realize the quality control of pentafluorophenol impurities to ensure product quality and safety.
  • the method provided in this application is highly versatile, efficient, and fast. Since the detected pentafluorophenol has nothing to do with the structure of the test sample, this detection method can be used as a platform method to take into account the detection of ADC drugs and small molecules and other drugs.
  • Figure 1 shows the HPLC spectrum of Test Example 1 of Embodiment 1;
  • Figure 2 shows the HPLC pattern of Test Example 2 of Embodiment 1;
  • Figure 3 shows the HPLC spectrum of Test Example 3 of Embodiment 1;
  • Figure 4 shows the HPLC chromatogram of elution procedure 3 of Example 2.
  • Figure 5 shows the HPLC chromatogram of elution procedure 4 of Example 2.
  • Figure 6 shows the HPLC pattern of Example 4.
  • the maximum tolerated daily dose of pentafluorophenol in injections, PDE permitted daily exposure
  • PDE permitted daily exposure
  • the maximum tolerated concentration of PFP is 0.134 ⁇ g/ml.
  • Example 1-1 Screening of mobile phase and chromatographic column
  • Diluent 30% acetonitrile water solution, used for solution preparation.
  • PFP reference substance mother solution Weigh the pentafluorophenol standard, dissolve it in acetonitrile, and prepare it to a concentration of approximately 1 mg/ml. solution as the PFP reference substance mother solution.
  • PFP standard working solution Use 30% acetonitrile aqueous solution to dilute the PFP reference substance mother solution to 0.1 ⁇ g/ml, 0.2 ⁇ g/ml, 0.5 ⁇ g/ml, 1.0 ⁇ g/ml, 2.0 ⁇ g/ml, 5.0 ⁇ g/ml respectively, as PFP Standard working fluid. Use the 1.0 ⁇ g/ml solution as the system suitability solution.
  • Preparation of the liquid to be tested Take an appropriate amount of the ADC drug prepared with reference to CN108697809A into the injection vial.
  • the molecular weight of the ADC drug is 150 ⁇ 50kD, and prepare the liquid to be tested.
  • Standardized solution Take the liquid to be tested, add a certain amount of PFP standard to it in the form of standard addition, and prepare spiking solutions with PFP concentrations of 0.1 ⁇ g/ml, 1.0 ⁇ g/ml and 5.0 ⁇ g/ml. Concentration: Make 3 servings.
  • Mobile phase A 0.1% trifluoroacetic acid-water solution (v/v), preparation method: measure 1000ml of ultrapure water, add 1ml of trifluoroacetic acid, and mix;
  • Mobile phase B 0.1% trifluoroacetic acid-acetonitrile solution (v/v), preparation method: measure 1000ml of acetonitrile, add 1ml of trifluoroacetic acid, and mix.
  • Mobile phase A phosphate buffer, preparation method: weigh 2.5g potassium dihydrogen phosphate and 2.5g dipotassium hydrogen phosphate, add 1000ml ultrapure water to dissolve, adjust the pH to 7.5, mix well, and filter through a 0.22 ⁇ m filter membrane;
  • Diluent 30% acetonitrile water solution, used for solution preparation.
  • PFP reference substance mother solution Weigh the pentafluorophenol standard, dissolve it in acetonitrile, and prepare a solution with a concentration of approximately 1 mg/ml as the PFP reference substance mother solution.
  • PFP standard working solution Use 30% acetonitrile aqueous solution to dilute the PFP reference substance mother solution to 0.1 ⁇ g/ml, 0.2 ⁇ g/ml, 0.5 ⁇ g/ml, 1.0 ⁇ g/ml, 2.0 ⁇ g/ml, 5.0 ⁇ g/ml respectively, as PFP Standard working fluid. Use the 1.0 ⁇ g/ml solution as the system suitability solution.
  • Preparation of the liquid to be tested Take an appropriate amount of the ADC drug I-1 prepared with reference to CN106729743A into the injection vial.
  • the molecular weight of the ADC drug is 150 ⁇ 50kD, and prepare the liquid to be tested.
  • Standardized solution Take the liquid to be tested, add a certain amount of PFP standard to it in the form of standard addition, and prepare spiking solutions with PFP concentrations of 0.1 ⁇ g/ml, 1.0 ⁇ g/ml and 5.0 ⁇ g/ml. Concentration: Make 3 servings.
  • Mobile phase A 0.1% trifluoroacetic acid-water solution (v/v), preparation method: measure 1000ml of ultrapure water, add 1ml of trifluoroacetic acid, and mix;
  • Mobile phase B 0.1% trifluoroacetic acid-acetonitrile solution (v/v), preparation method: measure 1000ml of acetonitrile, add 1ml of trifluoroacetic acid, and mix.
  • Mobile phase A phosphate buffer, preparation method: weigh 2.5g potassium dihydrogen phosphate and 2.5g dipotassium hydrogen phosphate, add 1000ml ultrapure water to dissolve, adjust the pH to 7.5, mix well, and filter through a 0.22 ⁇ m filter membrane;
  • Test Example 1-2 uses mobile phase 1 and chromatographic columns 1 and 2.
  • the target peaks all emit normal peaks, have good peak shapes, and have a certain degree of separation from impurity peaks.
  • Test example 3 uses mobile phase 2, and the chromatographic peaks of the system suitability solution are basically consistent with those of the diluent. It can be seen that under this condition, PFP does not emit peaks. Therefore, mobile phase A: 0.1% trifluoroacetic acid-aqueous solution and mobile phase B: 0.1% trifluoroacetic acid-acetonitrile solution were used as the mobile phases for subsequent investigations.
  • the chromatographic column Xselect CSH C18 has a smaller pore size and requires pretreatment (ACN precipitation) of the test sample, so Agilent Zorbax 300 SB-C8 was used for subsequent inspections.
  • elution program 3 When elution program 3 is used, the absorption of the PFP peak is higher, the response is sensitive, and the separation between the PFP peak and other impurity peaks is better, and the protein is completely eluted; shortening the elution time causes the separation between the target peak and other impurity peaks to change. Poor, the protein cannot be completely eluted, so elution program 3 is selected for subsequent tests.
  • Example 2 Use the mobile phase 1, the chromatographic column 1 of the above-mentioned Example 1, and the elution program 3 of Example 2 as the chromatographic conditions. Take the system suitability solution and use the following injection volumes to examine the sample detection. The test plan and results are shown in the table below.
  • the baseline signal-to-noise ratio at the quantitative limit concentration needs to be greater than or equal to 10.
  • the signal-to-noise ratio is around 15-30, which can meet the quantitative detection requirements. Therefore, a 100 ⁇ l injection volume is used. sample size.
  • the process intermediate solution is an intermediate product in the process of preparing ADC drugs, which contains PFP, toxin small molecules and related impurities.
  • chromatographic column 1 and elution procedure 3 of Example 2 as chromatographic conditions, take 3 spiked solutions with a PFP concentration of 0.1 ⁇ g/ml prepared in Example 1, and accurately measure 100 ⁇ l of each solution. Inject into the liquid chromatograph and examine the ratio of the measured concentration to the theoretical concentration (recovery rate), recovery rate RSD% and signal-to-noise ratio.
  • Example 1 Use the mobile phase 1 and chromatographic column 1 of Example 1 and the elution program 3 of Example 2 as chromatographic conditions to examine the peaks of the sample to be tested under different chromatographic column temperatures (57°C, 60°C and 63°C). Condition.

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Abstract

A method for measuring the content of pentafluorophenol. HPLC is used for performing the measurement. Chromatographic conditions of the HPLC comprise: a chromatographic column is a C8 chromatographic column or a C18 chromatographic column; a mobile phase A is a trifluoroacetic acid-aqueous solution, and a mobile phase B is a trifluoroacetic acid-acetonitrile solution; and a gradient elution program comprises: 0-5 min: the volume percents of the mobile phase A and the mobile phase B are respectively 70%-80% and 30%-20%; 5-26 min: the volume percents of the mobile phase A and the mobile phase B are respectively 50%-45% and 50%-55%; 26-33 min: the volume percents of the mobile phase A and the mobile phase B are respectively 0% and 100%; and 33-40 min: the volume percents of the mobile phase A and the mobile phase B are respectively 70%-80% and 30%-20%. The method has good specificity and high sensitivity and universality, and can be used as a platform method.

Description

一种测定五氟苯酚含量的方法A method for determining pentafluorophenol content 技术领域Technical field
本发明属于分析化学领域，具体涉及一种测定五氟苯酚含量的方法，尤其涉及一种采用高效液相色谱测定药物中五氟苯酚含量的方法。The invention belongs to the field of analytical chemistry, and specifically relates to a method for determining the content of pentafluorophenol, and in particular to a method for determining the content of pentafluorophenol in drugs using high-performance liquid chromatography.
背景技术Background technique
新化学和偶联技术的开发对于构建功能增强的蛋白质(例如已成功用作癌症治疗剂的抗体-药物偶联物(ADC))具有重要意义。由于溶剂可及性和蛋白质表面上这些残基的高丰度，蛋白质修饰最通用的方法之一是通过赖氨酸缀合，其通过在赖氨酸残基和活化酯如N-羟基琥珀酰亚胺(NHS)之间形成酰胺键来实现。The development of new chemistry and conjugation techniques is important for the construction of proteins with enhanced functionality, such as antibody-drug conjugates (ADCs) that have been successfully used as cancer therapeutics. Due to solvent accessibility and the high abundance of these residues on the protein surface, one of the most versatile methods for protein modification is through lysine conjugation, which is achieved by binding a lysine residue to an activated ester such as N-hydroxysuccinyl. This is achieved by forming amide bonds between imines (NHS).
多肽合成反应中，酰胺键形成的反应中常采用氨基酸保护基的烷基酯，但该类反应速率非常慢。肽键形成反应的目标是反应高效且无副反应。五氟苯酚酯由于具有较高的反应活性和稳定性，以及较高的偶联效率，是一种潜在的蛋白偶联活泼酯。在多肽合成中得到了广泛的应用，由于其在体内循环中的稳定性，常作为ADC药物的连接头，属于不可切割的类型，依靠细胞内的溶酶体降解来释放ADC中的有效小分子载荷。五氟苯酚(PFP)，是制备多肽合成中五氟代苯基活性酯的常用原料。In peptide synthesis reactions, alkyl esters of amino acid protecting groups are often used in the reaction to form amide bonds, but the reaction rate of this type of reaction is very slow. The goal of peptide bond formation reactions is to be efficient and without side reactions. Pentafluorophenol ester is a potential active ester for protein coupling due to its high reactivity, stability, and high coupling efficiency. It has been widely used in peptide synthesis. Due to its stability in body circulation, it is often used as a connector for ADC drugs. It is a non-cleavable type and relies on intracellular lysosomal degradation to release effective small molecules in ADC. load. Pentafluorophenol (PFP) is a commonly used raw material for the preparation of pentafluorophenyl active esters in peptide synthesis.
ADC终产品的质量控制对于药物体内药效的发挥非常关键。然而采用五氟苯酚酯连接头的反应中，会产生游离的PFP杂质，例如小分子合成工艺或者ADC药物偶联过程中，受小分子反应条件或者ADC药物接头与毒素连接子的稳定性影响，过程中产生的PFP杂质具有致畸毒性，细胞毒性有效载荷的过早释放也会导致脱靶毒性，存在一定的安全隐患，需要进行严格控制。在ADC药物中PFP残留的检测受其残留量少及大分子蛋白的影响，现有报道的方法不适用于ADC药物PFP残留的检测。 The quality control of the final ADC product is very critical to the efficacy of the drug in vivo. However, reactions using pentafluorophenyl ester linkers will produce free PFP impurities. For example, during the synthesis process of small molecules or the coupling process of ADC drugs, it is affected by the reaction conditions of small molecules or the stability of the ADC drug linker and toxin linker. The PFP impurities produced during the process have teratogenic toxicity, and premature release of the cytotoxic payload can also lead to off-target toxicity, which poses certain safety risks and requires strict control. The detection of PFP residues in ADC drugs is affected by the small amount of residues and large molecular proteins. The existing reported methods are not suitable for the detection of PFP residues in ADC drugs.
目前国内企业对PFP的检测，采用气相色谱法对其纯度进行分析，但将PFP作为药物杂质成分进行定量分析鲜有报道，特别是将PFP作为重组抗体偶联药物杂质成分进行定量分析。研究建立PFP残留量的检测方法对于小分子以及ADC药物的质量控制尤为必要。At present, domestic companies use gas chromatography to analyze the purity of PFP when testing it. However, there are few reports on the quantitative analysis of PFP as an impurity component of drugs, especially the quantitative analysis of PFP as an impurity component of recombinant antibody-conjugated drugs. Research and establishment of detection methods for PFP residues are particularly necessary for the quality control of small molecules and ADC drugs.
发明内容概述Summary of the invention
本申请的目的是建立一种测定PFP残留量的反相-高效液相检测方法。通过对检测方法的验证，证明本方法操作简便，优化后的色谱条件容易操作控制，耐用性好，具有非常高的灵敏度，能够准确定量测定含量极低的PFP杂质，可广泛应用于小分子和大分子药物的质量分析控制，实现质量控制高效化、平台化、环境友好化的目标，有利于产业化使用。The purpose of this application is to establish a reversed-phase high-performance liquid phase detection method for determining the residual amount of PFP. Through the verification of the detection method, it is proved that this method is easy to operate, the optimized chromatographic conditions are easy to operate and control, has good durability, has very high sensitivity, can accurately and quantitatively determine extremely low levels of PFP impurities, and can be widely used in small molecules and The quality analysis and control of macromolecular drugs can achieve the goals of efficient, platform-based and environmentally friendly quality control, which is conducive to industrial use.
本申请提供了一种采用HPLC测定五氟苯酚含量的方法，通过对色谱柱、流动相和洗脱程序等色谱条件的筛选调整，能够准确有效地实现五氟苯酚的含量测定。尤其是针对ADC大分子药物，即使在五氟苯酚残留量非常低的情况下，本申请提供的方法也具有非常高的检测灵敏度，能够将其准确定量测定。This application provides a method for determining the content of pentafluorophenol using HPLC. Through the screening and adjustment of chromatographic conditions such as chromatographic columns, mobile phases, and elution procedures, the content of pentafluorophenol can be determined accurately and effectively. Especially for ADC macromolecule drugs, even when the residual amount of pentafluorophenol is very low, the method provided in this application has very high detection sensitivity and can accurately measure it quantitatively.
本申请提供了一种测定五氟苯酚含量的方法，其特征在于，采用HPLC进行检测，所述HPLC的色谱条件包括：This application provides a method for determining the content of pentafluorophenol, which is characterized in that HPLC is used for detection, and the chromatographic conditions of the HPLC include:
色谱柱为C8色谱柱或C18色谱柱；The chromatographic column is C8 column or C18 column;
流动相A和B分别选自三氟乙酸-水溶液或者三氟乙酸-乙腈溶液；且Mobile phases A and B are respectively selected from trifluoroacetic acid-aqueous solution or trifluoroacetic acid-acetonitrile solution; and
梯度洗脱程序为：The gradient elution procedure is:
0～5min：流动相A与流动相B的体积百分比分别为70％～80％和30％～20％；0～5min: The volume percentages of mobile phase A and mobile phase B are 70%～80% and 30%～20% respectively;
5～26min：流动相A与流动相B的体积百分比分别为50％～45％和50％～55％；5～26min: The volume percentages of mobile phase A and mobile phase B are 50%～45% and 50%～55% respectively;
26～33min：流动相A与流动相B的体积百分比分别为0％和100％；26~33min: The volume percentages of mobile phase A and mobile phase B are 0% and 100% respectively;
33～40min：流动相A与流动相B的体积百分比分别为70％～80％和30％～20％。33～40min: The volume percentages of mobile phase A and mobile phase B are 70%～80% and 30%～20% respectively.
发明详述Detailed description of the invention
本申请提供了一种测定五氟苯酚含量的方法，其特征在于，采用HPLC进行检测，所述HPLC的色谱条件包括： This application provides a method for determining the content of pentafluorophenol, which is characterized in that HPLC is used for detection, and the chromatographic conditions of the HPLC include:
色谱柱为C8色谱柱或C18色谱柱；The chromatographic column is C8 column or C18 column;
流动相A和B分别选自三氟乙酸-水溶液或者三氟乙酸-乙腈溶液；且Mobile phases A and B are respectively selected from trifluoroacetic acid-aqueous solution or trifluoroacetic acid-acetonitrile solution; and
梯度洗脱程序为：The gradient elution procedure is:
0～5min：流动相A与流动相B的体积百分比分别为70％～80％和30％～20％；0～5min: The volume percentages of mobile phase A and mobile phase B are 70%～80% and 30%～20% respectively;
>5～26min：流动相A与流动相B的体积百分比分别为50％～45％和50％～55％；>5～26min: The volume percentages of mobile phase A and mobile phase B are 50%～45% and 50%～55% respectively;
>26～33min：流动相A与流动相B的体积百分比分别为0％和100％；>26～33min: The volume percentages of mobile phase A and mobile phase B are 0% and 100% respectively;
>33～40min：流动相A与流动相B的体积百分比分别为70％～80％和30％～20％。>33～40min: The volume percentages of mobile phase A and mobile phase B are 70%～80% and 30%～20% respectively.
在部分实施方案中，流动相A为三氟乙酸-水溶液，流动相B为三氟乙酸-乙腈溶液；优选地，流动相A为体积百分比为0.08％—0.12％的三氟乙酸-水溶液，流动相B为体积百分比为0.08％—0.12％的三氟乙酸-乙腈溶液；更优选地，流动相A为体积百分比为0.1％的三氟乙酸-水溶液，流动相B为体积百分比为0.1％的三氟乙酸-乙腈溶液。In some embodiments, the mobile phase A is a trifluoroacetic acid-aqueous solution, and the mobile phase B is a trifluoroacetic acid-acetonitrile solution; preferably, the mobile phase A is a trifluoroacetic acid-aqueous solution with a volume percentage of 0.08% to 0.12%. Phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.08%-0.12%; more preferably, the mobile phase A is a trifluoroacetic acid-water solution with a volume percentage of 0.1%, and the mobile phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.1%. Fluoroacetic acid-acetonitrile solution.
在部分实施方案中，采用HPLC进行检测，色谱条件包括：色谱柱为C8色谱柱，流动相A为体积百分比为0.1％的三氟乙酸-水溶液，流动相B为体积百分比为0.1％的三氟乙酸-乙腈溶液，梯度洗脱程序选自以下任一种：In some embodiments, HPLC is used for detection. The chromatographic conditions include: the chromatographic column is a C8 chromatographic column, the mobile phase A is a trifluoroacetic acid-water solution with a volume percentage of 0.1%, and the mobile phase B is a trifluoroacetic acid-water solution with a volume percentage of 0.1%. Acetic acid-acetonitrile solution, gradient elution program selected from any of the following:
(1) (1)
(2) (2)
(3) (3)
(4) (4)
在部分实施方案中，所述测定五氟苯酚含量的方法中，所述色谱柱为SB-C8色谱柱或CSH C18色谱柱；优选地，所述色谱柱为SB-C8，5μm 4.6×250mm色谱柱或CSH C18，3.5μm 4.6×150mm色谱柱；优选地，所述色谱柱为Agilent Zorbax 300 SB-C8，5μm 4.6×250mm色谱柱或Xselect CSH C18，3.5μm 4.6×150mm色谱柱；更优选地，所述色谱柱为Agilent Zorbax 300 SB-C8，5μm 4.6×250mm色谱柱。In some embodiments, in the method for determining pentafluorophenol content, the chromatographic column is an SB-C8 chromatographic column or a CSH C18 chromatographic column; preferably, the chromatographic column is SB-C8, 5 μm 4.6 × 250 mm chromatographic column column or CSH C18, 3.5μm 4.6×150mm chromatographic column; preferably, the chromatographic column is Agilent Zorbax 300 SB-C8, 5μm 4.6×250mm chromatographic column or Xselect CSH C18, 3.5μm 4.6×150mm chromatographic column; more preferably , the column is Agilent Zorbax 300 SB-C8, 5μm 4.6×250mm column.
在部分实施方案中，所述测定五氟苯酚含量的方法中，还可以选择与Agilent Zorbax300SB-C8柱性能相似的色谱柱。In some embodiments, in the method for determining pentafluorophenol content, a chromatographic column with similar performance to the Agilent Zorbax300SB-C8 column can also be selected.
在部分实施方案中，所述测定五氟苯酚含量的方法中，进样量为50—100μl；优选地，所述进样量为100μl。In some embodiments, in the method for determining pentafluorophenol content, the sample injection volume is 50-100 μl; preferably, the sample injection volume is 100 μl.
在部分实施方案中，所述测定五氟苯酚含量的方法中，五氟苯酚的定量限为0.1μg/ml。In some embodiments, in the method for determining the content of pentafluorophenol, the limit of quantification of pentafluorophenol is 0.1 μg/ml.
在部分实施方案中，所述测定五氟苯酚含量的方法中，色谱条件还包括：检测波长为254-265nm，流速为1-1.5ml/min，柱温为55-65℃；优选地，色谱条件还包括：检测波长为264nm，流速为1-1.5ml/min，柱温为57-63℃。In some embodiments, in the method for determining the content of pentafluorophenol, the chromatographic conditions also include: the detection wavelength is 254-265nm, the flow rate is 1-1.5ml/min, and the column temperature is 55-65°C; preferably, the chromatography The conditions also include: the detection wavelength is 264nm, the flow rate is 1-1.5ml/min, and the column temperature is 57-63°C.
在部分实施方案中，所述测定五氟苯酚含量的方法中，色谱条件还包括：检测波长为264nm，流速为1ml/min，柱温为60℃。In some embodiments, in the method for determining pentafluorophenol content, the chromatographic conditions also include: the detection wavelength is 264 nm, the flow rate is 1 ml/min, and the column temperature is 60°C.
在部分实施方案中，所述测定五氟苯酚含量的方法中，流动相A的配制方法为：量取超纯水1000ml，加入1ml三氟乙酸，混合均匀，经0.22μm滤膜过滤，即得；流动相B的配制方法为：量取乙腈1000ml，加入1ml三氟乙酸，混合均匀，经0.22μm滤膜过滤，即得。In some embodiments, in the method for determining the content of pentafluorophenol, the preparation method of mobile phase A is: measure 1000 ml of ultrapure water, add 1 ml of trifluoroacetic acid, mix evenly, and filter through a 0.22 μm filter membrane to obtain ; The preparation method of mobile phase B is: measure 1000ml of acetonitrile, add 1ml of trifluoroacetic acid, mix evenly, and filter through a 0.22μm filter membrane to obtain.
在部分实施方案中，所述测定五氟苯酚含量的方法包括以下步骤：In some embodiments, the method for determining pentafluorophenol content includes the following steps:
采用HPLC进行检测，所述HPLC的色谱条件包括：HPLC is used for detection, and the chromatographic conditions of HPLC include:
色谱柱为Agilent Zorbax 300 SB-C8，5μm 4.6×250mm色谱柱；The chromatographic column is Agilent Zorbax 300 SB-C8, 5μm 4.6×250mm column;
流动相A为体积百分比为0.1％的三氟乙酸-水溶液，流动相B为体积百分比为0.1％的三氟乙酸-乙腈溶液；Mobile phase A is a trifluoroacetic acid-water solution with a volume percentage of 0.1%, and mobile phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.1%;
进样量为100μl； The injection volume is 100μl;
检测波长为264nm；The detection wavelength is 264nm;
流速为1ml/min；The flow rate is 1ml/min;
’柱温为60℃；且’The column temperature is 60℃; and
梯度洗脱程序为
The gradient elution program is
在部分实施方案中，所述测定五氟苯酚含量的方法包括以下步骤：In some embodiments, the method for determining pentafluorophenol content includes the following steps:
(1)配制不同浓度的五氟苯酚标准品溶液、供试品溶液以及稀释液；(1) Prepare pentafluorophenol standard solution, test solution and diluent with different concentrations;
(2)设置上述HPLC条件；(2) Set the above HPLC conditions;
(3)按照上述进样量将步骤(1)中的各溶液注入液相色谱仪，采用紫外检测器记录色谱图。(3) Inject each solution in step (1) into the liquid chromatograph according to the above injection volume, and use a UV detector to record the chromatogram.
在部分实施方案中，所述五氟苯酚标准品溶液的浓度选自0.1μg/ml、0.2μg/ml、0.5μg/ml、1.0μg/ml、2.0μg/ml和5.0μg/ml。In some embodiments, the concentration of the pentafluorophenol standard solution is selected from the group consisting of 0.1 μg/ml, 0.2 μg/ml, 0.5 μg/ml, 1.0 μg/ml, 2.0 μg/ml and 5.0 μg/ml.
在部分实施方案中，采用稀释液溶解五氟苯酚对照品得到所述五氟苯酚标准品溶液；优选地，所述稀释液为体积浓度为30％的乙腈-水溶液。In some embodiments, a diluent is used to dissolve the pentafluorophenol reference substance to obtain the pentafluorophenol standard solution; preferably, the diluent is an acetonitrile-water solution with a volume concentration of 30%.
在部分实施方案中，所述供试品溶液为药物溶液。优选地，所述药物为ADC或小分子化合物；优选地，所述ADC的分子量为150±50kD，所述ADC在制备中可能会产生五氟苯酚；优选地，所述ADC药物中的毒素为奥瑞他汀类化合物，或者所述小分子化合物为生产过程中容易产生五氟苯酚的小分子化合物；优选地，所述小分子化合物为奥瑞他汀类化合物。In some embodiments, the test solution is a pharmaceutical solution. Preferably, the drug is an ADC or a small molecule compound; preferably, the molecular weight of the ADC is 150±50kD, and the ADC may produce pentafluorophenol during preparation; preferably, the toxin in the ADC drug is Auristatin compounds, or the small molecule compound is a small molecule compound that easily produces pentafluorophenol during the production process; preferably, the small molecule compound is an auristatin compound.
本申请提供的测定方法可用于测试药物中的五氟苯酚含量；优选地，所述药物为ADC偶联药物或小分子药物；优选地，所述ADC偶联药物的分子量为150±50KD；优选地，所述ADC偶联药物为偶联产物中会产生五氟苯酚游离分子的重组抗体偶联药物。The determination method provided by this application can be used to test the pentafluorophenol content in drugs; preferably, the drug is an ADC coupled drug or a small molecule drug; preferably, the molecular weight of the ADC coupled drug is 150±50KD; preferably Preferably, the ADC conjugated drug is a recombinant antibody conjugated drug that produces free molecules of pentafluorophenol in the conjugated product.
本申请提供了一种高效液相色谱测定药物，尤其是ADC大分子药物中五氟苯酚的含量的方法，该方法色谱条件简单，操作便捷，灵敏度高，耐用性好，可有效分离各成分并定量计算，快速准确地实现了五氟苯酚杂质的质控，确保产品质量安全。 This application provides a high-performance liquid chromatography method for determining the content of pentafluorophenol in drugs, especially ADC macromolecule drugs. The method has simple chromatographic conditions, convenient operation, high sensitivity, good durability, and can effectively separate the components and Quantitative calculations quickly and accurately realize the quality control of pentafluorophenol impurities to ensure product quality and safety.
本申请提供的方法通用性强、高效、快速，由于所检测的五氟苯酚与检测样品的结构无关，该检测方法可作为平台方法兼顾ADC药物及其小分子等药物的检测。The method provided in this application is highly versatile, efficient, and fast. Since the detected pentafluorophenol has nothing to do with the structure of the test sample, this detection method can be used as a platform method to take into account the detection of ADC drugs and small molecules and other drugs.
附图说明Description of drawings
图1显示了实施例1测试例1的HPLC图谱；Figure 1 shows the HPLC spectrum of Test Example 1 of Embodiment 1;
图2显示了实施例1测试例2的HPLC图谱；Figure 2 shows the HPLC pattern of Test Example 2 of Embodiment 1;
图3显示了实施例1测试例3的HPLC图谱；Figure 3 shows the HPLC spectrum of Test Example 3 of Embodiment 1;
图4显示了实施例2洗脱程序3的HPLC图谱；Figure 4 shows the HPLC chromatogram of elution procedure 3 of Example 2;
图5显示了实施例2洗脱程序4的HPLC图谱；Figure 5 shows the HPLC chromatogram of elution procedure 4 of Example 2;
图6显示了实施例4的HPLC图谱。Figure 6 shows the HPLC pattern of Example 4.
实施例Example
以下通过实施例的描述对本发明作进一步说明，但这并非是对本发明的限制，本领域技术人员根据发明的基本思想，可以做出各种修改或改进，但是只要不脱离本发明的基本思想，均在本发明的范围之内。The present invention will be further described below through the description of the embodiments, but this is not a limitation of the invention. Those skilled in the art can make various modifications or improvements according to the basic idea of the invention, but as long as they do not deviate from the basic idea of the invention, are within the scope of the present invention.
根据ICH指导原则(Q3C(R8)杂质：残留溶剂的指导原则)，按照大鼠皮下LD50计算得出注射剂中五氟苯酚每日最大耐受剂量PDE(permitted daily exposure)为1.932μg/天，PFP最大耐受浓度为0.134μg/ml。本申请通过摸索研究发现，待测定药物中PFP的定量限应不高于0.1μg/ml，才能满足定量检测的要求。According to the ICH guidelines (Q3C (R8) impurities: guidelines for residual solvents), the maximum tolerated daily dose of pentafluorophenol in injections, PDE (permitted daily exposure), was calculated as 1.932 μg/day based on the rat subcutaneous LD 50 . The maximum tolerated concentration of PFP is 0.134μg/ml. This application found through exploration and research that the quantitation limit of PFP in the drug to be measured should not be higher than 0.1 μg/ml in order to meet the requirements for quantitative detection.
实施例1-1.流动相及色谱柱的筛选Example 1-1. Screening of mobile phase and chromatographic column
1)溶液配制：1) Solution preparation:
稀释液：30％乙腈水溶液，用于溶液配制。Diluent: 30% acetonitrile water solution, used for solution preparation.
PFP对照品母液：称取五氟苯酚标准品，用乙腈溶解，配制成浓度约为1mg/ml的 溶液，作为PFP对照品母液。PFP reference substance mother solution: Weigh the pentafluorophenol standard, dissolve it in acetonitrile, and prepare it to a concentration of approximately 1 mg/ml. solution as the PFP reference substance mother solution.
PFP标准工作液：使用30％乙腈水溶液将PFP对照品母液分别稀释至0.1μg/ml、0.2μg/ml、0.5μg/ml、1.0μg/ml、2.0μg/ml、5.0μg/ml，作为PFP标准工作液。以1.0μg/ml的溶液作为系统适用性溶液。PFP standard working solution: Use 30% acetonitrile aqueous solution to dilute the PFP reference substance mother solution to 0.1μg/ml, 0.2μg/ml, 0.5μg/ml, 1.0μg/ml, 2.0μg/ml, 5.0μg/ml respectively, as PFP Standard working fluid. Use the 1.0μg/ml solution as the system suitability solution.
待测液制备：取适量参照CN108697809A制备的ADC药物至进样小瓶，ADC药物的分子量为150±50kD，配制待测液。Preparation of the liquid to be tested: Take an appropriate amount of the ADC drug prepared with reference to CN108697809A into the injection vial. The molecular weight of the ADC drug is 150±50kD, and prepare the liquid to be tested.
加标溶液：取待测液，以加标的方式向其中加入一定量的PFP标准品，配制成PFP浓度分别为0.1μg/ml、1.0μg/ml和5.0μg/ml的加标溶液，每个浓度配制3份。Standardized solution: Take the liquid to be tested, add a certain amount of PFP standard to it in the form of standard addition, and prepare spiking solutions with PFP concentrations of 0.1μg/ml, 1.0μg/ml and 5.0μg/ml. Concentration: Make 3 servings.
2)流动相：2) Mobile phase:
流动相1：Mobile phase 1:
流动相A：0.1％三氟乙酸-水溶液(v/v)，配制方法：量取超纯水1000ml，加入1ml三氟乙酸，混匀；Mobile phase A: 0.1% trifluoroacetic acid-water solution (v/v), preparation method: measure 1000ml of ultrapure water, add 1ml of trifluoroacetic acid, and mix;
流动相B：0.1％三氟乙酸-乙腈溶液(v/v)，配制方法：量取乙腈1000ml，加入1ml三氟乙酸，混匀。Mobile phase B: 0.1% trifluoroacetic acid-acetonitrile solution (v/v), preparation method: measure 1000ml of acetonitrile, add 1ml of trifluoroacetic acid, and mix.
流动相2：Mobile phase 2:
流动相A：磷酸缓冲液，配制方法：称量2.5g磷酸二氢钾，2.5g磷酸氢二钾，加入1000ml超纯水溶解，调pH至7.5，混匀，经0.22μm滤膜过滤；Mobile phase A: phosphate buffer, preparation method: weigh 2.5g potassium dihydrogen phosphate and 2.5g dipotassium hydrogen phosphate, add 1000ml ultrapure water to dissolve, adjust the pH to 7.5, mix well, and filter through a 0.22μm filter membrane;
流动相B：甲醇。Mobile phase B: methanol.
色谱柱1：Agilent Zorbax 300 SB-C8，(5μm 4.6×250mm)；色谱柱2：Xselect CSH C18，(3.5μm 4.6×150mm)；Column 1: Agilent Zorbax 300 SB-C8, (5μm 4.6×250mm); Column 2: Xselect CSH C18, (3.5μm 4.6×150mm);
流速：1.0ml/min(流动相1)或者1.5ml/min(流动相2)，检测波长：264nm，柱温：60℃； Flow rate: 1.0ml/min (mobile phase 1) or 1.5ml/min (mobile phase 2), detection wavelength: 264nm, column temperature: 60°C;
洗脱梯度1：
Elution gradient 1:
洗脱梯度2：
Elution gradient 2:
3)试验方法3)Test method
精密量取1)的系统适用性溶液100μl，注入液相色谱仪，考察检测情况。Precisely measure 100 μl of the system suitability solution of 1), inject it into the liquid chromatograph, and check the detection situation.
实施例1.流动相及色谱柱的筛选Example 1. Screening of mobile phase and chromatographic column
1)溶液配制：1) Solution preparation:
稀释液：30％乙腈水溶液，用于溶液配制。Diluent: 30% acetonitrile water solution, used for solution preparation.
PFP对照品母液：称取五氟苯酚标准品，用乙腈溶解，配制成浓度约为1mg/ml的溶液，作为PFP对照品母液。PFP reference substance mother solution: Weigh the pentafluorophenol standard, dissolve it in acetonitrile, and prepare a solution with a concentration of approximately 1 mg/ml as the PFP reference substance mother solution.
PFP标准工作液：使用30％乙腈水溶液将PFP对照品母液分别稀释至0.1μg/ml、0.2μg/ml、0.5μg/ml、1.0μg/ml、2.0μg/ml、5.0μg/ml，作为PFP标准工作液。以1.0μg/ml的溶液作为系统适用性溶液。PFP standard working solution: Use 30% acetonitrile aqueous solution to dilute the PFP reference substance mother solution to 0.1μg/ml, 0.2μg/ml, 0.5μg/ml, 1.0μg/ml, 2.0μg/ml, 5.0μg/ml respectively, as PFP Standard working fluid. Use the 1.0μg/ml solution as the system suitability solution.
待测液制备：取适量参照CN106729743A制备的ADC药物I-1至进样小瓶，ADC药物的分子量为150±50kD，配制待测液。Preparation of the liquid to be tested: Take an appropriate amount of the ADC drug I-1 prepared with reference to CN106729743A into the injection vial. The molecular weight of the ADC drug is 150±50kD, and prepare the liquid to be tested.
加标溶液：取待测液，以加标的方式向其中加入一定量的PFP标准品，配制成PFP浓度分别为0.1μg/ml、1.0μg/ml和5.0μg/ml的加标溶液，每个浓度配制3份。Standardized solution: Take the liquid to be tested, add a certain amount of PFP standard to it in the form of standard addition, and prepare spiking solutions with PFP concentrations of 0.1μg/ml, 1.0μg/ml and 5.0μg/ml. Concentration: Make 3 servings.
2)流动相：2) Mobile phase:
流动相1： Mobile phase 1:
流动相A：0.1％三氟乙酸-水溶液(v/v)，配制方法：量取超纯水1000ml，加入1ml三氟乙酸，混匀；Mobile phase A: 0.1% trifluoroacetic acid-water solution (v/v), preparation method: measure 1000ml of ultrapure water, add 1ml of trifluoroacetic acid, and mix;
流动相B：0.1％三氟乙酸-乙腈溶液(v/v)，配制方法：量取乙腈1000ml，加入1ml三氟乙酸，混匀。Mobile phase B: 0.1% trifluoroacetic acid-acetonitrile solution (v/v), preparation method: measure 1000ml of acetonitrile, add 1ml of trifluoroacetic acid, and mix.
流动相2：Mobile phase 2:
流动相A：磷酸缓冲液，配制方法：称量2.5g磷酸二氢钾，2.5g磷酸氢二钾，加入1000ml超纯水溶解，调pH至7.5，混匀，经0.22μm滤膜过滤；Mobile phase A: phosphate buffer, preparation method: weigh 2.5g potassium dihydrogen phosphate and 2.5g dipotassium hydrogen phosphate, add 1000ml ultrapure water to dissolve, adjust the pH to 7.5, mix well, and filter through a 0.22μm filter membrane;
流动相B：甲醇。Mobile phase B: methanol.
色谱柱1：Agilent Zorbax 300 SB-C8，(5μm 4.6×250mm)；色谱柱2：Xselect CSH C18，(3.5μm 4.6×150mm)；Column 1: Agilent Zorbax 300 SB-C8, (5μm 4.6×250mm); Column 2: Xselect CSH C18, (3.5μm 4.6×150mm);
流速：1.0ml/min(流动相1)或者1.5ml/min(流动相2)，检测波长：264nm，柱温：60℃；Flow rate: 1.0ml/min (mobile phase 1) or 1.5ml/min (mobile phase 2), detection wavelength: 264nm, column temperature: 60°C;
洗脱梯度1：
Elution gradient 1:
洗脱梯度2：
Elution gradient 2:
3)试验方法3)Test method
精密量取1)的系统适用性溶液100μl，注入液相色谱仪，考察检测情况。Precisely measure 100 μl of the system suitability solution of 1), inject it into the liquid chromatograph, and check the detection situation.
4)试验结果4)Test results
表1 流动相种类、色谱柱考察

Table 1 Mobile phase types and chromatographic column inspection

测试例1-2采用流动相1以及色谱柱1和2，目标峰均出峰正常，峰形较好，与杂峰具有一定分离度。测试例3采用流动相2，系统适用性溶液的色谱峰与稀释液的色谱峰基本一致，可见在该条件下PFP未出峰。因此，采用流动相A：0.1％三氟乙酸-水溶液、流动相B：0.1％三氟乙酸-乙腈溶液作为后续考察的流动相。色谱柱Xselect CSH C18的孔径较小，需要对供试品进行预处理(ACN沉淀)，因此采用Agilent Zorbax 300 SB-C8进行后续考察。Test Example 1-2 uses mobile phase 1 and chromatographic columns 1 and 2. The target peaks all emit normal peaks, have good peak shapes, and have a certain degree of separation from impurity peaks. Test example 3 uses mobile phase 2, and the chromatographic peaks of the system suitability solution are basically consistent with those of the diluent. It can be seen that under this condition, PFP does not emit peaks. Therefore, mobile phase A: 0.1% trifluoroacetic acid-aqueous solution and mobile phase B: 0.1% trifluoroacetic acid-acetonitrile solution were used as the mobile phases for subsequent investigations. The chromatographic column Xselect CSH C18 has a smaller pore size and requires pretreatment (ACN precipitation) of the test sample, so Agilent Zorbax 300 SB-C8 was used for subsequent inspections.
实施例2.洗脱程序筛选Example 2. Elution program screening
精密量取PFP浓度为1.0μg/ml的加标溶液100μl注入液相色谱仪，采用上述实施例1中筛选出的流动相1、色谱柱1，流速为1.0ml/min，并分别采用以下洗脱程序，考察样品检测情况。试验方案及结果如下表所示。Precisely measure 100 μl of the spiked solution with a PFP concentration of 1.0 μg/ml and inject it into the liquid chromatograph. Use the mobile phase 1 and chromatographic column 1 selected in the above Example 1, with a flow rate of 1.0 ml/min, and the following washing methods. Remove the procedure and inspect the sample testing status. The test plan and results are shown in the table below.
洗脱程序1
Elution procedure 1
洗脱程序2
Elution procedure 2
洗脱程序3
Elution procedure 3
洗脱程序4
Elution program 4
表2 洗脱程序考察
Table 2 Investigation of elution procedures
当采用洗脱程序3时，PFP峰吸收较高，响应灵敏，且PFP峰与其他杂质峰分离情况较好，蛋白洗脱完全；缩短洗脱时间导致目标峰和其他杂峰之间的分离度变差，蛋白不能完全洗脱，因此选用洗脱程序3进行后续试验。When elution program 3 is used, the absorption of the PFP peak is higher, the response is sensitive, and the separation between the PFP peak and other impurity peaks is better, and the protein is completely eluted; shortening the elution time causes the separation between the target peak and other impurity peaks to change. Poor, the protein cannot be completely eluted, so elution program 3 is selected for subsequent tests.
实施例3.进样量考察Example 3. Investigation of sample injection volume
采用上述实施例1的流动相1、色谱柱1以及实施例2的洗脱程序3作为色谱条件，取系统适用性溶液分别采用以下进样量，考察样品检测情况。试验方案及结果如下表所示。Use the mobile phase 1, the chromatographic column 1 of the above-mentioned Example 1, and the elution program 3 of Example 2 as the chromatographic conditions. Take the system suitability solution and use the following injection volumes to examine the sample detection. The test plan and results are shown in the table below.
表3.进样量考察
Table 3. Inspection of injection volume
根据《中国药典》要求，定量限浓度下的基线信噪比需要大于或等于10，当采用100μl进样量时，信噪比在15-30左右，能满足定量检测要求，因此选用100μl的进样量。 According to the requirements of the Chinese Pharmacopoeia, the baseline signal-to-noise ratio at the quantitative limit concentration needs to be greater than or equal to 10. When a 100 μl injection volume is used, the signal-to-noise ratio is around 15-30, which can meet the quantitative detection requirements. Therefore, a 100 μl injection volume is used. sample size.
实施例4.专属性考察Example 4. Specificity investigation
采用上述实施例1的流动相1、色谱柱1以及实施例2的洗脱程序3作为色谱条件，精密量取工艺中间品溶液100μl注入液相色谱仪，考察各成分的出峰情况。其中，工艺中间品溶液为在制备ADC药物过程中的中间产品，其含有PFP、毒素小分子及有关杂质。Using the mobile phase 1, chromatographic column 1 and elution procedure 3 of Example 2 as the chromatographic conditions, accurately measure 100 μl of the process intermediate solution and inject it into the liquid chromatograph to examine the peak elution of each component. Among them, the process intermediate solution is an intermediate product in the process of preparing ADC drugs, which contains PFP, toxin small molecules and related impurities.
结果见图6，在该色谱条件下，工艺中间品溶液中的PFP、毒素小分子及其杂质等各成分峰均能有效分离，峰形良好，符合专属性要求。The results are shown in Figure 6. Under these chromatographic conditions, the component peaks such as PFP, toxin small molecules and impurities in the process intermediate solution can be effectively separated, with good peak shape and meeting the specificity requirements.
实施例5.定量限考察Example 5. Quantitative limit investigation
采用实施例1的流动相1、色谱柱1以及实施例2的洗脱程序3作为色谱条件，取实施例1配制的PFP浓度为0.1μg/ml的3份加标溶液，精密量取各100μl注入液相色谱仪，考察实测浓度与理论浓度的比值(回收率)、回收率RSD％及信噪比。Using mobile phase 1, chromatographic column 1 and elution procedure 3 of Example 2 as chromatographic conditions, take 3 spiked solutions with a PFP concentration of 0.1 μg/ml prepared in Example 1, and accurately measure 100 μl of each solution. Inject into the liquid chromatograph and examine the ratio of the measured concentration to the theoretical concentration (recovery rate), recovery rate RSD% and signal-to-noise ratio.
表4.定量限考察
Table 4. Quantitative limit inspection
结果显示，浓度为0.1μg/ml的供试品加标溶液的信噪比分别为27.9、28.0、28.5；回收率分别为98％、99％、102％，回收率的RSD％为2.1％，满足方法开发的标准要求。可见，在该浓度下，供试品中的PFP能够被准确定量检测，因此将0.1μg/ml作为该方法的定量限浓度。The results showed that the signal-to-noise ratio of the test sample spiked solution with a concentration of 0.1 μg/ml was 27.9, 28.0, and 28.5 respectively; the recovery rates were 98%, 99%, and 102% respectively, and the RSD% of the recovery rate was 2.1%. Meets standard requirements for method development. It can be seen that at this concentration, PFP in the test sample can be accurately and quantitatively detected, so 0.1 μg/ml is used as the quantitative limit concentration of this method.
实施例6.线性考察Example 6. Linearity investigation
采用实施例1配制的0.1μg/ml、0.2μg/ml、0.5μg/ml、1.0μg/ml、2.0μg/ml、5.0μg/ml不同浓度的PFP标准工作液，采用实施例1的流动相1、色谱柱1以及实施例2的洗脱程序3作为色谱条件，精密量取上述6个不同浓度的PFP标准品溶液各100μg注入液相色谱仪， 记录色谱图，考察PFP浓度与峰面积的线性关系。结果显示，PFP浓度在0.1μg/ml～5.0μg/ml范围内，浓度与峰面积呈线性关系，标准曲线线性良好，线性函数为y＝19,707.52x-185.03，R2为0.999974，残差平方和为174110，满足R2≥0.99的要求，表明此方法的线性良好。Use PFP standard working solutions of different concentrations of 0.1 μg/ml, 0.2 μg/ml, 0.5 μg/ml, 1.0 μg/ml, 2.0 μg/ml, and 5.0 μg/ml prepared in Example 1, and use the mobile phase of Example 1 1. Chromatographic column 1 and elution procedure 3 of Example 2 are used as chromatographic conditions. Precisely measure 100 μg of each of the above 6 PFP standard solutions with different concentrations and inject them into the liquid chromatograph. Record the chromatogram and examine the linear relationship between PFP concentration and peak area. The results show that the PFP concentration is in the range of 0.1μg/ml~5.0μg/ml, the concentration and peak area are linearly related, the standard curve is linear, the linear function is y=19,707.52x-185.03, R2 is 0.999974, and the sum of squares of the residuals is 174110, meeting the requirement of R 2 ≥ 0.99, indicating that the linearity of this method is good.
实施例7.准确度考察Example 7. Accuracy investigation
取实施例1配制的PFP浓度分别为0.1μg/ml、1.0μg/ml、5.0μg/ml的加标溶液，每个浓度各3份，每份进样1针，进样量为100μl。采用实施例1的流动相1、色谱柱1；实施例2的洗脱程序3作为色谱条件，考察PFP实测浓度与理论浓度的比值(回收率％)及回收率的RSD％，判断该方法的准确度。Take the spiked solutions with PFP concentrations of 0.1 μg/ml, 1.0 μg/ml, and 5.0 μg/ml prepared in Example 1, and make 3 parts of each concentration. Inject 1 needle into each part, and the injection volume is 100 μl. Use the mobile phase 1 and chromatographic column 1 of Example 1 and the elution procedure 3 of Example 2 as chromatographic conditions. The ratio of the actual measured concentration of PFP to the theoretical concentration (recovery rate %) and the RSD% of the recovery rate are examined to determine the effectiveness of the method. Accuracy.
表5.准确度考察
Table 5. Accuracy inspection
上述低、中、高三个浓度水平的加标溶液中，PFP的回收率分别为低(98％、99％、102％)、中(90％、91％、90％)、高(86％、85％、84％)；回收率的RSD分别为2.1％、0.7％、1.2％，符合准确度验证要求，表明该方法在上述浓度范围内均能准确定量测定PFP。In the above-mentioned spiked solutions with three concentration levels of low, medium and high, the recovery rates of PFP were low (98%, 99%, 102%), medium (90%, 91%, 90%), high (86%, 85%, 84%); the RSDs of the recovery rates were 2.1%, 0.7%, and 1.2% respectively, which met the accuracy verification requirements, indicating that this method can accurately and quantitatively determine PFP within the above concentration range.
实施例8.耐用性考察(柱温)Example 8. Durability investigation (column temperature)
采用实施例1的流动相1、色谱柱1；实施例2的洗脱程序3作为色谱条件，考察在不同色谱柱温条件下(57℃、60℃和63℃)，待测样品的出峰情况。 Use the mobile phase 1 and chromatographic column 1 of Example 1 and the elution program 3 of Example 2 as chromatographic conditions to examine the peaks of the sample to be tested under different chromatographic column temperatures (57°C, 60°C and 63°C). Condition.
精密量取实施例1的系统适用性溶液100μl，注入液相色谱仪，记录色谱图，试验数据及结果见下表。Precisely measure 100 μl of the system suitability solution of Example 1, inject it into the liquid chromatograph, and record the chromatogram. The test data and results are shown in the table below.
表6柱温考察
Table 6 column temperature inspection
不同色谱柱温度(57℃、63℃)与方法设置柱温(60℃)下五氟苯酚检测值的差异百分比分别为1.4％、3.1％，符合耐用性验证要求。 The percentage differences in the detection values of pentafluorophenol at different column temperatures (57°C, 63°C) and method setting column temperature (60°C) were 1.4% and 3.1% respectively, which met the durability verification requirements.

Claims (10)

  1. 一种测定五氟苯酚含量的方法，其特征在于，采用HPLC进行检测，所述HPLC的色谱条件包括：A method for determining the content of pentafluorophenol, characterized in that HPLC is used for detection, and the chromatographic conditions of the HPLC include:
    色谱柱为C8色谱柱或C18色谱柱；The chromatographic column is C8 column or C18 column;
    流动相A和B分别选自三氟乙酸-水溶液或者三氟乙酸-乙腈溶液；且Mobile phases A and B are respectively selected from trifluoroacetic acid-aqueous solution or trifluoroacetic acid-acetonitrile solution; and
    梯度洗脱程序为：The gradient elution procedure is:
    0～5min：所述流动相A与所述流动相B的体积百分比分别为70％～80％和30％～20％；0～5min: the volume percentages of the mobile phase A and the mobile phase B are 70%～80% and 30%～20% respectively;
    5～26min：所述流动相A与所述流动相B的体积百分比分别为50％～45％和50％～55％；5～26min: The volume percentages of the mobile phase A and the mobile phase B are 50%～45% and 50%～55% respectively;
    26～33min：所述流动相A与所述流动相B的体积百分比分别为0％和100％；26~33min: The volume percentages of the mobile phase A and the mobile phase B are 0% and 100% respectively;
    33～40min：所述流动相A与所述流动相B的体积百分比分别为70％～80％和30％～20％。33-40 min: The volume percentages of the mobile phase A and the mobile phase B are 70%-80% and 30%-20% respectively.
  2. 根据权利要求1所述的测定方法，其特征在于，所述色谱柱为SB-C8色谱柱或CSH C18色谱柱；优选地，所述色谱柱为SB-C8，5μm 4.6×250mm色谱柱或CSH C18，3.5μm 4.6×150mm色谱柱；优选地，所述色谱柱为Agilent Zorbax 300 SB-C8，5μm4.6×250mm色谱柱或Xselect CSH C18，3.5μm 4.6×150mm色谱柱；更优选地，所述色谱柱为Agilent Zorbax 300 SB-C8，5μm 4.6×250mm色谱柱。The determination method according to claim 1, characterized in that the chromatographic column is an SB-C8 chromatographic column or a CSH C18 chromatographic column; preferably, the chromatographic column is an SB-C8, 5 μm 4.6×250mm chromatographic column or a CSH C18, 3.5μm 4.6×150mm chromatographic column; preferably, the chromatographic column is Agilent Zorbax 300 SB-C8, 5μm 4.6×250mm chromatographic column or Xselect CSH C18, 3.5μm 4.6×150mm chromatographic column; more preferably, the The column mentioned above is Agilent Zorbax 300 SB-C8, 5μm 4.6×250mm column.
  3. 根据权利要求1或2所述的测定方法，其特征在于，进样量为50-100μl；优选地，所述进样量为100μl。The assay method according to claim 1 or 2, characterized in that the sample injection volume is 50-100 μl; preferably, the sample injection volume is 100 μl.
  4. 根据权利要求1-3任一项所述的测定方法，其特征在于，五氟苯酚的定量限为0.1μg/ml。The assay method according to any one of claims 1 to 3, characterized in that the limit of quantitation of pentafluorophenol is 0.1 μg/ml.
  5. 根据权利要求1-4任一项所述的测定方法，其特征在于，所述色谱条件还包括：检测波长为254-265nm，流速为1-1.5ml/min，柱温为55-65℃；优选地，所述色谱条件还包括：检测波长为264nm，流速为1-1.5ml/min，柱温为57-63℃；优选地，所述色谱条件还包括：检测波长为264nm，流速为1ml/min，柱温为60℃。The determination method according to any one of claims 1 to 4, characterized in that the chromatographic conditions further include: the detection wavelength is 254-265nm, the flow rate is 1-1.5ml/min, and the column temperature is 55-65°C; Preferably, the chromatography conditions also include: the detection wavelength is 264nm, the flow rate is 1-1.5ml/min, and the column temperature is 57-63°C; preferably, the chromatography conditions also include: the detection wavelength is 264nm, the flow rate is 1ml. /min, the column temperature is 60°C.
  6. 根据权利要求1-5任一项所述的测定方法，其特征在于，所述流动相A为三氟乙酸-水溶液，所述流动相B为三氟乙酸-乙腈溶液；优选地，所述流动相A为体积百分 比为0.08％—0.12％的三氟乙酸-水溶液，所述流动相B为体积百分比为0.08％—0.12％的三氟乙酸-乙腈溶液；更优选地，所述流动相A为体积百分比为0.1％的三氟乙酸-水溶液，所述流动相B为体积百分比为0.1％的三氟乙酸-乙腈溶液。The measurement method according to any one of claims 1 to 5, characterized in that the mobile phase A is a trifluoroacetic acid-aqueous solution, and the mobile phase B is a trifluoroacetic acid-acetonitrile solution; preferably, the mobile phase Phase A is volume percentage The mobile phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.08%-0.12%; more preferably, the mobile phase A is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.1 % trifluoroacetic acid-water solution, and the mobile phase B is a trifluoroacetic acid-acetonitrile solution with a volume percentage of 0.1%.
  7. 一种测定五氟苯酚含量的测定方法，其特征在于，采用HPLC进行检测，所述HPLC的色谱条件包括：A method for determining the content of pentafluorophenol, characterized in that HPLC is used for detection, and the chromatographic conditions of the HPLC include:
    色谱柱为C8色谱柱或C18色谱柱；The chromatographic column is C8 column or C18 column;
    流动相A和B分别选自三氟乙酸-水溶液或者三氟乙酸-乙腈溶液；且Mobile phases A and B are respectively selected from trifluoroacetic acid-aqueous solution or trifluoroacetic acid-acetonitrile solution; and
    梯度洗脱程序选自以下中的任一种：The gradient elution procedure is selected from any of the following:
    (1)
    (1)
    (2)
    (2)
    (3)
    (3)
    (4)
    (4)
  8. 根据权利要求1-7任一项所述的测定方法，其包括以下步骤：The assay method according to any one of claims 1-7, which includes the following steps:
    (1)配制不同浓度的五氟苯酚标准品溶液、供试品溶液以及稀释液；(1) Prepare pentafluorophenol standard solution, test solution and diluent with different concentrations;
    (2)按照权利要求1-7任一项所述的测定方法设置HPLC条件； (2) Set HPLC conditions according to the assay method described in any one of claims 1-7;
    (3)按照权利要求3所述的进样量将步骤(1)中的各溶液注入液相色谱仪，采用紫外检测器记录色谱图。(3) Inject each solution in step (1) into the liquid chromatograph according to the injection volume of claim 3, and use a UV detector to record the chromatogram.
  9. 根据权利要求1-8任一项所述的测定方法，其中所述五氟苯酚标准品溶液的浓度选自0.1μg/ml、0.2μg/ml、0.5μg/ml、1.0μg/ml、2.0μg/ml和5.0μg/ml；The assay method according to any one of claims 1 to 8, wherein the concentration of the pentafluorophenol standard solution is selected from the group consisting of 0.1 μg/ml, 0.2 μg/ml, 0.5 μg/ml, 1.0 μg/ml, and 2.0 μg. /ml and 5.0μg/ml;
    优选地，采用稀释液溶解五氟苯酚对照品得到所述五氟苯酚标准品溶液；Preferably, the pentafluorophenol reference substance is dissolved in a diluent to obtain the pentafluorophenol standard solution;
    优选地，所述稀释液为体积浓度为30％的乙腈溶液。Preferably, the diluent is an acetonitrile solution with a volume concentration of 30%.
  10. 根据权利要求1-9任一项所述的测定方法，其用于测定化合物中的五氟苯酚含量；优选地，所述化合物为ADC或小分子化合物；优选地，所述ADC的分子量为150±50kD；优选地，所述ADC为制备过程中会产生五氟苯酚的ADC；所述小分子为制备过程可能产生五氟苯酚的小分子化合物。 The determination method according to any one of claims 1 to 9, which is used to determine the pentafluorophenol content in the compound; preferably, the compound is an ADC or a small molecule compound; preferably, the molecular weight of the ADC is 150 ±50kD; preferably, the ADC is an ADC that may produce pentafluorophenol during the preparation process; the small molecule is a small molecule compound that may produce pentafluorophenol during the preparation process.
PCT/CN2023/111922 2022-08-19 2023-08-09 Method for measuring content of pentafluorophenol WO2024037398A1 (en)

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