WO2024021509A1 - Nanohplc-titer system for quantitative determination of supernatant protein in culture medium - Google Patents

Nanohplc-titer system for quantitative determination of supernatant protein in culture medium Download PDF

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WO2024021509A1
WO2024021509A1 PCT/CN2022/142304 CN2022142304W WO2024021509A1 WO 2024021509 A1 WO2024021509 A1 WO 2024021509A1 CN 2022142304 W CN2022142304 W CN 2022142304W WO 2024021509 A1 WO2024021509 A1 WO 2024021509A1
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nanohplc
chromatographic column
tube
titer
column
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Chinese (zh)
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邵熙
杜青青
肖志华
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上海奥浦迈生物科技股份有限公司
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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
    • G01N30/60—Construction of the column
    • G01N30/6004—Construction of the column end pieces
    • 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
    • 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
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors

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  • the invention relates to the field of biochemical detection, and in particular to a nanoHPLC-Titer system used for quantification of culture medium supernatant protein.
  • High performance liquid chromatography (English: high performance liquid chromatography, abbreviation HPLC), also translated as high performance liquid chromatography, formerly referred to as high pressure liquid chromatography (high pressure liquid chromatography), is a chromatographic analysis technology that uses to separate mixtures to confirm and quantify the proportions of individual components. It relies on a pump to pressurize the sample through a pressure column filled with a stationary phase, causing the individual components of the sample to separate due to different forces of interaction with the stationary phase. High performance liquid chromatography is commonly used in biochemistry and analytical chemistry.
  • the existing HPLC-Titer system can accurately measure protein expression in cell culture supernatant, because it uses conventional chromatography, the required sample volume is generally in the milliliter level, and the quantitation limit is about 0.5 mg/mL.
  • the cell line screening stage 96-well plates are usually plated first. Since the culture medium in a 96-well plate is generally only about 100 ⁇ L, expression analysis based on HPLC-titer is generally not possible at the 96-well plate stage. In some projects, there will be samples with expression levels lower than 0.1 mg/mL, and these samples cannot be accurately quantified using conventional HPLC-titer systems.
  • the HPLC-Titer analysis system in the prior art requires a large sample volume and a high quantitation limit, which limits its role in quantitative analysis.
  • the present invention has developed a nanoHPLC for quantification of culture medium supernatant protein. -Titer system, the technical solution is as follows:
  • the first aspect of the invention discloses a new nanoHPLC-Titer system, which includes a nano affinity chromatography column, a nanoHPLC chromatography pump, an automatic sampling system and a detector. It is characterized in that the preparation of the nano affinity chromatography column The method is:
  • the material of the chromatography column tube in step (1) is a fused silica capillary tube, a polyetheretherketone tube or a stainless steel tube. Further, the inner diameter of the chromatography column tube in step (1) is less than or equal to 500 ⁇ m.
  • the length of the chromatography column tube in step (1) is 1-25cm.
  • the filler described in step (1) is Protein A filler, Protein G filler or Captol L filler.
  • the mobile phase in step (3) is selected from organic phases such as methanol, acetonitrile, ethanol or isopropyl alcohol; or a mixture of the aforementioned organic phase and water.
  • the dead volume between the automatic sampling system and the nano-affinity chromatographic column is less than 2 ⁇ L, and the dead volume between the nano-affinity chromatographic column and the detector is less than 1 ⁇ L.
  • the invention also discloses the use of the above-mentioned novel nanoHPLC-Titer system for quantification of culture medium supernatant protein.
  • the present invention has the following beneficial effects:
  • Figure 1 is a diagram of a packed chromatographic column in Example 1 of the present invention.
  • Figure 2 is a schematic diagram of the nanoHPLC-Titer system of the present invention, and the black connecting line is the connecting pipeline.
  • Figure 3 is a standard curve graph.
  • the nano affinity chromatography column is a self-made chromatography column, and its preparation method is as follows.
  • the material of the chromatography column tube used is fused silica capillary tube; the inner diameter of the chromatography tube used is less than or equal to 500 ⁇ m, and the tube length is between 1-25cm.
  • the packing used is Protein A packing. After the filling is completed, seal the sieve plate and union at the inlet end of the chromatographic column. Under the normal operating pressure of the corresponding chromatographic column, use the mobile phase to flush the installed chromatographic column.
  • the flushing time is greater than or equal to 1 hour.
  • the mobile phase used is methanol.
  • the nano affinity chromatography column is a self-made chromatography column, and its preparation method is as follows.
  • the material of the chromatography column tube used is polyetheretherketone tube; the inner diameter of the chromatography tube used is less than or equal to 500 ⁇ m, and the tube length is between 1-25cm.
  • the packing used is Protein G packing.
  • Under the normal operating pressure of the corresponding chromatographic column use the mobile phase to flush the installed chromatographic column.
  • the flushing time is greater than or equal to 1 hour.
  • the mobile phase used is acetonitrile. After the chromatographic column is flushed, seal the interfaces at both ends with plugs until use.
  • the nano affinity chromatography column is a self-made chromatography column, and its preparation method is as follows.
  • the material of the chromatography column tube used is stainless steel tube; the inner diameter of the chromatography tube used is less than or equal to 500 ⁇ m, and the tube length is between 1-25cm.
  • the packing used is Captol L packing. After the filling is completed, seal the sieve plate and union at the inlet end of the chromatographic column. Under the normal operating pressure of the corresponding chromatographic column, use the mobile phase to flush the installed chromatographic column.
  • the flushing time is greater than or equal to 1 hour.
  • the mobile phase used is a mixture of ethanol and water. After the chromatographic column is flushed, seal the interfaces at both ends with plugs until use.
  • the nano chromatographic column prepared in Example 1 is installed in a nano chromatograph, which at least includes a nano chromatography pump, an automatic sampling system and a detector. Through the connection of appropriate pipelines, ensure that the dead volume between the automatic sampling system and the nano chromatographic column is less than 2 ⁇ L, and the dead volume between the nano chromatographic column and the detector is less than 1 ⁇ L.
  • the nanoHPLC-Titer system diagram is shown in Figure 2.
  • phosphate buffer mobile phase to dilute the standard, which is the target protein to be analyzed: bevacizumab.
  • the standards were diluted to 0.50mg/mL, 0.20mg/mL, 0.10mg/mL, 0.05mg/mL, 0.03mg/mL, 0.02mg/mL, 0.01mg/mL, and Lunatic high-throughput microfluidic was used Spectral analyzer determines its accurate concentration.
  • the built nanoHPLC-Titer system was used to analyze the standard working solutions and samples of each concentration obtained in sequence, and the standard curve ( Figure 3) and sample titer value (Table 1) were obtained.
  • the correlation coefficient of the standard curve is >0.99
  • the signal-to-noise ratio of the peaks of the standard working solution at each concentration is greater than 10
  • the RSD of the three replicates of the samples at each concentration is less than 10%. That is, this system can use a volume of about 10 ⁇ L of cell culture supernatant to quantify the protein in the supernatant, and the quantification limit is as low as 0.01 mg/mL.

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Abstract

A nanoHPLC-Titer system for quantitative determination of supernatant protein in a culture medium, the system comprising a nano affinity chromatographic column, a nanoHPLC chromatographic pump, an automatic sample injection system and a detector. The nano affinity chromatographic column is prepared by: (1) taking a clean chromatographic column tube with a sieve plate and a two-way valve to block an end of the tube, and filling the chromatographic column tube with an affinity chromatographic filler; (2) after filling, blocking the inlet end of a chromatographic column with the sieve plate and the two-way valve; (3) rinsing the filled chromatographic column with a mobile phase at a normal operating pressure of the chromatographic column; and (4) after the rinsing of the chromatographic column is finished, sealing ports at the two ends with plugs for later use. The nanoHPLC system can determine the expression quantity of a low-concentration and small-volume supernatant sample, and has a wide application prospect in the field of protein quantitative analysis.

Description

一种应用于培养基上清蛋白定量的nanoHPLC-Titer系统A nanoHPLC-Titer system for quantification of culture supernatant protein 技术领域Technical field
本发明涉及生化检测领域，尤其涉及一种应用于培养基上清蛋白定量的nanoHPLC-Titer系统。The invention relates to the field of biochemical detection, and in particular to a nanoHPLC-Titer system used for quantification of culture medium supernatant protein.
背景技术Background technique
高效液相色谱法(英语：high performance liquid chromatography，缩写HPLC)，又译高效液相层析法，以前曾指高压液相层析法(high pressure liquid chromatography)，是一种色谱分析技术，用来分离混合物，以确认并量化各个成分的比例。它依赖泵加压样品以令其通过填充有固定相的压力柱，导致样品的各个成分因与固定相的相互作用力不同而分离。高效液相色谱法常用于生物化学和分析化学。High performance liquid chromatography (English: high performance liquid chromatography, abbreviation HPLC), also translated as high performance liquid chromatography, formerly referred to as high pressure liquid chromatography (high pressure liquid chromatography), is a chromatographic analysis technology that uses to separate mixtures to confirm and quantify the proportions of individual components. It relies on a pump to pressurize the sample through a pressure column filled with a stationary phase, causing the individual components of the sample to separate due to different forces of interaction with the stationary phase. High performance liquid chromatography is commonly used in biochemistry and analytical chemistry.
现有的HPLC-Titer系统，虽然能准确测定细胞培养液上清中的蛋白表达量，但是由于使用的是常规色谱，因此需要的样品体积一般为毫升级别，且定量限约0.5mg/mL。在细胞株筛选阶段，一般会先进行96孔板的铺板。由于96孔板中的培养基一般只有约100μL，所以在96孔板阶段，一般不能做基于HPLC-titer的表达量分析。在一些项目中，会出现表达量低于0.1mg/mL的样品，使用常规的HPLC-titer系统不能对这些样品准确定量。Although the existing HPLC-Titer system can accurately measure protein expression in cell culture supernatant, because it uses conventional chromatography, the required sample volume is generally in the milliliter level, and the quantitation limit is about 0.5 mg/mL. In the cell line screening stage, 96-well plates are usually plated first. Since the culture medium in a 96-well plate is generally only about 100 μL, expression analysis based on HPLC-titer is generally not possible at the 96-well plate stage. In some projects, there will be samples with expression levels lower than 0.1 mg/mL, and these samples cannot be accurately quantified using conventional HPLC-titer systems.
综上，目前还没有一种行之有效的仪器分析手段，能够使用约10μL体积的细胞培养液上清，对上清中的蛋白质进行定量，且定量限低于0.1mg/mL。In summary, there is currently no effective instrument analysis method that can use a volume of about 10 μL of cell culture supernatant to quantify the protein in the supernatant, and the limit of quantitation is lower than 0.1 mg/mL.
发明内容Contents of the invention
现有技术中的HPLC-Titer分析系统需要的样品体积大，定量限高，限制了其在定量分析中的作用，针对上述缺陷，本发明开发了一种应用于培养基上清蛋白定量的nanoHPLC-Titer系统，技术方案如下：The HPLC-Titer analysis system in the prior art requires a large sample volume and a high quantitation limit, which limits its role in quantitative analysis. In view of the above defects, the present invention has developed a nanoHPLC for quantification of culture medium supernatant protein. -Titer system, the technical solution is as follows:
本发明的第一个方面公开了一种新型nanoHPLC-Titer系统，包括nano亲和色谱柱、nanoHPLC色谱泵、自动进样系统和检测器，其特征在于，所述的nano亲和色谱柱的制备方法为：The first aspect of the invention discloses a new nanoHPLC-Titer system, which includes a nano affinity chromatography column, a nanoHPLC chromatography pump, an automatic sampling system and a detector. It is characterized in that the preparation of the nano affinity chromatography column The method is:
(1)取干净的色谱柱管，管末端使用筛板及两通封住，将亲和色谱填料装填至色谱柱管中；(1) Take a clean chromatography column tube, seal the end of the tube with a sieve plate and a union, and fill the affinity chromatography packing into the chromatography column tube;
(2)装填完成后，在色谱柱的入口端封上筛板及两通；(2) After the filling is completed, seal the sieve plate and union at the inlet end of the chromatographic column;
(3)在色谱柱的正常使用压力下，使用流动相冲洗装好的色谱柱；(3) Under the normal operating pressure of the chromatographic column, use mobile phase to flush the installed chromatographic column;
(4)色谱柱冲洗完成后，将两端接口使用堵头密封待用。(4) After flushing the chromatographic column, seal the interfaces at both ends with plugs for later use.
进一步地，步骤(1)所述色谱柱管的材质为熔融石英毛细管，聚醚醚酮管或不锈钢管。进一步地，步骤(1)所述色谱柱管的内径小于等于500μm。Further, the material of the chromatography column tube in step (1) is a fused silica capillary tube, a polyetheretherketone tube or a stainless steel tube. Further, the inner diameter of the chromatography column tube in step (1) is less than or equal to 500 μm.
进一步地，步骤(1)所述色谱柱管的管长为1-25cm。Further, the length of the chromatography column tube in step (1) is 1-25cm.
进一步地，步骤(1)所述的填料为Protein A填料，Protein G填料或Captol L填料。Further, the filler described in step (1) is Protein A filler, Protein G filler or Captol L filler.
进一步地，步骤(3)所述的流动相选自甲醇、乙腈、乙醇或异丙醇等有机相；或者前述有机相与水的混合物。Further, the mobile phase in step (3) is selected from organic phases such as methanol, acetonitrile, ethanol or isopropyl alcohol; or a mixture of the aforementioned organic phase and water.
进一步地，自动进样系统至nano亲和色谱柱之间的死体积小于2μL，nano亲和色谱柱至检测器之间的死体积小于1μL。Furthermore, the dead volume between the automatic sampling system and the nano-affinity chromatographic column is less than 2 μL, and the dead volume between the nano-affinity chromatographic column and the detector is less than 1 μL.
本发明还公开了根据上述的新型nanoHPLC-Titer系统用于培养基上清蛋白定量的用途。The invention also discloses the use of the above-mentioned novel nanoHPLC-Titer system for quantification of culture medium supernatant protein.
与现有技术相比，本发明具有的有益效果：Compared with the prior art, the present invention has the following beneficial effects:
(1)所需样品体积大幅下降，由1mL(HPLC-Titer)降低至10μL，缩小了100倍；(1) The required sample volume has been significantly reduced, from 1mL (HPLC-Titer) to 10μL, which is 100 times smaller;
(2)定量限由0.5mg/mL(HPLC-Titer)降低至0.01mg/mL，降低了50倍。(2) The limit of quantitation is reduced from 0.5 mg/mL (HPLC-Titer) to 0.01 mg/mL, which is a 50-fold reduction.
附图说明Description of drawings
图1为本发明实施例1所装填的色谱柱图。Figure 1 is a diagram of a packed chromatographic column in Example 1 of the present invention.
图2为本发明的nanoHPLC-Titer系统示意图，黑色连接线为连接管路。Figure 2 is a schematic diagram of the nanoHPLC-Titer system of the present invention, and the black connecting line is the connecting pipeline.
图3为标准曲线图。Figure 3 is a standard curve graph.
具体实施方式Detailed ways
下面结合实施例和附图对本发明的具体实施方式做进一步的详细描述。以下实施例和附图用于说明本发明，但不用来限制本发明的范围。Specific implementations of the present invention will be described in further detail below with reference to the examples and drawings. The following examples and drawings are used to illustrate the present invention but are not intended to limit the scope of the present invention.
实施例1 nano亲和色谱柱的制作Example 1 Preparation of nano affinity chromatography column
nano亲和色谱柱为自制色谱柱，其制备方法如下。使用的色谱柱管材质为熔融石英毛细管；使用的色谱管内径小于等于500μm，管长1-25cm之间。取一根干净的上述色谱柱管，管末端使用筛板及两通封住。将亲和色谱填料装填至色谱柱管中，使用的填料是Protein A填料。装填完成后，在色谱柱的入口端封上筛板及两通。在对应的色谱柱的正常使用压力下，使用流动相冲洗装好的色谱柱，冲洗时间大于等于1h，使用的流动相是甲醇。色谱柱冲洗完成后，将两端接口使用堵头密封待用。在显微镜下观察色谱柱柱床是否密实完整，装填完成的色谱柱显微镜图见图1。The nano affinity chromatography column is a self-made chromatography column, and its preparation method is as follows. The material of the chromatography column tube used is fused silica capillary tube; the inner diameter of the chromatography tube used is less than or equal to 500 μm, and the tube length is between 1-25cm. Take a clean chromatography column tube mentioned above, and seal the end of the tube with a sieve plate and a union. Pack the affinity chromatography packing into the chromatography column tube. The packing used is Protein A packing. After the filling is completed, seal the sieve plate and union at the inlet end of the chromatographic column. Under the normal operating pressure of the corresponding chromatographic column, use the mobile phase to flush the installed chromatographic column. The flushing time is greater than or equal to 1 hour. The mobile phase used is methanol. After the chromatographic column is flushed, seal the interfaces at both ends with plugs until use. Observe under the microscope whether the chromatographic column bed is dense and complete. See Figure 1 for the microscope picture of the completed chromatographic column.
实施例2 nano亲和色谱柱的制作Example 2 Preparation of nano affinity chromatography column
nano亲和色谱柱为自制色谱柱，其制备方法如下。使用的色谱柱管材质为聚醚醚酮管；使 用的色谱管内径小于等于500μm，管长1-25cm之间。取一根干净的上述色谱柱管，管末端使用筛板及两通封住。将亲和色谱填料装填至色谱柱管中，使用的填料是Protein G填料。装填完成后，在色谱柱的入口端封上筛板及两通。在对应的色谱柱的正常使用压力下，使用流动相冲洗装好的色谱柱，冲洗时间大于等于1h，使用的流动相是乙腈。色谱柱冲洗完成后，将两端接口使用堵头密封待用。The nano affinity chromatography column is a self-made chromatography column, and its preparation method is as follows. The material of the chromatography column tube used is polyetheretherketone tube; the inner diameter of the chromatography tube used is less than or equal to 500 μm, and the tube length is between 1-25cm. Take a clean chromatography column tube mentioned above, and seal the end of the tube with a sieve plate and a union. Pack the affinity chromatography packing into the chromatography column tube. The packing used is Protein G packing. After the filling is completed, seal the sieve plate and union at the inlet end of the chromatographic column. Under the normal operating pressure of the corresponding chromatographic column, use the mobile phase to flush the installed chromatographic column. The flushing time is greater than or equal to 1 hour. The mobile phase used is acetonitrile. After the chromatographic column is flushed, seal the interfaces at both ends with plugs until use.
实施例3 nano亲和色谱柱的制作Example 3 Preparation of nano affinity chromatography column
nano亲和色谱柱为自制色谱柱，其制备方法如下。使用的色谱柱管材质为不锈钢管；使用的色谱管内径小于等于500μm，管长1-25cm之间。取一根干净的上述色谱柱管，管末端使用筛板及两通封堵。将亲和色谱填料装填至色谱柱管中，使用的填料是Captol L填料。装填完成后，在色谱柱的入口端封上筛板及两通。在对应的色谱柱的正常使用压力下，使用流动相冲洗装好的色谱柱，冲洗时间大于等于1h，使用的流动相是乙醇和水的混合物。色谱柱冲洗完成后，将两端接口使用堵头密封待用。The nano affinity chromatography column is a self-made chromatography column, and its preparation method is as follows. The material of the chromatography column tube used is stainless steel tube; the inner diameter of the chromatography tube used is less than or equal to 500 μm, and the tube length is between 1-25cm. Take a clean chromatography column tube mentioned above, and seal the end of the tube with a sieve plate and a union. Pack the affinity chromatography packing into the chromatography column tube. The packing used is Captol L packing. After the filling is completed, seal the sieve plate and union at the inlet end of the chromatographic column. Under the normal operating pressure of the corresponding chromatographic column, use the mobile phase to flush the installed chromatographic column. The flushing time is greater than or equal to 1 hour. The mobile phase used is a mixture of ethanol and water. After the chromatographic column is flushed, seal the interfaces at both ends with plugs until use.
实施例4新型的nanoHPLC-Titer系统定量测定培养基上清蛋白Example 4 Novel nanoHPLC-Titer system for quantitative determination of culture medium supernatant protein
将实施例1中制备好的nano色谱柱安装在nano色谱仪中，nano色谱仪至少包括nano色谱泵、自动进样系统和检测器。通过合适管路的连接，确保自动进样系统至nano色谱柱之间的死体积小于2μL，nano色谱柱至检测器之间的死体积小于1μL。nanoHPLC-Titer系统图见图2。The nano chromatographic column prepared in Example 1 is installed in a nano chromatograph, which at least includes a nano chromatography pump, an automatic sampling system and a detector. Through the connection of appropriate pipelines, ensure that the dead volume between the automatic sampling system and the nano chromatographic column is less than 2 μL, and the dead volume between the nano chromatographic column and the detector is less than 1 μL. The nanoHPLC-Titer system diagram is shown in Figure 2.
将装有需要分析表达量样品的96孔板放入板式离心机，1000转离心10min，每个样品孔分别取10μL上清至锥形底色谱进样瓶。色谱分析时进样量1μL。Place the 96-well plate containing the expression samples to be analyzed into a plate centrifuge, and centrifuge at 1000 rpm for 10 minutes. Take 10 μL of supernatant from each sample well into a conical bottom chromatography injection bottle. The injection volume during chromatographic analysis was 1 μL.
使用磷酸盐缓冲液流动相稀释标准品，标准品为所预备分析的目标蛋白质：贝伐珠单抗。将标准品分别稀释至0.50mg/mL、0.20mg/mL、0.10mg/mL、0.05mg/mL、0.03mg/mL、0.02mg/mL、0.01mg/mL，并使用Lunatic高通量微流控光谱分析仪测定其准确浓度。使用搭建的nanoHPLC-Titer系统依次分析得到的各浓度标准品工作液和样品，得到标准曲线(图3)和样品滴度值(表1)。Use phosphate buffer mobile phase to dilute the standard, which is the target protein to be analyzed: bevacizumab. The standards were diluted to 0.50mg/mL, 0.20mg/mL, 0.10mg/mL, 0.05mg/mL, 0.03mg/mL, 0.02mg/mL, 0.01mg/mL, and Lunatic high-throughput microfluidic was used Spectral analyzer determines its accurate concentration. The built nanoHPLC-Titer system was used to analyze the standard working solutions and samples of each concentration obtained in sequence, and the standard curve (Figure 3) and sample titer value (Table 1) were obtained.
表1Table 1
Figure PCTCN2022142304-appb-000001
Figure PCTCN2022142304-appb-000001
结果显示标准曲线相关系数＞0.99，各浓度标准品工作液的谱峰的信噪比均大于10，且各浓度样品三重复RSD均小于10％。即本系统能够使用约10μL体积的细胞培养液上清，对上清中的蛋白质进行定量，且定量限低至0.01mg/mL。The results show that the correlation coefficient of the standard curve is >0.99, the signal-to-noise ratio of the peaks of the standard working solution at each concentration is greater than 10, and the RSD of the three replicates of the samples at each concentration is less than 10%. That is, this system can use a volume of about 10 μL of cell culture supernatant to quantify the protein in the supernatant, and the quantification limit is as low as 0.01 mg/mL.
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, etc. may be made without departing from the spirit and principles of the present invention. All simplifications should be equivalent substitutions, and are all included in the protection scope of the present invention.

Claims (8)

  1. 一种新型nanoHPLC-Titer系统，包括nano亲和色谱柱、nanoHPLC色谱泵、自动进样系统和检测器，其特征在于，所述的nano亲和色谱柱的制备方法为：A new type of nanoHPLC-Titer system includes a nano affinity chromatography column, a nanoHPLC chromatography pump, an automatic sampling system and a detector. It is characterized in that the preparation method of the nano affinity chromatography column is:
    (1)取干净的色谱柱管，管末端使用筛板及两通封堵，将亲和色谱填料装填至色谱柱管中；(1) Take a clean chromatography column tube, seal the end of the tube with a sieve plate and a union, and fill the affinity chromatography packing into the chromatography column tube;
    (2)装填完成后，在色谱柱的入口端封上筛板及两通；(2) After the filling is completed, seal the sieve plate and union at the inlet end of the chromatographic column;
    (3)在色谱柱的正常使用压力下，使用流动相冲洗装好的色谱柱；(3) Under the normal operating pressure of the chromatographic column, use mobile phase to flush the installed chromatographic column;
    (4)色谱柱冲洗完成后，将两端接口使用堵头密封待用。(4) After flushing the chromatographic column, seal the interfaces at both ends with plugs for later use.
  2. 根据权利要求1所述的新型nanoHPLC-Titer系统，其特征在于，步骤(1)所述色谱柱管的材质为熔融石英毛细管，聚醚醚酮管或不锈钢管。The new nanoHPLC-Titer system according to claim 1, characterized in that the material of the chromatographic column tube in step (1) is a fused silica capillary tube, a polyether ether ketone tube or a stainless steel tube.
  3. 根据权利要求1所述的新型nanoHPLC-Titer系统，其特征在于，步骤(1)所述色谱柱管的内径小于等于500μm。The new nanoHPLC-Titer system according to claim 1, characterized in that the inner diameter of the chromatographic column tube in step (1) is less than or equal to 500 μm.
  4. 根据权利要求1所述的新型nanoHPLC-Titer系统，其特征在于，步骤(1)所述色谱柱管的管长为1-25cm。The new nanoHPLC-Titer system according to claim 1, characterized in that the length of the chromatographic column tube in step (1) is 1-25cm.
  5. 根据权利要求1所述的新型nanoHPLC-Titer系统，其特征在于，步骤(1)所述的填料为Protein A填料，Protein G填料或Captol L填料。The novel nanoHPLC-Titer system according to claim 1, characterized in that the filler described in step (1) is Protein A filler, Protein G filler or Captol L filler.
  6. 根据权利要求1所述的新型nanoHPLC-Titer系统，其特征在于，步骤(3)所述的流动相选自甲醇、乙腈、乙醇或异丙醇这四种有机相；或者前述有机相与水的混合物。The new nanoHPLC-Titer system according to claim 1, characterized in that the mobile phase in step (3) is selected from the four organic phases of methanol, acetonitrile, ethanol or isopropanol; or the mixture of the aforementioned organic phase and water mixture.
  7. 根据权利要求1所述的新型nanoHPLC-Titer系统，其特征在于，自动进样系统至nano亲和色谱柱之间的死体积小于2μL，nano亲和色谱柱至检测器之间的死体积小于1μL。The new nanoHPLC-Titer system according to claim 1, characterized in that the dead volume between the automatic sampling system and the nano affinity chromatography column is less than 2 μL, and the dead volume between the nano affinity chromatography column and the detector is less than 1 μL. .
  8. 根据权利要求1-7任一项所述的新型nanoHPLC-Titer系统用于培养基上清蛋白定量的用途。Use of the novel nanoHPLC-Titer system according to any one of claims 1 to 7 for quantification of culture medium supernatant protein.
PCT/CN2022/142304 2022-07-27 2022-12-27 Nanohplc-titer system for quantitative determination of supernatant protein in culture medium WO2024021509A1 (en)

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