WO2020211584A1 - 组合物中泊洛沙姆188的检测方法 - Google Patents

组合物中泊洛沙姆188的检测方法 Download PDF

Info

Publication number
WO2020211584A1
WO2020211584A1 PCT/CN2020/079784 CN2020079784W WO2020211584A1 WO 2020211584 A1 WO2020211584 A1 WO 2020211584A1 CN 2020079784 W CN2020079784 W CN 2020079784W WO 2020211584 A1 WO2020211584 A1 WO 2020211584A1
Authority
WO
WIPO (PCT)
Prior art keywords
mobile phase
solution
detector
poloxamer
detection method
Prior art date
Application number
PCT/CN2020/079784
Other languages
English (en)
French (fr)
Inventor
曹宜力
李镭
王海彬
叶银萍
郭芮
Original Assignee
海正生物制药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 海正生物制药有限公司 filed Critical 海正生物制药有限公司
Priority to CN202080012695.9A priority Critical patent/CN113424057B/zh
Publication of WO2020211584A1 publication Critical patent/WO2020211584A1/zh

Links

Images

Classifications

    • 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
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation

Definitions

  • the invention belongs to the field of pharmaceutical preparations and relates to a method for detecting poloxamer 188 in a composition.
  • Poloxamer 188 is a polyoxyethylene polyoxypropylene ether block copolymer. It is a polymer nonionic surfactant and an excellent new excipient for pharmaceutical preparations. It is non-toxic, non-irritating, and allergic to skin and mucous membranes. It is widely used in the cell culture and fermentation process of biological products, mainly used as defoamer, solubilizer, dispersant, emulsifier and stabilizer.
  • the safety factor (ISF) of poloxamer 188 relative to the human body is 314, and the concentration of the production cell culture medium is not more than 1 mg/ml.
  • the half-lethal dose relative to a 90kg adult is 90g, and the intake of each dose is not more than 286mg.
  • Poloxamer 188 Since Poloxamer 188 has no ultraviolet absorption and the residual amount is too low, conventional detectors (UV) cannot effectively detect it.
  • the detection sensitivity of the differential detector (RID) is low, and gradient elution cannot be used, and it is not suitable for gradient elution in reversed phase methods.
  • the evaporative light detector (ELSD) has a detection sensitivity of ng level, but its repeatability is poor. Therefore, the conventional methods are not suitable for the detection of Poloxamer 188.
  • poloxamer 188 which is an antifoaming agent, is continuously removed from the purified solution through several purification methods, which makes the concentration of the target analyte extremely low, which increases the difficulty of detection.
  • the molecular weight of monoclonal antibody drugs is generally around 150KDa, and conventional C18 chromatographic columns cannot satisfy the separation of poloxamer 188, and the entry of monoclonal antibody drug molecules into conventional C18 chromatographic columns will cause the chromatographic column to block and damage the chromatographic column. While the size exclusion chromatography column can perform better separation for high-purity samples, but for complex samples, such as culture medium and partial purified liquid, the presence of other proteins will interfere with sample detection.
  • the detection sensitivity of the existing ELSD detector is low, and it cannot detect samples with a content of less than 200 ng or lower, which makes the use of drugs containing the excipient poloxamer 188 during the clinical period have certain safety risks. Therefore, inventing a sensitive and rapid method for detecting poloxamer 188 in an antibody composition is more conducive to improving the quality of antibody drugs.
  • the present invention provides a method for detecting poloxamer 188 in a composition.
  • the method is fast, simple, reproducible, and has higher detection sensitivity, and has good application prospects.
  • a method for detecting poloxamer 188 in a composition adopts a reversed-phase chromatography column combined with an electrospray detector (hereinafter referred to as a CAD detector) for detection, and the chromatography column is Cadenza HS-C18 reaction Phase chromatography column.
  • a CAD detector electrospray detector
  • the composition described in this application is a pharmaceutical composition, which can be any pharmaceutical composition containing Poloxamer 188.
  • adalimumab compositions refers to a mixture containing adalimumab, poloxamer 188 or/and other impurities, such as adalimumab culture medium, adalimumab purified liquid, adalimumab antigen solution, adalimumab Monoclonal antibody preparations, etc.
  • the specification of the Cadenza HS-C18 reverse phase chromatography column is 150*3mm, 3 ⁇ m.
  • the detection method adopts an external standard method to quantitatively detect the content of poloxamer 188 in the sample.
  • the mobile phase system used in the reversed-phase chromatography column includes mobile phase A and mobile phase B, wherein mobile phase A is 0.1M ammonium acetate solution, mobile phase B is acetonitrile; mobile phase before entering the high performance liquid chromatograph , Are filtered with 0.22 ⁇ m filter membrane.
  • the mobile phase A is a 0.1M ammonium acetate solution with a pH between 5.0 and 6.0, preferably pH 5.6.
  • the mobile phase elution procedure adopted by the reversed-phase chromatography column is: 0-5 min, mobile phase B 0%, 5-10 min, mobile phase B 10%; 10-18 min, mobile phase B 45%; 18 -22min, mobile phase B 100%; 22-28min, mobile phase B 0%.
  • the column temperature of the reverse phase chromatography column is 25°C-40°C, preferably 35°C.
  • the flow rate of the mobile phase is 0.4 ml/min to 0.8 ml/min, preferably 0.5 ml/min.
  • the injection volume of the reverse phase chromatography column is 10-100 ⁇ l, preferably 20 ⁇ l.
  • the parameter settings of the CAD detector include: the gas used by the CAD detector is high-purity nitrogen, the nitrogen purity is greater than or equal to 99.9%; the gas pressure is 0.5MPa ⁇ 0.05MPa; the CAD detector temperature mode is the High mode; CAD The temperature of the detector is 50°C; the sampling frequency is 5Hz, and the filter constant is 3.6S; the CAD detector opens the gradient compensation function, and the maximum liquid flow rate of the detector should not be higher than 2ml/min; the gradient compensation volume is 100 ⁇ l.
  • the present invention develops a method for detecting the content of poloxamer 188 in the composition by using a reverse phase chromatographic column combined with a CAD detector. Centrifuge the tested solution and take the supernatant directly into the chromatographic system for detection.
  • the method of the present invention has the advantages of quickness and simplicity, good reproducibility, high sensitivity, good selectivity, environmental protection, etc.
  • the culture medium or purified liquid does not need to be pretreated for protein removal, and the detection caused by the loss of the measured component due to sample pretreatment is reduced. A situation where the result is unreliable.
  • This method can be used for drugs produced by cell culture methods, such as adalimumab in cell culture, purification, and the detection of poloxamer 188 residues in the final product.
  • the target analyte can be quickly detected within 22 minutes, and the detection limit of poloxamer 188 obtained is less than 100 ng, which is significantly lower than the existing reported methods, and the quality control of adalimumab is more stringent.
  • Figure 1 shows the spectrum of the Candenza HS-C18 chromatographic column detecting the adamu stock solution added with the poloxamer 188 standard solution.
  • Figure 2 shows the poloxamer 188 standard and blank comparison chart detected by Candenza HS-C18 column.
  • Figure 3 shows the spectrum of adamu stock solution added with poloxamer 188 standard solution on the ZORBAX SB-C18 column.
  • Figure 4 shows the detection spectrum of the adamu stock solution added with the poloxamer 188 standard solution on the PolyRP-NP3 column.
  • Figure 5 shows the poloxamer 188 standard and blank comparison chart detected by the ZORBAX SB-C18 column.
  • Figure 6 shows the poloxamer 188 standard and blank control spectrum detected by the PolyRP-NP3 column.
  • Figure 7 shows the spectrum of the adamu stock solution added with poloxamer 188 standard solution on the CAPCELL PAK MF C8 column.
  • Figure 8 is the detection map of the test solution of the adalimumab culture solution.
  • Figure 9 is the detection spectrum of adalimumab purified C1P test solution.
  • Figure 10 is the detection spectrum of the C2P test solution of adalimumab purified solution.
  • Figure 11 is the detection spectrum of the C3P test solution of adalimumab purified solution.
  • Figure 12 is the detection map of the test solution of adalimin antigen solution.
  • Figure 13 is the detection spectrum of the test solution of the finished product of Adalimumab.
  • the reagents and equipment used in the following examples are all conventional and can be obtained through commercial channels; the methods used in the following examples are all conventional methods, and those skilled in the art can undoubtedly follow their descriptions. Repeat the experiment and obtain the corresponding results.
  • Mobile phase A is 0.1M ammonium acetate solution, pH 5.6; mobile phase B is acetonitrile.
  • Chinese hamster ovary (CHO) cells containing stable and highly efficient expression of adalimumab light and heavy chain genes were cultured at 37°C for 12 days with Permab basal medium and Cell boost medium, and adalimumab was obtained after deep filtration Anti-culture broth; the adalimumab culture broth was separated by MabSelect SuRe affinity chromatography, and eluted at pH 3.5 to obtain the adalimumab purified solution C1P; the adalimumab purified solution C1P was subjected to low pH virus inactivation (pH3.5, 120min), enter the QFF anion exchange chromatography to separate, and obtain the adalimumab purified C2P in the flow-through mode; then enter the adalimumab purified C2P into the SPFF cation exchange chromatography for separation, and linearly elute it.
  • CHO Chinese hamster ovary
  • the amino acid sequence of adalimumab and the light and heavy chain coding sequences are all conventional techniques in the art.
  • the materials and specific operation methods involved in the cell culture, various chromatographic separation, filtration, inactivation and other procedures in the above process are all conventional techniques in the field.
  • the gas used by the CAD detector is high-purity nitrogen, the nitrogen purity is greater than or equal to 99.9%; the gas pressure is 0.5MPa ⁇ 0.05MPa.
  • Mobile phase A is 0.1M ammonium acetate solution, pH 5.6.
  • Mobile phase B is acetonitrile.
  • the elution procedure is as follows: 0-5min, mobile phase B 0%; 5-10min, mobile phase B 10%; 10-18min, mobile phase B 45%; 18-22min, mobile phase B 100% ; 22-28min, mobile phase B 0%; column temperature is 35°C; injection volume is 20 ⁇ l; CAD detector temperature mode is High mode; detector temperature is 50°C.
  • the sampling frequency is 5Hz, and the filtering constant is 3.6S.
  • the CAD detector opens the gradient compensation function, and the maximum liquid flow rate of the detector should not be higher than 2ml/min; the gradient compensation volume is 100 ⁇ l; the gas pressure is 0.5MPa ⁇ 0.05MPa.
  • Mobile phase A 1 Candenza HS-C18 150*3mm, 3 ⁇ m 0.1M ammonium acetate solution pH 5.6 Acetonitrile 2 ZORBAX SB-C18 250*4.6mm, 5 ⁇ m 0.1M ammonium acetate solution pH 5.6 Acetonitrile 3
  • Adalimumab was completely separated from Poloxamer 188; at the same time, there was no blank peak to interfere with the detection of Poloxamer 188 ( Figure 2); while ZORBAX SB-C18 and Poly RP -NP3 can also completely separate Adalimumab from Poloxamer 188 ( Figure 3 and Figure 4); however, both ZORBAX SB-C18 and Poly RP-NP3 columns have blank peaks in the blank solution that interfere with Poloxamer 188 Detected ( Figure 5 and Figure 6), and the column pressure of the ZORBAX SB-C18 column was significantly increased after adalimumab was introduced, from 36 bar to 125 bar at the beginning, causing irreversible damage to the column.
  • Mobile phase B 1 0.05M ammonium acetate solution pH 5.6 Acetonitrile 2 0.1M ammonium acetate solution pH 5.6 Acetonitrile 3 0.2M ammonium acetate solution pH 5.6 Acetonitrile 4 0.05M ammonium formate solution pH 5.6 Acetonitrile
  • the ultrapure water and 50 ⁇ g/ml poloxamer 188 standard solution were tested and analyzed.
  • the results showed that the baseline signal and noise fluctuations of ammonium acetate and ammonium formate showed that the baseline noise range of ammonium acetate was 0.3 ⁇
  • 0.1M ammonium acetate solution has the smallest baseline noise fluctuation, which is between 0.5 and 1.3.
  • the baseline noise of ammonium formate is about 0.9-2.3, which fluctuates more than the baseline noise of ammonium acetate solution. Therefore, the choice of ammonium acetate is more conducive to high-sensitivity detection requirements and reduces signal noise interference caused by mobile phase.
  • the mobile phase A is 0.1M ammonium acetate solution pH 5.6
  • the mobile phase B is acetonitrile.
  • the elution procedure is as follows: 0-5min, mobile phase B 0%; 5-10min, mobile phase B 10%; 10-18min, mobile phase B 45%; 18-22min, mobile phase B 100% ; 22-28min, mobile phase B 0%; column temperature is 35°C; injection volume is 20 ⁇ l.
  • the CAD detector opens the gradient compensation function, and the liquid flow rate of the detector should not be higher than 2ml/min; the gradient compensation volume is 100 ⁇ l; the gas pressure is 0.5MPa ⁇ 0.05MPa.
  • the mobile phase A is 0.1M ammonium acetate solution pH 5.6
  • the mobile phase B is acetonitrile.
  • the elution procedure is as follows: 0-5min, mobile phase B 0%; 5-10min, mobile phase B 10%; 10-18min, mobile phase B 45%; 18-22min, mobile phase B 100% ; 22-28min, mobile phase B 0%; column temperature is 35°C; injection volume is 20 ⁇ l.
  • the CAD detector and the ELSD detector are compared with two different detectors, see Table 6 below. Determine the detector selection.
  • CAD detector parameter setting CAD detector temperature mode is High mode; detector temperature is 50°C. The sampling frequency is 5Hz, and the filtering constant is 3.6S.
  • the CAD detector opens the gradient compensation function, and the liquid flow rate of the detector should not be higher than 2ml/min; the gradient compensation volume is 100 ⁇ l; the gas pressure is 0.5MPa ⁇ 0.05MPa.
  • ELSD detector parameter settings gas flow rate 1.0SLM, atomization temperature 60°C, evaporation light temperature 105°C, data collection frequency 10H, detector gain PTM10.
  • CAD detector Under the same chromatographic conditions such as chromatographic column, mobile phase composition and elution gradient, CAD detector and ELSD were used to detect and analyze ultrapure water and 50 ⁇ g/ml poloxamer 188 standard solution. The results show that the data is shown in Table 7 below.
  • the detection limit using the CAD detector method is less than 100ng, while the detection limit using the ELSD detector method is less than 200ng. It shows that the CAD detector has higher inspection sensitivity, which is conducive to the lower limit analysis, so the CAD detector is selected as the detector of this method.
  • the mobile phase A is 0.1M ammonium acetate solution pH 5.6
  • the mobile phase B is acetonitrile.
  • the elution procedure is as follows: 0-5min, mobile phase B 0%; 5-10min, mobile phase B 10%; 10-18min, mobile phase B 45%; 18-22min, mobile phase B 100% ; 22-28min, mobile phase B 0%; column temperature is 35°C; injection volume is 20 ⁇ l; CAD detector temperature mode is High mode; detector temperature is 50°C.
  • the sampling frequency is 5Hz, and the filtering constant is 3.6S.
  • the CAD detector opens the gradient compensation function, and the liquid flow rate of the detector should not be higher than 2ml/min; the gradient compensation volume is 100 ⁇ l; the gas pressure is 0.5MPa ⁇ 0.05MPa.
  • the linearity, limit of detection (LOD), limit of quantification (LQD), repeatability, precision and accuracy of the method of the present invention were verified, see Table 8.
  • the results showed that when the concentration of poloxamer 188 was in the range of 5-50 ⁇ g/ml, the peak area and the concentration of poloxamer 188 standard solution showed a good linear range, and the square of the correlation coefficient was higher than 0.997.
  • the detection limit (signal-to-noise ratio of 2 ⁇ 4) and quantification limit (signal-to-noise ratio of 8 ⁇ 10) are 100ng and 280ng, respectively, and the sensitivity is high.
  • the experimental repeatability is expressed by repeating 6 times to obtain the relative standard deviation (RSD) of the peak area and retention time.
  • the peak area repeatability RSD is 2.7%, and the retention time repeatability RSD is 0.06%; the RSD of the day and the day are 1.1 respectively. % And 1.7%, the precision is good; the accuracy of the method is between 101.3% and 104.8%, and the relevant results are shown in Table 8.
  • the mobile phase A is 0.1M ammonium acetate solution pH 5.6
  • the mobile phase B is acetonitrile.
  • the elution procedure is as follows: 0-5min, mobile phase B 0%; 5-10min, mobile phase B 10%; 10-18min, mobile phase B 45%; 18-22min, mobile phase B 100% ; 22-28min, mobile phase B 0%; column temperature is 35°C; injection volume is 20 ⁇ l; CAD detector temperature mode is High mode; detector temperature is 50°C.
  • the sampling frequency is 5Hz, and the filtering constant is 3.6S.
  • the CAD detector turns on the gradient compensation function, and the liquid flow rate of the detector must not be higher than 2ml/min; the gradient compensation volume is 100 ⁇ l.
  • the gas pressure is 0.5MPa ⁇ 0.05MPa.
  • the test results are shown in Table 9.
  • the concentration of Poloxamer 188 in the adalimumab culture solution ( Figure 8) is 17.3 ⁇ g/ml, and the recovery rate is 96.8%.
  • Adalimumab purified solution C1P test product solution ( Figure 9), C2P test product solution ( Figure 10), C3P test product solution ( Figure 11), original test product solution ( Figure 12) and finished product test product
  • the content of Poloxamer 188 in the solution was below the detection limit of 5 ⁇ g/ml.
  • Test solution content Add concentration Measured concentration Sample recovery rate Culture medium 17.3 ⁇ g/ml 25 ⁇ g/ml 41.5 ⁇ g/ml 96.8% Purified solution C1P /* 25 ⁇ g/ml 25.4 ⁇ g/ml 101.6% Purified solution C2P /* 25 ⁇ g/ml 25.8 ⁇ g/ml 103.2% Purified solution C3P /* 25 ⁇ g/ml 24.4 ⁇ g/ml 97.6% Stock solution /* 25 ⁇ g/ml 24.1 ⁇ g/ml 96.4% Finished product /* 25 ⁇ g/ml 25.6 ⁇ g/ml 102.4%

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Treatment Of Liquids With Adsorbents In General (AREA)

Abstract

组合物中泊洛沙姆188的检测方法，检测方法利用反相色谱柱联合电喷雾检测器检测组合物中泊洛沙姆188含量，该方法具备快速简洁、灵敏度高、重复性好的优势。

Description

组合物中泊洛沙姆188的检测方法 技术领域
本发明属于药物制剂领域，涉及组合物中泊洛沙姆188的检测方法。
背景技术
泊洛沙姆188为聚氧乙烯聚氧丙烯醚嵌段共聚物，是一种高分子非离子表面活性剂，是优良的药物制剂新辅料，无毒、对皮肤黏膜无刺激性、过敏性，广泛应用于生物制品的细胞培养和发酵过程中，主要用作消泡剂、增溶剂、分散剂、乳化剂和稳定剂。
据报道泊洛沙姆188相对于人体的安全因子(ISF)为314，生产细胞培养基的浓度不大于1mg/ml。相对于90kg成人的半数致死量为90g，每剂量摄入不大于286mg，曾经有报道称日本发生过消毒剂中表面活性剂致死输液病人事件。因此，检测药物组合物中泊洛沙姆188含量，对于该药物的使用安全性非常必要。
由于泊洛沙姆188其无紫外吸收，且残留量过低，使得常规的检测器(UV)无法对其进行有效检测。示差检测器(RID)其检测灵敏度低，且无法采用梯度洗脱，不适用于反相方法梯度洗脱。蒸发光检测器(ELSD)其检测灵敏度有ng级别，但其重复性差。因此，常规的方法均不适用于泊洛沙姆188的检测。另一方面，抗体培养过程中，会通过几步纯化手段不断的除去纯化液中的作为消泡剂的泊洛沙姆188，使得目标分析物的浓度极低，增加了检测难度。同时由于大分子抗体的存在，其检测进一步受到了干扰。现有文献报道中，有使用Poly RP-100色谱柱与ELSD检测器结合，测定注射液中泊洛沙姆188含量，检测灵敏度约400ng左右(李响等.蒸发光散射检测法测定重组人IL-12注射液中泊洛沙姆188.药物评价研究2018年09期)。单抗药物分子量普遍在150KDa左右，常规C18色谱柱无法满足与泊洛沙姆188分离，且单抗药物分子进入常规C18色谱柱会造成色谱柱堵塞，损坏色谱柱。而分子排阻色谱柱对于高纯度样品可以进行较好分离，但是对于复杂样品，例如培养液和部分纯化液，由于其他蛋白的存在，会干扰样品检测。现有的ELSD检测器的检测灵敏度低，无法检测到200ng以下或者更低含量的样品检测，使得在临床期间使用含辅料泊洛沙姆188的药物存在一定安全隐患。因此，发明一种灵敏快速的检测抗体组合物中泊洛沙姆188的方法，更有利于提高抗体药物的质量。
发明内容
针对现有技术存在的灵敏度不足问题，本发明提供了一种检测组合物中的泊洛沙姆188的方法。该方法快速、简便、重复性好、检测灵敏度更高，具有很好的应用前景。
本发明具体技术方案如下：一种组合物中泊洛沙姆188的检测方法，采用反相色谱柱联合电喷雾检测器(以下简称CAD检测器)进行检测，所述色谱柱为Cadenza HS-C18反相色谱柱。
本申请中所述的组合物为药物组合物，可以是任何含泊洛沙姆188的药物组合物。特别是抗体组合物，例如阿达木单抗组合物。阿达木单抗组合物是指含有阿达木单抗、泊洛沙姆188或/和其它杂质的混合物，如阿达木单抗培养液、阿达木单抗纯化液、阿达木单抗原液、阿达木单抗制剂等。
作为优选，所述Cadenza HS-C18反相色谱柱的规格为150*3mm，3μm。
作为优选，所述检测方法采用外标法对样品中泊洛沙姆188含量进行定量检测。
作为优选，所述反相色谱柱采用的流动相体系包括流动相A和流动相B，其中流动相A为0.1M乙酸铵溶液，流动相B为乙腈；流动相在进入高效液相色谱仪前，均用0.22μm的滤膜过滤。
具体的，所述的流动相A为0.1M乙酸铵溶液，pH在5.0～6.0之间，优选pH5.6。
作为优选，所述反相色谱柱采用的流动相的洗脱程序为：0-5min，流动相B 0％，5-10min，流动相B 10％；10-18min，流动相B 45％；18-22min，流动相B 100％；22-28min，流动相B 0％。
具体的，所述反相色谱柱的柱温为25℃～40℃，优选35℃。
具体的，所述流动相的流速为0.4ml/min～0.8ml/min，优选0.5ml/min。
具体的，所述反相色谱柱的进样体积为10～100μl，优选20μl。
具体的，所述CAD检测器的参数设置包括：CAD检测器使用的气体为高纯度氮气，氮气纯度大于等于99.9％；气体压力为0.5MPa±0.05MPa；CAD检测器温度模式为High模式；CAD检测器温度为50℃；采样频率5Hz，过滤常数3.6S；CAD检测器开启梯度补偿功能，检测器液体流速最高不得高于2ml/min；梯度补偿体积为100μl。
本发明开发了一种利用反相色谱柱结合CAD检测器检测组合物中泊洛沙姆188含量的方法。将被测溶液离心后取上清液直接进入色谱体系检测。本发明方法具有快速简便、重复性好、灵敏度高、选择性好、环保等优点，培养液或纯化液无需进行除蛋白的预处理，减少因样品前处理使得被测组分损失而导致的检测结果不可信的情况。该方法可用于以细 胞培养方法生产的药物，如阿达木单抗在细胞培养、纯化及最终产品中的泊洛沙姆188的残留检测。在最佳条件下，目标分析物在22min内快速检测，获得的泊洛沙姆188检测限小于100ng，明显低于现有报道过的方法，在阿达木单抗的质量控制上更加严谨。
附图说明
图1为Candenza HS-C18色谱柱检测添加了泊洛沙姆188标准溶液的阿达木原液所得的图谱。
图2为Candenza HS-C18色谱柱检测所得的泊洛沙姆188标准品与空白对照图谱。
图3为ZORBAX SB-C18色谱柱检测添加了泊洛沙姆188标准溶液的阿达木原液所得的图谱。
图4为PolyRP-NP3色谱柱检测添加了泊洛沙姆188标准溶液的阿达木原液所得的图谱。
图5为ZORBAX SB-C18色谱柱检测所得的泊洛沙姆188标准品与空白对照图谱。
图6为PolyRP-NP3色谱柱检测所得的泊洛沙姆188标准品与空白对照图谱。
图7为CAPCELL PAK MF C8色谱柱检测添加了泊洛沙姆188标准溶液的阿达木原液所得的图谱。
图8为阿达木单抗培养液供试品溶液检测图谱。
图9为阿达木单抗纯化液C1P供试品溶液检测图谱。
图10为阿达木单抗纯化液C2P供试品溶液检测图谱。
图11为阿达木单抗纯化液C3P供试品溶液检测图谱。
图12为阿达木单抗原液供试品溶液检测图谱。
图13为阿达木单抗成品供试品溶液检测图谱。
具体实施方式
下面通过具体实施例子对本发明原理及优势进行解释和说明，以便本领域技术人员更好的理解本发明。下述说明为示例性的，并不对其内容进行限定。
如未明确说明，以下实施例中使用试剂和仪器设备都是常规的，可以通过商购途径获得；以下实施例中使用的方法都是常规方法，本领域技术人员根据其描述可以毫无疑义地重复所述实验并获得相应结果。
仪器设备
高效液相色谱仪，型号：Thermo U3000；
CAD检测器，型号：Dionex Corona Veo；
色谱柱：Imtakt Cadenza HS-C18，规格：150*3mm，3μm；
高速离心机，型号：LEGEND MICRO 17R。
试剂
泊洛沙姆188标准品，来源：BASF公司；
乙酸铵，分析级试剂，来源Merck KGaA公司；
乙腈，HPLC级试剂，来源：Merck KGaA公司；
乙酸，分析级试剂，来源：国药集团化学试剂有限公司。
流动相组成
流动相A是0.1M乙酸铵溶液，pH 5.6；流动相B是乙腈。
标准线性制备
称取泊洛沙姆188标准品25.0mg，加入超纯水适量溶解并定容至25ml，避免气泡形成，作为标准储备液。根据需要浓度，分别用超纯水将标准品储备液进行稀释，得到50μg/ml、40μg/ml、30μg/ml、20μg/ml、10μg/ml和5μg/ml的标准线性溶液，超纯水作为空白溶液。
供试品来源
由含稳定、高效表达阿达木单抗轻、重链基因的中国仓鼠卵巢(CHO)细胞，经Permab基础培养基和Cell boost补料培养基于37度培养12天，经深层过滤后获得阿达木单抗培养液；将阿达木单抗培养液进行MabSelect SuRe亲和层析分离，pH3.5条件下洗脱得到阿达木单抗纯化液C1P；将阿达木单抗纯化液C1P进行低pH病毒灭活(pH3.5，120min)，进入QFF阴离子交换层析分离，流穿模式下得到阿达木单抗纯化液C2P；再将阿达木单抗纯化液C2P进入SPFF阳离子交换层析分离，线性洗脱得到阿达木单抗纯化液C3P；然后将阿达木单抗纯化液C3P除病毒过滤及超滤置换缓冲液后得到阿达木单抗原液；往原液中添加适量辅料，经0.22μm滤膜两级除菌过滤后制成阿达木单抗成品。阿达木单抗的氨基酸序列以及轻、重链编码序列均为本领域常规技术。上述过程中出现的细胞培养、各种层析分离、过滤、灭活等程序所涉及的材料和具体操作方式都是本领域常规技术。
以下实施例中，CAD检测器使用的气体为高纯度氮气，氮气纯度大于等于99.9％；气体压力为0.5MPa±0.05MPa。
实施例1色谱柱的筛选
1、色谱条件
流动相A是0.1M乙酸铵溶液，pH 5.6。流动相B是乙腈。采用梯度洗脱模式，洗脱程序如下：0-5min，流动相B 0％；5-10min，流动相B 10％；10-18min，流动相B 45％；18-22min，流动相B 100％；22-28min，流动相B 0％；柱温为35℃；进样体积为20μl；CAD检测器温度模式为High模式；检测器温度为50℃。采样频率5Hz，过滤常数3.6S。CAD检测器开启梯度补偿功能，检测器液体流速最高不得高于2ml/min；梯度补偿体积为100μl；气体压力为0.5MPa±0.05MPa。
2、标准品制备
称取泊洛沙姆188标准品用超纯水将标准品储备液稀释得到50μg/ml泊洛沙姆188标准溶液。
3、添加泊洛沙姆188标准溶液的阿达木原液溶液制备
取阿达木单抗原液1ml，于12000r/min高速离心机离心10min，吸取上清500μl，加入50μg/ml的泊洛沙姆188标准溶液500μl，混匀。等待进样。
4、HPLC-CAD检测
取添加泊洛沙姆188标准溶液的阿达木原液溶液、超纯水和50μg/ml泊洛沙姆188标准溶液，分别用微量进样器取样，进入高效液相色谱体系进行HPLC分析，并用CAD检测器进行检测。
5、实验设计
为了得到更好的分离效果，对不同的色谱柱进行试验，见下表1，选择最佳色谱柱。
表1 不同色谱柱对比表
编号 色谱柱 规格 流动相A 流动相B
1 Candenza HS-C18 150*3mm，3μm 0.1M乙酸铵溶液pH5.6 乙腈
2 ZORBAX SB-C18 250*4.6mm，5μm 0.1M乙酸铵溶液pH5.6 乙腈
3 CAPCELL PAK MF C8 150*4.6mm，5μm 0.1M乙酸铵溶液pH5.6 乙腈
4 PolyRP-NP3 250*4.6mm，3μm 0.1M乙酸铵溶液pH5.6 乙腈
6、实验结果
按照上表1的色谱条件，对添加泊洛沙姆188标准溶液的阿达木原液溶液、超纯水和50μg/ml泊洛沙姆188标准溶液进行检测分析，结果显示，只有Candenza HS-C18色谱柱展现了最佳的分离效果(图1)，阿达木单抗与泊洛沙姆188完全分离；同时无空白峰干扰 泊洛沙姆188检测(图2)；而ZORBAX SB-C18和Poly RP-NP3也能将阿达木单抗与泊洛沙姆188完全分离(图3和图4)；但是ZORBAX SB-C18和Poly RP-NP3色谱柱都存在空白溶液中空白峰干扰泊洛沙姆188检测(图5和图6)，而且ZORBAX SB-C18色谱柱在进了阿达木单抗后色谱柱柱压明显升高，由开始的36bar，上升到125bar，造成色谱柱不可逆损坏。而CAPCELL PAK MF C8检测含阿达木单抗样品时，已经存在干扰泊洛沙姆188的物质，无法满足将阿达木单抗与泊洛沙姆188完全分离(图7)。因此，最佳的色谱柱是Candenza HS-C18。
实施例2流动相组成的筛选
1、色谱条件
色谱柱：Candenza HS-C18(150*3mm，3μm)。采用梯度洗脱模式，洗脱程序如下：0-5min，流动相B 0％；5-10min，流动相B 10％；10-18min，流动相B 45％；18-22min，流动相B 100％；22-28min，流动相B 0％；柱温为35℃；进样体积为20μl；CAD检测器温度模式为High模式；检测器温度为50℃。采样频率5Hz，过滤常数3.6S。CAD检测器开启梯度补偿功能，检测器液体流速最高不得高于2ml/min；梯度补偿体积为100μl；气体压力为0.5MPa±0.05MPa。
2、标准品制备
称取泊洛沙姆188标准品，用超纯水将标准品储备液稀释得到50μg/ml标准溶液。
3、HPLC-CAD检测
取超纯水和50μg/ml标准溶液，分别用微量进样器取样，进入高效液相色谱体系进行HPLC分析，并用CAD检测器进行检测。
4、实验设计
为了得到更好的分离效果，对不同的流动相组成进行试验，见下表2。选择最佳流动相组成。
表2 不同流动相组成对比表
编号 流动相A 流动相B
1 0.05M乙酸铵溶液pH5.6 乙腈
2 0.1M乙酸铵溶液pH5.6 乙腈
3 0.2M乙酸铵溶液pH5.6 乙腈
4 0.05M甲酸铵溶液pH5.6 乙腈
5 0.1M甲酸铵溶液pH5.6 乙腈
6 0.2M甲酸铵溶液pH5.6 乙腈
7 0.1M乙酸铵溶液pH5.6 甲醇
8 0.1M甲酸铵溶液pH5.6 甲醇
9 0.1％TFA水溶液 0.1％TFA乙腈
5、实验结果
按照上表2的色谱条件，对超纯水和50μg/ml泊洛沙姆188标准溶液进行检测分析，结果显示，乙酸铵与甲酸铵基线信号噪声波动对比发现，乙酸铵基线噪声范围在0.3～1.8之间，0.1M乙酸铵溶液基线噪声波动最小，在0.5～1.3之间。而甲酸铵基线噪声在0.9～2.3左右，比乙酸铵溶液基线噪声波动大，因此选择乙酸铵，更有利于高灵敏度检测要求，降低流动相带来的信号噪声干扰。
对比甲醇和乙腈，见表3，发现泊洛沙姆188在乙腈体系下的响应值比甲醇更高。而在0.1％TFA水溶液和0.1％TFA乙腈体系下，发现泊洛沙姆188响应值更低于乙酸铵乙腈体系。因此，根据试验结果我们最终选择流动相A为0.1M乙酸铵溶液，流动相B为乙腈。
表3 不同流动相试验条件对比结果
编号 流动相A 流动相B 峰高 峰面积 基线噪声
1 0.05M乙酸铵溶液pH5.6 乙腈 24.190 2.593 0.3～1.5
2 0.1M乙酸铵溶液pH5.6 乙腈 24.689 2.838 0.5～1.3
3 0.2M乙酸铵溶液pH5.6 乙腈 24.430 2.621 0.6～1.8
4 0.05M甲酸铵溶液pH5.6 乙腈 27.141 3.360 0.9～1.9
5 0.1M甲酸铵溶液pH5.6 乙腈 27.541 3.430 1.2～2.1
6 0.2M甲酸铵溶液pH5.6 乙腈 26.974 3.213 1.3～2.3
7 0.1M乙酸铵溶液pH5.6 甲醇 7.072 2.019 0.2～1.1
8 0.1M甲酸铵溶液pH5.6 甲醇 8.802 2.575 0.2～1.4
9 0.1％TFA水溶液 0.1％TFA乙腈 18.101 1.844 0.2～1.4
实施例3 CAD检测器参数的筛选
1、色谱条件
色谱柱：Candenza HS-C18(150*3mm，3μm)。流动相A为0.1M乙酸铵溶液pH5.6，流动相B为乙腈。采用梯度洗脱模式，洗脱程序如下：0-5min，流动相B 0％；5-10min， 流动相B 10％；10-18min，流动相B 45％；18-22min，流动相B 100％；22-28min，流动相B 0％；柱温为35℃；进样体积为20μl。CAD检测器开启梯度补偿功能，检测器液体流速最高不得高于2ml/min；梯度补偿体积为100μl；气体压力为0.5MPa±0.05MPa。
2、标准品制备
称取泊洛沙姆188标准品，用超纯水将标准品储备液稀释得到50μg/ml标准溶液。
3、HPLC-CAD检测
取超纯水和50μg/ml标准溶液，分别用微量进样器取样，进入高效液相色谱体系进行HPLC分析，并用CAD检测器进行检测。
4、实验设计
为了得到更好的检测效果，对CAD检测器的参数进行试验，见下表4，选择最佳的CAD检测器参数设定。
表4 CAD检测器不同参数对比
编号 温度模式 采样频率(Hz) 过滤常数(S)
1 Low(35℃) 10 1
2 Low(35℃) 10 2
3 Low(35℃) 10 3.6
4 High(50℃) 10 1
5 High(50℃) 10 2
6 High(50℃) 10 3.6
7 High(50℃) 5 1
8 High(50℃) 5 2
9 High(50℃) 5 3.6
5、实验结果
按照上表4的色谱条件，对超纯水和50μg/ml泊洛沙姆188标准溶液进行检测分析，结果显示，数据见下表5。温度选择越高，流动相雾化越完全，检测信号基线噪声波动越小，因此选择50℃作为雾化温度。采样频率越小，过滤常数越大，信号噪声越小，基线越平滑。因此，选择采样频率5Hz，过滤常数3.6S作为本方法的CAD检测器参数。
表5 CAD检测器不同参数对比结果
编号 温度模式 采样频率(Hz) 过滤常数(S) 基线噪声
1 Low(35℃) 10 1 0.8～2.6
2 Low(35℃) 10 2 0.9～2.1
3 Low(35℃) 10 3.6 0.7～1.8
4 High(50℃) 10 1 0.7～1.9
5 High(50℃) 10 2 0.8～1.6
6 High(50℃) 10 3.6 0.7～1.6
7 High(50℃) 5 1 0.6～1.6
8 High(50℃) 5 2 0.5～1.6
9 High(50℃) 5 3.6 0.5～1.3
实施例4检测器的筛选
1、色谱条件
色谱柱：Candenza HS-C18(150*3mm，3μm)。流动相A为0.1M乙酸铵溶液pH5.6，流动相B为乙腈。采用梯度洗脱模式，洗脱程序如下：0-5min，流动相B 0％；5-10min，流动相B 10％；10-18min，流动相B 45％；18-22min，流动相B 100％；22-28min，流动相B 0％；柱温为35℃；进样体积为20μl。
2、标准品制备
称取泊洛沙姆188标准品，用超纯水将标准品储备液稀释得到50μg/ml标准溶液。
3、实验设计及方法
为了对比与现行检测方法的检测效果，对CAD检测器和ELSD检测器(Alltech 3300ELSD)两种不同检测器进行比较，见下表6。确定检测器选择。
表6 CAD检测器和ELSD检测器对比试验
Figure PCTCN2020079784-appb-000001
3.1、HPLC-CAD检测
取超纯水和50μg/ml标准溶液，分别用微量进样器取样，进入高效液相色谱体系进行HPLC分析，并用CAD检测器进行检测。CAD检测器参数设置：CAD检测器温度模式为High模式；检测器温度为50℃。采样频率5Hz，过滤常数3.6S。CAD检测器开启梯度补偿功能，检测器液体流速最高不得高于2ml/min；梯度补偿体积为100μl；气体压力为0.5MPa±0.05MPa。
3.2、HPLC-ELSD检测
取超纯水和50μg/ml标准溶液，分别用微量进样器取样，进入高效液相色谱体系进行HPLC分析，并用ELSD检测器进行检测。ELSD检测器参数设置：气体流速1.0SLM，雾化温度60℃，蒸发光温度105℃，数据采集频率10H，检测器增益PTM 10。
4、实验结果
在色谱柱、流动相组成和洗脱梯度等色谱条件相同的情况下，分别用CAD检测器和ELSD对超纯水和50μg/ml泊洛沙姆188标准溶液进行检测分析，结果显示，数据见下表7。使用CAD检测器方法的检测限低于100ng，而使用ELSD检测器方法的检测限低于200ng。表明CAD检测器检验灵敏度更高，有利于更低限度的分析，因此选择CAD检测器作为本方法的检测器。
表7 CAD检测器和ELSD检测器对比结果
Figure PCTCN2020079784-appb-000002
实施例5方法验证
1、色谱条件
色谱柱：Candenza HS-C18(150*3mm，3μm)。流动相A为0.1M乙酸铵溶液pH5.6，流动相B为乙腈。采用梯度洗脱模式，洗脱程序如下：0-5min，流动相B 0％；5-10min，流动相B 10％；10-18min，流动相B 45％；18-22min，流动相B 100％；22-28min，流动相B 0％；柱温为35℃；进样体积为20μl；CAD检测器温度模式为High模式；检测器温度为50℃。采样频率5Hz，过滤常数3.6S。CAD检测器开启梯度补偿功能，检测器液体流速最高不得高于2ml/min；梯度补偿体积为100μl；气体压力为0.5MPa±0.05MPa。
2、标准线性溶液制备
称取泊洛沙姆188标准品25.0mg，加入超纯水适量溶解并定容至25ml，避免气泡形成，作为标准储备液。根据需要浓度，分别将标准品储备液进行稀释，得到50μg/ml、40μg/ml、30μg/ml、20μg/ml、10μg/ml和5μg/ml的标准线性溶液。
3、对本发明方法的线性、检测限(LOD)、定量限(LQD)、重复性，精密度和准确度进行了验证，见表8。结果显示，泊洛沙姆188在浓度5～50μg/ml的范围内时，峰面积和泊洛沙姆188标准溶液浓度呈良好的线性范围，相关系数平方高于0.997。检测限(信噪比为2～4)和定量限(信噪比8～10)的分别为100ng和280ng，灵敏度高。实验重复性通过重复 6次来获得峰面积和保留时间的相对标准偏差(RSD)表示，峰面积重复性RSD为2.7％，保留时间重复性RSD为0.06％；日间和日内的RSD分别为1.1％和1.7％，精密度好；方法准确度在101.3％～104.8％之间，相关结果见表8。
表8 方法验证结果
Figure PCTCN2020079784-appb-000003
实施例6阿达木单抗样品检测
1、色谱条件
色谱柱：Candenza HS-C18(150*3mm，3μm)。流动相A为0.1M乙酸铵溶液pH5.6，流动相B为乙腈。采用梯度洗脱模式，洗脱程序如下：0-5min，流动相B 0％；5-10min，流动相B 10％；10-18min，流动相B 45％；18-22min，流动相B 100％；22-28min，流动相B 0％；柱温为35℃；进样体积为20μl；CAD检测器温度模式为High模式；检测器温度为50℃。采样频率5Hz，过滤常数3.6S。CAD检测器开启梯度补偿功能，检测器液体流速最高不得高于2ml/min；梯度补偿体积为100μl。气体压力为0.5MPa±0.05MPa。
2、标准线性溶液制备
称取泊洛沙姆188标准品25.0mg，加入超纯水适量溶解并定容至25ml，避免气泡形成，作为标准储备液。根据需要浓度，分别将标准品储备液进行稀释，得到50μg/ml、40μg/ml、30μg/ml、20μg/ml、10μg/ml和5μg/ml的标准线性溶液。
3、供试品上清溶液制备
取阿达木单抗培养液、纯化液C1P、纯化液C2P、纯化液C3P、原液和成品各1ml，于12000r/min高速离心机离心10min，吸取上清，分别得到阿达木单抗培养液上清溶液、纯化液C1P上清溶液、纯化液C2P上清溶液、纯化液C3P上清溶液、原液上清溶液和成品上清溶液。
4、供试品溶液配制
取阿达木单抗培养液上清溶液用超纯水稀释10倍，混匀，作为阿达木单抗培养液供试品溶液；取纯化液C1P上清溶液、纯化液C2P上清溶液、纯化液C3P上清溶液、原液上清溶液和成品上清溶液各200μl，作为纯化液C1P供试品溶液、纯化液C2P供试品溶液、纯化液C3P供试品溶液、原液供试品溶液和成品供试品溶液。
5、加样回收溶液制备
取50μg/ml标准线性溶液分别与阿达木单抗培养液供试品溶液、纯化液C1P供试品溶液、纯化液C2P供试品溶液、纯化液C3P供试品溶液、原液供试品溶液和成品供试品溶液1:1(v:v)混合，分别作为阿达木单抗培养液加样回收溶液、纯化液C1P加样回收溶液、纯化液C2P加样回收溶液、纯化液C3P加样回收溶液、原液加样回收溶液和成品加样回收溶液。
6、HPLC-CAD检测
取超纯水、各线性标准溶液、各供试品溶液和各加样回收溶液，分别用微量进样器取样，进入高效液相色谱体系进行HPLC分析，并用CAD检测器进行检测。
7、实验结果
检测结果见表9，阿达木单抗培养液供试品溶液(图8)中含泊洛沙姆188浓度为17.3μg/ml，回收率为96.8％。阿达木单抗纯化液C1P供试品溶液(图9)、C2P供试品溶液(图10)、C3P供试品溶液(图11)、原液供试品溶液(图12)和成品供试品溶液(图13)中泊洛沙姆188含量均低于检测限5μg/ml。
表9 阿达木单抗各供试品溶液中泊洛沙姆188检测数据
供试品溶液 含量 加入浓度 测得浓度 加样回收率
培养液 17.3μg/ml 25μg/ml 41.5μg/ml 96.8％
纯化液C1P /* 25μg/ml 25.4μg/ml 101.6％
纯化液C2P /* 25μg/ml 25.8μg/ml 103.2％
纯化液C3P /* 25μg/ml 24.4μg/ml 97.6％
原液 /* 25μg/ml 24.1μg/ml 96.4％
成品 /* 25μg/ml 25.6μg/ml 102.4％
/*：未检出，低于检测限5μg/ml。

Claims (10)

  1. 一种组合物中泊洛沙姆188的检测方法，其特征在于：采用反相色谱柱联合电喷雾检测器进行检测，所述反相色谱柱为Cadenza HS-C18反相色谱柱；优选地，所述组合物为药物组合物；更优选地，所述组合物为阿达木单抗组合物。
  2. 根据权利要求1所述的检测方法，其特征在于所述Cadenza HS-C18反相色谱柱的规格为150*3mm，3μm。
  3. 根据权利要求1或2所述的检测方法，其特征在于采用外标法对样品中泊洛沙姆188含量进行定量检测。
  4. 根据权利要求1至3任一项所述的检测方法，其特征在于所述反相色谱柱采用的流动相体系包括流动相A和流动相B，其中流动相A为0.1M乙酸铵溶液，流动相B为乙腈。
  5. 根据权利要求4所述的检测方法，其特征在于所述的流动相A为0.1M乙酸铵溶液，pH在5.0～6.0之间，优选pH 5.6。
  6. 根据权利要求1至5任一项所述的检测方法，其特征在于所述反相色谱柱采用的流动相的洗脱程序为：0-5min，流动相B 0％，5-10min，流动相B 10％；10-18min，流动相B 45％；18-22min，流动相B 100％；22-28min，流动相B 0％。
  7. 根据权利要求1至6任一项所述的检测方法，其特征在于所述反相色谱柱采用的柱温为25℃～40℃，优选35℃。
  8. 根据权利要求6所述的检测方法，其特征在于所述流动相的流速为0.4ml/min～0.8ml/min，优选0.5ml/min。
  9. 根据权利要求1至8任一项所述的检测方法，其特征在于所述反相色谱柱的进样体积为10～100μl，优选20μl。
  10. 根据权利要求1至9任一项所述的检测方法，其特征在于所述CAD检测器的参数设置包括：检测器使用的气体为高纯度氮气，氮气纯度大于等于99.9％；气体压力为0.5MPa±0.05MPa；检测器温度模式为High模式；检测器温度为50℃；采样频率5Hz，过滤常数3.6S；检测器开启梯度补偿功能，检测器液体流速最高不得高于2ml/min；梯度补偿体积为100μl。
PCT/CN2020/079784 2019-04-19 2020-03-17 组合物中泊洛沙姆188的检测方法 WO2020211584A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202080012695.9A CN113424057B (zh) 2019-04-19 2020-03-17 组合物中泊洛沙姆188的检测方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910315638.7 2019-04-19
CN201910315638 2019-04-19

Publications (1)

Publication Number Publication Date
WO2020211584A1 true WO2020211584A1 (zh) 2020-10-22

Family

ID=72836861

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/079784 WO2020211584A1 (zh) 2019-04-19 2020-03-17 组合物中泊洛沙姆188的检测方法

Country Status (2)

Country Link
CN (1) CN113424057B (zh)
WO (1) WO2020211584A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115326736B (zh) * 2022-08-10 2023-11-24 无锡生基医药科技有限公司 一种定量检测Pluronic F-68的方法
CN116609460A (zh) * 2023-05-25 2023-08-18 中山未名海济生物医药有限公司 一种注射液中泊洛沙姆188含量的检测方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101262918A (zh) * 2005-09-14 2008-09-10 阿雷斯贸易股份有限公司 泊洛沙姆类的检测方法
US20080280316A1 (en) * 2003-12-11 2008-11-13 Kazuhiro Imai Method of Detection, Separation and Identification for Expressed Trace Protein/Peptide
CN103399115A (zh) * 2013-08-13 2013-11-20 河北圣雪大成制药有限责任公司 一种基于液相色谱仪检测莫西克汀含量的方法
CN106559983A (zh) * 2015-07-28 2017-04-05 株式会社三养生物制药 具有改善的储存稳定性的药物组合物及其制备方法

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR112019002979B1 (pt) * 2016-08-15 2023-10-03 Genentech, Inc Método de cromatografia para quantificação de um tensoativo não iônico em uma composição compreendendo o tensoativo não iônico e um polipeptídeo

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080280316A1 (en) * 2003-12-11 2008-11-13 Kazuhiro Imai Method of Detection, Separation and Identification for Expressed Trace Protein/Peptide
CN101262918A (zh) * 2005-09-14 2008-09-10 阿雷斯贸易股份有限公司 泊洛沙姆类的检测方法
CN103399115A (zh) * 2013-08-13 2013-11-20 河北圣雪大成制药有限责任公司 一种基于液相色谱仪检测莫西克汀含量的方法
CN106559983A (zh) * 2015-07-28 2017-04-05 株式会社三养生物制药 具有改善的储存稳定性的药物组合物及其制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TAKATS, Z. ET AL.: "Qualitative and quantitative determination of poloxamer surfactants by mass spectrometry", RAPID COMMUNICATIONS IN MASS SPECTROMETETRY, vol. 15, no. 10, 31 December 2001 (2001-12-31), XP055743582, ISSN: 0951-4198, DOI: 20200416152447A *

Also Published As

Publication number Publication date
CN113424057B (zh) 2024-04-05
CN113424057A (zh) 2021-09-21

Similar Documents

Publication Publication Date Title
WO2020211584A1 (zh) 组合物中泊洛沙姆188的检测方法
CN113866337B (zh) 一种分离与测定磷酸奥司他韦同分异构体的质量分析方法
US8465935B2 (en) Detection and quantitation of cyclodextrins
Genzel et al. Amino acid analysis in mammalian cell culture media containing serum and high glucose concentrations by anion exchange chromatography and integrated pulsed amperometric detection
CN108072717B (zh) 一种精氨酸溶液的检测方法
Ogasawara et al. HIGH PERFORMANCE LIQUID CHROMATOGRAPHIC DETERMINATION OF ACLACINOMYCIN A AND ITS RELATED COMPOUNDS II. REVERSE PHASE HPLC DETERMINATION OF ACLACINOMYCIN A AND ITS METABOLITES IN BIOLOGICAL FLUIDS USING FLUORESCENCE DETECTION
RU2670965C1 (ru) Способ измерения количества полисорбата-80 с применением щелочного гидролиза образца с последующей вэжх
CN110208419B (zh) 一种用于检测比伐卢定中杂质的方法
CN110806447B (zh) 百日咳杆菌气管细胞毒素的筛查方法和含量测定方法
CN114965761A (zh) 聚乙二醇化蛋白药物中n-羟基琥珀酰亚胺的检测方法
US20190339236A1 (en) Methods of detecting glycosaminoglycans
Wasito et al. Comparison of mean centering of ratio spectra based spectrophotometric approach and HPLC method for quantitative determination of pirenoxine in the presence of methylparaben and propylparaben
CN115728412B (zh) 一种发酵虫草菌粉(Cs-4)中虫草酸含量的检测方法
CN115728433A (zh) IgG4型单克隆抗体电荷异质性的离子交换色谱检测方法
Sávoly et al. Method development for the clearance study of the Pluronic F-68 nonionic surfactant used in the upstream process of monoclonal antibody production
CN115684393A (zh) 一种注射用头孢哌酮钠舒巴坦钠中杂质的二维检测方法
CN115078598A (zh) 直接进样测试血药浓度样本的试剂盒及应用
CN116381094A (zh) 一种个人护理品中泛醇含量的检测方法
CN117630263A (zh) 基于液相色谱串联质谱检测血浆中多种游离氨基酸的方法
CN116297904A (zh) 一种测定丙氨酰谷氨酰胺原料药中有关物质的高效液相色谱方法
CN117517435A (zh) Pcsk9单克隆抗体的电荷异质性检测方法
CN117849240A (zh) 一种硫酸多粘菌素b的有关物质分析检测方法
CN118090973A (zh) 一种同时测定His、TMH和EGT的方法
CN117347519A (zh) 检测抗体偶联药物中残留sn38小分子含量的液相色谱法
CN117451901A (zh) 一种基于imac-hplc测定组氨酸标签蛋白浓度的分析方法及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20790592

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20790592

Country of ref document: EP

Kind code of ref document: A1