發(fā)布時間：2018-01-06 09:35

  本文關鍵詞：FMU100抗體的超高效液相色譜法定量檢測方法的建立　出處：《福建醫(yī)科大學學報》2017年05期 　論文類型：期刊論文

  更多相關文章： 色譜法 高壓液相 抗體

【摘要】：目的建立UPLC-protein A定量檢測FMU100單克隆抗體的方法。方法使用Waters UPLC超高效液相色譜儀,Applied Biosystem的PA ImmunoDetection Sensor Cartridge(2.1×30mm,20μm)色譜柱,柱溫30℃,檢測波長280nm,進樣體積10μL,以流動相A(10mmol/L PBS含0.15mmol/L NaCl,pH 7.2)-流動相B(0.15mmol/L NaCl,pH 2.6)進行梯度洗脫,流速0.5mL/min。結(jié)果在0.034~2.200mg/mL濃度,線性相關系數(shù)r~2為0.999 9,濃度與峰面積間呈良好的線性關系;檢測限為0.01mg/mL,定量限為0.07mg/mL;回收率為98.18%~99.16%;重復性RSD為0.1%~1.9%;在8h內(nèi),每個2h測定發(fā)酵液中抗體的含量,FMU100抗體峰面積RSD為1.8%;在流動相A pH變化±0.2、流動相B NaCl比例變化±5%、柱溫變化±5℃、檢測波長變化±5nm、流速相對值變化±20%時,FMU100抗體峰峰面積RSD為1.7%(n=12);檢測方法符合系統(tǒng)適應性要求。結(jié)論建立的UPLC-protein A定量檢測FMU100抗體的方法簡單快速、方法可靠,符合方法學驗證要求。
[Abstract]:Objective to establish a UPLC-protein method for quantitative detection of A FMU100 monoclonal antibody. Methods using Waters UPLC ultra high performance liquid chromatography, Applied Biosystem PA ImmunoDetection Sensor Cartridge (2.1 x 30mm, 20 m) column, the column temperature was 30 degrees, the detection wavelength of 280nm, sample volume 10 L, mobile phase in A (10mmol/L PBS with 0.15mmol/L NaCl, pH 7.2 B (0.15mmol/L) - mobile phase NaCl, pH 2.6) by gradient elution, flow rate of 0.5mL/min. results in the concentration of 0.034~2.200mg/mL, the linear correlation coefficient r~2 is 0.9999, the concentration and peak area showed a good linear relationship; the detection limit is 0.01mg/mL, the limit of quantitation was 0.07mg/mL; the recovery rate is 98.18%~99.16%; repeat RSD 0.1%~1.9%; in 8h, determination of antibody in fermentation broth of each 2h, FMU100 antibody peak area RSD was 1.8%; in the mobile phase A pH + 0.2, NaCl + 5% B phase change in the proportion of flow, column temperature of - 5 DEG C, detection wavelength When the relative value of change is + 20%, the peak area of FMU100 antibody is 1.7% (n=12), and the detection method accords with the requirement of system adaptability. Conclusion the method of UPLC-protein A for the quantitative detection of FMU100 antibody is simple, fast, reliable, and accords with the requirement of 5nm validation.

【作者單位】： 福建醫(yī)科大學藥學院免疫治療研究所;
【基金】：福建省自然科學基金(2016J01154)
【分類號】：R392-33
【正文快照】： 活化的T細胞上表達有許多的抑制受體[1],在腫瘤組織中可能被腫瘤利用形成免疫逃逸[2],利用封閉性抗體能調(diào)節(jié)T細胞活性,對腫瘤的治療有非常好的效果[3]。FMU100為本單位基于此原理研制的新型人源化單克隆抗體。高效液相色譜法(high performance liquidchromatography,HPLC)因具，

本文編號：1387317