1.Study on separation and purification process of C1 esterase inhibitor
Jiabin XU ; Yawen LI ; Jiali GONG ; Erhua LUO ; Meng ZHANG
Chinese Journal of Blood Transfusion 2022;35(11):1121-1124
【Objective】 To study the technology of separating and purifying C1 esterase inhibitor (C1-INH) by using the waste washing liquid as raw materia during the preparation of human prothrombin complex (PCC) l. 【Methods】 C1-INH was isolated and purified by a two-step method of polyethylene glycol (PEG) 4000 precipitation and cation chromatography. The pH of raw materials and the concentration of PEG4000 were adjusted to investigate the optimal conditions of PEG4000 precipitation method. After PEG was precipitated and centrifuged, the supernatant is treated as the loading solution for cation exchange chromatography, using Fractogel EMD SE HiCap(M) gel and CM Sepharose FF gel for ion exchange chromatography. The most suitable gel and separation conditions were selected by comparing the C1-INH antigen yield, activity yield and specific activity. 【Results】 Under the condition of pH 6.1, when the mass fraction of PEG4000 was 14%, the recovery rate of C1 esterase inhibitor was close to 70%, and the removal rate of ceruloplasmin was more than 95% after stirring for 10 minutes. As fractogel EMD SE HiCap(M) gel was used for cation exchange chromatography, when the eluent salt concentration was 0.25 M sodium chloride, the activity yield of C1 esterase inhibitor was greater than 80%, and the specific activity was greater than 5 IU/mg. 【Conclusions】 Using the waste washing liquid as the raw material during the preparation of PCC, the C1 esterase inhibitor with high specific activity can be prepared through PEG precipitation and purification by Fractogel EMD SE HiCap(M) ion exchange chromatography.
2.Determination of tromethamine residues in human coagulation factor Ⅷ by high performance liquid chromatography method
Xiuliang CHEN ; Erhua LUO ; Xiaoming LIANG ; Jiafei CHEN
Chinese Journal of Blood Transfusion 2022;35(10):1020-1023
【Objective】 To develop and verify high performance liquid chromatography (HPLC) detection for tromethamine (Tris) residues in human coagulation factor Ⅷ. 【Methods】 Alanine was used as internal standard, and AQC for pre-column derivation. Inertsil® ODS-SP was adopted, and acetic acid-sodium acetate buffer and acetonitrile were used for gradient elution at the flow rate of 1 mL/min and column temperature of 37℃. The 25 μL sample was loaded and determined by UV-detector with detection wavelength at 248 nm. This method was then verified. 【Results】 Glycine, sodium citrate and calcium chloride showed no interference with the detection of tromethamine (Tris) residues. The recovery rate of spike samples was within 90.0%~100.8%. The RSD in repeatability test were 2.5%, 0.7% and 1.2%, respectively, and in intermediate precision test 0.8%. The tromethamine (Tris) at concentrates of (0.5~4.0)μg/mL showed good linear relationship to the peak area to internal standard, of which the regression equation was Y=0.012 9X-0.000 782, R=0.999 97.The quantitative detection limit was 0.04μg/mL. 【Conclusion】 The HPLC for determination of tromethamine (Tris) residues in human coagulation factor Ⅷ was successfully developed, which showed good linearity as well as high specificity, precision and accuracy.