Effect of Q Chromatography on the Recovery of Human Plasminogen in Affinity Chromatography
10.13303/j.cjbt.issn.1004-549x.2025.10.013
- VernacularTitle:Q层析的加入对人纤维蛋白溶酶原亲和层析回收率的影响
- Author:
Shenglan YUE
1
;
Taojing LI
2
;
Juan LI
1
;
Yan PENG
1
;
Lianzhen LIN
1
;
Yanxiang ZHOU
1
;
Feifei WANG
1
;
Chen ZHU
1
;
Shang WANG
1
;
Deming JI
1
;
Shuangying ZENG
1
;
Yong HU
1
;
Zhijun ZHOU
1
Author Information
1. Sinopharm Wuhan Bio-Pharmaceutical Co., LTD., Wuhan 430207, China
2. Chengdu Rongsheng Pharmaceutical Co., LTD., Chengdu 610200, China
- Publication Type:Journal Article
- Keywords:
human plasminogen;
full chromatography process;
relative purity;
potency recovery;
antigen recovery;
specific activity
- From:
Chinese Journal of Blood Transfusion
2025;38(10):1382-1388
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To compare quality control (relative purity and specific activity) and process control [plasminogen (Pg) antigen recovery and potency recovery] indexes of samples before and after adding the Q chromatography step to the full chromatography process of human Pg, thereby determining whether the addition of this step could improve Pg recovery by affinity chromatography. Methods: A Q chromatography step was added before the Pg affinity chromatography in the original Pg chromatography process. The loading solution, flow through solution and eluate of Q chromatography and Pg affinity chromatography were collected. The potency of coagulation factor Ⅱ (FⅡ), Ⅶ (FⅦ), Ⅷ (FⅧ), Ⅸ (FⅨ), and Ⅹ(FⅩ) were detected by the coagulation method, the total protein content was detected by the BCA method, and the Pg potency was detected by the chromogenic substrate method. The content of specific plasma proteins was detected by immunoturbidimetry, the potency recovery of coagulation factors was calculated, and the flow direction of coagulation factors was analyzed. The recovery of different plasma protein antigens were calculated, and the distribution of impurity proteins was analyzed. The relative purity and specific activity of Pg, antigen content, and potency recovery in the target fractions were calculated and compared with the original process indicators, so as to determine the effect of adding Q chromatography on the original process. Furthermore, the reproducibility after process modification was assessed. Results: 100% of FⅡ, FⅩ, and FⅨ, 87.81% of FⅧ, and 40.44% of FⅦ in filtered plasma were removed by Q chromatography. The residual FⅦ (53.26%) and FⅧ (13.30%) in Q flow-through fraction were completely removed by Pg affinity chromatography. In both the original process (without Q-chromatography) and the modified process (with Q-chromatography), non-target plasma proteins mainly existed in the flow-through fraction of Pg affinity chromatography. The antigen recovery of IgM, ceruloplasmin (CER), and fibronectin (FNC) in Q-chromatography flow-through fraction were reduced. In contrast, antigen recovery of other plasma proteins [IgG, IgA, Pg, albumin (AlB), alpha-1-antitrypsin (AAT), and fibrinogen (Fg)] were all >90%, which were consistent with the protein composition and proportion in the original affinity chromatography loading solution. Compared with the recovery rate of Pg antigen in the original process (74.4%), the total recovery of Pg antigen in the modified process was significantly increased (89.97%). Compared with the recovery of IgG (97.48%) and Fg (95.32%) in the Pg affinity flows-through fraction of the original process, the modified process resulted in a slight reduction in the recovery of IgG (94.60%), while the recovery of Fg was not affected (95.05%). The potency recovery rate, specific activity, and relative purity of Pg after Q chromatography were 99.3%, 0.016 U/mg, and 0.15%. These values were the same as those of Pg affinity chromatography loading solution by the original process, indicating that introduction of Q chromatography did not affect subsequent Pg affinity chromatography. Compared with the recovery of Pg antigen in three batches of the original process (66.49±1.02)%, the recovery of Pg antigen in the affinity chromatography eluent of the modified process [five batches; (77.43±4.43)%] was significantly improved. Furthermore, the potency recovery was (86.80±4.28)%, the relative purity was (81.99±1.25)%, the specific activity was (8.679±1.073)U/mg, and the process was reproducible. Conclusion: The addition of Q chromatography could improve the recovery of Pg affinity chromatography in the full chromatography process.
