Abstract
Objective To compare quality control(relative purity and specific activity)and process control[plasmino-gen(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 recov-ery 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 af-finity chromatography were collected.The potency of coagulation factorⅡ(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 po-tency was detected by the chromogenic substrate method.The content of specific plasma proteins was detected by immuno-turbidimetry,the potency recovery of coagulation factors was calculated,and the flow direction of coagulation factors was an-alyzed.The recovery of different plasma protein antigens were calculated,and the distribution of impurity proteins was ana-lyzed.The relative purity and specific activity of Pg,antigen content,and potency recovery in the target fractions were cal-culated and compared with the original process indicators,so as to determine the effect of adding Q chromatography on the o-riginal 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,ceruloplas-min(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 mod-ified 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 recov-ery 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 origi-nal 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 rel-ative 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 chroma-tography process.关键词
人纤维蛋白溶酶原/全层析工艺/相对纯度/效价回收率/抗原回收率/比活性Key words
human plasminogen/full chromatography process/relative purity/potency recovery/antigen recovery/specific activity分类
临床医学
