Enjolras N, Perot E, Le Quellec S, Indalecio A, Girard J, Negrier C, Dargaud Y
Unite Hemostase, Inflammation and Sepsis EA 4174, Faculte de Medecine Laennec, Universite Lyon 1-Hospices Civils de Lyon, Lyon, France.
Haemophilia. 2015 Jul;21(4):e317-21. doi: 10.1111/hae.12688. Epub 2015 May 15.
Post-translational modifications of the CHO-cell-derived-recombinant human factor IX (FIX) currently used for the treatment of hemophilia B (HB) are different from plasma derived FIX. Our previous studies described a rFIX (HIX) having better profile of post-translational modifications than rFIX produced by CHO cells. The aim of the study consisted to verify the improved post-translational modifications effect of HIX on in vivo recovery.
HIX has been produced in a bioreactor and then purified from supernatants. In vitro activation and activity were evaluated measured by thrombin generation tests (TGT) and compared to commercial molecules, Benefix(®) , Mononine(®) . The three molecules were then administrated (i.v.) to FIX-knockout mice and two minutes after injection, blood samples were collected and subjected to human FIX-specific-ELISA and TGT.
The clotting function of HIX, activation courses of HIX by FXIa and FVIIa-TF complex appear normal as did activation of Benefix(®) , Mononine(®) and TG constants of each FIX were equivalent. After injection to HB mice, circulating HIX did not present any significant difference in term of antigen value with Benefix(®) . Intriguingly, TGT were clearly exhibiting a better velocity for HIX than Benefix(®) and Mononine(®) . These data suggested that HIX may improve in vivo coagulant efficacy in comparison with the two commercial FIX injected at the same dose.
The study shows that HuH-7-derived-rFIX has better in vivo haemostatic activity in hemophilia B mice compared to the reference rFIX molecule despite similar in vivo recovery rates, suggesting that HuH-7 cells could represent an effective cellular system for production of rFIX.
目前用于治疗B型血友病（HB）的中国仓鼠卵巢细胞（CHO）衍生的重组人凝血因子IX（FIX）的翻译后修饰与血浆来源的FIX不同。我们之前的研究描述了一种重组FIX（HIX），其翻译后修饰谱比CHO细胞产生的重组FIX更好。本研究的目的是验证HIX对体内回收率的翻译后修饰改善效果。
HIX在生物反应器中产生，然后从上清液中纯化。通过凝血酶生成试验（TGT）评估体外活化和活性，并与商业分子Benefix®、Mononine®进行比较。然后将这三种分子静脉注射给FIX基因敲除小鼠，注射两分钟后，采集血样并进行人FIX特异性酶联免疫吸附测定（ELISA）和TGT。
HIX的凝血功能、FXIa和FVIIa - TF复合物对HIX的活化过程似乎正常，Benefix®、Mononine®的活化情况以及每种FIX的TG常数也相当。注射到B型血友病小鼠体内后，循环中的HIX在抗原值方面与Benefix®相比没有任何显著差异。有趣的是，TGT清楚地显示HIX的速度比Benefix®和Mononine®更快。这些数据表明，与以相同剂量注射的两种商业FIX相比，HIX可能会提高体内凝血效果。
该研究表明，尽管体内回收率相似，但HuH - 7衍生的重组FIX在B型血友病小鼠中的体内止血活性比参考重组FIX分子更好，这表明HuH - 7细胞可能是生产重组FIX的有效细胞系统。
