马抗破伤风毒素免疫球蛋白F(ab′)_2新制备工艺的建立

Development of a Novel Procedure for Preparation of Equine Immunoglobulin F(ab′)_2 against Tetanus Toxin

目的建立马抗破伤风毒素免疫球蛋白F(ab′)2新的制备工艺。方法制备并纯化破伤风类毒素(Tetanustoxoid,TT)及重组破伤风毒素c片段(Recombinant tetanus toxin C fragment,rTT-C),将TT/rTT-C(2∶1)免疫马匹,待血清抗体效价达3 500 IU/ml时,采血,分离血清,灭活,去除外源蛋白,胃蛋白酶消化制备F(ab′)2,经DEAE柱层析纯化,并对其进行中和抗体效力、安全性和稳定性检测。结果纯化的TT和rTT-C的浓度分别为70 000 Lf/L和4～5 mg/L,纯度分别达95%以上和96%以上;纯化的TAT-F(ab′)2收率为1.4%,纯度达91%以上,中和抗体效价>15 000 IU/ml;其安全性及稳定性均符合《中国药典》三部(2005版)要求,有效期暂定为18个月。结论建立了马抗破伤风毒素免疫球蛋白F(ab′)2新的制备工艺,制备的制品达到甚至超过了国外的质量标准,为马抗血清同类制品的升级换代提供了技术支持。

Objective To develop a novel procedure for preparation of equine immunoglobulin F （ab＇）2 against tetanus toxin. Methods Tetanus toxoid （TT） and recombinant tetanus toxin C fragment （rTY-C） were prepared and purified, then mixed at a ratio of 2 ： 1. Horse were immunized with the mixture TF/rTT-C and, when the serum antibody titer reached 3 500 IU/ml, the sera were collected and inactivated, from which the foreign protein was removed, then digested with pepsin. The prepared F（ab＇ ）2 was purified by DEAE column chromatography, and analyzed for neutralizing efficacy, safety and stability. Results The concentrations of purified TY and rTT-C were 70 000 Lf/L and 4 ～ 5 rag/L, while the purities were more than 95% and more than 96%, respectively. The recovery rate, purity and neutralizing antibody fiter of purified TAT- F （ab＇）2 were 1.4%, more than 91% and more than 15 000 IU/ml respectively, while the safety and stability met the requirements in Chinese Pharmacopeia （Volume B[, 2005 edition）. The validity period was determined as 18 months temperately. Conclusion A novel procedure for preparation of equine immunoglobulin F （ ab＇ ）2 against tetanus toxin was developed, and the quality of product was equal to or even higher than that of imported product, which provided a technical support for upgrading of equine antiserum of the same kind.