医用胶原修复膜病毒灭活/去除工艺的验证和评价

Validation and evaluation of procedure for inactivation/removal of viruses in medical collagen repair membranes

目的验证和评价医用胶原修复膜制备工艺中过氧化氢溶液、低p H孵放和γ射线辐照灭活/去除病毒的可行性。方法以水疱性口炎病毒(vesicular stomatitis virus,VSV)、伪狂犬病病毒(pseudorabies virus,PRV)、呼肠弧病毒Ⅲ(respiratory enteric orphan virusⅢ,Reo 3)、猪细小病毒(porcine parvovirus,PPV)作为指示病毒,采用非洲绿猴肾细胞(Vero)和猪肾细胞(PK-15)进行病毒增殖和感染性评价,Karber法测定病毒滴度;分别对牛腱片样品进行3%过氧化氢、低p H孵放处理(pH 3.4±0.3),检测病毒灭活/去除效果;将医用胶原修复膜中间品进行^(60)Coγ射线辐照(剂量为25 k Gy),检测病毒灭活/去除效果。结果经3%过氧化氢溶液处理1 h后,样品VSV和PRV的病毒灭活值均大于4 Logs;经低p H孵放处理14 d后,样品中已检测不到VSV和PRV,病毒灭活值分别为6.55和5.59 Logs;经25 k Gy剂量^(60)Coγ射线辐照处理后,样品中VSV、PRV、Reo3和PPV均被有效灭活,病毒灭活值分别为5.64、4.35、4.87和5.36 Logs。结论医用胶原修复膜制备工艺中的3种灭活/去除病毒步骤均有效,经灭活/去除病毒后的产品质量安全可靠。

Objective To validate and evaluate the feasibility of inactivation / removal of viruses in medical collagen repair membrane by hydrogen peroxide solution, low p H incubation and γ-irradiation. Methods Vesicular stomatitis virus（VSV）, pseudorabies virus（PRV）, respiratory enteric orphan virus Ⅲ（Reo 3）and porcine parvovirus（PPV）were selected as indicator viruses. Vero and PK-15 cells were used for the evaluation of virus proliferation and infection. The virus titer was determined by Karber method. The bovine tendon samples were soaked, treated with 3% hydrogen peroxide solution and low pH（3. 4 ± 0. 3） incubation respectively, and verified for virus inactivation / removal efficacy. The intermediate products of medical collagen repair membrane were treated with 25 kGy ^60Co γ-irradiation, and verified for virus inactivation / removal efficacy. Results After the samples were soaked in 3% hydrogen peroxide solution for 1 h, both the titers of VSV and PRV decreased by more than 4 Logs. After low p H incubation for 14 d, neither of VSV and PRV was detected in samples, of which the mean inactivation efficacies were 6. 55 and 5. 59 Logs respectively. After 25 kGy ^60Coγ-irradiation, VSV, PRV, Reo3 and PPV were effectively inactivated, of which the mean inactivation efficacies were 5. 64,4. 35, 4. 87 and 5. 36 Logs respectively. Conclusion All the three procedures of inactivation / removal of viruses in medical collagen repair membrane were effective, and the product after virus inactivation / removal was safe and reliable.