肝水解肽的制备及其病毒灭活/去除工艺的验证

Preparation of Heparolysate and Verification of Procedure for Inactivation /Removal of Virus

目的制备肝水解肽,并对其病毒灭活/去除工艺进行验证。方法以鸡新城疫病毒(NDV)和鸡传染性法氏囊病毒(IBDV)为指示病毒,在100℃,pH6.0～7.0煮沸20min灭活病毒,用截留相对分子质量为10000的中空纤维柱,在进压0.15Mpa、回流压0.05Mpa和进压0.20Mpa、回流压0.10Mpa各循环超滤3次去除病毒,用无特定病原(SPF)的鸡胚和鸡成纤维细胞进行病毒灭活/去除的效果验证。结果加热煮沸后,指示病毒未检出,滴度降低NDV≥7.4logEID50/0.2ml、IBDV≥5.45logCCID50/0.1ml;超滤后IBDV未检出,滴度降低≥5.43logCCID50/0.1ml。盲传复壮均未检出病毒。制备的肝水解肽多肽含量均在20mg/ml以上。结论制备的肝水解肽工艺中两种灭活/去除病毒方法均有效,经灭活/去除病毒后的产品质量稳定。

Objective To prepare heparolysate and verify its virus inactivation /removal procedure.Methods The Newcastle disease virusn（NDV）and infectious bursal disease virus（IBDV）as indicators were inactivated by boiling at 100℃,pH 6.0 ～ 7.0 for 20 min,then removed by ultrafiltration using hollow fiber column with a cut-off relative molecular mass of 10 000 at feeding pressure of 0.15 MPa,retention pressure of 0.05 MPa,and at feeding pressure of 0.20 MPa,retention pressure of 0.10 MPa,3 cycles for each.The virus inactivation /removal efficacies were verified by using SPF chick embryo and chick fibroblast.Results No indicator viruses were detected after boiling.The decreases of titers of NDV and IBDV were not less than 7.4 logEID50 /0.2 ml and not less than 5.45 log CCID50 /0.1 ml respectively.No IBDV was detected after ultrafiltration,and its titer decreased by not less than 5.43 logCCID50 /0.1 ml.After blind passage and recovery to normal,no virus was detected in chick embryos.The polypeptide content in prepared heparolysate was more than 20 mg /ml.Conclusion Both the procedures for virus inactivation /removal during preparation of heparolysate were effective,and the quality of obtained heparolysate was stable.