The methods for determining the purity and in vitro or in vivo activity of recombinant human endostatin.
In order to establish the methods of high-performance liquid chromatography (HPLC) for determining the purity of recombinant human endostatin (rhEndostatin) and in vitro or in vivo activity of rhEndostatin, two columns were firstly used in HPLC analysis for determining the purity of rhEndostatin, including Waters Symmetry 300C4 (4.6 mm x 250 mm, 5 microm) and the Superdex75 HR 10/30. Cell lines, bovine capillary endothelial cells (BCEs) or human umbilical vein endothelial cells (HUVECs) expression human vascular endothelial growth factor (hVEGF) were used in method MTT or LDH as substrate, respectively. The bioactivity in vivo was assayed by the anti-tumor proliferation rate in H22 liver tumor-bearing mice. The results showed that the retention time of rhEndostatin sample was stable at 19.066 min or 11.506 min in reverse phase HPLC (RP-HPLC) or gel filtering HPLC (GF-HPLC). The stableness, repeat and recovery rates were over 99% in both methods and there was no statistical difference between these two methods (p > 0.05). In nonserum culture medium, rhEndostatin can sensitively and stably inhibit the proliferation of the HUVEC cells that were transfected with plasmid encoding hVEGF. LDH substrate methods is the most sensitive and stable method. The anti-tumor activity in H22 tumor-bearing mice was also highly repeatable and had an inhibition rate over 50% at 20 mg kg(-1) weight. As a conclusion, the RP-HPLC and GF-HPLC set up in this paper are highly repeatable, accurate and sensitive for detecting the purity of rhEndostatin. The bioactivity of rhEndostatin can be measured through detection the proliferation-inhibition on HUVECs transfectants with hVEGF in vitro or on H22 liver tumor in vivo.
Zhang
Hong-Ying
HY
Department of Biological Science and Technology and the State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China.
Han
Qing-Wang
QW
Xu
Ye-Fen
YF
Fan
Yan-Rong
YR
Dai
Quan-Min
QM
Xu
Jia-Ming
JM
Zhu
Hao-Wen
HW
Rong
Zhi-Gong
ZG
Wang
Jian-Jun
JJ
Xu
Gen-Xing
GX
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D002417:Cattle; D049109:Cell Proliferation / Q000187:drug effects; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D043169:Endostatins / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000187:drug effects*; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008813:Mice, Inbred ICR; D011994:Recombinant Proteins / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000502:physiology; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D043169:Endostatins; D011994:Recombinant Proteins; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A