Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography.
The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn(2+) ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.
Lee
Dong-Sop
DS
Department of Life Science and Gachon BioNano Research Institute, Kyungwon University, 65 Bokjung-dong, Sujeong-gu, Seongnam-si, Kyeonggi-do 461-701, Republic of Korea.
Kim
Byong-Moon
BM
Seol
Dai-Wu
DW
D000256:Adenoviridae / Q000235:genetics / Q000302:isolation & purification*; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005822:Genetic Vectors / Q000302:isolation & purification*; D005998:Glycine / Q000737:chemistry; D006801:Humans; D008566:Membranes / Q000737:chemistry; D011995:Recombination, Genetic; D015032:Zinc / Q000737:chemistry*
D015032:Zinc; D005998:Glycine