Scalable purification of adeno-associated virus serotype 1 (AAV1) and AAV8 vectors, using dual ion-exchange adsorptive membranes.
In vivo gene transduction with adeno-associated virus (AAV)-based vectors depends on laborious procedures for the production of high-titer vector stocks. Purification steps for efficient clearance of impurities such as host cell proteins and empty vector particles are required to meet end-product specifications. Therefore, the development of alternative, realistic methods to facilitate a scalable virus recovery procedure is critical to promote in vivo investigations. However, the conventional purification procedure with resin-based packed-bed chromatography suffers from a number of limitations, including variations in pressure, slow pore diffusion, and large bed volumes. Here we have employed disposable high-performance anion- and cation-exchange membrane adsorbers to effectively purify recombinant viruses. As a result of isoelectric focusing analysis, the isoelectric point of empty particles was found to be significantly higher than that of packaged virions. Therefore, AAV vector purification with the membrane adsorbers was successful and allowed higher levels of gene transfer in vivo without remarkable signs of toxicity or inflammation. Electron microscopy of the AAV vector stocks obtained revealed highly purified virions with as few as 0.8% empty particles. Furthermore, the membrane adsorbers enabled recovery of AAV vectors in the transduced culture supernatant. Also, the ion-exchange enrichment of retroviral vectors bearing the amphotropic envelope was successful. This rapid and scalable viral purification protocol using disposable membrane adsorbers is particularly promising for in vivo experimentation and clinical investigations.
Okada
Takashi
T
Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan. t-okada@ncnp.go.jp
Nonaka-Sarukawa
Mutsuko
M
Uchibori
Ryosuke
R
Kinoshita
Kazue
K
Hayashita-Kinoh
Hiromi
H
Nitahara-Kasahara
Yuko
Y
Takeda
Shin'ichi
S
Ozawa
Keiya
K
D000327:Adsorption; D000818:Animals; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods*; D000229:Dependovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D004285:Dogs; D005822:Genetic Vectors; D006801:Humans; D007474:Ion Exchange; D007525:Isoelectric Focusing; D007668:Kidney / Q000166:cytology / Q000821:virology; D008297:Male; D008566:Membranes; D032446:Myoblasts / Q000166:cytology / Q000821:virology; D051381:Rats; D017208:Rats, Wistar; D011995:Recombination, Genetic; D012703:Serotyping; D014162:Transfection