title: Calcium-ion-modulated ceramic hydroxyapatite resin for the scalable purification of recombinant Adeno-Associated Virus serotype 9.
abstract: Column chromatography has been widely used as a scalable purification strategy for recombinant adeno-associated virus (rAAV) vectors. The rAAV1, 2, 4, 5, 6, 8 and 9 serotypes could be separated using affinity resins, ion exchange resins or other types of resins. Apatite resin has displayed outstanding performance in protein purification in the past 10 years, and ceramic hydroxyapatite (CHT) chromatography resin with a polyethylene glycol (PEG) modulation has recently been used for rAAV1 and rAAV9 vectors. This study reports the use of CHT chromatography modulated by calcium ions instead of PEG for rAAV9 purification. Calcium-ion-containing buffers effectively improve the inclusion of CHT as a capture resin, the resin-binding capacity and the yield. The optimum calcium ion concentration is 30ppm, and the optimum pH is 7.0. A frontal analysis indicated that the binding capacity of CHT at 2ml/min reaches 65.1mg total protein per ml of resin. A previously developed purification strategy consists of CHT followed by ANX anion exchange chromatography. The vector yield of this approach is approximately 70%, and a software analysis indicated a vector purity exceeding 98%. The residual host cell (HEK293) protein contents are 24.75±2.32ng and 67.21±2.10ng, and the Benzonase residue contents are 1.55±0.10pg and 1.95±0.16ng per 10(13) vector genome copies (G.C.) separated by CHT/ANX and CsCl. In addition, CHT/ANX yields 798.44±50.10pg of plasmid DNA and 2.17±0.11ng of HEK293 DNA, while CsCl purification yields 840.27±76.14pg of plasmid DNA and 2.43±0.19 of HEK293 DNA. The two methods produce vectors with similar in vitro and in vivo potencies. The results indicated that the CHT/ANX method is suitable for the scalable purification of the rAAV9 vector.
authors.affiliation: School of Medicine and Institute of Molecular Medicine, Huaqiao University, Quanzhou, Fujian 361021, China; College of Medicine and Life Science, Jiujiang University, Jiujiang, Jiangxi 332000, China.
authors.forename: Weihong
authors.initials: W
authors.lastname: Qu
authors.affiliation: School of Medicine and Institute of Molecular Medicine, Huaqiao University, Quanzhou, Fujian 361021, China; Engineering Research Centre of Molecular Medicine, Ministry of Education, Xiamen, Fujian 361021, China. Electronic address: mxwang@hqu.edu.cn.
authors.forename: Mingxi
authors.initials: M
authors.lastname: Wang
authors.affiliation: School of Medicine and Institute of Molecular Medicine, Huaqiao University, Quanzhou, Fujian 361021, China; Engineering Research Centre of Molecular Medicine, Ministry of Education, Xiamen, Fujian 361021, China.
authors.forename: Yaqing
authors.initials: Y
authors.lastname: Wu
authors.affiliation: School of Medicine and Institute of Molecular Medicine, Huaqiao University, Quanzhou, Fujian 361021, China; Engineering Research Centre of Molecular Medicine, Ministry of Education, Xiamen, Fujian 361021, China.
authors.forename: Yinghui
authors.initials: Y
authors.lastname: Lv
authors.affiliation: School of Medicine and Institute of Molecular Medicine, Huaqiao University, Quanzhou, Fujian 361021, China; Engineering Research Centre of Molecular Medicine, Ministry of Education, Xiamen, Fujian 361021, China.
authors.forename: Qizhao
authors.initials: Q
authors.lastname: Wang
authors.affiliation: School of Medicine and Institute of Molecular Medicine, Huaqiao University, Quanzhou, Fujian 361021, China; Engineering Research Centre of Molecular Medicine, Ministry of Education, Xiamen, Fujian 361021, China. Electronic address: ruianxu@hqu.edu.cn.
authors.forename: Ruian
authors.initials: R
authors.lastname: Xu
mesh_terms: D000818:Animals; D001709:Biotechnology; D002118:Calcium / Q000737:chemistry; D002460:Cell Line; D002516:Ceramics; D002846:Chromatography, Affinity / Q000379:methods*; D000229:Dependovirus / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017886:Durapatite / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000032:analysis / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D015843:Serpins; C051324:kallistatin; D017886:Durapatite; D002118:Calcium
keywords: Calcium ion; Ceramic hydroxyapatite resin; Scalable purification; rAAV9