Calcium-ion-modulated ceramic hydroxyapatite resin for the scalable purification of recombinant Adeno-Associated Virus serotype 9

• CaCl2 can improve the bind capacity of CHT resin for AAV9.
• The binding capacity of CHT at 2 ml/min is 65.1 mg total protein per ml of resin.
• When CHT combining with ANX, the yield comes to 70% and the purity is up to 98%.
• There is no difference between CHT/ANX and CsCl in the potency in vivo and in vitro.

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 30 ppm, and the optimum pH is 7.0. A frontal analysis indicated that the binding capacity of CHT at 2 ml/min reaches 65.1 mg 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 ± 2.32 ng and 67.21 ± 2.10 ng, and the Benzonase residue contents are 1.55 ± 0.10 pg and 1.95 ± 0.16 ng per 1013 vector genome copies (G.C.) separated by CHT/ANX and CsCl. In addition, CHT/ANX yields 798.44 ± 50.10 pg of plasmid DNA and 2.17 ± 0.11 ng of HEK293 DNA, while CsCl purification yields 840.27 ± 76.14 pg 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.