1. INTRODUCTION
Recombinant adeno-associated virus (rAAV) vectors have demonstrated significant promise for in vivo gene delivery due to their unique features, including non-pathogenicity to humans, low immunogenicity, and long-term gene expression.[1,2] Although scalable manufacturing of rAAVs has been demonstrated using mammalian and insect cell culture,[3,4] the rAAV production systems inherently form empty-rAAV particles that do not contain the desired therapeutic gene sequences. These empty-rAAV can pose several clinical challenges. A cell-mediated immune response that targets the rAAV capsid can cause the clearance of transduced tissue infected with therapeutic rAAV. This cytotoxic effect has been attributed to the introduction of high ratios of empty- to full-rAAV particles.[5]In addition to immune response challenges, the excess number of empty particles limits the rAAV genome titer in a drug product. This is particularly important for the treatment of diseases which require high doses of the therapeutic rAAV.[6]
The traditional approach of using density gradient ultracentrifugation methods has proven to be effective in removing empty particles.[7–9] However, the ability to scale and validate gradient ultracentrifugation methods to consistently deliver large doses of full-rAAV particles is challenging. In contrast, ion-exchange chromatography, particularly anion-exchange (AEX) chromatography, has been reported as a means of separating empty- and full-rAAV particles.[10] The ability for an AEX chromatography process to enrich full-rAAV particles is dependent on various factors including the properties of AEX stationary phase,[11] AEX mobile phase,[12] and properties of rAAV, such as serotype,[13] genome size,[14] and surface charge alteration.[15] AEX chromatography exploits the surface charge difference between empty- and full-rAAVs, whereby full-AAVs have a slightly lower isoelectric point (pI) than empty-rAAVs, therefore a shallow, linear-gradient elution (LGE) by increasing the salt (e.g ., sodium chloride [NaCl]) concentration could provide the resolution required for separation. In cases where sufficient resolution between Empty and Full populations was achieved in LGE, step elution methods have been reported.[10,16]
In addition to the elution methodology, various mobile phase salts have been used to enhance the separation between empty- and full-rAAVs in AEX chromatography. The strength of the eluent salt is adjusted by either increasing the concentration of the elution buffer or through the selection of a modifier with specific physicochemical properties that promote/decrease specific molecular interactions.[17] Reported salts include ammonium acetate (NH4Ac),[18] magnesium sulfate (MgSO4),[10] sodium acetate (NaAc),[19] and various quaternary ammonium (QA) salts.[11,19–21] Separation of Empty and Full peaks using a single solution gradient consisting of NaCl and MgCl2 has been previously reported, hypothesizing an interaction between Mg2+ and rAAV capsids.[16] In line with this notion, Namet. al . reported the cryo-EM structure of AAV8 suggested that there is a potential divalent ion interaction position in the 2-fold symmetry axis region.[22] Furthermore, Gagnonet al . were able to separate empty-rAAV from full-rAAV using a novel multimodal metal (including Mg2+) affinity chromatography method,[23] corroborating the interaction between Mg2+ and rAAV capsids. In instances where QA salts are compared with other salts, the QA salt tends to outperform their counterparts in the ability to resolve empty- and full- rAAV.[11,19,21]However, the role of the QA salt in the interaction between the rAAV particles and AEX stationary phase remains unclear. Wang et al . explored tetraalkylammonium chlorides with various alkyl-chain lengths and observed improved peak-to-peak resolution with increasing alkyl-chain length in analytical AEX chromatography.[11] While it is encouraging to see QA salts contribution in analytical AEX chromatography, its utilization in preparative AEX methods is not yet realized.[20]
Herein, we present a preparative AEX chromatography method for the separation of empty- and full-rAAV8 particles using the CIMmultus-QA monolith column. The method has a combination of wash1 step with QA salt to remove empty-rAAVs, wash2 step with NaCl salt to remove QA salt, and elution step with NaCl to elute full-rAAVs. Eventually, a scalable and manufacturing-friendly AEX process was developed and demonstrated to provide improved full-rAAV particle enrichment in rAAV downstream purification process.
2. MATERIALS AND METHODS