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