Figure 5 . Regression plot depicting the tradeoff between GOI
genome recovery and full-rAAV enrichment. Circles correspond to step
elution, whereas the squares correspond to gradient elution. Open
markers correspond to AEX chromatography runs using NaCl in process
buffers. Closed markers correspond to AEX chromatography runs that
employ an isocratic TEA-Ac wash, followed by either a step or gradient
elution using NaCl and MgCl2. The genome recovery is
calculated using the genome copy determined by qPCR assay, while the
full-rAAV percentage is determined by AUC assay.
4. DISCUSSION
Chromatographic separation of empty- and full-AAV particles is a
resolution challenge. This combination of TEA and Ac displays the
behavior of a weak eluent and is observed to be more effective to
discriminate empty- and full-rAAV species in AEX chromatography. In a
qualitative sense, if the eluting power of a modifier is too high, the
eluent may fail to distinguish the species retained on the stationary
phase. In addition, a strong eluent may produce retention times that are
too short with the result of poorly resolved elution peaks. Conversely,
a weaker eluent is more likely to discriminate between the species
retained on the stationary phase and increase the retention time, and in
return increase resolution. Similarly, the weak AEX stationary phase,
whose ligand is derived from a weak acid and partially ionized in a
narrow pH range, may also be able to provide
enhanced selectivity between empty-
and full-rAAV particles. In line with this notion, Zhou et al.reported that a DEAE-based weak AEX stationary phase, POROS 50D,
provided markedly improved resolution between empty and full-rAAV
particles of AAV-6, when compared with other strong AEX stationary
phases.[27] It is foreseeable that a more
systematic study around AEX stationary phase may provide further insight
on the delicate separation of empty and full-rAAV in AEX chromatography.
Most recently, it was reported that the empty-rAAV species may have
preferential binding to a process-related impurity, chromatin, in Sf9
cell line generated rAAV viral vector product.[28]As chromatin consists of histone octamers whose
protruded tails carry positive
charged lysine residues, it is possible that these positively charged
histones could preferentially bind
empty-rAAV. In line with this thought, it is also possible that TEA-Ac
preferentially interacts with empty-rAAV, either through electrostatic
interaction using TEA positive amine group or through enforced charge
pairing [24] using both TEA positive amine group
and TEA hydrophobic alkyl-chain. Both of these interactions may lead to
the decrease of net charge of the empty-rAAV species and thus benefiting
empty- and full-rAAV separation in AEX chromatography.
An important consideration of using QA
salt in purification process is
that its sufficient clearance needs to be demonstrated to assure product
safety. As aforementioned, since sodium ion has stronger elution power
than that of TEA ion, the second wash with NaCl could replace and flush
out residual TEA-Ac salt from AEX column prior to elution phase. In
addition, the volume of the second wash buffer could be further
optimized to achieve extra clearance of TEA ions. Furthermore, the final
concentration and buffer exchange step in downstream process usually
could achieve >99.9% buffer exchange
efficiency,[29] which offers at least 3 more log
reduction value (LRV) for TEA-Ac, providing feasibility for TEA-Ac to be
used as a potent process buffer salt to enhance full-rAAV percentage.
Regarding the implementation of AEX
step elution method, the lot-to-lot variability (in terms of peak
elution retention volume) of monolithic column may add one more layer of
complexity when converting LGE to isocratic elution, as the isocratic
elution buffer may not consistently elute full-rAAV species when AEX
column changed, resulting in either low full-rAAV percentage or low
genome recovery. As a mitigation, packed column with AEX resin may
provide more consistent peak elution behavior, although the resolution
of empty- and full-rAAV separation using AEX resin may suffer certain
level of decrease when compared to AEX monolithic column. In addition,
it has been communicated that a new line of AEX monolithic column
product will be lunched by CIMultus-QA vendor whose manufacturing
procedure was specifically improved to reduce lot-to-lot variability
(personal communication), which may offer process developer an extra
option to achieve enhanced process robustness while still maintaining
optimal empty full separation in AEX monolithic column operation.
Furthermore, AEX chromatography is
usually employed to clear process related impurities, such as host cell
protein (HCP).[30] In
AEX LGE run, the HCP species are
eluted off column by higher ionic strength post full peak, thus delicate
peak cutting criteria need to be implemented to exclude HCP from
entering product stream. Similarly, in AEX step elution run, elution
salt concentration needs to be carefully optimized to ensure equivalent
HCP clearance as achieved by LGE. When implementing TEA-Ac in an AEX
step, due to the unique property of TEA ion comparing to other cations
in process salts, TEA ion may be able to interact with HCP through its
hydrophobic alkyl-chain. Therefore, the HCP reduction capability of AEX
step needs to be re-evaluated to assure both product-related impurity
and process-related impurity are in control.
Lastly, certain challenges still remain in purification of certain AAV
serotype. For instance, Joshi et al . reported that AAV9 empty and
full peaks could be separated using POROS HQ column with 0.93 peak
resolution, compared to 0.91 for rAAV8 serotype, indicating that the
rAAV8 and rAAV9 viruses behave similarly in their study in terms of
empty full separation.[10] However, Lock and
Alvira found that their rAAV9 product possess two empty species with
distinct chromatography behavior on CIMmultus QA column using salt LGE,
with the major empty peak (early elution species) almost co-eluted with
full peak.[31] The discrepancy observed in rAAV9
Empty and Full separation may be contributed by the variation of
multiple factors between research labs, including DNA genome sequence,
DNA genome size, viral protein amino acid sequence,
viral protein post-translation
modification,[13] and other factors, which may
warrant researchers to consider a case-by-case implementation and
optimization of this TEA-Ac and step elution AEX technology.
AUTHOR CONTRIBUTIONS
D.P.C. : Conceptualization,
Methodology, Investigation, Writing – Original Draft, Writing – Review
Editing. C.H. : Supervision,
Resources, Writing – Original Draft, Writing – Review Editing.J.C.W. : Resources, Writing
– Review Editing.
CONFLICTS OF INTEREST
D.P.C. , C.H., and J.C.W. are inventors on a
patent that includes the work in this manuscript
Data Availability Statement
The original contributions presented in the study are included in the
article. Further inquiries can be directed to the corresponding author.
ACKNOWLEDGEMENTS
The authors thank the following members of Ultragenyx’s Pharmaceutical
Development, Analytical Development team for assay support: Will Beyer
and Sara Forman for supporting vector genome titer quantification
through qPCR assays; Brittany Brancato and Adriana Kita for supporting
capsid content quantification through SV-AUC; and Matt Lotti for
supporting AAV-particle concentration quantification through Gyrolab
xPand assays. The authors are grateful to Ultragenyx’s Pharmaceutical
Development, Pilot Plant team for their support in generating the AAV8
samples used in this study; in particular, the authors thank Amy
Medeiros for coordinating the in-process intermediate materials
generation and sample handoff.
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