2.3 Anion Exchange Chromatography
To prepare the AEX load, 1.5 mL of
affinity eluate was diluted 8-fold (to 15 mL) with 20 mM
Bis-tris-propane (BTP) to adjust pH to 9.0 and to reduce the load
conductivity to approximately 4 mS/cm. The AEX experiments were
performed on a 1-mL strong-base, CIMmultus-QA monolith (Lot:
20-GE03-005-001A-P1-2A-8-MI-2-MQ17, BIA Separations) with a nominal
channel size of 2 μm, using three basic buffering formats:
Linear-gradient Elution (NaCl, with and without MgCl2)
– 10 mL of the diluted affinity eluate was loaded on AEX media
pre-equilibrated with pH-matched equilibration buffer
(NaCl buffer: 20 mM BTP, 25 mM
NaCl, 0.001% (w/v) Pluronic F-68, pH 9.0). After washing with 10 column
volumes (CV) of equilibration buffer, the column-bound material was
eluted with a linear gradient from 25 to 182.5 mM NaCl generated over 90
CVs. AEX chromatography experiments
utilizing Mg-containing buffers were performed identically except for
the constituents of the equilibration (Buffer A) and end-point buffer
(Buffer B) contained 2 mM MgCl2. AEX chromatography
experiments utilizing 10% (v/v) isopropyl alcohol buffers were
performed identically except for the constituents of the Buffer A and
Buffer B contained 10% (v/v) isopropyl alcohol. The eluate was
collected in fractions and fractions corresponding to “Empty” and
“Full” peaks were pooled accordingly.
Linear-gradient Elution (QA, with
and without MgCl2) – 10 mL of the diluted affinity
eluate was loaded on AEX media pre-equilibrated with
pH-matched equilibration buffer (QA buffer: 20 mM BTP, 25 mM QA, 0.001%
(w/v) Pluronic F-68, pH 9.0). After washing with 10 CVs of equilibration
buffer, the column-bound material was eluted with
a linear gradient from 25 to 340 mM
QA generated over 90 CVs. AEX
chromatography experiments utilizing Mg-containing buffers were
performed identically except for the constituents of the Buffer A and
Buffer B contained 0.2, 1, 5, or 10 mM MgCl2. The eluate
was collected in fractions and fractions corresponding to “Empty” and
“Full” peaks were pooled accordingly.
Isocratic Wash and Elution – 10 mL
of the diluted affinity eluate was loaded on AEX media pre-equilibrated
with pH-matched equilibration buffer (TEA-Ac buffer: 20 mM BTP, 25 mM
TEA-Ac, 0.001% (w/v) Pluronic F-68, pH 9.0). After washing with 10 CVs
of equilibration buffer, the column was washed with 190–200 mM TEA-Ac
and the corresponding concentration of MgCl2 (either 0,
1, or 5 mM). The QA buffer was then removed in a second wash step (NaCl
buffer: 20 mM BTP, 25 mM NaCl, 2 mM MgCl2, 0.001% (w/v)
Pluronic F-68, pH 9.0). Elution was carried out in two manners: 1) a
linear gradient was generated
over
30 CVs from 25 to 200 mM NaCl; or 2) a step elution was employed using a
buffer containing 20 mM BTP, 117 mM NaCl, 2 mM MgCl2,
0.001% (w/v) Pluronic F-68, pH 9.0. In gradient operation, the eluate
was collected in fractions and fractions corresponding to “Empty” and
“Full” peaks were pooled accordingly. In isocratic mode, the full
elution peak was collected, which typically corresponded to a 3 CVs
pool.
Following elution, the column was then washed with 10 CVs of a high-salt
strip solution (20 mM BTP, 2 M NaCl, pH 9.0), followed by 10 CVs of a
sanitation solution (3 M NaCl, 1 M NaOH), and subsequently 10 CVs of a
column-storage solution (50 mM Tris, 150 mM NaCl, 20% ethanol, pH
7.5). The column is refrigerated in 2–8 °C after use.
2.4 Analysis of
Chromatography Fractions
Quantitative Polymerase Chain
Reaction (qPCR) – The quantity of encapsulated vector genome was
determined using a qPCR method applicable to rAAV samples that contain
the SV40 Poly-A sequence. To quantify only encapsulated vector genome,
rAAV samples were treated with salt-activated nuclease (SAN) to remove
non-encapsulated polynucleotides. The SAN were then inactivated and the
rAAV samples were treated with Proteinase K to digest the capsid
protein. This is followed by qPCR using the primer/probe set that is
specific to the SV40 Poly-A sequence of the rAAV genomes in the rAAV
therapeutic products prepared at Ultragenyx. The qPCR is performed on an
Applied Biosystems Flex 7 instrument and quantification is made using
the QuantStudio 7 software. For a given experiment (e.g., alkyl series
study or anion series study) the qPCR measurements were performed on the
same multi-well plate.
Gyrolab xPand – The quantity of rAAV capsid particles was determined
using the Gyrolab xPand (Gyros Protein Technologies) nanoliter-scale,
high-throughput immunoassay system, in combination with the
Gyrolab AAVX Titer kit (Gyros
Protein Technologies, P0020695). The AAVX ligand included in Gyrolab
AAVX Titer kit is based on the selective affinity ligands developed with
CaptureSelect technology (Thermo Fisher Scientific). Typically, a
dilution plate containing rAAV samples, calibrants required to generate
a standard curve, controls, and wash buffers are prepared ahead of time.
The xPand’s liquid handling system transfers these solutions into
microfluidic channels that contain biotin-labelled CaptureSelect AAVX
ligand functionalized on streptavidin-coated particles. As the solutions
pass through the channels, rAAV capsid particles are bound to the
capture media. Then, fluorescent-conjugated material is passed across
the channel to generate a fluorescence signal in the form of response
units. The signal obtained from the calibration solutions are then
fitted with a four-parameter logistic regression to generate a standard
curve to convert response units to particle titer units [vp
mL−1]. For a given study (e.g., alkyl series study
or anion series study) particle titer measurements were performed on the
same Gyrolab disc.
Sedimentation Velocity Analytical
Ultracentrifugation (SV-AUC) – The capsid content in rAAV preparations
and AEX elution fractions were quantified by SV-AUC using absorption
optics at 230 nm. SV-AUC was performed using the ProteomeLab LX-A
analytical ultracentrifuge from Beckman Coulter (Indianapolis, IN).
Samples were loaded into AUC cells equipped with sapphire windows and 12
mm double-sector charcoal-filled EPON centerpieces. 420
μL of sample was loaded in the
sample sector, whereas 430 μL of the buffer matrix was loaded in the
reference sector. AUC cells containing the samples were equilibrated at
20 °C for 1.5 h before the rotor was brought up to 12,000 rpm. UV scans
were collected at 230 nm every 20 s at a radial step-size of 3 μm.
Sedimentation coefficients and relative amounts of each species present
in solution were determined by processing the data with SEDFIT (v15.01b)
using a c(s) model. The empty, intermediate, and full species were
identified using established reference sedimentation
coefficients.[34]
3. RESULTS
3.1
Effect of Quaternary Ammonium Salt in Preparative AEX Chromatography
The AEX salt LGE runs were performed on CIMmultus-QA column with either
baseline condition (using NaCl) and QA condition (using
tetramethylammonium chloride, TMAC).
As shown in Figure 1A and 1B , in both conditions, the
empty peak elutes earlier than the full peak, which is then followed by
a third peak (attributed as empty capsids or capsid
debris,[23]). As TMAC requires a higher ionic
strength (191 mM) to elute rAAV than that of NaCl (124 mM), TMAC salt is
considered a weaker eluent than NaCl. In terms of chromatographic
separation, the AEX run with TMAC has higher empty and full peak
resolution (1.06) than that of the NaCl run (0.75), which is consistent
with a previous report.[21] However, the work from
Yang et al . was conducted using
0.1-mL CIMac column with analytical
loading and high operational pressure to ensure high resolution. In
contrast, we employed a 1-mL CIMmultus-QA column with preparative
loading and low operational pressure (<50 psi). As 1-mL
CIMmultus-QA column is a qualified scale-down model for up to 8000-mL
CIMmultus-QA (internal data, not shown), the AEX method established in
this work is appropriately scalable.