Human Gene Therapy - April 2023 - 327

ANALYTICAL DEVELOPMENT WITH PRECLINICAL MATERIAL
327
from American Type Culture Collection (Manassas, VA).
Dulbecco's phosphate-buffered saline (DPBS) and pluronic
F-68 were purchased from Gibco (Grand Island, NY).
Ultra-pure 10% sodium dodecyl sulfate and Trizma were
purchased from Invitrogen (Waltham, MA). Fetal bovine
serum (FBS) was purchased from HyClone (Logan, UT).
Tween 20, EDTA, 100% ethanol, NaOH, Accelagen,
Turbonuclease, and Maxisorp nunc plates were purchased
from Fisher Scientific (Waltham, MA). HCl was purchased
from Honeywell Fluka (Charlotte, NC). Proteinase
K, deoxycholic acid, Blocker Casein in phosphatebuffered
saline (PBS), POROS CaptureSelect AAVX
affinity resin, CaptureSelect Biotin AAV9, CaptureSelect
horseradish peroxidase (HRP) AAV9, 3,3¢5,5¢tetramethylbenzidine
(TMB) substrate, and sulfuric acid
were purchased from Thermo Scientific (Waltham, MA).
Dulbecco's modified Eagle's medium (DMEM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES), and
10·PBSwerepurchasedfromCorning (Corning,NY).Liquid
chromatography-mass spectrometry grade water and streptavidin
were purchased from Pierce (Waltham, MA). Bovine
serum albumin (BSA) was purchased from USP (Rockville,
MD). 10·PBS Tween (1%) was purchased from BostonBio
Products (Milford, MA). Automated droplet generation cartridges,
ddPCRsupermix for probes, ddPCR droplet reader oil,
pipet tips for automated droplet generator, automated droplet
generation oil for probes, ddPCR plate seals, and PCR plates
were purchased from Bio-Rad (Carlsbad, CA).
STRX-330 production
The STRX-330 material used here was produced using a
proprietary cell line and process. In brief, a HEK cell suspension
of the listed volumes was transfected with three
plasmids containing a plasmid with the gene ofinterest for the
treatment ofARVCflanked by ITR, a plasmid containing the
AAV2 Rep and STRV84 Cap, and a proprietary helper virus
plasmid. The gene cassette present on the ITR plasmid was
designed to minimize off-target DNA packaging (i.e., residual
and overpackaging). rAAV was produced and the cell
culture was lysed using a commercially available detergent,
treated with turbo nuclease (Accelagen), and then incubated
at 37Cfor several hours.Clarified andfiltered lysatematerial
was loaded onto an affinity resin (AAVX) column for purification
of the rAAV.
Capsids were eluted with a developed low pH buffer and
subsequently neutralized followed by loading onto cesium
chloride gradient sample tubes. Tubes were centrifuged at
approximately 220,000·g fora minimumof16h under
temperature control using a Beckam Coulter Optima XE-90
Ultracentrifuge. Full vector and empty capsid fractions were
purified using a 21-gauge needle, formulated in a PBS containing
buffer, additionally processed to achieve the target
VG/mL (or capsid particles [CP]/mL for the empty capsid
fraction), and aliquoted until subsequent testing. Test articles
were stored at -80C until used.
Size exclusion chromatography with UV
and multiangle light scattering
An Agilent 1260 Infinity II LC system (Agilent, Santa
Clara, CA) with binary pump, multisampler maintained at
4C, multicolumn thermostat maintained at 25C, and
diode array detector was coupled to a DAWN HELEOS
multiangle light scattering and Optilab refractometer for
detection (both from Wyatt Technology Corporation,
Santa Barbara, CA). An isocratic mobile phase of 1 ·PBS
(from 10 · PBS) containing 0.01% (vol/vol) pluronic F-68
was used after passing through a 0.2 lm filter. Liquid
chromatography-mass spectrometer grade water was used
for dilution of stock solutions. Two separate SEC analytical
columns were used: Wyatt 5 lm 500A˚
4.6mm ID ·
300mm (Wyatt Technology Corporation catalog WTC050N5)
and a SRT 5 lm 1,000A˚
4.6mm ID · 300mm
(Sepax Technologies, Newark, DE; catalog 2159504630).
Columns
were equilibrated for *20 h before injection
and flow rates (0.3 mL/min) and injection volumes (45 lL)
were kept constant for direct comparisons. Absorbance at
260 and 280nm were performed during testing and capsid
and vector extinction coefficients for STRX-330 were
determined using Optilab and UV detection. The extinction
coefficients at 260 and 280nm for the STRV84 were
1.40 and 2.01. In addition, the extinction coefficients at
260 and 280nm for STRX-330 were 25.09 and 14.56. Peak
detections, molar mass determinations and peak statistics
were performed in ASTRA 8.1.1.
For assessment of analytical performance of SECMALS,
linearity was performed by diluting the process
confirmation test article across nine levels with triplicate
volume transfers and singlicate injections. To achieve
accuracy, five volume transfers at seven different levels
were performed. Mobile phase was used as the diluent. For
routine testing of rAAV, 10 lL of sample was diluted into
40 lL of mobile phase. Sample volumes were added into
2mL polypropylene vials with 0.2mL glass insert with
vial screw caps (Agilent). Triplicate injections of BSA
were used as a system check during each run.
ddPCR and stunner testing
Design of experiment (DOE) approaches were used to
develop and optimize a titer assay for the genome quantification
of STRX-330. Multiple sets of primers and
TaqMan probes were ordered from Invitrogen, and conditions
were screened for desired response factors. The
developed assay included a single set of primers with
probe, a minimum of three serial dilution preparations of
samples and five levels prepared in the presence of
pluronic, treatment with Turbo DNase (Ambion), heat and
chelator inactivation of the DNase, and dilution in water.
DNase treated, diluted samples were mixed with ddPCR
supermix for probes and gene-specific primers/probe.
Droplets were generated using an Auto-droplet generator.
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