Human Gene Therapy - April 2023 - 329

ANALYTICAL DEVELOPMENT WITH PRECLINICAL MATERIAL
329
ratios were determined using the Spearman-Ka¨rber's
method from publicly available spreadsheets that were
modified for use.35
Sedimentation velocity analytical
ultracentrifugation
Purified vector from the 500L production was considered
as the full vector material. In addition, the empty
capsid fraction from this production was also separately
purified. A six-point curve was generated using the full
vector (sample 1) followed by spiking the full vector with
volumes of empty capsid material (samples 2 through 5).
In addition, the empty capsid material was included in the
data collection (sample 6). Samples 1-6 were analyzed by
SV-AUC at BioAnalysis, LLC (Philadelphia, PA). In
brief, formulation buffer was used for sample dilution to a
final concentration range of 0.5-0.9 OD at 230 nm. Samples
with lower than 0.5 OD were analyzed without dilution.
Four hundred twenty microliters was loaded into the
reference and 410 lL into the sample sectors (two sector
charcoal, Epon centerpiece; quartz/sapphire windows).
The samples were allowed to achieve temperature
equilibrium 1.5 h after the vacuum stabilized at 0 lm. The
experiments were collected using a Beckman Coulter XLI
and sedimentation was followed at 230 nm. Rotor speed
was 12,000 rpm and the scanning frequency was set to
150-200. SEDFIT (16.36) was used to generate the c(s)
and ls-g*(s) distributions from the SV-AUC data. Figures
were generated using GUSSI (1.4.2). SEDNTERP
(v.3.0.3; March 14, 2021) was used to determine the
density and viscosity of the buffer. Sedimentation coefficients
were uncorrected (s,S) for buffer, water, and 20C.
Integration of the c(s) distributions was performed using
both GUSSI and OriginLab (2015) or directly in Excel.
Cryogenic electron microscopy
The dilution series of full and empty capsids from above
were analyzed by cryo-EM at the University ofFlorida ICBR
Electron microscopy core. In brief, for each sample, 3.5 lL
was applied to a glow-dischargedQuantifoil copper grid with
2nm continuous carbon support over holes (Quantifoil R 2/4
400 mesh), blotted, and vitrified using a Vitrobot Mark 4
(FEI) at 95% humidity and 4C. Images were collected using
an FEI Tecnai G2 F20-TWIN microscope (FEI) operated
under low-dose conditions (200kV, *20e-/A˚ 2) on a GatanUltraScan4000CCDcamera
(Gatan). Thenumber ofempty
and full capsids in these images were counted manually.
pH stress studies
The study of STRX-330 in response to pH stress conditions
was performed using concentrated HCl or NaOH.
In brief, vials ofSTRX-330 were removed from -80Cand
thawed overnight at 4C. To initiate the stress conditions,
HCl or NaOHwas added to achieve a final concentration of
0.01 N. The volume of acid or base was*1%of the sample
volume to avoid dilution of formulation buffer. Samples
were kept at room temperature for 3 h and then neutralized
with the opposite treatment. For instance, acid-treated
samples were neutralized with equimolar base and vice
versa. As a control, equimolar base and acid were mixed to
neutralize and then added to STRX-330. The final pH of the
stressed conditions were *4.0 for acid treated, *11 for
base treated, and *7.5 for neutralized treated.
Data analysis and documentation
Data sets were analyzed in Microsoft Excel and
GraphPad Prism (9.0). Graphs were generated with
GraphPad Prism (linear regression) and coefficient of
variance (%CV) was determined using Excel. Experiments
were documented in near-time within the electronic
laboratory notebook of StrideBio.
RESULTS
Method development for SEC-MALS
Development of the method for testing STRX-330 was
performed using an isocratic mobile phase with two separate
SEC analytical columns, a Wyatt brand SEC column
(5 lm, 500A˚ ) and a Sepax Technologies SRT SEC column
(5 lm, 1,000A˚ ). A representative chromatogram from
these injections is shown in Fig. 1.
Monomeric AAV elution times were *8.5 and
*11 min for the Wyatt and SRT columns, respectively.
The dimer peak eluted at *7 min for the Wyatt column
and*10 min for the SRT column. Interestingly, there was
third peak that eluted at *6.5 min for the Wyatt column
and *7.5 min for the SRT column. While the monomeric
and dimer peaks both exhibited VG/CP ratios consistent
with AAV, this third peak exhibited pronounced absorbance
at 260nm with minimal absorbance at 280 nm.
Importantly, the monomeric peak statistics demonstrated
acceptable analytical performance for asymmetry, tailing
factor, and resolution between the dimer peak when either
column was used (Supplementary Table S1).
Both columns demonstrated strong agreement in vector
titer (capsid and genome), vector molar mass (within 1%
of theoretical), and DNA molar mass without any additional
optimization required (Supplementary Table S1).
These data sets were compared to internally developed
ddPCR and capsid ELISA methods and are shown in
Supplementary Table S2. The DNase-resistant mean
ddPCR value was within 20% while the mean capsid
ELISA value was within 5% of the SEC-MALS VG/mL
and CP/mL measurements, respectively. Comparable to
SEC-MALS precision, both ddPCR and ELISA values
produced intra-assay measurements <3%. The Wyatt
500A˚ column was used for subsequent experiments.
To determine whether the SEC-MALS approach could
achieve ICH and USP recommendations for analytical
methods, linearity and accuracy by recovery tests were
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