Human Gene Therapy - April 2023 - 337

ANALYTICAL DEVELOPMENT WITH PRECLINICAL MATERIAL
337
the molar mass data) within the monomer peak. Since the
protein molar mass did not change, this would suggest that
an alkaline pH condition has a profound impact on the
encapsidated DNA content. The acidic pH stress condition
impacted the capsid more so than DNA by promoting
aggregate formation. Surprisingly, the ddPCR/ELISA data
for the HCl-treated sample exhibited only a modest reduction
in the CP/mL and DNase-resistant VG/mL values.
However, the NaOH-treated material reduced the CP/mL
(*twofold) and the VG/mL values (>20-fold; Table 5).
Following treatment with 0.01N HCl, TCID50 data demonstrated
only a modest change in the infectivity. Following
treatment with 0.01N NaOH, infectivity measured
by TCID50 was markedly reduced to undetectable levels
(Table 5).
CONCLUSION
A successful gene therapy program requires robust and
adaptable analytical capabilities. Primary areas of analytical
focus for preclinical and early-stage programs are
establishing the dose-defining assay(s), transduction assays
to monitor expression of the DNA payload, and solidifying
the safety and stability-indicating assays. Shown
here is that SEC-MALS is a valuable analytical tool that
assists in meeting these areas of need. A product-specific
SEC-MALS method required little development and optimization
and could achieve the recommended ICH/USP
guidance for validation of chromatography methods.
Comparison of full-to-empty methods demonstrated
strong linearity and accuracy with values obtained by SVAUC.
The latter is considered the ''gold standard'' for
quantification of full particles.
However, we note that agreement between SEC-MALS
and SV-AUC may rely on the level of partial genome
concentrations within the purified vector. AAV containing
partial genomes may influence the SEC-MALS full-toempty
ratio leading to inaccurate measurements. In addition
to SEC-MALS and other approaches, we believe that
methods capable of quantifying partial genomes, such as
SV-AUC,18 be utilized early in program development in
accordance with an enhanced approach to analytical procedure
development.
The VG/mL values obtained with SEC-MALS were
within 20% of the DNase-resistant ddPCR values, which is
considered the primary dose-defining assay for AAV.
When used in combination with other stability-indicating
assays, SEC-MALS was capable of providing insight into
AAV degradation in response to pH stress. In addition to
pH stress, 10 · freeze-thaws and temperature stresses were
also performed. These data sets demonstrated that SECMALS
was capable of detecting modest to more extreme
changes in the AAV monomer peak (data not shown). This
supports the utility of SEC-MALS early in program development
since minimal sample handling and consumption
are needed to generate analytical data that are
supported with orthogonal methods.
Moreover, these results support that methods developed
with SEC-MALS can meet the analytical testing guidelines
and are capable of being transferred into a quality
control laboratory. Perhaps more interesting is the ability
of SEC-MALS to provide fundamental understanding of
the response of AAV to environmental stress conditions.
This was demonstrated with the pH stress studies that
determine acidic and alkaline pH negatively impact capsid
and genome content, respectively. This information may
yield additional knowledge of how AAV responds to
conditions experienced during its life cycle and the
manufacturing process.
ACKNOWLEDGMENTS
We are thankful to Lake Paul, PhD for the SV-AUC experiments.
We are thankful to Mario Mietzsch, PhD, and
Robert McKenna, PhD, for conducing the cryo-EM studies.
AUTHORS' CONTRIBUTIONS
B.T.-conceptualization, investigation, formal analysis,
resources, writing-original draft, visualization, and
project administration; I.-W.T.-methodology and resources;
K.S.-investigation, methodology, formal analysis,
and writing; C.I.K.-investigation, formal analysis;
S.S.-investigation, formal analysis, and writing; K.L.-
investigation, formal analysis, and writing; S.M.B.C.-
investigation and formal analysis; T.R.-investigation and
formal analysis.
AUTHOR DISCLOSURE
Authors have equity within StrideBio.
FUNDING INFORMATION
Funding for this work was provided by StrideBio.
SUPPLEMENTARY MATERIAL
Supplementary Figure S1
Supplementary Figure S2
Supplementary Figure S3
Supplementary Table S1
Supplementary Table S2
Supplementary Table S3
Supplementary Table S4
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