Poster
Optimizing the Method Lifecycle of GMP Manufacturing for AAV
Optimizing the Method Lifecycle of
GMP Manufacturing for AAV
Murali Jujjavarapu; Aparna Nandakumar; Delicia Henriques, PhD; Minqi Jiang; Aneri Maniar; Ryan Rubino, Eric Gliniak; Gelsey Garcia; Philemon Asfeha; Wei Cao, PhD.
INTRODUCTION
PROBIO ANALYTICAL OPERATIONS STRATEGY
REFERENCE
CONCLUSION
The analytical control of adeno-associated virus (AAV) products presents unique challenges due to the
structural complexity of viral vectors, evolving regulatory expectations, and the need to apply phase-
appropriate validation (PAV) across development and GMP manufacturing. Limited early-phase material
availability, platform adaptability across serotypes, and the need to balance assay rigor with program
phase and timeline are critical considerations for a successful project. In this abstract, we present our
capabilities through the successful execution of a current GMP project in which phase-appropriate
validation (PAV) principles were applied across the full analytical method lifecycle—from early
development through method qualification—to support
downstream transfection applications. We demonstrate analytical requirements were aligned with
program phase, cost considerations, and critical quality attributes (CQAs) to enable efficient and
compliant decision-making. comprehensive panel of orthogonal methods was developed and
implemented to characterize product quality, safety, and consistency. These included aggregation
assessment by UPLC, host cell DNA and residual plasmid quantification by droplet digital PCR (ddPCR),
vector genome (VG) titer determination by ddPCR, capsid titer measurement by ELISA, and host cell
protein (HCP) analysis by ELISA. Each method followed a structured lifecycle approach encompassing
development, optimization, and phase-appropriate qualification to ensure fitness for purpose under
GMP conditions. Emphasis was placed on assay robustness, sensitivity, and reproducibility, while
maintaining flexibility to accommodate evolving program needs. The analytical strategy enabled timely
lot disposition and supported delivery of a GMP final AAV product within a client-driven timeline. Our
results demonstrate integrated analytical and quality capabilities, highlighting how PAV-based method
lifecycle management can accelerate GMP programs while maintaining regulatory compliance and
product quality. This novel approach underscores the value of early client alignment and adaptable
validation strategies in the successful advancement of viral vector therapeutics.
Using this PAV approach, ProBio successfully delivered client X project for tox study for AAV within time
frame with key deliverables:
EQUIPMENT
STATUS
STANDARD/SYSTEM
SUITABILITY
(FOR METHODS)
PHASE
METHOD STATUS
DELIVERABLES
QA SCOPE
AD Method Development
Reports
Qualification
Protocols*
Qualification
Reports*
Developing Analytical
Method, FIO Testing,
QC/AD-Released
COA/COT
Minimum
Calibration
NA (only Veeva
Docs Approval)
R&D
AD Method
System Suitability Only
* Based on client requirements
a
high quality AAV product suitable for
CASE STUDY HIGHLIGHT
Minimal (Veeva
Docs Approval,
Crosscheck LDS
(ALCOA+) and
AD- Fully or Partially Calibration &
Developed Method Preventative
(Fit for purpose) Maintenance
A
PRECLINCAL
(TOX)
Method Development System Suitability, Assay
Report (optional)
Orthogonal characterization
provided additional insight into the
sample by differentiating and
quantifying full, empty and partial
capsids.
Control (optional)
CoT/CoA Approval.)
Full
Capsid
Orthogonal
Characterization
Empty
Capsid
Partial
Capsid
Development Reports,
Qualification/
Verification Protocols &
Reports, Raw Material
Qualifications
QC
PHASE I & II Qualified/Verified
Methods
Techniques & Analyses Used:
Calibration,
Preventative
Maintenance,
IQ/OQ/PQ,
21 CFR-
This provided a more complete
understanding and stronger
characterization of the AAV sample.
System Suitability, Internal
Reference Standards,
Specifications
Complete QA
Oversight
AUC
SEC-MALS
LC-MS/MS
TEM
qPCR
QC Project Specific
Validated Methods
PHASE III &
Development Reports,
Validation Protocols
& Reports
ELISA
Compliance
(FMEA &
Spreadsheet
Validations
COMMERCIAL
Table 1: ProBio’s phase-appropriate analytical operations strategy is designed to align analytical support with each stage of development,
ensuring the right level of rigor and efficiency throughout a project. Client deliverables can be customized based on specific program needs
and requirements.
FUTURE WORK
GOAL: Implement phase-appropriate, orthogonal assays to ensure a comprehensive understanding of
AAV quality attributes throughout development
WHY PHASE APPROPRIATE APPROACH
CASE STUDY: PERCENTAGE FULL CAPSID RATIO
AAV Titer and Capsid Characterization Summary
PRECLINCAL
(TOX)
PHASE I
PHASE II
PHASE III
COMMERCIAL
CE-SDS
(release)
SDS-PAGE
(routine)
CE-SDS
(higher resolution)
SDS-PAGE
vs
CE-SDS
Robust, high-resolution
method
for release
Capsid integrity
& purity
Enhanced resolution of capsid
species and fragments
ASSAY
RESULT
% FULL CAPSID
SEC-MALS
(initial
characterization)
2.8 x 1012
vg/mL
72%
(includes partial)
SEC-MALS
(continued characterization)
SEC-MALS
(release)
VG TITER
SEC-MALS
VG TITER
Molecular weight,
assembly state, and
heterogeneity
Ongoing assessment of capsid assembly,
heterogeneity, and stability
Orthogonal confirmation
of product quality
3.9 x 1012
capsids/mL
CAPSID TITER
–
ddPCR
(GOI)
qPCR
(generic target)
ddPCR
(generic target)
ddPCR
(GOI)
Qualifional release
method for potency
assessment
64.4%
(full only)
Screening and early- Increased sensitivity
stage titering
Product-specific
quantification
SV-AUC (% FULL)
–
& precision
ORTHOGONAL COMPARISON
(Phase I-II)
Integrated
Approach
DLS
(Pre-Clinical)
SEC-HPLC (FL)
(Phase I-II)
Table 2: Orthogonal comparison of the VG titer–to–capsid titer ratio is used to estimate full capsid percentage during toxicology studies,
while for Phase and later stages, this assessment is complemented or confirmed using SV-AUC to provide more direct and robust
measurement of capsid content and quality.
I
a
SV-AUC
Orthogonal methods
support robust
characterization
and control
SEC-MALS
Size and molecular weight
of aggregates
AGGREGATION
Rapid assessment of Fluorescence detector for
particle size and aggregate and variant
aggregation profiling
Sedimentation-based
orthogonal assessment of
aggregates
Figure 2: SV-AUC Data for
Characterization of Capsid
Population
ACKNOWLEDGEMENTS
Figure 1: Lifecycle Progression of Analytical and Process Validation Across Development Phases
•
•
This research was made possible through the support of the Analytical Department at ProBio Inc.,
Hopewell NJ.
Phase-Appropriate Qualifica6on
Data Integrity and Traceability
• QA oversight ensures traceability and
compliance
•
Implement qualification of analytical
methods tailored to each study phase for
appropriate support.
We thank the Process Development (PD) and Manufacturing (MFG) teams—Eric Lin, PhD; Haibo
Zhang, PhD; Andy Tran; Ang Chen Tsai, PhD; Lance Marquardt; and James Kuciel—for their
collaboration, technical expertise, and contributions to project execution.
Project Timelines and Efficiency
Fit-for-Purpose Methods
FDA & ICH (Q14, Q2(R2)) endorse fit-for-
purpose methods for early phases.
•
•
We would also like to acknowledge the support of the Quality Assurance (QA) team, Julie Erwin, PhD,
and Taruj Patel for their guidance and oversight throughout this work.
•
Maintain project timelines and optimize
resource efficiency throughout the
qualificaAon process.
•
Special thanks to Daniel Savad for all single handedly tackling all our documents.
ProBioCDMO.com
Contact: Murali Jujjavarapu | murali.jujjavarapu@probiocdmo.com