QIAcuity solutions for Biopharma

Vicki Vania
Sales Application Speciaist SEA-CP
Agenda
❑ Background
❑ QIAcuity in Biopharma
❑ Assays
❑ CGT Assays
❑ ResDNA Quant Assays
Agenda
❑ Background
❑ QIAcuity in Biopharma
❑ Assays
❑ CGT Assays
❑ ResDNA Quant Assays
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AAV vectors are a gene therapy tool of increasing importance

Profile Characteristics
• Dependoparvovirus with a 4.7 kb genome • Replication deficient without helper virus
• ssDNA genome flanked by 2 ITRs • Low integration efficiency
• Variable tropism • Prolonged transgene persistence

Vector utilization Quality control

• Delivery vehicle e.g., for gene • Detection of impurities (e.g., host cell DNA,
replacement, silencing and editing plasmids)
• Pre-clinical applications • Titer quantification for reproducible dosing
• Clinical applications • Determination of genome integrity

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Analytical Method Development
“Even in non GxP-settings an in-house evaluation establishing the above criteria (with documentation) concluding
development is good business practice and facilitates subsequent tech transfer and validation.”
Patrick Starremans, Dir Analytical Development National Resilience

R&D Preclinical Phase l Phase ll Phase lll BLA

IND Early Efficacy Confirmatory Efficacy &

Proof of Concept Safety Commercialization
Enabling & Safety Safety

• Dose range for preclinical
studies
• Dose finding • Max tolerated dose • Potentially efficacious • Efficacious dose • Dose indicated on label of
• Detailed SOP IND Contents: dose • Assay is validated released product
• Assay qualification across • Detailed assay description • Assay validation • Submitted to IND, include • Validation report, SOP
sample types • Qualification results • Variability sources changes • RM info included
• System suitability • Batch comparability

Assay development is critical throughout product development

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Bioprocessing Workflow

February 15, 2023 8

Key Customer Profiles in who are responsible for GMP

CRO/CDMO
Biotech
•Research & Development
•Collaboration
•All aspects of R&D, Analytical
Development, Process Sciences Industry
•Out-licensing and Quality Control
•Research & Development •Build portfolio through licensing •Fastest growing area due to
•Clinical Trial
•Heavily rely on contract and acquisition expertise
•Vector Core services for method •18 month project backlog
•Heavy use of CROs/CDMOs
•Small Scale GMP development, vector •Will take in methods or develop
Manufacturing production & manufacturing them for clients
•Out-licensing and collaboration •Drives system adoption out to
•Often targeted for acquisition by clients
Pharma

Academic Pharma

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Viral Titer

What is it?
• An absolute measurement of vector genomes per mL. Various
assay designs can be employed or multiplexed, including one of
more targets directed at the Gene of Interest (GOI), assays
measuring the ITR or promoter, or all of the above..

Why is it important?
• The amount of virus in the dose can influence safety and
efficacy of the therapy. It is also measured throughout
manufacturing to ensure titers align with expected values.

Why is it difficult?
• Secondary structure and accessibility of primer binding sites
near the ITRs can wreak havoc on PCR efficiency

• Well-characterized reference materials are hard to come by

• Assays don’t often include adequate coverage

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Importance of Viral Titer Measurements
Luxturna Zolgensma

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Old School

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Old School

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Middle School

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Middle School Troubles

Why would qPCR have precision issues?

• Poorly quantified or prepared standards

• What standard should be used?
• Inability to discern small changes in expression, 10%
• PCR inhibition

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New School - dPCR

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Agenda
❑ Background
❑ QIAcuity in Biopharma
❑ Assays
❑ CGT Assays
❑ ResDNA Quant Assays
Quantification via digital PCR (dPCR) eliminates the need for
standard curves
qPCR dPCR

Relative/absolute quantification: Cq Absolute quantification: Copies/µl

• dPCR quantification is based on many independent measurements whereas qPCR is based on a bulk measurement
• In dPCR, Poisson statistics is used for absolute quantification of the sample

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The QIAcuity Digital PCR System provides a qPCR-like workflow
with higher accuracy and precision
Partitioning and end-point PCR

Absolute target quantification

Sample Partitions No need for references or standard curves

Higher tolerance to inhibitors

Due to partitioning and end-point measurement

Precision
Detect very small fold-change differences

Increased analytical sensitivity

Detect rare mutations and low-abundance
targets
→ copies/µl
High reproducibility
Target of interest
Eliminate amplification efficiency bias
Interfering compounds

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dPCR is a more robust assay and provides tighter intra and inter-assay
precision

• qPCR may require a significant number of replicates to achieve

inter-assay precision required by the FDA

• qPCR precision may not be adequate as manufacturing scales

across many users, systems, sites

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How can Physical Viral Titer be measured?
Method Pros Cons
• Not affected by secondary structure • Laborious, time consuming, not
Dot Blot/Southern Blot
scalable
Pico Green Fluorimetry/UV Abs • Simple, fast • Non-specific, measures impurities
• Simple workflow • Poor rxn efficiency due to
• Fast secondary structure
• Sometimes robust with well- • Not inhibitor tolerant
designed assays • Variable across users
• Wide dynamic range/less dilution • Subjective data analysis
qPCR req • Reliant on a standard curve
• Well-established/known by FDA • Well-characterized ref. materials
for only AAV2 and AAV8 serotypes

• Accurate and precise • Cost (Inst, consumables, service)

• Inhibitor tolerant
• Less variability
dPCR
• Absolute quantification
• Simple workflow
• Fast

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Software Qualification – Computer System Validation

How do GMP customers qualify software?

What? • Assessment of a software/computer system used in a process to ensure

adequate safeguards and ability to comply with GMP guidance

How? • Form fill – Software architecture, data security, compliance to aspects of

21 CFR Part 11
• SOP - Test scripts to challenge software functionality

Who? • Vendor (20%)/Customer (80%)

• Pharma – internal CSV team, QA, QC-IT
• Biotech – outsourced to consultant

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Key Aspects of 21 CFR Part 11

Data Security

User Management

Audit Trails

Reports

Electronic Signature

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Positioning QIAcuity

GMP Compliance 21 CFR Part 11, IQOQ

Downtime 24-48hr Service TAT

“Robustness” of Methods Fixed partitioning, Automation-friendly, VPF

Time to Results 8X the throughput w/8.5K plate

Scalability Consistency across platforms, Footprint

Training/transient workforce FAS Support Infrastructure

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QIAcuity Features, Advantages & Benefits vs Bio-Rad
What problems faced by QX users do we solve?
Bio-Rad QX Problems QIAcuity Feature QIAcuity Advantage QIAcuity Benefit

Low Throughput 8-plate system format that runs Process more samples on fewer Generate 6X more data within a similar timeframe,
in 5.5 hours systems purchase and maintain fewer systems

Cumbersome workflow Fully integrated platform Less user intervention Conserve tech resources and reduce error

QX200 is not scalable 1, 4 and 8 plate formats Use same system across process No need to bridge methods to achieve higher
throughput
Quality/Reliability QIAGEN is known for quality Reliable, can trust data Less service intervention, greater confidence

Cost of Ownership Significantly lower system, Affordable high quality dPCR data Reduce manufacturing costs, be more competitive in
service and consumable costs securing client projects

Multiplexing True 5 color platform Enable higher multiplexing of 5 or more Measure all targets of interest in one sample, reduce
targets time to results and cost
ddPCR systems take up too QIAcuity has a small footprint Less than half the size 1:1 compared to Conserve valuable space in the lab and manufacturing
much space (~2x2x2ft) QX suite

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New School Problems

• Traceability
• Throughput
• Footprint
• Cost
• Time to data

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Agenda
❑ Background
❑ QIAcuity in Biopharma
❑ Assays
❑ CGT Assays
❑ ResDNA Quant Assays
Accurate and precise AAV titer quantification requires well-designed
assays

Design considerations Validation process (examples)

• GC content • Specificity and reproducibility

• Probe system • Optimal PCR cycling and imaging conditions

• Primer and probe placement • Linearity

• Length of primers and probes • Signal-to-noise ratio

• Melting temperatures • Ability to multiplex

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CGT dPCR Assays

Validated assays for precise AAV quantification using digital PCR

Isolate AAVs Setup reaction mix Pipet sample into a Run and analyze results in the
and add to in PCR pre-plate Nanoplate QIAcuity Digital PCR System
mastermix,
including the
restriction
enzyme

• 10 single assays available with different dye combinations for multiplexing

• 3-plex capacity (can be extended to 5-plex with customer-chosen GOIs)
• Complimentary, easy-to-use analysis in the QIAcuity Software Suite
• Used in combination with QIAcuity Probe PCR Kit

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CGT dPCR Assays – choose from ten validated targets

Pack sizes:
CGT dPCR Assays (20x), 500 x 12 µl reactions

Different dye configurations:

Kit components
FAM, HEX and Cy5
CGT dPCR Assays (20X) (GOI)
Quencher Zen and TAO IOWA Black (double-quenched)

For singleplex, duplex and multiplex use

Restriction enzyme compatibility: MspI, HpaII, SmaI Product Description Cat. No.
CGT dPCR Assays One tube of target 250230–250256
sequence dPCR Assay, 500
Ordering via QIAGEN website for 12 µl reaction in 8.5k
Nanoplate
QIAcuity Probe PCR Kit 1 ml or 5 ml Mastermix for 250101, 250102
Manufactured and stocked in Hilden (1 ml, 5 ml) the QIAcuity Digital PCR
instrument; water

For ordering, visit: www.qiagen.com/qiacuity-cell-and-gene-therapy-dpcr-assays

For ordering, visit: www.QIAGEN.com

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Dedicated CGT assays enable AAV quantification on the QIAcuity

CGT dPCR assay Target Fluorophore

ITR Inverted terminal repeats FAM, HEX, Cy5

CMV promoter Cytomegalovirus promoter FAM, HEX, Cy5

CMV enhancer Cytomegalovirus enhancer FAM, HEX, Cy5

SV40 promoter Simian virus 40 promoter FAM, HEX, Cy5

SV40 poly A Simian virus 40 polyadenylation signal FAM, HEX Channel Excitation (nm) Emission (nm) Example fluorophores

bGH poly A Bovine growth hormone polyadenylation FAM, HEX, Cy5

signal Green 463–503 518–548 FAM, EvaGreen

hGH poly A Human growth hormone polyadenylation FAM, HEX

signal Yellow 514–535 550–564 HEX, VIC, JOE, Cal Fluor 560

GFP Green fluorescent protein sequence FAM, HEX, Cy5 Orange 543–565 580–606 TAMRA, Alexa Fluor 546, Atto 550
WPRE Woodchuck hepatitis virus posttranscriptional FAM, HEX, Cy5
regulatory element Red 570–596 611–653 ROX, TexasRed

Amp resistance Ampicillin/carbenicillin resistance gene in FAM, HEX

bacteria Crimson 590–640 654–692 Cy5, Quasar 670

• CGT dPCR assays are validated and can be used in singleplex and multiplex reactions
• Assays can be additionally combined with gene-of-interest assays

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A simple and fast protocol
Step 1: Reaction setup in pre-plate, strips Step 2: Transfer to single or multiple Step 3: Load single or multiple Nanoplates into
or tubes Nanoplates QIAcuity and start the run

Volume per
reaction

Nanoplate 26k Nanoplate 8.5k Final

Component (24-well) (24-well, 96-well) concentration
Step Time Temperature
4x QIAcuity Probe PCR Master Mix 10 µl 3 µl 1x
Initial heat activation 2 min 95°C
20x dPCR CGT Assay-1 2 µl 0.6 µl 1x
2-step cycling (40 cycles)
Optional: Additional CGT Assays (2, 3, 4, 5) 2 µl 0.6 µl 1x
for up to fiveplex reaction* Denaturation 15 s 95°C

RNase-free water Variable Variable Combined annealing and extension 30 s 60°C

Template DNA (added at step 4) Variable Variable

Total reaction volume 40 µl 12 µl

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Assay properties and
applications
CGT dPCR Assays provide:

• Novel assay design for each backbone target

• Double-quenched probes to reduce background
• 3-plex capacity (can be extended to 5-plex with customer-chosen GOIs)
• High- to low-throughput applications with different QIAcuity instruments
• Compatible with QIAcuity Probe PCR Kit
• Single-tube format, easy handling
• Genome integrity

Applications include:

• Cell and gene therapy development – AAV quantification

• Gene editing using AAV as vector

CGT dPCR Assays are also compatible with digital PCR instruments
from other suppliers

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Workflow comparisons

QIAcuity Digital PCR System

Time to result

Pipette Load the

reaction plate(s) Priming,
thermocycling, Data
mixture to analysis ~2.5 hours
dPCR plate; readout
seal plate

Bio-Rad – QX200

Load Transfer
cartridges droplets Plate Data
and sealing Readout ~5.6 hours
to 96-well analysis
generate and PCR
plate
droplets

Thermo Fisher Scientific – Absolute Q

Load
cartridges Thermocycling Data
into 20k and readout analysis
~1.5 hours
fixed
partitions

Maximum 16 samples

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Outstanding CGT dPCR assay performance with the QIAcuity
Digital PCR System

High linearity from 2.5 copies/µl to 15,000 copies/µl on 8.5k Nanoplates

Data obtained through experiments conducted by QIAGEN R&D, Hilden, Germany

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Host cell DNA (HCD) quantification during biologics manufacturing

• In the manufacturing of biologics using

expression systems, host cell DNA levels
should be low in the final product to meet
regulatory guidelines appropriate for the
product’s intended use.
Cell Raw Final
culture Purification • Manufacturers of biologics use different
material steps product
approaches to show low residual DNA in the
purification process and final substance.

• Getting rid of impurities is very important.

Residual host cell DNA (HCD) monitoring is an
Residual DNA/RNA important step in the process of manufacturing
proteins and vaccines. The potential carryover
of HCD poses a safety concern.

• Levels of HCD must not exceed levels

established by regulatory agencies such as the
U.S. Food and Drug Administration and the
World Health Organization.

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Host cell models

Digital PCR-based host cell DNA monitoring

Three different residual DNA quant kits for use with the QIAcuity
Digital PCR System

• Human embryonic kidney cells (HEK)

• Chinese hamster ovary cells (CHO)

• Escherichia coli (E.coli)

The golden standard today is qPCR, however, digital PCR offers a

more robust, accurate and sensitive detection of host cell DNA

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QIAcuity resDNA Quant Kits

Validated assays for highly sensitive residual DNA quantification using digital PCR

Add sample to Setup reaction mix Pipet sample into a Run and analyze results in the
QIAcuity in PCR pre-plate Nanoplate QIAcuity Digital PCR System
resDNA quant
kit

• Two premixed master mixes for (CHO, E.coli) (HEK293 coming soon) available with
standards and internal controls
• Analysis performed with dPCR Software Suite
• Easy workflow
• Down to femtograms (fg) of residual DNA detection in a single reaction

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QIAcuity resDNA Quant Kits – CHO, E.coli and HEK293

Pack sizes: Product Description Cat. No.

dPCR resDNA Quant kit 4 x 24 reactions (96 reactions) with
QIAcuity CHO resDNA Quant Master Mix (2X),
positive and internal controls 500 μl (24 reactions), QIAcuity CHO resDNA
QIAcuity CHO resDNA Quant kit Quant Positive Control lyophilized, QIAcuity 250222
Different host cell DNA targets: CHO resDNA Quant Internal Control lyophilized,
dPCR Qualified Water 1.8 ml
CHO, E. coli (HEK293 coming soon) labeled with FAM
CHO resDNA Quant Standard 250223
Internal control with HEX
QIAcuity E.coli resDNA Quant Master Mix (2X),
500 μl (24 reactions), QIAcuity E.coli resDNA
For singleplex and duplex use QIAcuity E.coli resDNA Quant kit Quant Positive Control lyophilized, QIAcuity 250220
E.coli resDNA Quant Internal Control lyophilized,
Kit components dPCR Qualified Water 1.8 ml

E. coli resDNA Quant Standard 250221

QIAcuity CHO/E.coli/HEK293 (2X) 500 µl, 25 reactions
dPCR Qualified water (1.8 ml)
QIAcuity CHO/E.coli/HEK293 (2XresDNA Quant Positive Control lyophilized)
QIAcuity CHO/E.coli/HEK293 (2XresDNA Quant Internal Control lyophilized)
QIAcuity HEK293 resDNA Quant Kit
HEK293 resDNA Quant Standard
QIAcuity HEK293 resDNA Quant Master Mix
(2X), 500 μl (24 reactions), QIAcuity HEK293
resDNA Quant Positive Control lyophilized, 250224
QIAcuity HEK293 resDNA Quant Internal Control
lyophilized, dPCR Qualified Water 1.8 ml
250225

For ordering, visit: www.qiagen.com/qiacuity-residual-dna-quantification-kits

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Workflow comparisons
QIAcuity Digital PCR System
Time to result

Pipette Load the

reaction plate(s) Priming,
thermocycling, Data
mixture to analysis ~2.5 hours
dPCR plate; readout
seal plate

Bio-Rad – QX200

Load Transfer
cartridges droplets Plate Data
and sealing Readout ~5.6 hours
to 96-well analysis
generate and PCR
plate
droplets

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February 15, 2023 46
The dMIQE guidelines and ISO 20395-2019

The QIAcuity Digital PCR System is dMIQE and

ISO 20395-2019 compatible

dMIQE Group, & Huggett, J. F. (2020). The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020. Clinical chemistry, 66(8), 1012–1029.
ISO 20395:2019: Biotechnology — Requirements for evaluating the performance of quantification methods for nucleic acid target sequences — qPCR and dPCR.

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Update

February 15, 2023 48

Thank you
Questions?