The next generation of AAV analytics

Tony Bou KheirSenior Analytical Scientist

Development of AAV therapeuticsKTN - Cambridge 30 Jan 2019

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It is our vision for the UK to be a global leader in the development, delivery and commercialisation of cell and gene therapies.

Where businesses can start, grow and confidently develop advanced therapies, delivering them to patients rapidly and effectively.

Who we are

Part of a world-leading network of technology and innovation centres

Bridge the gap between businesses, academia, research and government

Provide access to unique technical facilities and expertise to help adopt, develop and exploit innovations

Were established by Innovate UK as a not-for profit, independent centre

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Who we are

2018 201920172012 2016

Cell Therapy catapultCentre of excellence in innovation

Cell and gene therapy Catapult - CGT

Manufacturing centre

Synpromics

Sartorius

Therapy provider 1

Combigene

COBRA / PALL

Therapy provider 2

CGT Core projects

Kings

qPCR

MADLS

ddPCR

ELISA

DLS

Western blot

HPCE

Auto-mated WES

FACS

LC-MS

Ovisio

Sterili-ty

Physi-cal

titre

Impe-dence

Empty/full

Aggregation

Raman

Purity

VCN

Infect-ioustitre

Trans-ducingunits

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CGT analytical capabilities and vision

Case study

Application of developed methods on an in-house production test run

Purity

Full / Empty ratio

Quantity - Physical capsid titre

Testing of ELISA commercial kits MADLS

Quantity - Vector genome titre

Commercial qPCR testing kits In-house ddPCR development In-house qPCR development

Presentation workflow

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Quantity – vector genome measure

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Quantitative real-time PCR (qPCR)

• Primer and probes targeting gene of interest and/or ITRs

• Use of a digested plasmid as a standard curve

Limitations

1. Highly sensitive to PCR inhibitors – viral proteins and/or vector diluent → decrease in amplification efficiency → Under-estimation of viral titre

2. Bias from amplification efficiency – especially if targeting ITR region → under-estimation of viral titre

3. Bias introduced from the standard curve – amplification of dsDNA vs ssDNA → Over-estimation of viral titre

4. Steps above →High inter/intra-assay variability

Quantity – vector genome titre

Traditional methods

A T C C r e f .

1 09

1 01 0

1 01 1

1 01 2

Co

pie

s/m

L

7

Develop a robust, accurate in-house method for measuring AAV2 vector genomes (and AAV2 derived serotypes)

Quantity – vector genome titre

Aim

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qPCR

qPCR alternatives - ddPCR

ddPCR

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1. Highly sensitive to PCR inhibitors – viral proteins and/or vector diluent → decrease in amplification efficiency →Under-estimation of viral titre

✓ Less sensitive to PCR inhibitors → suitable for in process vector genome measurement

2. Bias from amplification efficiency – especially if targeting ITR region → under-estimation of viral titre

✓ End product measurement – less dependent on amplification efficiency → Suitable for targeting ITRs – Universal assay

3. Bias introduced from the standard curve – amplification of dsDNA vs ssDNA → Over-estimation of viral titre

✓ Absolute quantification – no standard curve required → Improved precision

4. Steps above → High inter/intra-assay variability

✓ Robust and accurate method for in-process control and product characterisation

Scope

qPCR vs ddPCR

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ddPCR vg titre assay

0 . 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 1 0 0 0 0 0

1

1 0

1 0 0

1 0 0 0

1 0 0 0 0

1 0 0 0 0 0

d d P C R a s s a y l i n e a r i t y

E x p e c t e d c o p ie s

Ca

lcu

late

d c

op

ies

R square 0.9969Deviation from linearity non significantLLoD 16.16 copiesLLoQ 125 copiesIntra/inter assay CV < 20%

Primer/probe set targeting ITR2 sequence – matching against internal positive control primer/probe set

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ddPCR and Commercial qPCR method comparability

d d P C R q P C R

0

1 1 01 0

2 1 01 0

3 1 01 0

4 1 01 0

5 1 01 0

S e l e c t i v i t y a n d C o m p a r a b i l i t y

Ca

lcu

late

VR

16

16

Tit

re

ddPCR results within 95% CI

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1. ITR sequence detection → Applicable to any AAV2 and AAV2 derived serotypes

2. In-house designed primers and extraction method

3. Higher sensitivity → Suitable for in-process sample measurement

4. Increased precision and reproducibility over commercial and current available qPCR titration methods

To overcome ddPCR limited market coverage, CGT has adapted in-house primers/probe set to a qPCR platform

Summary

Fully functional assay offering

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Intra-assay CV < 10%

Inter-assay CV = 22%

Adapted in-house qPCR method

1 2 1 2 1 2 A v e r a g e A T C C

r e f .

1 09

1 01 0

1 01 1

1 01 2

Co

pie

s/m

L

A A V R e p l i c a t e

C V 7 % 1 8 % 7 % 6 % 5 % 9 %

E x p e r im e n t 1 2 3

Inter assay variability greater than seen on a ddPCR platform, but still meeting acceptance criteria

Quantity/puritytotal particle measureEmpty to full ratio

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Evaluation of 5 commercial kits

Inter assay CV <8%

Intra assay CV <5%

Limitations

1. Expensive

2. Labour intensive

3. Time consuming

4. Antibody specific/serotype dependent

Quantity/purity – total particle measure/ Empty to full ratio

D 3

1 : 1 0 0 0

D 4

1 : 1 0 0 0 0

D 1

1 : 1 0 0 0

D 2

1 : 1 0 0 0 0

D 1

1 : 1 0 0

D 2

1 : 1 0 0 0

D 1

1 : 1 0 0

D 2

1 : 1 0 0 0

0

1 1 08

5 . 0 0 1 1 01 1

1 . 0 0 0 1 1 01 2

V R - 1 6 1 6 A A V 2 r e f e r e n c e m a t e r i a l

Pa

rtic

les

/mL

E x p t 1 E x p t 2 E x p t 3 E x p t 4

ELISA – Commercial kit

qPCR ddPCR

% full particles 13.69% 6.72%

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Dynamic Light Scattering technology

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1. Expensive

✓ Long term cost efficient

2. Labour intensive

✓ No sample prep required

3. Time consuming

✓ Fast, readings in under a minute

4. Antibody specific/serotype dependent

✓ Physical measure of particle content, universal serotype measure

✓ Measures impurities

✓ Measures aggregates

Quantity – total particle number, alternative methods

MADLS and ELISA method comparability

Case study

Overview of downstream developmental scope

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AKTA™ AvantChromatography purification development and optimisation

Nuclease treatmentDNA removal development and optimisation

TFF KrosFloUF/DFConcentration & buffer exchange development and optimisation

ClarificationHarvest filtration development

Final filtrationFinal filtration development

Source nucleasesOptimise treatment.

Capture and polishing

Formulationconcentration

Sterile filtrationFill and FinishCell lysis methods.

Cell lysis methods.Filter options

Cell disruptionViral vector release development and optimisation

Cell Lysis Experiment – Study Design & Workflow

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Harvest

Centrifugation

Extraction buffer

Freeze/thaw cycles

DNase treatment

Lysate

Harvest

Physical lysis

Physical lysate

Chemical lysis

Chemical lysate

DNase treatment

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AAV titre and purity check

1 01 0

1 01 1

1 01 2

1 01 3

P a r t i c l e m e a s u r e

AA

V2

tit

re

V g ( d d P C R )

V g ( q P C R )

T o t a l ( E L I S A )

P h y s i c a l l y s i s

Ch

em

ica

l ly

sis

Fre

ez

e /

th

aw

0

5 0

1 0 0

1 5 0

F u l l t o e m p t y r a t i o

% F

ull

/ E

mp

ty

V g ( d d P C R )

V g ( q P C R )

P h y s c ia l l y s i s

Ch

em

ica

l ly

sis

Fre

ez

e /

th

aw

Particle number produced Sample purity

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Total particle measure - MADLS

LysisPrimary

clarificationSecondary

clarificationAffinity capture

4.67E+11

1.61E+20

CGT analytical capabilities and vision

CGT analytical capabilities

fullEmpty

Plasmids

Free genomes

VP1,2,3

Cell debris

Packaging Ratio ELISA/PCR HPCE

Viral capsid proteins Western blot Automated WES

Aggregation DLS MADLS

Infectious titre FACS ddPCR

Functional titre In-vitro FACS/Impedance

Packaged genomes qRT-PCR ddPCR

Physical titre ELISA MADLS

1st Gen 2nd Gen

Total protein Coomassie LC-MS

Sterility Growth based ddPCR

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Purity ddPCR / Seq

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CGT vision

The data analytics continuum

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CGT vision

Raw Material Inputs Variable product qualityConstrained Process

Now

FutureAdaptable ProcessRaw Material Inputs Predictable product quality

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CGT Strategy

Analyser

LC/MS

Raman / NIRRaman / NIR

Feedback controls

At-Line

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Julie Kerby

Damian Marshall

Mike Delahaye

Gregory Berger

Nicole Nicolas

Anusha Seneviratne

Nishanthi Weeratunge

Florian Leseigneur

Quentin Bazot

Elena Sokolskaja

Acknowledgements