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AAV next generation quality control

Tony Bou Kheir

Cell and Gene Therapy Manufacturing Workshop

March 12th 2019

AAV mediated gene therapy

AAV mediated gene therapy characteristics

Genetic capacity of ~4.5kbNon integratingTargets both dividing & non-dividing cellsBroad tissue tropism

The scientist, Lucy Reading-Ikkanda

GeneCopoeia

Setback for gene therapy for safety reasons in the 90s

Discovery of novel, safer and more efficient AAV vectors

• Exponential growth

• Funding influx

• High profile deals

• > 100 clinical trial

• 2 launched products

• Glybera, 2012

• Luxturna, 2017

Cell and Gene Therapy Manufacturing Workshop 3

AAV mediated gene therapy

Vector Design

IP, capsid, promoter

Manufacture

Scaleup


Challenges of AAV gene therapies

Disease vg/patientEstimated patient number (EU/US) Potential uptake

Estimated Equivalent culture volume (L)

DMD 1.00E+15 1000000 50% 250,000,000

SMA1 6.00E+14 2000 50% 6,000,000

Haemophilia A 4.20E+15 25000 25% 28,000,000

Haemophilia B 7.00E+14 100000 25% 7,000,000

Wet AMD 1.00E+11 1000000 10% 10,000

Chloroidermia 6.00E+09 25000 50% 750

Vector Design

IP, capsid, promoter

Manufacture

Scaleup

Standardization

International standards

QC (IPC, Product/batch release, process validation)

Regulatory


Challenges of AAV gene therapies

Physical size

120nm Lentivirus

90nm Adenovirus

25nm AAV

10nm Antibody

AAV mediated next generation quality control at CGT

7

AAV projects at CGT

2018 201920172012 2016

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


8

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

Purity ddPCR / Seq


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

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

9

Traditional methods

Quantity – vector genome titre

A T C C r e f .

1 09

1 01 0

1 01 1

1 01 2

Co

pie

s/m

L


10

qPCR alternatives - ddPCRq

PC

R

dd

PC

R


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

11

Scope

qPCR vs ddPCR


12

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


13

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


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

2. Novel 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

14

Fully functional assay offering

Summary


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

15

ELISA

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

qPCR ddPCR

% full particles 13.69% 6.72%


16

Dynamic Light Scattering technology


Case study

18

Overview of downstream developmental scope

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 Finish

Cell disruptionViral vector release development and optimisation

Physical lysis

Freeze/thaw lysis

Chemical lysis


19

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

AKTA™ AvantNuclease treatment

TFF KrosFloUF/DF

Clarification

Final filtration

Cell disruption


20

Sample purity - MADLS

4.67E+11

1.61E+20

AKTA™ AvantNuclease treatment

TFF KrosFlo UF/DFClarification

Final filtration

Cell disruption


21

Gene therapy – Overcoming challenges



Overcoming AAV therapy challenges

Disease vg/patientEstimated patient number (EU/US) Potential uptake

Estimated Equivalent culture volume (L)

DMD 1.00E+15 1000000 50% 250,000,000

SMA1 6.00E+14 2000 50% 6,000,000

Haemophilia A 4.20E+15 25000 25% 28,000,000

Haemophilia B 7.00E+14 100000 25% 7,000,000

Wet AMD 1.00E+11 1000000 10% 10,000

Chloroidermia 6.00E+09 25000 50% 750


Continuous processing - consortium


Acknowledgements

• Julie Kerby

• Damian Marshall

• Mike Delahaye

• Gregory Berger

• Nicole Nicolas

• Anusha Seneviratne

• Nishanthi Weeratunge

• Florian Leseigneur

• Quentin Bazot

• Elena Sokolskaja

• Hadi Mirmalek-Sani

Cell and Gene Therapy Catapult is a trading name of Cell Therapy Catapult Limited, registered in England and Wales under company number 07964711, with registered office at 12th Floor Tower Wing, Guy’s Hospital, Great Maze Pond, London, SE1 9RT. VAT number 154 4214 33.

12th Floor Tower WingGuy’s Hospital

Great Maze PondLondon SE1 9RT

[email protected]

Twitter: @CGTCatapultCell and Gene Therapy Catapult is committed to ensuring high standards of research integrity and research best practice in the activities we carry out. We subscribe to the principles described in the UK concordat to support research integrity.


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