Upstream Scientist and Project Manager

Dr Helen Young and Dr Natasha Lethbridge

THE ANALYTICAL TOOLBOX FOR VIRAL VECTOR CHARACTERISATION: PROGRESS AND CHALLENGES

4th October 2018

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CPI BIOLOGICS COLLABORATIVE PROJECT EXAMPLES

IUK Medicines Manufacturing

Cell free expression of dDNA

Ipsen Biopharm, Touchlight Genetics

IB Catalyst

Combinatorial genome editing to create enhanced

biomanufacturing platforms

Horizon Discovery, University of Manchester

H2020

Development of Sustainable

Manufacturing Process Platforms for Solid Core

NPs

Midatech, Prochimia, Galchimia, Applus, EPFL,

UCD (CBNI), IFOM

IB Catalyst

Improving therapeutic window of glycosylated drugs and developing a

novel, HT analytical methodology

Glythera, GlycoSeLect

Regen Med

Development of an industrial manufacturing

platform for AAV production

Cobra Biologics

AMSCI

Harnessing UK innovation to streamline the biologics

supply chain from molecule to medicine

UCB, Lonza, Sphere Fluidics, Horizon

Discovery, Alcyomics

IB Catalyst

UK Continuous, integrated biologics manufacturing

project

Pall UK, GSK, Medimmune, Allergan,

Siemens

H2020

Continuous template assisted membrane

crystallisation of MAbs

7 Academic Institutes

Viral vector product characterisation and challenges

titreidentity

and purity

potency safety

Particle titre

Genomic titre

Infectious titre

Serotype confirmation

Empty particles

Process impurities

Genomic impurities

Aggregation

Infectious titre

Transgene expression

Protein activity

Sterility

Mycoplasma

Genotoxicity

NGS for characterising misincorporated DNA

• Viral vector encapsidated nucleic acid impurities may arise from any of the

sources of DNA in the production process, including DNA from producer cells

or helper components

• Assessment of misincorporated DNA has traditionally been done through gel

electrophoresis and qPCR methods

– A major limitation with qPCR is the requirement for sequence specific

primers the identity of a contaminant must be known in order to detect

it

– This leads to poor coverage of contaminants leading to underestimation

of contamination

• Use of next-generation sequencing (NGS) enables the detection of all DNA in

within a sample allowing identification of all contaminating DNA sequences

Credit: www.thermofisher.com

Workflow

• Started with 20 x 1012 AAV capsids and 100ng of λ DNA

Monday, 08 October 2018

DNAse treatment of intact capsid

Viral DNA extraction

2nd strand synthesis

Production of library (including

shearing)

Templating on Ion Chef

Sequencing (FASTQ output)

Start of λ

bacteriophage

DNA workflow

(J02459, dsDNA)

Start of AAV

workflow

(pAAV-LacZ)

Analysis of sequence

• Analysis of sequences using ContaVect:

– Reference against:

1. rAAV insert

2. rAAV backbone

3. Helper plasmid

4. RepCap plasmid

5. E. coli genome (MG1655)

6. λ bacteriophage DNA (J02459)

7. hg19 (human genome)

- as surrogate for HEK293 genome

Monday, 08 October 2018

Analysis of sequence

Monday, 08 October 2018

• Full data set has been aligned against all

references

– Whole data represents ~ 50 million reads

• Data indicates that the majority of mapped

reads are against the expected input

sequences

– In the AAV sample, only around 4% of

sequence didn’t map to the insert region

0

10

20

30

40

50

60

70

80

90

100

Mapped r

eads a

gain

st

each r

efe

rence (

%) rAAV-LacZ

λ DNA

Mapped reads against each reference (%)

rAAV-LacZ λ DNA

pAAV-LacZ genome 96.09 0.07

pAAV-LacZ backbone 3.31 0.00

Helper 0.00 0.00

Repcap 0.47 0.00

E. coli (U00096.3) 0.00 10.65

λ DNA (J02459) 0.00 89.28

Human gDNA (GRCh38) 0.13 0.00

Figure 1: Relative alignments against references

with the different input samples

Mapped reads against human DNA

• Less than 0.15% of mapped reads in

rAAV-LacZ DNA mapped against the

human genome

• Of these reads, chromosomes 1 and

17 were most highly represented.

• No reads aligned against the y

chromosome, consistent with the

female provenance of HEK293 cells

• In the Lambda DNA, essentially no

human alignments were detectable

Monday, 08 October 2018

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

1 2 3 4 5 6 7 8 9

10

11

12

13

14

15

16

17

18

19

20

21

22 X Y

Mitocho

ndri

al

Human chromosome (GRCh38)

Mapped r

eads a

gain

st

refe

rence (

%)

rAAV-LacZ

λ DNA

Conclusions

Monday, 08 October 2018

• Method developed to assess misincorporation in rAAV (SSV-Seq)

• Sequencing on Ion Torrent System generated good quality reads and gave significant sequencing depth due to the small size of the

genome being sequenced

• Results demonstrated ~4% misincorporation in rAAV sample, with the majority of contamination coming from backbone of genome

pDNA as expected

• The additional benefit of sequencing was shown by the ability to characterise the genomic region for mutations

• Contaminants could also be fully characterised through further analysis (eg. determining source of human DNA misincorporation

down to base level)

• The use of this approach on manufactured material would enable regulatory hurdles to be met regarding product characterisation and

quality

Acknowledgements

Daniel Smith

Kevin Bowes

Robert Seymour

Vera Lukashchuk

George Prout

Monday, 08 October 2018

Funding from

Sam Stephen

Philip Probert

Jade Tuck

Jonathan Welsh

Jayan Senaratne

Bethany Kerr

Samantha Ward

Grace Last

Helen Young

Natasha Lethbridge

www.uk-cpi.com

http://www.uk-cpi.com/