THE ANALYTICAL TOOLBOX FOR VIRAL VECTOR CHARACTERISATION: PROGRESS AND CHALLENGES

Transcription

1 AMC Technical Meeting 4 th October 2018 THE ANALYTICAL TOOLBOX FOR VIRAL VECTOR CHARACTERISATION: PROGRESS AND CHALLENGES Dr Helen Young and Dr Natasha Lethbridge Upstream Scientist and Project Manager

2

3 Research and Invention Innovation Public / Private Collaboration Commercial Market Innovation Ready Investment Ready

4 FUNDING ACCESS TO FINANCE Our expert bid team can support you in scoping projects, finding partners, building consortia, developing project concepts and writing compelling proposals to bring you the right funding at the right time. Regional National International Private

5 CPI BIOLOGICS COLLABORATIVE PROJECT EXAMPLES IUK Medicines Manufacturing IB Catalyst AMSCI H2020 Cell free expression of ddna Combinatorial genome editing to create enhanced biomanufacturing platforms Harnessing UK innovation to streamline the biologics supply chain from molecule to medicine Continuous template assisted membrane crystallisation of MAbs Ipsen Biopharm, Touchlight Genetics Horizon Discovery, University of Manchester UCB, Lonza, Sphere Fluidics, Horizon Discovery, Alcyomics 7 Academic Institutes H2020 IB Catalyst Regen Med IB Catalyst Development of Sustainable Manufacturing Process Platforms for Solid Core NPs Improving therapeutic window of glycosylated drugs and developing a novel, HT analytical methodology Development of an industrial manufacturing platform for AAV production UK Continuous, integrated biologics manufacturing project Midatech, Prochimia, Galchimia, Applus, EPFL, UCD (CBNI), IFOM Glythera, GlycoSeLect Cobra Biologics Pall UK, GSK, Medimmune, Allergan, Siemens

6 Viral vector product characterisation and challenges Particle titre Genomic titre Infectious titre Infectious titre Transgene expression Protein activity titre identity and purity potency safety Serotype confirmation Empty particles Process impurities Genomic impurities Aggregation Sterility Mycoplasma Genotoxicity

7 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 Credit: Use of next-generation sequencing (NGS) enables the detection of all DNA in within a sample allowing identification of all contaminating DNA sequences

8 Workflow Started with 20 x AAV capsids and 100ng of λ DNA Start of AAV workflow (paav-lacz) DNAse treatment of intact capsid Viral DNA extraction 2 nd strand synthesis Start of λ bacteriophage DNA workflow (J02459, dsdna) Production of library (including shearing) Templating on Ion Chef Sequencing (FASTQ output) Monday, 08 October 2018

9 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

10 Mapped reads against each reference (%) Analysis of sequence 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 raav-lacz λ DNA Mapped reads against each reference (%) raav-lacz λ DNA paav-lacz genome paav-lacz backbone Helper Repcap E. coli (U ) λ DNA (J02459) Human gdna (GRCh38) Figure 1: Relative alignments against references with the different input samples Monday, 08 October 2018

11 X Y Mitochondrial Mapped reads against reference (%) Mapped reads against human DNA Less than 0.15% of mapped reads in raav-lacz DNA mapped against the human genome raav-lacz λ DNA Of these reads, chromosomes 1 and 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 Human chromosome (GRCh38) Monday, 08 October 2018

12 Conclusions 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 Monday, 08 October 2018

13 Acknowledgements Sam Stephen Philip Probert Jade Tuck Jonathan Welsh Jayan Senaratne Bethany Kerr Samantha Ward Grace Last Helen Young Natasha Lethbridge Funding from Daniel Smith Kevin Bowes Robert Seymour Vera Lukashchuk George Prout Monday, 08 October 2018

14