Guido Krupp & the AmpTec team AmpTec GmbH Hamburg, Germany

1. Synthetic mRNAs as optimised tools for stem cell generation and for manipulating cellular phenotypes Guido Krupp & the AmpTec team AmpTec GmbH Hamburg, Germany

2. An independent Start-up company Founded in 2005 with 3 employees 2016: 14 employees 2 of 21

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4. Many Sample Types From picograms to micrograms Tissues/Cells/Body Fluids Degraded RNA – FFPE RNA – Bacteria RNAready isolation kits & ExpressArt mRNA Amplification kits Microarray Platforms & qPCR & NGS 4 of 21

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6. AmpTec High Quality Provider of IVT-RNAs since 1998 ISO 9001 in 2008 ISO 13485 since 2010 Compliance with cGMP (FDA 21 CFR Part 820) since 2012 References: [1] Guido Krupp et al. (2003) Nucleic acid preparations of pathogens from biological samples for real-time PCR analysis. In: Peter Dürre & Botho Bowien (eds) Nucleic Acids Isolation Methods. American Scientific Publishers, Stevensom Ranch, USA, 2003, pp.95-135. [2] Qiagen Diagnostics: Real-time artus RT-PCR & Real-time artus PCR Kit Handbooks. Syn-mRNA [3] Quabius & Krupp (2015) Synthetic mRNAs for manipulating cellular phenotypes: an overview. New Biotechnol. 32: 229-235. 6 of 21

7. Customers for IVD RNAs and DNAs Qiagen Germany – UK -USA Insight Genetics USA Novartis Germany - USA Sysmex-InosticsGermany – USA - Italy Altona Diagnostics Germany InstitutfürHämatopathologie GmbH Germany Bundeswehr Germany PEI Paul-Ehrlich-InstitutGermany Exosome Diagnostics Germany - USA University & Research Institutions Germany & WW Syn-mRNA Pharma Companies in Europe, USA, Canada Research Institutions in Europe, USA, Australia, Singapore, China 7 of 21

8. Elgar Susanne Quabius and Guido Krupp 8 of 21

9. C THE Synthetic mRNA world Applications of Synthetic mRNA mRNA mediated expression of dysfunctional protein 9 of 21

10. standard nucleotides (A) VVV n THE Synthetic mRNA world Required Features of Synthetic mRNA Immunogenic Synthetic mRNA A1 A2 A2 Non-Immunogenic Synthetic mRNA B1+C1 B2+C2 10 of 21

11. THE Synthetic mRNA world TECHNOLOGY ADVANTAGES * Unlike DNA-based Gene Therapy mRNA acts as cellular software, transient activity like a drug * Therapeutic mRNA can address myriad of serious diseases  * New drug modalities * Directing ribosomes to express protein therapeutics within targeted tissues * mRNA encodes intracellular proteins in compartments unreachable by injected biologics * Proven success Multiple clinical trials in cancer immunotherapy Repair and regeneration of tissue, retina Replacement of non-functional proteins, fatal lung disease 11 of 21

12. THE Synthetic mRNA world FINANCE CureVac, Germany, founded in 2000 Investors (>$500M) & Gates Foundation ($52M) BioNTech, Germany, founded in 2008 Investors (>$500M) Moderna, USA, founded in 2011 (>$950M) & DARPA ($25M) Pharma Investments: Alexion, AstraZeneca, Boehringer Ingelheim, Johnson&Johnson, Merck, Sanofi-Aventis, Shireand many more! 12 of 21

13. Synthetic mRNAs at AmpTec • Intelligent design at initial stageKnow-how about implications of 3’- and 5’-end structures, codon usage adaptations,nucleoside modifications is put into mRNA product design and manufacture • Regulatory-friendly manufacturing processHigh throughput approach from DNA template to mRNA product • Scalable and reproducible • Stringent quality control for cell therapy applications 13 of 21

14. Conventional Standard Process for Synthetic mRNA manufacturing Linearised plasmid as Template for IVT A120-GAAGAGC Potential Problems Hompolymeric A-120 is unstable/poor reproducibility Internal Sap I site not acceptable Incomplete cleavage is difficult to trace/poor reproducibility long, poorly detectable trailing Plasmid linearised with Sap I A120 3‘-A120 14 of 21

15. AmpTec Process for Synthetic mRNA manufacturing Insert-Spanning PCR Product as Template for IVT Heel-Primer 5‘-T120 Advantages Hompolymeric A-120 is well defined by synthetic primer Internal restriction sites are no problem PCR product is well defined / good reproducibility INSERT-PCR M13-fwd / 3‘-gene-T120 Option: 5‘-Biotin-M13-fwd 3‘-A120 3‘-A120 15 of 21

16. Example with MS analysis of a 136-mer Heel-Primer 16 of 21

17. Improved Procedures for IVT and for PCR Example 2 Example 1 Conventional Conventional Conventional New IVT New IVT Example 3 New PCR + New IVT 17 of 21

18. Schematic Work-Flow for Synthetic mRNAs The Full Service includes: Synthesis of DNA, plasmid cloning, purification, complete bidirectional DNA sequencing documentation, PCR amplification of insert, purification and characterisation of PCR product, with subseque nt RNA synthesis by in vitro transcription, purification and characterisation. The customer provides text files with the desired sequence data. Production steps: 1. Chemical synthesis of dsDNA fragments 2. Cloning of the DNA fragments in a plasmid 3. Quality con trol by sequencing of plasmid inserts 4. Synthesis of PCR products: 5’ - primer is based on the plasmid sequence, 3‘-Heel primer combines a 5’-terminal stretch of T120 with a 3‘-terminal gene-specific sequence 5. PC R amplification of the cloned insert with the primers described in step 4 6. Purification of the PCR products with spin columns 7. Quality control of PCR products by capillary electrophoresis (Agilent Bioanalyzer) 8. PC R templates for production of m RNA by i n vitro transcription with T7 RNA polymerase , including a cap analogue (ARCA), Option: with modified NTP’s, like m5CTP and Pseudo-UTP 9. DNase treatment for removal of the DNA template 10. Purification of m RNA with spin columns Option: 11. Phosphatase treatment to remove 5’-triphosphates 12. Quality control of the in vitro transcripts by capillary electrophoresis (Agilent Bioanalyzer) Test IVT red: unmodified RNA blue: with 5‘-ARCA,100% m5C, 100% Ψ mRNA with 5‘-ARCA, 100% m5C, 100% Ψ, 3‘-A120 18 of 21

19. Overview workflow IVT production Gene Synthesis Primer Dilution IPC-Plasmid OOS Plasmid Dilution positive Insert PCR Insert PCR Cleanup IPC-Insert PCR OOS positive IVT IVT Cleanup Quantification IVT IVT Dilution Quality Control IVT OOS positive Reference Sample IVT IVT Release Packaging Version 1.0 March 2012 19 of 21

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21. Thank you! www.amp-tec.comkrupp@amp-tec.com