Fraunhofer Institute for Cell Therapy and Immunology IZI
Fraunhofer Institute for Cell Therapy and Immunology IZI

Automated production technologies for mRNA-based drugs

mRNA research services

Doctor hand holds syringe and vaccine
© Ekaterina_1525 - stock.adobe.com / Fraunhofer IZI / Alernon77 - stock.adobe.com / CoreDESIGN - stock.adobe.com / Fraunhofer IZI

Design of DNA templates

  • mRNA construct design and optimization at the DNA level:
    • Codon optimization
    • Flanking regulatory elements (5‘/3‘ UTRs)
    • Encoded poly(A) tail

Plasmid synthesis and linearization

  • Cloning strategies:
    • Restriction / ligation
    • Gibson assembly
    • Golden gate cloning
  • Endotoxin-free plasmid isolation from E. coli
  • Enzymatic linearization of plasmid DNA
  • Optional: PCR to generate linear template DNA

Co- and posttranscriptional modifications

  • Co-transcriptional capping
  • Incorporation of chemically modified nucleotides
  • Optional: Post-transcriptional addition of poly(A) tail
  • Phosphatase treatment

In vitro RNA production

  • In vitro transcription for mRNA generation:
    • Kit-based
    • Single component based
    • Use of optimized polymerases
    • Customized protocols applicable
    • Screening of different conditions in μL to mL scale possible

Purification

  • mRNA purification for μL scales:
    • Spin-column purification
    • Oligo(dT) bead purification
  • mRNA purification for mL scales:
    • Column chromatographic purification methods

Quality control

  • Concentration (UV / Vis)
  • Integrity (gel electrophoresis, bioanalyzer)
  • Identity (sequencing)
  • Purity:
    • ELISA / DotBlot for dsRNA
    • ELISA for enzyme residues
  • In vitro translation
  • Expression test in various cell types

Relevant publications

  • Kitte R, Rabel M, Geczy R, Park S, Fricke S, Koehl U, Tretbar US. Lipid Nanoparticles outperform Electro­poration in mRNA-based CAR T cell Engineering. Mol Ther Methods Clin Dev. 2023 Oct 18:31:101139. doi: 10.1016/j.omtm.2023.101139. eCollection 2023 Dec 14.
  • von Auw N, Serfling R, Kitte R, Hilger N, Zhang C, Gebhardt C, Duenkel A, Franz P, Koehl U, Fricke S, Tretbar US. Comparison of two lab-scale protocols for enhanced mRNA-based CAR-T cell generation and functionality. Sci Rep. 2023 Oct 24;13(1):18160. doi: 10.1038/s41598-023-45197-x
  • Lu D, Chatterjee S, Xiao K, Riedel I, Huang CK, Costa A, Cushman S, Neufeldt D, Rode L, Schmidt A, Juchem M, Leonardy J, Büchler G, Blume J, Gern OL, Kalinke U, Wen Tan WL, Foo R, Vink A, van Laake LW, van der Meer P, Bär C, Thum T. A circular RNA derived from the insulin receptor locus protects against doxorubicin-induced cardio­toxicity. Eur Heart J. 2022 Nov 7;43(42):4496-4511. doi: 10.1093/eurheartj/ehac337