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Plants as Recombinant Protein Expression Vehicles

Plants as Recombinant Protein Expression Vehicles. Inga Hitzeroth. Molecular Farming. Requirements and Expectations for Molecular Farming Transgenic plants, Plant Viral Vectors, Transient infiltration Transfer of DNA from Agrobacterium tumefaciens into plants, Gene silencing, Glycosylation

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Plants as Recombinant Protein Expression Vehicles

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  1. Plants as Recombinant Protein Expression Vehicles Inga Hitzeroth

  2. Molecular Farming • Requirements and Expectations for Molecular Farming • Transgenic plants, Plant Viral Vectors, Transient infiltration • Transfer of DNA from Agrobacteriumtumefaciens into plants, Gene silencing, Glycosylation • Biopharming of: Antibodies Vaccines

  3. Requirements and Expectations for Molecular Farming • Friedrich Bischoff • Chapter 17 of Molecular Farming • Additional Reading • Approaches to achieve high-level heterologous protein production in plants • S.J. Streatfield • Plant Biotech Journal (2007) 5, 2-15

  4. Other heterologous production systems • Bacteria – non-glycosylated proteins: insulin, interleukin, G-CSF • Fungi – Hepatitis B vaccine • Insect cells – HPV vaccine • Mammalian cells – CHO and murine myeloma lines • Production Platforms

  5. Recombinant Proteins • First recombinant product – Insulin 1982 • Monoclonal Ab • Annual market for biopharmaceuticals ~ 30 billion US$ • 500 in clinical evaluation • 40 will reach market

  6. Plant Made Pharmaceuticals (PMPs) Plant made pharmaceuticals are medically-relevant proteins produced in plant cells or whole plantsvia the expression of recombinant DNA

  7. Host Plants for Molecular Farming: Acceptance Favourable Acceptable Unfavourable Maize Potato Rapeseed Rice Soybean Sunflower Tomato Sugarcane Alfalfa Wheat Tobacco Cotton Sugar beet Lettuce

  8. Host Systems for the Manufacturing of PMPs Yield Scale Stability Biosafety Leafy +++ +++ + ++/+ Seeds ++ ++ +++ ++ Fruit/veg + ++ ++ ++ Non-food + ++ ++ + Lemna ++ + ++ +++ Cells +/++ + ++ +++ Mushroom ++ ++ ? ++

  9. Why Plants • New production platform • Shorten time • Cost of production - low • Eukaryotic post-translational modifications – glycosylation • Safety - no mammalian contaminants (serum) – Bacteria, viruses, prions (BSE), endotoxins

  10. Plant transformation • Viral vectors • Use recombinant virus (TMV) • Spreads to all cells • Few weeks • Transgenic plants • Takes 6-12 month to get first results • Transient expression of proteins • Use Agrobacterium tumefaciens • T-DNA vector with GOI • Transferred to host cell • Takes 2 weeks

  11. Recombinant Protein Production in Plant Cell Culture • Advantages: • containment • less space required • better growth parameter control • homogeneity and sterility • complies with cGLP and cGMP standards • improved purification procedure

  12. Protein Production in Plant Cells Frozen master and working cell banks Transgenic plant Product Product formulation, Filling, QA, QC, Regulatory Establish cell culture Archive/ Recover Recover culture supernatant Chromatography Purification (DSP) Select stable, high-performance cell lines Scale up Filter, clarification Co-culture with Agrobacterium tumefaciens, or transform by particle b. Filter, clarification Homogenize cells Centrifuge and filter Wastemanagement Homogenate without cell debris BY2 cell culture Crude homogenate

  13. Recombinant Protein Production in Plant Cell Culture 100L fermentation of an anti-HIV mAb in transgenic BY-2 cells

  14. Cost of Production • Manufacturing facility ~ 150 – 400 million $ • 4 years to build • Equipment can only be used for one product • Normally only produce one molecule • Limited capacity • Generics – patents run out, • Patent on production methods

  15. Production costs

  16. Upscaling • Transgenic seeds • Can plant as necessary • Proteins expressed in seed - store • Production on demand • Low initial capital investment • Greenhouse as opposed to fermentation plant • Big decisions when product reaches Phase III of development • With plants can easily double production

  17. Dispersion generators for plant protein extraction Benchtop scale for ~ 500 g of plant material Pilot scale for ~ 15 kg of plant material

  18. Upscaling plant processing capacity Fraunhofer Institute Germany • „Integrated Plant Processing System“ • processing of ~ 500kg of biomass • surface cleaning, dispersion, extrac-tion and filtration in a custom designed integrated pipeline

  19. Requirements • Plant produced proteins must be same as the one produced in traditional cell cultures • Biochemically • Pharmacologically • Clinically • SDS-PAGE, protein sequencing, isoelectric focusing, carbohydrate mapping, • Potency assays • Batch to batch consistency

  20. Key Contaminants • host proteins • enzymes (e.g. proteases) • nucleic acids • secondary metabolites (e.g. alkaloids)

  21. Processing of Proteins • ER –folding, disulfide bridges • Golgi – glycosylation Targeting proteins to ER increases yield Add KDEL sequence to C-terminal

  22. Protein stabilization through targeting • apoplast • cytosol • ER • chloroplasts • vacuoles • protein bodies • oilbodies • membranes

  23. Problems Degradation Low expression levels Gene silencing Solutions Addition of inhibitors Target proteins to ER or storage organs Target to seeds, or cell compartments Codon usage Transcriptional and translational enhancers Silencing inhibitors/transgenic plants Improvement of PMP

  24. Gene delivery & integration method • stable integration: • biolistic (particle gun) • agrobacterium tumefaciens • microinjection • transient: • viral vectors • vacuum infiltration • microinjection

  25. Improvement: species/tissue related issues • Benefits Limitations • Leafy crops Large amount of biomass Low protein stability • Seed crops Proteins very stable Lower protein yields, and • Fruit/veg crops Oral application food chain contamination • Oil crops Simple extraction Lower biomass yields • Simple plants Containment, extraction & cell suspension cGMP production, batch Running costs cultures consistency

  26. Improvement of PMPs • biosafety issues, approval by regulatory authorities • protein accumulation & stabilization: • targeting to different compartments • protein complexes • plant species, monocot versus dicot • controlled copy number and gene silencing • promoter selection: • leaf, seed, fruit or organ specific, inducible • transcriptional and translational enhancers, codon usage

  27. Development Pipeline for Molecular Farming Production systems Production technologies Protein purification Quality control Pre/clinical development • Plant species (stable, transient) • increase of yield and stability • leaf, seed, fruit • promoters • transformation technologies • vectors • target proteins • biosafety & public acceptance • - Suspension cultures • - Algae & mosses

  28. Development Pipeline for Molecular Farming Production systems Production technologies Protein purification Quality control Pre/clinical development • Yield improvements (targeting, stability, silencing) • Yield improvement (breeding programes) • Protein modification • Containment (labeling, biological safety) • Post harvest production • Quick scale up (green house, field, fermenter, GAP) • Controlled biomass production (GAP, GMP)

  29. Development Pipeline for Molecular Farming Production systems Production technologies Protein purification Quality control Pre/clinical development • Use existing technologies (food & beverage industries) for processing of leaves, seeds and fruits • Processing economics • Process safety • Avoid plant-inherent problems • cGMP conformity • Formulation • Sustainability, environmental friendliness

  30. Escenario No 2: Producción de 1 kg/año { Molienda 9 procesos, Batidora de 25 L • Escala de purificación de trabajo: Escala No 3( 3g/corrida) • Esquema de trabajo: { - filtraciones por sublotes de molienda usando el filtro de manga Filtración { Congelación/descongelación Dos tanques de 100 L { 1 o mas proceso por el Filtro Prensa Filtración { 1 corrida Proteína A { Concentración 1 proceso de DC30 { G-25 2 proceso G-25 Purification of PMP Tobacco Plant Biomass Grinding Filtration Centrifugation Affinity Chromatography Gel Filtration Sterile Filtration

  31. Production costs

  32. M M Man-7 1581.2 M M-GN-GN 1000 M-M-M 800 M-M M 600 M M-GN-GN SPR signal RU -1 400 M-M-M Man-8 1743.1 200 0 0 100 200 300 400 Man-9 1905.0 time s -1 M-M M M-M M-GN-GN M-M-M Development Pipeline for Molecular Farming Protein purification Quality control Pre/clinical development Production systems Production technologies cGMP - Functionality - Purity - Stability - Identity -

  33. Development Pipeline for Molecular Farming Protein purification Quality control Pre/clinical development Production systems Production technologies QC - Efficacy - Toxicology - Immunogenicity - Pharmacology - Phase I-III -

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