1. Efficient and Reliable Production of Pharmaceuticals in Alfalfa (Marc-André D’Aoust, Patrice Lerouge, Ursula Busse, Pierre Bilodeau, Sonia Trépanier,Véronique Gomord, Loïc Faye and Louis-Philippe Vézina).

1.1 Introduction.

1.2 Alfalfa-specific Expression Cassettes.

1.3 Alfalfa Transformation Methods.

1.4 Characteristics of Alfalfa-derived Pharmaceuticals.

1.5 Industrial Production of Recombinant Proteins in Alfalfa.

1.5.1 Ramping Up Alfalfa Biomas.

1.5.2 Alfalfa Harvest, and Recovery of Recombinant Molecules.

1.6 Conclusions.

References.

2. Foreign Protein Expression Using Plant Cell Suspension and Hairy Root Cultures (Fiona S. Shadwick and Pauline M. Doran).

2.1 Foreign Protein Production Systems.

2.2 Production of Foreign Proteins Using Plant Tissue Culture.

2.2.1 Suspended Cell Cultures.

2.2.2 Hairy Root Cultures.

2.2.3 Shooty Teratoma Cultures.

2.2.4 Scale-up Considerations for Different Forms of Plant Tissue Culture.

2.3 Strategies for Improving Foreign Protein Accumulation and Product Recovery in Plant Tissue Culture.

2.3.1 Expression Systems.

2.3.1.1 Modifications to Existing Expression Constructs.

2.3.1.2 Transient Expression Using Viral Vectors.

2.3.2 Secretion of Foreign Proteins.

2.3.3 Foreign Protein Stability.

2.3.3.1 Stability Inside the Cells.

2.3.3.2 Stability Outside the Cells.

2.3.3.3 Medium Additives.

2.3.3.4 Medium Properties.

2.3.4 Bioprocess Developments.

2.3.4.1 Product Recovery from the Medium.

2.3.4.2 Oxygen Transfer and Dissolved Oxygen Concentration.

2.4 Conclusions.

References.

3. Novel Sprouting Technology for Recombinant Protein Production (Kimmo Koivu).

3.1 Introduction.

3.2 Biology of Sprouting.

3.2.1 Structure and Content of Dicotyledonous and Monocotyledonous Seeds.

3.2.2 Germination.

3.2.3 The Sprout.

3.2.4 Rubisco Synthesis.

3.2.5 Rubisco Promoters.

3.2.6 Inhibition of Endogenous Gene Expression.

3.3 Expression Cassette Design.

3.4 Sprouting Equipment.

3.5 Sprouting Conditions.

3.5.1 Sterilization.

3.5.2 Sprouting Time and Temperature.

3.5.3 Light.

3.5.4 Inhibition of Endogenous Gene Expression.

3.5.5 Growth Regulators.

3.5.6 Nitrogen Fertilizer.

3.5.7 Seed Production.

3.6 Yield Estimates and Benefits of Sprouting Technology in Protein Production.

3.6.1 Yield Estimates.

3.6.2 Quality and Environmental Aspects.

References.

4. Monocot Expression Systems for Molecular Farming (Paul Christou, Eva Stoger and Richard M. Twyman).

4.1 Introduction.

4.2 Cereal Production Crops.

4.3 Technical Aspects of Molecular Farming in Cereals.

4.3.1 Cereal Transformation.

4.3.2 Expression Construct Design.

4.3.3 Production Considerations for Cereals.

4.4 Examples of Recombinant Proteins Produced in Cereals.

4.4.1 ProdiGene and Maize.

4.4.2 Recombinant Proteins Expressed in Rice.

4.4.3 Recombinant Proteins Produced in Wheat.

4.4.4 Recombinant Proteins Produced in Barley.

4.5 Conclusions.

References.

5. The Field Evaluation of Transgenic Crops Engineered to Produce Recombinant Proteins (Jim Brandle).

5.1 Introduction.

5.2 Regulation of Field-testing.

5.3 Design of Field Trials.

5.4 Results of Field Trials.

References.

6. Plant Viral Expression Vectors: History and New Developments (Vidadi Yusibov and Shailaja Rabindran).

6.1 Introduction.

6.2 Plant RNA Viruses as Expression Vectors.

6.2.1 Tobacco mosaic virus (TMV).

6.2.2 Potato virus X (PVX).

6.2.3 Cowpea mosaic virus (CPMV).

6.2.4 Alfalfa mosaic virus (AlMV).

6.3 Biological Activity of Target Molecules.

6.4 Efficacy of Plant Virus-produced Antigens.

6.4.1 Vaccine Antigens.

6.4.2 Particle-based Vaccine Antigen Delivery.

6.4.3 Other Uses of Plant Virus Particles.

6.5 Plant Viruses as Gene Function Discovery Tools.

6.6 New Approaches to the Development of Viral Vectors.

6.7 Conclusion.

References.

7. Production of Pharmaceutical Proteins in Plants and Plant Cell Suspension Cultures (Andreas Schiermeyer, Simone Dorfmüller and Helga Schinkel).

7.1 Introduction.

7.2 Plant Species Used for Molecular Farming.

7.3 Cell Culture as an Alternative Expression System to Whole Plants.

7.4 From Gene to Functional Protein: Processing Steps in Plants.

7.5 Case Studies of Improved Protein Yields.

7.6 Downstream Processing.

7.7 Market Potential of Plant-derived Pharmaceuticals.

7.8 Containment Strategies for Molecular Farming.

7.9 Concluding Remarks.

References.

8. Chloroplast Derived Antibodies, Biopharmaceuticals and Edible Vaccines (Henry Daniell, Olga Carmona-Sanchez and Brittany E. Burns).

8.1 Introduction.

8.2 Expression of Therapeutic and Human Proteins in Plants.

8.3 The Transgenic Chloroplast System.

8.3.1 Chloroplast-derived Human Antibodies.

8.3.2 Chloroplast-derived Biopharmaceuticals.

8.3.2.1 Human Serum Albumin.

8.3.2.2 Human Insulin-like Growth Factor-1.

8.3.2.3 Human Interferon (IFNα2b).

8.3.2.4 Anti-Microbial Peptides (AMPs): MSI-99.

8.3.3 Chloroplast-derived Vaccine Antigens.

8.3.3.1 Cholera Toxin B Subunit (CTB).

8.3.3.2 Bacillus anthracis Protective Antigen.

8.3.3.3 Yersinia pestis F1~V Fusion Antigen.

8.3.3.4 Canine Parvovirus (CPV) VP2 Protein.

8.4 Advances in Purification Strategies for Biopharmaceuticals.

8.5 Conclusion.

Acknowledgements.

References.

9. Plant-derived vaccines: progress and constraints (Guruatma Khalsa, Hugh S. Mason, Charles J. Arntzen).

9.1 Introduction.

9.2 Strategies for Vaccine Production in Plants.

9.3 The Biomanufacture of Vaccines.

9.3.1 Advantages of Plants.

9.3.2 Oral Delivery and Mucosal Immune Responses.

9.3.4 Examples of Antigens Produced in Plants.

9.3.5 Targeting Antigens to Specific Tissues.

9.3.6 Expression Systems.

9.3.7 Mucosally-targeted Fusion Proteins.

9.3.8 Forming Multivalent and Multicomponent Vaccines.

9.3.9 Stability and Processing.

9.4 Clinical Trials with Plant-derived Vaccines.

9.4.1 Enterotoxic E. coli and Vibrio cholerae.

9.4.2 Norwalk Virus.

9.4.3 Hepatitis B Virus.

9.4.4 Rabies Virus.

9.5 Issues and Challenges.

9.5.1 Development and Licensing of Plant-derived Vaccines.

9.5.2 Confronting GM Food Issues.

References.

10. Production of Secretory IgA in Transgenic Plants (Daniel Chargelegue, Pascal M.W. Drake, Patricia Obregon and Julian K.-C.Ma).

10.1 Introduction.

10.2 Antibodies.

10.2.1 Mucosal Antibodies.

10.2.2 Structure and ‘Natural’ Production of SIgA.

10.2.3 Passive Immunization with SIgA.

10.2.4 Production of Recombinant SIgA.

10.3 Production of Recombinant SIgA in Plants.

10.3.1 Production of Full-length Antibodies in Plants.

10.3.2 Production of Multimeric Antibodies: SIgA.

10.3.3 Glycosylation of Antibodies in Transgenic Plants.

10.3.4 Plant Hosts.

10.4 Conclusions.

References.

11. Production of Spider Silk Proteins in Transgenic Tobacco and Potato (Jürgen Scheller and Udo Conrad).

11.1 Introduction.

11.1.1 Structure and Properties of Spider Silk.

11.1.2 Strategies for the Production of Recombinant Spider Silk Proteins.

11.1.3 Applications of Spider Silk Proteins.

11.1.3.1 Synthetic Spider Silk Fibers: ‘Natural’ vs Artificial Spinning Strategies.

11.1.3.2 Synthetic Spider Silk Proteins for the In Vitro Proliferation of Anchoragedependent Cells.

11.1.4 Molecular Farming: Plants as Biofactories for the Production of Recombinant Proteins.

11.2 Spider Silk and Spider Silk-ELP Fusion Proteins from Plants: Expression, Purification and Applications.

11.2.1 Spider Silk-ELP Expression in Transgenic Tobacco and Potato.

11.2.2 Purification of Spider Silk-Elastin Fusion Proteins by Heat Treatment and Inverse Transition Cycling.

11.2.3 Applications of Spider Silk-ELP Fusion Proteins in Mammalian Cell Culture.

11.3 Discussion.

References.

12. Gene Farming in Pea Under Field Conditions (Martin Giersberg, Isolde Saalbach and Helmut Bäumlein).

12.1 Introduction.

12.2 Procedures for Foreign Protein Expression in Transgenic Pea Seeds.

12.2.1 Plant Material, Transformation and Field Growth.

12.2.2 Transformation Vectors and Analysis of Transgenic Plants.

12.3 Expression of α-Amylase in Transgenic Pea Seeds.

12.4 Conclusions.

12.5 Acknowledgements.

References.

13. Host Plants, Systems and Expression Strategies for Molecular Farming (Richard M. Twyman).

13.1 Introduction.

13.2 Host Species for Molecular Farming.

13.2.1 Leafy Crops.

13.2.1.1 Tobacco (Nicotiana tabacum).

13.2.1.2 Tobacco (Nicotiana benthamiana).

13.2.1.3 Alfalfa (Medicago sativa).

13.2.1.4 White clover (Trifolium repens).

13.2.1.5 Lettuce (Lactuca sativa).

13.2.1.6 Spinach (Spinacia oleracea).

13.2.1.7 Lupin (Lupinus spp.).

13.2.2 Dry Seed Crops.

13.2.3 Fruit and vegetable crops.

13.2.4 Oilcrops.

13.2.5 Unicellular Plants and Aquatic Plants Maintained in Bioreactors.

13.2.6 Non-cultivated Model Plants.

13.3 Expression systems for molecular farming.

13.3.1 Transgenic plants.

13.3.2 Transplastomic plants.

13.3.3 Virus-infected plants.

13.3.4 Transiently transformed leaves.

13.3.5 Hydroponic cultures.

13.3.6 Hairy roots.

13.3.7 Shooty teratomas.

13.3.8 Suspension cell cultures.

13.4 Expression strategies and protein yields.

13.5 Conclusions.

References.

14. Downstream Processing of Plant-derived Recombinant Therapeutic Proteins (Juergen Drossard).

14.1 Introduction.

14.2 Similarities and Differences in the Processing of Pharmaceutical Proteins from Different Sources.

14.3 Process Scale.

14.4 The Individual Steps of a Downstream Process.

14.4.1 Initial Processing and Extraction.

14.4.2 Chromatographic Purification.

14.5 Regulatory Requirements for Downstream Processing of Plant-derived Pharmaceutical Products.

References.

15. Glycosylation of Plant-made Pharmaceuticals (Véronique Gomord, Anne-Catherine Fitchette, Patrice Lerouge and Loïc Faye).

15.1 Introduction.

15.2 Plant Cells can Reproduce the Complexity of Mammalian Proteins.

15.3 Plant-made Pharmaceuticals and their Native Mammalian Counterparts Contain Structurally-distinct N-linked Glycans.

15.4 Plant-made Pharmaceuticals Possess Immunogenic N-glycans.

15.5 Current Strategies to Eliminate Immunogenic N-glycans from Plant-made Pharmaceuticals.

15.6 Towards Humanized N-glycans on PMPs Through the Expression of Mammalian Glycosyltransferases in the Plant Golgi Apparatus.

15.7 Concluding Remarks.

15.8 Acknowledgements.

References.

16. Biosafety Aspects of Molecular Farming in Plants (Ulrich Commandeur and Richard M. Twyman).

16.1 Introduction.

16.2 Transgene Spread.

16.2.1 Classes of Foreign DNA Sequences in Transgenic Plants.

16.2.2 Mechanisms of Transgene Pollution –Vertical Gene Transfer.

16.2.3 Mechanisms of Transgene Pollution – Horizontal Gene Transfer.

16.3 Combating the Vertical Spread of Transgenes.

16.3.1 Choosing an Appropriate Host.

16.3.2 Using Only Essential Genetic Information.

16.3.3 Elimination of Markers After Transformation.

16.3.4 Containment of Essential Transgenes.

16.4 Unintended Exposure to Recombinant Proteins.

16.4.1 Environmental Risks of Unintended Exposure.

16.4.2 Addressing the Risks of Unintended Exposure.

16.4.2.1 Controlling Transgene Expression.

16.4.2.2 Controlling Protein Accumulation and Activity.

16.4.2.3 Contamination of the Food Chain During Processing.

16.5 Conclusions.

References.

17. A Top-down View of Molecular Farming from the Pharmaceutical Industry: Requirements and Expectations (Friedrich Bischoff).

17.1 Introduction.

17.2 Industrial Production: The Current Situation.

17.3 Expectations.

17.4 Requirements.

17.4.1 Equivalence of the Recombinant Product to the Original Protein.

17.4.2 Processing in the Endoplasmic Reticulum (ER).

17.4.3 Glycosylation in the Golgi.

17.4.4 Differential Glycosylation – Implications on Immunogenicity of vaccines.

17.4.5 Glycosylation and Stability.

17.4.6 Equivalence of Enzymes.

17.4.7 Degradation.

17.4.8 Efficacy in Clinical Trials.

17.4.9 The Optimal Production System.

17.4.10 Post-harvest expression.

17.4.11 Purification.

17.5 Conclusions.

References.

18. The Role of Science and Discourse in the Application of the Precautionary Approach (Klaus Ammann).

18.1 Introduction.

18.2 Other Roots to Problems with the Precautionary Approach.

18.2.1 The Roots of the Precautionary Approach and Environmental Debate.

18.2.2 Discussion About the PA is Too Closely Related to Factual Knowledge Alone.

18.3 The First and Second Generation Systems Approaches.

18.3.1 First Generation Systems Approach.

18.3.2 Second Generation Systems Approach.

18.4 How to Solve Wicked Problems in Biotechnology and the Environment.

18.5 How to Achieve Such Demanding Planning Goals.

18.6 There is no Scientific Planning.

18.7 Outlook.

Bibliography.

Subject Index.