Molecular farming : plant-made pharmaceuticals and technical proteins

1 Efficient and Reliable Production of Pharmaceuticals in Alfalfa 1 Marc-André D’Aoust, Patrice Lerouge, Ursula Busse, Pierre Bilodeau, Sonia Trépanier,Véronique Gomord, Loïc Faye and Louis-Philippe Vézina 1.1 Introduction 1 1.2 Alfalfa-specific Expression Cassettes 2 1.3 Alfalfa Transformation Methods 3 1.4 Characteristics of Alfalfa-derived Pharmaceuticals 6 1.5 Industrial Production of Recombinant Proteins in Alfalfa 9 1.5.1 Ramping Up Alfalfa Biomass 9 1.5.2 Alfalfa Harvest, and Recovery of Recombinant Molecules 10 1.6 Conclusions 11 References 11 2 Foreign Protein Expression Using Plant Cell Suspension and Hairy Root Cultures 13 Fiona S. Shadwick and Pauline M. Doran 2.1 Foreign Protein Production Systems 13 2.2 Production of Foreign Proteins Using Plant Tissue Culture 14 2.2.1 Suspended Cell Cultures 15 2.2.2 Hairy Root Cultures 20 2.2.3 Shooty Teratoma Cultures 20 2.2.4 Scale-up Considerations for Different Forms of Plant Tissue Culture 21 2.3 Strategies for Improving Foreign Protein Accumulation and Product Recovery in Plant Tissue Culture 22 2.3.1 Expression Systems 22 2.3.1.1 Modifications to Existing Expression Constructs 22 2.3.1.2 Transient Expression Using Viral Vectors 23 2.3.2 Secretion of Foreign Proteins 25 2.3.3 Foreign Protein Stability 26 2.3.3.1 Stability Inside the Cells 26 IX Molecular Farming. Edited by Rainer Fischer, Stefan Schillberg Copyright
2004 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim ISBN: 3-527-30786-9 2.3.3.2 Stability Outside the Cells 26 2.3.3.3 Medium Additives 28 2.3.3.4 Medium Properties 31 2.3.4 Bioprocess Developments 33 2.3.4.1 Product Recovery from the Medium 33 2.3.4.2 Oxygen Transfer and Dissolved Oxygen Concentration 33 2.4 Conclusions 34 References 34 3 Novel Sprouting Technology for Recombinant Protein Production 37 Kimmo Koivu 3.1 Introduction 37 3.2 Biology of Sprouting 38 3.2.1 Structure and Content of Dicotyledonous and Monocotyledonous Seeds 38 3.2.2 Germination 39 3.2.3 The Sprout 40 3.2.4 Rubisco Synthesis 40 3.2.5 Rubisco Promoters 41 3.2.6 Inhibition of Endogenous Gene Expression 42 3.3 Expression Cassette Design 43 3.4 Sprouting Equipment 44 3.5 Sprouting Conditions 45 3.5.1 Sterilization 46 3.5.2 Sprouting Time and Temperature 46 3.5.3 Light 47 3.5.4 Inhibition of Endogenous Gene Expression 47 3.5.5 Growth Regulators 49 3.5.6 Nitrogen Fertilizer 49 3.5.7 Seed Production 49 3.6 Yield Estimates and Benefits of Sprouting Technology in Protein Production 50 3.6.1 Yield Estimates 50 3.6.2 Quality and Environmental Aspects 52 References 53 4 Monocot Expression Systems for Molecular Farming 55 Paul Christou, Eva Stoger and Richard M. Twyman 4.1 Introduction 55 4.2 Cereal Production Crops 56 4.3 Technical Aspects of Molecular Farming in Cereals 57 4.3.1 Cereal Transformation 57 4.3.2 Expression Construct Design 59 4.3.3 Production Considerations for Cereals 61 4.4 Examples of Recombinant Proteins Produced in Cereals 61 X Contents 4.4.1 ProdiGene and Maize 62 4.4.2 Recombinant Proteins Expressed in Rice 63 4.4.3 Recombinant Proteins Produced in Wheat 64 4.4.4 Recombinant Proteins Produced in Barley 64 4.5 Conclusions 64 References 65 5 The Field Evaluation of Transgenic Crops Engineered to Produce Recombinant Proteins 69 Jim Brandle 5.1 Introduction 69 5.2 Regulation of Field-testing 69 5.3 Design of Field Trials 73 5.4 Results of Field Trials 74 References 75 6 Plant Viral Expression Vectors: History and New Developments 77 Vidadi Yusibov and Shailaja Rabindran 6.1 Introduction 77 6.2 Plant RNA Viruses as Expression Vectors 78 6.2.1 Tobacco mosaic virus (TMV) 80 6.2.2 Potato virus X (PVX) 80 6.2.3 Cowpea mosaic virus (CPMV) 81 6.2.4 Alfalfa mosaic virus (AlMV) 81 6.3 Biological Activity of Target Molecules 81 6.4 Efficacy of Plant Virus-produced Antigens 83 6.4.1 Vaccine Antigens 83 6.4.2 Particle-based Vaccine Antigen Delivery 84 6.4.3 Other Uses of Plant Virus Particles 86 6.5 Plant Viruses as Gene Function Discovery Tools 87 6.6 New Approaches to the Development of Viral Vectors 87 6.7 Conclusion 88 References 89 7 Production of Pharmaceutical Proteins in Plants and Plant Cell Suspension Cultures 91 Andreas Schiermeyer, Simone Dorfmüller and Helga Schinkel 7.1 Introduction 91 7.2 Plant Species Used for Molecular Farming 92 7.3 Cell Culture as an Alternative Expression System to Whole Plants 99 7.4 From Gene to Functional Protein: Processing Steps in Plants 102 7.5 Case Studies of Improved Protein Yields 104 7.6 Downstream Processing 105 7.7 Market Potential of Plant-derived Pharmaceuticals 106 7.8 Containment Strategies for Molecular Farming 107 Contents XI 7.9 Concluding Remarks 108 References 109 8 Chloroplast Derived Antibodies, Biopharmaceuticals and Edible Vaccines 113 Henry Daniell, Olga Carmona-Sanchez and Brittany E. Burns 8.1 Introduction 113 8.2 Expression of Therapeutic and Human Proteins in Plants 114 8.3 The Transgenic Chloroplast System 114 8.3.1 Chloroplast-derived Human Antibodies 116 8.3.2 Chloroplast-derived Biopharmaceuticals 118 8.3.2.1 Human Serum Albumin 118 8.3.2.2 Human Insulin-like Growth Factor-1 119 8.3.2.3 Human Interferon (IFN
2b) 119 8.3.2.4 Anti-Microbial Peptides (AMPs): MSI-99 122 8.3.3 Chloroplast-derived Vaccine Antigens 123 8.3.3.1 Cholera Toxin B Subunit (CTB) 123 8.3.3.2 Bacillus anthracis Protective Antigen 124 8.3.3.3 Yersinia pestis F1~V Fusion Antigen 126 8.3.3.4 Canine Parvovirus (CPV) VP2 Protein 127 8.4 Advances in Purification Strategies for Biopharmaceuticals 129 8.5 Conclusion 131 Acknowledgements 131 References 131 9 Plant-derived vaccines: progress and constraints 135 Guruatma Khalsa, Hugh S. Mason, Charles J. Arntzen 9.1 Introduction 135 9.2 Strategies for Vaccine Production in Plants 138 9.3 The Biomanufacture of Vaccines 139 9.3.1 Advantages of Plants 139 9.3.2 Oral Delivery and Mucosal Immune Responses 140 9.3.4 Examples of Antigens Produced in Plants 140 9.3.5 Targeting Antigens to Specific Tissues 140 9.3.6 Expression Systems 141 9.3.7 Mucosally-targeted Fusion Proteins 142 9.3.8 Forming Multivalent and Multicomponent Vaccines 143 9.3.9 Stability and Processing 151 9.4 Clinical Trials with Plant-derived Vaccines 151 9.4.1 Enterotoxic E. coli and Vibrio cholerae 152 9.4.2 Norwalk Virus 152 9.4.3 Hepatitis B Virus 153 9.4.4 Rabies Virus 153 9.5 Issues and Challenges 154 9.5.1 Development and Licensing of Plant-derived Vaccines 154 XII Contents 9.5.2 Confronting GM Food Issues 154 References 155 10 Production of Secretory IgA in Transgenic Plants 159 Daniel Chargelegue, Pascal M.W. Drake, Patricia Obregon and Julian K.-C.Ma 10.1 Introduction 159 10.2 Antibodies 159 10.2.1 Mucosal Antibodies 160 10.2.2 Structure and ‘Natural’ Production of SIgA 160 10.2.3 Passive Immunization with SIgA 162 10.2.4 Production of Recombinant SIgA 162 10.3 Production of Recombinant SIgA in Plants 163 10.3.1 Production of Full-length Antibodies in Plants 163 10.3.2 Production of Multimeric Antibodies: SIgA 165 10.3.3 Glycosylation of Antibodies in Transgenic Plants 166 10.3.4 Plant Hosts 167 10.4 Conclusions 167 References 168 11 Production of Spider Silk Proteins in Transgenic Tobacco and Potato 171 Jürgen Scheller and Udo Conrad 11.1 Introduction 171 11.1.1 Structure and Properties of Spider Silk 171 11.1.2 Strategies for the Production of Recombinant Spider Silk Proteins 173 11.1.3 Applications of Spider Silk Proteins 174 11.1.3.1 Synthetic Spider Silk Fibers: ‘Natural’ vs Artificial Spinning Strategies 174 11.1.3.2 Synthetic Spider Silk Proteins for the In Vitro Proliferation of Anchoragedependent Cells 175 11.1.4 Molecular Farming: Plants as Biofactories for the Production of Recombinant Proteins 175 11.2 Spider Silk and Spider Silk-ELP Fusion Proteins from Plants: Expression, Purification and Applications 176 11.2.1 Spider Silk-ELP Expression in Transgenic Tobacco and Potato 176 11.2.2 Purification of Spider Silk-Elastin Fusion Proteins by Heat Treatment and Inverse Transition Cycling 177 11.2.3 Applications of Spider Silk-ELP Fusion Proteins in Mammalian Cell Culture 178 11.3 Discussion 179 References 180 12 Gene Farming in Pea Under Field Conditions 183 Martin Giersberg, Isolde Saalbach and Helmut Bäumlein 12.1 Introduction 183 12.2 Procedures for Foreign Protein Expression in Transgenic Pea Seeds 184 Contents XIII 12.2.1 Plant Material, Transformation and Field Growth 184 12.2.2 Transformation Vectors and Analysis of Transgenic Plants 185 12.3 Expression of
-Amylase in Transgenic Pea Seeds 185 12.4 Conclusions 188 12.5 Acknowledgements 189 References 190 13 Host Plants, Systems and Expression Strategies for Molecular Farming 191 Richard M. Twyman 13.1 Introduction 191 13.2 Host Species for Molecular Farming 194 13.2.1 Leafy Crops 194 13.2.1.1 Tobacco (Nicotiana tabacum) 194 13.2.1.2 Tobacco (Nicotiana benthamiana) 195 13.2.1.3 Alfalfa (Medicago sativa) 195 13.2.1.4 White clover (Trifolium repens) 195 13.2.1.5 Lettuce (Lactuca sativa) 196 13.2.1.6 Spinach (Spinacia oleracea) 196 13.2.1.7 Lupin (Lupinus spp.) 196 13.2.2 Dry Seed Crops 196 13.2.3 Fruit and vegetable crops 199 13.2.4 Oilcrops 201 13.2.5 Unicellular Plants and Aquatic Plants Maintained in Bioreactors 203 13.2.6 Non-cultivated Model Plants 204 13.3 Expression systems for molecular farming 205 13.3.1 Transgenic plants 206 13.3.2 Transplastomic plants 207 13.3.3 Virus-infected plants 207 13.3.4 Transiently transformed leaves 208 13.3.5 Hydroponic cultures 209 13.3.6 Hairy roots 209 13.3.7 Shooty teratomas 210 13.3.8 Suspension cell cultures 210 13.4 Expression strategies and protein yields 210 13.5 Conclusions 212 References 213 14 Downstream Processing of Plant-derived Recombinant Therapeutic Proteins 217 Juergen Drossard 14.1 Introduction 217 14.2 Similarities and Differences in the Processing of Pharmaceutical Proteins from Different Sources 218 14.3 Process Scale 220 XIV Contents 14.4 The Individual Steps of a Downstream Process 221 14.4.1 Initial Processing and Extraction 222 14.4.2 Chromatographic Purification 224 14.5 Regulatory Requirements for Downstream Processing of Plant-derived Pharmaceutical Products 228 References 230 15 Glycosylation of Plant-made Pharmaceuticals 233 Véronique Gomord, Anne-Catherine Fitchette, Patrice Lerouge and Loïc Faye 15.1 Introduction 233 15.2 Plant Cells can Reproduce the Complexity of Mammalian Proteins 233 15.3 Plant-made Pharmaceuticals and their Native Mammalian Counterparts Contain Structurally-distinct N-linked Glycans 238 15.4 Plant-made Pharmaceuticals Possess Immunogenic N-glycans 241 15.5 Current Strategies to Eliminate Immunogenic N-glycans from Plant-made Pharmaceuticals 242 15.6 Towards Humanized N-glycans on PMPs Through the Expression of Mammalian Glycosyltransferases in the Plant Golgi Apparatus 245 15.7 Concluding Remarks 248 15.8 Acknowledgements 248 References 248 16 Biosafety Aspects of Molecular Farming in Plants 251 Ulrich Commandeur and Richard M. Twyman 16.1 Introduction 251 16.2 Transgene Spread 252 16.2.1 Classes of Foreign DNA Sequences in Transgenic Plants 252 16.2.2 Mechanisms of Transgene Pollution –Vertical Gene Transfer 253 16.2.3 Mechanisms of Transgene Pollution – Horizontal Gene Transfer 253 16.3 Combating the Vertical Spread of Transgenes 254 16.3.1 Choosing an Appropriate Host 254 16.3.2 Using Only Essential Genetic Information 255 16.3.3 Elimination of Markers After Transformation 257 16.3.4 Containment of Essential Transgenes 259 16.4 Unintended Exposure to Recombinant Proteins 261 16.4.1 Environmental Risks of Unintended Exposure 261 16.4.2 Addressing the Risks of Unintended Exposure 262 16.4.2.1 Controlling Transgene Expression 262 16.4.2.2 Controlling Protein Accumulation and Activity 263 16.4.2.3 Contamination of the Food Chain During Processing 263 16.5 Conclusions 264 References 265 Contents XV 17 A Top-down View of Molecular Farming from the Pharmaceutical Industry: Requirements and Expectations 267 Friedrich Bischoff 17.1 Introduction 267 17.2 Industrial Production: The Current Situation 267 17.3 Expectations 270 17.4 Requirements 273 17.4.1 Equivalence of the Recombinant Product to the Original Protein 273 17.4.2 Processing in the Endoplasmic Reticulum (ER) 274 17.4.3 Glycosylation in the Golgi 275 17.4.4 Differential Glycosylation – Implications on Immunogenicity of vaccines 277 17.4.5 Glycosylation and Stability 277 17.4.6 Equivalence of Enzymes 279 17.4.7 Degradation 279 17.4.8 Efficacy in Clinical Trials 280 17.4.9 The Optimal Production System 283 17.4.10 Post-harvest expression 285 17.4.11 Purification 285 17.5 Conclusions 287 References 287 18 The Role of Science and Discourse in the Application of the Precautionary Approach 291 Klaus Ammann 18.1 Introduction 291 18.2 Other Roots to Problems with the Precautionary Approach 292 18.2.1 The Roots of the Precautionary Approach and Environmental Debate 292 18.2.2 Discussion About the PA is Too Closely Related to Factual Knowledge Alone 294 18.3 The First and Second Generation Systems Approaches 295 18.3.1 First Generation Systems Approach 295 18.3.2 Second Generation Systems Approach 295 18.4 How to Solve Wicked Problems in Biotechnology and the Environment 298 18.5 How to Achieve Such Demanding Planning Goals 299 18.6 There is no Scientific Planning 299 18.7 Outlook 300 Bibliography 301 Subject Index 303 XVI Contents