Pharmaceutical Biotechnology: A Focus on Industrial Application

Description

Pharmaceutical Biotechnology: A Focus on Industrial Application covers the development of new biopharmaceuticals as well as the improvement of those being produced. The main purpose is to provide background and concepts related to pharmaceutical biotechnology, together with an industrial perspective. This is a comprehensive text for undergraduates, graduates and academics in biochemistry, pharmacology and biopharmaceutics, as well as professionals working on the interdisciplinary field of pharmaceutical biotechnology. Written with educators in mind, this book provides teachers with background material to enhance their classes and offers students and other readers an easy-to-read text that examines the step-by-step stages of the development of new biopharmaceuticals. Features: Discusses specific points of great current relevance in relation to new processes as well as traditional processes Addresses the main unitary operations used in the biopharmaceutical industry such as upstream and downstream Includes chapters that allow a broad evaluation of the production process Dr. Adalberto Pessoa Jr. is Full Professor at the School of Pharmaceutical Sciences of the University of S�o Paulo and Visiting Senior Professor at King's College London. He has experience in enzyme and fermentation technology and in the purification processes of biotechnological products such as liquid-liquid extraction, cross-flow filtration and chromatography of interest to the pharmaceutical and food industries. Dr. Michele Vitolo is Full Professor at the School of Pharmaceutical Sciences of the University of S�o Paulo. He has experience in enzyme technology, in immobilization techniques (aiming the reuse of the biocatalyst) and in the operation of membrane reactors for obtaining biotechnological products of interest to the pharmaceutical, chemical and food industries. Dr. Paul F. Long is Professor of Biotechnology at King's College London and Visiting International Research Professor at the University of S�o Paulo. He is a microbiologist by training and his research uses a combination of bioinformatics, laboratory and field studies to discover new medicines from nature, particularly from the marine environment. Cover Half Title Title Page Copyright Page Table of Contents Preface Editors Contributors Chapter 1 Fundamentals of Biotechnology 1.1 Introduction 1.2 Biological Molecules 1.2.1 Introduction 1.2.2 Proteins 1.2.3 Nucleic Acids 1.2.4 Virus 1.3 Basic Technologies of the Biotechnological Processes 1.3.1 Monoclonal Antibody Technology 1.3.2 Bioprocessing Technology 1.3.3 Cell Culture Technology 1.3.4 Tissue Engineering Technology 1.3.5 Biosensors Technology 1.3.6 Genetic Engineering Technology 1.3.7 Protein Engineering Technology 1.3.8 Antisense RNA Technology 1.3.9 Chip DNA Technology 1.3.10 Biocomputing Technology 1.4 Biotechnology and Applications 1.4.1 Medical 1.4.1.1 Diagnostics 1.4.1.2 Therapeutics 1.4.2 Environment 1.4.3 Livestock Raise and Agriculture 1.5 Matters of Business 1.5.1 Structuring of a Biotechnology Enterprise 1.5.2 Biopharmaceutical Industry 1.6 Social Aspects of Biotechnology 1.6.1 Use of Bioproducts 1.6.2 Genetic Privacy and Laboratory Prognostic Testing 1.6.3 Stem Cells and Cloning 1.6.4 Use of Test Subjects 1.6.5 Agriculture 1.7 Final Considerations References Chapter 2 Thermodynamics Applied to Biomolecules 2.1 Introduction 2.2 Thermodynamic Concepts 2.3 Mathematical Modelling 2.3.1 Reaction Thermodynamics 2.3.1.1 Activation Energy and Standard Enthalpy Variation of the Unfolding Equilibrium 2.3.1.2 Enthalpy, Entropy and Gibbs Free Energy of the Reaction 2.3.2 Thermodynamics of Biomolecules Thermo-inactivation 2.3.2.1 Enthalpy, Entropy and Gibbs Free Energy of Biocatalyst Thermo-Inactivation 2.3.2.2 Complementary Kinetic Parameters 2.4 Experimental Procedures and Case Studies 2.4.1 Thermodynamics of Enzyme-Catalysed Reactions 2.4.1.1 Experimental Procedure 2.4.1.2 Practical Example 2.4.2 Thermodynamics of Biocatalyst Thermal Denaturation 2.4.2.1 Experimental Procedure 2.4.2.2 Practical Example 2.5 Physical and Chemical Factors Influencing Thermodynamic Parameters 2.5.1 Temperature 2.5.2 pH 2.5.3 Substrate Concentration 2.5.4 Techniques of Biomolecule Improvement 2.6 Final Considerations References Chapter 3 Expression Systems for the Production of Therapeutic Recombinant Proteins 3.1 Introduction 3.2 Differences between Synthetic Drugs and Biopharmaceuticals Based on Recombinant Proteins 3.3 Main Systems for Expression of Therapeutic Recombinant Proteins 3.3.1 Bacteria 3.3.2 Yeasts 3.3.3 Insect Cells 3.3.4 Plant Cells 3.3.5 Mammalian Cells 3.4 Development of Bioprocesses for the Production of Therapeutic Recombinant Proteins 3.5 Final Considerations References Chapter 4 Molecular Biology: Tools in Industrial Pharmaceutical Biotechnology 4.1 Introduction 4.2 Therapeutic Targets 4.3 Cellular Communication 4.4 Expression Gene Regulation 4.5 Transcriptional System in Prokaryotes 4.5.1 Promoter Structure and Operon 4.5.2 Transcription Termination 4.6 Transcription Regulation in Eukaryotes 4.7 RNA Processing 4.8 Final Considerations References Chapter 5 Molecular Biology Tools: Techniques and Enzymes 5.1 Introduction 5.2 Isolating a Gene Sequence-DNA Polymerase 5.3 Manipulating the Gene Obtained by PCR 5.4 Restriction Enzymes and Expression Vectors 5.5 Final Considerations References Chapter 6 Bioinformatics Applied to the Development of Biomolecules of Pharmaceutical Interest 6.1 Introduction 6.2 Databases 6.2.1 Databases of Small Molecules 6.2.2 Natural Products Databases 6.2.3 Nucleic Acid Databases 6.2.4 Peptide Databases 6.2.5 Protein Databases 6.2.6 Other Relevant Biological Databases 6.3 Structural Protein Analysis 6.3.1 Visualization Programmes and Analysis of Three-Dimensional Structures 6.3.2 Protein Modelling. Why Model? 6.3.2.1 Basis of Methods 6.3.3.1 MD 6.3.3.2 NMA: Normal Modes Analysis 6.3.3.3 Comparison: MD vs NMA 6.3.3.4 Use 6.3.3.5 Interaction Models: Protein vs Ligand 6.3.3.6 Docking: Protein vs Small Molecule 6.3.3.7 Docking: Protein vs Protein 6.4 Examples 6.4.1 Bioinformatics in Vaccine Discovery 6.4.2 Use of Peptides in New Drug Development 6.4.3 Human Immunodeficiency Virus Reverse Transcriptase Inhibitors 6.4.4 Applied Bioinformatics Synthetic Biology: Production and Discovery of New Biopharmaceuticals 6.5 Final Considerations References Chapter 7 Bioprocesses: Microorganisms and Culture Media 7.1 Introduction 7.2 General Characteristics of Microorganisms 7.2.1 Cell Concentration Measurement 7.2.2 Standard Dry Mass Curve 7.2.2.1 Results 7.2.3 Duplication and Generation Time 7.3 Cultivation Media of Industrial Interest 7.3.1 Trace Elements and Minerals 7.3.2 Growth Factors 7.3.3 Synthetic or Chemically Defined Media 7.3.4 Complex Media 7.4 Antifoam Agents 7.5 pH Control in the Bioprocess 7.6 Precursors 7.7 Primary and Secondary Metabolites 7.7.1 Overproduction of Secondary Metabolites 7.8 Final Considerations References Chapter 8 Sterilization in Pharmaceutical Biotechnology 8.1 Introduction 8.2 Terms and Definitions 8.3 Types of Sterilization 8.3.1 Physical Sterilization Methods 8.3.1.1 Heat 8.3.1.2 Ionizing Radiation 8.3.1.3 Filtration 8.3.2 Chemical Sterilization Methods 8.4 Sterilization in Upstream Operations 8.4.1 Culture Media and Its Sterilization 8.4.2 Batch Heat Sterilization 8.4.3 Heat Sterilization in a Continuous System 8.4.4 Sterilization Kinetics of Microorganisms 8.4.5 Air Sterilization 8.5 Sterilization in Downstream Operations 8.5.1 Terminal Sterilization and Aseptic Processing 8.5.2 Sterilization of Biopharmaceuticals in Downstream Processes 8.5.3 Sterilization of Biopharmaceuticals 8.6 Validation of Sterilization Processes 8.7 Final Considerations References Chapter 9 Kinetics of Cell Cultivation 9.1 Introduction 9.2 Typical Batch Cultivation Phases 9.3 With What Does μ Vary? (Model for Cell Growth) 9.4 What Does S Vary with? (Model for Substrate Consumption) 9.5 How to Calculate the Productivity of a Process? 9.6 How Does the Product Concentration Vary (Model for Production)? 9.7 Final Considerations References Chapter 10 Bioreactors: Modes of Operation 10.1 Introduction 10.2 General Characteristics 10.2.1 Batch Mode 10.2.2 Fed-Batch Mode 10.2.3 Continuous Mode 10.3 Mass Balances for Different Modes of Operation 10.4 Batch Operation 10.4.1 Mass Balance for the Cells in the Batch Mode 10.4.2 Mass Balance for the Limiting Substrate in the Batch 10.4.3 Mass Balance for the Product in Batch Mode 10.5 Fed-Batch Operation 10.5.1 Mass Balance for Cells in Fed-Batch Mode 10.5.2 Mass Balance for the Limiting Substrate in Fed-Batch Mode 10.5.3 Mass Balance for the Product in Fed-Batch Mode 10.6 Continuous Mode Operation 10.6.1 Mass Balance for Cells in Continuous Mode without Cell Recycling 10.6.2 Mass Balance for a Limiting Substrate in Continuous Mode without Cell Recycling 10.6.3 Mass Balance for the Product in Continuous Mode without Cell Recycling 10.6.4 Mass Balance for Cells in Continuous Mode with External Cell Recycling 10.6.5 Mass Balance for the Limiting Substrate in Continuous Mode with External Cell Recycling 10.6.6 Mass Balance for the Product in Continuous Mode with External Cell Recycling 10.6.7 How to Calculate the Volumetric Productivity of a Continuous Process? 10.7 Final Considerations References Chapter 11 Agitation and Aeration: Oxygen Transfer and Cell Respiration 11.1 Introduction 11.2 Gas Pressure and Oxygen Partial Pressure 11.3 Aeration Systems 11.3.1 Superficial Aeration 11.3.2 Submerged Aeration 11.3.2.1 Aeration by Sparging with Mechanical Agitation (Stirred Tank Reactor) 11.3.2.2 Aeration by Bubbling with No Mechanical Agitation 11.4 Mechanical Agitation Systems in Bioreactors 11.5 Oxygen Transfer and Respiration: From Gas Bubbles to Cells 11.6 Gas-Liquid Mass Transfer (Oxygen Supply): OTR 11.7 Oxygen Uptake in Cell Cultures (Oxygen Demand): OUR 11.8 Integrating Oxygen Supply and Demand 11.9 Dissolved Oxygen Concentration (C) Profile in a Culture with Constant Oxygen Transfer Conditions 11.10 Control of C 11.11 Measurement of k[sub(L)]a 11.11.1 Dynamic Method without Cells 11.12 Measurement of Q[sub(O2] X 11.12.1 Dynamic Method 11.12.2 Gas Balance 11.12.3 Liquid Phase Mass Balance 11.13 Criteria for Scaling Up the Bioprocess 11.14 Final Considerations References Chapter 12 Mammalian Cell Culture Technology 12.1 Introduction 12.2 Brief History 12.3 Main Product Categories 12.3.1 Vaccines 12.3.2 Monoclonal Antibodies 12.3.3 Glycoproteins 12.3.4 Cells and Tissues 12.3.5 Gene Therapy 12.4 Mammalian Cells 12.4.1 Basic Characteristics 12.4.2 Post-Translational Processes 12.4.3 Cell Types 12.4.4 Hybridoma Cells 12.4.5 Cell Culture Derivatives 12.4.5.1 Immunogenicity of Recombinant Proteins 12.4.5.2 Basic Structure of Antibodies or Immunoglobulins 12.4.5.3 Types of Monoclonal Antibodies 12.4.5.4 Phage Display Technology 12.4.5.5 Culture Media Used in Mammalian Cell Technology 12.4.5.6 Culture Medium Optimization 12.4.6 Basic Cell Culture Laboratory 12.4.7 Bioprocess and Mammalian Cells 12.4.8 Main Guidelines for Obtaining Cell Banks 12.4.8.1 Obtaining and Controlling the ‘Original’ Cell 12.4.8.2 Cell Bank Preparation 12.4.8.3 Qualification Tests of the Cell Bank 12.4.8.4 Karyotyping (Cytogenetic Analysis) 12.4.8.5 Analysis of Isoenzymes 12.4.8.6 DNA Fingerprinting 12.4.8.7 Genetic Stability of the Recombinant Mammalian Cell 12.4.9 Best Practices in Cell Culture 12.4.10 Development of a Cell Line 12.4.10.1 Transient Expression 12.4.10.2 Stable Expression 12.4.10.3 Construction of Expression Vector 12.4.10.4 Promoters 12.4.10.5 Enhancer 12.4.10.6 Elements that Stabilize and Increase the Translation of the Primary Transcript 12.4.10.7 Selection Markers 12.4.10.8 Selection of Clones 12.4.10.9 Transfection 12.5 Production Scale-Up 12.6 Bioreactors and Mammalian Cell Cultivation 12.6.1 Small Scale Culture 12.6.2 Scaling-Up Problems 12.7 Anchorage-Dependent Cell Systems 12.7.1 Roller Bottles 12.7.2 Stacked-Plate or Multitray Systems 12.7.3 Microcarriers 12.7.4 Bed Bioreactors 12.8 Systems for Cells in Suspension (or Cells Attached to Microcarriers) 12.8.1 Spinner Bottles 12.8.2 Shake Flasks 12.8.3 Culture Bags 12.8.4 Single-Use Bioreactors (SUBs) 12.9 Bioreactors 12.9.1 Hollow Fibre Bioreactors Applied to the Cultivation of Mammalian Cells 12.9.2 Type of Bioprocesses in Cell Culture 12.9.3 Cell Metabolism 12.9.4 Glucose, Glutamine and Amino Acids as Sources of Energy and Carbon 12.9.5 Effects of Lactate and Ammonia 12.9.6 Role of Oxygen and CO[sub(2)] in Cellular Metabolism of Mammalian Cells 12.10 Monitoring and Control of Mammalian Cell Culture 12.10.1 Partial Pressure of O[sub(2)] (pO[sub(2)]) 12.10.2 Partial Pressure of Carbon Dioxide 12.10.3 Metabolites and Products 12.10.4 Cell Concentration and Viability 12.11 Final Considerations References Chapter 13 Purification Process of Biomolecules 13.1 Introduction 13.2 Cell-Liquid Separation 13.2.1 Filtration 13.2.2 Centrifugation 13.3 Cell Disruption 13.4 Concentration of Biomolecules 13.4.1 Precipitation 13.4.2 Tangential Filtration 13.4.3 Liquid-Liquid Extraction 13.5 Chromatographic Processes 13.5.1 Molecular Exclusion 13.5.2 Ion Exchange 13.5.3 Hydrophobic Interaction 13.5.4 Affinity Chromatography 13.5.5 Scale Up 13.5.6 Expanded Bed Adsorption 13.6 Final Treatment 13.7 Purification of Monoclonal Antibodies 13.8 Endotoxin Removal 13.9 Yield and Purity 13.10 Identification of Biomolecules 13.11 Trends in Processes Applied to the Purification of Biomolecules 13.12 Final Considerations References Chapter 14 Lipopolysaccharides: Methods of Quantification and Removal from Biotechnological Products 14.1 Introduction 14.2 LPS Characteristics and Properties 14.3 LPS Functions and Mechanisms of Action 14.4 Main Techniques for LPS Quantification in Biotechnological Products 14.4.1 LPS Test in Rabbits 14.4.2 LPS Test – Gel Clot 14.4.3 LPS Test – Chromogenic and Turbidimetric Kinetics 14.4.4 New Methods for LPS Quantification 14.5 Techniques Used to Remove LPS from Biotechnological Products 14.5.1 Chromatographic Techniques 14.5.1.1 Affinity Chromatography 14.5.1.2 Ion-Exchange Chromatography 14.5.1.3 Hydrophobic Interaction Chromatography 14.5.2 Membrane-Based Filtration 14.5.3 Ultrafiltration 14.5.4 Microfiltration 14.5.5 Depth Filtration 14.5.6 Novel Methods for LPS Removal 14.5.6.1 Aqueous Two-Phase Systems 14.6 Final Considerations References Chapter 15 Enzymes: The Catalytic Proteins 15.1 Introduction 15.2 Enzyme Specificity 15.3 Enzyme Activity 15.3.1 Quantification of Enzyme Activity 15.3.2 Expression of Enzyme Activity 15.3.3 Factors Affecting Enzyme Activity 15.3.3.1 Physical-Chemical Factors 15.3.3.2 Chemical Factors 15.3.3.3 Physical Factors 15.3.4 Thermodynamics of Enzyme Catalysis 15.4 Final Considerations References Chapter 16 Enzymes as Drugs and Medicines 16.1 Introduction 16.2 Enzymes in Medicines 16.2.1 Enzymes Bioavailability 16.2.1.1 Transport across Endothelium 16.2.2 Therapeutic Enzyme Delivery 16.2.3 Acylation of Enzymes 16.2.4 Molecular Modelling of Enzymes 16.2.5 Important Aspects of Therapeutic Enzymes 16.3 Enzymes in Clinical Analysis and Cosmetics 16.3.1 Clinical Analysis 16.3.2 Cosmetics 16.4 Final Considerations References Chapter 17 Aspects of the Immobilization Technique 17.1 Introduction 17.2 Types of Immobilization 17.2.1 Entrapment 17.2.1.1 Cross-Linked Matrices 17.2.1.2 Encapsulation 17.2.1.3 Microencapsulation 17.2.2 Bonding Formation 17.2.2.1 Adsorption 17.2.2.2 Covalent Binding 17.2.2.3 Cross-Linking 17.2.3 Supports 17.2.4 Effects Caused by Immobilization 17.2.4.1 Steric and Conformational Effects 17.2.4.2 Diffusion and Mass Transport Effects 17.2.4.3 Effects of Microenvironment 17.2.4.4 Advantages and Disadvantages of the Immobilization Technique 17.2.4.5 Applications 17.3 Fundamentals of Enzyme Reactors 17.3.1 Types of Enzyme Reactors 17.3.2 Enzyme Reactor Kinetics 17.3.3 Operation of Enzyme Reactors 17.4 Final Considerations References Chapter 18 Biomolecules in Analytical Methods 18.1 Introduction 18.2 Enzymes as Diagnostic Tools 18.2.1 Diagnosis of Gastric Disorders 18.2.2 Neonatal Screening 18.2.3 Cancer Diagnosis 18.2.4 Enzyme Detection of Micronutrients, Microorganisms and Cholesterol 18.3 Test Strips 18.4 Biosensors 18.4.1 General Characteristics 18.4.2 Electrochemical Detectors 18.4.2.1 Amperometric Biosensors 18.4.2.2 Potentiometric Biosensors 18.4.2.3 Optical Detectors 18.4.2.4 Thermal Detectors 18.4.2.5 Piezoelectric Detectors 18.4.2.6 Immunodetection Biosensors 18.5 Novel Trends in Biosensing Technology 18.5.1 Microfluidic Integrated Biosensors: Towards Lab-on-a-Chip 18.5.2 Wearable Biosensors for Healthcare Monitoring 18.6 Final Considerations References Chapter 19 Nanotechnology and Biopharmaceuticals 19.1 Introduction 19.2 Nanobiotechnology for Biopharmaceuticals 19.3 Liposomes 19.4 Polymersomes 19.5 Polymeric Micelles 19.6 Polymeric Nanoparticles: Nanocapsules and Nanospheres 19.7 Nanoemulsions and Microemulsions 19.8 Solid Lipid Nanoparticles and Nanostructured Lipid Carriers 19.9 PEGylation 19.10 Characterization Techniques 19.10.1 Dynamic Light Scattering (DLS) 19.10.2 Nanoparticle Tracking Analysis (NTA) 19.10.3 Zeta Potential (ZP) 19.10.4 Microscopy 19.11 Nanotoxicity 19.12 Regulatory Aspects 19.13 Final Considerations References Chapter 20 Biosafety Applied in Pharmaceutical and Biotechnological Processes 20.1 Introduction 20.2 Definitions of Biosafety 20.3 Classification of Microorganisms by Risk Group 20.4 Biosafety Levels 20.4.1 Biosafety Level 1 (BSL-1) 20.4.1.1 Laboratory Practices for BSL-1 20.4.1.2 Safety Equipment for BSL-1 20.4.1.3 BSL-1 Laboratory Facility 20.4.2 Biosafety Level 2 (BSL-2) 20.4.2.1 Laboratory Practices for BSL-2 20.4.2.2 Safety Equipment for BSL-2 20.4.2.3 BSL-2 Laboratory Facility 20.4.3 Biosafety Level 3 (BSL-3) 20.4.3.1 Laboratory Practices for BSL-3 20.4.3.2 Safety Equipment for BSL-3 20.4.3.3 BSL-3 Laboratory Facility 20.4.4 Biosafety Level 4 (BSL-4) 20.4.4.1 Laboratory Practices for BSL-4 20.4.4.2 Safety Equipment for BSL-4 20.4.4.3 BSL-4 Laboratory Facility 20.5 Laboratory Accidents 20.5.1 Historical Accidents 20.5.2 Accident Prevention 20.5.2.1 Proper Laboratory Supervision 20.5.2.2 Training and Awareness 20.5.2.3 Continued Education 20.5.2.4 Proper Laboratory Techniques 20.5.2.5 Proper Laboratory Equipment 20.5.2.6 Laboratory Organization 20.5.2.7 Vaccination 20.6 Activity Planning 20.7 Safety Equipment 20.8 Final Considerations References Chapter 21 Pharmaceutical Quality System for Biotechnology Products 21.1 Introduction 21.2 Pharmaceutical Quality System (PQS) 21.3 Quality-by-Design in Biotechnology Product and Process Development 21.3.1 Historical Context 21.3.2 QbD-based Development 21.3.2.1 Product Design Space 21.3.2.2 Process Design Space 21.3.2.3 Control Strategy 21.3.2.4 Lifecycle Management Plan 21.4 Practical Notes on Process Development 21.4.1 Expression Systems 21.4.1.1 Bacterial Systems 21.4.1.2 Filamentous Fungi and Yeast Systems 21.4.1.3 Mammalian Cell Systems 21.4.2 Where Does Biopharmaceutical Scale-Up Start? 21.5 Good Manufacturing Practices (GMP) 21.5.1 Validation – Production Process 21.5.2 Process Validation in Bioreactors (Upstream) 21.5.3 Validation of Extraction and Purification Processes (Downstream) 21.5.4 Validation – Viral Safety 21.5.4.1 Physical Methods 21.5.4.2 Chemical Methods 21.5.5 Validation – Analytical Methods 21.5.5.1 Reference Standard 21.5.5.2 Method Development 21.5.6 Validation – Biological Assays 21.5.7 Change Control 21.6 Analytical Methods for Laboratory Quality Control 21.6.1 Physicochemical Tests 21.6.1.1 High-Performance Liquid Chromatography (HPLC) 21.6.1.2 Gel Electrophoresis 21.6.1.3 NMR – Nuclear Magnetic Resonance 21.6.1.4 Capillary Electrophoresis 21.6.1.5 Spectrophotometric Methods 21.6.2 Others (pH, Total Solids, Preservatives, Isotonicity) 21.6.3 In vitro Tests 21.6.3.1 Cytotoxicity 21.6.3.2 Isoenzymes 21.6.3.3 Microbiological Tests 21.6.3.4 Mycoplasma 21.7 Preclinical Safety Evaluation 21.7.1 Acute Toxicity 21.7.2 Subacute Toxicity or Repetitive Doses 21.7.3 Chronic and Subchronic Toxicity 21.7.4 Teratogenicity and Reproductive Disorders 21.7.5 Immunotoxicity 21.7.6 Pharmacokinetics 21.7.7 Carcinogenicity or Oncogenicity 21.7.7.1 In vitro Preclinical Studies – Mutagenicity 21.7.7.2 In Vitro Mammalian Chromosomal Aberration Test (OECD 473) 21.7.7.3 In Vitro Mammalian Cell Gene Mutation Test (OECD 476) 21.7.7.4 Recombinant Bacterial Testing (OECD 471) 21.8 Legal Aspects for Clinical Trials 21.8.1 Phases of the Clinical Trial 21.8.1.1 Phase I 21.8.1.2 Phase II (Pilot Therapeutic Study) 21.8.1.3 Phase III 21.8.1.4 Phase IV 21.8.2 Ethical Aspects of Human Trials 21.9 Final Considerations References Chapter 22 Techno-Economic Evaluation of Biotechnological Processes and Pharmacoeconomic Analysis 22.1 Introduction 22.2 Bioprocess Design and Economics 22.2.1 Design Basis 22.2.2 Upstream Section 22.2.3 Bioreaction Section 22.2.4 Downstream Section 22.2.5 Process Flow Diagram and Process Simulation 22.2.6 Economic Analysis 22.2.7 Cost Analysis 22.2.8 Profitability Analysis 22.3 Examples of Techno-Economic Analysis 22.3.1 Comparison of the Insulin and BGL Production Processes 22.4 Beyond Techno-Economic Analysis 22.5 Costs Considerations in Biopharmaceuticals 22.6 Pharmacoeconomic Analysis 22.7 Biopharmaceutical Pharmacoeconomic Study Example 22.8 Final Considerations References Chapter 23 Perspectives for Pharmaceutical Biotechnology 23.1 Introduction 23.2 Some Neurodegenerative Diseases 23.3 Schistosomiasis 23.4 AIDS 23.5 Cancer 23.6 Stem Cells 23.7 Biotechnology Interfaces 23.8 Administration of Bioactive Molecules 23.9 Individualized Therapeutics 23.10 Synthetic Biomolecules 23.11 Electronics in Pharmaceutical Biotechnology 23.12 Final Considerations References Index