This book is the most comprehensive and complete treatise on nucleic acid therapeutic
products, including mRNA vaccines, their manufacturing, formulations, and
testing for safety and efficacy. Details include cGMP-compliant manufacturing and
regulatory filing steps. A new concept of “biosimilar” mRNA vaccine is presented
to secure fast approval of copies of mRNA vaccines. Projections of financial plans
to establish RNA manufacturing facilities are provided, along with details of supply
chain management. Finally, the future of nucleic acid products in gene therapy and
other newer applications is presented, along with a perspective that all new vaccines
will be the nucleic acid type that will further provide first-time prevention of autoimmune
disorders. It is projected that both big pharma and start-ups will enter this field,
and we can expect significant additions to our drug armamentarium soon.
Author(s): Sarfaraz K. Niazi
Publisher: CRC Press
Year: 2022
Language: English
Pages: 325
City: Boca Raton
Cover
Half Title
Title Page
Copyright Page
Dedication
Table of Contents
Preface and Perspective
Acknowledgments
Author
Background
B.1 The RNA Lead
B.2 Development perspective
B.3 Intellectual Property Perspective
B.4 Publications
Chapter 1 The Genome Machine
1.1 Background
1.2 Molecular Clock
1.3 Evolution
1.4 Life
1.4.1 Aging
1.4.2 Humans
1.4.3 Biological Cell
1.5 Proteins
1.6 Conclusion
Chapter 2 Understanding Nucleic Acids
2.1 Background
2.2 The DNA
2.3 The RNA
2.3.1 Replication
2.3.2 Transcription
2.3.3 Codons
2.3.4 Translation Process
2.4 Leventhal Paradox
2.5 Conclusion
Chapter 3 RNA Therapeutics
3.1 Background
3.2 DNA Therapeutics
3.2.1 DNA Plasmids
3.2.2 Viral Vectors
3.3 Types of RNAs
3.4 Approved Therapies
3.5 RNA Therapies
3.6 Noncoding RNA
3.6.1 Small interfering RNA
3.6.2 Micro RNA
3.6.3 Long Non-coding RNA
3.6.4 Antisense RNA
3.6.5 Interfering RNA
3.6.6 Aptamers
3.7 Coding RNA
3.7.1 mRNA as Therapeutics
3.7.2 mRNA as Replacement Therapy
3.7.3 mRNA Vaccines
3.7.4 mRNA-Enhanced Cell Therapies
3.8 Delivery of RNA Therapeutics
3.8.1 Lipid Nanoparticles
3.8.2 Liposomes
3.8.2.1 Leukosomes
3.8.3 Nanotechnology
3.8.3.1 Therapeutic Modules
3.8.3.2 Construction of Multifunctional RNA Nanoparticles
3.8.4 Polymer Nanomaterials
3.8.5 Silica Nanoparticles
3.8.6 Carbon and Gold Nanomaterials
3.8.7 N-Acetylgalactosamine (GalNAc)
3.8.7.1 Testing
3.9 Conclusion
Chapter 4 Nucleoside Vaccines
4.1 Background
4.2 History
4.3 Immunogenicity
4.3.1 Antigens
4.3.2 Dendritic Cells
4.3.3 RNA Virus
4.4 Messenger RNA
4.4.1 Types
4.4.2 Chemical Modifications
4.4.2.1 Sugar Modifications
4.4.2.2 Backbone Modifications
4.4.2.3 Base Modifications:
4.4.3 MHC
4.4.4 Stability
4.4.5 Production
4.4.6 Delivery
4.4.6.1 Lipid Nanoparticles
4.5 Development Strategies
4.5.1 Vaccine Design
4.5.2 Process Development
4.5.3 Construction of Plasmids for RNA Transcription
4.5.4 Plasmid Purification and Linearization
4.5.5 In Vitro Transcription of RNA
4.5.6 RNA Capping
4.5.7 RNA Quality Control
4.5.8 Immunization and Allergic Sensitization of Mice
4.5.9 Measurement of Antibody Subclasses
4.5.10 Basophil Release Assay
4.5.11 Culture of RBL Cells
4.6 Nucleoside Vaccines Perspective
4.6.1 SARS-CoV-2 Vaccine
4.6.2 Antiallergy mRNA Vaccine
4.6.3 Anti-infective mRNA Vaccines
4.6.4 Rabies
4.6.5 Influenza
4.6.6 Respiratory Syncytial Virus
4.6.7 Human Metapneumovirus and Type 3 Parainfluenza Virus
4.6.8 Human Cytomegalovirus
4.6.9 Zika Virus
4.6.10 Epstein–Barr Virus
4.6.11 HIV
4.6.12 Streptococcus
4.6.13 Ebola Virus
4.7 Conclusion
Chapter 5 cGMP mRNA Vaccine Manufacturing
5.1 Background
5.2 cGMP Process
5.3 Manufacturing Process
5.3.1 Protocol 1 Steps
5.3.1.1 IVT Steps
5.3.1.2 Final Quality Testing Steps:
5.3.2 Protocol 2 Steps
5.3.3 Facility
5.3.4 Product Selection
5.3.5 Reverse Transcription of the Target RNA Sequence
5.3.6 Synthesis of the Template DNA by PCR
5.3.7 Selection and Design of a Plasmid DNA Vector Backbone
5.3.8 Synthesis of a Template Plasmid DNA Vector by Cloning
5.3.9 Synthesis of the Template Plasmid DNA Vector by Oligonucleotide Annealing
5.3.10 Amplification of the Template Plasmid DNA Vector: Transformation
5.3.11 Quality control: Isolation of plasmid DNA and subsequent analysis of the plasmid DNA
5.3.12 Isolation of Plasmid DNA by Mini Preparation kit
5.3.13 Plasmid Preparation
5.3.14 Linearization
5.3.15 Purification of the Linearized Template Plasmid DNA:
5.3.16 Test Transcription
5.4 RNA transcription
5.4.1 Removal of Template DNA
5.4.2 Enzymatic Capping
5.4.2.1 Enzymatic polyadenylation
5.4.3 Quality Control: Size Determination of Poly(A)-Tail Via Poly(A) Binding Protein Assay
5.4.4 Transcription
5.4.4.1 Capping
5.4.4.2 In Vitro Synthesis
5.4.5 Purification
5.4.5.1 Reversed-Phase Chromatography
5.4.5.2 TFF
5.4.5.3 Ion Exchange Chromatography
5.4.5.4 Ion Pair Reverse Phase Chromatography
5.4.5.5 Affinity Purification (oligo-dT)
5.4.5.6 Hydroxyapatite Chromatography
5.4.5.7 Core Bead Chromatography (does not require prior DNA digest)
5.4.5.8 Lyophilization of the Purified RNA Transcript
5.4.5.9 Resuspension of Lyophilized RNA and Adjustment of the RNA Concentration
5.4.6 Formulation
5.4.7 Release Testing
5.4.8 Fill and Finish
5.5 Quality Control
5.5.1 Determination of Template Plasmid DNA Content
5.5.2 Determination of the Identity of the DNA Sequence Encoding the Target RNA Sequence
5.5.2.1 Determination of the identity of the DNA sequence encoding the target RNA sequence by PCR
5.5.2.2 Determination of the identity of the DNA sequence encoding the target RNA sequence by restriction analysis
5.5.3 Restriction Reaction
5.5.4 Analysis of the DNA Fragments Resulting from Restriction Reaction
5.5.4.1 Determination of the Identity of the DNA Sequence Encoding the Target RNA Sequence by DNA Sequencing
5.5.4.2 Determination of Purity of Template Plasmid DNA Preparation
5.5.5 Determination of the RNA Concentration/RNA Content/RNA Amount
5.5.5.1 Determination of RNA Identity
5.5.5.2 Determination of RNA Identity by Reverse Transcription Sequencing:
5.5.5.3 Determination of RNA Identity by Oligonucleotide Mapping
5.5.5.4 Determination of RNA Identity by RNA Sequencing
5.5.5.5 Determination of RNA Integrity
5.5.5.6 Determination of pH
5.5.5.7 Determination of Osmolality
5.5.5.8 Determination of Bioburden/Microbial Content
5.5.5.9 Determination of Endotoxin Contamination
5.5.5.10 Determination of Protein Contamination
5.5.5.11 Determination of Plasmid DNA Contamination
5.5.5.12 Determination of Bacterial DNA Contamination
5.6 Conclusion
Chapter 6 Regulatory Guidance
6.1 Background
6.2 Regulatory Background
6.2.1 Requirements for Safety/Toxicity Testing
6.2.2 Study Report (GLP Compliant)
6.3 Testing Methods
6.3.1 Prerequisites
6.4 Animal Testing
6.4.1 Testing Strategy for the Safety Assessment
6.4.2 Single Dose Toxicity
6.4.3 Repeated Dose Toxicity
6.5 Safety Pharmacology
6.5.1 Immunogenicity
6.6 Environmental Risk Assessment (ERA)
6.7 Nonclinical Data
6.8 Analytical Testing
6.8.1 Capability and Capacity
6.8.2 Technical Dossier
6.9 The European Regulatory Framework
6.9.1 Advanced Therapies for the Approval of mRNA-Based Medicines
6.9.1.1 Classification of Medicinal Products by the CAT
6.9.2 Quality Regulatory Requirements
6.9.3 Preclinical Regulatory Requirements for mRNA Medicinal Products
6.9.4 Clinical Regulatory Requirements for mRNA Medicines
6.9.5 Clinical Batch Records
6.9.5.1 Substance Used to Make Drugs (2.2.1.S)
6.9.5.2 Investigational Medicinal Product Under Test (2.2.1.P)
6.9.5.3 Control of Drug Substances/Impact Factors
6.10 FDA Regulatory Guidance for mRNA Vaccines
6.10.1 CMC Considerations
6.10.1.1 Manufacture of Drug Substances and Drug Products
6.10.2 Facilities and Inspections
6.10.3 Nonclinical Testing
6.10.3.1 General Considerations
6.10.3.2 Toxicity Studies
6.10.3.3 Characterization of the Immune Response in Animal Models
6.10.3.4 Vaccine-associated Enhanced Respiratory Disease
6.10.4 Clinical Trials
6.10.4.1 General Considerations
6.10.4.2 Trial Design
6.10.4.3 Efficacy Considerations
6.10.4.4 Statistical Considerations
6.10.4.5 Safety Considerations
6.10.5 Post-License Safety
6.10.5.1 General Considerations
6.10.5.2 Pharmacovigilance Activities for Vaccines
6.10.5.3 Required Post-Marketing Safety Studies
6.10.6 Additional Considerations
6.11 Biosimilar mRNA Vaccines
6.12 Conclusion
Appendix 1: COVID-19 mRNA Vaccine Manufacturing Feasibility
Appendix 2: Pharmacopeial Testing
Appendix 3: Suggested Reading
Index
