FINAL L4 - Vaccines Flashcards
Polio Vaccine, A whole virus vaccine
In 1955, more than half of the first publicly available vaccine bore the Lilly Label
Lilly’s sales soared
Vaccine was produced until 1968. By then, Polio cases in the US. once numbering in the tens of thousands had dropped to 53
Know the idealized view of RNA in the core
Slide 12
Recognize RNA hydrolysis in Blood or cells
Slide 13 mechanism
Slide 23
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RNA Vaccine Manufacture by CMOs
-CMOs are contract manufacturing orgs.
-There is a major effort by these orgs to provide vaccine manufacture. services
-Companies who have new vaccines do not have to develop manufacturing capability - can use this org
-Similar to a company Byrn co-founded -provided contract research services in difficult replicate areas
RNA Vaccine workflow
-Important to note this level of detail is not available from pharm. companies because they WANT to keep their methods a trade secret
-Many approaches for manufacture of vaccines are discussed at meetings and with the FDA
-Documents like the Cytiva one are generally reliable because they are controlled by the FDA and in this way ultimately become public
Making Plasmid DNA - ThermoFischer
-Process Development
-QC and Analytical
-cGMP Manufacturing
Drug substance Manufacture 1: Plasmid linearization
-Objective: Produce DNA template
-Considerations: Restriction enzyme digest of plasmid DNA (pDNA) to create a linear template
Quality of ingoing pDNA to control isomeric form
-Strategies: Choose appropriate scale and vessel with controlled environment
Select plasmid source and use analytics with quality control in mind
Drug substance Manufacture 2: In Vitro transcription (IVT)-
-Objective: Enzymatic synthesis and capping of mRNA
-Considerations: choice of enzymes, dNTPS, and nucleosides
Whether to cap before or during transcription
DNase treatment and removal of residual DNA and RNA isoforms
Scale and use of organic solvents
-Strategies: Identify enzymes and nucleotides specific to target mRNA: replace uridine/cytosine with modified release nucleosides
Precipitation and wash step - remove residual impurities
-Optimize reaction conditions for capping (ie temperature)
Drug Substance Manufacture 3: Buffer Exchange
Objective: Prepare feed for purification step
-Considerations: Avoiding loss of titer for best product recovery
Mitigating risk of failure in multistep process
Avoiding filter fouling, clogging, and contamination
-Strategies: Select the right filter type and sizing for scale-up
Use a scalable, automated TFF system
-Consider single-use closed systems
Drug Substance Manufacture 4- Capture
-Objective - mRNA purification
-Considerations: Molecule size
Selecting resin that purifies full-length mRNA + removes contaminants
Elution conditions for max recovery
-Strategies: Uses resins w/specific ligand for faster throughput
Control bioburden + speed changeover w/prepacked resin formats
-Use scalable column platform from PD to full-scale manufacturing
Drug Substance Manufacture 5: Concentration
-Objective: Volume reduction and change to stable salt conditions
-Considerations: Avoiding loss of titer for best product recovery
-Strategies: Choose a scalable, automated, single-use, closed TFF system
Tailor buffer conditions to control secondary mRNA structures
Optimize concentration w/filter cut off
Drug Product Manufacture 11: LNP formation
-Objective: LNO formation as an mRNA drug delivery vehicle
-Considerations: Optimizing mix of mRNA with lipids to generate desired LNP size
Optimizing ratio of empty vs filled LNPs
Avoiding excess lipid monomer formation
-Strategies: Monitor particle size, polydispersity index, LNP formation efficiency
Validate chem, MW cut-off/pore sizes of filters
Validate parameters (eg. flow rate, path length)
Ensure scalability - optimized parameters from bench to process scale
