design and operation of large scale rna production daniel jacinski, phd candidate college of...
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![Page 1: Design and Operation of Large Scale RNA production Daniel Jacinski, PhD candidate College of Pharmacy](https://reader036.vdocument.in/reader036/viewer/2022062407/56649c7b5503460f9492ee4e/html5/thumbnails/1.jpg)
Design and Operation of
Large Scale RNA production
Daniel Jacinski,
PhD candidate
College of Pharmacy
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Problem
• For our customers we need to produce RNA in large scale.
• High cost and high amounts of waste are preventing scaling up of production.
• For large scale synthesis, the goal is to generate 1 gram of RNA with one synthesis run
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RNA Background
Replication
Translation
Transcription miRNA
Central Dogma
DNA
RNA
Protein
Non
codi
ng R
NA
(doe
s no
t co
de fo
r pr
otei
n)
Blocks protein production
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siRNA Background
• siRNA
• Small Interfering RNA
• Primarily 21 nucleotides in length
• Double stranded
• Mimics miRNA and specifically controls gene regulation
• Degrades mRNA preventing protein production
• Great interest in using siRNA in therapeutics for cancers and viral infections
• New class of Drug currently being discovered and developed
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Why produce siRNA• As a new class of drugs there is a high interest to
use siRNA for the treatment of disease
• Large production of siRNA strands can be used by
• Researchers
• Clinicians for clinical trials
• Eventually as a pharmaceuticals in humans
• We want to produce large amounts of RNA, while reducing cost, to supply to potential customers.
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Large Scale RNA synthesis – RNAi in Big Pharma
• Big Pharma is actively exploring RNAi for novel cancer treatments
• Is seen as the next phase in drug discovery
• RNAi as the therapy or RNA as the target for treatment
http://seekingalpha.com/article/2329785-celsions-egen-acquisition-brings-immunotherapy-and-rna-therapeutics-to-its-cancer-platform
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RNA in Clinical Trials
Watts JK, Corey DR. Silencing disease genes in the laboratory and the clinic. Journal of Pathology; 2012, 226: 365-379. DOI: 10.1002/path.2993
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RNA synthesis
• This scheme is for DNA, for RNA there is a 2’ TBDMS protecting group to protect the highly reactive 2’OH on RNA
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RNA synthesis
• Basic Synthetic Cycle Steps
• The Detritylation Step (deblocking)
• The Coupling (Activation) Step
• The Capping Step
• The Oxidation Step
• Monomers used in synthesis are different from what is used for natural RNA production
• Phosphoramidite chemistry
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RNA synthesis – Most Important Steps for high yield• Detritylation
• N-1 nucleotide must have the 5’ group deprotected to add the next monomer
• Coupling
• Addition of the next monomer – RNA takes much longer than DNA
• At least 5-6 minutes per base
• WATER KILLS COUPLING EFFICIENCY
• Probably the most important aspect for synthesis efficiency
• Argon gas normally used to keep synthesis in moisture free environment
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Post Synthesis Procedure• Cleavage off the beads is the first step of the
post-synthesis procedure
• RNA has many reactive groups that must be protected during synthesis, and then removed afterwards to generate biologically active RNA
• Base deprotection is done simultaneously with cleavage from the beads
• For RNA, the 2’TBDMS protecting group must also be removed
• Additional deprotection step to base deprotection and cleavage from the beads
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Desalting and Purification
• All of the deprotecting groups that were removed from the oligonucleotide need to be separated out
• This can be done by large scale FPLC or HPLC
• Reverse phase
• Anion exchange
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Problems of RNA synthesis
• RNA production results in three major problems from reagents used:
• Many chemicals that are used are hazardous to health and must be handled with care
• Large production of hazardous wastes, mainly organic solvents
• Acetonitrile
• Iodine
• THF (Tetrhydrofuran)
• Acetic Anhydride
• Trichloracetic acid
• Cost of large amount of reagents
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Problems of RNA synthesis
• Reagents and their waste are a major problem.
• If we could recycle and reuse these organic wastes, it would significantly reduce cost of waste reagents bought and waste removal costs.
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Special considerations for RNA synthesis• RNA is easily degraded by enzymes called
nucleases found everywhere.
• To keep the product stable all equipment must be cleaned of nucleases and sterile.
• We normally autoclave any materials that will touch RNA products
• RNA degraded at elevated temperatures faster
• Should be stored at -20 °C for short term
• Should be stored at -80 °C for long term
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Regulations
• Good manufacturing practices (GMP) must be followed for synthesis of pharmaceuticals
• These are the minimum requirements that must be met to assure that the products are of high quality and do not pose any risk to the consumer or public.
• Things to consider
• Hygiene
• Cross contamination
• Consistency of manufacture
• Records of manufacture
• Recall system (in case bad batches are made)
• http://www.fda.gov/food/guidanceregulation/cgmp/default.htm
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The Goal
• The goal is to scale up a synthesis that will generate 1 gram of siRNA
• Luciferase siRNA
• A standard siRNA used to assay efficiency of gene knockdown
• Used in most labs that work with RNAi
• 5’-UCGAAGUACUCAGCGUAAGUU-3’
• In a typical 1 μmole scale synthesis we generally get about 1 milligram