Design and Operation
of Large Scale RNA
production
Daniel Jacinski,
PhD candidate
College of Pharmacy
Problem
• For our customers we need to produce RNA in large scale.
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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
RNA Background
Central Dogma
Replication
DNA
Transcription
miRNA
RNA
Translation
Protein
Blocks protein production
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
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
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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.
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-bringsimmunotherapy-and-rna-therapeutics-to-its-cancer-platform
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
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
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
RNA synthesis – Most Important Steps for
high yield
• Detritylation
•
N-1 nucleotide must have the 5’ group deprotected to add the
next monomer
• Coupling
•
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Addition of the next monomer – RNA takes much longer than
DNA
At least 5-6 minutes per base
• WATER KILLS COUPLING EFFICIENCY
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Probably the most important aspect for synthesis efficiency
Argon gas normally used to keep synthesis in moisture free
environment
Post Synthesis Procedure
• Cleavage off the beads is the first step of the postsynthesis 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
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
Problems of RNA synthesis
• RNA production results in three major problems from
reagents used:
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Many chemicals that are used are hazardous to health and must
be handled with care
Large production of hazardous wastes, mainly organic solvents
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Acetonitrile
Iodine
THF (Tetrhydrofuran)
Acetic Anhydride
Trichloracetic acid
Cost of large amount of reagents
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.
Special considerations for RNA
synthesis
•
RNA is easily degraded by enzymes called nucleases found
everywhere.
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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
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Should be stored at -20 °C for short term
Should be stored at -80 °C for long term
Regulations
•
Good manufacturing practices (GMP) must be followed for
synthesis of pharmaceuticals
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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
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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.ht
m
The Goal
• The goal is to scale up a synthesis that will generate 1
gram of siRNA
• Luciferase siRNA
•
•
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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