Optimizing mRNA Purification
eBook
Last Updated: October 20, 2023
(+ more)
Published: October 16, 2023
The approval of mRNA vaccines against COVID-19 has highlighted its potential as a therapeutic tool. Today, scientists are using mRNA to develop novel treatments for cancer, genetic disorders, and infectious diseases.
This eBook highlights innovative purification tools that support scientists in the production of high-quality mRNA for therapeutic applications.
Download this eBook to discover:
• The history of mRNA-based therapeutics
• The latest manufacturing methods and purification solutions
• Innovative tools that can streamline your mRNA workflows
SPONSORED BY
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Optimizing mRNA
Purification
The revolution of mRNAbased
therapeutics
mRNA manufacturing
workflow
Tools to optimize
mRNA purification
The Revolution of mRNA-Based Therapeutics 4
Engineering the Future: Unraveling the Manufacturing
Workflow of mRNA Therapeutics 10
Enabling mRNA-Based Therapeutics Development Through
Efficient and Scalable mRNA Purification Methods 11
The mRNA Therapeutics Boom 12
New Bead Technology Enables Commercial-Scale
mRNA Purification 17
Optimizing mRNA Purification Conditions by Using a
High-Throughput Approach 19
Curated content to boost your mRNA purification process 20
Contents
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Foreword
Optimizing mRNA Purification
The potential of mRNA as a therapeutic tool has been recognized by
researchers ever since its discovery in 1961. However, it was not until 2020,
when mRNA vaccines for COVID-19 received approval, that the therapeutic
promise of mRNA was fully realized.
Building on this success, researchers are today actively working on harnessing
the power of mRNA to create new therapies for a broad range of medical
conditions, including cancer, genetic disorders and other infectious
diseases. As this field rapidly advances, mRNA therapies hold the promise of
revolutionizing modern healthcare and opening new avenues for personalized
and precise treatments.
Nevertheless, before these groundbreaking therapies can be scaled up and
made available for patients, manufacturing processes need to be optimized.
Solving this challenge is essential to maximize efficiency and ensure
the production of adequate quantities of mRNA-based therapeutics for
widespread application and accessibility.
This eBook reviews the history of mRNA-based therapeutics, examines
the upstream and downstream steps in the manufacturing process of these
products and presents innovative purification tools. Altogether, this eBook is
an essential read for anyone interested in this revolutionary therapeutic tool.
4 TECHNOLOGYNETWORKS.COM
Optimizing m Optimizing mRRNNAA P Puurirfiicficaatiotinon
Messenger RNA (mRNA) is a type of single-stranded
ribonucleic acid that can be translated into functional
proteins. Researchers have acknowledged mRNA’s
therapeutic potential since its discovery in 1961.
However, it wasn’t until 2020, when mRNA vaccines
against COVID-19 were approved, that mRNA’s
therapeutic promise was finally fulfilled. Following this
success, other mRNA therapies to treat a broad range
of diseases are now under development. The first step in
any mRNA-based therapy is the generation of a synthetic
mRNA encoding the gene of interest using the in vitro
transcription (IVT) process. This mRNA is usually
encapsulated within a delivery system before being
administered to the patient. Once inside a cell, the mRNA
directs the translation of the desired protein (Figure 1).1
This article summarizes the advances in mRNA research
since its discovery, examines its therapeutic potential and
discusses the future perspectives of the field.
Six decades of research and development
Basic research (1961–1990)
The discoveries leading to the development of mRNA-based
therapeutics began more than 60 years ago. Knowledge
accumulated during the 1950s on the structure and function
of DNA contributed to the discovery of mRNA in 1961 by
two independent research groups.2 The timeline in Figure
2 presents some of the milestones in mRNA research. For
example, the development of mRNA delivery systems
started in the same year as mRNA discovery.3 Some years
later, researchers successfully achieved in vitro transcription
of mRNA in a mammalian cell-free system for the first
5’ Cap
5’UTR
GOI
3’UTR
3’ poly(A)
tail
Protein
release
In vitro transcription
Optimal modifications
Modified mRNA
Plasmid
DNA
Gene of
interest
In vivo
administration
Integration with
the cell
Upload into
delivery system
(e.g., LNPs)
Nucleus
Protein
expression
Translation
Ribosome
1
2
5
3 4
Figure 1. Steps involved in mRNA-based therapies.1
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Optimizing mRNA Purification
time.4 Soon after, scientists successfully delivered mRNA
into cells of different animal species, which led to protein
translation and expression.5,6 The commercialization of
various materials for in vitro mRNA production began in
the 1980s, making IVT more accessible and paving the
way for numerous applications. Finally, in 1990, it was
demonstrated that synthetic mRNA also leads to successful
protein expression in in vivo models.7 This milestone can be
considered the end of the first phase in the development of
mRNA-based therapeutics. Researchers now had the tools
to produce mRNA in vitro, different methods for mRNA
delivery and were able to successfully express proteins both
in vitro and in vivo.
Exploring clinical applications (1990–2019)
Following this first phase, researchers in subsequent years
used the knowledge accumulated to develop the first mRNA
clinical applications (Figure 2).8
Protein replacement
The first study on mRNA-based protein replacement was
reported in 1992. Researchers injected mRNA coding
for vasopressin into the hypothalamus of rats unable to
produce this protein – a condition that leads to diabetes
insipidus. The injected mRNA was successfully translated
CRISPR-Cas9 mRNA
for gene editing
mRNA-based company founded
and discovery that 3’-UTR
regulates mRNA localization
Preclinical study with
intranodally injected
(DC-targeted) mRNA
First clinical trial with
mRNA using ex vivo
transfected DCs
1961 1969 1975 1978 1983 1985 1989
Discovery of mRNA
and using protamine
for RNA delivery
mRNA cap was
discovered
Cap analogue
commercialized
Cationic
lipid-mediated
mRNA delivery
1990
In vitro transcription
of isolated mRNA
Liposome-entrapped
mRNA delivery
T7 RNA polymerases
commercialized
Naked mRNA is
translated in vivo by
direct injection
2017 2013 2012 2010 2009 2002 1999 1997 1995
Using mRNAs for
cancer
immunotherapy
Antitumor T cell
response induced
by mRNA
mRNA-based
immunotherapy for
human cancer
Protective mRNA
vaccination in
influenza and RSV
Personalized mRNA
cancer vaccine for
clinical trials
2019 2020 2022 2023
Clinical trials of mRNA
vaccines for cancer and
infectious disease
mRNA-based therapeutics:
powerful and versatile tools to
combat diseases
mRNA-1273 and BNT162b
emergency use for
SARS-CoV-2 pandemic
Successful clinical trial for
personalized mRNA
vaccine for melanoma
Figure 2. Representative milestones in mRNA research and mRNA-based therapeutics development.8
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Optimizing mRNA Purification
to vasopressin and temporarily reversed diabetes insipidus
within hours.9
Cancer vaccines
In 1995, the use of mRNA as a cancer vaccine was evaluated
for the first time.10 This preclinical study demonstrated
that intramuscular injection of naked RNA encoding
carcinoembryonic antigen elicited antigen-specific antibody
responses in mice. Another milestone in mRNA-based
cancer therapy occurred in 1999 when researchers induced
antitumor T-cell responses by mRNA injection in vivo.11
In this case, mRNA coding for a human melanomaassociated
antigen was encapsulated in liposomes and
injected into mice’s spleens, inducing both specific antibody
and T-cell responses. Moreover, this injection delayed
tumor growth and significantly prolonged survival.11 These
and other preclinical studies demonstrated the potential
of mRNA-based tumor antigen vaccines for human
cancer treatment.
In 2002, the first clinical trial for an mRNA-based cancer
vaccine took place. Dendritic cells (DCs) transfected with
mRNA encoding prostate-specific antigens (PSA) were
injected into patients with metastatic prostate cancer.
Following this treatment, patients displayed PSA-specific
T-cell responses.12 Similar results were obtained when
metastatic melanoma patients were administered with
protamine-protected mRNA coding for different tumorassociated
antigens.13 These studies confirmed that
mRNA-based cancer vaccination was feasible and safe,
encouraging further clinical investigation. In 2017, the first
personalized mRNA vaccine was administered to patients
with melanoma.14 The vaccine induced specific immune
responses, decreased the metastatic rate and prolonged the
patients’ progression-free survival.14
Vaccines for infectious diseases
The potential of mRNA-based prophylactic vaccines
for infectious diseases was also explored. In 2012, two
preclinical studies demonstrated the feasibility of mRNATable
1. Examples of mRNA-based therapeutics and their applications.
Application mRNA encoding Examples
Prophylactic vaccines for
infectious diseases
Pathogen-specific antigens SARS-CoV-2 vaccines (approved)
HIV (phase I clinical trial)27
Influenza (phase III clinical trial)28
Cancer vaccines Immunostimulant molecules
Specific-tumor antigens
Solid tumors (phase I clinical trial)29
Personalized vaccines for metastatic
melanoma or epithelial cancer (phase II
clinical trial)30
Protein replacement Normal copy of the deficient/
absent protein
Cystic fibrosis (phase II clinical trial)31
Methylmalonic acidemia (phase II
clinical trial)32
Antibody production Antigen-specific antibodies Bi-specific antibodies against cancer
(preclinical)33
Antibodies against Chikungunya virus
(preclinical).34
Gene editing CRISPR/Cas9 gene editing
system
Transthyretin amyloidosis (phase I
clinical trial)35,36
Hereditary angioedema (phase II
clinical trial)37
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Optimizing mRNA Purification
based protective vaccines for influenza and respiratory
syncytial viruses.15,16 In 2017, the first clinical trial for a
prophylactic mRNA-based vaccine against rabies was
performed.17 This study served as a proof of concept and was
followed by other trials in the subsequent years.
Gene editing
Yet another modality of mRNA-based therapeutic emerged
in 2012 with the description of Cas9 as an RNA-guided
endonuclease (RGEN).18 The study described a family of
endonucleases that use dual-RNAs for site-specific DNA
cleavage and highlighted the potential of this system for
RNA-programmable genome editing. The following year,
it was shown that RGENs can be used to program genome
editing in human cells.19
mRNA as a disruptive therapeutic technology
(2019–present)
Since 2019, numerous clinical trials have been testing the
efficacy and safety of mRNA-based vaccines for various
types of cancer and infectious diseases.20 The first approved
therapeutics arrived in 2020, during the COVID-19
pandemic, with two mRNA vaccines against the SARSCoV-
2 virus (BNT162b2 and mRNA-1273).21,22 These
vaccines proved that mRNA is a powerful tool to combat
disease and further boosted the number of clinical trials
taking place. In April 2023, the results of a phase IIb clinical
trial showed that a personalized mRNA-based cancer
vaccine developed by Moderna, in combination with the
immune checkpoint inhibitor pembrolizumab developed
by Merck, cut the risks of recurrence or death by 44% in
patients with high-risk melanoma.23 Almost certainly, the
next few years will witness the approval of new mRNAbased
therapies.
mRNA-based therapeutics today
Currently, mRNA therapeutics fall into four basic
categories: prophylactic vaccines, therapeutic vaccines,
protein-encoding therapies (protein replacement and
antibody production) and gene editing therapies (Table 1).
There are hundreds of ongoing clinical trials investigating
mRNA-based therapies for different types of diseases,
including cancer, infectious diseases and genetic disorders.24
All these applications require effective delivery of the
mRNA to the target cells and different types of delivery
systems have been developed to achieve this. At present,
there are around 60 types of delivery systems, including
various nanoparticles, polymers and viruses.8,25 mRNA
(naked or encapsulated in a delivery system) can be
administered directly to the patient using different routes
(e.g., intravenous, intramuscular, inhalation).25 Yet, another
strategy is to first transfect cells ex vivo (e.g., dendritic cells)
and then inject these cells containing the therapeutic mRNA
into the patient.26
Future perspectives
mRNA-based therapeutics are a novel and versatile
alternative to traditional drugs and biologics, with enormous
potential. They offer numerous advantages compared
to other biologics. For example, mRNA overcomes the
obstacles of post-translational modification, folding and
assembly of protein-based therapies. The technology also
allows for the delivery of “instructions” to synthesize
multiple proteins (or proteins with multiple subunits) in a
dose dependent manner. mRNA is safer than DNA-based
therapies as it does not integrate into the host’s genome,
making the risk of insertion mutagenicity negligible. In
addition, as mRNA itself is only transiently active, the
burden to the host homeostasis is minimized. mRNA-based
therapeutics are also more flexible, as the safety, efficacy,
delivery and duration of action can be manipulated by
altering the structure of the original molecule.38
Despite all these advantages, challenges still exist. For
example, further research is needed to optimize delivery
systems to improve mRNA delivery, activity and cell
targeting.1 This is particularly important for applications
that need cell- or tissue-specific delivery. Improving mRNA
stability (to avoid early degradation) and ensuring prolonged
expression patterns (especially in chronic diseases requiring
sustained protein expression) is also crucial.1 Another
important challenge to overcome is synthetic mRNA’s high
innate immunogenicity, which is undesired in applications
such as protein-replacement therapies. Although modified
nucleosides can suppress mRNA immunogenicity, this
approach is expensive and imposes extra constraints in
sequence design.39 Hence, further research is needed
to address this hurdle. Exploring the most appropriate
administration route for each mRNA therapeutic is also
essential to ensure the best results.17
The mRNA manufacturing process is cell-free, making
it simpler, faster and cheaper to produce than other
biologics. However, as it is still an emerging technology,
manufacturers face a lack of clear and comprehensive
regulatory guidance.40 For instance, the impurity profile
and critical quality attributes (CQAs) of mRNA-based
products are not yet clearly defined – US Pharmacopeia
has published some guidelines for CQAs and analytical
techniques but there are no defined critical limits to
any of the CQAs.41 Moreover, establishing robust and
scalable production processes to meet the growing
demand for mRNA therapeutics is a priority in the
industry. Process development and optimization to
increase yield and quality, and to reduce costs, are still
in their early stages. Researchers and manufacturers are
actively exploring various approaches, such as improving
mRNA synthesis methods and optimizing purification
techniques. In addition, new formulations are being
developed to over-come storage and shipping drawbacks.40
Refining these processes and leveraging technological
advancements are essential to ensure the accessibility and
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widespread adoption of mRNA therapeutics for the benefit
of patients worldwide.
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mR
NATherapeuticsEngineering the Future:Unraveling the Manufacturing Workflow of How are mRNAs manufactured?UpstreamDownstreamFill and finishBreakthroughs in vaccine technology during the COVID-19 pandemic expanded the potential for mRNA-based therapeutics, opening the door to new disease treatment options. In this scenario, optimization of themRNA manufacturing workflow became essential to sustain the increasing demand of these therapeutics.This infographic provides valuable insights into the bioprocessing steps involved in the production ofmRNA therapeutics.The field of mRNA-based therapeutics is rapidly expanding and projected to reach a global market size of USD 37.76 billionby 2030.1 Various types of mRNA-based therapies are currently under development, including vaccines, mRNA-enhancedcell therapy, mRNA-based antibody production and mRNA-based protein replacements.2 These innovative therapies holdthe potential to treat a diverse range of diseases.mRNA manufacturing begins with the production of plasmids containing a gene of interest. These plasmid templates are thenused to synthesize the therapeutic mRNA through the process of in vitro transcription (IVT). Although IVT is a cell-free system, reaction mixtures may still contain impurities that can reduce translation efficiency and increase immunogenicity if delivered to the cells. Hence, impurities must be removed by chromatographic methods during downstream processing. The purified mRNA is encapsulated in a delivery vehicle to be administered to patients. A variety of technologies are employed throughout the bioprocessing workflow to ensure the quality and safety of the final therapeutic product.ApplicationsUSD 37.76billion20232022USD 33.02billion43%40%4%5%6%2%GeneticdiseasesNeurologicaldisordersMetabolicdiseasesOthersCancerInfectiousdiseasesmarket forecast to growat a CAGR of 1.7%.*percentage of clinical trials.3Target genediscoveryPlasmid creation: the geneof interest is integrated intoa plasmid DNA (pDNA)pDNA amplification: performedin host cells (typically E. coli)Purification ofsupercoiled pDNALinearization ofthe pDNAPurification of thelinearized plasmidIn vitrotranscription (IVT)Polyadenylation: theaddition of a PolyAtail enhancesmRNA stabilityCapping: the additionof the Cap1 structurepromotes translation,prevents degradationand reducesimmunogenicityThe final mRNA (capped and with the PolyA tail) is then ready fordownstream purification to remove impurities from the IVT reactionEncapsulation ofpurified mRNA withina delivery vehicle(e.g. lipid nanoparticle)Remove process-related components(truncated mRNA, DNA template,buer, nucleoside triphosphates)Remove double-stranded(ds)RNA and uncapped mRNAReduce volume andremove impuritiesReduce volume andfinal 0.2 μm filtrationConcentration adjustmentand 0.2 μm sterile filtrationPackaging of the final productin a sterile environment andstorage at -80 °CTemplatedesign andplasmidproduction1RNA polymeraseLinearizedpDNAmDNAPolyA polymerasePolyA tailmRNAsynthesis357924Polishing6810AnitychromatographyUltrafiltrationand buerexchangePlasmidpurificationUltrafiltrationand buer exchangeFinal formulationand filtrationEncapsulation Filling andpackagingPharmaceutical Grade Reagent. For Manufacturing and Laboratory Use Only. © 2023 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.Discover more about mRNA purification solutions References1. Global mRNA therapeutics market report 2022. Research and Markets. https://www.researchandmarkets.com/reports/5644939/global-mrna-therapeutics-market-size-share-and?utm_source=GNOM&utm_medium=PressRelease&utm_code=zh2p66&utm_campaign=1787683+-+Insights+on+the+mRNA+Therapeutics+Global+Market+to+2030+-+Featuring+Translate+Bio%2c+GSK%2c+Pfizer+and+AstraZeneca+Among+Others&utm_exec=jamu273prd. Published: July, 2022. Accessed: 29 June, 2023.2. Qin S, Tang X, Chen Y, et al. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022;7(1):166. doi: 10.1038/s41392-022-01007-w. 3. Webb C, Ip S, Bathula NV, et al.. Current status and future perspectives on mRNA drug manufacturing. Mol Pharm. 2022;19(4):1047-1058. doi: 10.1021/acs.molpharmaceut.2c00010.Applications of mRNA therapeuticsGlobal mRNAtherapeutics marketPlasmid identity and quality οAgarose gel electrophoresis οSequencingAnalyticsPlasmid quantitation οUV absorbancePlasmid purity (Quantitation of host cell DNA, RNA and proteins) οqPCR, οRT-qPCR οRP-HPLCAnalyticsIdentity (sequence confirmation) οRT-qPCRRNA content οRT-qPCR οRT-dPCR οUV absorbance οFluorescence-based RNA assaysPurity (detection of residual DNA, proteins and dsRNA) οqPCR οImmunoblotIntegrity οQuantitation of fragmented mRNA using capillary gel electrophoresis οQuantitation of uncapped mRNA using UPLC, RP-HPLC, LC/MS οQuantitation of mRNA with no PolyA tail using RP-HPLCSafety οEndotoxins οBioburdenAnalyticsParticle size οDynamic Light ScatteringmRNA encapsulation οFluorescence-based RNA assaysLipid identity, lipid content and impurities οHPLC οLC/MS
11 TECHNOLOGYNETWORKS.COM
Optimizing mRNA Purification
Enabling mRNA-Based Therapeutics
Development Through Efficient and
Scalable mRNA Purification Methods
The possibilities of mRNA-based therapeutics appear
limitless, with a growing number of applications swiftly
populating the clinical pipeline. As a result, the global
market for these products is experiencing remarkable
growth. To keep up with this surge in demand, the industry
is actively exploring efficient and scalable manufacturing
methods to support the commercial production of mRNAbased
therapeutics.
A significant breakthrough in this field comes from the
development of affinity resins that can bind mRNA through
a simple AT base-pairing mechanism. These innovative
resins serve as a purification platform for the purification of
all mRNA molecules that contain a polyA tail.
Watch Now
Watch this webinar to learn more about the advantages of affinity capture of
mRNA and how it maximizes the efficiency of mRNA purification.*
*webinar originally cited from BPI international
12 TECHNOLOGYNETWORKS.COM
A SPONSORED PUBLICATION FROMThe mRNA Therapeutics BoomPhoto: Getty Images, NANOCLUSTERING/SCIENCE PHOTO LIBRARY© Gen Publishing – April 2021Scalable Purification of InVitro Transcribed mRNA Accelerates mRNA-Based Therapy Development mRNAis beautifully simple; it provides your body instructions. Analogous to computer code, mRNA programs the body to produce specific proteins giving this molecule utility in a myriad of therapeutic approaches, including vaccines against common and rare infectious diseases, oncology indica-tions, and protein replacement treatments for genetic disorders. The wide diversity of mRNA-based thera-peutic applications has led to increased interest in using synthetic mRNA.In the early 90s, scientists demonstrated efficacy when using mRNA as a potential therapy.1,2 But interest in antibodies, whose potential was more broadly accepted, took over in prominence. Today, mRNA How to Simplify Workflows and Maximize the Efficiency of the mRNA Purification Process with POROS™ Oligo (dT)25 Affinity Resin.
13 TECHNOLOGYNETWORKS.COM
2 | GENengnews.com
The mRNA Theraptutics Boom
is back in focus with the general acknowledgment
that these types of therapies do not target and manipulate
genes and DNA.
To date, no mRNA-based therapy has been commercialized
though some are in late-stage trials or
approved for emergency use, such as vaccines
targeting SARS-CoV-2. The vast majority of therapies
in development apply to relatively small populations,
thousands to hundreds of thousands of patients. Even
mRNA-based cancer immunotherapies would serve
a significantly smaller subset of patients than a global
vaccination campaign.
Since preclinical and early clinical pipelines of most
of these mRNA therapies only required a few liters
of materials, traditional laboratory-scale approaches,
such as precipitation, were leveraged for production.
“We are going to see mRNA therapies start to move
quicker to the clinic,” says Kelly Flook, PhD, Senior
Product Manager, Purification Products, Thermo
Fisher Scientific, ”and a greater acceptance of protein
replacement therapies and varied immunological
approaches than are currently being evaluated.”
Scaling-Up Options
The drive to rapidly develop a COVID vaccine put a
focus on large-scale mRNA manufacturing. The limits
of research-scale purification techniques were realized,
and available purification methods became a bottleneck
for commercialization.
To resolve this challenge, different options are under
investigation. For example, scaling up reverse-phase
chromatography is of interest. “It is scalable but not
as efficient as an affinity approach in purifying the
product and removing process impurities,” says Flook.
In addition, reverse-phase chromatography uses flammable
solvents requiring the removal of detrimental
post-purification impurities. Safety is a concern, as well
as the necessity and expense of building a chemical
manufacturing site to handle the solvents.
“Aqueous-based techniques, ion exchange, and
affinity, are commonly used in research, and a similar
solution is desirable for scale-up production of mRNA,”
says Flook. Process speed also plays an important role.
“A few years ago, as more companies began working
on therapeutics in this space, we saw an increase in
inquiries about large-scale mRNA purification,” says
Flook. “Most resins on the market were research-scale
technology, such as our popular Dynabead option
with a polyT on the surface. Initially, we provided
custom resins until the momentum grew, and it made
sense to develop a generic product. So we took the
polyT technology and applied it to our bioprocessing
POROS™ resins.”
Producing mRNA
“RNA is made using a process called in vitro transcription
(IVT). During the IVT process, DNA is converted
to RNA,” explains Venkata Indurthi, PhD, Vice President,
Research and Development, Aldevron. “It is
critical to get rid of all the impurities after the reaction
is complete, including any residual raw materials,
because they can trigger nonspecific immune
responses.”
Compared to DNA, RNA is fragile; harsh purification
techniques are unsuitable. RNA also has secondary
structures that can impact purification.
“There are multiple ways of purifying RNA, chargebased
methods, precipitation-based methods, and
others like hydrophobic interaction chromatography
(HIC),” says Indurthi. “The specific advantage of the
affinity oligo dT approach is that you can easily get rid
of the impurities generated during IVT.”
Affinity chromatography, a highly-scalable method,
has earned its credits in the development of biologics,
such as the use of Protein A for the purification of therapeutic
antibodies and, more recently, anti-AAV resins
in gene therapy workflows. An effective affinity purification
step can help to simplify biomolecule downstream
processing, reduce the number of purification
steps, and lower the overall cost of goods in biotherapeutic
manufacturing.
Thermo Fisher’s new affinity-based mRNA chroma14
TECHNOLOGYNETWORKS.COM
3 | GENengnews.com
The mRNA Theraptutics Boom
tography resin, POROS Oligo (dT)25, was specifically
developed for the scalable purification and isolation of
mRNA from the IVT manufacturing processes.
“We worked closely with our customers to develop
the resin, including AmpTec, a leading RNA CRO, in
Europe,” says Flook. “They were tasked to develop a
scalable, efficient method for manufacturing mRNA
that would allow them to take on large-scale vaccine
manufacturing.”
The Oligo dT Affinity Approach
POROS Oligo (dT)25 is based on POROS resin technology,
a poly(styrene-divinylbenzene) base bead
coated with a proprietary functional hydrophilic
coating to reduce nonspecific binding. A dT-25–
poly-deoxythymidine ligand is attached to the bead
surface.
Since every mRNA has a polyA tail for molecular
stability, the resin is a platform solution. “Across a
range of mRNA sizes and constructs, you get equivalent
recovery, purity, and yield,” says Flook. “The size or
sequence does not matter; the resin can be used to
purify anything that has a polyA tail.”
Use of the resin is straightforward; the polyT ligand on
the bead binds to the polyA tail of the mRNA. In brief,
hydrogen bonding occurs as salt neutralizes the backbone
of the mRNA and the polyT, allowing flushing
and removal of the non-bound IVT components. After
the salt is removed, the hydrogen bonds break, and
the polyA containing mRNA is eluted.
Typically, this affinity approach is used at the beginning
of the purification scheme to remove process-related
impurities, such as DNA templates, nucleotides,
enzymes and buffer components, and other constituents
such as mRNA without a polyA tail.
In some cases, a product-related impurity can result
from IVT, such as double-stranded RNA (hairpin) or
another undesirable species that has a polyA tail.
“Then we suggest adding a second polishing step
with ion exchange or HIC. The loop-back doublestranded
effect can also be engineered out during the
IVT process,” says Flook. “Another optional way to use
the resin is downstream as a final polishing step or for
buffer exchange. You can elute in water and formulate
directly from that.”
Depending on the application, mRNA will vary in size
and design of the backbone. “As a CDMO, Aldevron
has been supplying RNA for a couple of years at
all quality grades from RUO to GMP, and we are
continuing to invest heavily in the space,” says Indurthi.
“RNA has a 5’ UTR and a 3’UTR and varying sequences.
THERMO SCIENTIFIC™ POROS™ OLIGO (dT)25 AFFINITY RESIN
Designed for the isolation and purification of mRNA from in vitro
transcription manufacturing processes
Simplified workflow to maximize efficiency and reduce complexity of
subsequent polish steps
LINKER
A A A A A A A A A A A A A A A A
Poly-A mRNA
Poly-dT ligand Fig. 1 POROS Oligo (dT)25
affinity resin consists of a polydeoxythymidine
(dT-25) ligand
attached to a 50μm rigid, porous
bead through a proprietary
linker. The poly-dT ligand allows
binding with poly-A tailed mRNA
molecules through AT base pairing.
Simple mRNA capture through AT base pairing
Easy to use: load in NaCl and elute in water
Dynamic Binding Capacity up to 5 mg/mL for 4000nt mRNA
> 90% recovery
Excellent scalability
Non-animal derived
OPTIMIZING PROCESS CONDITIONS FOR RESIN USE
Precipitation point determination Salt type and concentration
POROS Oligo (dT)25 for mRNA production
Affinity
purification
Removal of process related
components such as DNA
template, nucleotides, enzymes
and buffer components
Removal of product related
components such as mRNA
without a polyA tail
POROS Oligo(dT) 25
Affinity
polish
Polishing of final product
Buffer exchange/formulation
POROS Oligo(dT) 25
IP-RP /
HIC / IEX
Removal of dsRNA and
uncapped RNA from the final
product
Removal of secondary RNA
structures if needed (e.g.
hairpin)
POROS HIC or IEX
Fig.1: POROS Oligo (dT)25
affinity resin consists of a
polydeoxythymidine (dT-25)
ligand attached to a 50μm
rigid, porous bead through a
proprietary linker. The poly-dT
ligand allows binding with
poly-A tailed mRNA molecules
through AT base pairing.
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The mRNA Theraptutics Boom
We do not control the design, and different designs
can trigger complexity in the purification process.”
The POROS Oligo (dT)25 resin, as a purification platform,
can be cross applied to different constructs, emphasizes
Flook. The resin addresses the current challenges
involved with large-scale mRNA purification for potential
clinical use by simplifying the downstream process,
increasing purity and yield, and allowing for scale-up
without the use of toxic chemicals.
Validating the POROS Oligo (dT)25 Resin
Standard mRNA contains between 1000-5000 nucleotides,
which is the size the resin was designed to optimally
operate in. Optimized conditions can maximize
the binding capacity, even for larger RNAs, to achieve
a more efficient purification process.
“From a binding perspective, we have seen customers
achieving up to 5mg/ml of 4000 bp mRNA,” says Flook.
“This is significantly higher than what you would see
with some of the research products.”
A standardized experiment looked at the binding
capacity of three different sizes of mRNA without optimizing
conditions for each independently. Size did not
impact recovery even with samples straight from an
IVT mixture. Low nonspecific binding and the affinity
approach only allow polyA species to bind. Recovery
rates are greater than 90% and, in most cases, greater
than 95%. Adjusting the column size according to
need makes the process flexible and scalable.
From a purity perspective, evaluation of the proteinaceous
load showed that primarily enzymes from
the IVT mixture are seen in the flow-through but not
detected within the elution peak. “If we analyze the
fractions from the elution peak starting with about
17% product-related impurities, a reverse-phase spin
column slightly reduces it to 13%,” says Flook.
“All of the process-related components are removed
with the POROS Oligo (dT)25 resin, and we see a
significant reduction in product-related impurities,”
continues Flook. “This means our affinity resin does a
much better job of removing non polyA species than
reverse phase.” All remaining product-related impurities
are polyadenylated, as expected.
Overall, the POROS Oligo (dT)25 resin demonstrated
efficient elution at different load concentrations and
excellent recovery with high purity regardless of
sample type.
The POROS Oligo (dT)25 affinity resin demonstrates:
Efficient elution at different load concentrations
Excellent recovery with high purity independent of sample type used
Below
detection
limit
Purification with POROS Oligo (dT)25 resin leads to significant
reduction of impurities
Fig. 4 Chromatogram showing efficient separation of a 2000nt mRNA from an IVT mixture at a
load concentration of 2 mg/mL. Elution was performed using H2O and yielded in >95% recovery.
Fig. 5 High recovery and purity independent of sample type used. Recovery of mRNA from pure
mRNA and unpurified IVT mixture, showed no differences (left). Amount of protein was determined in
load, flow through (FT) and elution pools (right). No proteins were detected in the elution pool, indicating
excellent impurity removal of IVT mixture products.
CONCLUSIONS
The POROS Oligo (dT)25 resin addresses the current challenges involved
with large scale mRNA purification used in potential mRNA-based therapies.
Impurity analysis by HPLC
Fig. 6 Efficient removal
of impurities compared
to the spin column
method. HPLC analysis of
unpurified IVT mixture,
spin column purified
mRNA and mRNA purified
with POROS Oligo (dT)25
resin.
7.0 8.0 9.0
13.4%
15.7%
6.2%
0.0 2.0 4.0 6.0 8.0 10.0
0
100
200
300
Time (min)
0
50
100
150
Current spin column purification method
Absorbance, 260nm (mAU)
0
200
400
600
Unpurified IVT mixture
Purified with POROS Oligo (dT)25 resin
mRNA
Improved impurity
clearance
0
5
10
0
500
1000
0 10 20 30 40 50 60 70 80
Elution Volume, CV
Sample load Washing Elution Base Equilibrate/Flush clean Equilibrate
• DNA – by pass
• Enzymes – by pass
IVT mixture:
• ss-mRNA
• ds-mRNA
• DNA
• Enzymes
Fig. 2: High recovery and purity independent of sample type used. Recovery of mRNA from pure mRNA and unpurified IVT mixture,
showed no differences (left). Amount of protein was determined in load, flow through (FT) and elution pools (right). No proteins were
detected in the elution pool, indicating excellent impurity removal of IVT mixture products.
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The mRNA Theraptutics Boom
“In reverse-phase, as the column is reused, the impurities
start to follow the column, and that can change
the column’s effectiveness,” says Flook. With POROS
Oligo (dT)25 resin, the impurities are removed without
first binding them to the column, rendering the
number of impurities irrelevant. The resin is reusable
and base stable for in-place sanitization.
“Each mRNA is designed for a specific application; the
Cadillac version of purification is not always required.
We have different purification strategies for different
applications,” says Indurthi. “As an example, if mRNA is
being used for a vaccine, you do not need to get rid
of certain impurities, but if you are making RNA for
therapeutic use, you do because they will trigger an
unwanted immune response.”
In general, oligo dT purification can be used as a
stand-alone purification. “We have seen pure RNA
using this approach; it is a better way. The purer the
end product, the less you need to get a response,” says
Indurthi. “Secondary structure, size, and other factors
affect the recovery, not the resin itself.”
“With all of the oligo dT resins we have tested, we
observed that POROS Oligo (dT)25 has a very high
binding capacity and provides the ability to purify
larger mRNAs as compared to other products,” adds
Indurthi. “It works really well for our applications and
will be added to our repertoire for mRNA production.”
Looking to the Future
The COVID mRNA vaccines are expanding the already
significant interest in the RNA space and taken mRNA
manufacturing to a new level. Going forward, the
biggest bottlenecks will be the DNA templates and
the enzymes needed for synthesis. Luckily, the boom
has also catapulted development activity in resins,
nucleotides, and enzymes.
Hurdles still remain for mRNA therapeutics for different
indications. One of the challenges is how to get the
mRNA to the right cells, especially when targeting
specific cancers. “We are going to see a lot of development
around delivery systems,” says Flook.
However, these challenges will not change the molecule’s
current trajectory. mRNA therapeutics are poised
to become an important element in the healthcare
landscape in the coming years. n
References:
Wolff J.A. et al. Science 247, 1465-1468 (1990)
Martinon F.et al. Eur J Immunol 23, 1719-1722 (1993)
Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use Only.
Fig. 3: Efficient removal
of impurities compared
to the spin column
method. HPLC analysis of
unpurified IVT mixture,
spin column purified
mRNA and mRNA
purified with POROS
Oligo (dT)25 resin.
TRADEMARKS/LICENSING
© 2020 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.
This information is not intended to encourage use of these products in any manner that might infringe the intellectual property rights of others.
The POROS Oligo (dT)25 affinity resin demonstrates:
Efficient elution at different load concentrations
Excellent recovery with high purity independent of sample type used
Purification with POROS Oligo (dT)25 resin leads to significant
reduction of impurities
load, flow through (FT) and elution pools (right). No proteins were detected in the elution pool, indicating
excellent impurity removal of IVT mixture products.
CONCLUSIONS
The POROS Oligo (dT)25 resin addresses the current challenges involved
with large scale mRNA purification used in potential mRNA-based therapies.
Use of this resin will:
Simplify your mRNA downstream process
Increase purity and yield
Allow for scalable mRNA purification process without the use of toxic
chemicals
Customer testimonial – “This promising technology will allow us to meet the increasing
demands of mRNAs from our customers” – Peter Scheinert, CEO AmpTec
Samples used were kindly provided by AmpTec
Impurity analysis by HPLC
Fig. 6 Efficient removal
of impurities compared
to the spin column
method. HPLC analysis of
unpurified IVT mixture,
spin column purified
mRNA and mRNA purified
with POROS Oligo (dT)25
resin.
7.0 8.0 9.0
13.4%
15.7%
6.2%
0.0 2.0 4.0 6.0 8.0 10.0
0
100
200
300
Time (min)
0
50
100
150
Current spin column purification method
Absorbance, 260nm (mAU)
0
200
400
600
Unpurified IVT mixture
Purified with POROS Oligo (dT)25 resin
mRNA
Improved impurity
clearance
Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use Only.
17 TECHNOLOGYNETWORKS.COM
18 | 2022 | GENengnews.com
mRNA Vaccines and Therapeutics
mRNA-based therapies catapulted
to the forefront of public
consciousness in the form of vaccines
against the SARS-CoV-2 virus. After that
success, mRNA therapeutics are now
being developed for an ever-growing
number of indications and applications
that include cancer, cystic fibrosis, and
infectious diseases, as well as gene and
stem cell therapies based upon either
gene replacement or gene editing.
However, the purification bottleneck
must be solved before these therapies can be
scaled up and produced in adequate quantities
for clinical trials or commercialization.
“Traditionally, small-scale tools and products
have been used to purify mRNA, such
as reverse-phase high-performance liquid
chromatography (HPLC), precipitation, and
in some cases, cellulose-based
chromatography,” says Sirat Sikka, field
application scientist at Thermo Fisher
Scientific (Thermo Fisher). Those methods
can be used to purify a few grams of mRNA
and are adequate for bench work and
some applications. Scale-up for clinical trials
and commercialization, however, requires
the ability to purify tens of grams or even
tens of kilograms of mRNA.
“At Thermo Fisher, we understood the
importance of mRNA and knew, even
before the pandemic, that mRNA would
be widely used,” Sikka recalls. Since then,
scientific and trade journals alike have cited
mRNA therapeutics and vaccines as disruptive
advances that can change the future of
medicine and ease of manufacturing, and
the ability to target pathways that otherwise
are undruggable. Industry analyst Research
and Markets predicts the global segment
for mRNA therapeutics will grow from $46.7
billion in 2021 to $101.3 billion by 2026.
That’s a compound annual growth rate of
16.8%. (https://www.researchandmarkets.
com/reports/5441159/mrna-therapeuticsand-
global-markets-2021-2026).
Relieving the bottleneck
To be ready for such rapid growth in
mRNA development, Thermo Fisher began
developing a new affinity chromatography
resin to isolate and purify mRNA long before
the technology became a “household word.”
The team sought to develop a resin enabling
improved recovery, increased purity, and
enhanced reproducibility.
The POROS™ Oligo (dT)25 Affinity
Resin—the resulting product—is a 50 μm
poly(styrene-co-divinylbenzene) crosslinked
porous bead functionalized with
deoxythymidine (dT) strands that bind
to mRNA via the poly-A tail (a chain of
adenine nucleotides) that is on the threeprime
end of all mRNA molecules.
One of the challenges is the size of mRNA.
It is a large molecule —20 to 50 nm or greater
in size that varies with construct length and
solution composition—so there can be limitation
to diffusion through the chromatography
media and, therefore, hindrance to mass transfer,
Sikka explains. Because the POROS™ beads
have large throughpores the surface area
available for interaction between the resin and
mRNA molecule is increased leading to higher
capacity. The large pores also result in a reduced
mass transfer resistance, which helps to
improve process efficiency and productivity.
“The POROS Oligo (dT)25 Affinity Resin
minimizes the need to deal with organic solvents
that are often used with HPLC systems,”
Sikka continues. “Using organic solvents in
large volumes becomes an issue for manufacturing.”
She cites safety concerns regarding
solvent disposal as well as the need to
retrofit facilities to deal with them.
Instead of using toxic chemicals, after
mRNA synthesis the column is loaded “with
mRNA plus salt (for example, NaCl).” This neutralizes
the negative charges on the RNA molecules
so the poly-A tail can bind with the dT
strands on the beads. Then, she says, “Elution
can be performed using a low-conductivity
buffer, or even water in some cases.” Impurities
and salt ions are washed away. With the
sodium removed, the negative charges on
poly-dT and the poly-A tail repel each other,
freeing the purified mRNA and generating a
recovery, typically above 90%, depending on
the elution buffer and mRNA construct size.
From bench to manufacturing
The POROS Oligo (dT)25 Affinity Resin is
designed for scalable purification processes,
New Bead Technology Enables Commercial-Scale
mRNA Purification
By Gail Dutton
Affinity chromatography beads designed specifically for mRNA and FPLC
eliminate toxic chemicals and purify tens of grams, removing a key bottleneck
18 TECHNOLOGYNETWORKS.COM
GENengnews.com | 2022 | 19
so it is used to pack fast protein liquid chromatography
(FPLC) columns. “The columns
can be packed to multiple column size according
to customers’ needs, based on their
process development and optimization,”
Sikka says. “We also have small-volume prepacked
columns and Robocolumns. The 1
mL and 5 mL prepacked columns could be
used with HPLC if needed, but that would
only allow customers to purify very small
sample volumes, may require re-plumbing
and is usually not ideal for process development,
so switching to FPLC is preferred. What
customers mostly are looking for when they
choose this resin is to scale-up purification,
so they use it with FPLC systems.”
Sikka says this affinity resin is a good option
for scientists interested in developing a
platform process that can be implemented
for a variety of mRNA constructs. One of the
benefits of using mRNA is that the same
construct backbone could potentially be
used to express different proteins. As a result,
scientists can potentially use a platform process
for multiple mRNA programs.
While researchers may switch out the gene
of interest, “they could still be working with
mRNA of comparable sizes,” Sikka explains.
“For example, depending on the protein they
are trying to express, if the size range of all the
constructs is between 4000 to 6000 bases,
they could use this as the first capture step
and develop a platform process.” Working with
much larger mRNA, such as self-amplifying
could require some additional development.
As a platform technology, the first purification
step with POROS Oligo (dT)25 would
remove digested DNA template, nucleotides,
enzymes, and buffer components.
This could be the only step in the process
before concentration and buffer exchange. If
needed, a second chromatography step can
be developed with POROS™ hydrophobic
interaction chromatography (HIC) or anion
exchange chromatography (AEX) resins to
remove double-stranded RNA and uncapped
or residual incomplete RNA transcripts.
“Starting with this resin during the research
and discovery phase lets scientists continue
using the same purification resin all the way to
commercial manufacturing,” she says. “This also
eases the process of transitioning from one
mRNA construct to another of similar size.”
As she elaborates, “Once used in a process,
the resin is already in the system and
accepted by the customer’s quality team.”
Additionally, scientists needn’t redevelop
their purification steps during each phase of
scale-up, which minimizes the need to onboard
a variety of chemicals and solutions
or develop different buffer compositions,
thus accelerating process development and
reducing time to market. The resins are also
reusable, which reduces the cost of goods.
“Importantly, POROS Oligo (dT)25
Affinity Resin beads are available for GMP
production, and we provide the regulatory
support package,” Sikka says.
Transitioning to a new bead
Switching to the POROS Oligo (dT)25
Affinity Resin is just a matter of ordering the prepacked
columns if customers already use FPLC.
“A lot of our customers, however, are
still at the research scale and are interested
in scaling up,” Sikka points out. “They don’t
necessarily have FPLC systems, and are
trying to understand their options.”
In those instances, she recommends
ordering loose POROS Oligo (dT)25
Affinity Resin, which can be used in spin columns
or microfuge tubes in a batch mode.
If that works well for their purposes, they
may consider investing in an FPLC for further
purification optimization and scale-up.
“Thermo Fisher is very focused on the
modern day,” Sikka says, with solutions that
address current and emerging purification
challenges. Today, that means an intense
focus on mRNA purification.
As interest in mRNA therapeutics continues
to increase, the company’s R&D is
focusing on understanding the complexity
associated with purifying self-amplifying
mRNA, removal of product related impurities
such as double-stranded RNA and abortive
transcripts, and use POROS Oligo (dT)25
Affinity Resin and other technologies to resolve
existing and emerging challenges.
mRNA Vaccines and Therapeutics
Before mRNA-based therapeutics can be produced in adequate quantities for clinical trials and
commercial distribution, it will be necessary to remove a key bottleneck: mRNA purification. Existing
methods usually purify just a few grams of mRNA, not the tens of grams or even the tens of
kilograms needed. To improve mRNA purification, Thermo Fisher has developed the POROS Oligo
(dT)25 Affinity Resin. Unlike alternate purification approaches, chromatography with a bead-based
resin has excellent scalability. Notably, the resin selectively captures mRNA via the polyadenylated
tail using simple salt and water purification steps. Artur Plawgo/Getty Images
19 TECHNOLOGYNETWORKS.COM
Optimizing mRNA Purification
Optimizing mRNA Purification
Conditions by Using a High-
Throughput Approach
The advancement of chromatography solutions for
purifying mRNA is of utmost importance in optimizing the
manufacturing process of mRNA-based therapeutics and
meeting the growing demand for these products.
Affinity resins capable of specifically binding mRNA offer
a valuable tool that helps to address the selectivity and
capacity requirements for the large-scale manufacturing of
mRNA therapeutics.
Watch Now
Watch this webinar to discover how you can implement a simplified mRNA
purification workflow and enhance the efficiency of affinity chromatography
in the mRNA manufacturing process. Don’t miss this opportunity to discover
the latest techniques and strategies to improve mRNA purification for
therapeutic applications.
Optimizing mRNA Purification
Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use Only. © 2023 Thermo Fisher Scientific Inc.
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boost your mRNA
purification process
Access to expert industry insights can help you streamline your
mRNA process development and manufacturing.
That’s why our virtual Chromatography Learning Lab offers
a broad collection of mRNA webinars, articles, eBooks,
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Here, you can discover expert approaches to topics
such as:
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• Addressing large-scale purification challenges
• Scalable purification methods
• Optimizing mRNA purification conditions
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