mRNA Vaccines

A New Era of Biotherapeutic Development

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TABLE OF CONTENTS

mRNA Vaccines
A New Era of Biotherapeutic Development

04
Introduction

05
Changing the Status
Quo of Vaccine Production

10
Increasing mRNA Capabilities and
Capacity For Growing Market Needs

16
Key Insights Into Overcoming
mRNA Process Challenges

24
Translation of RNA
Medicines from Design to Clinic

© GEN Publishing Inc., January, 2022

GENengnews.com |3
Introduction
mRNA Vaccines: A New Era of
Biotherapeutic Development
2020 was devastating. It was also a year of firsts. Fueled by
decades of research and widespread global cooperation,
the first mRNA-based vaccines against SARS-CoV-2 were
produced, tested, and injected mere months after COVID-19
hit. Science spoke, telling us these vaccines—the first
mRNA drug products—were effective at protecting against this prolific killer. Production
processes were put into action at lightning speed, but these were far from optimized.

With mRNA technology proven, the floodgates are now open to a deluge of possibilities. Can we
use mRNA to make vaccines to other infectious agents? To treat cardiovascular disease or cystic
fibrosis? To target one person’s unique cancer? Countless firsts are on the horizon. But to realize the
sheer volume and breadth of mRNA uses being considered, we as an industry need to do what
we’ve done with mAbs to bring yields and efficiencies up and costs down. And we must continue
to adapt to the trends for a greater number of biologics at smaller scales, produced locally.

That means developing platform processes for all steps, from making the DNA template and mRNA
molecules through purifying, encapsulating, filling, and testing. And ensuring that these processes are
scalable to dose one patient or millions. And finally, baking in the flexibility to manufacture anywhere
in the world and to adopt innovations waiting in the wings to shave time, labor, and cost. Modular
manufacturing solutions based on single-use technology – with operations integrated and automated
across the workflow – were one answer for mAbs. And they offer the same promise for mRNA.

We’ll learn quickly from our collective experience with other biologics. And we’ve got some of
the brightest and most industrious on the case to solve the challenges unique to mRNA. Read
this collection of articles to join the mRNA evolution and bring more ‘firsts’ to patients.
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

Changing the Status

Quo of Vaccine Production
Precision NanoSystems maintains that mRNA and
LNP technologies, microfluidics, and distributed
manufacturing will revamp vaccine production

The rapid onset and spread of SARS-CoV-2 COVID-19 has enhanced existing disparities
resulting in the global COVID-19 pandemic in wealth and resources where high-income
has had devastating effects on global health, countries have over 200% population coverage
education, and economies. The long-term of vaccine doses, leaving developing countries
fallout of this truly catastrophic event has struggling to gain access to supply even with
yet to be fully realized as many countries efforts by the COVID-19 Vaccines Global Access
are still battling rising infection rates, the Facility (COVAX Facility) to facilitate equitable
emergence of more infectious variants, vaccine global distribution.1 The focus on self-recovery
supply and distribution issues, and more. has overshadowed the need for global

GENengnews.com |5
 Changing the Status Quo of Vaccine Production

immunization to overcome this pandemic. industry to replace outdated technologies

Despite the admitted early failures in the and embrace new innovations can help better

response to SARS-CoV-2, retrospective reports prepare the world for future pandemics and

applaud the unprecedented speed at which democratize global access to vaccines.

COVID-19 vaccines were developed and The mRNA technology behind COVID-19 vaccines
deployed—speed to clinic had never been more is poised to disrupt the status quo where speed,
important. The Moderna vaccine (mRNA-1273) versatility, and flexible platform production
went from sequence selection to preclinical represent significant advantages to improve
evaluation in 63 days and was in commercial global vaccine manufacturing capabilities.
production in just 10 months.2
Convergence of innovative technologies

The disease-agnostic Decades of mRNA research have been

brought to fruition by the Moderna and
mRNA/LNP platform can be Pfizer-BioNTech COVID-19 vaccines, which rely
easily adapted to produce on mRNA to deliver the genetic instructions
encoding the SARS-CoV-2 spike protein to
a wide range of RNA-based cells.4 In contrast to classical vaccines, RNA
treatments that can expand technology leverages the cells’ own transla-

the scope of the technology tional machinery to produce the viral proteins
that will activate the immune system.
beyond infectious diseases A critical technology that was essential to the
to broader disease targets. successes of the mRNA vaccines is the lipid
nanoparticle (LNP) delivery system used to
By comparison, the development of the mumps get the mRNA inside cells. LNPs encapsulate
vaccine, which previously held the fastest and protect the mRNA to facilitate its entry
record, took four years from the initial isolation into cells where it is translated and presented
of the virus to regulatory approval in 1967.2,3 as a membrane-bound spike protein antigen

Lessons learned during the development of that can elicit an immune response.

COVID-19 vaccines underscore the need to The modularity of mRNA and LNP technolo-
reimagine the current paradigm of vaccine gies can provide the agility for rapid, iterative
production from design to manufacturing prototyping of vaccine variants without the
methods, which has lagged severely behind. need for process modification or revalidation
Reinvigorated investment in the vaccine since common manufacturing processes can be

6| GENengnews.com
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

Precision NanoSystems has

developed a microfluidic-based
continuous flow manufacturing
platform for the development
and manufacture of nanoparticle
drug delivery systems. The
platform, called NanoAssemblr®,
includes Blaze™ (shown here), a
system that allows nanoparticle
formulations to be scaled rapidly
in late preclinical development.

leveraged.5 The disease-agnostic platform can are needed. A manufacturing technology

be easily adapted to produce a wide range of that scales easily and practically from the
RNA-based treatments that can expand the bench to commercial manufacturing is a
scope of the technology beyond infec- critical issue in translating RNA medicines.
tious diseases to broader disease targets, Next-generation microfluidic mixing devices
which could make alternate manufacturing that easily integrate into existing workflows and
models more economically feasible. rapidly scale across all stages of development
As RNA-LNP technology continues to mature, and manufacturing are improving vaccine time
the following factors will provide efficiencies to market, formulation robustness, and repeat-
to allow more flexible manufacturing designs: ability. Innovative technologies like these are
better mRNA constructs; LNP modifications helping address uncertainties related to both
to improve stability; increases in in vivo the development and operation of large-scale
efficacy; and reduced dosing requirements. production processes, as RNA-LNP technology
becomes more widespread and readily adopted.
The success of mRNA vaccines has driven an
acceleration of other RNA-enabled treatments Decentralized and integrated
that will only exacerbate the already strained manufacturing pave the way forward
capacity to produce the COVID-19 vaccines; Centralized, single-product, single-facility
therefore, new solutions to address bottle- manufacturing, while providing economies of
necks in development and manufacturing scale for classical and current COVID-19 mRNA

GENengnews.com |7
 Changing the Status Quo of Vaccine Production

vaccine production, is inherently inflexible more economical. The availability of modular-

and difficult to pivot quickly in pandemic ized GMP production suites and advances in
responses.6 This model presents single points bioproduction technologies like microfluidics,
of failure in the supply chain that are vulnerable digital or “4.0” automated bioprocess capabil-
to materials and personnel shortages, export ities, and inclusion of single-use equipment,
bottlenecks, and complex cold chain logistics. constitute key components to build out such
All impact production and distribution, resulting manufacturing designs. As well, once produc-
in incomplete geographical coverage. tion processes are established and validated,
the technology could be adopted by other
Techno-economic assessments suggest that
facilities to form a network of manufacturing
the facility footprint required to produce
sites with harmonized processes to grow the
RNA vaccines could be two to three orders of
global vaccine production capabilities.5
magnitude smaller than conventional vaccine
production processes with 1/20th to 1/35th the A report by the World Economic Forum lists the

upfront capital investment.5 This could make establishment of a consortium of biofoundries

a geographically distributed, decentralized to foster accelerated development and large-

manufacturing model more feasible. Moreover, scale vaccine production as a critical element to

integrated manufacturing designs where RNA combating pandemics.7 The concept of foundries

drug substance production, LNP formulation, has revolutionized manufacturing in other indus-

analytical testing, and fill/finish operations trial sectors (such as the semiconductor sector)

are localized in a single facility aligns with the and is a logical path forward for RNA vaccine

desire of many countries to establish their own production. Support for this is evidenced by

domestic vaccine manufacturing capabilities. initiatives such as R3 (a $60 million project jointly
funded by CEPI and Wellcome Leap), which
In the face of current COVID-19 vaccine aims to establish a global network of biofound-
shortages, localized manufacturing can support ries to democratize access to state-of-the-art
national vaccine requirements as well as offer the manufacturing centers that will accelerate the
capability to handle emerging regional variants. pace and diversity of RNA biologics.8 This could

Additionally, the modular, disease-agnostic open up a new era of biomanufacturing with

nature of RNA-LNP means integrated manu- the agility to pivot rapidly for the emergency

facturing facilities could be used to produce capacity needed for rapid pandemic responses.

a number of RNA therapeutics where shared Of course, continued collaboration and commu-
resources (such as equipment and personnel) nication among all stakeholders from researchers,
and costs could make decentralized facilities developers, manufacturers, and regulatory

8| GENengnews.com
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

agencies will be paramount to support these Pandemic_final.pdf. Published: May 12, 2021. Accessed:
November 5, 2021.
endeavors. It is clear that investment in these
2. Bloom DE, Cadarette D, Ferranna M, Hyer RN, Tortorice
innovations is needed to ensure preparedness in DL. How New Models Of Vaccine Development For
COVID-19 Have Helped Address An Epic Public Health
the event of future outbreaks and pandemics. Crisis. Health Aff. (Millwood). 2021; 40(3): 410–418. DOI:
10.1377/hlthaff.2020.02012.
Precision NanoSystems is leveraging years 3. Ball P. The lightning-fast quest for COVID vaccines—
of experience in LNP formulation, vaccine and what it means for other diseases. Nature 2021;
589(7840): 16–18. DOI: 10.1038/d41586-020-03626-1.
manufacturing platform development, and 4. Dolgin E. The tangled history of mRNA vaccines. Nature
industry know-how to usher in the new 2021; 597(7876): 318–324. DOI: 10.1038/d41586-021-
02483-w.
frontier of vaccine production. Through the 5. Kis Z, Kontoravdi C, Dey AK, Shattock R, Shah N. Rapid
company’s active involvement in the novel development and deployment of high-volume vaccines
for pandemic response. J. Adv. Manuf. Process. 2020;
COVID-19 mRNA vaccines, this specialized 2(3): e10060. DOI: 10.1002/amp2.10060.
knowledge and expertise, paired with highly 6. Sell TK, Gastfriend D, Watson M, et al. Building the global
vaccine manufacturing capacity needed to respond to
scalable instrument platforms and LNP pandemics. Vaccine 2021; 39(12): 1667-1669. DOI:
10.1016/j.vaccine.2021.02.017.
reagents, can be leveraged by researchers,
7. Freemont P, Curach N, Friedman D, Lee SY. These
developers, and manufacturers alike. n ‘biofoundries’ use DNA to make natural products we
need. World Economic Forum. https://www.weforum.
org/agenda/2019/10/biofoundries-the-new-factories-
References for-genetic-products/. Published October 28, 2019.
1. The Independent Panel for Pandemic Preparedness Accessed: October 20, 2021.
and Response. COVID-19: Make It the Last Pandemic. 8. R3: RNA Readiness & Response Program. Wellcome
https://theindependentpanel.org/wp-content/ Leap. https://wellcomeleap.org/r3. Published July 14,
uploads/2021/05/COVID-19-Make-it-the-Last- 2020. Accessed November 5, 2021.

Additional Resources...

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Learn how Precision NanoSystems
can help accelerate development
of genomic medicines.

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GENengnews.com |9
 Increasing mRNA Capabilities And Capacity for Growing Market Needs

Increasing mRNA
Capabilities And Capacity for
Growing Market Needs

By Sebastian Almeida, Director, CMC, Akron Biotech; Lloyd Jeffs, Director, Clinical
Manufacturing Solutions, Precision NanoSystems; John Harmer, Head of Marketing,
Aseptic Filling, and Katarina Stenklo, Enterprise Solutions Activation Leader, Cytiva

After more than a year of enduring myriad on messenger RNA (mRNA), which delivers
economic and social disruptions and a instructions to human cells on how to fight
devastating loss of life from the COVID-19 COVID-19 through a biological Trojan horse.
crisis, the pharmaceutical industry was able The ability to “program” mRNA with genetic
to deliver its first tools in the fight against the code to treat/prevent disease has been
pandemic. The catalyst for this milestone was known for decades with clinical testing of
the rapid development of innovative vaccines. some mRNA vaccines underway even prior
The emergency use authorization (EUA) of to the rise of COVID2; however, challenges
the Moderna and Pfizer-BioNTech vaccines, associated with transitioning mRNA into a
which offer 94% efficacy for fully vaccinated drug product and an industry averse to change
adults, was impressive.1 Both vaccines rely have historically hindered its potential.

10 | GENengnews.com
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

Fast forward to late 2020, when the urgency such as smallpox and influenza,4 even industry
to stop the spread of coronavirus led to the experts were skeptical in the early stages of the
groundbreaking EUAs for the Moderna and pandemic about whether vaccine manufacturers
Pfizer-BioNTech COVID-19 vaccines. Now, the could produce an approved vaccine quickly
success of the mRNA vaccine market—recently enough to change the course of the COVID-19
projected to reach $1273 billion by 20273—is outbreak. And under typical circumstances,
fueling high demand for mRNA, which has their concerns would’ve been justified.
resulted in a critical need to address mRNA Traditional viral vector production, where animal
manufacturing bottlenecks. Innovation and cell culture infected with a weakened virus is
expertise in multiple areas of the industry are grown in chicken eggs or a fermenter, can take
available to address these issues, clearing a path four to six weeks to achieve adequate biomass
for the rise of mRNA therapeutics to treat other to begin manufacturing, with an additional week
indications as well. As manufacturers across needed for growth and production.5 However,
the industry continue to pursue mRNA-based this months-long process can be reduced to
therapies, it is important they understand the just minutes with mRNA, due to its reliance
challenges they may face in their journey and on an in vitro cell-free transcription reaction.
what solutions could help overcome them. Eliminating the reliance on live-attenuated or
viral-vectored vaccines during development
and manufacturing offers several advantages
An mRNA Bottleneck: Yesterday’s Facilities
Cannot Manufacture Tomorrow’s Products to safety, speed, cost, and efficacy. Reaping the

As the industry raced to answer the call for a benefits, though, is possible only with access to

COVID-19 vaccine, it seemed, at least to those critical raw materials and flexible facilities armed

unfamiliar with mRNA, that the clinical success with specialized equipment and expertise.

and expedited production of the Moderna For Moderna, which had never produced or sold
and Pfizer-BioNTech vaccines were nothing a commercial drug prior to receiving the EUA
short of a marvel of modern medicine. Public for its COVID vaccine, this meant relying on a
perception was that vaccines cannot be web of outsourcing partners to scale up produc-
made in a matter of months, or even years, of tion.6 This approach is inherently risky and can
development and clinical testing before they reduce speed to market if there isn’t seamless
can be safely manufactured to meet large- coordination among all partners. And in the end,
scale demand. And considering the lengthy Moderna is ultimately responsible for ensuring its
timeframes it took for the medical community vaccine is made in compliance with regulatory
to respond to infectious diseases in the past, requirements, which can be challenging when

GENengnews.com | 11
 Increasing mRNA Capabilities And Capacity for Growing Market Needs

production doesn’t occur in-house. Even Pfizer strategy, the therapeutic type, and target patient
ran into complications when working with population. For example, current mRNA vaccines
BioNTech to manufacture its vaccine, as they are generated in large batches and then filled
faced a supply shortage of key raw materials in single units, whereas mRNA-based personal-
and starting components.7 Therefore, if you’re ized therapies are produced in a large number
interested in or already pursuing mRNA-based of smaller batches. It is essential to establish a
therapies and vaccines, you cannot rely on tradi- flexible platform, such as the Cytiva FlexFactory™
tional, often rigid, manufacturing platforms and single-use platform, that can adapt to scale
filling operations for the production of mRNA. and product modality with minimal downtime.
Independent of scale, the mRNA manufac-
turing site design is unique. Each phase of the
Outside of manufacturing, complex workflow requires separate suites for
lipid nanoparticle production of various key starting materials and
components as well as the careful completion
encapsulation is critical for of critical process steps. Since mRNA is a cell-free
establishing the potency process, it is incredibly challenging to manufac-
ture in facilities where traditional mammalian
of the mRNA drug by cell culture systems are in operation and may
providing appropriate present contamination risks. Alternatively, if you
do not have the ability or capacity to execute the
targeting and release. mRNA workflow in-house, you can work with an
outsourcing partner. Yet, given the rising demand
Instead, you must work with solutions that are for mRNA manufacturing capabilities, you will
designed to support the unique characteristics likely need to get in line with your competitors.
and manufacturing needs of the mRNA workflow.
For example, plasmid DNA (pDNA), which
To understand why this is important, consider
provides the DNA template for gene expression
the most critical areas of the mRNA workflow
during the cell-free process of in vitro tran-
and why flexibility is necessary for success.
scription (IVT), is an essential building block to
produce the viral vectors needed for cell and
Using Flexibility To Navigate The gene therapies. With both markets on the rise,
Complex Workflow For mRNA demands for production of high quality pDNA
Batch size for mRNA is one of the crucial consid- are increasing, and the industry is facing limited
erations and can vary based on a company’s availability with long lead times for this crucial

12 | GENengnews.com
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

ingredient. Research-grade pDNA is an alternative the burden of legacy facilities and embracing a
option, but the methods to produce it are not digitized, standardized solution, Akron Biotech
validated, resulting in varying product quality is able to position themselves for a place
from batch to batch as well as the potential in a growing field of novel therapeutics.
risk of cross contamination due to less rigid
cleaning requirements in research laboratories. Encapsulating mRNA For Potent Delivery

Modular facilities, such as the Cytiva KUBio™ One of the biggest technical challenges in

modular environments, which are capable of developing mRNA therapies is encapsulation

rapidly deploying GMP-level manufacturing using specialized lipid nanoparticles, which both

capabilities, are one solution to overcome the protect the mRNA and enable delivery to the

shortage in pDNA supply and address the unique targeted cell cytoplasm. Conventional methods

needs of mRNA across its entire workflow. These for encapsulation present considerable chal-

predesigned, prefabricated manufacturing lenges for maintaining the efficiency of mRNA

platforms offer a quicker route to operational manufacturing, such as limited control over

availability as well as rapid product changeover particle size; significant batch-to-batch variability;

and reduced cleaning requirements through substantial material loss from low encapsulation

the use of single-use technology (SUT). As efficiency; and a labor-intensive production

opposed to constructing a dedicated greenfield process that is difficult to scale up.9 Outside
of manufacturing, encapsulation is critical for
production facility for the mRNA workflow that
establishing the potency of the mRNA drug by
would likely take years to build, a new modular
providing appropriate targeting and release.
facility can be up and running in about 12
months. Akron Biotech recently announced its Consistency in particle size requires a tech-
use of Cytiva’s FlexFactory™ single-use platform nology that ensures robust and reproducible
for the manufacture of pDNA, which it sees as lipid nanoparticle production that can control
an opportunity to support innovative therapies how the environment and concentration of
with scalable solutions.8 Utilizing FlexFactory ™ RNA and lipids come together to form the
Figurate™ automation and connectivity, Akron final particle. Microfluidics, a standard tool in
Biotech is able to maintain GMP compliance development settings, offers this control by
using a fully validated manufacturing platform using non-turbulent, time-invariant mixing
that facilitates regulatory filings. This is critical conditions to create highly reproducible
in an evolving area of the industry where both mRNA lipid nanoparticles. Microfluidics
manufacturers and regulatory authorities are technology offers encapsulation efficiency
continuing to learn and grow. By eliminating of >90% throughout manufacturing.10 Using

GENengnews.com | 13
 Increasing mRNA Capabilities And Capacity for Growing Market Needs

technologies, such as Precision Nanosystems’ development candidates just by changing the

NxGen™ platform, microfluidics is now available payload of the RNA within the lipid nanopar-
at manufacturing scales. Next, tangential flow ticle to deliver a different outcome or treat a
filtration is used to remove the ethanol as well different indication. Standardized robotic filling
as any unwanted solvents and then replace systems that rely on presterilized, disposable
them with the desired buffer for storage. Finally, components within a closed system, such as
any bioburden that is in the bulk mRNA lipid the Cytiva Microcell vial filler, facilitate faster
nanoparticle formulation is removed through product changeover and enable improved
sterile filtration, ensuring sterility and preserving process control by limiting exposure to any
the inherently unstable mRNA molecule. external forms of contamination, regardless of
Protecting the mRNA molecule in the final stages the processes and formulation used upstream.11
of production will also drive storage stability, This means you can quickly produce your full

another challenge in the mRNA workflow. product portfolio in any dosage presentation

Ultra-cold chain requirements for mRNA have as needed, driving clinical candidates forward

made widespread distribution of the Moderna faster and ultimately improving speed to market.

and Pfizer-BioNTech COVID-19 vaccines difficult There is also a reduction in startup time, with

and costly. An ultracold storage box for the robotic filling systems requiring only six months

-20 C and -70 C temperature requirements, to one year for installation as opposed to the 18

respectively, typically cost between $10,000 to 24 months needed for conventional systems.

and $20,000.11 Recent readiness assessments for

COVID-19 vaccine distribution found that only mRNA Readiness From Idea To Injection
about 50% of countries assessed had the cold While the need to move quickly to slow the
chain capacities necessary to deploy them.12 pandemic required utilization of traditional
Another way to protect mRNA stability is by and less efficient processes and technologies,
reducing the risk of microbial contamination a limitless future for mRNA means exploring
during fill finish using advanced technologies new and innovative solutions that can open the
that eliminate human intervention, such as door for improvement in many areas. Progress
robotic aseptic filling systems utilizing SUT. is being made across the entire workflow,
Traditional filling machines require downtime for with an increased focus on securing starting
cleaning and sterilization, which is not feasible materials, optimizing large-scale manufacturing
for small batch processing, where flexibility platforms, and improving technical skills for
and speed are key. For example, the science mRNA process development and manufac-
of mRNA allows a company to target multiple turing. As you consider your strategy and the

14 | GENengnews.com
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

tools you’ll need to be successful, look for 5. Verga, David. (February 10, 2021). mRNA and the future
of vaccine manufacturing. PATH. https://www.path.org/
experienced and knowledgeable vendors that articles/mrna-and-future-vaccine-manufacturing/
have already begun to equip their arsenal with 6. Trefis Team, Great Speculations. How Is Moderna’s
Vaccine Production Scaling Up?. Forbes. https://www.
products designed to accommodate the mRNA forbes.com/sites/greatspeculations/2021/01/07/
workflow from idea all the way to injection. how-is-modernas-vaccine-production-scaling-
up/?sh=5be1a52b6e68
Doing so will position you at the forefront of this 7. Langreth, Robert. (December 3, 2020). Pfizer Scaled
biopharmaceutical revolution, where mRNA is Back 2020 COVID-19 Vaccine Production Targets From
100 Million to 50 Million Doses. Time. https://time.
emerging as an unstoppable force in fighting com/5917847/pfizer-cut-covid-19-vaccine-targets/
and preventing disease around the world. n 8. Akron Biotech. (October 2020). Akron Biotech Acquires
Cytiva FlexFactory for the Manufacture of Plasmid
DNA. https://www.akronbiotech.com/newsroom/
References akron-biotech-acquires-cytiva-flexfactory-for-the-
1. Center for Disease Control. (May 7, 2021). Effectiveness manufacture-of-plasmid-dna/
of Pfizer-BioNTech and Moderna Vaccines Against 9. Precision Nanosystems. Areas of Interest: Messenger
COVID-19 Among Hospitalized Adults Aged >65 Years RNA. https://www.precisionnanosystems.com/
– United States, January-March 2021. https://www.cdc. workflows/payloads/mrna
gov/mmwr/volumes/70/wr/mm7018e1.htm
10. Precision Nanosystems. (2020). Accelerating the
2. Kwon, Diana. (November 25, 2020). The Promise
Development of Transformative Nanomedicines with
of mRNA Vaccines. The Scientist. https://www.
NxGen™ Microfluidics Technology. https://www.
the-scientist.com/news-opinion/the-promise-of-mrna-
precisionnanosystems.com/docs/default-source/
vaccines-68202
pni-files/app-notes/model-mrna-lnp-therapeutic5139
3. Globe News Wire. (June 23, 2021). Global mRNA 4821a9754311bfebb59ab1e88149.f?sfvrsn=125053a8
Vaccines Market to Reach $127.3 Billion by _0therapeutic51394821a9754311bfebb59ab1e88149.
2027. https://www.globenewswire.com/news- pdf?sfvrsn=125053a8_0
release/2021/06/23/2251546/0/en/Global-mRNA-
Vaccines-Market-to-Reach-127-3-Billion-by-2027.html 11. Chakamba, Rumbi. (May 13, 2021). The cold chain
storage challenge. Devex. https://www.devex.com/
4. Colarossi, Natalie. (July 18, 2020). How long it took to news/the-cold-chain-storage-challenge-99869
develop 12 other vaccines in history. Business Insider.
https://www.businessinsider.com/how-long-it-took-to- 12. Cytiva. Meet the SA25 Aseptic Filling Workcell. https://
develop-other-vaccines-in-history-2020-7#influenza-5 vanrx.com/products/sa25-aseptic-filling-workcell/

Additional Resources...

mRNA Manufacturing
Strategies
Solutions for large-scale
production of mRNA molecules.

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GENengnews.com | 15
 Key Insights Into Overcoming mRNA Process Challenges

Key Insights Into Overcoming

mRNA Process Challenges

As the COVID-19 pandemic began to wreak However, advances in science and an upheaval
havoc on the world in 2020, the biopharmaceu- of focus and resources dedicated to mRNA R&D
tical industry started working tirelessly to develop during the response to the COVID-19 outbreak
a vaccine to stop this global threat. Emerging have catapulted it into the spotlight as a disrup-
from this effort were the vaccines by Moderna tive technology that could change the future
and Pfizer-BioNTech using messenger RNA, or of medicine. The scientific community still faces
mRNA. Originally targeting primarily therapeutic hurdles, though, when it comes to efficient
cancer vaccines, mRNA has had a slow uptake and effective process development of mRNA
in the industry until now due to challenges vaccines and therapies. Realizing the potential of
with distribution, specificity, and stability within mRNA requires focus on key areas and strategies
the body. In an industry averse to change, that could help alleviate the bottlenecks in this
these limitations have left mRNA brewing on growing market segment, ultimately leading to
the back burner of innovation for decades. a transformative breakthrough in patient care.

16 | GENengnews.com
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

Growing Interest In mRNA Capabilities 2. Speed. It is produced more rapidly by cell-

free processes than other biologics and is
Although many people did not know about
readily standardized and scaled up, improving
mRNA until it ushered us toward a return to
responsiveness to large emerging outbreaks.
normalcy during the COVID-19 pandemic,
researchers have been studying the capabili- 3. Efficacy. It induces expression of specific
antigens that give rise to both humoral
ties of RNA-based vaccines and therapeutics
(antibodies) and cell-mediated immunity
for almost 30 years. The potential of mRNA
(T cells), which results in efficient and effective
was first discovered by Hungarian scientist
immune response.
Katalin Kariko in the 1990s1; yet, advancing it
beyond an idea proved difficult, due to the Dr. Jing Zhu, associate director of mRNA devel-

body’s natural immune response to synthetic opment at GeneLeap Biotech, says the success

RNA. Kariko, along with immunologist Drew of the Moderna and Pfizer-BioNTech vaccines

Weissman, eventually overcame this hurdle drove significant changes in his company’s

by incorporating modified nucleosides into plans for the future. “Last year, GeneLeap

mRNA, laying the foundation for its use in the Biotech had three areas of focus when it came

vaccines later developed for COVID-19.1 to developing the vaccines and therapies in
our pipeline: AAV [adeno-associated virus],
The expected growth of the mRNA market signals
oligonucleotides, and mRNA,” he explains. “But
the success of the Moderna and Pfizer-BioNTech
because of the success with the COVID-19
vaccines as only the beginning of a new era in
vaccines, we strategically shifted our main efforts
the biopharmaceutical industry. In 2019—just
to mRNA. Not only does it have the power to
before the COVID-19 outbreak—the mRNA
do things traditional modalities do not, but the
vaccines and therapeutics market was valued
development timelines and investment needed
at almost $600 million.2 Now, recent reports
for mRNA are much less than other biologics.”
show this number could be as high as $2,911.9
million by 2026,2 with 155 therapies based on The impact of mRNA’s success in 2020 was
mRNA already in today’s clinical pipeline.3 also felt at the Centre for Process Innovation
(CPI), a part of the UK government’s High Value
Manufacturing network which has an objective
There are three parameters
of providing process development support
driving interest in mRNA:
and manufacturing expertise to the biopharma
1. Safety. mRNA vaccines do not involve
infectious elements like many conventional industry. Over the last year, CPI was a key part
vaccines. mRNA is also degraded rapidly after of the UK government response to support
injection by normal cellular processes. rapid vaccine development against COVID-19.

GENengnews.com | 17
 Key Insights Into Overcoming mRNA Process Challenges

As such it lead the work stream to develop achieve four or five grams per liter within a few
scalable manufacturing solutions for Imperial hours. These advantages mean that a company
College London’s saRNA (an mRNA type) vaccine with access to the necessary capabilities and
candidate and was awarded £5 million early in raw materials to produce a new mRNA vaccine
2021 to support the development of a COVID-19 could do so in a matter of weeks, while other
variant mRNA vaccine library in the U.K.4,5 “Prior approaches will take significantly longer.”
to the pandemic, CPI was studying lipid nanopar- Overall, the characteristics of mRNA
ticles and also working with other companies make it ideal for rapid response to infec-
tious disease as well as for precision, i.e.,
The implications of what personalized, medicine, a growing focus
in a changing biopharma landscape—but
mRNA could achieve for only if the industry can overcome existing

modern medicine are far mRNA process development challenges.

reaching; yet, advancing Where Do We Go From Here?

its capabilities means The implications of what mRNA could achieve for
modern medicine are far reaching; yet, advancing
establishing a development its capabilities means establishing a development

toolbox that is fit for toolbox that is fit for purpose, rather than relying
on legacy methods designed for monoclonal
purpose, rather than relying antibodies (mAb) or other gene therapies. And
while some aspects of mRNA seem simplified,
on legacy methods. the reality is that, technically, everything is new.
“Compared to protein products, RNA is far more
and organizations to develop cell-free expression sensitive to degradation – by several orders of
platforms, which prepositioned us for what we’re magnitude. It is very prone to RNase [ribonu-
doing now,” says Dr. John Liddell, chief technolo- clease] degradation,” explains Dr. Liddell. “RNase
gist at CPI. “The attraction of mRNA as a vaccine is activity is ubiquitous and will destroy all of your
that it’s highly potent, and you can generate a lot products before you even know they’ve been
of vaccine product from small volumes without destroyed. Therefore, we have to eliminate RNase
requiring a large manufacturing footprint. It also activity during development and manufacture
offers a high transcription yield and reaction of mRNA-based products, which is very different
through enzymatic synthesis, allowing you to from working with proteins. mRNA has the

18 | GENengnews.com
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

Plazmid In vitro Buffer

linearization transcription exchange Capture Concentration Encapsulation Polishing Drug product

Figure 1: mRNA process development steps

making of a platformable process, similar to what the mRNA compared to traditional recombinant
we have at the moment with mAbs. In that case, proteins. There is also a lack of experience and
this has arisen from the advantage of having 20+ knowledge of scaling up mRNA processes as well
years of knowledge accumulated in that space, as associated perceived regulatory uncertainties.
whereas with mRNA, there is still much to learn.”
A Closer Look At mRNA Process
Many of the challenges associated with the devel- Development Challenges
opment and manufacture of mRNA are reminis- A more detailed examination of mRNA process
cent of the early days of mAbs, when the industry development (Figure 1) uncovers several areas
struggled to overcome low titer and poor puri- for improvement across multiple steps.
fication yields, leading to costly and inefficient
commercial manufacturing. A focused effort to Plasmids
overcome those production challenges resulted An integral raw material for mRNA-based
in mAbs becoming the fastest growing class of therapies and vaccines is plasmid DNA (pDNA),
biopharmaceutical products.6 A critical factor in which acts as a template for the mRNA. Yet,
driving mRNA forward is likely to be achieving pDNA is also used for the production of viral
consistency in process development of mRNA- vector-based therapies—another growing area
based products. “The processes used for mRNA of biopharma. This has led to a significant strain
today have all been created by independent on GMP-quality pDNA supply. CPI has been
developers, each of which applied their own skills fortunate to secure long-standing agreements
and knowledge,” says Dr. Liddell. “It should be that with companies that make pDNA, but emerging
the same methods used to make a COVID vaccine companies like GeneLeap Biotech struggle to
are the same ones used for an influenza vaccine, get the attention they need in an increasingly
which thus requires standardization across a competitive environment. However, Dr. Liddell
range of areas.” One of the main challenges in highlights a possible solution, “For DNA template
mRNA process development and scale-up is the production, there are a number of alternative
lack of dedicated equipment and consumables fit cell-free technologies to generating pDNA, such
for the relatively small volumes and large size of as rolling circle DNA amplification approaches,

GENengnews.com | 19
 Key Insights Into Overcoming mRNA Process Challenges

which seem very promising for reducing process Dr. Zhu says the need for additional optimiza-
timelines and improving product quality,” he tion has led to issues and costs in GeneLeap
explains. “There has been a lot of pressure on Biotech’s work with mRNA. “We have found
plasmid manufacture, particularly to GMP, to that each vendor’s T7 RNA polymerase has a
support gene therapy and mRNA applica- different ‘flavor,’ which means we will optimize
tions. Hence with these supply chain issues one IVT system using the T7 from one vendor
alternative approaches such as rolling circle but then have to optimize again if we switch
amplification may see their use accelerated.” to a different one. That is why sustainable
supply from a reliable vendor is critical for us.”
In Vitro Transcription Equipment fit for the unique characteristics
of mRNA is also essential. “For the upstream,
After pDNA is manufactured in an E.coli-
we need equipment that is designed for
based fermentation process, it is harvested,
cell-free expression platforms,” adds Dr. Zhu.
linearized, purified, and used as templates
“We can use some of the functionality on
for the enzymatic in vitro transcription (IVT)
the bioreactors available now, but they are
process, yielding the desired mRNA molecule.
missing some important functions we need,
IVT is currently the cost-driving step in mRNA
like certain inline monitoring, which will help
process development. “IVT is what generates
with scale-up by monitoring the different
the product, but it is a highly complex step. It
parameters that vary with mRNA models.”
requires the careful addition of a number of
diverse components in addition to the DNA
Purification
template, such as enzymes and nucleotides to
synthesize the mRNA,” explains Dr. Liddell. “You Compounding raw material variability challenges

also need a capping reagent that will be added is the impurity profile of mRNA molecules that

to the five-prime end of the mRNA. Design of can vary with each project, requiring different

Experiments approaches are typically used for purification steps from case to case. “mRNA

process optimization of these different compo- is a very large molecule—30 to 50 nanome-

nents to optimize yield and quality. Currently, ters—which is way beyond the size of proteins

reactions are batch based, but alternate reactor and comparable to viral vectors. That means

designs could be devised to reduce inventory they do not interact well with conventional

of expensive raw materials, eventually even chromatography resins, where you will likely get

moving to a continuous reaction scheme. only surface adsorption,” explains Dr. Liddell.

This may be harder to develop, but it could The varying impurity profiles of mRNA from
make a big difference in overall productivity.” the IVT step calls on options in purification

20 | GENengnews.com
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

technologies that would allow process devel- For some mRNA variants, though, we found
opment scientists to mix and match media very strong double-stranded RNA product
based on the specific characteristics of the impurities, which required a specific polishing
molecule. Dr. Zhu points to an internal case step to separate. That is why a mix-and-match
where GeneLeap Biotech used the Oligo dT approach based on the impurity profile
purification platform for mRNA. “For some would be ideal to form the final process. More
of our projects, the mRNA we worked on adapted purification solutions for mRNA, such
was very clean, with a purity profile higher as Cytiva’s fiber-based Fibro chromatography,
than 90% after the IVT. Therefore, we applied could also offer a potential alternative to help
facilitate purification of mRNA in the future.”

The varying impurity Additional considerations must also be made

profiles of mRNA from based on whether the mRNA is conventional or

self-amplifying, with the latter having a much
the IVT step calls on bigger structure that creates additional chal-

options in purification lenges in the purification step. “mRNA can be

made using the four conventional nucleotides
technologies that would as well as modified mRNA, where you are using
allow process development base analogs to increase the half-life, which

scientists to mix and typically means swapping out uridine for pseu-
do-uridine,” says Dr. Liddell. “For self-amplifying
match media based on the mRNA, you are using sequences derived from
specific characteristics certain viruses to generate what is called a
replicon, constructed from four non-structural
of the molecule. proteins coded by the saRNA, which then makes
copies of the transgene protein that the RNA
TFF [tangential flow filtration] or SEC [size is coding for.8 It’s quite cunning technology
exclusion chromatography] to remove the but is at an earlier stage of development.”
small amount of remaining impurities,” he
explains. “For other projects, the impurity Encapsulation
profile was more complex and finding a Another critical step in mRNA processing is
common purification platform has been encapsulation using lipid nanoparticles. The
more difficult than we anticipated. Oligo specialized lipids used for mRNA provide
dT mediated purification often works fine. a delivery system that protects the nucleic

GENengnews.com | 21
 Key Insights Into Overcoming mRNA Process Challenges

acid from degradation as the drug makes deployment,” explains Dr. Liddell. “We had
its way through the patient’s body. Prior to to move very quickly for COVID, so people
encapsulation, the lipids must all first be used what was available, but I really think
dissolved in an organic solvent, which is there’s ample scope for improvement by
typically ethanol. Because ethanol is highly selecting appropriate additives and excip-
flammable, facilities must be properly equipped ients to achieve storage stability at higher
to ensure safe use of those types of materials, temperatures. In addition to alternate lipid
such as using flame-proof equipment. and excipient formulations, we may be able

If a company does not have these capabilities to utilize other formulation technologies, such

in-house, they may look to an outsourcing as lyophilization to achieve better stability.”

partner for encapsulation services; however, this

The Future Of mRNA Beyond COVID
could be challenging due to the limited number
of CDMOs with experience in this area. There While the COVID-19 pandemic took so much
is also a significant number of patents in this from us—most importantly, over 3.8 million
area, leading to a complex intellectual property lives globally7—the white knight of mRNA that
landscape. Dr. Liddell says the lipids used for offers eligible vaccine recipients with invaluable
mRNA today were originally developed to deliver protection will likely open the door to new
small interfering RNA (siRNA) therapies. There opportunities to improve patient care; however,
may be better LNP formulations or alternative gaps in knowledge could slow progress in not
delivery technologies that might improve only development but also regulatory approval.
conditions required for storage stability, which Both Drs. Liddell and Zhu agree that we will
is another challenge for mRNA products. likely see continued focus on using mRNA
for vaccines, such as replacing the egg-based
Storage production methods for vaccines, and even

Due to the sensitivity of mRNA, an ultra- oncology applications, but we must address

cold chain is necessary to deliver COVID-19 these process development issues and more

vaccines across the world. Paving a way toward to make the possibilities of mRNA a reality.

a future with mRNA by addressing limita- Wherever the industry does go from here,

tions during process development, though, though, is possible only through collaboration,

could help minimize mRNA’s temperature communication, and a concerted effort to

requirements. “The low temperature needed pave the way for the exciting future of mRNA.

to stabilize mRNA comes from the rough Click here to learn more about equipment and
edges arising from rapid development and solutions for efficient mRNA production. n

22 | GENengnews.com
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

References www.uk-cpi.com/news/cpi-joins-national-taskforce-to-
1. Garde, Damian. (November 10, 2020). StatNews. The develop-covid-19-vaccine
story of mRNA: How a once-dismissed idea became a 5. Genetic Engineering & Biotechnology News. (June
leading technology in the COVID vaccine race. https:// 2020). U.K. Government-Backed Centre for Process
www.statnews.com/2020/11/10/the-story-of-mrna- Innovation (CPI) Scaling Up Imperial’s Coronavirus
how-a-once-dismissed-idea-became-a-leading- Vaccine. https://www.genengnews.com/topics/
technology-in-the-covid-vaccine-race/ bioprocessing/u-k-government-backed-centre-
2. PR Newswire. (April 5, 2021). mRNA Vaccines and for-process-innovation-cpi-scaling-up-imperials-
Therapeutic Market Size is Projected To Reach USD coronavirus-vaccine/
2911.9 Million 2026, Says Brandessence Market
6. Ecker, Dawn M., et. al. (October 2020). The Therapeutic
Research. https://www.prnewswire.com/in/news-
Monoclonal Antibody Product Market. https://
releases/mrna-vaccines-and-therapeutics-market-size-
bioprocessintl.com/business/economics/the-market-
is-projected-to-reach-usd-2911-9-million-2026-says-
for-therapeutic-mab-products/
brandessence-market-research-885706800.html
3. Roots Analysis. (January 2021). mRNA Therapeutics and 7. WHO Coronavirus Dashboard. (June 2021). https://
Vaccines Market, 2020-2030. https://www.rootsanalysis. covid19.who.int/
com/reports/mrna-therapeutics-and-vaccines-market.html 8. Blakney A et al. (Vaccines, Jan 2021). An Update on Self-
4. Centre for Process Innovation. (April 2020). CPI joins Amplifying mRNA Vaccine Development DOI: 10.3390/
national taskforce to develop COVID-19 vaccine. https:// vaccines9020097

Additional Resources...

mRNA Manufacturing
Workflow DNA
From plasmid linearization
to the final drug product, this
mRNA 5’ G UTP
illustrated diagram will show 5mCTP
CH3

a concise picture of the

View Here Cap

entire process.

GENengnews.com | 23
 Translation of RNA Medicines from Design to Clinic

Translation of RNA Medicines

from Design to Clinic
During development of pharmaceutically-applicable
mRNA therapeutics, synthesis, optimization, and
formulation must be taken into consideration.

RNA therapeutics comprise a rapidly expanding for COVID-19 marked 2020 as a breakout
category of drugs that will speed clinical year for mRNA technology platforms.
solutions, actualize personalized medicine, and mRNA therapeutics are applicable to cancer
make the term “undruggable” obsolete, according immunotherapies, infectious diseases, and other
to a March 2021 Frontiers in Bio-engineering indications that require protein replacement
and Biotechnology review.1 Different classes therapy or antibodies. After cellular uptake, the
of RNA-based therapeutics include antisense therapeutic molecule translates genetic informa-
oligonucleotides, aptamers, small interfering tion into protein, an antigen, antibody, or other
RNAs, microRNAs, and messenger RNA (mRNA). therapeutic protein. During development of the
The widespread use of mRNA vaccines therapeutics several aspects must be taken into

24 | GENengnews.com
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

consideration–synthesis, optimization, and formu-

lation--to deliver the cargo to the target site.

Although currently best known for their work

with Pfizer on the BNT162b2 mRNA vaccine for
SARS-CoV-2, according to Heinrich Haas, PhD, Vice
President RNA Formulation and Drug Delivery,
BioNTech, one of the company’s key areas of
development is cancer immunotherapies. Heinrich Haas, PhD, Vice
President RNA Formulation and
To achieve this particular type of vaccination an Drug Delivery, BioNTech
mRNA that codes for a tumor-associated antigen
(TAA) is transferred into dendritic cells, which and the cap structure are added. Once the
have a key function in tumor therapy. Inside mRNA is transcribed, the DNA is digested.
the cells the mRNA is translated and stimulates
Then, importantly, the RNA needs to be
a cascade of immune responses specific to the
formulated for delivery. “Here the challenges
TAA in addition to a systemic immune response.
are a bit higher than for small molecules as
many functions need to be fulfilled by the
R&D Formulation Development
formulation,” said Haas. The formulation has
The coding region of mRNA contains the infor- to control serum interactions and protect the
mation required to synthesize a protein. Other RNA from degradation in circulation, control
important elements include the 5’ and 3’ UTR biodistribution and deliver the RNA to the target
regions, a 5’ cap and a 3’ polyA tail. Synergistically, site, enable and improve cellular uptake and
the elements make a pharmaceutically-ap- translation at the target site, and, finally, ensure
plicable molecule and need to be optimized protein expression and release into circulation.
individually to facilitate increased intercellular
Most lipid nanoparticle formulations consist
half-life, translation, and MHC presentation.
of four components: the ionizable lipid (e.g.
“The good thing is that mRNA can be manu- DODMA, Dlin-MC3-DMA), cholesterol, helper
factured in a cell-free in vitro transcription (IVT) lipid (DSPC, DOPE) and grafted lipid (PEG-lipid).
enzymatic reaction,” said Haas. A DNA plasmid The ionizable lipid is positively charged at low
containing the DNA template is linearized, and pH and neutral at higher pH. The ionizable
RNA polymerase, nucleotide triphosphates lipids bind the RNA in the positively charged
(NTPs), modified UTP substrates, inorganic state through electrostatic interactions and
pyrophosphatase, ribonuclease inhibitors, their pKa values are tailored to favor release

GENengnews.com | 25
 Translation of RNA Medicines from Design to Clinic

from the endosome following cellular uptake. mixing lipids (an ionizable lipid, a helper lipid,
and a grafted lipid) in ethanolic solution with
To manufacture the nanoparticles, the RNA
RNA in an acidic buffer to complex the RNA and
dissolved in buffer, and lipids dissolved in ethanol
form the LNP in one step. Permanently cationic
are rapidly mixed at precise ratios, to induce
LPXs are thought to be more cytotoxic than LNPs,
self-assembly into particles with the desired
which are largely uncharged at physiological pH.
properties. Further process steps, comprising
This reduces serum interactions and thus also
buffer adjustment, addition of stabilizers,
reduces one main cause of potential toxicity.
concentration adjustment and sterile filtration
may be involved to obtain the end product. Alnylam’s Onpattro™ for the treatment of
After determining various physico-chemically polyneuropathy caused by hereditary ATTR
characteristics for quality control, the biological (hATTR) amyloidosis and the Moderna
activity is measured in cell or animal models. The mRNA-1273 and Pfizer-BioNTech BNT162b2
correlation between particle characteristics and SARS-CoV-2 vaccines all use ionizable cationic
biology is used as a basis for system optimization. lipids formed by precipitation in ethanol.

In lipid-based delivery systems some general The two systems differ in their internal structure,
features are considered to be related to activity. which can be demonstrated by small angle x-ray
One is the capacity to undergo transitions scattering (SAXS) where the sample is irradiated
between phase states, e.g., hexagonal, inverse with an x-ray beam and the scattered light
hexagonal, or lamellar. Lipids where the transi- collected. Scattering profiles are displayed as a
tion between lamellar and hexagonal phases function of the angle or the momentum transfer.
is facilitated are considered to be particularly “From this profile you can determine if a Bragg
helpful for uptake or release across bilayer peak is present along with its height and area
membranes. Another important aspect is to get quantitative information on the internal
the pKa value of ionizable lipids, which is organization of your particle to assist system
decisive for pH dependent changes of the specification and optimization,” explained Haas.
particle characteristics in circulation and
The LPX structure is characterized by a narrow and
during endosomal processing after uptake.
well-defined Bragg peak whereas the LNP peak
Two examples of lipid-based delivery systems are is broader, indicating a lower degree of internal
lipoplexes (LPXs) and lipid nanoparticles (LNPs). organization. A further difference between
LPXs are made by mixing preformed cationic LNPs and LPXs is that typically in the former
liposomes with RNA to form a lamellar-like ionizable lipids are used, while LPXs typically
lipoplex stack. In contrast, LNPs are formed by comprise permanently charged cationic lipids.

26 | GENengnews.com
 mRNA VACCINES: A NEW ERA OF BIOTHERAPEUTIC DEVELOPMENT

“This is something that might deserve a closer (RNA) showed high expression in the spleen. “Just
evaluation,” cautioned Haas. “For example, by changing that one physical parameter you
homologous lipids may be considered, which can change organ efficacy,” emphasized Haas.
are identical in structure except for one Such insight allowed initiation of various clinical
nitrogen atom in the head group which can studies in the field of cancer immunotherapy.
be permanently charged or ionizable. If they
Thorough characterization of the particles by
are ionizable the charge can be switched by
SAXS and other measurements were helpful to
adjusting the pH. This impacts packing.”
accurately determine critical quality attributes as
“SAXS provides accurate information on the pH a basis for manufacturing of the drug products.
dependent structural changes inside nanopar-
ticles comprising either permanently charged Clinical Product Development
or ionizable lipid. This allows one to determine “After you have identified your formulation
a structural equivalent to pKa values with high the development work begins. Now you
resolution and purpose,” said Haas. “By variation have to refine all of the parameters and
of mixing ratios between lipids and RNA these justify them,” said Haas. He recommends
values can be accurately fine-tuned. This can be review of the FDA Liposome Drug Products
helpful along with chemical synthesis of a lipid guidance that details the requirements for
library. You can tailor the library based on compo- characterization and data generation.
sition and other aspects of the LPX system, which
“For liposome manufacturing, when it comes to
allows easier and more accurate adjustment of
process development, you start with something
parameters to optimize endosomal processing.”
adapted for lab-scale experiments,” said Haas.
There is also another straightforward approach “Since this process may change as it is scaled
to modulate particle characteristics and activity, up into a GMP environment you need to
added Haas. “If you mix RNA and liposomes in control, detail, and justify all process parame-
different ratios, you can find conditions where ters with more accuracy and care than what
you obtain colloidally-stable particles either with was done for the formulation experiments.”
an excess of negative or positive charge,” he said.
In order to follow up with the requirements
Animal experiment using luciferase as a reporter in the guidances, extensive characterization
gene demonstrated that LPXs formed at an and thorough understanding of structural
excess of positive charge (liposomes) resulted in and functional coherencies inside the product
high transfection efficacy in the lung, whereas in development is mandatory. Here, SAXS
those formed with an excess of negative charge and other measurements for extended

GENengnews.com | 27
 Translation of RNA Medicines from Design to Clinic

characterization can be useful to generate A thorough understanding of the coherencies

a sound database. By performing many inside the delivery systems, such as structure
measurements for all parameters, the process and function as derived from advanced
conditions needed to manufacture and store characterization, including SAXS analysis,
a pharmaceutical product can be defined. assists performance of rational formulation
development. This also helps define the critical
quality attributes required to specify the
Summary
product that will be important for successful
RNA is labile, highly charged, and complex. translation of RNA into clinical development.
As a result, pharmaceutical development
Listen to Dr. Haas’ full keynote presentation from
becomes a greater challenge than that for small
Precision NanoSystems’ Virtual Symposium:
molecules. CMC (chemistry, manufacturing,
Genetic Medicine from Concept to Clinic. n
and controls) aspects are extremely important
for successful development of complex Reference
1. Damase TR, Sukhovershin R, Boada C, Taraballi
nanoparticles that consist of RNA and vehicular F, Pettigrew RI., Cooke JP. The Limitless Future of
molecules. Formulation is of key importance RNA Therapeutics. Frontiers in Bioengineering and
Biotechnology 9, 161 (2021). https://doi.org/10.3389/
because it may affect the broader qualities. fbioe.2021.628137

Additional Resources...

Application Notes
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data, tools and resources
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