B I O P R O C E S S TECHNICAL

A New Runway for Purification

of Messenger RNA

LY
N
Pete Gagnon, Špela Peršič, Blaž Goričar, Urh Černigoj, Aleš Štrancar

O
A

N
high-performing capture method Figure 1: Analytical
Analytical size-exclusion chromatography (SEC) of bovine serum albumin (BSA)
marketed for processing nucleic acids; sample prestained with PicoGreen dye; TSKgel

is a critical bedrock asset for
developing industrial
purification processes. This is
IO
G4000SWxl, 7.8 mm × 30 cm, 50 mM MES, 150 mM NaCl, 0.05% Pluronic reagent F68,
pH 6.5; flow rate: 0.5 mL/min.

S
especially true for extended families of 70 12.5
products that share highly similar

IS
chemical composition. Therapeutic UV mAU Pico
260 nm FL
monoclonal IgG is an example. The 280 nm Bovine chromatin BSA
ability of protein A affinity DNA + histones
M 7.5
chromatography to achieve 95% purity 42
in one simple step was the runway that
5.0
R
got recombinant immunotherapy off the
ground and made it available to 28
LMW
E

millions. debris 2.5

In fact, protein A did more. Beyond
14
P

giving the industry a foundation 0

HMW Polymers
manufacturing method, it made IgG
aggregates
purification accessible to immunologists 0
H

without the need for advanced 5 10 15 20 25 30

purification skills. It allowed them to Elution Time (min)
IT

screen efficacy, stability,

pharmacokinetics, and various aspects
W

of manufacturability for hundreds of clones, then objectively select the best But it’s not. Transcription does not
qualified clinical candidates, far in always proceed to completion. In vitro
advance of commercial product transcription (IVT) mixtures commonly
PRODUCT FOCUS: VACCINES
FOCUS: VACCINES
T

development. contain a substantial proportion of

(COVID-19) RNA needs this kind of asset to make incomplete transcripts.
IN

gene therapy and vaccines available to Posttranscriptional variants include

PROCESS FOCUS: DOWNSTREAM, billions. This article shows how anion- subpopulations of double-stranded RNA
PURIFICATION exchange chromatography can fulfill (dsRNA). This refers to species in which
R

AUDIENCE: PROCESS
AUDIENCE: PROCESS that need. part of the sequence doubles back on
DEVELOPMENT AND itself and forms hydrogen-bonded
P

MANUFACTURING The Reality of mRNA Purification bridges to complementary sequences on

In vitro synthesis of RNA requires a the same molecule and/or with other
E

KEYWORDS: MRNA, purified DNA plasmid, purified RNA RNA molecules. When dsRNA is
PLASMID DNA, PURIFIED RNA
R

polymerase, metal-ion enzyme cofactors, administered in vivo, recipient cells

POLYMERASE, ANION-EXCHANGE
CHROMATOGRPHY, METAL-ION
ENZYMES, IN VIVO TRANSCRIPTION,
CHROMATINS, AGGREGATES
LEVEL: INTERMEDIATE
and raw-material nucleotides. RNA in
the completed transcription mixture is
massively more abundant than the
plasmid or enzymes. Purification should
be simple.
sense it as an invading virus. It must be
absent from a final product to prevent
triggering a toxic cytokine storm and to
facilitate proper translation of the
single-stranded RNA (ssRNA) (1).

36 BioProcess International 18(10) October 2020

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Figure 2: Analytical SEC of commercial T7 RNA polymerase prestained with PicoGreen
dye; TSKgel G3000SWxl, 7.8 mm × 30 cm, 0.5 mL/min; note that the fractionation range of aggregates also is a concern because
this column is lower than in the column of Figure 1. high–molecular weight contributes
further to their immunogenicity (9).
70 12.5 12.5
The UV profile reveals high–
UV mAU Trp Pico molecular weight aggregates
260 nm Enzyme FL FL corresponding with the chromatin DNA.
280 nm
Non- It also reveals a series of peaks on the
protein 7.5 7.5
42 leading side of the main BSA peak. They
BSA low represent albumin homo- and hetero-
Trp FL 5.0 5.0
HMW polymers. A subset of albumin contains
28 DNA free sulfhydryl groups that enable
Fragments 2.5 2.5 covalent bonding to similarly endowed
14 plasma proteins, including IgA, alpha-1
0.0 0 protease inhibitor, and antitrypsin-3
(10). Such protein hybrids may have a
0 legitimate biological role in mammalian
5 10 15 20 25 30 physiology, but not in IVT mixtures.
Elution Time (min) Non–protein-based enzyme-stabilizing
formulations are discussed elsewhere
(11).
Figure 3: Overlay of SEC profiles for T7 RNA polymerase from two different suppliers;
the orange profile is from Figure 2, TSKgel G3000SWxl. Figure 2 illustrates results from
analytical SEC of a commercially
15 available T7 RNA polymerase on a
TSKgel G3000SWxl column. This single-
Trp Enzyme
FL subunit enzyme has a mass of 99 kDa.
BSA The sample was prestained with
PicoGreen dye and additionally
9
monitored for intrinsic tryptophan
fluorescence. Like many enzymes, RNA
6 Fragments
polymerase is sold by the concentration
HMW Formulation of activity units. That can leave protein
aggregates additives concentration too low for accurate
3
monitoring by UV absorbance.
Tryptophan fluorescence increases
0 sensitivity of protein detection 15–20-
5 10 15 20 25 30 fold over UV. It does not detect nucleic
Elution Time (min) acids.
DNA contamination is obvious, but
its source is uncertain. It could have
Plasmids and enzymes can be run on a TSKgel G4000SWxl column come from the host cells used to
sources of contaminants and (Tosoh Bioscience). The intercalating produce the enzyme or it could have
adventitious agents. Plasmid fluorescent dye PicoGreen (Thermo come with the BSA, or both. Aside from
preparations can be contaminated with Fisher Scientific) was added to the that, the enzyme is not homogeneous
host DNA, RNA, and proteins. Purity sample in advance to increase but, instead, distributed in two peaks.
among commercially available sensitivity of DNA detection (2). Elution Heterogeneity among RNA polymerases
polymerase enzymes is variable, and was monitored with UV and is known (2–14). Although heterogeneity
some enzymes are stabilized with fluorescence. affects activity, it’s correlation with the
bovine serum albumin (BSA). The The most obvious feature of the two peaks separated by SEC is unknown.
animal origin of BSA presents a liability chromatogram is heavy contamination Figure 3 overlays the tryptophan
from the start, but it is accompanied by by DNA. This is not pure DNA. It is the fluorescence profile of RNA polymerase
other contaminants that amplify the remnant of bovine chromatin: from a second supplier on the profile of
safety risk. nucleosomal debris containing DNA the first. The relative distribution of the
Figure 1 illustrates results from fragments of various sizes, complexed two enzyme peaks is markedly
analytical size-exclusion with histones and other host proteins. different. Aggregate and fragment
chromatography (SEC) of a commercial Viruses tend to associate with contents are higher. BSA either is absent
BSA preparation marketed for enzyme chromatin (3–5). Endotoxins do so as or present in a reduced amount. This is
stabilization in conjunction with well (6), and chromatin is antigenic (7, an advantage, considering the
processing nucleic acids. The assay was 8). The size of the DNA-protein immunogenic potential of extracellular

38 BioProcess International 18(10) October 2020

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Figure 4: Coordination bonding of magnesium ions to phosphatidic acid residues on
RNA; negative charges indicated in red with yellow halos and coordination bonds in
green. The metal-ion positive charge can attract other RNAs and create coordination The ssRNA needs to be
bonds with them as well.
EXTRACTED from
H
N
H its associations with
H H
O
O – contaminants before it can
N P O
N O O
O
be fractionated from
O O P O
– them.
N
+ + O
Mg
N O
mixture, it outcompetes other species
O
O
– O for the available metal ions, or it shares
H P O
O O them in stable complexes. This is why
O – O P O N
the use of EDTA is virtually universal in
O H
H
O
O
– O
O
O N N
chromatography buffers for purification
N N P H H
O
O
of nucleic acids.
O
O Figure 5 illustrates results from
O
H O nanoparticle tracking analysis (NTA,
N using NanoSight instruments (Malvern
N Panalytical) of an IVT mixture
N containing ssRNA with a size of 1,200
N
bases (1.2 kb). It might be hoped that
N such a mixture would be dominated by
H
H a single peak. Instead, there are many
peaks. Those with median size values of
37, 68, and 107 nm represent incomplete
Figure 5: Nanoparticle-tracking analysis (NTA) of an in vitro transcription mixture transcripts. The peak at 163 nm
containing 1,200 bases ssRNA represents free ssRNA. The later peaks
E5 7 at 220 and 315 nm represent aggregates,
NTA E2 4 and they contain more RNA than the
6 107 apparent monomer population. To the
Intensity (AU)
Concentration (particles/mL)

3 extent that nucleic acid behavior in

5 mammalian cell culture harvests can be
163
trusted as a guide, these aggregates are
4 2
likely to represent stable associations
3 among RNA, DNA, proteins, and metal
1 ions (15).
220
2 The NTA profile highlights the
greatest purification challenge of all the
315 0
1 68 foregoing figures. Purification is
0 125 250 375 500
37 Size (nm) difficult enough if all the components in
0 a sample exist as independent entities.
0 200 400 600 800 1,000 It becomes much more difficult if
Size (nm) contaminants are stably associated with
a product. Probability of poor recovery
increases in parallel. The ssRNA needs
chromatin, nonhuman proteins, and transcription become contaminants in to be extracted from its associations
aggregates. It also leads to some the finished IVT mixture. The with contaminants before it can be
practical conclusions: Enzymes in this phosphatidic acid residues along the fractionated from them.
field are not interchangeable across ribose backbone of RNA have high
suppliers, and variations in enzyme affinity for such metals. RNA-metal Current Tools for Industrial
heterogeneity could affect the outcome binding can alter charge, conformation, Purification of mRNA
of in vitro transcription. Lot-to-lot stability, and function (Figure 4). An affinity method for RNA purification
testing is highly recommended to Metal-ion coordination also has the already exists. Hybridization-affinity
document quality and consistency of ability to create crosslinks to other chromatography with an oligo dT (poly-
supply. nucleic acids and proteins, forming thymidine) ligand captures mRNA by its
One more contaminant class requires stable intermolecular hybrids that poly-A tail. About 250 mM sodium
consideration. The multivalent metal contribute to aggregate formation. When chloride suppresses electrostatic
cations included as enzyme cofactors for RNA becomes the dominant species in a repulsion between the negatively

40 BioProcess International 18(10) October 2020

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For research use only. Not intended for animal or human therapeutic or diagnostic use.
Figure 6: Hybridization-affinity chromatography of an IVT mixture on a CIMmultus Oligo
dT column; equilibration/Wash1 with 50 mM sodium phosphate, 250 mM sodium chloride, are prone to fouling by crude IVT
5 mM EDTA, pH 7.0; Wash2 with 50 mM sodium phosphate, 5 mM EDTA, pH 7.0; mixtures. They both tolerate extended
elute = 10 mM Tris, pH 8.0. cleaning with 1 M NaOH, so they can be
2,500 50 restored to original condition, but
fouling within a run can interfere with
mAU mS purification performance. This makes
260 /cm
nm Non-poly-A both of them better suited for polishing.
contaminants Poly-A
mRNA RNA Purification by Anion-
1,500 30
Exchange Chromatography
Ion-exchange chromatography has
1,000 20
served protein and plasmid DNA
Wash1 Wash2 Elute
purification for decades. Traditional
500 10 exchangers such as diethylaminoethyl
(DEAE) and quaternary amine (QA) work
for RNA transcripts smaller than 500
0 0
0 4 8 12 16 20 bases (22, 23), but not for large
Elution Time (min) transcripts. At ambient temperature,
their elevated hydrogen bonding
capacity prevents them from being
Figure 7: Hydrophobic interaction chromatography of an IVT mixture on a CIMmultus C4 eluted with anything less than sodium
HLD column; sample loaded in 1.8 M NaCl, pH 7.0, and eluted with a descending salt hydroxide (Figure 9). Heating the
gradient. The red trace shows a separate dsRNA sample loaded under identical buffers and columns into the range of
conditions. 50–70 °C suppresses hydrogen bonding
sufficiently to enable elution with
2,000 2.0
sodium chloride gradients (24).
UV mAU M Heating imposes a burden on process
260 nm ssRNA NaCl development and manufacturing, but it
also provides a clue: An exchanger with
1,200 1.2 less hydrogen bonding capacity should
DNA and be able to elute RNA at ambient
unreacted temperature. Figure 10 verifies this
nucleotides
800 0.8 prediction by illustrating anion-
exchange fractionation of mRNA from
Short
400 dsRNA transcripts 0.4 plasmid DNA at ambient temperature on
13.3 kb a CIMmultus PrimaS column (BIA
Separations). The sample was bound at
0 0 neutral pH and eluted with an
0 3 6 9 ascending pH gradient. DNA eluted
Elution Time (min)
before and well-separated from ssRNA.
Double-stranded RNA elutes slightly
charged backbone phosphatidic acid cleaned with 100 mM NaOH, but higher after DNA but still earlier than ssRNA
residues on the ligand and on RNA. That concentrations are not recommended. (11).
enables them to approach each other This is a concern because of the high Both double-stranded species can be
closely. RNA is captured by A-T base fouling potential of IVT mixtures. removed instead by a neutral pH wash
pairing. Removing the salt reestablishes Hydrophobic-interaction step with 1 M NaCl and 10 mM
their mutual charge repulsion, chromatography (HIC) has shown ethylenediaminetetraacetic acid (EDTA).
overwhelms A-T hydrogen bonding, and valuable utility for RNA purification. Proteins are eliminated with them.
elutes the RNA (Figure 6) (11, 16, 17). With the correct choice of binding salt, Single-stranded RNA remains bound.
Hybridization-affinity with oligo dT DNA and dsRNA fail to bind (Figure 7) The column is washed with another
can be used for capture, but it bears (11). Incomplete transcripts elute before buffer to clear the excess salt, then
some limitations. It cannot discriminate intact ssRNA, and NaOH is required to eluted with a pH gradient. Overall
ssRNA from dsRNA, and it has no ability remove the majority of proteins. purification potential of the pH gradient
to fractionate mRNA according to size. Reversed-phase chromatography (RPC) is increased by eliminating most of the
Intact product, incomplete transcripts, using styrenedivinylbenzene (SDVB) contaminants in advance. That leaves
oligomers, fragments, aggregates; any media also has proven useful to remove the gradient better able to polish out
species with an accessible poly-A tail dsRNA and fractionate ssRNA by size their last traces. Figure 11 shows the
elutes with all the rest. Oligo dT can be (Figure 8) (18–21). HIC and RPC media results of this approach with the DNA

42 BioProcess International 18(10) October 2020

 Figure 8: Reversed-phase chromatography of dsRNA and ssRNA ladders on a CIMmultus
plasmid and ssRNA from the previous SDVB column at 65 °C; equilibration/wash buffer 0.1 M triethylaminoacetate, pH 7.0;
figure. Figure 12 shows results with elution buffer 0.1 M triethylaminoacetate, 25% acetonitrile, pH 7.0
dsRNA and ssRNA ladders. Partial size 600
fractionation of ssRNA is evident.
The wash step can be intensified by mAU
260 dsRNA, 21–500b
substituting guanidine-hydrochloride
nm 4kb
for sodium chloride. Combining the
chaotropic salt with EDTA 3kb
360 dye marker 6kb
simultaneously relaxes nonspecific 2kb
electrostatic and hydrophobic 200b 1.5kb
interactions, hydrogen bonding, and 240 1kb
500b
metal coordination. Single-stranded
RNA remains bound, which creates an 120
opportunity to scrub and remove ssRNA
contaminants that may have become
associated with ssRNA during 0
transcription. 0 2 4 6 8 10
The pH of the eluted ssRNA should be Elution Time (min)
neutralized as soon as possible after
elution. As in the field of protein-
Figure 9: Distribution of charged residues, hydrogen donors, and hydrogen acceptors on
affinity chromatography, this is easily
RNA. Negative charges are indicated in red with yellow halos. Hydrogen acceptors are
done by pre-aliquoting a neutralizing indicated in red with pairs of red dots to indicate free lone-pairs of electrons. Hydrogen
buffer in the fraction vessels or donors are in blue. Compared with the four negative charges, there are 11 hydrogen
neutralizing the ssRNA fractions donors and 75 acceptors (one acceptor for each lone pair).
immediately after completion of the run.
Brief exposure to alkaline pH produces
O – O – O – O –
O O O O
P P P P
no evidence of modification. O O O O
O O O O
Like most anion exchangers, the
O O O O
column can be cleaned for extended O O O O
H H H H
periods with 1 M NaOH. Exposure to N N N N
large-volume IVT mixtures generally N O N O
N N N
requires treatment for at least an hour. N
O N H
Cleaning can be enhanced by combining H N N
H
N
O N H
1–3 M NaCl and 10–20 mM EDTA with H
H H H
the NaOH. Badly fouled columns can be
restored by treatment for 16–24 hours.
Figure 10: Anion-exchange fractionation of plasmid DNA from ssRNA by pH gradient
Purification of Research- and elution on a CIMmultus PrimaS pH column; Buffer A = 20 mM Tris, 20 mM bis-tris-
Clinical-Grade ssRNA propane, 20 mM glycine, 50 mM NaCl, 10 mM EDTA, pH 7.0; Buffer B: 20 mM Tris, 20 mM
For purification of research-quality bis-Tris-propane, 20 mM glycine, 50 mM NaCl, 10 mM EDTA, pH 11.0. Nucleic acids are
from New England BioLabs.
ssRNA, a CIMmultus PrimaS column
with a salt wash before pH elution 250 10.5
provides one-step purification
mAU
performance comparable to what protein
260 pH
A affinity has been providing to nm
immunologists since the 1990s. This
gives researchers a simple protocol to 150 9.5
ssRNA
obtain small amounts of good quality 5,000 b
ssRNA quickly and easily to advance
100 9.0
their studies. It gives upstream process sc pDNA
developers an easy tool to evaluate the 6,000 bp
effects of different variables in 50 8.5
optimizing their IVT protocols.
It gives downstream developers the
0 8.0
high-performing capture foundation
0 3 6 9 12 15
needed for purification of clinical-
Elution Time (min)
quality ssRNA. It provides high initial
purity and removes the foulants that

October 2020 18(10) BioProcess International 43

Figure 11: Elution of plasmid DNA and ssRNA from a CIMmultus PrimaS column. In one 3 McPhillips M, Ozato K, McBride A.
experiment, ssRNA with a size of 5,000 b was applied and eluted in a pH gradient. In the Interaction of Bovine Papillomavirus E2
other experiment, plasmid DNA was bound then washed off the column with 1 M NaCl, Protein with Brd4 Stabilizes Its Association
10 mM EDTA. Note its absence from the pH gradient except for a small doublet at about with Chromatin. J. Virol. 79(14) 2005: 8920–
11 minutes. Nucleic acids are from New England BioLabs. 8932; https://dx.doi.org/10.1128%2FJ
VI.79.14.8920-8932.2005.
1,000 10.5 4 Knipe D, et al. Snapshots: Chromatin
Control of Viral Infection. Virol. 435(1) 2013:
mAU 141–156; https://dx.doi.org/10.1016%2Fj.
260 pH
virol.2012.09.023.
nm
5 Lieberman P. Chromatin Regulation of
1 M NaCl pH gradient mRNA Virus Infection. Trends Microbiol. 14(3)
600 wash elution 5 kb 9.5
2006: 132–140; https://doi.org/10.1016/j.
tim.2006.01.001.
400 9.0 6 Augusto L, et al. Histones: A Novel
pDNA
6 kbp Class of Liposaccharide-Binding Molecules.
Biochem. 42(13) 2003: 3929–3938; https://
aggregates doi.org/10.1021/bi0268394.
20 8.5
7 Goldblatt D, Bustin M. Antigenicity of
Histones in Various Chromatins. Biochim.
0 8.0 Biophys. Acta 606(2) 1980: 304–315; https://
0 3 6 9 12 15 doi.org/10.1016/0005-2787(80)90040-4.
Elution Time (min) 8 Goldblatt D, Bustin M. Exposure of
Histone Antigenic Determinants in
Chromatin. Biochem. 14(8) 1975: 1689–1695;
https://doi.org/10.1021/bi00679a022.
Figure 12: Elution of a dsRNA ladder and a ssRNA ladder from a CIMmultus PrimaS
column. In one experiment, a ssRNA ladder containing species ranging in size from 200 9 Rosenberg A. Effects of Protein
to 6,000 bases was applied and eluted in a pH gradient. In the other experiment, a Aggregates: An Immunologic Perspective.
dsRNA ladder containing species ranging from 21 to 500 bases was bound, then washed AAPS J. 8(3) 2006: E501–507; https://doi.
off the column with 1 M NaCl, 10 mM EDTA. Note its absence from the pH gradient. org/10.1208/aapsj080359.
Nucleic acids are from New England BioLabs. 10 Schultze HE, Heremans JF. Molecular
Biology of Human Proteins with Special
250 10.5 Reference to Plasma Proteins, Vol. 1: Nature
and Metabolism of Extracellular Proteins.
mAU Elsevier: Amsterdam, The Netherlands, 1966.
260 pH 11 Gagnon P. Purification of Nucleic
nm Acids: A Handbook for Purification of Plasmid
1 M NaCl pH gradient ssRNA DNA and mRNA for Gene Therapy and
wash elution 200b
150 –6kb 9.5 Vaccines. BIA Separations: Ajdovščina,
Slovenia, 2020: https://www.biaseparations.
dsRNA com/en/products/monolithic-columns/books.
100 21–500b 9.0 12 Coban O, et al. Conformational
Heterogeneity in RNA Polymerase Observed
By Single-Pair FRET Microscopy. Biophys. J.
50 8.5 90(12) 2006: 4605–4617; https://dx.doi.org/
10.1529%2Fbiophysj.105.078840.
13 Fukuda R, Iwakura Y, Ishihama A.
0 8.0 Heterogeneity of RNA Polymerase in
0 3 6 9 12 15 Escherichia coli: A New Holoenzyme
Elution Time (min) Containing a New Sigma Factor. J Mol Biol.
83(3) 1974: 361–367; https://doi.
org/10.1016/0022-2836(74)90284-8.
could interfere with polishing methods. Acknowledgments 14 Westpheling J, Ranes M, Losick R. RNA
Polishing methods that also support Some of the figures in this article were Polymerase Heterogeneity in Streptomyces
removal of dsRNA, DNA, and proteins adapted from 11, with permission from the coelicolor. Nature 313, 3 January 1985: 22–27;
publisher. https://doi.org/10.1038/313022a0.
and achieve size fractionation suggest
themselves as effective partners. The 15 Gagnon P, et al. Nonspecific
Interactions of Chromatin with
low salt concentration of the ssRNA References Immunoglobulin G and Protein A, and Their
coming off anion exchange enables 1 Gantier M, Williams B. The Response
Impact on Purification. J. Chromat. A. 1340,
smooth transition to RPC. It also of Mammalian Cells to Double-Stranded RNA.
2014: 68–78; https://doi.org/10.1016/j.
Cytokine Growth Factor Rev. 18(5–6) 2007:
provides a smooth workflow with HIC. chroma.2014.03.010.
363–371; https://doi.org/10.1016/j.
Just add salt. Both platforms should cytogfr.2007.06.016. 16 Satterfield B, et al. Microfluidic
consistently deliver clinical quality Purification and Preconcentration of mRNA
2 Tan L, et al. Characterization of DNA
By Flow-Through Polymeric Monolith. Anal.
ssRNA. in Cell Culture Supernatant By Fluorescence-
Chem. 79(16) 2007: 6230–6235; https://doi.
Detection Size-Exclusion Chromatography.
org/10.1021/ac0709201.
Anal. Bioanal. Chem. 407(14) 2015: 4173–
4181; https://doi.org/10.1007/s00216-015- 17 Slater R. The Purification of Poly(A)-
8639-9. Containing RNA By Affinity Chromatography.
44 BioProcess International 18(10) October 2020

Vaccines and Beyond: The Future of Therapeutic RNA Beckons
The COVID-19 pandemic has focused the
international biopharmaceutical community on
the need to develop effective vaccine
candidates quickly. RNA-based vaccines offer
potential to accelerate development, simplify
manufacturing, and lower costs compared
with traditional approaches.
One of the particular benefits of RNA-based
vaccines is that they represent the ultimate
platform-manufacturing candidates.
Chemically and structurally, they are so similar
to each other that a unified development
template can be applied to all of them. This
lays the foundation for multivalent vaccines,
for which each component can be prepared
by essentially the same process. Indeed,
some such products include up to six
components to ensure a broad immune
response to a particular pathogen. Similarity
among those RNAs offers potential for
manufacturing all components in a single
production facility.
Clinical trials already have shown efficacy
against COVID-19 and other viruses including
hepatitis, Zika, influenza, and rabies. More
than 50 clinical trials also are proceeding
using RNAs as anticancer vaccines. These
properly represent immunotherapeutics
rather than vaccines in the traditional sense,
but they still help to illustrate RNA’s potential,
and there are many therapeutic candidates
beyond vaccines.
Single-gene disorders in which a critical
protein is not translated or is dysfunctional
represent another potential application.
Hemophilia B (clotting Factor IX deficiency)
and alpha-1 antitrypsin deficiency, which
elevate risk of liver and lung disease, are
obvious candidates, especially because they
particularly affect pediatric patients.
Methylmalonic acidemia is an inherited
disorder that prevents proper processing of
proteins and lipids. It presents in early infancy
with mild to life-threatening symptoms.
Clinical trials for such RNA therapies have
been fast-tracked by the FDA.
In the United States alone, over 30 million
people suffer from rare diseases. Those tend
to be under-addressed by recombinant-
protein–based therapeutics because of their
high development costs. The relative
simplicity of developing RNA-based therapies
offers hope.
As in any discussion concerning the
biopharmaceutical industry, antibodies
eventually come into the picture, and
discussion of RNA is no exception. It enables
patient cells to produce their own therapeutic
antibodies with absolutely authentic human
glycosylation. Every therapeutic antibody
presently approved for immunotherapy, every
antibody currently in clinical trials, and every
antibody yet to come is a potential candidate
for replacement by RNA.
Challenges remain, however. One challenge
is delivery: getting the RNA to the site of
therapy. Free RNA is broken down quickly
in vivo. Incorporation into larger molecules
and packaging inside liposomes or
nanoparticles have shown promise. Storage
is another challenge. Freezing or refrigeration
currently is required. Alternatives are under
development to make RNA therapies
available in regions where refrigeration is
unavailable. Purification represents a third
challenge. Double-stranded RNA conformers
and heteroaggregates of RNA with residual
synthesis reagents can elicit antagonistic
immune responses. Development of a
purification platform that can remove those
species simply, scalably, reproducibly, and
with good process economy remains an
important goal.
In this context, COVID-19 may yet offer a
positive outcome that outweighs its negative
impact: By elevating the impetus to solve
those remaining challenges, it can help to
usher the full range of RNA-based therapies
into a transformed, better prepared, and
healthier new world.
For Further Reading
About mRNA. Moderna Therapeutics: Cambridge,
MA, 2020; https://www.modernatx.com/about-
mrna.
Michel T, Wendel H-P, Krajewski S. Next-
Generation Therapeutics: mRNA as a Novel
Therapeutic Option for Single-Gene Disorders.
Modern Tools for Genetic Engineering. Kormann
M, ed. IntechOpen: London, UK, 2016; https://doi.
org/10.5772/62243.
Pardi N, et al. mRNA Vaccines: A New Era in
Vaccinology. Nat. Rev. Drug Discov. 17(4) 2018:
261–279; https://doi.org/10.1038/nrd.2017.243.
RNA Vaccines: An Introduction. PHG Foundation:
Cambridge, UK, 2020; https://www.
phgfoundation.org/briefing/rna-vaccines.
Schlake T, et al. mRNA: A Novel Avenue to
Antibody Therapy? Mol. Therapy 27(4) 2019:
https://doi.org/10.1016/j.ymthe.2019.03.002.
Tang X, et al. Therapeutic Prospects of mRNA-
Based Gene Therapy for Glioblastoma. Front.
Oncol., 8 November 2019; https://doi.
org/10.3389/fonc.2019.01208.
Tavernier G, et al. mRNA as Gene Therapeutic:
How to Control Protein Expression. J. Contr.
Release 150(3) 2011: 238–247; https://doi.
org/10.1016/j.jconrel.2010.10.020.
The Advantages of mRNA Vaccines. Moderna
Therapeutics: Cambridge, MA, 2020; https://
www.modernatx.com/pipeline/therapeutic-areas/
mrna-therapeutic-areas-infectious-diseases.
Van Hoecke L, Roose K. How mRNA
Therapeutics Are Entering the Monoclonal
Antibody Field. J. Transl. Med. 17, 2019: 54;
https://doi.org/10.1186/s12967-019-1804-8.
October 2020
Meth. Molec. Biol. 2, 1984: 117–120; https://
doi.org/10.1385/0-89603-064-4:117.
18 Karikó K, et al. Generating the
Optimal mRNA for Therapy: HPLC
Purification Eliminates Immune Activation
and Improves Translation of Nucleoside-
Modified, Protein-Encoding mRNA. Nucleic
Acid Res. 39(21) 2011: e142; https://doi.
org/10.1093/nar/gkr695.
19 Weismann D, et al. HPLC Purification
of In Vitro Transcribed Long RNA. Meth.
Molec. Biol. 969, 2013: 43–54; https://doi.
org/10.1007/978-1-62703-260-5_3.
20 Azarani A, Hecker KH. RNA Analysis
By Ion-Pair Reversed-Phase High
Performance Liquid Chromatography. Nucleic
Acid Res. 29(2) 2001: e7; https://dx.doi.
org/10.1093/nar/29.2.e7.
21 Majors RE. The Cleaning and
Regeneration of Reversed-Phase HPLC
Columns. LC-GC Europe 21(1) July 2003:
19–26; https://cdn.sanity.io/files/0vv8moc6/
chroma/1ab2a45ce4f7c16aa08f343c168ed8a1
2a4aa.pdf.
22 Prazeres D, Schluep T, Coony C.
Preparative Purification of Supercoiled
Plasmid DNA Using Anion Exchange
Chromatography. J. Chromatog. A 806(1)
1998: 31–45; https://doi.org/10.1016/s0021-
9673(97)01254-5.
23 Koubek J, et al. Strong Anion-
Exchange Fast Performance Liquid
Chromatography as a Versatile Tool for
Preparation and Purification of RNA
Produced By In Vitro Transcription. RNA
19(10) 2013: 1449–1459; https://doi.
org/10.1261/rna.038117.113.
24 World Patent Application
WO2014144767. Ion Exchange Purification of
mRNA. WIPO: Geneva, Switzerland, 15 March
en/detail.jsf?docId=WO2014144767. c
2013; https://patentscope.wipo.int/search/
Corresponding author Pete Gagnon is chief
scientific officer at BIA Separations, Ajdovščina,
Slovenia, and a member of BPI’s Editorial Advisory
Board; pete.gagnon@biaseparations.com.
Špela Peršič and Blaž Goričar are members of the
DNA plasmid and mRNA purification process
development team, and Urh Černigoj is the head
of new product development. Aleš Štrancar is chief
executive officer, BIA Separations.
To share this in PDF or professionally printed
format, contact Jill Kaletha: jkaletha@
mossbergco.com, 1-574-347-4211.
18(10) BioProcess International 45