TrAC Trends in Analytical Chemistry,
Journal Year:
2024,
Volume and Issue:
176, P. 117758 - 117758
Published: May 14, 2024
Therapeutic
oligonucleotides
(ONs)
are
a
new
generation
of
drugs
that
have
captured
the
attention
pharmaceutical
industry
over
past
decade,
as
evidenced
by
FDA
approval
14
ONs
since
2018.
Although
therapeutic
complex
to
analyze,
there
is
an
unprecedented
need
for
powerful
analytical
methods
serve
this
scope,
and
liquid
chromatography
coupled
mass
spectrometry
(LC-MS)
one
leading
techniques.
Ion-pairing
reversed-phase
(IP-RPLC)
gold
standard
ON
characterization,
but
hydrophilic
interaction
(HILIC)
has
clearly
emerged
alternative
option.
This
review
critically
examines
recent
advances
in
HILIC-MS
developments
analysis
their
impurities.
In
particular,
critical
selection
optimization
chromatographic
conditions
discussed,
together
with
latest
technological
required
coupling.
Finally,
several
applications
context
bioanalysis
reported.
Analytical Chemistry,
Journal Year:
2025,
Volume and Issue:
unknown
Published: Jan. 21, 2025
Phosphorothioate
(PS)
modifications
in
single-guided
RNA
(sgRNA)
are
crucial
for
genome
editing
applications
using
the
CRISPR/Cas9
system.
These
may
enhance
sgRNA
stability,
pharmacokinetics,
and
binding
to
targets,
thereby
facilitating
desired
genetic
alterations.
Incorporating
multiple
PS
groups
at
varying
positions
introduce
chiral
centers
into
backbone,
resulting
a
complex
mixture
of
constitutional-
stereoisomers
that
challenges
current
analytical
capabilities
reliable
identification
quantification.
In
this
study,
we
developed
an
innovative
methodology
combines
endonuclease
digestion
with
ion
pairing
reversed-phase
liquid
chromatography
coupled
cyclic
mobility
mass
spectrometry
(IPRP-LC/cIMS)
fully
distinguish
PS-induced
isomers
mixture.
The
relative
abundance
each
isomer
was
quantified
two-step
method,
wherein
sequentially
extracted
from
LC/MS
chromatogram
LC/cIMS
two-dimensional
mobiligram.
This
quantification
method
thoroughly
evaluated,
demonstrating
excellent
sensitivity,
precision,
dynamic
range,
repeatability,
accuracy.
addition,
enables
investigation
kinetics
forming
phosphodiester
(PO)
impurities
under
oxidative
stress
conditions,
offering
unprecedented
insights
stability
single-residue
resolution.
context,
highlights
in-depth
characterization
PS,
its
capability
support
biomedical
research,
development,
production
products.
Journal of Separation Science,
Journal Year:
2025,
Volume and Issue:
48(1)
Published: Jan. 1, 2025
ABSTRACT
Oligonucleotides
(ONs)
are
an
increasingly
popular
category
of
molecules
in
the
pharmaceutical
landscape,
particularly
attractive
for
treatment
genetic
and
rare
diseases.
However,
analyzing
these
presents
significant
challenges,
due
to
their
highly
hydrophilic
nature,
multiple
negative
charges,
presence
closely
related
impurities
resulting
from
complex
solid‐phase
synthesis
process.
Ion
pairing
reverse‐phase
liquid
chromatography
(IP‐RPLC)
is
preferred
technique
ONs
analysis
but
not
ideal
mass
spectrometry
(MS)
coupling.
Consequently,
there
a
growing
interest
exploring
alternative
strategies
with
interaction
(HILIC)
emerging
as
one
most
promising
options.
As
HILIC
yet
fully
established
ONs,
we
have
prepared
this
protocol
paper
facilitate
entry
into
field.
It
only
provides
best
practices,
opportunities,
potential
advantages
also
caveats
other
important
considerations
using
characterize
ONs.
The
addresses
selection
stationary
mobile
phases,
optimization
gradient
conditions,
MS
coupling,
key
aspects
consider
when
manipulating
ON
samples.
We
hope
will
help
establish
more
universal
solution
analysis.
Analytical Chemistry,
Journal Year:
2025,
Volume and Issue:
97(5), P. 2837 - 2844
Published: Jan. 30, 2025
Single-stranded
guide
RNAs
(sgRNAs)
are
important
therapeutic
modalities
that
facilitate
selective
genome
editing
by
the
CRISPR/Cas9
system.
While
these
synthesized
through
solid
phase
oligonucleotide
synthesis
similar
to
small
interfering
RNA
(siRNAs)
and
antisense
(ASOs)
therapeutics,
their
sequence
length
complex
secondary
tertiary
structure
hinder
analytical
characterization.
The
resulting
current
sgRNA
methodologies
have
limited
chromatographic
selectivity
near
FLP
MS
compatibility.
Here,
we
perform
a
systematic
evaluation
of
parameters
develop
highly
ion-pairing
reversed-phase
chromatography
separation
for
sgRNAs.
We
identify
stronger
more
hydrophobic
reagents
promote
long
truncation
impurities.
Further,
reduced
flow
rates
temperatures
FLP.
Together,
this
study
reports
first
single-nucleotide
resolution
method
an
modality
while
employing
MS-compatible
mobile
phase.
This
work
provides
further
evidence
is
independent
on-column
mass
transfer.
methodology
will
enable
high-resolution
characterization
providing
insights
into
impurity
profiles
toxicological
studies
process
development
genetic
medicines.
Analytical Chemistry,
Journal Year:
2025,
Volume and Issue:
unknown
Published: Feb. 27, 2025
Recently,
there
has
been
emerging
interest
in
the
characterization
of
higher
order
structure
(HOS)
oligonucleotide
therapeutics
because
its
potential
impact
on
function.
However,
many
existing
experimental
and
computational
methods
face
challenges
with
respect
to
throughput,
cost,
resolution
for
large
ribonucleic
acids
(RNAs).
In
this
study,
we
present
use
two
orthogonal
analytical
methods,
size-exclusion
chromatography
(SEC)
microfluidic
modulation
spectroscopy
(MMS),
which
are
used
investigate
conformational
changes
100
mer
single
guide
RNAs
(sgRNAs)
complex
HOS
aggregation
specie
profiles.
SEC,
coupled
multiangle
light
scattering
(MALS),
mass
spectrometry
(MS),
isothermal
MMS
revealed
various
forms
interactions.
We
also
developed
temperature-course
SEC
thermal
ramping
monitor
real-time
from
room
temperature
RNA
melting
point.
Through
experiments,
observed
discrete
steps
thermally
induced
dissociation
aggregates,
namely
aggregates
(HOA)
dimer
dissociation.
Temperature-course
allows
thermodynamic
analysis
enthalpy
entropy
reaction.
identified
spectral
regions
infrared
(IR)
spectra
MMS,
1665
cm-1
between
1700
1720
cm-1,
closely
correlated
Watson-Crick
base
pairing
related
change
RNA.
The
combination
offers
a
comprehensive
biophysical
toolkit
under
native
conditions,
providing
valuable
insights
candidate
optimization
formulation
screening
development
therapeutics.
Deleted Journal,
Journal Year:
2025,
Volume and Issue:
unknown, P. 20 - 26
Published: March 1, 2025
Hydrophilic
interaction
liquid
chromatography
(HILIC)
has
emerged
as
a
promising
alternative
to
traditional
ion-pair
reversed
phase
(IP-RPLC)
methods
for
separating
oligonucleotides
(ON).
This
work
investigates
the
application
of
HILIC
separation
ON
sequence
and
length
variants,
duplexes,
single-stranded
components.
Method
variables,
including
ionic
strength,
temperature,
mobile
composition,
gradient
optimization,
are
investigated
in
detail.
A
sequential
injection
procedure
is
also
proposed
eliminate
breakthrough
effects
peak
splitting.
Some
considerations
generic
(platform)
suggested.
