Protein Quality Control

Application Note NT-PR-004

Rapid Quantification of Unfolded Proteins for Quality Control

and Optimization of Storage Conditions
Melanie Maschberger, Dennis Breitsprecher

NanoTemper Technologies GmbH, Munich, Germany

quality can therefore have severe and expensive

Abstract consequences (Raynal et al, 2014).
The rigorous control of the quality of biological
Protein unfolding and denaturation is one of the
samples is of major importance in
major causes for protein loss-of-function, and must
pharmaceutical research. On the one hand,
therefore be carefully controlled in drug discovery
drug discovery projects with purified drug
and drug development. For instance, therapeutic
targets such as kinases, receptors or integral
antibodies must maintain their functionality when
membrane proteins require a consistent quality
stored under different conditions, and must also
of the proteins to ensure successful screening
tolerate moderate physical stress (e.g. shaking) as
campaigns. On the other hand, a consistent
well as changes in temperature. Moreover, many
quality of biologicals such as antibodies or
potential drug targets such as integral membrane
other therapeutic proteins is essential in later
proteins, cell surface receptors or kinases, must
stages of the drug development process,
maintain their functionality during cost-intensive
especially for tests in clinical trials, and is
screening campaigns, and might react very
finally required for approval of new drugs by
differently to physical and thermal stress.
federal agencies.
Here we demonstrate how the Prometheus
NT.48 can be used to investigate the long-term
stability of proteins as well as to determine
optimal handling conditions by quantifying the
fraction of unfolded proteins within seconds.

Introduction
The number of purified proteins that are used for
diagnostic, academic and therapeutic applications
is constantly increasing. Especially the use of more
challenging proteins such as integral membrane
proteins or conjugated antibodies is becoming more
and more widespread. Biophysical downstream
analysis in drug discovery and the in development
of biologicals however is expensive, time
consuming and often requires a variety of different
instruments. Failure in those downstream or
upstream processes due to an insufficient protein
Figure 1 Example of a typical thermal unfolding curve by
nanoDSF. The fluorescence ratio F350/F330 can be expressed
as % unfolded if the fluorescence ratios of the folded and
unfolded states are known.
Here we show that the Prometheus NT.48 allows
for a precise determination of the fraction of
unfolded protein in just a few µl of protein
formulations within seconds.
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The Prometheus NT.48 measures thermal calculated and experimentally determined
unfolding profiles of proteins by detecting even percentage of unfolded IgG versus the detected
minute changes in the emission properties of the F350/F330 ratio displayed a clear linear correlation
amino acid tryptophan upon unfolding. By following as well as a very good agreement between
the shift in the fluorescence emission wavelength, calculated and experimental data. Thus, the
an unfolding curve can be generated Prometheus NT.48 can pick up even small fractions
(Figure 1).Importantly, the specific fluorescence of unfolded IgG (< 0.5 %) in formulations, and could
ratios (F350/F330) of the folded and unfolded thus be used as a quick and reliable method to
states can be directly correlated to the respective follow the stability of antibodies, e.g. during long-
fraction of unfolded protein at any point during the term storage tests.
unfolding process (Figure 1). This also means that
a single fluorescence scan of a protein sample
contains information about the overall amount of
unfolded protein in the protein sample, once the
respective F350/F330 values of the folded and
unfolded state are known. This information is
valuable for protein quality control, either for
evaluating long-term stabilities of biologicals or the
effects of physical or thermal stress on protein
stability.
The Prometheus NT.48 uses a capillary “dip-and-
read” format, meaning that the capillaries can be
directly loaded by dipping into a protein solution
without additional pipetting steps. The
measurement of the F350/F330 of up to 48
capillaries at a given temperature takes 7 seconds,
and the Prometheus NT.48 instrument also does
not require any calibration times, so that quality
control measurements as we describe them here
can be performed in less under a minute.

Results
Unfolding of an IgG-antibody as proof-of-
concept
In order to verify the capability of the Prometheus
NT.48 to exactly quantify the fraction of unfolded Figure 2. Establishing a protein unfolding standard.
protein in a given formulation based on the Unfolded IgG at different concentrations was mixed with folded
F350/F330 ratio, we created an unfolding standard IgG and subjected to thermal unfolding. The percentage of
unfolded IgG in the solution was quantified based on the
solution in which IgG antibodies were subjected to F350/F330 ratio measured at 25 °C.
controlled thermal unfolding at 80 °C. Unfolded IgG
was then mixed with folded IgG to different
Assessing long-term stability of integral
extends, resulting in formulations with defined
membrane proteins
amounts of unfolded antibody.
Next, we asked whether this approach can even be
In thermal unfolding experiments, all formulations
used for long-term storage tests of detergent-
showed similar thermal unfolding profiles with
solubilized membrane proteins. For this, we used
unfolding transition temperatures around 74 °C.
the slow-anion channel protein HiTeHa (which was
However, the initial fluorescence ratio (F350/F330)
also used for a detergent-based stability screen in
at 25 °C increased with increasing concentrations
the Application Note NT-PR-002 (Maschberger et
of unfolded antibody (Figure 2). A plot of the
al, 2015)) and incubated the protein at 4 °C and at
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RT, respectively. The, we determined F350/F330
ratios and measured thermal unfolding curves over
a period of 34 days. Again, a clear storage
temperature- and incubation time-dependent shift in
the F350/F330 ratio could be observed, while the
Tm-values of the thermal unfolding curve did not
change significantly. The results show that HiTeha
is rather stable, but that it tends to degrade slowly
when stored at room temperature, and that the
F350/F330 ratio can serve as a measure of the
amount of unfolded protein in solution.
Figure 3 Long-term storage test on HiTeha. Aliquouts of the
integral membrane protein HiTeha were stored at 4 °C and at
RT, respectively, and thermal unfolding curves were measured
over a time period of 34 days. %unfolded protein was calculated
based on the F350/F330 ratio.
Forced denaturation of the drug target MEK1
Last, we asked whether this approach can also be
applied to evaluate the effects of forced
degradation on proteins. Such an approach can be
valuable to identify critical handling steps during
protein purification, storage or setup of biochemical
assays. Information about the stability of a protein
under different conditions is particularly important
for biophysical screening campaigns, in which
multiple biochemical assays are applied. We chose
the popular drug target kinase MEK1, which
already has been used in a large number of
different drug discovery screening campaigns,
including a MST-based fragment screen (see
Application Note NT-MO-021 (Breitsprecher et al,
2014) and (Amaning et al, 2013; Oyarzabal et al,
2010)). MEK1 solutions were subjected to a
number of thermal and physical stresses, namely
incubation at different temperatures, heavy
vortexing, and repeated freeze-thaw cycles. Again,
the fluorescence ration F350/F330 served as an
indicator for the fraction of unfolded protein
(Figure 4).
Figure 4 Forced-degradation stress-test on MEK1
MEK1 protein was subject to the indicated stresses, and the
fraction of unfolded protein was calculated based on the
F350/F330 ratio at 25 °C. Error bars are s.d. from three
measurements.
Incubation of MEK1 for 1 h on ice or at RT resulted
in no significant difference in the F350/F330 ratio,
showing that the protein can be handled at RT over
this time period without risking protein denaturation.
This finding is in agreement with the previously
performed MST-based fragment screening
campaign, in which the MEK1-nucleotide
interaction shown to be stable for > 12 h at RT
(Breitsprecher et al, 2014). In contrast, incubation
at an elevated temperature of 40 °C for 1 h, which
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is above the unfolding onset temperature, already
resulted in a fraction of unfolded protein of ~ 65 %.
Incubation of the protein solution for 15 minutes at
60 °C led to a complete unfolding of the protein, as
expected, indicated by the absence of a unfolding
transition in the thermal unfolding curve.
Interestingly, the protein was rather resistant
towards mechanical stress, as vortexing for
1 minute at maximum rpm resulted in only a small
fraction of unfolded protein (< 5 %). In contrast,
repeated freeze-thaw cycles strongly denatured the
protein, leading to > 75 % unfolded protein
(Figure 4).
Thus, this set of experiments quickly revealed that
MEK1 is stable at ambient temperature and rather
insensitive against mechanical stress such as
vortexing, whereas freeze-thaw cycles and
elevated temperatures promote the denaturation of
the protein and should be avoided.
Conclusion
The presented data demonstrate that the
Prometheus NT.48 can be employed to quickly
detect and quantify unfolded proteins for quality
control purposes with unmatched speed, at the
same time offering unique ease of use. The
presented quality control experiments can be
performed by filling capillaries directly from stock
solutions without laborious sample preparation or
loading. F350/F330 values of up to 48 samples are
then recorded in parallel using a one-button routine,
providing stability data within seconds.
Material and Methods
Protein preparation
IgG unfolding standards were created by incubating
mouse IgG from serum (Sigma Aldrich) at a
concentration of 2 mg/ml in PBS, pH 6.2 was
heated to 75°C with shaking for 3 h. The fully
unfolded IgG was then cooled on ice and mixed
with folded IgG in the same buffer to create
formulations of 2 mg/ml IgG with the fractions of
unfolded IgG shown in Figure 2.
HiTeha long-term storage experiments were
performed by incubating aliquots of HiTeHa at a
concentration of 2 mg/ml in 50 mM Tris, 200 mM
NaCl, 0.02 % DDM, pH 8 at 4 °C and at RT. For
each experiment, 5 nanoDSF grade capillaries per
condition were filled with 10 µl of sample,
respectively, and subject to a thermal ramp from
25 °C to 90 °C with a heating rate of 1 °C/min.
The MEK1 forced degradation screen was
performed with 2.9 µM MEK1 in 20 mM HEPES
pH 7.4, 300 mM NaCl, 2 mM DTT. For each
treatment, aliquots of 50 µl were used and subject
to the treatments described in the results section.
Samples from each condition were filled into
3 nanoDSF grade capillaries, respectively, and
subject to a thermal ramp from 25 °C to 90 °C with
a heating rate of 1 °C/min.
References
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© 2015 NanoTemper Technologies GmbH
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