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Advancing Monoclonal Antibody Stability Studies With Mass Photometry
App Note / Case Study
Last Updated: June 21, 2024
(+ more)
Published: April 16, 2024
Credit: Technology Networks
Antibody aggregation severely impacts a biopharmaceutical product’s efficacy and safety. Hence, an array of techniques are used to assess the physiochemical stability of candidate therapeutic antibodies throughout the biopharmaceutical development cycle.
Mass photometry is an innovative biophysical analysis tool used to study the native behavior of biomolecules with minimal sample preparation.
This application focus explores how mass photometry can be used for biophysical characterization, antibody stability studies and the analysis of antibody aggregates.
Download this application focus to learn more about:
Accurate, label-free mass measurements of single molecules in solution
How mass photometry complements size exclusion chromatography data
Mass photometry instruments that perform measurements and analysis in less than five minutes
Antibody aggregation can severely impact a biopharmaceutical product’s efficacy and safety. Hence, researchers apply an array of techniques to assess the physiochemical stability of candidate therapeutic antibodies throughout the biopharmaceutical development cycle.
Mass photometry is a robust method to characterize the biophysical properties of antibodies. It can also be applied to antibody stability studies and the characterization of antibody–antigen interactions.
Biophysical characterization using
mass photometry
Mass photometry is an innovative biophysical analysis tool that can be used for sample characterization, interaction studies and the analysis of protein complexes. The technique builds on the principles of interference reflection microscopy and interfero-metric scattering microscopy to measure the molecular mass of proteins and protein assemblies in solution.1,2
®
APPLICATION NOTE
Advancing monoclonal
antibody stability studies with mass photometry
DR. AMY CHAU
Field Applications Scientist
Figure 1. The principles of mass photometry. A detector measures the scattered light and creates an interference pattern that can be visualized and analyzed to identify the species in the sample, based on their mass, and quantify their relative abundance.
Biomolecules approaching a glass surface are illuminated by a laser
The biomolecules scatter light, which interferes with the
light reflected
at the interface
The resulting interference contrast scales linearly with the mass of the biomolecule
A mass histogram is generated from the single molecule measurements
Molecules in solution
Incident light Scattered light
Mass photometry contrast
Molecules in solution
Expected mass (kDa)
200 400 600 800 1000
400
200
800
1000
600 Mass measured by
mass photometry (kDa)
Incident light Scattered light
Mass photometry contrast
Molecules in solution
Mass photometry contrast
Counts
1
2
3
4
APPLICATION NOTE
ADVANCING MONOCLONAL ANTIBODY STABILITY STUDIES WITH MASS PHOTOMETRY 2
Instruments measure the light scattered by a biomolecule in
solution, which is linearly scaled with the molecule’s volume and
refractive index (Figure 1). The optical properties and density
vary by only a few percent within a class of biomolecules (e.g.,
proteins). This means that the scattering signal directly correlates
to the molecule’s mass.
Mass photometry enables accurate mass measurements of
single molecules in solution without the need for labels. This
makes it a rapid and cost-effective method to study the native
behavior of biomolecules with minimal sample preparation. Mass
photometry can be used to determine complex stoichiometry
or oligomeric states and monitor macromolecular assembly or
other complex processes.
This technique can also be used to characterize and monitor
sample heterogeneity – this is particularly important for antibody
studies – providing insights into sample purity and allowing the
observation of stability under different conditions. Two case
studies are presented below to illustrate mass photometry
applications in therapeutic antibody development.
Characterizing antibody stability
Mass photometry is an ideal way to assess sample purity and
monitor aggregate formation, owing to its wide mass range and
high dynamic range. In this case study, researchers used mass
photometry to conduct forced degradation assays to screen
candidate therapeutic antibodies and select the right candidates
for further development.3
Forced degradation assays monitor how samples react under
different stress conditions. This study tested 14 different
antibodies to establish whether changes in exposure to light, pH
and hydrogen peroxide (oxidative stress) resulted in antibody
aggregation or fragmentation. For this reason, the researchers
used an automated mass photometer (TwoMP Auto) to expedite
their workflow, reduce the amount of hands-on time required
and improve the precision of the results by removing human
error from the pipetting and mixing steps.
For “antibody 1” (Figure 2), the results showed the presence of
the intact antibody monomer. However, a low molecular mass
species (LMM) and high molecular mass species (HMM) were
also identified, representing the fragmented antibody and
aggregated antibody respectively. The percentage of abundance
for each peak was quantified for each form of antibody 1,
indicating that stress condition 2 induces the highest degree of
fragmentation, whereas stress condition 3 induces the highest
degree of aggregation.
For “antibody 2” (Figure 3), the aggregation region was further
defined with trimer formation. Comparison of relative abundance
showed that stress conditions 1 and 3 promote aggregation (the
formation of dimers and trimers).
This study demonstrated that analytical tools spanning a wide
mass range may be required to capture the complete data set
for in-depth characterization of all products or variants in
Condition % Fragmented
antibody
% Intact
antibody
% Aggregated
antibody
Control 9% (± 0.7) 90% (± 0.6) 1% (± 0.3)
Stress 1 10% (± 1.1) 88% (± 1.1) 2% (± 0.1)
Stress 2 27% (± 1.8) 70% (± 1.0) 4% (± 0.9)
Stress 3 14% (± 2.2) 81% (± 2.1) 5% (± 0.3)
Figure 2. Fragmentation and aggregation products from “antibody 1” upon exposure to
three stress conditions.
Figure 3. Fragmentation and aggregation products from “antibody 2” upon exposure to
three stress conditions.
Condition
%
Fragmented
antibody
% Intact
antibody
%
Aggregated
antibody
(dimer)
%
Aggregated
antibody
(trimer)
Control 4% (± 0.8) 95% (± 0.7) 1% (± 0.1) 0%
Stress 1 5% (± 0.4) 85% (± 0.3) 8% (± 0.4) 2% (± 0.2)
Stress 2 7% (± 1.5) 91% (± 1.4) 2% (± 0.1) 0%
Stress 3 5% (± 0.8) 86% (± 0.5) 8% (± 0.3) 1% (± 0.1)
APPLICATION NOTE
ADVANCING MONOCLONAL ANTIBODY STABILITY STUDIES WITH MASS PHOTOMETRY 3
degradation analysis. Mass photometry can quickly screen a
large number of candidates, supporting rapid decision making
in biopharmaceutical development.
Assessing antibody aggregation
When assessing the aggregation levels of a biologic, mass
photometry can complement other techniques such as size
exclusion chromatography (SEC). A study was designed to assess
the aggregation levels of the monoclonal antibody trastuzumab
(a biologic used to treat cancer under the commercial name
Herceptin) and four biosimilars (clones 3, 8, 9 and 10), using
both mass photometry (TwoMP system) and SEC.4
Although SEC separates molecules based on their molecular
sizes and mass photometry identifies molecules based on their
mass, the results from both techniques should lead to the same
conclusion. The SEC chromatogram produced in the study
(Figure 4a) showed a small secondary peak – indicating the
presence of an antibody dimer – for clones 3 and 10. When the
same five samples were measured using mass photometry (Figure
4b), clones 3 and 10 also showed notable secondary peaks,
indicating the presence of dimers.
SEC results must be quantified appropriately to achieve
standardization. Absorbance cannot be directly compared to the
number of counts measured in mass photometry (i.e., the height
of the histogram bar), because the absorbance data is both a
function of the molecule’s concentration and is dependent on the
samples’ UV absorbing properties. The specifications of the UV
detector itself (e.g., its sensitivity at a particular UV wavelength)
also influence the data. Therefore, the Beer–Lambert law must
be applied to the absorbance values generated from SEC
chromatograms in order to calculate relative abundance values
and compare the data to mass photometry results.
For this type of experiment, the results generated from SEC
and mass photometry are very similar (Table 1). However, the
advantage of using mass photometry is that these results can be
achieved in a shorter time – 1 minute per measurement and less
than 3 mins including analysis. Mass photometry also requires a
smaller sample for analysis – only nanograms compared to the
micrograms required for SEC. If researchers don’t need sample
separation or want to be more strategic in selecting the right
candidate for these longer separation experiments, then mass
photometry is the right choice.
Figure 4. (a) SEC chromatogram showing the elution of molecules over time, where
larger molecules elute first. Below is a magnified version highlighting a secondary peak
between 7 and 8 minutes. (b) Mass photometry histogram using the same samples.
(a)
(b)
Table 1. Quantified percentage abundance values calculated from SEC-UV (top) and mass
photometry (bottom).
SEC-UV
Monomer 99.9 97.7 99.8 99.2 97.6
Dimer 0.1 2.3 0.2 0.8 2.4
Abundance (%)
Sample Trastuzumab Clone 3 Clone 8 Clone 9 Clone 10
originator
Mass
photometry
Monomer 99.3 96.8 98.8 98.7 97.1
Dimer 0.7 3.2 1.2 1.3 2.9
Abundance (%)
Sample Trastuzumab Clone 3 Clone 8 Clone 9 Clone 10
originator
Mass photometry systems
from Refeyn
Refeyn is a company specializing in the development and
manufacture of instrumentation for the characterization of
biomolecules using mass photometry technologies. The case
studies highlighted in this application use the TwoMP system
offered by Refeyn, which is quick and easy to use; from
measurement to analysis, users can obtain the results in less
than five minutes.
The TwoMP is a mass photometry instrument that requires
manual pipetting during sample preparation, and the TwoMP
Auto features the same internal system plus a pipetting robot.
With the TwoMP Auto, the user can design experimental
protocols with the number of repeats and dilutions that are
necessary, and the instrument performs the dilutions and
transfers samples to the measurement stage. Advantageously,
the TwoMP system can be retrofitted with the automation unit
to upgrade when the need arises.
APPLICATION NOTE
ADVANCING MONOCLONAL ANTIBODY STABILITY STUDIES WITH MASS PHOTOMETRY 4
Conclusion
Whether it is using a manual or automated system, researchers
can gain quick insights by conducting mass photometry on a
dedicated instrument. When used as an initial screening tool
to streamline analyses, it is an easy and cost-effective way to
accelerate biologics development.
In the development of therapeutic antibodies, mass photometry
is well suited for the characterization of antibody aggregation and
fragmentation. It can also be used to characterize antibody–
antigen interactions and quantify binding site occupancy under
different conditions. Furthermore, binding affinity between
proteins can be determined using single-shot Kd measurements.5
Mass photometry is already making a big impact in furthering
the understanding of biomolecule behaviors. Since 2018, Refeyn
has installed over 350 mass photometers worldwide, with
users in both academia and industry, and more than 300 peer
reviewed papers have used these techniques to answer pressing
biological questions.
Advance your research
with mass photometry
References
1. Verschueren H. Interference reflection microscopy in cell biology:
methodology and applications. J Cell Sci. 1985;75:279-301. doi:10.1242/
jcs.75.1.279
2. Ortega-Arroyo J, Kukura P. Interferometric scattering microscopy
(iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopy.
Phys Chem Chem Phys. 2012;14(45):15625-15636. doi:10.1039/
c2cp41013c
3. Characterization of forced antibody degradation with automated mass
photometry. Application Note. Refeyn. https://www.refeyn.com/
characterizing-antibody-aggregation-with-mass-photometry.
4. Verscheure L, De Vos J, Sadowska W, et al. Characterizing antibody
aggregation with mass photometry. Technical Note. Refeyn. https://
www.refeyn.com/mass-photometry-vs-sec-in-protein-analysis.
5. Soltermann F, Foley EDB, Pagnoni V, et al. Quantifying Protein-Protein
Interactions by Molecular Counting with Mass Photometry.Angew
Chem Int Ed Engl. 2020;59(27):10774-10779. doi:10.1002/
anie.202001578
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