Simplifying Intact Molecular Weight Determination for ADCs
Key Benefits
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Any help in reducing sample preparation, analysis time and effort can move products through
the biopharmaceutical pipeline more easily
Tools to facilitate the analysis and review of complex, high molecular weight species such as
ADCs have been produced by AB SCIEX
These tools are accessible to scientists at all stages of biopharmaceutical development
Introduction
Heterogeneity and high molecular weight species pose challenges for analytical scientists and
both of these problems converge with Antibody Drug Conjugates (ADCs). ADCs are one of the
fastest growing segments of the biotherapeutic pipeline, with hundreds of therapeutics in
development. Early in development, analysts are tasked with providing rapid feedback to their
synthetic chemists on how well a conjugation strategy may have worked. Later in development
as process development accelerates, analysts need means to rapidly confirm that the product
has maintained its integrity, for example in formulation. Therefore there is always pressure to
respond rapidly to the demands of multiple departments.
In this Technical Brief we illustrate how
TripleTOF® technology coupled with
SelexION™, Eksigent ekspert™ MicroLC, and
BioPharmaView™ software or PeakView®
software can provide answers where they are
needed, fast. Assays that may have needed
days to complete are now ready to report
within less than an hour.
Discussion
The tasks involved in determining the average
molecular weight of an Antibody Drug
Conjugate (ADC) appear to be straightforward:
• Determine the intact molecular weight of
the construct
• Obtain Drug-Antibody Ratio (DAR)
• Determine the range of the number of
drugs linked
Figure 1a (upper trace): ESI-MS spectrum of an Intact ADC
on a TripleTOF® 5600+.
Figure 2b (lower trace): ESI-MS spectrum of an Intact ADC on
a TripleTOF® 5600+ with a CoV applied on the DMS device
prior to entry into the flight path of the mass spectrometer.
[Values anonymized]]
In reality, the task may be complicated by the heterogeneity of the constructs, the number of
impurities or fragments if the synthesis is still at an early stage of optimization, or interfering
compounds such as those found in formulation. Overcoming these difficulties might involve
careful and time-consuming sample preparation, involve reducing the construct to its
component parts to simplify analysis, or may require fraction collection. All of these may cost
time, or modify the sample, and in the worst cases make it harder to draw firm conclusions
about the stability.
AB SCIEX needs to provide its partners in a competitive space with tools that help them
maintain a competitive lead. By innovating in technology, AB SCIEX can provide easier
methodologies to mitigate some of these challenges. The solution in this case was to provide a
combination of tools that fit seamlessly together for the analysis to take place.
Figure 1 shows the ESI-MS spectrum of an intact ADC based on an IgG1 molecule. In the
spectrum there are a number of interfering species, some of which are visible at the lower end
of the m/z scale below approximately 2500 m/z. The deconvolution of this spectrum provided
only a gross, uninformative reconstruction which did not conclusively clarify detail.
However, the application of
SelexION™ technology
provided much needed
clarity. SelexION™ is a
simple, differential ion
mobility device that fits at
the source of the mass
spectrometer. SelexION™
does not interfere with the
analysis, and provides an
orthogonal separation based
on ion mobility. A number of
attributes make this an ideal
addition to the analysis:
• Simple Voltage
changes are applied
instantly
• No user expertise is
Figure 3: Data processed in BioPharmaView™ for an ADC in development. The
required, and no
panels at the bottom show the raw (left) and automatically deconvoluted (right)
need for complex
spectra for this sample. [Values anonymized].
tuning
• Separation occurs PRIOR to detection, so no interference with data processing
• An identical (LC) method can be used for all cases
Results
The Lysine-linked ADC studied here eluted from the DMS device at a CoV around -5V. Smaller
contaminants and contaminating fragments elute from the DMS device approaching positive
CoV values. Retention time and all other instrument parameters were held constant for all
sample acquisitions. Figure 2 shows the raw spectrum of the ADC without the interfering
species, and Figure 3 shows the deconvoluted MS Spectrum in BioPharmaView™ software, used
for automated data processing and reporting.
Conclusions
For complex biologics
species, SelexION™
on a Triple Tof™
instrument from AB
SCIEX may provide a
simple, elegant
solution to cleaning
up intact species from
an ESI spectrum.
The rapid, simple
application of Ion
Mobility facilitates
Figure 4a: ADC processed through BioPharmaView™ software to show raw data,
deconvolution, and peak assignments automatically. The in-built comparability of
the analysis of Intact
BioPharmaView™ is demonstrated in the mirror plots in panels on the right hand side of
ADCs and
the graphic. [Values anonymized].
• Has no effect
on other experimental conditions
• Uses identical processing parameters/ informatics
• Is applicable with no expert training
• Is transferable across ion-mobility platforms with SelexION™ such as QTrap® technology.
For Research Use Only. Not for use in diagnostic procedures.
© 2014 AB SCIEX. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license.
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