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.

1. Simplifying Intact Molecular Weight
Determination for ADCs
For Research Use Only. Not for use in diagnostic procedures

2. 2 © 2015 AB Sciex
•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 SCIEX
•These tools are accessible to scientists at all stages of
biopharmaceutical development
KEY BENEFITS

3. 3 © 2015 AB Sciex
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.

4. 4 © 2015 AB Sciex
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
DISCUSSION

5. 5 © 2015 AB Sciex
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.
SCIEX needs to provide its partners in a competitive space with tools that
help them maintain a competitive lead. By innovating in technology, 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.
DISCUSSION

6. 6 © 2015 AB Sciex
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.
DISCUSSION

7. 7 © 2015 AB Sciex
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 required, and no need for complex
tuning
• Separation occurs PRIOR to detection, so no interference
with data processing
• An identical (LC) method can be used for all cases
DISCUSSION

8. 8 © 2015 AB Sciex
DISCUSSION

9. 9 © 2015 AB Sciex
DISCUSSION
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.

10. 10 © 2015 AB Sciex
CONCLUSIONS
For complex biologics species, SelexION™ on a Triple Tof™ instrument
from SCIEX may provide a simple, elegant solution to cleaning up intact
species from an ESI spectrum.
The rapid, simple application of Ion Mobility facilitates the analysis of
Intact ADCs and
• 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.

11. 11 © 2015 AB Sciex
Trademarks/Licensing
For Research Use Only. Not for use in diagnostic
procedures.
© 2015 AB Sciex. SCIEX is part of 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.