Streamlining the Analysis of Free Drug Content in ADCs
App Note / Case Study
Published: January 29, 2025

Credit: iStock
Antibody–drug conjugates (ADCs) are complex therapeutic biomolecules engineered to specifically target and eliminate cancerous cells while preserving healthy cells.
However, the presence of free drug and related species in the final ADC product could lead to compromised product efficacy and increased toxicity.
This application note presents a method that integrates protein elimination and free drug identification into one analysis using a two-dimensional liquid chromatography/quadrupole time-of-flight mass spectrometry (2D-LC/Q-TOF) approach.
Download this application note to discover:
- An innovative method for the identification of free drug content in ADCs
- A sensitive analysis of free drug content without manual protein precipitation
- An integrated approach to save time and achieve superior chromatographic separation
Application Note
Biopharma/Pharma
Author
Yulan Bian
Agilent Technologies Inc.
Abstract
Antibody-drug conjugates (ADCs) are complex therapeutic biomolecules composed
of an antibody linked to a potent cytotoxic small molecule drug. ADCs are
engineered to specifically target and eliminate cancerous cells while preserving
healthy cells. One critical quality attribute (CQA) of ADCs is the free drug content,
which is the unconjugated small molecule drug. This free drug content could be
caused by incomplete conjugation or formation of a degradation product. The
presence of free drug and related species could lead to compromised product
efficacy and increased toxicity.
This application note demonstrates a two-dimensional liquid
chromatography/quadrupole time-of-flight mass spectrometry (2D-LC/Q-TOF)
approach to identify the free drug content of an ADC sample. The method
combines size exclusion chromatography (SEC) in the first dimension (1
D) and
reversed-phase (RP) separation in the second dimension (2
D). This method enables
sensitive and straightforward analysis of free drug content in ADC without manual
protein precipitation.
Analysis of Free Drug Content in
Antibody-Drug Conjugate Using
2D-LC/Q-TOF
2
Introduction
ADCs are complex therapeutic biomolecules composed of an
antibody linked to a potent cytotoxic small molecule drug. The
antibody enables targeted delivery of the cancer-killing drug
to the tumor site while limiting the toxicity to healthy cells.
There are three components within an ADC: a monoclonal
antibody (mAb), a small molecule drug, and a linker. The linker
reacts with the free drug first, transforming it into a linker-drug
compound. This compound is subsequently conjugated to
specific amino acid sites on the antibody. The process is
illustrated in Figure 1.
Incomplete conjugation results in the presence of unbound
drugs within the ADC, potentially leading to heightened
toxicity. Therefore, measurement of free drug content is a
unique CQA of ADCs. Reversed-phase liquid chromatography
(RPLC) can be used for this CQA analysis.1
However, ADC
samples need to be pretreated to remove protein content
before injection onto RP columns. Otherwise, the irreversible
binding of antibodies to the stationary phase will damage
the HPLC columns. Cleanup approaches include solid phase
extraction (SPE)2
and protein precipitation3
with organic
solvent. However, these manual and offline procedures are
tedious and time consuming.
In the recent years, 2D-LC technology has been proven
to be reliable and efficient in bioseparation of mAbs,
ADCs, oligonucleotides, and related impurities. Different
combinations of separation mechanisms have been
reported.4,5 Heart-cutting mode is the most commonly used
2D-LC mode whereby only the eluent of interest from 1
D is
cut and transferred into 2
D for further separation. This mode
largely reduces the complexity of the analysis.
In this study, we used a heart-cutting 2D-LC coupled to an
LC/Q-TOF MS to identify free drug content in an ADC sample.
The analytical components are depicted in Figure 2. This
approach involved initially separating the ADC from small
molecular species by SEC in the 1
D, and then achieved
effective separation of free drug and associated impurities by
RPLC in 2
D. This automated online protein removal procedure
enhanced operational efficiency.
Figure 1. ADC conjugation process.
SMCC
Linker
DM1
Cytotoxic drug
+
SMCC-DM1
Linker drug
ADC
mAb
-NH
3
Experimental
Materials and methods
Ammonium acetate and acetonitrile (ACN, LC/MS grade)
were purchased from Merck Millipore (Burlington, MA, USA).
Formic acid (FA, LC/MS grade) was purchased from Fisher
Scientific (Pittsburgh, PA, USA). Ultrapure water was collected
from an in-house Merck Millipore Milli-Q system (Burlington,
MA, USA). The ADC sample was purchased from Alliance
Pharm (Singapore, SG). The free drug (DM1) and linker drug
(SMCC-DM1) standards were purchased from BroadPharm
(San Diego, CA, USA).
Sample preparation
The ADC sample was desalted and dissolved in 100 mM
ammonium acetate buffer (pH 7.0). The concentration was
adjusted to 5 mg/mL before injection.
The DM1 and SMCC-DM1 were separately weighed and
dissolved in 50% ACN, creating two individual stock solutions
at a concentration of 5,000 µg/mL. Both compounds were
then spiked to the ADC sample, resulting in a final spike
concentration of 100 µg/mL for each compound.
Instrumentation
– Agilent 1290 Infinity II Bio 2D-LC including:
– Two Agilent 1290 Infinity II Bio High Speed Pumps
(G7132A) with Agilent Bio Jet Weaver mixer kit, 35 µL
volume (G7132-68135)
– Agilent 1290 Infinity II Bio Multisampler (G7137A) with
Agilent InfinityLab Sample Thermostat (option #101,
G4761A)
– Agilent 1290 Infinity II Multicolumn Thermostat
(G7116B) equipped with Agilent Quick-Connect
Bio Heat-Exchanger, standard flow (option #065,
G7116-60071)
– Three Agilent 1290 Infinity Valve Drives (G1170A)
equipped with 1x Agilent InfinityLab Bio 2D-LC ASM
Valve (G5643B), 2x Agilent Multiple Heart-Cutting
Valves with biocompatible 40 μL loops
– Agilent 1290 Infinity II Variable Wavelength Detector
(G7114B) equipped with an Agilent Bio Standard Flow
Cell for VWD (option #028, G1314-60188)
– Agilent 1290 Infinity II Diode Array Detector FS
(G7117A) equipped with biocompatible InfinityLab
Max-Light Cartridge Cell (G7117-60020)
– Agilent 6545XT AdvanceBio LC/Q-TOF with Agilent Dual
Jet Stream ESI source
Figure 2. Analytical components of the Agilent Bio 2D-LC/Q-TOF analysis of ADC free drug content.
Cleanup & Separate Detect Analyze
Agilent 1290 Infinity II
Bio 2D-LC System
Agilent 6545XT AdvanceBio
LC/Q-TOF System
Agilent MassHunter Software Suite
Agilent AdvanceBio SEC
(first dimension)
Agilent Poroshell C18
(second dimension)
4
Software
– Agilent MassHunter Acquisition software 11.0
– Agilent MassHunter Qualitative Analysis software 11.0
2D-LC/MS analysis
Parameter Value
First Dimension
Column Agilent AdvanceBio SEC 200 Å, 4.6 × 150 mm, 1.9 µm
(PL1580‑3201)
Thermostat 6 °C
Solvent A 100 mM ammonium acetate
Solvent B Acetonitrile
Gradient Isocratic, 40% B
Column Temperature 25 °C
Flow Rate 0.25 mL/min
Injection Volume 10 µL
UV Detection 252 nm at 20 Hz data rate
Second Dimension
Column Agilent Poroshell ECC18, 3.0 × 50 mm, 1.9 µm
(699675302)
Solvent A 0.1% Formic acid
Solvent B 0.1% Formic acid + 95% acetonitrile/H2
O
LC Mode Heartcutting
Flow Rate 0.5 mL/min
Stop Time 17 min
Sampling Table 9.0 min, Timebased heart cut, HiRes 3 × 7.68 s,
multiinject: yes
Cycle Time Analysis: 5 min
Equilibration: 0.7 min
Gradient 38 to 65% B in 5 min
Flush Gradient
Time (min) %B
0 38
0.05 65
Duration 0.8 min, equilibration 0.7 min
Column Temperature 40 °C
UV Detection 252 nm at 20 Hz data rate
Table 1. Liquid chromatography parameters.
Agilent 6545XT AdvanceBio LC/Q-TOF
Parameters Value
Source Agilent Jet Stream ESI
Polarity Positive
Drying Gas Temperature 300 °C
Drying Gas Flow 11 L/min
Nebulizer 35 psi
Sheath Gas Temperature 350 °C
Sheath Gas Flow 11 L/min
Capillary Voltage 3,500 V
Nozzle Voltage 0 V
Fragmentor 135 V
Skimmer 65 V
Quad amu 750 V
Mass Range m/z 100 to 1,700
Acquisition Rate 1 spectra/s
Acquisition Mode Positive, extended dynamic range (2 GHz)
Reference Mass 922.009798
Table 2. MS data acquisition parameters.
Results and discussion
First dimension SEC method development
SEC is a widely used technique to separate size variant
molecules inside a given protein sample. A typical elution
order is aggregates, followed by monomers, and then low
molecular weight (Mw) species. Therefore, SEC is an ideal
separation mechanism to be employed as the first dimension
to set free drug content apart from the ADC.
Buffer solution at a neutral pH is usually adopted as an
SEC mobile phase for protein analysis. Considering the
compatibility with MS detection, ammonium acetate was
selected over phosphate buffer as the SEC mobile phase in
this study.
However, SEC separation of ADCs poses unique challenges
because the conjugated small molecule drug increases the
hydrophobicity of ADCs. This results in longer retention of
ADC in the SEC column due to hydrophobic interactions
with the column. What made it more difficult was that the
drug and drug-linker compounds, which were naturally small
and hydrophobic, would not elute with pure aqueous mobile
phase. In fact, DM1 and SMCC-DM1 were fully retained on the
column under aqueous conditions.
5
Introduction of a low percentage of organic solvent such
as methanol, isopropanol, or acetonitrile can mitigate the
hydrophobic interactions and facilitate the elution of both
protein and small molecules. In our case, ACN was chosen as
the organic modifier because of the low pressure generated
in the column. The concentration of ACN was scouted from
20 to 40% in increments of 5% for the analysis of the DM1
standard. Figure 3 showed that 40% acetonitrile rendered
the best peak shape of the DM1. This percentage of organic
modifier falls within the organic solvent tolerance limit of 50%
for the AdvanceBio SEC column. Therefore, it is safe to use
without worry of column damage.
Second dimension sampling
Using the 40% ACN in 100 mM ammonium acetate pH 7.0 as
SEC mobile phase, the separation between ADC and free drug
content was achieved in the 100 µg/mL spiked sample at the
1
D as illustrated in Figure 4. However, DM1 and SMCC-DM1
coeluted as a broad peak from 8 to 10 minutes in the spiked
samples. This is partly because the Mw difference between
the two molecules was not significant enough for separation
under SEC. Moreover, the spiked DM1 and SMCC-DM1 could
have had interactions with ADC at the column head during
the initial stage of separation, which may have caused the
broadening of the peaks.
Three fractions of the broad peak, as shaded in Figure 4, were
sampled into three loops through multi-inject sampling mode.
All three cuts were sequentially transferred to the 2
D in one
shot and analyzed within one single 2
D gradient cycle. The
multi-inject mode enabled sampling of a broad 1
D peak using
standard 40 µL loops without hardware modification. It also
reduced the run time by analyzing multiple cuts within one 2
D
cycle. The total method time was only 17 minutes.
Acquisition time (min)
Absorbance (mAU) ×101
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
40%
35%
30%
20%
25%
Figure 3. Scouting of ACN concentration from 20 to 40% in 1
D SEC for the
DM1 standard.
Figure 4. Overlaid UV chromatograms of first dimension SEC separation of desalted ADC (green) and 100 µg/mL spiked desalted ADC (blue). The shaded areas
represent the three continuous cuts sampled into 2D using multi-inject mode.
–0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
Acquisition time (min)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Absorbance (mAU)
Desalted ADC
100 µg/mL spike
1 2 3
×102
6
Second dimension reversed-phase LC/MS analysis of
DM1 and SMCC-DM1
Since RPLC has a proven track record of separating
hydrophobic small molecules, it was chosen as the 2
D
separation mechanism. A total of 96 µL of 1
D effluent was
transferred onto a 2
D RP column. Three peaks were detected
and separated in both UV and total ion chromatogram (TIC)
as shown in Figure 5.
Peak 1 was confirmed as DM1 based on the measured
mass of 738.2839 Da. The mass accuracy of it was
2.30 ppm from theoretical mass 738.2822 Da. Sodium
and potassium adducts presented high abundance, which
could be attributed to the use of buffer salt in 1
D SEC and
its subsequent introduction to the 2
D during sampling. The
neutral loss of water fragment was also detected due to
in-source fragmentation.
Peak 2 and 2' have identical MS spectra. The mass of
the [M+H]+
ion was 1,072.3985 Da, which was confirmed
as SMCC-DM1. The mass accuracy was 0.18 ppm from
the theoretical mass of 1,072.3987 Da. This doublet
peak phenomenon was caused by the presence of a
stereocenter in the SMCC-DM1 molecule.6
The two peaks are
diastereomers that have identical mass.
Applying the 2D-LC/Q-TOF technique, the free drug content
was separable from ADC in SEC, and then the individual
components were successfully separated from each other in
RP and confirmed by MS.
0.1
0
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Acquisition time (min)
2.5 3.0 3.5 4.0
Response (%)
UV
TIC
1
2
2’
A ×102
0
0.5
1.0
1.5
2.0
600 650 700 750 800 850 900 950
0
1
2
3
4
5
6
7
950 1,000 1,050 1,100 1,150 1,200 1,250
Peak 1
Peak 2 and 2’
[M-H2
O+H]+
[M+H]
+
[M+Na]+
[M+K]
+
[M-H2
O+H]+
[M+H]
+
[M+Na]+
[M+K]
+
B
C
×106
×101
Figure 5. Identification of DM1 and SMCC-DM1 by second dimension LC/MS analysis. (A) UV and total ion chromatogram, (B) MS spectra of DM1, and (C) MS
spectra of SMCC-DM1.
www.agilent.com
DE17060666
This information is subject to change without notice.
© Agilent Technologies, Inc. 2024
Printed in the USA, February 26, 2024
59947182EN
Conclusion
Agilent has developed an innovative and effective
two-dimensional liquid chromatography/quadrupole
time-of-flight mass spectrometry (2D-LC/Q-TOF) method
for the identification of free drug content in antibody-drug
conjugates (ADCs). This solution uses the Agilent 1290
Infinity II Bio 2D-LC with the Agilent AdvanceBio SEC column,
the Agilent Poroshell EC-C18 column, and the Agilent 6545XT
AdvanceBio LC/Q-TOF. The Agilent MassHunter Workstation
for LC/TOF and LC/Q-TOF 11.0 and Agilent MassHunter
Qualitative 11.0 software were used for data acquisition
and analysis.
This method integrates protein elimination and free drug
identification into one analysis. Automated protein removal
saves time and safeguards the RP column from deterioration.
Moreover, the method achieves superior chromatographic
separation between the drug and the linker-drug and enables
reliable identification through accurate mass detection.
References
1. Li, Y.; et al. Limiting Degradation of Reactive Antibody Drug
Conjugate Intermediates in HPLC Method Development.
Journal of Pharmaceutical and Biomedical Analysis 2014,
92, 114–118.
2. Hurwitz, E.; et al. The Covalent Binding of Daunomycin
and Adriamycin to Antibodies With Retention of Both
Drug And Antibody Activities. Cancer Research 1975, 35,
1175–1181.
3. Chari, R. V.; et al. Enhancement of the Selectivity and
Antitumor Efficacy of a CC-1065 Analogue Through
Immunoconjugate Formation. Cancer Research 1995, 55,
4079–4084.
4. Wong, D. Characterization of Antibody-Drug Conjugates
Using 2D-LC and Native MS. Agilent Technologies
application note, publication number 5994-4328EN, 2021.
5. Stoll, D. R. Recent Advances in 2D-LC for Bioanalysis.
Bioanalysis 2015, 7(24), 3125–3142.
6. Singh, R.; et al. A New Triglycyl Peptide Linker for
Antibody–Drug Conjugates (ADCs) with Improved
Targeted Killing of Cancer Cells. Molecular Cancer
Therapeutics 2016, 15(6), 1311–1320.
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