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Development of Integrated Informatics Workflows for the Automated Assessment of Comparability for
Antibody Drug Conjugates (ADCs) Using LC/UV and LC/UV/MS
Henry Shion,1 Robert Birdsall,1 Frank W. Kotch,2 April Xu,3 Thomas J. Porter,4 Weibin Chen1
1. Pharmaceutical Business Operations, Waters Corporation, 34 Maple Street, Milford MA 01757
4. Pfizer Analytical Research & Development, 1 Burtt Road, Andover, MA 01810
CONCLUSIONS
INTRODUCTION
OVERVIEW
Integrated Informatics Workflow for ADC
Comparability Assessment
2. Pfizer Bioprocess Research & Development, 401 N. Middletown Road, Pearl River, NY 10965
3. Pfizer Analytical Research & Development, 401 N. Middletown Road, Pearl River, NY 10965
 An integrated informatics workflow with UNIFI was developed to streamline data acquisition, processing and
 An
reporting for ADC analysis by LC/UV and LC/MS so that information from multiple analytical techniques can be
integrated informatics workflow is presented for automatically assessing ADC
and increasing the productivity during process development.
molecules
 DAR
effectively synergized for rapid and quantitative assessment of comparability of ADCs.
 Critical quality attributes (CQAs), such as average drug-to-antibody ratio (DAR) and drug load distribution for
multiple batches of ADCs was acquired using the informatics workflow, and a comparability study was
performed based on DAR for the different ADC preparations.
values and drug loading distributions for cysteine-conjugated ADCs are automatically
acquired from HIC-LC analysis and from native SEC-LC/MS analysis, and the results show
excellent agreement.
Automated Analysis of Lysine-Conjugated ADCs for
Determination of DAR Values Based on RP-LC/MS
Intact Mass Analysis
 The
workflow automatically produces DARs and drug loading distributions for lysineconjugated ADCs from RP-LC/MS analysis.
RESULTS AND DISCUSSION
Automated Analysis of Cysteine-Conjugated ADCs for Determination of DAR Value
Based on Hydrophobic Interaction Chromatography (LC/UV-based method)
Predicated
drug load
Positional
isomers
Figure 1. Antibody Drug Conjugate (ADC) analysis workflow with
the UNIFI Scientific Information System
=
0
2
4
6
8
Predicated
drug load
{
Low
drug load
conjugation
METHODS
Automated Analysis of Cysteine-Conjugated ADCs for Determination of DAR Value
Based on Native Intact Mass Analysis (SEC-LC/MS based method)
Positional
isomers
Figure 3. Integrated informatics for ADC analysis.
Custom fields are used to calculate the DAR value using peak area and display the results in the component summary pane.
=

0
2
4
6
8
Low
drug load
conjugation
Low drug load
conjugation
Moderate drug
load conjugation
High drug load
conjugation
Naked mAb, unconjugated
Instrumentation
LC: Waters ACQUITY H-Class Bio
MS: Waters ACQUITY® Xevo G2-S QTof
Columns
Moderate
drug load
conjugation
Moderate
drug load
conjugation
HIC (LC/UV)
Mobile Phase:
Mobile Phase:
A: 125 mM Sodium Phosphate Buffer, pH 6.7, 2.5M
ammonium sulfate
B: 125 mM Sodium Phosphate Buffer, pH 6.7
C: Isopropanol
D: 18 MΩ water
A: 50, 100 or 200 mM NH4OAc in
H2O (Isocratic)
High
drug load
conjugation
Naked mAb
Mobile Phase:
Naked unconjugation
(un-conjugated)
A: Water, B: Acetonitrile
C: 1% Formic Acid in waters
D: 1% TFA in Waters
Capillary: 3kV; Sample Cone voltage: 150 v; Source Temp: 500°C;
Desolvation Temp: 350 °C; Desolvation Gas Flow: 800 L/h
High
drug load
conjugation
High
drug load
conjugation
LC-MS (RP-LC/MS)
MS Conditions
Figure 6. Native SEC-LC/MS raw spectra of cysteineconjugated ADC samples from different drug loading.
Figure 2. Cysteine conjugated ADC analysis using HIC. Drug
distribution was determined for three different cysteine-conjugated
ADC samples with increasing drug load.
Low
drug load
conjugation
Moderate
drug load
conjugation
Waters ProteinPak Hi Res HIC (available 3Q, 2014)
ACQUITY UPLC Protein BEH C4 Column, 300Å, 1.7 µm, 2.1 mm X 50
mm (p/n: 186004495)
ACQUITY UPLC Protein BEH SEC Column, 200Å, 1.7 µm, 4.6 mm X
150 mm (p/n: 1860005225)
LC-MS (Native SEC-LC/MS)
Figure 8. The component summary table in Unifi review pane displays identified drug
loading distribution based on deconvoluted spectra peaks (as shown in Figure 9),
automatically calculated DARs, integrated chromatogram and DAR values bar chart for
the three level drug loading lysine-conjugated samples. DAR determined by LCMS can
be used for lot to lot comparison for lysine conjugates. However, the absolute DAR
value needs to be verified by an orthogonal technique.
Figure
4.
Automated
reporting
using
UNIFI
informatics. A report of peak area, DAR value, and
associated statistics was automatically generated
after data acquisition and analysis.
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Figure 5. Deconvoluted intact mass spectra for cysteine-conjugated
ADCs from native SEC-LC/MS after deglycosylation. Drug distribution
was compared for three different cysteine-conjugated ADC samples with
increasing drug load.
Figure 7. DARs comparison between HIC and native
SEC-LC/MS experiments in table (top) and 3-D graph
format (bottom) shown excellent agreement between
the two methods for all three drug loading levels.
Figure 9. The deconvoluted MS spectra for lysine-conjugated ADCs. The spectra
compare the difference in drug distribution for three batches of ADC preparation at
various levels of drug loadings and can be automatically generated in the report.
©2014 Waters Corporation