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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. TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS 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