Simplifying Intact Molecular Weight Determination for ADCs
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This document discusses simplifying the determination of intact molecular weight for antibody drug conjugates (ADCs) using advanced SCIEX technology. It highlights the challenges of heterogeneity and the need for rapid analysis in biopharmaceutical development, emphasizing the integration of Selexion technology for improved clarity and speed in results. The combination of tools offered by SCIEX allows for efficient data processing and analysis of complex biologics, aiding researchers in maintaining a competitive edge.
Simplifying Intact Molecular Weight Determination for ADCs
- 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.