Friday, August 5, 2011

Overcoming Hurdles in Mass Spectrometry-based mAb Quantification with Xiaotao Duan

Cell Based Assays Speaker Spotlight:
Xiaotao Duan, Assistant Professor, State University of New York – Buffalo
Presenting: Overcoming Hurdles in Mass Spectrometry-based mAb Quantification
For more on Xiaotao Duan's presentation, download the Cell Based Assays  Bioanalytical Method Development Brochure here.

What are you currently working on at University of Buffalo. What impact does this research have on the future of pharmaceutical / medical development?
My research in SUNY Buffalo has been focused on:
a) developing bioanalytical methods for characterization, identification and quantification of therapeutic proteins/peptides
b) the application of mass spectrometry to the quantitative and structural analysis of endogenous markers and small‐molecule drugs/metabolites.
These analytical efforts have contributed greatly to a variety of PK/PD studies and clinical investigations.

What interested you in this line of work in the first place?
LC/MS based targeted protein quantification, which has shown great potential in the development of protein therapeutics.

How do you use LC‐MS applications in your own work?
Our lab is equipped with a variety of state-of-the-art LC/MS instruments. We have a high resolution / mass accuracy platform (e.g. LTQ/Orbitrap XL with ETD) which is largely engaged in large-scale proteome profiling and PTM identification. We also have several triple-quadruple MS dedicated for targeted protein quantification, as well as sensitive measurement of small- molecule biomarkers/drugs.

How much of your protein characterization and quantification work for monoclonal antibodies depends on use of MS techniques, and how much depends on ligand‐binding assays (or other assay designs)? Do you find that these approaches compliment one another? Or is one strictly better than the other?
For mAb quantification, in most cases we prefer MS techniques, because the specific reagents for ligand-binding assays (LBA) are usually not available and the MS method development is faster and more cost-effective. Moreover, MS methods often provide superior sensitivity and specificity as well as reproducibility. Nonetheless, I would by no means conclude that MS is strictly better than LBA. In fact, a decent LBA, once successfully developed, can be run at higher throughput and offers even better sensitivity in specific applications.

Have you come across any surprising or unusual results?
With regards to mAb quantification using LC/SRM-MS, we did observe several “hidden risks” that are often overlooked by others. For example, in most applications for protein drug/biomarker quantification, synthesized signature peptides are typically used as the reference standards to prepare both calibration solutions and quality control samples. Nevertheless, our finding suggests this peptide-referenced calibration may introduce significant biases for mAb quantification, as a complete digestion of mAb is unachievable most of the time. Therefore, pure protein standards are always preferable to enable an accurate quantification of mAb

What are some of the biggest challenges that you face in your work, and how do you manage to address them?
When you pave the road to a more refined LC/MS analysis, challenges are always there. A recent case occurred in the context of tissue mAb quantification. A big concern of this work was how to ensure assay accuracy, which is often compromised by nonquantitative sample preparation, unforeseeable instability of signature peptides, and potential pre-analytical variations (e.g. dissociation of light and heavy chains). To address these challenges, we put a lot of efforts to optimize the extraction/digestion protocol, and to evaluate peptide stability in targeted tissues. We also monitored more than one peptide for each mAb to strengthen our confidence on the quantification results.

What would you consider to be the most exciting news or recent developments in LCMS applications? Are there new discoveries in particular that you’ve heard of and tried to incorporate into your own work—and if so, how did that turn out?
There have been tremendous technical advances in LC/MS analysis over the last few years. A significant one is the implementation of ETD on high-resolution / mass accuracy hybrid instrumentation such as the Orbitrap. We have successfully applied this strategy to improve the characterization of therapeutic proteins/peptides and the identification of important PTMs.

What will you be discussing at the CBA‐BAMD conference? Who do you think would benefit the most from hearing you speak?
While LC/MS holds great promise for therapeutic protein quantification, developing a sensitive and specific LC/SRM-MS method for mAb quantification in complex matrix remains challenging. In the upcoming CBA-BAMD conference, I will discuss in detail the critical issues in the implementation of MS / MS-based mAb quantification, including signature peptide selection and SRM optimization. I will also introduce a novel, on-the-fly optimization approach to facilitate method development. Demonstrative applications to mAb PK/PD studies will be reported as well. I believe this topic will be of great interest to the audience—in particular, to anyone working directly on MS-based targeted protein quantification.

What are you most looking forward to learning at the CBA‐BAMD conference?
I will be especially interested in the latest applications of novel immunoaffinity approaches combined with mass spectrometry.

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