Using HRMS in Bioanalysis – A conversation with Rand Jenkins and Patrick Bennett

Bioanalysis Zone interviewed two established experts using high-resolution mass spectrometry (HRMS) technology. In this short interview we caught up with PPD’s Rand Jenkins and Patrick Bennett to find out about their experience using HRMS in their work, including some of the technology’s advantages and challenges, providing insight into future developments of the field.

Rand Jenkins is Scientific Director for the Chromatographic Sciences Department (CSD) of PPD® Laboratories’ bioanalytical lab in Richmond (VA, USA) and Middleton (WI, USA). Since obtaining a BS in chemistry from the University of Nevada Reno in 1972, Rand has been involved in the development and application of GC– and LC–MS technologies in the environmental and pharmaceutical fields. He joined PPD in 1994 and currently provides scientific guidance to the R&D teams in bioanalytical methods development and validation. Rand’s major focus in recent years has been to establish and promote bioanalysis of peptides and proteins using LC–MS technology.

Patrick Bennett is the Executive Director of PPD Laboratories’ biomarker lab. Patrick has nearly 30 years of experience in pharmaceutical analysis. This includes Bristol-Myers Squibb Department of Metabolism and Pharmacokinetics, Advion Biosciences, where he was the Laboratory Director and Tandem Labs where he was Vice President. In 2010, Patrick joined Thermo Fisher Scientific as Director of Global Strategic Marketing within the Chromatography and Mass Spectrometry Division. In 2014, Patrick joined PPD to create a new Biomarker Division, which has dedicated laboratory facilities providing Molecular Genomics, flow cytometry, ligand binding and LC–MS based biomarker services. Patrick earned a BS degree in Toxicology and a MS degree in Pharmacology St. John’s University and an MBA from Syracuse University.

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Could you introduce yourself and tell us a bit about your work background?

My name is Rand Jenkins, Scientific Director with PPD Laboratories’ bioanalytical lab. I have been with the company and its predecessors for 26 years primarily focusing on chromatography and mass spectrometry technologies applied to regulated bioanalysis.  I first began working with MS in 1972 using early single quad GC-MS instruments initially in biomedical research, then later as an Applications Chemist for Finnigan Instruments (now Thermo), an environmental Research Chemist at NMF/NOAA, and as a Lab Director with ETC/APBI, before transferring to the Pharmaco division (now part of PPD) in 1994.

In the early years we conducted extensive bioanalysis work using capillary GC–MS to analyse steroids and other small molecules. However, it wasn’t long before we got into LC–MS/MS, starting with the original Sciex API-3 platform, eventually converting nearly all of our small molecule assays over from GC, GC–MS and HPLC to LC–MS/MS technology. In recent years, we have undergone a major expansion into large molecule bioanalysis, particularly using hybrid LBA/LC–MS techniques with both triple quadrupole (QQQ) and HRMS instrumentation.

My name is Patrick Bennett, Executive Director for PPD Laboratories’ biomarker lab.  I have been involved in bioanalytical mass spectrometry since 1988 as a Research Scientist in the DMPK group at BMS.  From 1993 to 2010, I managed bioanalytical mass spectrometry groups at Advion and Tandem Labs.  This included a biomarker group that performed analysis using both QQQ mass spectrometers as well as TOF-based instruments.

From 2010 to 2014, I was the Director of Global Strategic Marketing for Thermo Scientific Life Sciences Mass Spectrometry.   I was responsible for the development and application strategies of both triple quadrupole and high resolution accurate mass instruments for the pharma and biopharma industry.

Could you tell us a little about some of the work you carry out at PPD using HRMS technology?

The power and versatility of HRMS technology have dramatically increased in recent years. While already well-established as a qualitative analysis tool, newer generation HRMS systems are allowing us to explore realistic quantitative bioanalysis approaches for a variety of small and large molecules, both digested and intact. HRMS assays can sometimes be a superior alternative to conventional SRM assays. Other times HRMS may be applied to facilitate method development and confirm the performance of an SRM assay, intended to be used for routine analysis.

From a biomarker perspective, HRMS technology allows us to quantify both large endogenous molecules, similar to biotherapeutic drugs, as well as very small molecules like Bile Acids.  If a biomarker assay shows an increased background using a triple quadrupole instrument, the HRMS instruments usually can reduce or eliminate that background.

What drew you to working with/using HRMS?

We first began using HRMS a few years ago when we started moving into LC–MS analysis of biotherapeutics. At that time our primary use was protein/peptide characterization to support method development of protein assays, mainly using the trypsin digestion and surrogate peptide approach on conventional QQQ instruments. HRMS was invaluable for mapping, sequence confirmation, and selection of appropriate surrogate and monitoring peptides for SRM analysis.

The ability to perform both qualitative and quantitative analysis using HRMS is very appealing. From a drug discovery perspective, we could obtain very rapid information on not just an analyte, but also metabolites.  Also, the ability to use a single platform for both targeted quantitation and high throughput screening is appealing.  From a drug development perspective, the qualitative and quantitative capabilities described above are a significant draw to the technology.  And, from a biomarker perspective, the ability to use the instrument for both biomarker discovery (-omics based) and quantitation makes it an ideal platform for both research and CRO labs.

How have recent developments in HRMS transformed LC–MS analysis?

Major strides have been made by vendors, particularly in sensitivity, stability, and overall system ruggedness. On the small molecule side, the resolving power to overcome interference issues can be a major advantage. For large molecules, the high mass range allows direct intact detection of large proteins, like monoclonal antibodies. In addition to potential quantification, it is likely that biotherapeutic drug development will significantly benefit from applying established proteomics techniques (e.g., top-down, middle-down, bottom-up) using HRMS to characterize in vivo biotransformation changes.

The focus on ease of use and software for these instruments is also important – particularly for the characterization of proteins.

Why do you think the field is starting to gain so much interest?

The greatly increased sensitivity and overall capabilities of the latest generation of instruments is gaining attention. We also see many HRMS discussions taking place at industry conferences and a growing number of HRMS bioanalysis application articles being published.

Some people have speculated the significance of HRMS as potentially replacing conventional LC–MS/MS technology (i.e., using QQQ instruments) in large molecule analysis – what are your views on this?

Looking forward a few years, we would agree that it is very likely that hybrid or tandem HRMS-based systems (i.e., Q-Tofs and Q-Orbitraps) will take over for many large (and small) molecule bioanalysis applications. However, it is doubtful that QQQ instruments will be completely replaced. There is such a large installed base, with many experienced users, and tandem quads are still very capable for targeted analysis. A mix is more likely, with the systems being applied to cost-effectively obtain the desired measurements with appropriate scientific rigor.

As mentioned, sometimes HRMS will be the best way to go to quantify an analyte (and obtain qualitative information), while other times it will be applied as a complimentary tool to aid development and application of a QQQ assay (or even an LBA).  It’s also likely that triple quadrupoles (and software) will continue to evolve and become engineered for specific uses.

What do you see as some of the main benefits of using HRMS?

The short answers, as alluded to earlier, are power and versatility. In comparison to conventional ‘unit-resolution’ quadrupole-based instruments, HRMS offers significantly greater specificity, along with the ability to determine elemental formulae, sequence peptides, and establish molecular weights of multiply charged molecules, using high resolution accurate mass data.

Current generation hybrid HRMS systems already rival the sensitivity of QQQ instruments, while offering versatile modes of operation, such as PRM, and targeted SIM. Of course these systems can also provide data for quan/qual applications, although not without some practical challenges.

Lastly, the expanded mass range of HRMS platforms also opens up the potential for intact analysis of large molecules, both for characterization using software deconvolution techniques and for direct quantification using highly selective XICs.

One of the highlights of HRMS is its versatility in providing quantitative/qualitative data. How do you think this will change the current workflow practice in labs?

That will likely depend a number of factors and practical issues. Quantitative /qualitative data acquisition produces very large data files, which can be a problem for practical storage. Being able to go back at a later time and “mine” the data for additional metabolites or biomarkers could be very valuable. However, this would likely be a qualitative or semi-quantitative situation at best, because sample collection and/or the preparation method may not have been optimum for the new analyte of interest. There would not have been a calibrator or IS analysed along with these new ‘targets’ either.

A major concern for human samples will be obtaining appropriate informed consent to cover potential future expanded uses of the clinical sample or data. Clinical protocols would also need to be flexible to allow future data mining.

What are the implications of these changes?

This is difficult to say yet, due to the variety of issues. Certainly there will be value in having more data available for examination without having to reanalyse the sample. But there will be limitations, including ensuring that informed consent is available to interrogate this data retrospectively as well as ensuring that the sample curation is known.  Assuming that the sample is viable and can be re-interrogated, the ability to identify potential biomarkers from samples that have a known outcome is powerful.

There are being concerns voiced about collection and use of quantitative/qualitative data when the samples being analysed are from human clinical trials. How are CROs like PPD Laboratories addressing issues like this?

Where possible, informed consent documents are being modified to allow more open-ended use of a patient’s sample and data. We must also anticipate that regulators may ask sponsors to go back and look for something, such as a potential metabolite, in the data if they know that full scan data were collected.  SOP’s can be used as the basis for performing this analysis.  These can provide procedures and controls to ensure appropriate data processing is performed on the full scan data.

What are some of the ways you support vendors in improving software of HRMS technology?

Current users of a vendor’s HRMS system have the opportunity to provide feedback on their experiences and unmet needs in using the instrument. This is particularly important with software, which typically lags behind the development of the hardware. Sometimes it may be a bug or a user (in)convenience situation. Other times, it may be a desire to do something different than originally designed by the vendor.

A case of this we are working on is the use of deconvolution algorithms to ‘compress’ full scan HRMS data for quantitation of intact proteins, as a potential alternative to using conventional XIC chromatograms.

What are some of the regulatory concerns of applying HRMS?

In many respects LC–HRMS may be considered another “flavour” of LC–MS. Regulators are very familiar and comfortable with the use of conventional LC–MS/MS for regulated bioanalysis, particularly with small molecules. We should remember that prior to LC–MS, the techniques of GC, GC–MS and HPLC with various detection methods were being used.  This is just another step in that progression.

Of course with HRMS there are many differences relative to standard practices using unit-resolution QQQ instruments. The effects of applied resolution (vs. cycle time), mass extraction window, signal-to-noise (when there is none), are a few. New data acquisition modes are also possible with HRMS, such as PRM and tSIM, and survivor SIM.

Why do you think some people are still sceptical about using HRMS?

A lot of this has to do with misinformation.  HRMS, as a composite technique (typically hybrid LC–tandem MS/HRMS), is viewed by many as exotic and ‘for research use only’ by some. Others think HRMS instruments are much more expensive and difficult to use compared to QQQ systems. Of course others worry about reliability and potential acceptance by regulators. In reality, most of these concerns are exaggerated or not even real.

What advice would you give to investigators and researchers who are considering switching to HRMS?

Attend presentations by instrument vendors and users at scientific meetings and in webinars. Read application articles, such as those published in the July 2016 special themed issue of Bioanalysis on HRMS in DMPK.

Another useful exercise is to engage the applications labs of instrument vendors to analyze some test samples that you may have already evaluated using conventional triple quads. Comparing the data between platforms can be very enlightening.

As they say, “try it, you’ll like it.” Make the leap to HRMS; you will be impressed by its amazing power and versatility.