LC–MS and small molecule analysis: an interview with Liam Moran

Liam Moran
Charles River Laboratories (OH, USA)

Liam Moran is currently the Director of Bioanalytical chemistry at Charles River Laboratories. He leads a bioanalytical department that performs regulated and nonregulated analysis of small molecule, peptides, proteins, antibody–drug conjugates (ADCs), anti-drug antibodies, oligonucleotides and hybrid therapies. His former roles include Head of Bioanalytical Chemistry at Lexicon Pharmaceuticals (TX, USA), Principal Scientist at Battelle (OH, USA) and LC–MS/MS Application Scientist for proteomics and metabolomics at Thermo Fischer Scientific (MA, USA).

1. Please describe some nontraditional ways in which LC–MS has emerged as a key enabler of drug development.

When I think of how LC–MS is used in a traditional sense in drug development, it is the measurement of the API and metabolites in plasma and tissues to correlate exposure to efficacy and/or toxicity.

We are getting an increasing number of studies that are intended to answer questions about the pharmacodynamic outcome to help ‘tune’ the dosing regimen or delivery scheme. Some oligonucleotide therapies are designed to minimize exons skipping and promote the expression of full-length proteins by drugging mutations in introns. We use MS to measure full-run length and truncated proteins as a pharmacodynamic marker for target engagement of these therapies. A second example is the measurements of antitumor drugs in voxelated brains to create a three-dimensional map of drug distributions in the brain. This data is used to guide the development of delivery systems that can spatially target delivery. A third example is the measurement of specific mutational variants of proteins that are down-regulated by gene therapies. This allows investigators to determine if the therapy has a stronger response based on the mutational variant in individual patients.

2. Instrumentation continues to evolve towards increased sensitivity and resolution. What are you most excited about with upcoming releases of MS technology?

The rate of change on traditional triple-quadrupole systems is slowing. Where I see the exciting developments happening are in the high-resolution systems hybridized with quadrupole mass selection and fragmentation. There have been new fragmentation schemes implemented on Q-TOF devices that allow for electron dissociation-type fragmentation on singly change molecules. The orbi-trap systems have had a large jump in resolution and sensitivity while becoming more user-friendly with internally generated lock masses.

3. More frequently, traditional large molecule ELISA assays are inadequate for monitoring bioengineered drug constructs. What unique role is LC–MS playing to fill that gap?

There are three key areas where MS is making inroads into therapies that are typically served by LBA assays. The first is nanoparticles. In some cases, LBA assays can be created for nanoparticles but the regulators want to see data on individual elements of the particle like the core material, surface payload or surface proteins. We usually create multiple MS assays for nanoparticle programs. Another area where MS excels is in biologic therapies (either protein or oligonucleotide), where the therapy is very similar in sequence homology to an endogenous construct. When the protein or oligo is one or two point mutations different than the endogenous form, ELISA cannot distinguish them whereas MS can. A third area is in pro-drug biologics. Several innovators we have worked with have protein-based therapies that cleave to become an active form after they enter a cell, tissue or tumor. It is difficult to come up with a series of ligand binding reagents to distinguish between the full therapy and the truncated form. MS can measure signature peptides from both domains, and a peptide at the linker region is used as a readout of the full-run length construct.

4. Where are there still challenges with ‘traditional’ small molecule bioanalysis? Are there any?

Generally small molecule active pharmaceutical ingredients (APIs) are getting easier to work with as the sensitivity of mass spectrometers increase. When a molecule has drug-like properties, it lends itself to extraction with organic solvent, reverse phase chromatography in MS friendly mobile phases, ionization by atmospheric ionization and it is big enough to have a fragmentation pattern for MS/MS detection.

The two ‘problem-sources’ for small molecules I see are biomarkers and non-GRAS (generally regarded as safe) excipients used in nanoparticle encapsulation schemes. Many of the biomarkers from the small ‘molecule-ome’ do not have drug-like properties and need special considerations for both separations and MS. Specifically, small polar molecules like amino acids, their metabolites and molecules in the energy transfer pathways require reverse-phase chromatography or derivatization. Very small molecules that lack adequate fragmentation patterns require detection by single-stage HRMS.

The proprietary excipients used in nanoparticle formulations have properties similar to either lipids or homopolymers with polydisperse weight distribution. The lipid like molecules that lack ionizable groups can be done with GC–MS/MS. We often resort to HRMS to analyze polymeric materials because the intensity of all the MS lines can be summed.

5. What advice do you have for aspiring bioanalytical scientists just starting their career?

20 years ago, I would have told a young scientist to get a position in big pharma or biotech.  Back then big pharma had the best technology and the largest teams of scientists. Now, with more and more of the drug discovery/development work being done at CROs, the trend is CROs have increasing talent pools, capital equipment outlays and the latest technology in both software and hardware. In addition, CROs work on the pipelines of hundreds or thousands of companies. This gives the aspiring bioanalyst the opportunity to work on a wide variety of therapeutic classes and assay formats that support them. As someone that has worked in big pharma, biotech and CROs, I see very clearly that the development of new bioanalytical staff is greatly accelerated in the CRO space.

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