Panel discussion on large molecule quantification by LC–MS
The increasing importance of large molecule therapeutics has been coupled with advancements in LC–MS technology, opening up new opportunities. Typically, large molecules are analyzed using ligand-binding assays but with advanced progress in MS technology the community has seen increased success in overall sensitivity and selectivity. However, what does this mean for future analysis and what challenges remain for the use of LC–MS for large molecules.
In this free panel discussion, our experts will provide insights into their own research with large molecules including the challenges they have had to overcome, key trends they have seen and their future outlook of the development of this field. There is also an opportunity for your own questions to be answered in the live Q&A.
What will you learn?
- Best practices of protein quantification and digestion
- Considerations for top-down vs bottom-up approaches
- Future developments (including enhancing assay sensitivity)
QWhat are the dose levels in a preclinical study that can be handled by an LC-MS assay? Michael: Depends on the assay and the requirement of the project team? Generally, at a preclinical stage, it should be possible to reach the desired LLOQs using modern top end LCMS, eg Xevo, 6500, Altis.
Michael: No, because there are different stages of molecule development which require different sensitivity and specificity levels, and therefore different kinds of approach. But at different stages there generally are common approaches. E.g. for say preclinical testing of human antibodies for example, approaches are now pretty standard. Also we have creative companies always looking for better approaches, leading to innovation.
QOne of the deficiencies of LBA assays is the inability to incorporate an internal standard. So if you can include a suitable internal standard, then why not stay with chromatographic criteria (15%)? Michael: For hybrid assays we are using ligand binding as a part of the approach, so it is logical to use ligand binding acceptance levels. Also hybrid assays are more complex than simple small molecule approaches. I recommend AAPS (Rand Jenkins et al) and EBF publications (eg Schmidt and Knuttson) which discuss this in detail.
QWhich kind of 2D-LC did you use? Heart-cut, multiple heart-cut or comprehensive? Rob: We used single heart cutting, although with modern LC systems that are capable of fast gradient delivery, multiple heart-cutting is possible without the need for parking peaks in a loop or trap.
Q LC-MS instruments constantly get better (even ToF technology that is around for decades), when will the instruments be good enough in terms of sensitivity, acquisition speed and resolution? Or are we there already? Rob: As bioanalysts we are all too aware of the constant demands from our colleagues to achieve better assay sensitivity, with more potent and targeted drugs and a shift toward intact/sub-unit analysis I see the demand for more sensitivity remaining for some time. QqQ acquisition speed is now very fast and with scheduled MRM we can monitor hundreds of transitions in one run if we require, so I don’t feel there is a demand for more speed here. The speed of ToF detection has increased quite significantly recently, with some vendors now effectively decoupling sensitivity from acquisition speed. Although we have seen some good improvements over the years there still remains a trade-off for orbitrap technology however between acquisition speed and resolution. The major advantage to orbitrap technology is the level of resolution it can offer over the competitors, now offering up to 1 million. As mentioned earlier, with a trend toward intact or sub-unit analysis, we are going to require this resolving power and more in quantifying these molecules in the future.
Michael: Although I do not have direct experience, from presentations viewed recently at ASMS and other venues, I would suggest we are there already, apart from perhaps the most demanding clinical assays where ligand binding assays are still prevalent and probably preferred.
QWhen quantifying large multi-subunit protein analyte, how can an LC–MS method be sure the molecule is intact? Rob: The nature of a bottom up approach doesn’t owe itself to this task. However we can measure different surrogate peptides originating from the different sub-units and monitor ratio between them, which would be indicative of presence of sub-units but not necessarily infer the protein is intact. To ensure the molecule is intact we can use an immunoprecipitation. If we pull the protein down through capturing one of the sub-units, any sub-units not intact will be removed through subsequent washing. We then monitor the presence of the other sub-units to provide confidence that the protein is intact.
QFor labs suffering from high turnover, how do you justify developing the necessary experience to develop large molecule assays and hybrid approaches? Is it better to bring in the experience instead? Michael: Such a difficult question: The vendors e.g Thermo, Waters, are good at providing training now in the use of their kits, and how to optimize their methodologies. We are going to use them to help us develop our skills. ASMS for example do good value training courses in protein quan which can help with understanding. You have to convince management that this training will bring benefits. Large molecule analysis is mainstream now, so it is a necessary skillset for example, CROs.
QAre there dis/advantages to using chemical extraction (protein crash) and sample clean-up (LLE) over IA approaches to sample prep prior to LC-MS quantitation? Michael: The big disadvantage is that the extracts (PPt or LLE) will contain peptides and proteins. These cause interferences. This may be OK for discovery or low sensitivity assays, but as you move up the development chain greater sensitivity is required and hybrid assays will generally give better sensitivity due to their more specific clean up.
Meet our panelists
Bioanalytical Scientist, Method Development
ARCINOVA (Alnwick, UK)
Michael completed postgraduate work at the University of Wales (Bangor, UK) and began work for the government in the UK, measuring the concentrations of pollutants in aquatic samples by gas and later liquid chromatography-mass spectrometry. He later joined the pharmaceutical industry with Sanofi (Guildford, UK), as a mass spectrometrist, doing bioanalytical and drug metabolism studies. Since then he has worked as a Principle Investigator and Head of Method Development at Covance (Alnwick, UK) and latterly for ARCINOVA as a method developer. The development of better insulin assays using hybrid MS approaches has been a major interest for about 10 years.
PRA (NC, USA)
Senior R&D Scientist
MilliporeSigma (MA, USA)
Cory is a Senior R&D Scientist in the Liquid Separations R&D group at MilliporeSigma, Bellefonte, PA. He completed his graduate studies at Youngstown State University in 2013, focusing on the analysis and characterization of oxidized proteins. Upon graduation, Cory joined MilliporeSigma in 2013, first joining the chemical standards R&D group, then transferring to the liquid separations R&D group. His current role at MilliporeSigma is to research, develop, and present on new particle technology for improved chromatographic separations of both small and large molecules as well as to develop new methodologies for characterizing biomacromolecules by several modes of chromatography.
Senior Research Scientist
PPD (NC, USA)
Dr Omnia Ismaiel is a Senior Research Scientist at Bioanalytical Labs, Pharmaceutical Product Development, USA. She is also an Associate Professor of Pharmaceutical Analytical Chemistry at Faculty of Pharmacy (Zagazig University, Egypt) and Affiliate Assistant Professor at School of Pharmacy (Virginia Commonwealth University, VA, USA). She was a Postdoctoral Fellow at Virginia Commonwealth University and a Postdoctoral Research Associate at University of Georgia, USA. She has been in the Bioanalysis field for more than 12 years. and has many years of teaching experience too. She is currently working on development and validation of Bioanalytical LC–MS methods for therapeutic peptides/proteins, glycan analysis and high-resolution MS.
LGC (Cambridge, UK)
Robert is a principal scientist at LGC where he leads of a group of scientists focused on providing a large molecule LC-MS bioanalytical service. He has expertise in the bioanalysis of small molecules, oligonucleotides, peptides and proteins utilizing LC-MS/MS and ligand binding assay technologies. He has developed a keen interest in the implementation of immunoaffinity, 2D-LC and HR-MS techniques for protein LC-MS quantification workflows.
Boehringer Ingelheim Pharmaceuticals (CT, USA)
Hongbin Yu is the Director of Bioanalytical Mass Spectrometry in the DMPK department at Boehringer Ingelheim Pharmaceuticals in Ridgefield (CT, USA). He obtained his Ph.D in organic chemistry from the University of Missouri-Columbia (MO, USA). He joined Boehringer Ingelheim in 2006 and has supported drug development for both small and large molecules. For small molecules, his responsibilities focused on biotransformation and bioanalysis utilizing LC–MS. For large molecules, his responsibilities included supervising immunoassay development/validation, critical reagent generation and pharmacokinetic analysis. He currently oversees regulated and non-regulated bioanalysis for small molecules by LC–MS and large molecules by hybrid LBA/LC–MS.
Janssen Biotherapeutics (PA, USA)
Sanjeev is working as a Senior Scientist in PK/PD Assay Development group of Biologics Discovery sciences at Janssen Biotherapeutics (PA, USA). He obtained his PhD in Protein Mass Spectrometry from the University of Cincinnati (OH, USA). He started his professional career with Applied Biosystems (now Sciex) as a scientist developing MS methods for proteins and peptides. His next job was at Merck (NJ, USA) developing assays for protein/peptide biomarkers of toxicity in animal models utilizing LC–MS and hybrid LC–MS approaches. In his current job at Janssen, he is leading the development of hybrid LBA/LC–MS assays for mAbs/proteins/peptides to support in vivo PK studies to support early drug development.