The Rise of Low-flow Liquid Chromatography Mass Spectrometry in Biopharma

The biopharmaceutical industry is finally coming of age and we are seeing more biologic therapies reaching the clinic to treat a variety of ailments. These range from rare, previously undruggable conditions, like spinal muscular atrophy, to more common diseases, such as cancer and cardiovascular disease. Gene, cell and immune-based therapies are being developed and applied in new and exciting ways, such as precision and personalized medicines. The industry has gained momentum and biopharmaceutical innovation is accelerating. To keep pace with this progress, other technologies have also needed to advance. Mass spectrometry (MS) and liquid chromatography (LC) have similarly gained ground, innovating rapidly to meet the challenges and opportunities that this new type of therapeutic brings. These technologies are providing increasingly precise, sensitive, flexible and fast quantitative capabilities.

All biologics and traditional small-molecule pharmaceuticals need to be analyzed, at several points throughout their lifecycle, from drug discovery and research and development (R&D) through to clinical trials and post-marketing monitoring. Biologic therapies have however introduced new challenges for such analyses, as these compounds are produced using living biological systems, and are much larger and more complex than conventional small-molecule drugs. The bioanalysis of these compounds has traditionally been performed using ligand-binding assays (LBAs), such as enzyme-linked immunosorbent assays (ELISAs). Although these are still in routine use, LC-MS methods are coming to the fore, to complement LBA results. This is because although ELISAs are rapid, sensitive and easy to use, they are prone to cross-reactivity with matrix components, as can be the case with complex matrices such as biological fluids. Such issues with selectivity can hinder the R&D of a biopharmaceutical, even delaying it during late-stage development. Therefore, LC-MS methods are increasingly being employed to confirm ELISA findings, particularly in the analysis of compounds such as immunoglobulins (e.g. monoclonal antibodies), antibody-drug conjugates (ADCs), oligonucleotides, and other complex, large-molecule therapeutics.

The sensitivity advantage phenomenon with low-flow LC-MS

A highly sensitive and selective LC-MS method for analyzing large-molecule biopharmaceuticals in complex matrices is with microflow LC and tandem MS (MS/MS). There is a move within the biopharmaceutical industry to not only incorporate LC-MS/MS bioanalysis methods, but to prefer ones with low flow rates. This is because of a phenomenon referred to as the sensitivity advantage, which is the most profound feature of low-flow LC-MS/MS. The enhanced sensitivity of microflow LC-MS/MS typically extends to well over an order of magnitude beyond that attainable with conventional flow rates. Compared with conventional flow LC-MS, the signal with microflow can be as much as 4–20 times higher while the noise often remains the same (Figure 1). Moreover, this sensitivity advantage is especially useful in the analysis of large molecules because the larger the molecule, the greater the increase in relative signal.

Figure 1. A 4-fold improvement of signal intensity in the signal:noise ratio using microflow LC-MS/MS quantitation of an ADC in mammalian plasma. Extracted ion chromatogram (XIC) data for microflow and conventional flow LC for 10 μg/ml (top) and 5 ng/ml (bottom) shows improved signal:noise ratio. Credit: SCIEX.

It is no wonder that biopharmaceutical companies are taking advantage of this phenomenon for the bioanalysis of their biologic compounds, particularly as the reproducibility achieved with microflow LC-MS/MS is very high and easily surpasses the minimum standards set by regulatory authorities, such as the US Food and Drug Administration (FDA). Other benefits of low-flow LC-MS/MS are that it is more efficient, cheaper and greener. The smaller injection volumes required means that ion sources last longer, leading to more uptime. This also means less resources and solvent are needed, so running costs are lower and more ecologically sound.

Incorporating low-flow LC-MS/MS to maximize bioanalytical capabilities

The timely introduction of instruments and methods for low-flow LC-MS/MS can dramatically improve the operational function and capabilities of a bioanalytical laboratory. This is exemplified by Alturas Analytics, a global expert LC-MS/MS bioanalytical laboratory and contract research organization (CRO) that relatively recently incorporated a state-of-the-art triple quadrupole mass spectrometer into their suite of MS instruments. They decided upon the SCIEX Triple Quad 6500+ LC-MS/MS System due to its sensitivity and dynamic range and opted to include the OptiFlow ion source, an electrospray ionization (ESI) source designed and optimized for microflow LC-MS/MS.

“We are seeing an increasing need for microflow LC-MS/MS. We use SCIEX instruments because of the high sensitivity, rugged sources, and accepted technologies – especially for quantitative analysis. We use the Triple Quad 6500+ when we want ultimate sensitivity and/or have strictly limited sample volumes, for example, for analyzing most of our clinical samples. It’s allowed us to streamline our workflow as well as develop more microflow methods and assays, which wouldn’t have been possible otherwise.” – Dr Shane Needham, PhD, Chief Scientific Officer, Alturas Analytics, Inc.

With the new instrument, the laboratory was able to perform more sensitive and selective bioanalyses, across a greater dynamic range and at faster scan speeds. Samples required less dilution and so were easier to prepare, while the OptiFlow source was easy to switch, enabling convenient changing between conventional and low flow rates. Although the majority of LC-MS/MS bioanalysis for (bio)pharmaceutical laboratories is conducted using conventional flow rates, the proportion of work performed using microflow LC-MS/MS is increasing. Academic laboratories are already using microflow relatively routinely for research, and clinical laboratories are beginning to use it for a few specific applications. With regulatory authorities getting on board, it is largely a matter of time before microflow is used routinely in (bio)pharmaceutical analytical testing. This shift towards low-flow LC-MS/MS will also include nanoflow rates, especially as all the advantages conferred by microflow vs conventional flow are amplified. Therefore, now is the time for bioanalytical laboratories to gear up and incorporate LC-MS instruments that can perform and easily switch between conventional and low flow LC-MS/MS.