The rate of innovation in mass spectrometry (MS) is rapidly growing. Faster than ever before, manufacturers are introducing technologies that are more robust, sensitive and flexible, to deliver superior quality in molecular quantification and characterization. This is especially true for laboratories responsible for the bioanalysis of samples from the pharmaceutical and biotechnology industries. Bioanalysis is needed for all therapeutics and plays a key role in their development and beyond. As such, innovations have been developed that can be variously applied to analyze the full spectrum of therapeutics, from small molecules through to large complex ones, such as oligonucleotides for gene therapies and proteins for immunotherapies.
After several years of setbacks, gene and cell therapies are finally coming to the fore, providing new treatments for previously undruggable diseases. They are at the forefront of medical research aimed at the realization of precision medicines and personalized health. These and other biologic therapies are not only manufactured in a radically different way but also incorporate larger molecules than traditional small-molecule drugs. At some point throughout the research and development (R&D) process, all these compounds will need to be analyzed: detected, identified and quantified, in biological samples such as blood and urine, which contain many other types of molecules.
Being able to perform such precise bioanalysis for the full spectrum of therapeutics in complex matrices presents new challenges and opportunities for analytical facilities involved in performing the bioanalysis of these compounds, from drug discovery through to Phase IV clinical trials and post-marketing monitoring. This means laboratory managers need to gear up, both in terms of new equipment as well as upskilling members of their team to support and train users. Selecting the right equipment requires the judicious balance of multiple factors, including benefits, risks, best value, and the current and future needs of users. Futureproofing the laboratory capabilities and operations is critical as the pace of progress continues to accelerate.
Although the traditional approach to bioanalysis, using ligand-binding assays, is still very much the workhorse of (bio)pharmaceutical industry, rapid advances in liquid chromatography (LC) and tandem mass spectrometry (MS/MS) technology means that we are seeing a shift towards LC-MS/MS methods. While immunoassays, particularly enzyme-linked immunosorbent assays (ELISAs), are easy to use, rapid and sensitive, they are prone to cross-reactivity with components of complex sample matrices. Serious selectivity issues like these can hinder the R&D of a therapeutic, even delaying progression during late-stage development. Despite this and the growing interest in LC-MS/MS methods, it is unlikely that they will ever fully replace ligand-binding assays. However, they are being increasingly employed to complement and confirm ELISA results, especially for the analysis of monoclonal antibodies (mAbs; immunoglobulins), antibody-drug conjugates (ADCs), and other large-molecule therapeutics.
A highly sensitive and selective MS method for analyzing large-molecule biopharmaceuticals is with microflow LC-MS/MS. This is because low flow rates like microflow confer a sensitivity advantage compared with conventional flow rates. This phenomenon is the most profound feature of microflow, with the enhanced sensitivity typically extending to well over an order of magnitude beyond that attainable with conventional flow [1]. The signal, in the signal-to-noise ratio, can go up by as much as 4–20 times while the noise often remains the same, compared with conventional flow LC-MS/MS (see Figure 1). This sensitivity advantage is especially useful in the analysis of large molecules because the larger the molecule, the greater the increase in relative signal. The reproducibility achieved with microflow is very high and easily surpasses the minimum standards defined in bioanalysis guidelines of regulatory authorities, such as the US Food and Drug Administration (FDA). There are also other benefits to low flow rates in LC-MS/MS. It requires the injection of smaller sample volumes. Low-flow LC-MS/MS also uses fewer resources and solvent, so it is both cheaper and more ecologically sound. Aware of these advantages, particularly that of the enhanced sensitivity, biopharmaceutical companies are choosing to use microflow LC-MS/MS more often for the detection and quantitation of their large-molecule therapeutics.
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 [2].
For all these reasons, it is likely that microflow LC-MS/MS will become routinely used in the biopharmaceutical industry in the foreseeable future. Therefore, it would be prudent for laboratories working in the bioanalysis of therapeutics to consider the adoption of MS technologies capable of performing microflow LC-MS/MS, if they do not already possess that capability. An example of how such an addition can substantially improve the function and operation of a laboratory and what it can offer to users is provided by Alturas Analytics, a bioanalytical laboratory that offers MS/MS services worldwide and relatively recently integrated a state-of-the-art triple quadrupole mass spectrometer into their suite of MS instruments. They chose the SCIEX Triple Quad 6500+ LC-MS/MS System because it was judged to be one of the most sensitive instruments on the market, with a very wide dynamic range, and opted to include the OptiFlow ion source, an electrospray ionization (ESI) source designed and optimized for microflow LC-MS/MS.
“We chose SCIEX because they have the most rugged source, most sensitive instruments, and most accepted technologies – especially for quantitative analysis. We obtained the latest machine, the Triple Quad 6500+ because we want to be leaders, and as such, we need to have the best instruments. We find the 6500+ is the most sensitive on the market and has the best dynamic range, and the OptiFlow source is ideal for microflow LC-MS. We use it for 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.” according to Dr. Shane Needham, Ph.D., Chief Scientific Officer, Alturas Analytics, Inc.
With the new instrument, the laboratory service was able to improve the bioanalytical sensitivity and selectivity that they offer their customers. The faster scan speeds, greater dynamic range, and less demanding sample preparation (which required less sample dilution) contributed to more efficient workflows. The OptiFlow ion source also helped because it is easy to switch, allowing for more convenient changing between set-ups for microflow and conventional flow rates. Although the majority of LC-MS/MS work in most (bio)pharmaceutical laboratories are performed using conventional flow rates, the proportion of microflow-rate work 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 trend towards lower flow rates may eventually encompass nanoflow LC-MS/MS. All the advantages conferred by microflow vs conventional flow are not only true of nanoflow but are amplified due to its even lower flow rate. Nanoflow LC-MS/MS is particularly useful for applications such as proteomic analyses because it provides the sensitivity and selectivity needed to detect and quantify a variety of peptides and proteins present across a wide dynamic range, including some at trace levels. These applications are increasingly vital for biomedical research, as the understanding of health and disease moves beyond the genomic realm. As was the case with genomics, the development of advanced high-throughput analytical technologies will facilitate the acceleration of proteomics, metabolomics, and other omics research.
The combination of omics analyses can provide a clearer picture of biological processes and systems. Such a multi-omics approach may be exemplified by proteogenomics, which is being investigated as a way of better characterizing human tumors for personalized medicine [3]. By including data from the proteome, which encodes information (such as post-translational modifications and epigenetic inactivation) that is not discerned through genomic analysis alone, tumor phenotypes can be more fully annotated. This can lead to more successful matching of cancer patients to treatments that will be efficacious for them, resulting in higher therapeutic response rates and prolonged patient survival with precision medicine. It is expected that the integration of personalized genome information, coupled with data regarding its execution at the protein level, will soon become an accepted component in the practice of precision oncology.
Through the timely introduction and integration of new innovations such as MS systems capable of handling microflow and nanoflow LC-MS/MS, laboratories can optimize and future proof their operations and services. Analyses can be performed with substantially improved selectivity and sensitivity, with lower limits of quantitation (LLOQ) being as much as 10 times lower. Efficiency can be increased by streamlining workflows and productivity can grow by 50 percent. Microflow LC-MS/MS is likely to be relatively routine in (bio)pharmaceutical laboratories in the next 10 to 20 years, with nanoflow following soon after. To prepare and make the most of these advances and innovations occurring in MS and the (bio)pharmaceutical industry, it is critical that laboratory managers consider the incorporation of MS instruments and microflow and nanoflow methods now. The future is bright and these low-flow LC-MS technologies will play a key role in the quantitative bioanalysis required for the realization of precision and personalized medicine, such as gene and cell therapies.
References
- Needham SR, Valaskovic GA. Microspray and Microflow LC–MS/MS: The Perfect Fit for Bioanalysis. Bioanalysis. 2015; 7: 1061–1064.
- Motamedchaboki M, van Soest R, Moore I. Sensitive and Accurate Quantitation of the Antibody-Drug Conjugate Ado-Trastuzumab Emtansine in Rat Plasma. SCIEX Tech Note. https://sciex.com/Documents/tech%20notes/ADC-quantitation-trastuzumab-Rat-Plasma.pdf (accessed October 2019).
- Zhang B, Whiteaker JR, Hoofnagle AN, et al. Clinical Potential of Mass Spectrometry-based Proteogenomics. Nat Rev Clin Oncol. 2019; 16: 256–268.
Neil Walsh is the Global Marketing Manager, Pharma at SCIEX, a Danaher operating company and a global leader in the precise quantification of molecules.