Small molecule drugs account for the majority of the pharmaceuticals market in terms of number of products, but the fastest growing segment of the market is biologics. Unlike small molecules, biologics are large proteins that require different approaches to development and manufacturing. Created via biological processes rather than being chemically synthesised, biologics are more complex and this makes them more difficult to manufacture and characterise.
At present, the majority of research and development for biologics is focused on recombinant proteins and monoclonal antibodies. Biologics are used for the treatment of a range of conditions, including cancer, infectious diseases and immune disorders. By their nature, biologics are more targeted than small molecule therapies. The targeted distribution of a biologic drug is particularly useful in cancer, in that the drug can selectively bind to the receptors on cancer cells, leaving healthy cells undamaged. This in turn means fewer side-effects for patients.
Development of targeted therapies allows pharmaceutical companies to offer drugs or products to those patient groups for whom the drugs will be most effective. It may also be that a targeted product can progress through clinical trials more easily than a drug given to a general disease population. Small molecule drugs generally hit sites all over the body, giving greater potential for side-effects than more targeted biologics.
MS in discovery and development
Mass spectrometry platforms for analysing small molecules are well developed because small molecules have been the focus of the pharmaceutical industry for many years. Mass spectrometry grew in importance for drug discovery during the late 1980s and early 1990s, when affordable bench top LC-MS systems became available. These systems allowed easy access for non-expert users and became an essential part of laboratories within the pharmaceutical industry.2
During this time triple quadrupole systems became established as the preferred method for quantitative studies, with the AB SCIEX API III LC/MS/MS system and subsequent models (from the same supplier) being the gold standard for the industry. The latest in this family, the QTRAP 6500 LC/MS/MS system, has a dual or switchable mass range, meaning that the radio frequency (RF) value can be switched on the quadrupole. Tuning the quadrupole on a mass spectrometer allows ions of different mass to pass through. Thus sensitive detection and analysis of small molecules or large molecules can be performed using the same system.
Other types of mass spectrometers such as ion traps, quadrupole-time-of-flight systems and Fourier transform ion cyclotron resonance mass spectrometers were developed primarily as qualitative tools.
Small molecule drug development is typically a linear and predictable process from discovery to development and finally production, whereas the pipeline for a biologic is more dynamic, with the development cycle continuing for the entire lifetime of the product. Improvements in the development of a biologic might include increased product yields or drug efficacy. The iterative modifications of the biologic have to be characterised to ensure that no adverse changes are introduced. Alterations in manufacturing processes can lead to heterogeneity or impurities; therefore comparability studies have to be performed each time a new batch of drug is produced.
Comparability studies examine the mass, sequence and structural conformation of the biologic. The structure is important because it can affect the efficacy (e.g. bioavailability) or the safety (e.g. antigenicity) of the compound. The risk of structural modifications is also increased for biologics because of their mode of production and large structure.3 Methods are therefore required that can qualitatively and quantitatively analyse these complex molecules, in terms of structure and the level of post-translational (such as glycosylation) or post-production modifications (such as deamidation or oxidation).
The importance of MS for characterisation of biologics: The fact that the characterisation of biologics requires more complex analysis has led the industry to look for suitable analytical platforms. These include simple techniques, such as protein gels, or HPLC methods with optical detection, as well as MS techniques.
Historically, the use of MS has been driven by the ability to connect HPLC to MS. Combining the separation capabilities of LC with the mass analysis capabilities of MS gives high sensitivity and selectivity, which is effective for the characterisation of small molecules. However, this technique is not always sufficient for the characterisation of the more complicated biologic molecules.
These large molecules can be so complex that simply introducing them into a mass spectrometer can be problematic. Biologics are mixtures rather than pure compounds, making the results more difficult to understand and interpret than for relatively pure compounds such as small molecules. Mass spectrometry instruments that are currently used for the analysis of complex molecules have not changed much in terms of hardware. However, specific techniques have emerged, such as hydrogen/deuterium exchange and electron transfer dissociation, that have been developed for better characterisation of biologics.1
Limitations of existing MS technologies for characterisation of biologics: Biologics are natural compounds, and their shape has an impact on their efficacy. Introducing a protein into a mass spectrometer in its native form can be difficult. To hold a complex natural molecule in solution requires a salt and buffer concentration that is not generally amenable to MS. Capillary electrophoresis (CE)-MS can help circumvent this problem, but has its limitations. Separations by CE can be very powerful but traditionally in CE-MS the separations are diluted with a make-up flow, decreasing the sensitivity of the method.
Currently, software solutions for MS are limited. New solutions are needed to fully characterise post-translational or post-production modifications. This requires software that can deconvolute the proteins. Manufacturers are working on software tools to make the process easier. Finally, another limitation of MS methods for biologics analysis is that of the human resource – i.e. the difficulty of finding individuals who have an understanding of MS, chemistry and biology.
CESI-MS the new approach
A new MS front-end separation approach, known as capillary electrospray ionisation-mass spectrometry (CESI-MS), allows the combination of high efficiency CE with electrospray ionisation (ESI), and can help improve and simplify the analysis of biologics by MS. The recent launch of the CESI 8000 supplied by Sciex Separation (a part of AB SCIEX) is a significant improvement on the CE-MS technique.
With CESI-MS the sensitivity of the assay is increased and ion suppression is reduced. The separation goes directly into the mass spectrometer with no need for mixing. The system runs at an ultra low-flow rate, which gives high sensitivity for detecting post-translational modifications. It also uses very low volumes in a relatively short cycle time (16–30min), allowing high throughput. CESI-MS is far more robust than the past iterations of the technology.
Advantages of CESI for characterisation of biologics: One advantage of the CESI 8000 system is that it is relatively simple to use. The capillary plugs directly into the source adaptor, with no mixing of gas, or make-up flow, making it easy to connect.
Peptides are optimally separated and detected at lower flow rates,4 and therefore nano LC has been the preferred choice for characterisation of proteins in the past. Nano LC requires that the column used to do the separation should be connected to a nano emitter which then plugs into the source.4 These connections can be difficult, due to the need to minimise the dead volume in any connection at these lower flow rates, which means an expert is required.
There are no difficult connections in CESI-MS. The end of the capillary is etched so that it acts as the emitter. This allows scientists to perform complete sequence coverage and modification mapping of a whole protein in one injection, which cannot always be done using standard LC technology. Whole proteins can be analysed using CESI-MS because the capillaries do not have a stationary phase. The capillaries are open tubes, which means that larger peptides that would normally have been retained on the stationary phase are able to flow through and be detected. The other important advantage of CESI-MS is that there is no void volume, as in standard LC. Therefore smaller peptides that would have been lost can be detected resulting in improved sequence coverage. Thus proteins can be separated and characterised in a much faster time.
Novel applications for MS are continuously being developed. The sensitivity, specificity, dynamic range and throughput of MS techniques are of great importance to scientists and can always be enhanced. But to further advance MS techniques for biologics characterisation, efforts are being made to further ‘clean up’ the front-end of the mass spectrometer. Front-end clean up is required to remove anything that may interfere with analysis of the sample. In future sample preparation and sample clean up will also help with improving biologics characterisation.
References
1. Bobst, C. E., and Kaltashov, I. A. 2011. Curr. Pharm. Biotechnol. 12(10): 1517–1529
2. Grebe, S. K., and Singh, R. J. 2011. Clin. Biochem. Rev. 32(1): 5–31
3. Kaltashov, I. A., Bobst, C. E., Abzalimov, R. R., Wang, G., Baykal, B., and Wang, S. 2012. Biotechnol. Adv. 30(1): 210–222
4. Kelly, R. T., Page, J. S., Marginean, I., Tang, K., and Smith, R. D. 2008. Anal Chem. 80(14): 5660–5665
QTRAP 6500 is registered to, and AB SCIEX API III a trade mark of, AB Sciex