Therapies for diseases such as cancer, Alzheimer’s disease, and lupus have been significantly impacted by the introduction of biotherapeutics. These large molecule drugs include monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), and fusion proteins and have the ability to uniquely target specific biomarkers or processes instead of systemic action. For this reason, they are touted as more effective and potentially safer than conventional small molecule counterparts.
Because large molecule drug discovery and development is still maturing, systems to test and measure the safety and efficacy of a new biotherapeutic tend to better accommodate small molecule drugs. Defining optimal large molecule drug metabolism and pharmacokinetics (DMPK) characteristics, in particular, with tools that analyze biotherapeutic metabolism, behavior, and target binding in difficult matrices is important in determining the ideal drug dosage required to be effective in a certain population. The integration of more pointed technologies such as mass spectrometry (MS) has improved assay sensitivity, speed and accuracy, and quality of results, so much so that MS approaches have become a forerunner among biotherapeutic testing techniques that successfully determine the validity of a new large molecule drug.
MS for effective DMPK studies
Once biotherapeutics are administered and circulate through the body, they will be metabolized, potentially modifying or even destroying drug potency. DMPK studies that focus on identifying drug molecule metabolites and monitoring the rate of molecule circulation can confirm that the drug is still effective and reaching its intended target. Yet, even though many large molecule drugs show efficacy in early drug discovery using in vitro cell-based platforms, these same drug candidates could be differentiated based on their DMPK profile when introduced into an in vivo system.
“Because biotherapeutics have a larger chemical structure, they have a higher number of potential outcomes that can be observed when studying their DMPK profile. Understanding how these DMPK outcomes effect the specificity and potency of a large molecule drug will increase the chances of a drug discovery hit being advanced to a drug development candidate,” explains Stephen Kurzyniec, pharma biopharma sales specialist at Shimadzu Scientific Instruments.
Conventional tests such as ligand binding assays still tend to reign supreme for large molecule bioanalysis. However, advancements in MS techniques are more recently allowing large molecule drug developers to better identify multiple attributes that contribute to a drug’s effectiveness, even linking molecular details to function. With more complex structures, modern MS tools have made this process more effective, efficient, and reproducible, notes Kurzyniec.
Bringing together a wide variety of MS platforms that can be utilized in DMPK studies offers orthogonal analytical approaches required to get an accurate drug profile. Initial sample preparation is key with MS-based large molecule techniques to ensure proper results from complex matrices. Applying companion systems that can be used to automate and streamline the sample preparation process and can be directly connected to LC-MS/MS such as the Perfinity online protein digestion system and the CLAM-2000 (Clinical Lab Automation Module) provides the speed and precision for large molecule drug optimization. Shimadzu recommends their LC/MS/MS 8060 as highly effective in molecule quantitation in a wide variety of biological matrices or the new LC-MS 9030 Q-ToF, which can be used to obtain a range of large molecule structural details using high mass accuracy, high mass resolving power, and high signal sensitivity.
Quality results for complex assays
Not only must early drug discovery apply DMPK studies to instill confidence in a drug target for further development, but also bioproduction, where small post-translational modifications can affect the viability and activity of the developed drug. “The hybridization of existing techniques and the development of new techniques will be essential as the number and complexity of large molecule drug discovery projects continue to increase and move through the pipeline,” explains Keeley Murphy, senior marketing specialist for pharma/biopharma at Thermo Fisher Scientific. Mass spectrometry approaches aimed at biotherapeutic DMPK analysis offer a unique level of target specificity when profiling large molecule analytes throughout discovery and development, resolving and detecting small differences or changes in target mass.
Murphy comments that although high-resolution mass spectrometry is available on multiple platforms, the quality of spectra produced must be considered to help confidently direct decisions on whether a molecule should continue through discovery to development. “Thermo Fisher’s line of benchtop Orbitrap mass analyzer systems, for example, offer the highest level of spectral data quality with robust resolving power and high mass accuracy,” he says.
Recent product introductions offer new capabilities that make MS ideal for complex large molecule profiling. The Thermo Scientific™ Q Exactive™ UHMR Hybrid Quadrupole-Orbitrap™ mass spectrometer combines high resolution, high sensitivity, MS2 and pseudo-MS3 capabilities, while the newly enhanced Thermo Scientific Orbitrap™ Fusion™ Lumos™ Tribrid mass spectrometer has the ability to select the most promising precursor ions, create new sequence- or structure-informative fragments, and characterize them to the level of fine isotope structure.
Biotherapeutics design will continue to advance as we learn more about their interactions with other molecules and their environment. Improvements in chromatographic separation, hybrid immuno-affinity technologies, high-resolution mass spectrometry, and data analysis software will all most likely play a role in supporting these future analyses. “Thermo Fisher Scientific continuously strives to not only improve these technological platforms, but also to develop technology to enable discoveries for the next therapeutics to be found. We are focused on applying improvements in intelligent data acquisition, integrated data processing tools and algorithms to ease the adoption of LC-MS into more applications,” Murphy offers.
Quantitative and qualitative monitoring
Discovery and development of new biotherapeutics with increasing complexity and high molecular weight present additional quantitative and qualitative analytical challenges. Intact level protein analysis using high-resolution mass spectrometry (HRMS) offers direct quantitative measurement of the whole molecule for DMPK profiling, potentially including sub-species and variant information, as well as qualitative insights into in vitro and in vivo molecular stability and modification sites. This data is not easily attained by traditional ligand binding assays, which can lack specificity and are costly and time consuming to develop at early stages when the molecular structure of the biotherapeutic may not be fully defined.
“DMPK and development labs are working more closely together using HRMS to provide better quantitative and qualitative information, leading to more informed decisions earlier in development and ensuring product safety, efficacy and quality in clinical trials,” explains Dr. Ian Edwards, business development manager for the pharmaceuticals business at Waters. Dr. John Mehl, senior research investigator at Bristol Myers Squibb, recently presented on the use of the Xevo® G2-XS Q-Tof high resolution mass spectrometer for the bioanalysis of antibody-based therapeutics using intact mAb and subunit detection. He concluded that the combination of affinity capture with mass spectrometry provides valuable information about the molecular behavior and stability of antibody-based therapeutics.
In addition to drug clearance, investigators are concerned with the catabolism of large molecule drugs, particularly those that contain non-natural amino acids such as therapeutic peptides. Metabolites can be tracked and accurately resolved using data independent ion mobility data collection (HDMSE) approaches with the Waters VionTM IMS Q-Tof, which can resolve chromatographic co-elutions that would otherwise result in mixed product ion spectra. Measurement of the collisional cross section (CCS) or shape of a molecule, provided by ion mobility, enables tracking and identification of metabolites regardless of chromatographic conditions or matrices. A challenge remains with the few software packages available to characterize biotherapeutic clearance and metabolism. Waters recently announced a new interface with Mass-MetaSite, a drug discovery metabolite identification software, to complement the Waters UNIFI metabolite identification application solution. Using intelligent software platforms, data can be routinely collected, processed, and reviewed across time courses and treatments. “Waters helps our customers accelerate drug discovery and development, to bring life-changing therapeutics to market. Coupling our high-resolution, ion mobility spectral data acquisition capabilities with the industry’s leading drug discovery metabolite identification software allows us to deliver new capabilities for DMPK scientists,” Dr. Edwards said.
Speed and simplicity
Mass spectrometry has been growing as the technique of choice in large molecule DMPK studies for a reason. These assays are more sensitive and reliable even with high sample complexity and matrix difficulty. Furthermore, MS allows for higher throughput, leading to faster analysis in both early and later DMPK studies. By achieving valid and high-quality results, even with mounting workloads and progressively complex biotherapeutic characterization demands, MS approaches are able to surpass conventional systems that may no longer be up to the task.
SCIEX biologics characterization solutions are an example of how MS capabilities make it possible to extend analytical capability, increase lab throughput, and automate data processing to simplify biotherapeutic characterization for a more accessible approach.
Cyprotex Discovery collaborates with SCIEX to innovate new solutions for their work as a DMPK ADME contract research organization. The company investigates drug actions on the body as well as provides information to screen out undesirable compounds with poor DMPK parameters. The company uses the TripleTOF® 6600 Quadrupole Time-Of-Flight mass analyzer, a high-resolution accurate mass system with SWATH® Acquisition that comprehensively collects data throughout each run, eliminating the need for optimization. A large benefit to Cyprotex’s DMPK processes is that all compounds can be assayed in one experiment, saving time, improving efficiency, and allowing retrospective reprocessing of the data to examine different analytes without having to repeat analysis.
Cyprotex uses the TripleTOF 6600 for the analysis of biotherapeutic-type molecules including next-generation peptides, seeing it as an improvement over triple-quad instruments due to the molecule’s increased molecular weight, which makes it difficult to get selective MRM transitions and required sensitivity.
It is well understood that large molecules are unique compared to their small molecule counterparts. Thus, DMPK studies for biotherapeutics must be approached in accordance to these differences. This includes more in-depth and species-specific profiling to ensure safety and efficacy of a drug candidate and its metabolites in a target population. Innovations in MS approaches tailored to large molecule DMPK assays have driven its accelerated adoption. Improved sensitivity in complex matrices and increased selectivity for better indications of drug success has led MS to be more than a viable alternative for both qualitative and quantitative applications of large molecules.