Glycosylation of biologic drugs affects their mechanism of action, pharmacokinetics, and efficacy. Glycan analysis, as a result, is critical for determining the quality of these biotherapeutics. Until recently, analytical methods have been slow and tedious and largely limited to use in the later stages of product development. The introduction of high-throughput techniques is, however, creating opportunities for the application of glycan analysis early in bioprocess development.
Scott A. Siegel, chief operating officer, and Hidehisa Asada, vice president of research and development, both with Ezose Sciences Inc., spoke with Cynthia Challener, editor of the Pharmaceutical Sciences, Manufacturing & Marketplace Report, about the importance of glycan analysis for biotherapeutic development, the impact of recent advances in high-throughput analysis capabilities, and what further advances such glycan analysis technology might enable in the future.
Importance of glycosylation
Pharmaceutical Sciences, Manufacturing and Marketplace Report: Why is the characterization of the glycosylation patterns on glycoproteins important?
Siegel (Ezose): More than half the proteins expressed in humans are post-translationally modified by glycosylation. Differences in glycosylation can affect protein structure, stability, immunogenicity, and function. Thus, glycomics plays a significant part in health and disease. In fact, glycans play a key role in normal cellular function. They mediate interactions among cells and between cells and pathogens. They enable immune responses. They also have a role in tumor progression.
In addition, biotherapeutics, including monoclonal antibodies (mAbs), are often glycosylated, and the extent, type, and location of the glycan structures present on the proteins impact the mechanism of action, pharmacokinetics, and efficacy of these drugs. In some cases, when non-human glycan structures are present, they can trigger an undesirable immune response. Glycosylation is, therefore, often a critical quality attribute in bioprocessing, and these biotherapeutic molecules have become a mainstay of the biotech and pharmaceutical industries.
Role in biomarker development
Pharmaceutical Sciences, Manufacturing and Marketplace Report: How is glycan analysis related to the development of novel biomarkers?
Asada (Ezose): Glycomics is a natural extension of genomics and proteomics. Glycan patterns distinguish otherwise similar proteins, so we believe these patterns may serve to distinguish between healthy and diseased tissue samples, between a disease that is indolent and a disease that is aggressive, and between a patient in whom a drug is effective and a patient in whom it isn’t. There are already examples of marketed, glycan-related products, such as tests to manage cases of pancreatic and liver cancer. And yet the lack, until recently, of high-throughput techniques for the analysis of glycans has meant that the glycome today remains mostly uncharted territory.
Limited by slow and laborious techniques
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What are the challenges in completing effective glycan analyses?
Siegel (Ezose): Traditionally, glycan analysis has been a slow, labor-intensive process, and has been more routinely applied late in bioprocess development. Glycan analysis early in bioprocess development has been limited to the analysis of small numbers of samples. With the ever-growing focus on biotherapeutics, including the increased investment in “biosimilars” and “biobetters,” there is, however, a need to improve the process by monitoring glycosylation patterns across large sets of samples generated during cell-line selection and early bioprocess development. The challenge has always been to achieve speed and scope—a high-throughput technology platform. Glycan analysis and glycomics in general lacked laboratory techniques comparable to those that accelerated progress in genomics. But now we’re getting there.
New technologies enable high-throughput solution
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What recent advances in technology (in general) have been introduced that help address these issues?
Asada (Ezose): Mass spectrometry, automation, sample preparation, and separation methods continue to improve, permitting more rapid and sensitive glycan analysis. Our GlycanMap platform leverages several of these advantages. We can now analyze more than 150 samples a day, and that number is scalable as the demand grows. That kind of throughput would once have taken weeks or even months to complete.
Automated enrichment, mass spec detection, and bioinformatics
Pharmaceutical Sciences, Manufacturing and Marketplace Report: How did you develop your GlycanMap technology, and why did you combine automated chemoselective, bead-based, glycan enrichment with quantitative matrix-assisted laser desorption-ionization time-of-flight (MALDI-TOF) mass spectrometry coupled to custom bioinformatics in your system?
Asada (Ezose): The core GlycanMap technology was originally developed by researchers at Hokkaido University and has been further refined for commercial use by Ezose. The platform combines three basic components: automated glycan enrichment, detection by mass spectrometry, and custom bioinformatics that convert the mass spectrometric data into glycan identities and concentrations.
The chemoselective bead-based enrichment provides rapid clean-up and capture of glycans from complex samples, including cell culture media and biological fluids, such as serum, urine, and cell and tissue lysates. Automating this once time-consuming and tedious step accelerates the analysis and improves reproducibility, resulting in a more industry-friendly solution.
Mass spectrometry is used for glycan detection because its mass accuracy provides accurate identification of the oligosaccharide composition of each glycan, and it provides a throughput that cannot be attained today with high-performance liquid chromatography (HPLC). The mass spec data is then analyzed using our proprietary bioinformatics system, which identifies glycan compositions and converts peak intensities to absolute concentrations (in µM).
Facilitating biopharmaceutical production
Pharmaceutical Sciences, Manufacturing and Marketplace Report: What further advances might be expected in the near future? Longer term?
Siegel (Ezose): For the immediate future, we are focused on a new option that makes our GlycanMap platform services routinely available to researchers, particularly in bioprocessing, who need to rapidly screen major glycans for cell-line selection and early bioprocess development. At the same time, we have also driven down our cost for running the service, and we are passing this savings along to our clients in the form of lower pricing. We call our new CRO service GlycanMap Xpress analysis.
We continue to also see value in applying our technology services to discovering novel biomarkers, particularly those for use in developing new diagnostics for disease or companion diagnostics to stratify patient cohorts and monitor response to medicines. Whatever the application, our technology can be applied broadly to many different types of biomolecules and across therapeutic areas.
So, we have an ambitious research program that keeps us busy. We can, however, imagine still other avenues of investigation. Some of our partners are interested in applying our technology to vaccine development, for example, or using glycomics to develop a deeper understanding of disease biology and drug mechanisms, and potentially to discover new drug targets.