Professor Michael Butler, head of the National Institute for Bioprocessing Research and Training (NIBRT) Cell Technology Group (CTG), explains that the characterisation and analysis of glycans is important for the biopharma sector as it can help in the development of therapeutic agents such as monoclonal antibodies and other recombinant proteins. These are used in therapies in a range of serious conditions including cancer, inflammatory diseases, and cardiovascular diseases. The mechanism of action depends upon their strong affinity to molecular targets associated with disease.
The current global therapeutic market is the result of 30 years of sustained growth and development in the field of biotechnology. This progress includes the expansion of genomic sciences and the ability to design cell bioprocessing platforms for biotherapeutics production that can be used to address previously unmet medical needs. These breakthroughs are adding value to the biotech sector, but growth barriers remain an issue.
For example, the NIBRT 2021 annual report announced that global sales of biopharmaceuticals have topped $300 billion, with a compound annual growth rate of 12%1. The same data also reveals that approximately 60% of therapeutics in development are biopharmaceuticals. However, obstacles to large-scale production and usage of these biotherapeutics are hampering progress. NIBRT works to actively identify and address these bottlenecks through collaborations with biopharmaceutical companies allowing progress in the large-scale production of these valuable therapeutics.
Understanding glycan profiles
Glycan analysis and characterisation is an increasingly important area for biopharma as understanding glycan profiles helps in the development of new therapeutic agents. It is also important for the creation of biobetters when innovator molecules fall out of patent.
Large-scale production is made possible by the production and selection of mammalian cells with an ability for high cell-specific productivity of a targeted protein, such as a monoclonal antibodies (mAbs). However, it is well known that the glycosylation of these proteins is far from homogenous and depends both on the metabolic profile of enzymes within the cell as well as the environmental factors of the bioprocess, including the media composition. Although this glycosylation profile is only a small component of most glycoproteins, it is often critical to biological function.
A collaboration on glycan analysis between NIBRT’s CTG and Agilent is building on the CTG’s work on the glycan profiling of glycoprotein, an important step in improving the stability and efficacy of therapeutics. Given the critical importance of the glycan profile for the biotherapeutic activity of these glycoproteins, it is essential to have a method of analysis that is rapid, reliable and sensitive. This goal sits at the heart of NIBRT’s collaboration with Agilent.
An overview of the method
The appropriate glycosylation profile can improve protein stability, increase the half-life in blood circulation and decrease immunogenicity. As well as that, it has been found that glycosylation of mAbs is essential for the therapeutic efficacy in the destruction of cancer cells. The absence of a glycan may lead to little or no therapeutic benefit while a modification to a single glycoform may enhance the therapeutic benefit substantially.
InstantPC from Agilent (formerly ProZyme) is a Glycosamine labeling dye, with high FLD and MS signal that is applied to the rapid analysis of released N-glycans using a 96-well plate format for streamlined glycan profiling.
This initial work, which was published in Analytical Biochemistry and co-authored by scientists from both NIBRT and Agilent (Xie et al, 2021), includes the analysis of glycan profiles of two mAbs, human polyclonal antibodies and the total protein in human serum. The method involves separation of extracted glycans by hydrophilic interaction liquid chromatography with fluorescence detection using the InstantPC label.
The total preparation time for glycan labelling was shortened to less than 10 minutes, which is a significant improvement on alternative methods. Furthermore, the sensitivity of the fluorescent label was shown to be around x100 greater than 2-aminobenzamide, a reagent that has been used extensively in the past to label glycans.
The glycan database
As a result of the ongoing collaboration with Agilent, the CTG team continues to make significant progress in showing the benefits of the new method of analysis of glycans using InstantPC as a highly sensitive fluorescent label. They are currently working on an extensive InstantPC glycan library that now contains 264 unique glycan structures, while new application methods are being developed. The library has been used to analyse the changing profile of glycans attached to serum proteins following Covid infection with the possibility that this can be used as a biomarker for the short or long-term impact of Covid.
The database was established by analysing the glycan profiles of various glycoproteins including those from human serum and a range of specific proteins from various sources. Precise chemical structures were assigned and validated using a combination of chromatographic and mass spectrometry analysis.
The project has served up a range of challenges. For instance, the development of the analysis of glycans has been difficult due to the diversity of structures and multiple ways that individual monosaccharides can form linkages. It is therefore not easy to predict which structures will be synthesised in cellular processes, as their formation is not governed by sequential templates – as is the case with nucleic acids or proteins – but rather by the array of glycoenzymes present in the cell and the environmental conditions of culture. This complexity makes quantification challenging.
Areas for future collaboration
Agilent’s next round of collaboration will look at cell and gene therapy glycan applications. The InstantPC workflow has been shown to be suitable as a method for automated and rapid handling of multiple human serum samples that could be used in screening for cancer and other conditions that can be detected by aberrant changes in molecular profiles of glycan structures.
Further areas of application for sensitive glycan analysis are in the monitoring and control of therapeutic agents or in providing insights into pathways of viral infection in which glycans have a role in viral attachment and the immune response. Some of the most pathogenic viruses, including HIV, influenza, coronavirus, Ebola, and Lassa, are enveloped by glycoproteins that play a critical role in their ability to infect human cells. An understanding of these structures is important in their role as antigens and as candidate targets for vaccines.
NIBRT has commenced a large-scale building extension to increase NIBRT’s capability in cell and gene therapy research and training, that will provide a platform to build on current approaches. The CTG will form a key part of this development plan as will the continued collaboration with Agilent. In particular this collaboration will involve the critical aspects of glycan profiling in cell and gene therapy.
References
Everest-Dass AV, Moh ESX, Ashwood C, Shathili AMM, Packer NH. 2018. Human disease glycomics: technology advances enabling protein glycosylation analysis – part 1. Expert Rev Proteomics, 15:165-182.
Li JD. 2010. Functional glycomics : methods and protocols Humana:New York, N.Y.
Munkley J, Elliott DJ. Hallmarks of glycosylation in cancer. Oncotarget. 2016 Jun 7;7(23):35478-89. doi: 10.18632/oncotarget.8155
Oliveira-Ferrer L, Legler K, Milde-Langosch K. Role of protein glycosylation in cancer metastasis. Semin Cancer Biol. 2017 Jun;44:141-152. doi: 10.1016/j.semcancer.2017.03.002
Harleen Kaur (2021) Characterization of glycosylation in monoclonal antibodies and its importance in therapeutic antibody development, Critical Reviews in Biotechnology, 41:2, 300-315, DOI: 10.1080/07388551.2020.1869684
Jefferis, R. (2009). Glycosylation as a strategy to improve antibody-based therapeutics.
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Watanabe Y, Bowden TA, Wilson IA, Crispin M. Exploitation of glycosylation in enveloped virus pathobiology. Biochim Biophys Acta Gen Subj. 2019 Oct;1863(10):1480-1497. doi: 10.1016/j.bbagen.2019.05.012.
Yongjing Xie, Letícia Martins Mota, Adam Bergin, Roisin O’Flaherty, Aled Jones, Beth Morgan and Michael Butler. High-throughput and high-sensitivity N-Glycan profiling: a platform for biopharmaceutical development and disease biomarker discovery Anal Biochem 2021 Apr 20:114205. doi: 10.1016/j.ab.2021.114205.
Yongjing Xie and Michael Butler. Construction of InstantPC derivatized glycan GU database: a foundation work for high-throughput and high-sensitivity glycomic analysis. Glycobiology 2022 Mar 31;32(4):289-303. doi: 10.1093/glycob/cwab128.
