Recombinant monoclonal antibody therapeutics (mAbs) represents the largest group of therapeutic proteins. The efficacy of these therapeutics is highly dependent on the correct glycosylation patterns of the mAbs. The analysis of the glycan pattern is therefore an essential part of the mAb characterization process. However, glycan mapping is a complicated and multi-step process.
In the drive to discover the next promising biotherapeutic or develop a reliable biosimilar, researchers cannot afford to compromise on analytical accuracy or efficiency. Rapid, reproducible and high resolution glycan mapping is now achievable thanks to developments in column technology which help researchers overcome the challenges associated with glycan analysis.
The glycan mapping challenge
Labeled glycans are hydrophilic structures so hydrophilic interaction chromatography (HILIC) using fluorescence detection is a robust separation method for glycan analysis. HILIC/LC can also be coupled to mass spectrometry to obtain important mass and structure information. One of the biggest challenges in HILIC/LC is achieving high-resolution with fast analysis times without compromising performance. Because glycans include many closely related structures it is critical to achieve the highest resolution in the quickest amount of time possible.
In addition to long analysis times, achieving reproducible glycan results can be another challenge because glycan analytes are often of low abundance in complex biological samples and the glycans must be cleaved from the glycoprotein and labeled prior to analysis. Instrument limitations can also be another issue due to increased levels of backpressure with the sub 2 um particles.
Sample preparation for better Biochromatography
Sample preparation is an important step towards achieving accurate, reproducible, and meaningful results from Biochromatography. Proteins are expressed in complex matrices, cell cultures, and so must be characterized prior to characterization. With monoclonal antibodies the most efficient way to capture the product molecule is to use Protein A. With HPLC columns such as the Agilent Bio-Monolith Protein A column simple centrifugation is needed to separate the liquid containing the soluble protein form the insoluble cell debris. Due to the fact that proteins are present in multiple forms and can be found within different cell locations, no universal sample preparation procedure technique exists for proteins. Common protein sample preparation processes include desalting, concentration, centrifugation, affinity, dialysis, filtration and ultrafiltration, precipitation, and lyophilization. Since proteins are very fragile, care must be taken in the sample preparation process to avoid the introduction of unwanted modifications that change conformation and biological activity.
For the characterization of the glycan component of a glycoprotein then additional sample preparation is required to obtain the free glycans. The sample preparation method is dictated by the type of sample and the kind of separation required, based on the detector, instrument, and other factors. Automated biologic sample preparation enables quantitation and sample preparation in the same workflow, reduces errors and increases walkaway time with reliable, automated processes. Agilent and ProZyme have introduced an automated collaborative solution for automated N-glycan sample preparation. Agilent’s AssayMAP Sample Preparation enables the automation of complex sample preparation workflows with the power of precision flow control.
The glycan mapping workflow
Missing information in the early stages of the development process can cause major setbacks downstream. Production process inconsistencies can lead to changes in glycosylation which can negatively impact immunogenicity and efficacy. Reference standards should be used throughout the glycan mapping workflow that critical data has been captured and that every component throughout the workflow is working correctly to achieve optimum results. The Agilent Dextran ladder standard (labeled and unlabeled) facilitates data analysis and reporting and the Agilent Ig N-linked glycan standard (labeled and unlabeled) confirms workflow efficiency. Find out how you can optimize the glycan mapping workflow and use standards to validate the workflow by listening to our on-demand webinar “Glycan mapping – does it have to be complex and time consuming.”
Achieving high-speed, high-resolution glycan mapping
To help researchers overcome the challenges associated with glycan analysis, Agilent has introduced its AdvanceBio Glycan Mapping columns which have been engineered and manufactured to deliver rapid, reproducible, high-resolution glycan identification to increase throughput without sacrificing the quality of data. These innovative columns have a unique bonding which uses a HILIC mechanism to support glycan separations to achieve high speed performance. Glycan maps can now be obtained in less than ten minutes with 1.8µm particles which is a significant benefit when analyzing and characterizing a large volume of samples during process development.
High resolution separations enable the characterization of both labeled and unlabeled glycans and identify any glycosylation changes that occur during process variables. The AdvanceBio Glycan Mapping columns are also available with a 2.7µm superficially porous particles which allow users to achieve high resolution and efficiency with less backpressure and for UHPLC analysis where speed and data are paramount the columns with fully porous 1.8µm particles are suitable. To find out more about the benefits of using AdvanceBio Glycan Mapping columns download the flyer and application note outlining how fast and efficient glycan analysis can be achieved. Discover how you can take the puzzle out of protein characterization at www.agilent.com/chem/AdvanceBio
For Research Use Only. Not for use in diagnostic procedures.
Agilent AdvanceBio columns are designed to enhance the accuracy and speed of your biomolecule characterization, including monoclonal antibodies (mAbs), other proteins, peptides and synthetic oligonucleotides. Learn more