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Protein Glycosylation

Protein Glycosylation

Be Agilent Sure When Characterizing Intact Glycoforms and Released Glycans

Monoclonal antibody (mAb) based entities represent a rapidly growing class of biologics that require extensive characterization to obtain approval for clinical trials and subsequent market release. N-linked glycosylation of these molecules is a complex post-translational modification that plays important roles in many biological processes, and affects the therapeutics’ efficacy, stability, pharmacokinetics, and immunogenicity. Glycans are a challenging post-translational modification to characterize given the diversity in their composition, and the presence of many isomeric structures.

Glycans can be analyzed as intact glycoforms and as released glycans. Monitoring the glycoforms at the intact protein level provides fast results and allows efficient screening for major glycan content. This method provides rapid answers to glycosylation profile and is particularly useful when O-link glycosylation is present on the glycoprotein. The glycosylation pattern can also be profiled after the antibodies are reduced to light chain and heavy chain or digested with IdeS enzyme to produce antibody subunits. Subunit glycoform analysis provides higher resolution to the glycan profiles and other post translational modifications.

N-linked glycans can be released from glycoproteins by PNGase F. To enhance detection sensitivity, the released glycans are labeled with fluorescent or mass spectrometry sensitive tags and separated by HPLC or CE, and then detected by s fluorescence detector or mass spectrometer. This approach gives superior quantitation and allows detailed analysis by mass spectrometry for glycan structure identification.

Agilent provides techniques and technologies to enable you to streamline your glycan analysis without compromising data quality. Automation on the Agilent AssayMAP Bravo can reduce the probability of human error, increase reproducibility, and create more walk-away time. The 1290 Infinity II LC used with AdvanceBio Columns deliver fast, high resolution, reproducible glycan separation. The Agilent AdvanceBio 6545XT LC/Q-TOF was designed for biopharma and features large molecule or fragile compound autotune, allowing you to unlock the full potential of the system by flexibly optimizing for large proteins or small labile structures such as glycans. MassHunter BioConfirm software provides a Released Glycans workflow that allows for easy setup of the analysis and accommodates many commercial and custom tags.

Agilent offers a complete portfolio of samples to answers solutions to allow you to characterize released glycoforms and intact glycans efficiently and with confidence.

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For Research Use Only. Not for use in diagnostic procedures.