Biological drugs based on proteins and monoclonal antibodies are very complex molecules, and while the core of such molecules can be readily controlled using specific expression vectors, they are often modified after they are produced (post-translational modification) in the cell. While there are various different ways that cells modify these molecules post-translation one area that has become increasingly important to the development of new biological drugs is the addition of sugars (glycans) either by enzymes (glycosylation) or by carbohydrates (glycation). The latest research, published as an early view article in the journal Analytical Chemistry, by researchers from Amgen's Department of Analytical Sciences, used a combination of techniques that included affinity chromatography, high resolution mass spectrometry (MS) and a simple derivitisation procedure that simplifies the analysis of these modifications. The unwanted addition of glycans to proteins and monoclonal antibodies by carbohydrates during the biomanufacturing process can impair the quality of the product and its efficacy. Any variation can then impede a products approval by the regulatory authorities. Formulation variations can also affect the quality of a biologic drug as the hydrolysis of sucrose excipients can lead to glycan additions that affect the drugs half life. During the routine characterisation of an investigational MAb using MS techniques the Amgen researchers detected a minor species that hinted that an unwanted glycation event was occurring somewhere in the production process. Other methods such as peptide mapping had failed to detect the modification to the antibody and while the species could be detected using MS on the intact antibody, they were not easily observed in deconvoluted data due to its low abundance. The researchers force-glycosylated the antibody to increase the amount of the glycated antibody present in the samples. Interestingly, they found that even when intact mass MS analysis indicated that the major form of the MAb was the singly glycated protein, the reduced mass MS data showed indicated that the major form was the unmodified protein. They then used boronate affinity chromatography that adsorbed the glycated protein while enabling the unmodified proteins to be eluted to enable the ratio between the two forms to be quantified using a UV/vis spectrometer. "It may be necessary to show the specificity of the column for each new antibody or protein sample, as others have noted specificity issues when attempting to resolve serum and other proteins on this resin," write the authors. To determine the glycation site the researchers employed a derivitisation procedure that stabilised the molecule leading to improved mass spectral data from tandem MS analysis. This improved data led to increased confidence in the assignment of the glycation position. "This simple procedure addresses a data quality problem well documented in the literature and allows less ambiguous assignment of location of glycation on MS/MS data," write the authors "We show (in this paper) that a combination of methods is best for quantification and identification of glycation sites in our antibody and that no one method reveals a comprehensive analysis of the glycation by itself."