Peptide Mapping by LC-MS: A Multi-Attribute Method for the Characterization of Therapeutic Proteins

23rd July 2018

Category: Bioanalytical

Tags: , ,

By: Dr Martin De Cecco - Senior Scientist,

A hot topic in the field of biopharmaceutical analysis is the concept of a multi-attribute method (MAM)¹, i.e. the characterization of several different product quality attributes with a single assay. The appeal to developers of biotherapeutics is clear: more information from a smaller number of tests. Such an approach is in keeping with the principle of Quality by Design (QbD) which requires a good understanding of the product and process from an early stage. This enables critical quality attributes (CQAs) to be monitored throughout the development lifecycle and beyond.

Several leading pharmaceutical companies have developed MAMs based on liquid chromatography-mass spectrometry (LC-MS) technology². Analysis of protein subunits by LC-MS can be employed to assess a variety of CQAs, including glycosylation, glycation, oxidation and N/C-terminal modifications. Such methods generally feature simple sample preparation and relatively short run times, enabling data to be obtained quickly to facilitate process-related decisions.

The mass difference of some modifications – especially deamidation (approximately +1 Da) – can be too small to distinguish on large protein subunits. Yet a change in mass of this magnitude is readily detected at the peptide level. Hence the attractiveness of MAMs based on peptide mapping by LC-MS, which can be used to characterize deamidation in addition to the CQAs listed above.  This methodology also has the advantage of being able to discriminate between modifications at different sites on the same protein subunit.


Method considerations for MAMs based on peptide mapping by LC-MS

Traditional sample preparation procedures for peptide mapping often employ overnight digestions at 37 °C and near-neutral pH. It is known that these conditions are stressful enough to induce changes in the protein³. Care should be taken to minimize modifications that occur as a result of the method in order to distinguish them from real differences in the samples. Reducing the pH and/or digestion time may be necessary to minimize method-induced deamidation.

BioOutsource | Peptide Mapping by LC MS Blog

Figure 1: Levels of deamidation at different sites in a mAb before/after stress at 37 oC for 1 week.

Using a MAM based on peptide mapping by LC-MS, we were able to identify and quantify various modifications in a monoclonal antibody that had been subjected to different forms of stress. Higher levels of deamidation were observed (Figure 1) after storage at elevated temperature. The same method also detected oxidation at a number of different positions following oxidative stress (data not shown).

Thus peptide mapping by LC-MS is a powerful technique that provides much more information than just sequence confirmation.  Its use as multi-attribute method allows several CQAs to be characterized within the same run. Furthermore, it enables the identification of new peaks that may not have been observed or considered previously.

¹ R. S. Rogers et al., mAbs (2015), 7 (5), 881-890.

² Review by S. Berger and J. Fredette, The Column (2017), 13 (9), 9-13.

³ L. A. Marzilli et al., Curr. Trends Mass Spectrom. (2017), 15 (1), 6-14.

Sign-up to our newsletter to be the first to hear about our new services & offers

Sign-up to our newsletter to be the first to hear about our new services & offers

Enter your email address to subscribe to our newsletter which provides information about our services. By subscribing, you are agreeing to the processing of your personal information for this purpose as set out in our privacy policy