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Deamidation

The real benefit of peptide mapping by RPLC is the ability to not only identify the amount and type of PTMs present, but to also assign them to specific peptides. When RPLC is operated in conjunction with mass spectrometry (LC-MS), modifications can be isolated to specific amino acid residues in the peptide in question. In Figure 1 we revert back to the previously described trastuzumab peptide map following trypsin digestion, but this time PTMs specific to this product have been highlighted. These PTMs are common across most IgG isoforms and include deamidation (N → D), oxidation (M → Mox) and lysine truncation (+K).

Herceptin peptide map with specific PTMs

Figure 1: Herceptin peptide map with specific PTMs highlighted. 250 x 2.1 mm, 2.7 µm C18, mobile phase A: 0.05% TFA, mobile phase B: 0.05% TFA in acetonitrile, flow rate 300 µL/min, UV 214 nm, gradient 1-45 %B 2-35 minutes, 60 °C


Deamidation is probably the most commonly encountered PTM for protein based biopharmaceuticals. Whilst asparagine and glutamine residues can both undergo deamidation, it is far more common to see modification of the former. There are multiple pathways for asparagine deamidation to occur; by direct hydrolysis, as for peptide T3, or via a succinimide intermediate, as is the case for peptide T26. The latter produces both iso-aspartate and aspartate at a relative ratio of 3/4 : 1  (Figure 2).

Asparagine deamidation via a succinimide intermediate

Figure 2: Asparagine deamidation via a succinimide intermediate.


The higher structure, and therefore efficacy of a mAb can be dramatically affected when deamidation occurs via the succinimide intermediate and a substantial amount of isoaspartate is formed. As previously mentioned, deamidation can be an artefact of sample preparation; this can be investigated by performing all sample preparation in deuterated solvents, this allows native deamidations and the sample preparation induced occurrences to be distinguished.

Trastuzumab peptide T26 deamidates via a succinimide intermediate, forming both isoaspartate and aspartate at an approximate ratio of 3/4 : 1. The more polar isomeric version exhibits itself as a pre-peak, relative to the non-deamidated precursor, and the more hydrophobic aspartate variant as a post-peak in the RPLC peptide map. In the example below, a pH stressed trastuzumab sample, where PTMs such as deamidation are promoted, is shown with the deamidation of peptide T3 exploded.

Deamidation of peptide T26

Figure 3: Deamidation of peptide T26 under normal and pH stressed conditions.

 

When we examine peptide T3 of the same samples we only see the production of aspartate and no isomerization.

Deamidation of peptide T3

Figure 4: Deamidation of peptide T3 under normal and pH stressed conditions.

 
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