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Common mAb Post-Translational Modifications (PTMs)

Figure 1 highlights the characteristic ‘Y’ mAb representation of Herceptin and some of the common post translational modifications (PTMs). These include pyroglutamate formation at the N-terminal (E → pE), asparagine deamidation (N→ D), aspartate isomerization (D → isoD) and lysine truncation at the C-terminal (+K).

Herceptin

Figure 1: Common representation of Herceptin along with some common PTMs.

Although 62 is quite a large number of peaks to chromatographically resolve, it is not beyond the capability of RPLC with modern particle technology and instrumentation.

However, the hydrophobic similarity of a number of peptides, remember they are all built from the same 20 amino acid building blocks, mean that mass spectral resolution is required, by way of hyphenating a mass selective detector onto the liquid chromatograph; LC-MS.

Without this additional separation dimension, orthogonal modes of selectivity would be required to perform peptide mapping. Also, due to incomplete and missed cleavages, as well as sample prep related PTMs, significantly more peaks than the 62 predicted are produced, as seen in Figure 2 of a typical Herceptin peptide map.


Typical Herceptin Peptide Map

Figure 2: Typical Herceptin peptide map. 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

It should be noted that some peptides are unretained (T4, T12, T20, T36, T39, T48 and T49) – these are the most polar peptides and would require an alternative mode, such as HILIC (hydrophilic interaction liquid chromatography), for retention and separation. A number of PTMs associated with specific peptides are depicted in RED on the chromatogram and, as indicated, the peaks marked with asterisks are related to material from the digestion process.

Due to the precise and predictable nature of the hydrophobic retention of RPLC, estimates as to where the modified peptide will elute in relation to the native, unmodified variant can be made. This can be a helpful tool when trying to identify and assign unexpected peaks. Asparagine deamidation can produce both pre- and post-peaks due to deamidation occurring via the succinimide intermediate , iso-Asp (pre-peak) and Asp (post-peak) in a 3/4 : 1 ratio.

It is not always possible to predict the hydrophobic nature of the fragments, so this variability of retention cannot be predicted.


Modification RPLC Peptide
Aspartate isomerization Pre-peak
Asparagine deamidation* Post-peak + pre-peak
Oxidation Pre-peak
PyroGlu from Glu (-H2O) Post-peak
PyroGlu from Gln (-NH3) Post-peak
Succinimide Post-peak
Sugar Pre-peak
C-terminal Lysine Pre-peak
Fragmentation Variable

Table 1: Peptide PTM RPLC peak prediction relative to the unmodified parent peptide

 
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