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Typical Protein Level Conditions

The principles, and important parameters, are similar to those discussed at the peptide level with typical operating conditions
(Table 1).

Instrument:   UHPLC
Column:   100 mm L x 2.1 mm ID x < 2 μm FPP or < 4 μm SPP, ≥ 300 Å, C4 or C8
Mobile Phase A:   0.1% TFA
Mobile Phase B:   0.1% TFA in acetonitrile
Flow rate:   200 μL/min
Gradient:   0-2 min: 30% B, 2-40 min: 30 – 40%
Column temp.:   80 °C
UV:   214 and 280 nm

Table1: Typical protein analysis conditions.

The protein analysis method bears many similarities to that used for peptide mapping, however there are some differences in the critical and key parameters. These will be discussed in detail in the forthcoming section with specific examples given for optimizing methods for both intact, and protein fragments.

Once again, the precise nature of RPLC permits common PTMs to be predicted as eluting before (pre-peak), or following (post-peak), the principle peak. Please note that, as was the case with peptide mapping, when asparagine deamidation occurs via the succinimide intermediate both iso-aspartate (pre-peak) and aspartate (post-peak) are formed. It is also worth noting that fragmentation is near impossible to predict but as this typically involves a reduction in molecular weight, and therefore hydrophobicity, they are commonly observed as pre-peaks.

The common PTMs, alongside their expected retention time relative to the principal intact protein peak, are shown in Table 2.
The list is not exhaustive and peak identification should always be confirmed mass spectrometrically wherever possible.

Modification RPLC Protein
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
Aggregation -
Fragmentation Variable

Table 2: Common PTMs, with expected retention times relative to the principal intact protein peak.

Using the typical chromatographic conditions as detailed in Table 1, commercially available batches of Herceptin have been run at the intact level (Figure 1). This chromatogram is fairly clean, showing a principal peak with a small number of pre- and post-peaks resolved to varying degrees. The shortest alkyl chain stationary phases, typically C4, are employed for this type of analysis.

Herceptin chromatogram at the Fab and Fc level

Figure 1: Herceptin chromatogram at the intact level.

Following papain cleavage at the hinge region, the Fab and Fc regions of Herceptin are separated and a chromatogram with two predominant peaks will be acquired (Figure 2). Additional smaller peaks around the Fab and Fc peaks are also present.

These peaks will be representative of the different PTMs and glycoforms, as well as artefacts from the sample preparation step.

Care should always be exercised to identify which peaks are related to the true sample and which are sample preparation induced.  At this level the slightly longer C8 stationary phase is more commonly used.

Herceptin chromatogram at the Fab and Fc level

Figure 2: Herceptin chromatogram at the Fab and Fc level.

In the final chromatogram in this series, Herceptin has been analyzed following disulfide bridge reduction with dithiotreitol, generating peaks corresponding to the heavy and light chains (Figure 3).

As Herceptin is monospecific the two heavy and two light chains are identical and co-elute. Once again much more detail, such as the glycoprofiles around the heavy chain and some specific PTMs around the light chain, can be observed.

Herceptin chromatogram at the Hc and Lc level

Figure 3: Herceptin chromatogram at the Hc and Lc level.

The area around the Lc and the Hc is expanded in Figure 4. Although requiring accurate MS detection to identify the PTMs, the power of RPLC can be witnessed as the extent of disulfide bridge reduction can be detected. Where differing disulfide bridges have been reduced, full resolution can be observed and whilst not completely resolved, asparagine deamidation is present too. It should be noted that in this instance the specific asparagine deamidation has taken place via direct hydrolysis as only a post-peak is observed.

Detailed Herceptin chromatogram at the Hc and Lc level

Figure 4: Detailed Herceptin chromatogram at the Hc and Lc level.

Some of the commonly encountered challenges when analyzing at the intact protein and protein fragment level, and their associated solutions are shown in Table 3.

Issue Reason Solution
Peak tailing
  • Secondary ionic interactions
  • High number of positive charges on proteins
  • Stationary phase with limited access to residual silanols
  • Ion pairing reagent
  • Higher temperature
Peak broadening
  • Low diffusion of large molecules
  • Limited access to pores
  • Wide pore phases
  • Higher temperature
  • Efficient stationary phase (sub-2 μm superficially porous)
  • Hydrophobicity
  • Less hydrophobic stationary phases
  • Stronger solvent
  • High temperatures

Table 3: Challenges and their solutions when analyzing at the protein level.

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