5. Size exclusion chromatography and its orthogonal and complimentary modes for detection of heterogeneity

Two chromatographic modes are considered orthogonal techniques if the selectivity of the two modes are significantly different. Under ideal conditions without any secondary interaction, SEC should yield a characteristic Gaussian-shaped peak without the presence of any heterogeneity.

Figure 28. Analysis of papain digested IgG1 fragments. (A) Chromatogram of papain digested IgG1 fragments separated on a TSKgel SuperSW3000 column. (B) Individual peaks F (ab) 2 and Fab + Fc from the SEC separation (panel A) were applied to a reversed-phase chromatographic (RPC) TSKgel protein C4–300 column.

As shown in the figure below when individual peaks F (ab) 2 and Fab + Fc from SEC separation (panel A) were applied to the reversed-phase chromatographic (RPC) column, a number of hydrophobic variants eluted (panel B) in the increasing order of their hydrophobicity (Figure 28). Mechanism of papain digestion is discussed in Section 2.6. Though papain digestion yields primarily the Fab fragments, F(ab')2 fragment can be generated if the papain is first activated with 10 mM cysteine. Following the completion of the reaction, the excess needs to be removed by gel filtration. Size exclusion chromatography cannot differentiate these heterogenic impurities or hydrophobic variants, which are not sufficiently different in the size or hydrodynamic radii from each other. Similarly, a number of other chromatographic modes, other than RPC, can also be used as an orthogonal technique. The extent of the heterogeneity present in the SEC peak can only be confirmed by an orthogonal analysis.

Similarly, a reversed-phase chromatography column can also be used as a complimentary chromatography column along with SEC as shown below (Figure 29). The elution order of elution of the peaks is simply reversed as expected.

The PEG-conjugated species were more strongly retained by RPC, than the different forms of intact lysozyme. The order of elution in RPC is opposite to the order of size-based separation in SEC [35].

5. Size exclusion chromatography and its orthogonal and complimentary

Table 4. Analysis of retention time, peak area, peak height, as (peak asymmetry) and N (theoretical plates) of the monomers. The average (Avg), standard deviation (Std) and relative standard deviation (RSD) are also shown.

Two chromatographic modes are considered orthogonal techniques if the selectivity of the two modes are significantly different. Under ideal conditions without any secondary interaction, SEC should yield a characteristic Gaussian-shaped peak without the presence of any heterogeneity.

Figure 28. Analysis of papain digested IgG1 fragments. (A) Chromatogram of papain digested IgG1 fragments separated on a TSKgel SuperSW3000 column. (B) Individual peaks F (ab) 2 and Fab + Fc from the SEC separation (panel A) were

applied to a reversed-phase chromatographic (RPC) TSKgel protein C4–300 column.

modes for detection of heterogeneity

162 Antibody Engineering

Figure 29. Separation of PEG (MW 5000)-lysozyme and PEG (MW 30,000)-lysozyme on a SEC TSKgel SuperSW3000 column (A) followed by chromatography of the SEC fractions 1–4 using a reverse phase TSKgel protein C4–300 column (B).
