AL Results for large-molecule HPLC


Large-molecule RP-HPLC separations and loss of retention and efficiency in the hindered diffusion region are referred to as the exclusion effect. Serious performance problems usually result when solute size exceeds about one-tenth of the mean pore diameter.


The 160-Å C4 SPP column in Figure 4 is recommended for solutes up to about 15,000 D, so it would not be expected to perform well for a monoclonal antibody (150 kDa). The peak is too broad and too close to the exclusion limit of this 160-Å column. A calibration plot with a 200-Å SEC column would likely predict this result; this was confirmed by doing the same separation on a 400-Å C4 column. Note that peak width has become much narrower with the larger-pore column, which shows that pore crowding has been greatly reduced for the mAb, allowing better mass transfer and a much sharper peak. Although surface area is much smaller for the larger-pore column, retention is comparable, showing there is much more room in the larger pores for phase access.


Figure 5 shows a monoclonal antibody separation on a 1000-Å SPP C4 column and a 300-Å FPP C4 column. The larger-pore SPP particle exhibits greater retention and higher efficiency for the mAb. This is ex- pected for the 1000-Å particle, because the radius-ratio would be about 0.1 compared to only about 0.3 for the 300-Å particle, and peak broad- ening arises from slower mass transfer, described earlier by Giddings.


Figure 4 – Example of monoclonal antibody that is too large for a 160-Å-pore column, showing peak broadening corrected by changing to a 400-Å-pore column, which greatly improves efficiency and allows important peaks to be detected. Conditions—columns: 2.1 × 150 mm; mobile phase A: water/0.1% difluoroacetic acid; mobile phase B: ACN (acetonitrile)/0.1% difluoroacetic acid; gradient: 27–37% B in 20 min; flow rate: 0.4 mL/min; temperature: 80 °C; injection volume: 2 µL of 0.5 mg/mL Sigma MAb; instrument: Nexera; detection: PDA, 280 nm.


Although calibration curve results are not shown for 300-Å and 1000-Å SEC columns, mAbs should lie in the low-performance region for a 300-Å SEC column and in the high-performance region for a 1000-Å SEC column (see Figure 2).


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