5
large biomolecules to be able to more freely diffuse into and out of the particles to stagnant mobile phase and bulk mobile phase. Keeping in mind the suggestion that pore size should be at least ten times the diameter of the analyte in order to avoid restricted diffusion, the following calculation is presented. Ten times the value for the x-axis of the IgG in Figure 1 is about 1200 Å. The 1000 Å material has pores in this size range, and the advantages of these larger-diameter pores on reversed-phase separations of mAbs will be examined later in this paper.
Figure 5. Sample loading using trastuzumab. Conditions: Columns: 2.1 x 150 mm; Mobile phase A: water/0.1% DFA; Mobile phase B: ACN/0.1% DFA; Gradient: 27-37% B in 10 min; Flow rate: 0.5 mL/ min; Temperature: 80°C: Sample: trastuzumab; Injection volume: 0.1, 0.5, 1, 5, 10, or 20 µL of 7 mg/mL; Instrument: Shimadzu Nexera; Detection: 280 nm with 350 nm reference wavelength
Sample Loading
Sample loading of the 1000 Å SPPs has also been investigated. Results using trastuzumab are shown in Figure 5. The plotted points are the average of duplicate measurements. Sufficient loading capacity with this mAb on the 1000 Å column was observed until the peak width increased to about five times its initial level at around 70 µg on column. Notice that for all load levels the 1000 Å SPPs have smaller peak widths than those of the 300 Å FPPs. This result may be counterintuitive coupled with the knowledge that the absolute surface area of the 300 Å FPPs is about 90 m2 to about 20 m2
/g compared /g for the 1000 Å SPPs. Even
though the surface area of the 1000 Å SPPs is about four and half times smaller, there is not a significant difference in sample loading. This suggests that the trastuzumab sample has greater access to the surface/ pores of the 1000 Å SPPs.
Improved Resolution of Minor Variants and Increased Retention of MAbs
Figure 6. Intact trastuzumab separation using 1000Å SPPs and 300Å FPPs. Conditions: Columns: 2.1 x 150 mm; Mobile phase A: water/0.1% TFA; Mobile phase B: ACN/0.1% TFA; Gradient: 32-38% B in 12 min; Flow rate: 0.4 mL/min; Temperature: 80°C: Sample: trastuzumab; Injection volume: 2 µL of 0.5 mg/mL; Instrument: Shimadzu Nexera; Detection: 280 nm with 350 nm reference wavelength
technique. We have used the SEM size to refer to the 1000 Å particles throughout this paper, except when discussing the Coulter counter data. The particle size distribution shown in Figure 3 is narrow for the 1000 Å SPPs as has been previously reported for SPPs with smaller pore sizes [8]. For comparison, the particle size distribution of 1.7 µm 300 Å fully porous particles (FPPs) is included in Figure 3. The broad distribution of the FPPs can be quantified by the value of polydispersity which is more than three
times that of the 1000 Å SPPs.
The pore size distribution as measured by nitrogen adsorption is shown in Figure 4. The mode of the distribution is at 1000 Å for the SPPs. For comparative purposes, the pore size distribution of 300 Å FPPs is also shown in Figure 4. The shift in pore size distribution is apparent, with the 1000 Å material having a significantly larger population of greater than 400 Å pores. The larger pores of the SPPs allow for
The separation of intact (not digested or fragmented) trastuzumab using a column of 1000 Å SPPs is shown in Figure 6. Two distinct advantages are seen for the 1000 Å SPP column compared to the 300 Å FPP column: the peak width for the main trastuzumab peak is 43% smaller, and there is improved resolution of the minor variants that appear after the main peak. Another interesting observation is the increased retention of trastuzumab on the 1000 Å SPP column compared to the 300 Å FPP column despite the increased surface area for the 300 Å FPP column. Analysis of small molecule retention comparing these two columns demonstrated greater retention with the 300 Å FPP column [9], consistent with its greater surface area measurements.
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