4 February / March 2017
Experimental General
Chemicals: All solvents were HPLC grade. Acetonitrile (ACN) was from EMD (Gibbstown, NJ). Trifluoroacetic acid (TFA) was from Pierce Chemicals (Rockford, IL). Difluoroacetic acid (DFA) was purchased from SynQuest Laboratories (Alachua, FL).
Figure 2A. SEM image of a 2.7 µm superficially porous 1000Å particle. 2B. FIB image of a 1000Å SPP showing the 1.7 µm core with 0.5 µm shell.
Equipment: A Zeiss (Jena, Germany) Auriga 60 High Resolution Focused Ion Beam & Scanning Electron Microscope was used to image the 1000 Å particles at the University of Delaware (Newark, DE). A Micromeritics Tristar II (Norcross, GA) was used to collect pore size distributions through nitrogen adsorption. A Multisizer 4e Coulter Counter (Indianapolis, IN) was used to measure particle size. A Shimadzu Nexera liquid chromatography instrument (Tokyo, Japan) was used to collect all of the chromatographic measurements.
Detection parameters: Photodiode Array (PDA) detection was used at 280 nm with a reference wavelength of 350 nm for all of the results presented.
Sample preparation: All mAbs were commercially obtained and used as received. The mAbs were diluted in water to specified sample concentration listed in the Figure captions. Mobile phase composition is listed in the Figure captions.
Techniques Column liquid chromatography:
Figure 3. Particle size distributions of 1.7 µm 300Å FPPs and 2.7 µm 1000Å SPPs. The polydispersity is calculated as the ratio of the square root of the second moment to the first moment of the Coulter Counter data. These moments are obtained by curve fitting an exponentially modified Gaussian peak shape to the raw Coulter Counter data using a nonlinear least-squares program written in MATLAB.
Columns of 1000 Å SPPs with C4 bonded phase were obtained from Advanced Materials Technology, Inc. (Wilmington, DE). The Waters BEH 300 C4 column was obtained from Waters Corporation (Milford, MA). The MAbPac RP column was obtained from Thermo Fisher Scientific (Waltham, MA).
Results and Discussion 1000 Å Particle Characterisation
SEM images of the 1000 Å SPPs are shown in Figure 2. The individual particle shown in Figure 2A has uniform and spherical shape. The typical surface area of these particles is about 20 m2
/g. In Figure 2B, the particle has been Figure 4. Pore size distributions of 1.7 µm 300Å FPPs and 2.7 µm 1000Å SPPs.
cross-sectioned using a Focused Ion Beam (FIB) to reveal the 1.7 µm core and the 0.5 µm shell. The large 1000 Å pores are visible in the porous shell. The mean particle size of the 1000 Å SPPs measured by Coulter counter was found to be 2.5 µm. This is smaller than the size that was measured via SEM analysis. This difference in measured sizes may be due to the large pores which may disrupt the Coulter measurement
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