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Improved MAb Separations with 1000 Å Superficially Porous Particles
Stephanie A. Schuster*, Brian M. Wagner, Joseph J. DeStefano, Taylor J. Shields, William L. Miles, and Barry E. Boyes Advanced Materials Technology, Inc, 3521 Silverside Rd., Ste. 1-K, Quillen Bldg, Wilmington, DE 19810
*Corresponding Author: Stephanie A. Schuster, Advanced Materials Technology, Inc, 3521 Silverside Rd., Ste. 1-K, Quillen Bldg, Wilmington, DE, 19810 USA, 1-302-477-1526 (phone); 1-302-477-2514 (fax);
sschuster@advanced-materials-tech.com
The current state of the art in the analysis of monoclonal antibodies is to separate and characterise digested fragments of the antibody by reversed-phase HPLC. However, there are instances where intact analysis of the antibody is preferred. Separations of intact monoclonal antibodies using 1000 Å pore superficially porous particle columns are presented in comparison to previously reported HPLC column technologies. The advantage of the larger pores is evident in the narrow peak shapes, the resolution of minor variants, and increased retention.
Introduction
As more effort is spent on the development and characterisation of biopharmaceuticals, the analytical techniques used to characterise these molecules must provide more meaningful information and deeper levels of detail. Pharmaceutical companies are increasingly directing their research efforts toward monoclonal antibodies (mAbs), antibody-drug conjugates (ADCs), hybridised antigen binding structures, and biosimilars. These molecules are large in size and present additional analytical challenges over classic small-molecule drug therapeutics. Figure 1 shows the crystal structure of a human IgG antibody [1]. The x-axis is about 120 Å while the y-axis is about 158 Å. In order for these large biomolecules to efficiently interact with the internal surfaces of the analytical column particles, the particles must have sufficient pore size to accommodate the dimensions shown in the figure. Since proteins are dynamic structures, and exist with significant solvent association, it is reasonably assumed that the actual structures resolved by HPLC are at least as large as the structures observed in crystal structures. When biomolecules have free access to the surface within the particle pores, the peak shapes will be narrow and excellent resolution of minor variants will be obtained. The importance of matching particle pore size to analyte size has been previously described [2, 3]. It has been suggested [4, 5] that the pore size should be on the order of 10 times the diameter of the analyte for optimal chromatographic performance.
Figure 1. Crystal structure of human IgG [1] with the x and y lengths.
In addition to pore structure/size consideration, the use of superficially porous particles (SPPs) to improve the separations of biomolecules is advantageous because of the small diffusion coefficients of these large analytes. HPLC is inherently a method that operates away from kinetic equilibrium, but diffusional processes directly affect band broadening. The unique particle design of SPPs features a solid silica core surrounded by a thin, porous shell. The thin shell is ideal for the slowly diffusing biomolecules as the analyte does not have to traverse the entire
diameter of the particle. This reduction in diffusion path results in improved mass transfer, sharper peaks, and faster separations. For a review on superficially porous particles see Hayes, et al. [6], and for descriptions of the particles and advantages for larger protein separations, Kirkland, et al. [7]. This paper describes SPPs of a total particle size of 2.7 µm with a 0.5 µm shell containing 1000 Å pores and the improvements that can be achieved using these particles for reversed-phase HPLC separations of mAbs.
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