6 February / March 2017


variants, together with mass spectrometric characterisation techniques should greatly enhance biotherapeutic advancements going forward.


Acknowledgements


The authors would like to thank Mark R. Schure and Richard A. Henry for valuable suggestions and feedback and Conner McHale for chromatographic measurements.


This work was supported in part by National Institute of General Medical Sciences, [GM116224 to BEB]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Health.


References


Figure 7. Intact trastuzumab separation using 1000Å SPPs and 1500Å FPPs. Conditions: Columns: 2.1 x 100 mm; Mobile phase A: water/0.1% TFA; Mobile phase B: 80/20 ACN/water/0.085% TFA; Gradient: 40-47.5% B in 8 min; Flow rate: 0.4 mL/min; Temperature: 80°C: Sample: trastuzumab; Injection volume: 2 µL of 2 mg/mL; Instrument: Shimadzu Nexera; Detection: 280 nm with 350 nm reference wavelength


These data coupled with the load data shown in Figure 5 indicates that there is greater access for large molecules to enable uninhibited surface interaction with the 1000 Å column, compared to the 300 Å column. All of the analytical data suggests that the pore size and distribution play a significant role in the separation processes.


The 1000 Å SPP column was also compared to another large pore column. The separation of trastuzumab shown in Figure 7 compares the 1000 Å SPP column to a 1500 Å fully porous polymer particle column. This separation also demonstrates the increased retention and improved resolution of minor variants for the 1000 Å SPP column. In this case, the peak width of the major peak is 24% smaller on the 1000 Å SPP column. The peak may be sharper due to the combination of smaller particle size and thin


shell of the 1000 Å SPP column (2.7 µm with 0.5 µm shell) compared to the 4 µm particle size of the 1500 Å fully porous polymer particles. Enhanced resolution of the minor variants is again observed with the 1000 Å SPP column.


Conclusions


The large 1000 Å pore size of the SPPs used for RP-HPLC described herein enables full access to the surface for larger molecules such as intact mAbs. This improved access to the surface results in enhanced resolution of minor mAb variants and narrower peak widths and can lead to increased retention. As pharmaceutical companies develop more biotherapeutics, the need to completely characterise those molecules will continue to grow. The ability to separate intact monoclonal antibodies and their


1. Image from the RCSB PDB (HYPERLINK “http:// www.rcsb.org” www.rcsb.org) of PDB ID 1HZH E.O. Saphire, P.W. Parren, R. Pantophlet, M.B. Zwick, G.M. Morris, P.M. Rudd, R.A. Dwek, R.L. Stanfield, D.R. Burton, I.A. Wilson (2001) Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design Science 293: 1155-1159.


2. B.M. Wagner, S.A. Schuster, B.E. Boyes, J.J. Kirkland, J. Chromatogr. A 1264 (2012) 22.


3. S.A. Schuster, B.M. Wagner, B.E. Boyes, J.J. Kirkland, J. Chromatogr. A 1315 (2013) 118.


4. S. Fekete, J.-L. Veuthey, D. Guillarme, J. Pharm. Biomed. Anal. 69 (2012) 9.


5. R.A. Henry, S.A. Schuster, LCGC North America submitted.


6. R. Hayes, A. Ahmed, T. Edge, H. Zhang, J. Chromatogr. A 1357 (2014) 36.


7. J.J. Kirkland, S.A. Schuster, W.L. Johnson, B.E. Boyes, J. Pharm. Anal. 3 (2013) 303.


8. J.J. DeStefano, T.J. Langlois, J.J. Kirkland, J. Chromatogr. Sci. 46 (2008) 254.


9. B.M. Wagner, S.A. Schuster, B.E. Boyes, T.J. Shields, W.L. Miles, M.J. Haynes, R.E. Moran, J.J. Kirkland, M.R. Schure, J. Chromatogr. A (2017) accepted.


10. J. Wypych, M. Li, A. Guo, Z. Zhang, T. Martinez, M.J. Allen, S. Fodor, D.N. Kelner, G.C. Flynn, Y.D. Liu, J. Biol. Chem. 283 (2008) 16194.


11. Y. Zhang, R. Bailey, N. Nightlinger, A. Gillespie, A. Balland, R. Rogers, J. Chromatogr. B 997 (2015) 30.


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