40
May/June 2013 fully porous material was being used.
Large molecules diffuse much more slowly than small molecules, which results in the typical optimal flow rate for protein separations to be lower than for smaller molecules [6,7]. The lower diffusion rates result in a greater radial equilibration effect, so called ‘c’ term. Therefore, a column with a shorter diffusion path for protein migration becomes much more desirable. Agilent Poroshell 300 columns offer this shorter diffusion distance due to their superficially porous particles, making it possible to operate steeper gradients at higher flow rates without extra band effects [8,9]. Figure 3 shows a separation achieved in under five minutes, at 2.5 mL flow rates, demonstrating this advantage.
Figure 5: LC/MS analysis of Fc Region – Glycosylation Profiling. A and B show averaged mass spectrum and deconvoluted spectrum of the IgG1 Fc fragment from the separation.
In biotherapeutic development, the number of samples submitted for product quality analyses, such as glycosylation profiling during cell line and clone selection and cell culture optimisation studies, can be enormous. At the same time, data turn-around time needs to be fast, due to precise process development timelines [9]. The chromatogram and expanded view inset detail in Figure 4 show the rapid and efficient reversed-phase separation of papain digested IgG1 Fab/Fc fragments using a 2.1 x 75 mm Agilent Poroshell 300SB-C3 column which helps to facilitate the fast data turnaround times necessary. The Fab and Fc chromatogram details the excellent resolving power needed for profiling the glycosylated variant peaks [10]. Figures 5A and 5B show the averaged mass spectrum and deconvoluted spectrum of the IgG1 Fc fragment from the separation in Figure 4. Partial digestion of the intact IgG1 into Fc & Fab fragments allow for a more efficient analysis for enhance glycoform profiling.
Faster Peptide Mapping Figure 6: Tryptic Digest rhEPO Protein 2.1 x 250mm AdvanceBio Peptide Mapping Column
A peptide map is a fingerprint of a protein and the end product of several processes that provide a comprehensive understanding of the protein being analysed. It involves four major steps: isolation and purification of the protein; selective cleavage of the peptide bonds; chromatographic separation of the peptides; and validated analysis of the peptides.
Often peptide maps using a 2.1 x 250 mm column can take two hours or longer to produce the necessary resolution. Additionally, re-equilibration and run-to-run cycle times can add extended analysis time, significantly affecting laboratory production. The conditions outlined here enable a fast analysis of a rhEPO
Figure 7: 100% sequence coverage using MassHunter Molecular Feature Extractor (MFE).
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