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inert LC system with auto injector (HiP-ALS), binary pump, thermostatted oven (TCC) and diode array detector (DAD)
Columns Protein columns
The following columns were used for the analysis of the protein samples;
• Agilent Poroshell 300SB-C3, 2.1 x 75 mm, 5 µm (p/n 660750-909)
• Agilent Poroshell 300SB-C18, 2.1 x 75 mm, 5 µm (p/n 660750-902)
Peptide columns
The following columns were used for the analysis of the peptides samples;
• Agilent AdvanceBio Peptide Mapping Column, 2.1 x 150 mm, 2.7 mm (p/n 653750-902)
• Agilent AdvanceBio Peptide Mapping Column, 2.1 x 250 mm, 2.7 mm (p/n 651750-902)
• Agilent AdvanceBio Peptide Mapping Column, 3.0 x 150 mm, 2.7 mm (p/n 653950-302)
Optimising Methods for Improved Speed and Resolution for Intact Protein Separations
During the development of a protein biopharmaceutical, the need for rapid profiling methods are growing as throughput demands increase. A forced degradation study is one means to predict / evaluate changes in protein heterogeneity during monitoring and formulation. The advantage of the low back pressure experienced when using a superficially porous material is shown in Figure 2. The top chromatogram in Figure 2 shows a separation performed on a 3.6 mm superficially porous material. The low back pressure and high peak capacity associated with the superficially porous material allow for a substantial reduction in the analysis time [4,5], which is not always achievable with a fully porous material. The rapid separation of the degraded Insulin (Figure 2, lower chromatogram) was generated 10 times faster than a traditional separation (top chromatogram). This was achieved by using a shorter column, increasing the flow and reducing the gradient time. The fast separation maintained peak shapes of the degradant products which was achieved without the need to increase the backpressure above 300 bar, which would not be possible if a
Figure 2: An example of rapid HPLC profiling of degraded insulin using Agilent Poroshell 300SB-C18, 2.1 x 75 mm, 5 µm, column in comparison to an alternative superficially porous column, C18 phase, 2.1 x 150mm, 3.6 µm using the same instrumentation, Agilent 1260 Infinity Bio-Inert LC System.
Figure 3: Eight intact proteins resolved in under five minutes using Agilent Poroshell 300SB-C18
Figure 4: Reversed-phase separation of IgG1 after papain digestion showing two primary peaks of the Fc and Fab fragments. The inset details partially resolved peaks representing variants of the Fc and Fab fragments (arrows)
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