bioZen WidePore C4

In this application note, Phenomenex discusses the use of cysteine proteases is an increasingly useful sample preparation technique to gain insight on mAb heterogeneity in conjunction with a wide pore core-shell LC column, bioZen™ 2.6 µm WidePore C4.

In this application note, Phenomenex discusses the analysis of antibody fragments can provide key insights into sample heterogeneity. This application note highlights how the bioZen™ 2.6 µm WidePore C4 LC column can be used to separate variants such as lysine and deamidated variants by digestion using a site-specific cysteine protease.

Monoclonal antibodies (mAbs) are well-established therapeutics, with a variety of different analytical methods associated with purity analysis. An analytical technique common for mAb purity is reversed phase LC (RPLC). This is a primary technique utilized for mAbs because of its relatively short analysis time, with high resolution, and its ability to separate hydrophobic variants such as oxidation, glycoforms, and lysine variants. As such, intact reversed phase methods can be implemented during lead selection and optimization, and on to downstream, including stability-indicating methods and quality control lot release. One particularly appealing aspect of analysis by intact reversed phase, is the capability of implementing a “platform” method.

In this application note, Phenomenex discusses how the analysis of antibody fragments can provide key insights into sample heterogeneity. By using the cysteine protease IgdE, further characterization of the Fab region can be obtained.

A primary analytical technique for purity for recombinant proteins is reversed phase LC (RPLC). This is a simple, robust methodology with a relatively short analysis time, with high resolution, its ability to separate hydrophobic variants such as oxidation, glycoforms, and clipping. As such, RPLC is a common analytical approach for monoclonal antibodies (mAbs), especially considering it can be implemented at both intact and subunit levels without extensive method optimization when combined with a bioZen™ 2.6 µm WidePore C4 LC column.

The analysis of intact monoclonal antibodies by reversed phase HPLC is a common technique for assessing protein variation, such as clipping and heterogeneity due to post-translational modifications. In this technical note, Phenomenex demonstrates how utilizing a bioZen™ 2.6 µm WidePore C4 LC column allows for better resolution and reproducibility through a wider pore core shell particle combined with bioinert hardware. In order to optimize the method, the gradient profile is analyzed.

In this technical note, Phenomenex demonstrates using bioZen™ 2.6 µm WidePore C4 LC Columns how increases in temperature can lead to improvements in peak shape in large molecule reversed phase LC methods.

In this technical note, Phenomenex analyzes how changes in the flow rate effect the overall analysis when using a bioZen WidePore C4 column. It is shown that when choosing a flow rate, it should be selected for good peak shapes and to increase the throughput of the application.

In this application note, temperature, gradient program, and flow rates are explored and optimized for intact mass applications for mAbs when using bioZen WidePore C4 a core-shell wide pore column with bioTi hardware.