Multidimensional LC Approaches for Intact Protein Biopharmaceutical Characterization

Multidimensional LC can automate sample preparation and provide a convenient way to analyze a sample with complementary columns.
Sep 01, 2012
By LCGC Editors

This installment provides a brief overview of some multidimensional approaches targeted at intact protein analysis for life science applications, particularly for biopharmaceutical analysis.

Since the 1944 publication of a paper by Consden, Gordona, and Martin (1) describing multidimensional separations, the prospect of using this technique to address complex analytical challenges has sustained the interest of the analytical chemistry community. Analysts often use multidimensional techniques when they require higher separation capacity than single-dimension analyses can afford. By using dimensions of various selectivity for complex analytes such as food, polymers, and hydrocarbons, maximum resolution can be achieved among components that are generally difficult to separate using a single-dimension technique.

The multidimensional approach for separations has been — and continues to be — highly utilized. Not surprisingly, its use as an analytical technique has been expanded. It now includes various applications that do not necessarily result in greater resolution. In those applications, the technique serves as the means for conducting separate analyses of the same analyte without the need for multiple instruments. It can also reduce steps for sample preparation and provide a convenient way to perform automated assays.

In the past few years, an emerging area of focus in the pharmaceutical industry is the biopharmaceuticals. The compounds in this class are very complex and require a high degree of characterization. Indeed, the level of characterization necessary is increasing, largely because of improved analytical technologies and regulatory requirements. Also at play is the emergence of biosimilar approval pathways, in which protecting intellectual property for innovators or proving similarity for biosimilar producers is increasingly important (2).

Developing and manufacturing biopharmaceuticals requires amassing a variety of chromatographic and mass spectral data. The different chromatographic modes probe particular characteristics of the analyte. Some of these characteristics include size, charge, and mass profile. As such, they are related to, or are themselves, critical quality attributes.

For some biopharmaceutical applications, a multidimensional separation can reduce a practitioner's analytical burden, yielding greater assay reproducibility by dispensing with hands-on sample preparation. Note, however, that although adding chromatographic dimensions can prove useful, to do so without realizing additional benefit, such as increased resolution, sensitivity, or automation, introduces unnecessary complexity.

This column installment provides a brief overview of some multidimensional approaches targeted at intact protein analysis for life science applications, particularly for biopharmaceutical analysis. For the applications described here, adding a liquid chromatography (LC) dimension automates sample preparation and provides a convenient means of analyzing an analyte by different methods that require different chromatographic eluents. As such, these applications are meant for targeted analyses where sample complexity does not demand adding a dimension. Rather, the criterion for needing a multidimensional separation is the primary chromatographic method's incompatibility with the desired detection method. For example, because of high mobile-phase salt concentrations, ion-exchange chromatography (IEC) is incompatible with mass spectrometry (MS) detection directly. Capturing a peak of interest to a second-dimension reversed-phase column, however, allows for desalting the analyte and collecting MS data.