The Use of Light-Scattering Detection with SEC and HPLC for Protein and Antibody Studies, Part II: Examples and Comparison to Mass Spectrometry

Dec 01, 2012

Light scattering detection, particularly multiple-angle light scattering (MALS), can by very useful for the characterization of proteins and antibodies. In a number of applications, its performance exceeds that of mass spectrometry (MS) detection. In part I of this two-part article (1), we outlined the development of light-scattering detection in biotechnology, summarized its current uses and advantages, and explained the theory of how it works. Here in part II, we provide some detailed examples of its use and discuss how its performance compares to MS and other methods. As we will see in the examples, light-scattering analysis particularly excels at studying the bulk properties of proteins and antibodies in highly complex solutions.

Where Mass Spectrometry Falls Short, Light Scattering Steps In

When addressing the characterization of biopharmaceuticals or other complex biological samples, it is impossible to ignore analytical methods involving the combination of liquid chromatography and mass spectrometry (LC–MS) (2). These techniques range from high performance liquid chromatography (HPLC)–MS, where a relatively simple mixture is separated into several components and characterized by the intact masses and relative abundances (or even absolute concentrations, with the use of reference standards), to multidimensional LC techniques paired with high-resolution MS systems, which separate complex biological mixtures into thousands of peaks and perform in-depth analysis of individual species with multiple rounds of fragmentation. The information that is gained by in-depth LC–MS-MS analysis includes identification of post-translational modifications (PTM), oligosaccharide structure, and sequencing of peptides or even proteins with masses as large as 30 kDa.

In essence, LC–MS techniques are strongest for determining specific details about individual species. This ability is important for characterizing a substance with a limited number of variants, such as a small protein drug product. However, LC–MS falls short in quantitating the bulk properties of extremely complex substances, including PEGylated protein drug products (where PEG is polyethylene glycol), biologically derived substances such as low-molecular-weight heparin (LMWH), and inherently complex pharmaceutical substances such as copaxone. Light-scattering techniques pick up some slack in these areas.

Two additional challenges that cannot currently be addressed by LC–MS techniques are the determination of the tertiary or quaternary structure of a protein, and the study of protein–protein interactions (including aggregation). This information is irreversibly lost when proteins are ionized and vaporized by electrospray ionization or matrix-assisted laser desorption ionization (MALDI) for MS detection. Light-scattering techniques have the advantage of analyzing the protein in solution where the native conformations and protein–protein interactions are intact. In particular, size-exclusion chromatography (SEC)–MALS is especially valuable for very complex mixtures of proteins, antibodies, and aggregates (noncovalent or covalent), as well as highly derivatized proteins or antibodies as drug conjugates or PEGylated species.

Some Practical Biotechnology Applications of Light-Scattering Techniques

Below, we present several specific applications where light-scattering techniques adequately solve biotechnology challenges that are not currently fathomable by any LC–MS technique. All three involve studying highly complex solutions. Because many current protein-based pharmaceutical products are relatively large and complex, the development of therapeutic formulations at the high concentrations required can lead to a variety of complex protein stability issues (3). Many of these issues involve the interaction of protein therapeutics in vitro, leading to various degrees of mass-action associated and non-mass-action associated aggregation states.