The Use of Light-Scattering Detection with SEC and HPLC for Protein and Antibody Studies, Part I: Background, Theory, and Potential Uses - - Chromatography Online
The Use of Light-Scattering Detection with SEC and HPLC for Protein and Antibody Studies, Part I: Background, Theory, and Potential Uses


LCGC North America
Volume 30, Issue 9, pp. 842-849

Current Uses of Light Scattering in the Biotechnology Industry

The use of SEC–MALS and similar techniques is still growing within the biotechnology industry. The most common uses are outlined below.

Determining Absolute Molecular Weight

In general, today most applications of SEC–LS involve the determination of absolute molecular weights, as described above, plus either R h or R g. By absolute, we mean without reference to standards whose molecular weights are often known and used to calibrate the instrumentation. Nor does absolute refer to any technique that makes use of calibration curves such as SEC or intrinsic viscosity based on so-called "universal calibration."

That is, light scattering can be used to determine absolute molecular weights 2–3% (standard deviation) or better for individual proteins after HPLC or SEC separations, and for covalent (oligomers) or noncovalent aggregates of such proteins with one another or with their antibodies. As long as the conditions being used in HPLC or SEC do not change the nature of the protein samples through the chromatographic process, then light-scattering analysis will provide a true picture of what was in the injected sample in terms of monomer, dimer, or higher-order aggregates of the parent protein or antibody.

Of the four absolute methods for measuring molecular weights, light scattering is applicable to the greatest range of samples. The other three methods capable of absolute molecular weight determination are sedimentation equilibrium, vapor osmometry, and MS (although the latter method is not as useful if the "solution" properties of the molecules, such as noncovalent aggregates, are being measured). For light scattering, the range of accessible molecular weights is from several hundred to tens of millions of daltons (grams per mole). Vapor osmometry measurements span a much smaller range, up to only a few hundred thousand daltons under the most favorable of conditions. The lower limit, however, can be an order of magnitude smaller. Sedimentation equilibrium, also called analytical ultracentrifugation, often requires several days of measurement and has a range of applicability considerably smaller than light scattering. MS has an upper limit between 500,000 and 1,000,000 Da, although today, it can be considerably higher, in the tens of millions, with special instrumentation.

Light-scattering techniques also have another interesting advantage when combined with fractionation techniques such as SEC: They can be used to determine the molecular weight distribution and various other molecular properties, as we shall see in the following sections and in part II.

Studying PEGylated Proteins

PEGylation (adding a molecule of polyethylene glycol [PEG]) is a widely used technique for modifying protein drugs to make them more water soluble and provide different pharmacokinetic properties. HPLC–LS or SEC–LS can be used to determine the nature of PEGylated proteins, or of proteins that have been PEGylated and perhaps then modified with another, synthetic organic polymeric reagent (17,18). It is now possible to determine the molecular weight ratio of protein to PEG in a PEGylated protein. This is also true for virtually any polymer used to modify a protein by covalent or even noncovalent steps. Electron-transfer dissociation (ETD) in MS has also proven useful for identifying the specific site and nature of PEGylation in certain peptides (19). However, this technique does not work when trying to determine the molar ratio of protein to PEG for a mixture of PEGylated proteins.


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