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

Aug 31, 2012

Light-scattering detection, particularly combined with size-exclusion chromatography or high performance liquid chromatography, is very effective for the characterization of proteins and antibodies. Examples of applications include determining absolute molecular weight, studying PEGylated proteins, characterizing protein–drug and antibody–drug conjugates, and studying protein aggregation.

Light-scattering (LS) detection, particularly multiple-angle light scattering (MALS) can be very useful for the characterization of proteins and antibodies. In a number of applications, its performance exceeds that of mass spectrometry (MS) detection. Here in part I of this two-part column, we outline the development of light scattering in biotechnology, summarize its current uses and advantages, and explain the theory of how light scattering works. In part II, we provide a few detailed examples of its use and discuss how its performance compares to MS and other methods.

History of Light Scattering

In 1974, Ouano and Kaye ushered in a new era for macromolecular characterization with the first use of on-line light-scattering detection (1,2).

Despite the intrinsic appeal of this concept, however, many improvements in both the chromatography and detection were required for further implementation with relative ease. Important developments included the detection and quantitation of branching, precise measurement of the polydispersity of so-called "standards," conformation plots, precise extraction of M-H-S coefficients by combining light-scattering and viscosity measurements with reversed-phase chromatography to detect and quantify multimeric formation, field-flow fractionation (FFF) for an alternative separation combined with light scattering, temperature rising elution fractionation (TREF) measurements, capillary electrophoresis (CE) combined with light scattering, and the quantitation of aggregation phenomena and microgel formation.

Light Scattering in the Study of Biopolymers

In the mid-1980s there were few, if any, applications of chromatography combined with any form of light-scattering detection for the study of biopharmaceuticals described in the literature (3–6). This was true even for conventionally derived proteins from natural sources (that is, not recombinant proteins). Most detectors used in tandem with size-exclusion chromatography (SEC) or high performance liquid chromatography chromatography (HPLC) for biopolymers were ultraviolet (UV), differential refractive index (DRI), photodiode array (PDA), ultraviolet–visible (UV–vis), or fluorescent. Back then, MS was not yet conveniently interfaced with any form of SEC or HPLC. There was no general usage of electrospray ionization (ESI) or matrix-assisted laser desorption–ionization-time of flight (MALDI-TOF) MS developed or applied for biopolymers. Most vendors of light-scattering instrumentation had not yet developed instrumentation or applications aimed at the nascent, but rapidly growing biopharmaceutical industry. This situation would rapidly change.

Our own light-scattering work (Krull and colleagues) in those days was supported and encouraged by a firm known as Laboratory Data Control/Milton Roy (LDC/MR), in Florida, which no longer exists. This company was involved in both HPLC and light-scattering instrumentation, but it manufactured only a low-angle laser light scattering (LALLS) instrument and an off-line, differential refractometer for measuring the differential or incremental refractive index. Both instruments used a 633-nm laser. Of course, LALLS had been used for synthetic organic polymer characterizations but there very few, if any, applications in the literature for using LALLS or other light scattering, with biopolymers separated using HPLC or SEC (3–16) In our initial discussions with the management of LDC/MR, we proposed to study such new and novel applications, and to bring LALLS/DRI into a more common usage for biopharmaceutical proteins, antibodies, and related analytes. Herb Kenny and colleagues were influential in bringing about this collaboration, which lasted several years. Eventually LDC/MR was purchased by Thermo Electron Corporation, and the light-scattering product line was subsequently abandoned. Other vendors picked up the slack of course, and today there are at least two major companies offering such instrumentation with numerous applications for biopharmaceuticals: Wyatt Technology and Malvern Instruments. There are some other, smaller firms as well, but these do not offer the same extent of instrumentation diversity, in-house applications literature, or technical expertise.

In the late 1980s and early 1990s, Krull and others used SEC or HPLC with isocratic elution conditions interfaced on-line and in real-time with LALLS, using off-line DRI for dn/dc measurements of the protein analytes. Later, we moved to using either isocratic or gradient elution in ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC), or reversed-phase chromatography (3–16). We also began using on-line, 633-nm DRI with UV detection (concentration detector) to obtain at least three chromatograms (light scattering, UV, and DRI). Such approaches then provided direct light-scattering, dn/dc, and c (concentration) measurements on every injection of protein products and mixtures. With this approach, we could derive very accurate and precise measurements of molecular weights (weight-average, z-average, and n-average) but we could not yet calculate the radius of gyration (R g)or the hydrodynamic radius (R h). We could also obtain the second virial coefficient term (A 2) in the Zimm equation, suggesting the best solvents to use for nonaggregation or disaggregation prevention and overall stability or compatibility of the proteins with the HPLC solvents for their elution and separations. Eventually, we were able to use viscometry instrumentation, in an on-line format with the light-scattering, UV, and DRI detectors, and then derive R h in addition to the measurements previously described. These were studies in the late 1980s and early 1990s, using instruments that are by and large no longer commercially available. However, the techniques were soon readily adopted and adapted by various biotechnology firms for their own recombinant protein or antibody products, perhaps using other commercial instrumentation and methods, but especially SEC–MALS (17).