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
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
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).