Originally Published January 2000
A static and dynamic laser light-scattering device can detect both molecular weight and size.
Because cell-culture production is a batch process, characterizing new diagnostic antibodies is important for consistency and quality control of the product. To ensure that it meets quality standards, an antibody's size and molecular weight must be accurately assessed. Antibody aggregation state and molecular weight distribution must also be determined. Antibodies can aggregate as a function of temperature, acidity, ionic strength, and concentration. Even small amounts of aggregates (dimer, trimer, etc.) can be significant because they can cause conformational shifts in the molecular structure. This can alter the function of the biomolecules as an effective therapeutic or diagnostic agent. Monitoring and understanding such effects is fundamental not only for meeting quality control requirements, but also for research and development.
Traditional analytical methods for determining molecular weight include size-exclusion chromatography (SEC), gel electrophoresis, Rayleigh light scattering, analytical ultracentrifugation, and, to some extent, time-of-flight mass spectrometry. These technologies are time-consuming (some take days or weeks), provide data based on relative standards, or cannot characterize very high molecular weight aggregates.
Figure 2. The PD2000/DLS detector combines static and dynamic light-scattering technologies in a single detector that can be coupled to any SEC/FLPC currently in the lab.
A new method has very high sensitivity and can operate both in flow mode with an HPLC/SEC system and in batch mode. Dynamic laser light-scattering (DLS) technology combined with static laser light-scattering (SLS) technology provides a tool to obtain valuable information for developing new bioprocesses.
When a polarized, monochromatic laser beam passes through a solvent containing proteins, the amount of light scattered by the protein or antibody molecules at an angle to the incident beam divided by the amount scattered by the solvent alone is directly proportional to the molecular weight (Mw) of the biomolecule times the concentration of the molecule. The equation that expresses this relationship is as follows:
This is called static light scattering and is used for determining the absolute molecular weight of eluting biomolecules.1,2
Proteins also undergo diffusion-related movements known as Brownian motion. The motion is related to the protein's hydrodynamic radius (Rh), according to the Stokes-Einstein equation.
As biomolecules or a distribution of biomolecules diffuse around the laser beam coherence area, light scattered from them overlaps and interferes with the transmission of the laser light. A high-sensitivity detector can then record and compare the time-varying signal caused by scattered light to the constant signal emitted when no molecules are present. This process is known as dynamic light scattering (DLS) or quasi-elastic light scattering and photon correlation spectroscopy, and is analogous to the Doppler shift of sound frequencies emitted from a moving source. Small particles (or biomolecules) diffuse quickly, causing rapid fluctuations of the scattered light. Larger particles (e.g., antibody aggregates etc.) diffuse slowly, resulting in fewer fluctuations in the intensity of the scattered light.
Figure 1. A combined static and dynamic light-scattering platform. Axial design and small (10 µl) flow-cell provide high-sensitivity single- and dual-angle (90° and 15°) laser light-scattering signals for measuring absolute molecular weight from less than 1000 daltons to more than 10 million daltons. Dynamic light-scattering capabilities determine molecular size (hydrodynamic radius) data from 1.0 to 1000 nm.
Interpreting these information-rich signals can be accomplished by using a 1024-channel correlator and proprietary PrecisionDeconvolve software (Precision Detectors Inc.; Franklin, MA). Using a 100-mW laser with a fiber optic—coupled high-speed photon-counting detector mounted at a 90° angle from the incident laser beam, the diffusion constant can be calculated as follows. The software statistically compares the intensity fluctuation-set measured over several microseconds with the next set and plots the decay over time. This autocorrelation function is then used to define the diffusion constant. From the diffusion constant, the molecules' hydrodynamic radius is then calculated by using the Stokes-Einstein equation. Key advances of the DLS technology are its very high sensitivity and unique ability to operate in both a flow mode with an HPLC/SEC system and in a batch mode.3 A schematic of this high-sensitivity static and DLS platform is seen in Figure 1.
Recent innovations in modern high-speed electronic components such as high-performance diode lasers, high-speed digital-signal processors and modern avalanche photodiode detectors have led to the development of a combined static and dynamic laser light-scattering detector. The PD2000/DLS has a 10-µl flow-cell design and can characterize both the molecular weight and size of biomolecules (see Figure 2). The detector and its associated software provide the following.
A cuvette-based batch DLS molecular size—characterization instrument (PDDLS/batch molecular size analysis system) has been developed to measure the hydrodynamic radius and distribution of antibodies and other nanoparticles ranging in size from 1.0 to 1000 nm.
High-Sensitivity Antibody Cell-Culture Supernatant Assay
DLS detection can be applied to most soluble biomolecules such as proteins, large polypeptides, polysaccharides, oligonucleotides, and antibodies with a molecular weight generally greater than 5 kD. The detector can characterize molecules having a hydrodynamic radius between 1.0 and 1000 nm. This range easily covers the molecular sizes and weights of most recombinant proteins and antibodies.
Typical applications of DLS involve characterizing aggregation and size variations of antibodies as they are affected by time, temperature, and acidity. Chromatographic purity of the eluting protein or antibody from an SEC column can be elucidated by monitoring the hydrodynamic radius and molecular weight of all the eluting fractions. Any perturbation in the hydrodynamic radius across the peak, as it elutes, reflects a co-eluting contaminant or conformational structure variation of the molecule. The following example describes the characterization of a hollow-fiber cell-culture supernatant containing an antibody from a proprietary Chinese hamster ovary (CHO) cell line.
The first set of data is from the flow-mode detector in conjunction with an SEC separation. The supernatant is characterized by using a refractive index (RI) detector and simultaneous static and DLS detectors coupled to an HPLC running in the SEC mode.
The SEC system comprises a Waters 600-MS and 712 WISP autoinjector (Waters Corp.; Milford, MA); YMC-Pack Diol-120 column,
The sample is diluted 1:1 in a PBS buffer. The antibody concentration in the supernatant is determined to be 1.3 mg/ml by radial immunodiffusion (RID) assay. The final concentration in the SEC assay is then 650 µg/ml. The injection volume is 100 µl, providing 65 µg injected into the column.
Figure 3. Molecular weight and size data for this cell-culture supernatant clearly reveal the presence of a higher-molecular-weight material and the antibody monomer. The molecular weight of the monomer region was determined to be 224 kD.
The high sensitivity of the combined static and DLS detector (with only 65 µg injected) reveals two major large molecules (see Figure 3). The molecular weight calculation of 224 kD for the second peak indicates that it is the monomeric antibody. The presence of an aggregation component (the first peak) is clearly visible with both the RI detector and the LLS detector. However, the static LLS detector reveals the presence of a very-high-molecular-weight material at extremely low concentration (< 1%). Both show a shoulder that is characteristic of the mAb aggregate. The DLS detector calculates the hydrodynamic radius of the antibody to be 8.0 nm (see Figure 4). The aggregate area (1255 kD Mw) is approximately 18% of the total mass, representing only 12 µg of material being recognized and analyzed by DLS. The hydrodynamic radius of the aggregate is 15 nm, as determined by the flow-mode DLS detector (see Figure 5). The aggregate area percentage of 18% is determined by the RI signal. To resolve higher-order aggregates another SEC column with a larger pore size would be needed.
Figure 4. The dynamic light-scattering data for the antibody peak indicate a Rh of ~ 8 nm. The descending Rh across the early eluting peaks indicates that multiple species are present at very large sizes.
Figure 5. The high-molecular-weight peak (RI and LLS) was determined to have a Mw of 1.2 million daltons. Higher-order aggregates are clearly seen by the 90° static light-scattering detector.
Figure 6. A known standard, bovine serum albumin, was run under the identical conditions as the cell-culture supernatant.
A known standard material is prepared in the same manner as an unknown. Figure 6 is a chromatogram of bovine serum albumin (2.5 mg/ml) injected under the same conditions as the cell culture supernatant. The column clearly resolved the monomer and dimer with the monomer molecular weight calculated at 66.5 kD. Also visible are higher-order aggregates (trimer and more) at extremely low concentrations.
Rh Distribution Assay by DLS
Using the cuvette-based system, the raw cell-culture supernatant was assayed for the hydrodynamic radius distributions in the sample (see Figure 7). The supernatant was centrifuged at 14,000 rpm for 5 minutes to remove any large particles or dust that might be present. DLS technology is very sensitive to large particles and can cause errors when analyzing in the batch mode unless this centrifugation takes place. After centrifugation, 100 µl of the sample is placed in the cuvette for analysis. The sample showed a bimodal distribution of nanoparticles consisting of materials at 7.36 nm and a broad distribution of aggregates centered at 78 nm (see Figures 8 and 9).
Figure 7. The PDDLS/batch molecular size analysis system measures Rh and Rh distributions in a cuvette-based unit. Only 100 µl of sample is needed. The instrument range is from 1.0 to 1000 nm.
Figure 8. The cell-culture supernatant was assayed by batch dynamic light scattering and resolved two particle size distributions at 7.36 and 78 nm. The 7.36-nm area is consistent with the Rh determined in flow mode.
Figure 9. The large aggregate area reveals a particle or molecular size distribution centered at 78 nm.
The distribution centered at 7.36 nm is consistent with the hydrodynamic radius determined by the PD2000/DLS measurement in flow-mode for the IgG monomer. The presence of this higher-order aggregate at 78 nm is again consistent with the very early eluting materials from the SEC column. In flow mode, the DLS signal indicated a consistent reduction of hydrodynamic radius as the first peak in the chromatogram eluted (see Figure 4). Also, in this study the use of a batch DLS instrument further supported the flow-mode DLS data.
A combination static and dynamic laser light-scattering detector can be added to any HPLC/SEC/FPLC instrument that performs chromatographic separations of biomolecules. This high-sensitivity detection coupled with on-the-fly determination of hydrodynamic radius provides new insights for the aggregation studies of macrobiomolecules such as antibodies and proteins. Both absolute molecular weight and size (Rh) can be determined simultaneously. Combining both static and dynamic light scattering into one cell provides a new tool to obtain valuable information for research groups developing new bioprocesses. In addition, quality control operations within established bioprocesses can better ensure the quality and consistency of their process for regulatory documentation. Both flow-mode and batch DLS instruments can be used in characterizing raw cell-culture supernatants for antibody content, molecular weight, and hydrodynamic radius, all of which can be used for research and process quality control purposes.
1. JP Helfrich, "Dynamic Laser Light Scattering Technology for the Molecular Weight and Hydrodynamic Radius Characterization of Proteins," Pharmaceutical Laboratory 1, no. 4 (1998): 34–40.
2. JP Helfrich, "Flow-Mode Dynamic Laser Light Scattering Technology for 21st Century Biomolecular Characterization," American Biotechnology Laboratory 16, no. 11 (1998): 64–66.
3. JP Helfrich et al., "Hydrodynamic Radius Characterization of Biomolecules and Nanoparticles," American Laboratory News 31, no. 5 (1999): 6–7.
John P. Helfrich is vice president of the life science group, and William R. Jones is director of applications development, at Precision Detectors Inc. (Franklin, MA).