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Biophysical analysis, the use of physical techniques to investigate and characterize biological systems, has become increasingly important in the characterization of biologic drug candidates. A large number of analyses can be performed to determine 3D protein structure, including secondary, tertiary, and even quaternary structures of protein molecules such as monoclonal antibodies, to determine that the molecule is in a stable state.

It is also possible to determine buffer conditions and degradation pathways, yielding valuable information on the impact of formulation on the stability of the protein molecule. The techniques applied in biophysical analysis can also provide information for comparability studies, which is especially important in the area of biosimilars.

Biophysical analysis includes both traditional techniques for determination of secondary or tertiary structures and methods for studying the aggregation properties of biologics. These methods include site exclusion chromatography (SEC), which complements other techniques such as analytical ultracentrifugation (AUC), a very powerful but complex and slow technique. Using an orthogonal approach, techniques such as SEC, SEC-MALS (SEC with combined with multi-angle light scattering), sedimentation velocity AUC (SV-AUC), dynamic light scattering (DLS), sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-Page) and field flow fractionation (FFF) can be used for the determination of a wide range of colloidal features of biomolecules. These techniques generate valuable data that can assess bioavailability of biologic drugs and are becoming industry standards.


Choice of Technique: Experimental Strategy

In bioanalysis, the aim is to optimize experimental strategy to achieve the intended purpose of the study. However, each biophysical analysis technique has its limitations and no single technique provides all the information required. AUC provides information on quantification of aggregates in the absence of a stationary phase, but only gives average values of molecular weight. Thus, it is important to carefully plan which techniques to use to optimize the outcome of the study program. Most biophysical analysis techniques are orthogonal, providing information on the same properties of the compound, but each has its limitations, and therefore it is essential to build an analytical landscape that demonstrates the overall behaviour of the molecule.

In general, it is usual to employ a single technique in the very early stages of biopharmaceutical discovery research when small amounts of material are used. As the development program progresses, it is more common to perform further tests using orthogonal methods.

Characterization of Protein Aggregation

The aggregation of biopharmaceuticals is a complex physical process and its analysis requires the use of a selection of suitable methods to build up a comprehensive dataset that would then support appropriate interpretation and scientifically sound conclusions.

The orthogonal methods discussed (SV-AUC, SEC, SEC-MALS, DLS, SDS-Page and FFF) provide ways of characterizing soluble aggregates but have a number of limitations in their abilities to provide quantitative information. For efficient aggregation analysis, it is essential to make a careful selection of methods in order to minimize gaps in the resultant data because of the wide range of hydrodynamic sizes that an aggregate can have. Proper method selection ensures these data are both orthogonal and valuable, and that none of the data collected are redundant.

The following discusses a number of biophysical methods in detail and shows how apparently conflicting data from techniques such as SV-AUC, SEC-MALS and DLS can be integrated. The experiments also show how a combination of these data, with results obtained from further orthogonal methods, can provide a comprehensive description of protein aggregation.

The experiments describe the analysis of two proteins: a bovine IgG antibody in three different formulations, and a single formulation of α-amylase, an enzyme that catalyses the hydrolysis of polysaccharides.

Materials and Methods

Bovine IgG formulations A, B and C were analyzed using a range of biophysical methods, including SV-AUC (Beckman Coulter XL-A AUC), SEC-MALS (GE Ettan HPLC system and Wyatt Corporation detection technology) and DLS (Malvern Zetasizer Nano-S). Lyophilized bovine IgG was dissolved in the respective formulation buffers at concentrations of 1 mg/mL for the complete set of measurements.

The α-amylase, obtained from Bacillus subtilis, was formulated at pH 7 and tested using SV-AUC, SEC-MALS, DLS and SDS-Page. Lyophilized α-amylase was dissolved in the respective formulation buffer at concentrations of 0.6 mg/mL (SV-AUC and SEC-MALS) for the complete set of measurements; 0.5 mg/ml for DLS; and a range of concentrations (0.8-2.0 mg/mL) for the SDS-Page measurements. Further SV-AUC measurements were made in α-amylase in the range of concentrations 0.2 to 1.4 mg/mL in the absence and presence of NaCl in a 10:1 molar ratio of salt to protein.

Bovine IgG Formulation Case Study

Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC)

Figure 1 shows the results of SV-AUC analysis of bovine IgG in formulation buffers A, B and C, from left to right, respectively, and Table 1 shows the relative abundance of monomers to dimers of bovine IgG in the three formulations as determined by calculating the integrated areas of the C(s) distributions obtained by fitting the SV-AUC data with SEDFIT software. The advantages and disadvantages of the SV-AUC method are:


  • Excellent separation of oligomers – dimers, trimers, tetramers, etc.
  • Absence of a matrix or stationary phase
  • Compatible with most formulation buffers
  • Almost no sample preparation
  • Quantitative assessment of relative proportion of monomers to oligomers


  • Limit of detection greater than that of size exclusion chromatography (SEC)
  • Molecular weight determinations are estimates
  • High concentrations (> 10 mg/mL) are problematic
  • Low throughput
  • Impractical to use as a routine release method



Size-Exclusion Multi-Angle Light Scattering (SEC-MALS)

Table 2 shows the relative abundance of monomers to dimers of bovine IgG in formulations A, B and C as determined by SEC-MALS. The advantages and disadvantages of SEC-MALS are:


  • Widely accepted by regulatory authorities
  • Quantitative assessment of oligomers and aggregates
  • MALS detector allows direct molecular weight determination
  • Low detection limits and high precision
  • Rapid, economical and suitable as a release and stability method


  • Presence of stationary phase: potential adsorption of oligomers/aggregates
  • Limited size range
  • Dilution effects and shear forces affecting oligomers in equilibrium
  • Interference from surfactants
  • Large aggregates co-eluting in the void volume

Table 2

Dynamic Light Scattering (DLS)

Figure 2 shows DLS size distributions of bovine IgG in formulation buffers A, B and C (red, blue and green, respectively) by intensity (upper panel) and volume (lower panel), and Table 3 shows hydrodynamic parameters obtained in bovine IgG in the three formulations as determined by DLS. The advantages and disadvantages of DLS are:


  • Very sensitive: detects high-molecular-weight aggregation
  • Absence of a matrix or stationary phase
  • Compatible with most formulation buffers
  • Broad range of hydrodynamic diameters covered
  • Suitable for high concentrations


  • Qualitative
  • Poor resolution: oligomers are not easily detected
  • Surfactants and sugars can interfere with measurements
  • Medium throughput
  • Large aggregates co-eluting in the void volume



Figure 3 shows SGS-Page and DLS measurements of α-amylase at a concentration of 0.5 mg/mL in the absence of NaCl. The right upper panel corresponds to the intensity distribution and the right bottom panel is the volume distribution. Figure 4 shows SEC-MALS (left panel) and SV-AUC (right panel) measurements on α-amylase at a concentration of 0.6 mg/mL in the absence of NaCl, and Figure 5 shows SV-AUC concentration dependence measurements of α-amylase in the absence and presence of NaCl. The left panel shows a linear dependence on the level of dimerization of α-amylase as a function of concentration. Salt screening by NaCl mitigates the electrostatic interactions leading to the productive formation of dimers, resulting in a shallower linear oligomeric concentration dependence response.

Significance of Results

The bovine IgG small-scale formulation study in three different formulation buffers with different excipient compositions evaluated the stability of bovine IgG from a quaternary point of view. Bovine IgG in formulation buffer A showed a significant amount of dimerization (14.95% ± 0.35%) as depicted by the SV-AUC results in Figure 1 and Table 1. SV-AUC is a powerful technique that can quantify and resolve oligomeric states of proteins: Figure 1 clearly indicates the presence of sedimenting species and the sedimentation coefficient and estimated molecular weight (330kDa) correlated well with the presence of dimers of bovine IgG in formulation A.

On the other hand, formulations B and C, which were designed to minimize electrostatic interactions showed a decrease in the dimerization of bovine IgG (Figure 1) with corresponding relative abundances to those of the monomer of 3.93% ± 0.34% and 0.80% ± 0.27% as shown in Table 1. SV-AUC measurements in bovine IgG appeared to indicate that formulation C, and to a lesser degree formulation B, were successful in mitigating the electrostatic interactions leading to the formation of dimeric species.

A similar trend on the effects of the three different formulations of bovine IgG but with significant differences in the level of dimers was determined by SEC-MALS (10.87% ± 0.14%, 2.49% ± 0.41% and 0.03% ± 0.04% for formulation A, B and C, respectively as shown in Table 2) relative to those reported by SV-AUC. DLS measurements on bovine IgG did not give information on the presence of high-molecular-weight aggregates, but neither did it account for the presence of any oligomeric species or any differences in oligomeric state between the three formulations tested.

The testing of α-amylase by SV-AUC, SEC-MALS, DLS and SDS-Page had the purpose of determining the dimerization propensity of this protein. The α-amylase was formulated at pH 7, close to the isoelectric pH (pI 6.9) value reported in the literature, and, with a molecular weight of 58,380 Da, this enzyme is expected to populate dimers under these conditions. The initial SDS-Page measurements (Figure 3) suggest a monomeric population of α-amylase in a range of concentrations, from 0.8 to 2.0 mg/mL. Additional DLS measurements (Figure 3) at 0.5 mg/mL showed a monomodal size distribution, both by intensity and volume distribution, without any evidence of oligomerization or high-molecular-weight aggregation.

In contrast, however, SEC-MALS and SV-AUC measurements (Figure 4) at 0.6 mg/mL indicated the presence of low levels of dimerization (2.05% and 2.15% respectively). Figure 5 provides supporting evidence for dimerization in α-amylase; the SV-AUC measurements showing a linear concentration dependence on the level of dimerization at concentrations from 0.2 to 1.4 mg/mL both in the absence and presence of NaCl. NaCl mitigates the level of dimerization observed in α-amylase by disrupting electrostatic interactions, leading to the effective formation of α-amylase dimers.

In Summary

The most commonly used methods for the characterization of soluble oligomerization and aggregation are SEC and SDS-Page. These methods can be insensitive to subtle oligomerization changes in the formulation environment or can prove to be inadequate when the type of oligomerization of interest is easily disrupted by the analytical method itself. Data obtained from analyzing three different formulations of bovine IgG and an α-amylase formulation showed that the choice of methods used to build up an overall picture or ‘analytical collage’ is critical in order to give successful interpretation of the data.

The main conclusion from these representative experiments is that employing orthogonal studies using readily available techniques such as SV-AUC, SEC-MALS, DLS and FFF is the right approach for the correct interpretation of data in oligomerization and aggregation studies of proteins. This provides further confirmation that orthogonal studies provide the broadest range of valuable data when performing biophysical analysis of proteins in order to determine key quality attributes of biologic drugs.

For further information, please contact:

Iñigo Rodriguez-Mendieta
Biophysical Group of SGS Life Science Services
West Chester, PA, USA.
t: +1 610 696 8210