Shine a light on your proteins

During therapeutic protein development it is vital that the active ingredient remains stable in various conditions – the best way to examine stable protein formulations, says Sigrid Kuebler, is to use Dynamic Light Scattering

DURING the development of a peptide or protein as a therapeutic product the most crucial step is formulation development. An ideal environment for detergents, stabilisers and buffers needs to be premeditated in order to preserve the integrity of the intricate and often extremely vulnerable three-dimensional structure of the biopharmaceutical drug substances. The resulting formulation must be resilient both to physical and chemical degradation. Covalent and non-covalent aggregations, deamidation, cleavages, oxidation and surface denaturation are typical stability problems observed in protein dosage form. All of these may lead to loss of the biological activity of the protein therapeutic. Surfactants, sugars, salts, antioxidants and amino acids are screened to optimise protein stability during formulation development.

Static light scattering (SLS) detectors are frequently complemented by size exclusion chromatography, (SEC) which is a routine method for detecting and quantifying protein aggregation. However the potential loss of aggregates through filtering or binding to the column is a major disadvantage of SEC. Therefore, column-free analytical methods, such as, analytical ultracentrifugation (AUC), field-flow fractionation (FFF) and DLS are now increasingly used for aggregation analysis. DLS presents the benefit of being a rapid measurement in contrast to the other techniques above. Additionally, potential dissociation of reversible aggregates can be prevented as it is non-perturbing and non-diluting.

Dynamic light scattering (DLS) and static light scattering (SLS) are utilised to characterise macromolecules. Dynamic light scattering detectors measure the time-dependent fluctuations of the intensity of scattered light in the order of 100ns to 100ms, arising from the molecules undergoing Brownian motion (translational diffusion). Larger molecules progress more slowly than smaller ones, therefore the time scale of the light intensity fluctuations can be related to the molecular size. The larger the protein, the slower it diffuses, resulting in a slower decay of the autocorrelation function. Specifically, the parameter measured by DLS is the hydrodynamic radius, defined as the radius of a spherical particle with the same diffusion coefficient as the macromolecule of interest. Light scattering methods are highly sensitive for detecting small amounts of large aggregates, as the intensity of the scattered light is proportional to molecular weight.

SLS detectors and specifically multi-angle light scattering (MALS) detectors calculate the time average intensity of light scattered from the macromolecules in solution. The intensity of scattered light is proportional to both concentration and the absolute molecular weight (Mw) of the molecules. SLS measurements can be conducted in two
modes. Stand-alone or batch mode avoids the need for size separation and yields the average Mw of the molecules. Measurements can also be made in online mode. This requires a size exclusion chromatography (SEC) system to determine molecular weight and molecular weight distribution. Concentration is generally determined online with a UV or refractive index detector, regardless of the shape of the macromolecules. For larger molecules (usually much larger than a protein monomer) with a radius exceeding 10nm, the angular dependence of the static light scattering can also be used to determine their size. This technique additionally can be used to ascertain the second virial coefficient, A2 that expresses the interaction between the molecules and the solution. This can be utilised to screen the crystallisation behavior of biological macromolecules in solution.

The experiment

Therapeutic peptide from two different preparations was screened for aggregation behavior in different buffer formulations, using a DLS Plate Reader (Wyatt Technology). The samples were dissolved in 10µL of buffers at protein concentrations of approximately 1mg/mL. Samples were then transferred to a 1536 well plate and assessed at room temperature. The instrument is compatible with industry standard 96, 384 and 1536 format well plates with a minimum sample volume as low as 4µL per well. The 1536 well plate was selected to reduce the sample quantity required for each formulation and permit the screening of an extensive range of formulation conditions. The total measurement time per well was 100sec.

Figure 1. Autocorrelation function for the therapeutic protein prepared by method B in sodium acetate buffer at pH = 5

Five different formulation conditions ranging from pH 5.0 to ph 8.0 were explored for each protein preparation. To assess reproducibility, two wells were measured for each sample. Figure 1 shows a typical autocorrelation function for the therapeutic protein prepared by method B in sodium acetate buffer at pH = 5.

Figure 2 shows the corresponding size distribution by scattered light intensity calculated by the built-in regularisation algorithm, resulting in an average hydrodynamic radius (Rh) of 2.0nm and a size distribution (% polydispersity) of 14%. For globular proteins, the relationship of Rh and Mw is well known, which results in an estimated Mw of 17kDa of the protein above. This is slightly larger than the Mw of 12kDa measured by size exclusion chromatography, representing the presence of predominantly monomers with a small fraction of oligomers in this particular protein formulation.

Figure 2. Corresponding size distribution by scattered light intensity

A comparison of the particle sizes in different buffer conditions for protein formulation A is shown in Figure 3. The formulations show the least amount of aggregates in the size range >10nm and are thus the most stable in buffers ranging from pH 6 to pH 7.

The peaks at the smallest radii in the range of 0.1 to 0.5 that are often visible for low concentration samples are attributable to the presence of buffer salts and are not taken into account for the analysis. The protein monomer and its oligomers

Figure 3. Particle sizes for different buffer conditions for protein preparation A.

possess a hydrodynamic radius of approximately 2nm. This peak is the predominant peak in all formulation conditions. For pH 5 and pH 6 the protein peaks are shifted to slightly larger hydrodynamic radii, indicating the presence of a higher fraction of oligomers than for the other formulations. In addition, pH 5 and pH 8 conditions result in the presence of larger aggregates in the size range of 10-100nm. A small fraction of even larger aggregate peaks in the micron size range are present in all formulations and is due to particulates introduced during the protein purification process.
Figure 4 shows the particle sizes in the same buffer conditions for protein preparation B.

The formulations show the least amount of aggregates in the size range >10nm and are thus the most stable in buffers ranging from pH 5 to pH 7.

Figure 4.  Particle sizes for different buffer conditions for protein preparation B.

This preparation results in a more uniform size for the protein monomer across all buffer formulations due to a lesser degree of oligomerisation. Furthermore, fewer aggregates are observed in the size range of 10-100nm. Overall, protein preparation B is more stable than protein preparation A across a wider range of pH values. 

A detailed comparison of the two different protein preparations at pH 5 is depicted in Figure 5 the sample in wells A25 and A26 show excellent reproducibility of the particle size for protein preparation A, as do wells A29 and A30 for protein preparation B. The aggregation peak at 23nm observed for preparation A is completely absent in B, indicating a higher stability in the latter.

Figure 5.  Reproducibility and comparison of protein preparation A and B at pH 5.0.

The screening results are summarised in Figure 6 displays the protein monomer sizes vs, buffer conditions of the protein peak. The highest stability is found for protein preparation B in pH7 and PBS buffers as indicated by the smallest protein sizes and maximum percentage intensity.
 

This research established how the screening and characterisation of proteins can be accomplished through the use of dynamic light scattering (DLS). This technique allows rapid and automatic screening for aggregates present in protein formulation, and requires minimal sample volumes, as little as 10µL per well. 

Figure 6: Protein monomer sizes vs. buffer conditions

Automated temperature studies for stability screening are also possible.





By Sigrid Kuebler. Sigrrid is an Application Scientist for the Wyatt Technology Corporation