Using a DLS Plate Reader from Wyatt Technology (www.wyatt.com), a therapeutic peptide from two different preparations was screened for aggregation behavior in different buffer formulations. The samples were dissolved in 10 µL of buffers at protein concentrations of approximately 1 mg/mL. Samples were transferred to a 1,536-well plate and measured at room temperature.
The instrument is compatible with industry-standard 96-, 384-, and 1,536-format well plates with a minimum sample volume as low as 4 µL per well. The 1,536-well plate was chosen to minimize the sample amount needed for each formulation and enable the screening of a wide range of formulation conditions. The total measurement time per well was 100 seconds.
Five different formulation conditions, ranging from pH 5 to pH 8, 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 regularization algorithm, which results in an average hydrodynamic radius (Rh) of 2.0 nm 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 17 kDa of the protein above. This is slightly larger than the molecular weight of 12 kDa measured by SEC, indicating the predominance of monomers with a small fraction of oligomers in this particular protein formulation.
A comparison of the particle sizes in different buffer conditions for protein formulation A is shown in Figure 3. The formulation shows the least amount of aggregates in the size range >10 nm and is thus the most stable in buffers ranging from pH 6 to pH 7.
The peaks at the smallest radii in the range of 0.1–0.5 that are often visible for low concentration samples are caused by the presence of buffer salts and are not taken into account for the analysis. The protein monomer and its oligomers possess a hydrodynamic radius of approximately 2 nm. 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 10–100 nm. 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 comparison of particle sizes in the same buffer conditions for protein preparation B, which has the least amount of aggregates in the size range >10 nm and is thus the most stable in buffers ranging from pH 5 to pH 7.
This preparation results in a more uniform size for the protein monomer across all buffer formulations due to a lesser degree of oligomerization. Furthermore, fewer aggregates are observed in the size range of 10–100 nm. 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 23 nm observed for preparation A is completely absent in B, indicating a higher stability in the latter.
The screening results (summarized in Figure 6) display the protein monomer sizes vs. buffer conditions of the protein peak. The highest stability is found for protein preparation B in pH 7 and PBS buffers as indicated by the smallest protein sizes and highest percentage intensity.