The stability profile determines the shelf life of therapeutic proteins. The two primary degradation pathways of proteins are chemical degradation and physical degradation. Chemical degradation involves formation or breaking of chemical bonds: hydrolysis, oxidation, deamidation, etc.. Physical degradation involves unfolding and aggregation (Figure 1). Protein aggregates can form during production, storage, shipment or delivery to the patient. Many environmental conditions (i.e., pH, ionic strength, temperature, light, and oxidation) and processing conditions (i.e., lyophilization) lead to protein misfolding and subsequently to aggregation. It is very crucial to monitor protein stability during formulation development.

Dynamic light scattering (DLS) to evaluate the aggregation state

Dynamic light scattering (DLS) or quasi elastic light scattering (QELS) has been widely used to evaluate the aggregation state of proteins in solution. DLS is a non-destructive technique for the determination of size distribution of particles. DLS does not provide the absolute concentration of each fraction, but it is very sensitive to large aggregates and can detect aggregates at very low concentration. It is particularly useful for comparing samples in different media or at different time points for physical stability evaluation. Figure 2 shows different aggregation states of lysozyme in different buffers. At low pH (<4), lysozyme exists predominantly in a monomeric state with a hydrodynamic radius (Rh) of 1.7‒2 nm. At higher pH (>5), the molecules undergo self-association forming oligomers with a larger Rh (>100 nm). Self-association of lysozyme at higher pH has been well documented in the literature [1-3].

Figure 3 shows the heat induced aggregation of IgG. At ambient temperature, the Rh of IgG was determined to be approximately 5 nm, and the intact IgG remained stable up to 65 °C. Further heating resulted in aggregation as indicated by a larger Rh. Under reducing conditions, the disulfide bonds that stabilize the IgG molecules are destroyed, leading to self-association at lower temperature (~45 °C).

Size exclusion chromatography with multi-angle static light scattering to evaluate the aggregation state

Size exclusion chromatography with multi-angle static light scattering (SEC-MALS) is another powerful tool to characterize oligomerization/aggregation. Different aggregation states are separated by size, and the molecular weight is calculated from light scattering properties. Low amounts of aggregates can be detected due to increased light scattering from larger molecular weight species. Figure 4 illustrated a typical SEC-MALS chromatogram of bovine serum albumin. The chromatogram of BSA displayed a main peak with a molecular weight of 69 kDa, consistent with the molecular weight of the BSA monomer. A low intensity peak eluting before the monomer peak showed a molecular weight of 139 kDa, consistent with the dimeric form of BSA.

References

  1. Sophianopoulos, A. J.; van Holde, K. E. Biol. Chem. 239, 2516-2524 (1964).
  2. Bruzzesi, M. R.; Chiancone, E.; Antonini, E. Biochemistry 4, 1796-1800 (1965).
  3. Gottschalk, M and Halle, B. Phys. Chem. B 107, 7914-7922 (2003).

 

Figure 1. Illustration of protein aggregation.

Figure 2. Particle size distribution of lysozyme in different buffers

 

Figure 3. Thermal stability of a monoclonal antibody

 

Figure 4. SEC-MALS of bovine serum albumin