Parallel chromatography and in situ scattering to interrogate competing protein aggregation pathways

Area of Science:

• Biochemistry
• Protein Science
• Pharmaceutical Science

Background:

• Protein aggregation is a critical concern in pharmaceutical development, impacting drug stability and efficacy.
• Understanding aggregation pathways is essential for controlling protein behavior and preventing adverse outcomes.

Purpose of the Study:

• To quantitatively and qualitatively assess a combined ex situ size-exclusion chromatography (SEC) and in situ laser scattering (LS) approach for monitoring protein aggregation.
• To investigate the influence of initial dimer levels and temperature on aggregation kinetics using α-chymotrypsinogen A (aCgn) as a model system.

Main Methods:

• Utilized a dual approach combining SEC for detailed analysis and LS for real-time monitoring of protein aggregation kinetics.
• Investigated aggregation under initial-rate conditions (low monomer conversion) across various temperatures and initial dimer concentrations.

Main Results:

• Decreased initial dimer levels significantly reduced monomer loss rates but did not alter the activation energy for aggregation.
• Laser scattering (LS) indicated aggregation rates approximately one order of magnitude higher than those measured by SEC.
• LS signals became nonlinear under conditions of suppressed dimer seeding and increased reversible oligomer formation, indicating monitoring of different aggregation stages.

Conclusions:

• The combined SEC-LS methodology provides a comprehensive view of protein aggregation pathways, differentiating early oligomerization from stable aggregate formation.
• Relying on a single method for monitoring protein aggregation can lead to inaccurate kinetic profiles and misinterpretation of the underlying mechanisms.
• This study underscores the importance of employing orthogonal methods to fully characterize protein aggregation, particularly in pharmaceutical applications.