Agitation-induced aggregation and subvisible particulate formation in model proteins

Area of Science:

• Biopharmaceutical development
• Protein aggregation kinetics
• Subvisible particle formation

Background:

• Subvisible particles (SbVPs) are critical quality attributes in biopharmaceuticals.
• Understanding agitation-induced SbVP formation is crucial for drug product stability.

Purpose of the Study:

• Investigate the initial phase (<2%) of agitation-induced SbVP formation under low protein concentration and low agitation speed.
• Elucidate the mechanisms of SbVP formation for model proteins: human serum albumin (HSA), hen egg white lysozyme (HEWL), and a monoclonal antibody (IgG2).

Main Methods:

• Agitation of model protein solutions (HSA, HEWL, IgG2) at low concentration and speed.
• Analysis of soluble higher molecular mass species (HMMS) and subvisible particles (SbVPs).
• Assessment of protein structural changes (hydrophobic site exposure) and SbVP dissociation behavior.

Main Results:

• Agitation induced SbVP and HMMS formation at different rates and via distinct mechanisms.
• HSA showed enhanced hydrophobic site exposure upon agitation; HEWL and IgG2 did not.
• SbVP dissociation behavior varied significantly between HSA, HEWL, and IgG2 depending on buffer conditions.

Conclusions:

• Agitation-induced SbVP formation mechanisms differ among proteins and are influenced by solution conditions.
• Protein properties like hydrophobic surface area or melting temperature (Tm) are not reliable predictors of agitation-mediated SbVP formation.
• The study highlights the complexity of protein aggregation pathways under mechanical stress.