Native and Non-Native aggregation pathways of antibodies anticipated by cold-accelerated studies

Area of Science:

• Biopharmaceutical science
• Protein chemistry
• Physical chemistry

Background:

• Assessing cold stability is vital for biotherapeutic manufacturing and commercialization.
• Current methods using elevated temperatures and Arrhenius equations often underestimate protein aggregation during storage.
• Understanding aggregation kinetics across wide temperature ranges is needed.

Purpose of the Study:

• To investigate antibody aggregation rates across a broad temperature spectrum, from 60 °C to -25 °C.
• To develop a more accurate model for predicting protein aggregation, accounting for both hot and cold denaturation.
• To enable rapid assessment of storage stability at refrigeration temperatures.

Main Methods:

• Employed an isochoric cooling method to prevent freezing below 0 °C.
• Measured aggregation rates of two antibodies from 60 °C down to -25 °C.
• Utilized Arrhenius and Gibbs-Helmholtz equations to model temperature-dependent aggregation kinetics.

Main Results:

• Both antibodies showed increased aggregation rates at extreme temperatures (hot and cold denaturation).
• The developed model successfully deconvoluted unfolding and protein association kinetics.
• Accurate aggregation rates at 5 °C were predicted within 1-2 weeks using SEC-HPLC and DLS.

Conclusions:

• Standard accelerated stability testing underestimates cold-induced aggregation.
• A broader temperature range assessment combined with advanced modeling provides accurate predictions for biotherapeutic cold stability.
• This approach facilitates faster and more reliable shelf-life determination for biologics.