Quantitative Evaluation of Colloidal Stability of
Antibody Solutions using PEG-Induced Liquid–Liquid Phase Separation
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Abstract
Colloidal stability of antibody solutions, i.e., the propensity
of the folded protein to precipitate, is an important consideration
in formulation development of therapeutic monoclonal antibodies. In
a protein solution, different pathways including crystallization,
colloidal aggregation, and liquid–liquid phase separation (LLPS)
can lead to the formation of precipitates. The kinetics of crystallization
and aggregation are often slow and vary from protein to protein. Due
to the diverse mechanisms of these protein condensation processes,
it is a challenge to develop a standardized test for an early evaluation
of the colloidal stability of antibody solutions. LLPS would normally
occur in antibody solutions at sufficiently low temperature, provided
that it is not preempted by freezing of the solution. Poly(ethylene
glycol) (PEG) can be used to induce LLPS at temperatures above the
freezing point. Here, we propose a colloidal stability test based
on inducing LLPS in antibody solutions and measuring the antibody
concentration of the dilute phase. We demonstrate experimentally that
such a PEG-induced LLPS test can be used to compare colloidal stability
of different antibodies in different solution conditions and can be
readily applied to high-throughput screening. We have derived an equation
for the effects of PEG concentration and molecular weight on the results
of the LLPS test. Finally, this equation defines a binding energy
in the condensed phase, which can be determined in the PEG-induced
LLPS test. This binding energy is a measure of attractive interactions
between antibody molecules and can be used for quantitative characterization
of the colloidal stability of antibody solutions
