Formation of Stable Nanobubbles on Reconstituting Lyophilized Formulations Containing Trehalose

Abstract

During an investigation of subvisible particles found in lyophilized formulations of intravenous immunoglobulin, we used resonant mass measurement techniques and discovered the presence of nanobubbles (NBs) when a 5% trehalose formulation was reconstituted. This discovery prompted studies to characterize these NBs in placebo formulations as a function of processing conditions and solution compositions. Degassing the reconstituted solutions by applying vacuum removed micron-sized bubbles but did not substantially affect the concentration of NBs. Samples that were annealed in the frozen state before lyophilization had reduced surface areas and, on reconstitution, yielded fewer NBs. Trehalose formulations with added polysorbate 20 (PS20) and formulations with higher ionic strength also had smaller numbers of NBs. Zeta potentials of the bubbles were negative in each of the formulations tested, but the negative zeta potentials were decreased in magnitude with increasing ionic strength and with addition of PS20. When incubated at 4°C, the number of NBs was largely unchanged at the end of 11 days, whereas the number of micron-sized bubbles gradually decreased during the 11-day incubation. Because of their exceptional stability, NBs are expected to contribute to the numbers of submicron particles that can be detected in reconstituted lyophilized protein formulations.