Formation of Stable Nanobubbles on Reconstituting Lyophilized Formulations Containing Trehalose.
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.
Department of Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Aurora, Colorado 80045.
Chen
C
Zhou
Department of Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Aurora, Colorado 80045.
Derrick
D
Cleland
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309.
Jared
J
Snell
Malvern Instruments, Inc., Columbia, Maryland 21046.
Wei
W
Qi
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309.
Theodore W
TW
Randolph
Department of Pharmaceutical Sciences, University of Colorado, Anschutz Medical Campus, Aurora, Colorado 80045. Electronic address: John.Carpenter@ucdenver.edu.
John F
JF
Carpenter
D004339:Drug Compounding; D004355:Drug Stability; D004563:Electrochemistry; D005079:Excipients; D005612:Freeze Drying; D016756:Immunoglobulins, Intravenous / Q000737:chemistry; D053758:Nanoparticles / Q000737:chemistry*; D010316:Particle Size; D011136:Polysorbates; D013499:Surface Properties; D014199:Trehalose / Q000737:chemistry*
D005079:Excipients; D016756:Immunoglobulins, Intravenous; D011136:Polysorbates; D014199:Trehalose
nanobubble; particle analysis; protein lyophilization; protein particles; resonant mass measurement