Protein microbeadification to achieve highly concentrated protein formulation with reversible properties and in vivo pharmacokinetics after reconstitution

Abstract

A protein precipitation technique was optimized to produce biophysically stable 'protein microbeads', applicable to highly concentrated protein formulation. Initially, production of BSA microbeads was performed using rapid dehydration by vortexing in organic solvents followed by cold ethanol treatment and a vacuum drying. Out of four solvents, n-octanol produced the most reversible microbeads upon reconstitution. A Shirasu porous glass (SPG) membrane emulsification technique was utilized to enhance the size distribution and manufacturing process of the protein microbeads with a marketized human IgG solution. Process variants such as dehydration time, temperature, excipients, drying conditions, and initial protein concentration were evaluated in terms of the quality of IgG microbeads and their reversibility. The hydrophobized SPG membrane produced a narrow size distribution of the microbeads, which were further enhanced by shorter dehydration time, low temperature, minimized the residual solvents, lower initial protein concentration, and addition of trehalose to the IgG solution. Final reversibility of the IgG microbeads with trehalose was over 99% at both low and high protein concentrations. Moreover, the formulation was highly stable under repeated mechanical shocks and at an elevated temperature compared to its liquid state. Its in vivo pharmacokinetic profiles in rats were consistent before and after the 'microbeadification'.

Keywords: High concentration protein; Protein microbead; Protein precipitation.