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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'. [Display omitted] [ABSTRACT FROM AUTHOR]

The effects of the manufacturing process and the regeneration of Shirasu porous glass (SPG) membranes were investigated on the reproducibility of protein precipitants, termed protein microbeads. Intravenous immunoglobulin (IVIG) was selected as a model protein to produce its microbeads in seven different cases. The results showed that the hydrophobically modified SPG membrane produced finer microbeads than the hydrophilic SPG membrane, but this was inconsistent when using the general regeneration method. Its reproducibility was determined to be mostly dependent on rinsing the SPG membrane prior to the modification and on the protein concentration used for emulsification. The higher concentration could foul and plug the membrane during protein release and thus the membrane must be washed thoroughly before hydrophobic modification. Moreover, the membrane regenerated by silicone resin dissolved in ethanol had better reproducibility than silicone resin dissolved in water. On the other hand, rinsing the protein precipitant with cold ethanol after the emulsification was not favorable and induced protein aggregation. With the addition of trehalose, the purity of the IVIG microbeads was almost the same as before microbeadification. Therefore, the regeneration method, protein concentration, and its stabilizer are key to the success of protein emulsification and precipitation using the SPG membrane. [ABSTRACT FROM AUTHOR]