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

Lessons Learned in Protein Precipitation Using a Membrane Emulsification Technique to Produce Reversible and Uniform Microbeads

“…N 2 gas was purged at a pressure of 50 kPa to release the IVIG solution. Similar to the previous study [ 17 ], the volume ratio of IVIG solution and n -octanol was derived from a water saturation fraction ( f ) which was fixed at 0.5 for fast dehydration [ 26 , 27 , 28 ]. The volume of the IVIG solution, V w , was calculated according to the following equation: V w = f × V s × C s × ρ where V s is the volume of n -octanol, C s is the solubility of water in the organic solvent (water/solvent in grams), and ρ is the density of n -octanol.…”

“…The flow rate was fixed at 0.1 mL/min. The analysis was performed according to our previously reported procedures [ 12 , 17 ]. The lowest particle size measured was from 1 μm.…”

“…The recovery of IVIG was calculated using the following equation: Recovery of IVIG % = ( A s ÷ A 0 ) × 100 where ‘ A s ’ is the area of the monomeric peak after rehydration of the microbeads and ‘ A 0 ’ is the area of the monomeric peak of the reference before microbeadification. The analysis was performed according to our previously reported procedure [ 17 ].…”

“…Based on these findings, we hypothesized that organic solvents are promising for producing reversible protein precipitates, but they require new insights into their mechanisms for designing better preparation methods that are applicable for commercial production. In our previous study, a combination process of cold n -octanol precipitation with membrane emulsification was able to produce a uniform and reversible IgG precipitant (called a ‘microbead’) [ 17 ]. Briefly, IgG solution was injected into cold n -octanol through a Shirasu porous glass (SPG) membrane to produce a uniformly distributed water-in-oil (W/O) emulsion followed by vortexing for rapid precipitation (i.e., dehydration).…”

“…Then, it was centrifuged to remove the supernatant and vacuum dried under controlled vapor pressure to remove any remaining organic solvents. With this effort to improve the process development, the recovery of IgG upon rehydration exhibited almost the same content as before the precipitation process [ 17 ].…”