Abstract
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.
Highlights
Protein precipitation is gaining interest for downstream steps in bioprocesses due to its capability of purifying therapeutic proteins, including monoclonal antibodies and immunoglobulins (IgGs) in a scalable and cost-effective manner [1,2,3,4]
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
By Flow Imaging (FI) analysis, the mean size of the Intravenous immunoglobulin (IVIG) microbeads was water flushed) Shirasu porous glass (SPG) membrane affects the efficiency of microparticle formation, and a longer ejection time might induce a wider size deviation
Summary
Protein precipitation is gaining interest for downstream steps in bioprocesses due to its capability of purifying therapeutic proteins, including monoclonal antibodies (mAbs) and immunoglobulins (IgGs) in a scalable and cost-effective manner [1,2,3,4]. Pharmaceutics 2021, 13, 1738 and incubation temperature in 80% acetone with rapid precipitation have resulted in high protein recovery (around 98%) from complex proteome extracts [16]. 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. 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].
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