Enhanced protein aggregation suppressor activity of N-acetyl-l-arginine for agitation-induced aggregation with silicone oil and its impact on innate immune responses.

Previously, N-acetyl-l-arginine (NALA) suppressed the aggregation of intravenous immunoglobulins (IVIG) more effectively and with a minimum decrease in transition temperature (Tm) than arginine monohydrochloride. In this study, we performed a comparative study with etanercept (commercial product: Enbrel®), where 25 mM arginine monohydrochloride (arginine) was added to the prefilled syringe. The biophysical properties were investigated using differential scanning calorimetry (DSC), dynamic light scattering (DLS), size-exclusion chromatography (SEC), and flow-imaging microscopy (FI). NALA retained the transition temperature of etanercept better than arginine, where arginine significantly reduced the Tm by increasing its concentration. End-over-end rotation was applied to each formulation for 5 days to accelerate protein aggregation and subvisible particle formation. Higher monomeric content was retained with NALA with a decrease in particle level. Higher aggregation onset temperature (Tagg) was detected for etanercept with NALA than arginine. The results of this comparative study were consistent with previous study, suggesting that NALA could be a better excipient for liquid protein formulations. Agitated IVIG and etanercept were injected into C57BL/6J female mice to observe immunogenic response after 24 h. In the presence of silicone oil, NALA dramatically reduced IL-1 expression, implying that decreased aggregation was related to reduced immunogenicity of both etanercept and IVIG.

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

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.

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

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'.