Supercritical fluid precipitation of recombinant human immunoglobulin from aqueous solutions

Abstract

Supercritical carbon dioxide was used as an antisolvent for producing recombinant human immunoglobulin G (rIgG) particulate powders. Liquid carbon dioxide (CO2) was premixed with ethanol to create a single‐phase, modified supercritical fluid (SCF). The modified SCF was then vigorously mixed with a pharmaceutically acceptable, aqueous formulation of rIgG, and the mixture was immediately atomized into a pressurized vessel where rapid expansion of the modified SCF extracted the aqueous phase, resulting in precipitation of the protein powder. The process was reproducible, and resulting powder products were characterized by their aqueous solubilities, and the spectroscopic profile, molecular integrity, and antigen binding activity of the individual soluble fractions. Molecular integrity was assessed via size‐exclusion high‐performance liquid chromatography (SEC), whereas antigen binding activity was determined using an enzyme‐linked immunosorbent assay (ELISA). Attempts to characterize particle size and morphology were confounded due to the extremely deliquescent nature of the powders, causing them to absorb moisture rapidly and become gummy. Operational conditions were optimized to a point which yielded powders that were completely soluble, and had ultraviolet (UV) spectroscopic and SEC profiles indistinguishable from those of the reference standard starting solution from which the powders were derived. Antigen binding activities of the powders, however, were ≤50% of expected levels, revealing the need for improvement of this SCF processing approach for rIgGs. From this study, two important conclusions were drawn: (1) Biopharmaceutical product quality assessments must include definitive activity analyses, because profound activity losses, such as those suffered here by the rIgGs, may not be revealed when using standard physicochemical analyses alone; and, (2) A more comprehensive understanding and control of the range of SCF processing effects, particularly on aqueous‐based macromolecular protein therapeutics, is necessary for SCF precipitation to gain full acceptance as a viable option for secondary manufacturing of biopharmaceutical powders. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 457–464, 2000.

CO2 and Fluorinated Solvent‐Based Technologies for Protein Microparticle Precipitation from Aqueous Solutions

“…This higher retained activity suggests that adding the cosolvent with the antisolvent, or precipitating proteins directly from an aqueous solution, may be a better microparticle formation technique than introducing an organic cosolvent in the aqueous phase. In comparison, retained activities of proteins precipitated from aqueous solutions by ethanol-enhanced CO 2 using the SEDS process have been reported as 95% for lysozyme (7) and 38-48% for rIgG (13). Winters et al (1) have reported recovered activity for lysozyme precipitated from DMSO solutions using liquid and supercritical CO 2 to range between 88 and 100% and between 89 and 99%, respectively.…”

“…Only recently has PCA been used to precipitate solutes in aqueous solutions, including organic salts (11), salicylic acid, lactose (12), recombinant IgG (13), lysozyme, rhDNase, insulin, albumin (14), and plasmid DNA (15). Although the proteins precipitated from these aqueous solutions were micronized, the considerable aggregation of the protein particles led to a loss of activity, with the exception of lysozyme and insulin micropowders (13)(14)(15).…”

Recent Applications of Supercritical Fluid Technology to Pharmaceutical Powder Systems

“…The difficulty of applying gas antisolvent techniques to the processing of proteins is that they involve exposure of the protein to organic solvents, the latter being potential denaturants and very poor solvents for KONA No.l9 (2001) most therapeutic macromolecules. The ASES has been modified to enable the spraying of aqueous protein solution simultaneously with an organic solvent into carbon dioxide [24] or directly into carbon dioxide, which has been modified with an organic solvent [22,65].…”

“…Recombinant human immunoglobulin (rhiG) [65], lysozyme [22], albumin, recombinant human deoxyribonuclease (rhDNase), insulin [22], trypsin [53], a therapeutic peptide antibody Fv and Fab and plasmid DNA [54] have been precipitated from aqueous based solutions. Except for rhiG, which was not obtained as a stable powder, all the proteins precipitated as micron-sized particles or agglomerated nano-spheres.…”

Two Different Shapes of Insulin Microparticles Produced by Solution Enhanced Dispersion Supercritical Fluid (SEDS) Process

“…6 A SEDS process has also been applied to the precipitation of an aqueous formulation of recombinant human immunoglobin G using carbon dioxide with dimethyl sulfoxide as a modified supercritical fluid anti-solvent. 7 Sievers and coworkers consistently obtained particles of various pharmaceutical agents, peptides and proteins, with mean particle sizes in the range of 0.1-3 µm, from their respective aqueous solutions through rapid decompression of emulsions formed by mixing the solutions with carbon dioxide. 8,9 Product particle sizes and their distributions could be controlled to some extent by varying concentration of the protein solution.…”