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 (CO(2)) 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.

Fabrication of Poly(ethylene glycol) Hydrogel Microstructures Using Photolithography

“…CO 2 is an environmentally benign, nontoxic compound which is being used in the pharmaceutical industry as a reaction medium for synthesis of drug compounds 41,42 and as a medium for processing protein powders with retention of activity. 43,44 In addition, SC-CO 2 is being investigated as a possible medium for photoresist image development. 45,46 The motivating factor for the latter studies is the potential of this solvent in achieving high feature resolution as well as the ability to control solvating power by varying temperature or pressure.…”

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).…”

“…Ethanol, which is soluble in both water and CO 2 , increases the miscibility of the solvent and the antisolvent and, hence, provides a one‐phase operating region required for PCA. 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 (13–15).…”

Solution-enhanced dispersion by supercritical fluids: an ecofriendly nanonization approach for processing biomaterials and pharmaceutical compounds

“…59,90 In general, the SEDS process is not only used for operating poorly water-soluble drugs, but also widely applied for the precipitation of protein-and peptide-based drugs that are intended to be absorbed from noninvasive routes of administration such as pulmonary and transdermal delivery. 96,97 Despite the success and reliability in formulating the therapeutic proteins by the SEDS process, the longterm stability may be compromised due to the undesirable chemical degradation mechanisms and conformational alterations that can affect the primary, secondary, and/or tertiary structures of a protein. 96 However, extensive studies are required to overcome these limitations and explore the processing of sensitive biomacromolecules alone.…”