title: 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.
authors.affiliation: SmithKline Beecham Pharmaceuticals, Research and Development, King of Prussia, Pennsylvania 19406, USA. Doug_P_Nesta@SBPHRD.com
authors.forename: D P
authors.initials: DP
authors.lastname: Nesta
authors.forename: J S
authors.initials: JS
authors.lastname: Elliott
authors.forename: J P
authors.initials: JP
authors.lastname: Warr
mesh_terms: D001709:Biotechnology / Q000295:instrumentation / Q000379:methods; D002245:Carbon Dioxide / Q000737:chemistry; D011232:Chemical Precipitation; D002850:Chromatography, Gel / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007136:Immunoglobulins / Q000235:genetics* / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012995:Solubility; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet; D014867:Water
chemical_list: D007136:Immunoglobulins; D011994:Recombinant Proteins; D012996:Solutions; D014867:Water; D002245:Carbon Dioxide