title: Characterization of Protein-Excipient Microheterogeneity in Biopharmaceutical Solid-State Formulations by Confocal Fluorescence Microscopy.
abstract: Protein-stabilizer microheterogeneity is believed to influence long-term protein stability in solid-state biopharmaceutical formulations and its characterization is therefore essential for the rational design of stable formulations. However, the spatial distribution of the protein and the stabilizer in a solid-state formulation is, in general, difficult to characterize because of the lack of a functional, simple, and reliable characterization technique. We demonstrate the use of confocal fluorescence microscopy with fluorescently labeled monoclonal antibodies (mAbs) and antibody fragments (Fabs) to directly visualize three-dimensional particle morphologies and protein distributions in dried biopharmaceutical formulations, without restrictions on processing conditions or the need for extensive data analysis. While industrially relevant lyophilization procedures of a model IgG1 mAb generally lead to uniform protein-excipient distribution, the method shows that specific spray-drying conditions lead to distinct protein-excipient segregation. Therefore, this method can enable more definitive optimization of formulation conditions than has previously been possible.
authors.affiliation: Center for Molecular and Engineering Thermodynamics, Department of Chemical and Biomolecular Engineering, University of Delaware , Newark, Delaware 19716, United States.
authors.forename: Stijn H S
authors.initials: SH
authors.lastname: Koshari
authors.affiliation: DBI Bio-Imaging Center, Delaware Biotechnology Institute , Newark, Delaware 19711, United States.
authors.forename: Jean L
authors.initials: JL
authors.lastname: Ross
authors.affiliation: Eurofins Lancaster Laboratories Inc. , Lancaster, Pennsylvania 17605, United States.
authors.forename: Purnendu K
authors.initials: PK
authors.lastname: Nayak
authors.affiliation: Late Stage Pharmaceutical Development, Genentech Inc. , South San Francisco, California 94080, United States.
authors.forename: Isidro E
authors.initials: IE
authors.lastname: Zarraga
authors.affiliation: Drug Delivery Department, Genentech Inc. , South San Francisco, California 94080, United States.
authors.forename: Karthikan
authors.initials: K
authors.lastname: Rajagopal
authors.affiliation: Center for Molecular and Engineering Thermodynamics, Department of Chemical and Biomolecular Engineering, University of Delaware , Newark, Delaware 19716, United States.
authors.forename: Norman J
authors.initials: NJ
authors.lastname: Wagner
authors.affiliation: Center for Molecular and Engineering Thermodynamics, Department of Chemical and Biomolecular Engineering, University of Delaware , Newark, Delaware 19716, United States.
authors.forename: Abraham M
authors.identifier: 0000-0002-7831-219X
authors.initials: AM
authors.lastname: Lenhoff
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D001702:Biopharmaceutics / Q000379:methods; D002626:Chemistry, Pharmaceutical / Q000379:methods; D004355:Drug Stability; D005079:Excipients / Q000737:chemistry*; D005612:Freeze Drying / Q000379:methods; D007074:Immunoglobulin G / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry*
chemical_list: D000911:Antibodies, Monoclonal; D005079:Excipients; D007074:Immunoglobulin G; D011506:Proteins
keywords: lyophilization/freeze-drying; monoclonal antibody; protein−stabilizer microheterogeneity; spray-drying; three-dimensional reconstruction