title: Quality by design: impact of formulation variables and their interactions on quality attributes of a lyophilized monoclonal antibody.
abstract: The purpose of this study was to use QbD approaches to evaluate the effect of several variables and their interactions on quality of a challenging model murine IgG3κ monoclonal antibody (mAb), and then to obtain an optimized formulation with predefined quality target product profile. This antibody was chosen because it has a propensity to precipitate and thus represents a challenge condition for formulation development. Preliminary experiments were conducted to rule out incompatible buffer systems for the mAb product quality. A fractional factorial experimental design was then applied to screen the effects of buffer type, pH and excipients such as sucrose, sodium chloride (NaCl), lactic acid and Polysorbate 20 on glass transition temperature ( [Formula: see text] ), monoclonal antibody concentration (A(280)), presence of aggregation, unfolding transition temperature (T(m)) of the lyophilized product, and particle size of the reconstituted product. A Box-Behnken experimental design was subsequently applied to study the main, interaction, and quadratic effects of these variables on the responses. Pareto ranking analyses showed that the three most important factors affecting the selected responses for this particular antibody were pH, NaCl, and Polysorbate 20. The presence of curvature in the variables' effects on responses indicated interactions. Based on the constraints set on the responses, a design space was identified for this mAb and confirmed with experiments at three different levels of the variables within the design space. The model indicated a combination of high pH (8) and NaCl (50mM) levels, and a low Polysorbate 20 (0.008 mM) level at which an optimal formulation of the mAb could be achieved. Moisture contents and other analytical procedures such as size exclusion chromatography, protein A analysis and SDS-PAGE of the pre-lyophilized and final reconstituted lyophilized products indicated an intact protein structure with minimal aggregation after formulation and lyophilization. In conclusion, experimental design approach was effective in identifying optimal concentrations of excipients and pH for this challenging monoclonal antibody formulation.
authors.affiliation: Division of Product Quality Research, Office of Testing and Research, OPS, CDER, FDA, United States.
authors.forename: David
authors.initials: D
authors.lastname: Awotwe-Otoo
authors.forename: Cyrus
authors.initials: C
authors.lastname: Agarabi
authors.forename: Geoffrey K
authors.initials: GK
authors.lastname: Wu
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Casey
authors.forename: Erik
authors.initials: E
authors.lastname: Read
authors.forename: Scott
authors.initials: S
authors.lastname: Lute
authors.forename: Kurt A
authors.initials: KA
authors.lastname: Brorson
authors.forename: Mansoor A
authors.initials: MA
authors.lastname: Khan
authors.forename: Rakhi B
authors.initials: RB
authors.lastname: Shah
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002626:Chemistry, Pharmaceutical; D005079:Excipients / Q000737:chemistry*; D005612:Freeze Drying; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000276:immunology; D009345:Neisseria meningitidis / Q000276:immunology; D011786:Quality Control
chemical_list: D000911:Antibodies, Monoclonal; D005079:Excipients; D007074:Immunoglobulin G