title: Stirred batch crystallization of a therapeutic antibody fragment.
abstract: Technical-scale crystallization of therapeutic proteins may not only allow for a significant cost-reduction in downstream processing, but also enable new applications, e.g., the use of crystal suspensions for subcutaneous drug delivery. In this work, the crystallization of the antigen-binding fragment FabC225 was studied. First, vapor diffusion crystallization conditions from the literature were transferred to 10μL-scale microbatch experiments. A phase diagram was developed in order to identify the crystallization window. The conditions obtained from the microbatch experiments were subsequently transferred to parallelized 5mL-scale stirred-tank crystallizers. This scalable and reproducible agitated crystallization system allowed for an optimization of the crystallization process based on quantitative measurements. The optimized crystallization process resulted in an excellent yield of 99% in less than 2h by increasing the concentration of the crystallization agent ammonium sulfate during the process. The successful scalability of the Fab fragment crystallization process to 100mL-scale crystallizers based on geometric similarity was demonstrated. A favorable crystal size distribution was obtained. Furthermore, a wash step was introduced in order to remove unfavorable low-molecular substances from the crystals.
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Hebel
authors.forename: Sabine
authors.initials: S
authors.lastname: Huber
authors.forename: Bernd
authors.initials: B
authors.lastname: Stanislawski
authors.forename: Dariusch
authors.initials: D
authors.lastname: Hekmat
mesh_terms: D000645:Ammonium Sulfate / Q000737:chemistry; D011232:Chemical Precipitation; D003460:Crystallization / Q000379:methods; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry* / Q000302:isolation & purification; D009038:Motion; D009994:Osmolar Concentration; D010316:Particle Size; D013535:Suspensions; D013997:Time Factors
chemical_list: D007140:Immunoglobulin Fab Fragments; D013535:Suspensions; D000645:Ammonium Sulfate
keywords: Antibody fragment; Protein crystallization; Scale-up; Stirred tank