Stirred batch crystallization of a therapeutic antibody fragment

• Published in: Journal of biotechnology Vol. 166; no. 4; pp. 206 - 211
• Main Authors: Hebel, Dirk, Huber, Sabine, Stanislawski, Bernd, Hekmat, Dariusch
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 20-07-2013
• Subjects: ammonium sulfate; Ammonium Sulfate - chemistry; antibodies; Antibody fragment; biopharmaceuticals; biotechnology; Chemical Precipitation; crystallization; Crystallization - methods; crystals; Immunoglobulin Fab Fragments - chemistry; Immunoglobulin Fab Fragments - isolation & purification; Motion; Osmolar Concentration; Particle Size; Protein crystallization; quantitative analysis; Scale-up; Stirred tank; Suspensions; Time Factors; vapors; Stirred tank; Antibody fragment; Protein crystallization; Scale-up
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2013.05.010

•The crystallization of the antigen-binding fragment FabC225 was studied.•Conditions from microbatch experiments were transferred to mL-scale stirred tanks.•The stirred-tank crystallization system allowed for an optimization of the process.•Successful scalability of the crystallization process was demonstrated.•Protein crystallization can be a viable alternative to preparative chromatography.
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.