title: Development of an ELP-Z based mAb affinity precipitation process using scaled-down filtration techniques.
abstract: In this work, a proof of concept elastin-like polypeptide-Z domain fusion (ELP-Z) based monoclonal antibody (mAb) affinity precipitation process is developed using scaled-down filtration techniques. Tangential flow filtration (TFF) is examined for the recovery of ELP-Z-mAb precipitates formed during the mAb binding step and the ELP-Z precipitates formed during the mAb elution step. TFF results in complete precipitate recovery during both stages of the process and high host cell protein and DNA impurity clearance after diafiltration. Total recycle TFF experiments are then employed to determine permeate flux as a function of the precipitate concentration for both stages of the process. While the ELP-Z-mAb precipitate recovery step resulted in high permeate flux (550-600L/m(2)/h/bar), the ELP-Z precipitates are shown to severely foul the TFF membrane, causing rapid flux decay. Confocal microscopy of the ELP-Z-mAb and ELP-Z precipitates suggests significant differences in the morphology and the kinetics of formation of these precipitates, which is likely responsible for their different behavior during TFF. Finally, an alternative normal flow filtration strategy is developed for the ELP-Z precipitate recovery step during mAb elution, using a combination of 5μm and a 0.45/0.2μm filters. Using this approach, the ELP-Z precipitates are separated from the final mAb elution pool at high volumetric throughputs and high ELP-Z recovery (96%) is obtained after resolubilization from the filter. This study demonstrates that the ELP-Z affinity precipitation process can be readily scaled up using conventional membrane processing.
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, United States; Biologics Process Development, Bristol-Myers Squibb, East Syracuse, NY, United States.
authors.forename: Rahul D
authors.initials: RD
authors.lastname: Sheth
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, East Syracuse, NY, United States.
authors.forename: Bharat V
authors.initials: BV
authors.lastname: Bhut
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, East Syracuse, NY, United States.
authors.forename: Mi
authors.initials: M
authors.lastname: Jin
authors.affiliation: Biologics Process Development, Bristol-Myers Squibb, East Syracuse, NY, United States.
authors.forename: Zhengjian
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, United States.
authors.forename: Wilfred
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180, United States. Electronic address: crames@rpi.edu.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Cramer
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification; D000915:Antibody Affinity; D011232:Chemical Precipitation; D004549:Elastin; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005374:Filtration; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D010455:Peptides; D011993:Recombinant Fusion Proteins; D004549:Elastin
keywords: Affinity precipitation; Elastin-like polypeptides; Monoclonal antibodies; Normal flow filtration; Scale-up; Tangential flow filtration