title: Automated high-throughput purification of antibody fragments to facilitate evaluation in functional and kinetic based assays.
abstract: Screening antibodies from phage displayed in vitro libraries and from affinity maturation of lead antibodies requires testing of antibody fragments (scFvs and Fabs) for function and binding affinities. Crude scFv or Fab periplasmic preparations from Escherichia coli are often not pure and/or concentrated enough for use in functional and affinity assays. We have developed an automated high-throughput approach for small and large-scale expression and purification of His-tagged scFvs and Fabs using the Qiagen BioRobot 3000 LS with optimized application software. This automated procedure enabled us to rapidly evaluate antibody fragments in functional and surface plasmon resonance (SPR) assays. We have used these procedures to make thousands of purified scFv/Fabs for several antibody maturation campaigns and significantly decreased the time needed to select the best candidates. The assay results from these purified samples were used to prioritize candidates before converting them to IgG. This protocol can process up to 300 small-scale and up to 72 large-scale scFvs or Fabs per week per full-time employee (FTE).
authors.affiliation: Department of Vaccines and Biologics Research, Merck Research Laboratories, 770 Sumneytown Pike, P.O. Box 4, West Point, PA 19486, USA.
authors.forename: Bin
authors.initials: B
authors.lastname: Su
authors.forename: Renee
authors.initials: R
authors.lastname: Hrin
authors.forename: Barrett R
authors.initials: BR
authors.lastname: Harvey
authors.forename: Ying-Jie
authors.initials: YJ
authors.lastname: Wang
authors.forename: Robin E
authors.initials: RE
authors.lastname: Ernst
authors.forename: Richard A
authors.initials: RA
authors.lastname: Hampton
authors.forename: Michael D
authors.initials: MD
authors.lastname: Miller
authors.forename: William R
authors.initials: WR
authors.lastname: Strohl
authors.forename: Zhiqiang
authors.initials: Z
authors.lastname: An
authors.forename: Donna L
authors.initials: DL
authors.lastname: Montgomery
mesh_terms: D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007118:Immunoassay; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000302:isolation & purification*; D007141:Immunoglobulin Fc Fragments / Q000276:immunology / Q000302:isolation & purification*; D007700:Kinetics; D019151:Peptide Library; D012371:Robotics / Q000295:instrumentation*; D012984:Software; D020349:Surface Plasmon Resonance
chemical_list: D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D019151:Peptide Library