title: A case study on Sema3E-Fc aggregation and assay-dependent differences in quantitation.
abstract: BACKGROUND
  In evaluating the serum concentrations in mice of a Sema3E IgG1 Fc fusion protein, a possible antitumor agent, two ELISAs were developed: a generic assay detecting only the Fc portion of the therapeutic and a specific receptor-binding assay detecting intact protein.
  
  
  RESULTS
  An unexpected discrepancy was observed in the measured in vivo serum concentrations, with the generic ELISA yielding higher concentrations than the specific ELISA. Size-exclusion HPLC and SDS-PAGE analysis of in vitro serum stability samples revealed extensive aggregation of Sema3E-Fc. The generic assay recovered more Sema3E-Fc in the presence of aggregates than the specific assay.
  
  
  CONCLUSIONS
  Biophysical characterization combined with immunochemical analysis was key to elucidating not only the nature of the protein instability, but also the cause for the assay discrepancy.
authors.affiliation: Department of Pharmacokinetic & Pharmacodynamic Sciences, Genentech, Inc., CA, USA.
authors.forename: Daniela
authors.initials: D
authors.lastname: Bumbaca
authors.forename: Raymond K
authors.initials: RK
authors.lastname: Tong
authors.forename: Alexander W
authors.initials: AW
authors.lastname: Koch
authors.forename: Hong
authors.initials: H
authors.lastname: Xiang
authors.forename: Laura E
authors.initials: LE
authors.lastname: DeForge
authors.forename: Ben-Quan
authors.initials: BQ
authors.lastname: Shen
authors.forename: Yanmei
authors.initials: Y
authors.lastname: Lu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000097:blood* / Q000378:metabolism / Q000493:pharmacokinetics; D039961:Semaphorins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D013997:Time Factors
chemical_list: D000906:Antibodies; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins; C494123:SEMA3E protein, human; D039961:Semaphorins