title: Sensitive methods for evaluation of antibodies for host cell protein analysis and screening of impurities in a vaccine process.
abstract: BACKGROUND
  Host cell proteins (HCP) should be carefully monitored in vaccine production. To achieve a reliable HCP estimation, a mixture of polyclonal antibodies (pAbs) with broad affinity would be of preference. Sensitive evaluations of the pAbs are therefore of value.
  
  
  METHODS
  Column purification of specific HCPs with affinity to the anti-HCP pAbs was compared with Western blotting of the anti-HCP pAbs binding to filter bound total lysate. The anti-HCP pAbs were used in an HCP quantification analysis using surface plasmon resonance (SPR). Host cell derived impurities from an influenza vaccine process were analyzed using 2-D DIGE analysis.
  
  
  RESULTS
  The Western blotting showed a similar HCP binding pattern of anti-HCP pAbs from immunizations using two adjuvants: CFA/IFA and AbISCO(®). From the column purification of HCPs, total proteins detectable were similar for all anti-HCP pAbs; however the immune response pattern differed significantly for the anti-HCP pAbs from the AbISCO(®) immunization. In the SPR HCP quantification assay the standard curve ranged from 0.3 to 40 μg/ml. The advantage of SPR compared with ELISA was the decreased hands on time and that the sample number was not limiting. The 2-D DIGE showed that most of the HCPs were removed at the clarification and virus capture step.
  
  
  CONCLUSIONS
  Column purification of HCPs with affinity to the anti-HCP pAbs increased the sensitivity of affinity analysis compared with Western blotting and opened the possibility of further analysis. The anti-HCP pAbs did not interact with proteins in the virus; simplifying analysis of process samples using SPR. 2-D DIGE analysis gave a direct study of the impurity profile with the advantage of independence from antibody performance.
authors.affiliation: GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden.
authors.forename: Christine Sund
authors.initials: CS
authors.lastname: Lundström
authors.affiliation: GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden.
authors.forename: Anna
authors.initials: A
authors.lastname: Mattsson
authors.affiliation: Isconova, Kungsgatan 109, SE-753 18 Uppsala, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Lövgren
authors.affiliation: GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden.
authors.forename: Åsa
authors.initials: Å
authors.lastname: Eriksson
authors.affiliation: GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden.
authors.forename: Åsa Liljegren
authors.initials: ÅL
authors.lastname: Sundberg
authors.affiliation: GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden.
authors.forename: Mats
authors.initials: M
authors.lastname: Lundgren
authors.affiliation: GE Healthcare Bio-Sciences AB, Björkgatan 30, SE-751 84 Uppsala, Sweden. Electronic address: camilla.estmernilsson@ge.com.
authors.forename: Camilla Estmer
authors.initials: CE
authors.lastname: Nilsson
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D000915:Antibody Affinity; D015153:Blotting, Western; D002522:Chlorocebus aethiops; D011506:Proteins / Q000276:immunology* / Q000302:isolation & purification; D020349:Surface Plasmon Resonance; D013678:Technology, Pharmaceutical / Q000379:methods; D057825:Two-Dimensional Difference Gel Electrophoresis; D014612:Vaccines / Q000737:chemistry; D014709:Vero Cells
chemical_list: D000276:Adjuvants, Immunologic; D000906:Antibodies; D011506:Proteins; D014612:Vaccines
keywords: 2-D DIGE; Adjuvant; Biacore; Host cell protein; Influenza virus; SPR