title: A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins.
abstract: Rationale. Evaluation of the potential allergenicity of proteins derived from genetically modified foods has involved a weight of evidence approach that incorporates an evaluation of protein digestibility in pepsin. Currently, there is no standardized protocol to assess the digestibility of proteins using simulated gastric fluid. Potential variations in assay parameters include: pH, pepsin purity, pepsin to target protein ratio, target protein purity, and method of detection. The objective was to assess the digestibility of a common set of proteins in nine independent laboratories to determine the reproducibility of the assay when performed using a common protocol. Methods. A single lot of each test protein and pepsin was obtained and distributed to each laboratory. The test proteins consisted of Ara h 2 (a peanut conglutin-like protein), beta-lactoglobulin, bovine serum albumin, concanavalin A, horseradish peroxidase, ovalbumin, ovomucoid, phosphinothricin acetyltransferase, ribulose diphosphate carboxylase, and soybean trypsin inhibitor. A ratio of 10U of pepsin activity/microg test protein was selected for all tests (3:1 pepsin to protein, w:w). Digestions were performed at pH 1.2 and 2.0, with sampling at 0.5, 2, 5, 10, 20, 30, and 60min. Protein digestibility was assessed from stained gels following SDS-PAGE of digestion samples and controls. Results. Results were relatively consistent across laboratories for the full-length proteins. The identification of proteolytic fragments was less consistent, being affected by different fixation and staining methods. Overall, assay pH did not influence the time to disappearance of the full-length protein or protein fragments, however, results across laboratories were more consistent at pH 1.2 (91% agreement) than pH 2.0 (77%). Conclusions. These data demonstrate that this common protocol for evaluating the in vitro digestibility of proteins is reproducible and yields consistent results when performed using the same proteins at different laboratories.
authors.affiliation: ILSI Health and Environmental Sciences Institute, Washington, DC, USA. kthomas@ilsi.org
authors.forename: K
authors.initials: K
authors.lastname: Thomas
authors.forename: M
authors.initials: M
authors.lastname: Aalbers
authors.forename: G A
authors.initials: GA
authors.lastname: Bannon
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authors.lastname: Bartels
authors.forename: R J
authors.initials: RJ
authors.lastname: Dearman
authors.forename: D J
authors.initials: DJ
authors.lastname: Esdaile
authors.forename: T J
authors.initials: TJ
authors.lastname: Fu
authors.forename: C M
authors.initials: CM
authors.lastname: Glatt
authors.forename: N
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authors.lastname: Hadfield
authors.forename: C
authors.initials: C
authors.lastname: Hatzos
authors.forename: S L
authors.initials: SL
authors.lastname: Hefle
authors.forename: J R
authors.initials: JR
authors.lastname: Heylings
authors.forename: R E
authors.initials: RE
authors.lastname: Goodman
authors.forename: B
authors.initials: B
authors.lastname: Henry
authors.forename: C
authors.initials: C
authors.lastname: Herouet
authors.forename: M
authors.initials: M
authors.lastname: Holsapple
authors.forename: G S
authors.initials: GS
authors.lastname: Ladics
authors.forename: T D
authors.initials: TD
authors.lastname: Landry
authors.forename: S C
authors.initials: SC
authors.lastname: MacIntosh
authors.forename: E A
authors.initials: EA
authors.lastname: Rice
authors.forename: L S
authors.initials: LS
authors.lastname: Privalle
authors.forename: H Y
authors.initials: HY
authors.lastname: Steiner
authors.forename: R
authors.initials: R
authors.lastname: Teshima
authors.forename: R
authors.initials: R
authors.lastname: Van Ree
authors.forename: M
authors.initials: M
authors.lastname: Woolhiser
authors.forename: J
authors.initials: J
authors.lastname: Zawodny
mesh_terms: D019411:Clinical Laboratory Techniques / Q000592:standards*; D004063:Digestion; D004591:Electrophoresis, Polyacrylamide Gel; D005765:Gastrointestinal Agents / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D010434:Pepsin A / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D015203:Reproducibility of Results
chemical_list: D005765:Gastrointestinal Agents; D010446:Peptide Fragments; D011506:Proteins; D010434:Pepsin A