A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins

K. Thomas, M. Aalbers, G. A. Bannon, M. Bartels, R. J. Dearman, D. J. Esdaile, T. J. Fu, C. M. Glatt, N. Hadfield, C. Hatzos, S. L. Hefle, J. R. Heylings, Richard E Goodman, B. Henry, C. Herouet, M. Holsapple, G. S. Ladics, T. D. Landry, S. C. MacIntosh, E. A. Rice & 6 others L. S. Privalle, H. Y. Steiner, R. Teshima, R. Van Ree, M. Woolhiser, J. Zawodny

Research output: Contribution to journalArticle

191 Citations (Scopus)

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), β-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/μg 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.

Original languageEnglish (US)
Pages (from-to)87-98
Number of pages12
JournalRegulatory Toxicology and Pharmacology
Volume39
Issue number2
DOIs
StatePublished - Apr 1 2004

Fingerprint

Pepsin A
Digestion
Assays
Safety
Proteins
In Vitro Techniques
Genetically Modified Food
Ovomucin
Ribulose-Bisphosphate Carboxylase
Lactoglobulins
Trypsin Inhibitors
Ovalbumin
Horseradish Peroxidase
Concanavalin A
Bovine Serum Albumin
Soybeans

Keywords

  • Biotechnology
  • Digestive stability
  • Genetically modified foods
  • Pepsinolysis
  • Protein allergenicity

ASJC Scopus subject areas

  • Toxicology

Cite this

A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. / Thomas, K.; Aalbers, M.; Bannon, G. A.; Bartels, M.; Dearman, R. J.; Esdaile, D. J.; Fu, T. J.; Glatt, C. M.; Hadfield, N.; Hatzos, C.; Hefle, S. L.; Heylings, J. R.; Goodman, Richard E; Henry, B.; Herouet, C.; Holsapple, M.; Ladics, G. S.; Landry, T. D.; MacIntosh, S. C.; Rice, E. A.; Privalle, L. S.; Steiner, H. Y.; Teshima, R.; Van Ree, R.; Woolhiser, M.; Zawodny, J.

In: Regulatory Toxicology and Pharmacology, Vol. 39, No. 2, 01.04.2004, p. 87-98.

Research output: Contribution to journalArticle

Thomas, K, Aalbers, M, Bannon, GA, Bartels, M, Dearman, RJ, Esdaile, DJ, Fu, TJ, Glatt, CM, Hadfield, N, Hatzos, C, Hefle, SL, Heylings, JR, Goodman, RE, Henry, B, Herouet, C, Holsapple, M, Ladics, GS, Landry, TD, MacIntosh, SC, Rice, EA, Privalle, LS, Steiner, HY, Teshima, R, Van Ree, R, Woolhiser, M & Zawodny, J 2004, 'A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins', Regulatory Toxicology and Pharmacology, vol. 39, no. 2, pp. 87-98. https://doi.org/10.1016/j.yrtph.2003.11.003
Thomas, K. ; Aalbers, M. ; Bannon, G. A. ; Bartels, M. ; Dearman, R. J. ; Esdaile, D. J. ; Fu, T. J. ; Glatt, C. M. ; Hadfield, N. ; Hatzos, C. ; Hefle, S. L. ; Heylings, J. R. ; Goodman, Richard E ; Henry, B. ; Herouet, C. ; Holsapple, M. ; Ladics, G. S. ; Landry, T. D. ; MacIntosh, S. C. ; Rice, E. A. ; Privalle, L. S. ; Steiner, H. Y. ; Teshima, R. ; Van Ree, R. ; Woolhiser, M. ; Zawodny, J. / A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins. In: Regulatory Toxicology and Pharmacology. 2004 ; Vol. 39, No. 2. pp. 87-98.
@article{85797765c2384563a9b5530d030ced28,
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), β-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/μg 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.",
keywords = "Biotechnology, Digestive stability, Genetically modified foods, Pepsinolysis, Protein allergenicity",
author = "K. Thomas and M. Aalbers and Bannon, {G. A.} and M. Bartels and Dearman, {R. J.} and Esdaile, {D. J.} and Fu, {T. J.} and Glatt, {C. M.} and N. Hadfield and C. Hatzos and Hefle, {S. L.} and Heylings, {J. R.} and Goodman, {Richard E} and B. Henry and C. Herouet and M. Holsapple and Ladics, {G. S.} and Landry, {T. D.} and MacIntosh, {S. C.} and Rice, {E. A.} and Privalle, {L. S.} and Steiner, {H. Y.} and R. Teshima and {Van Ree}, R. and M. Woolhiser and J. Zawodny",
year = "2004",
month = "4",
day = "1",
doi = "10.1016/j.yrtph.2003.11.003",
language = "English (US)",
volume = "39",
pages = "87--98",
journal = "Regulatory Toxicology and Pharmacology",
issn = "0273-2300",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - A multi-laboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins

AU - Thomas, K.

AU - Aalbers, M.

AU - Bannon, G. A.

AU - Bartels, M.

AU - Dearman, R. J.

AU - Esdaile, D. J.

AU - Fu, T. J.

AU - Glatt, C. M.

AU - Hadfield, N.

AU - Hatzos, C.

AU - Hefle, S. L.

AU - Heylings, J. R.

AU - Goodman, Richard E

AU - Henry, B.

AU - Herouet, C.

AU - Holsapple, M.

AU - Ladics, G. S.

AU - Landry, T. D.

AU - MacIntosh, S. C.

AU - Rice, E. A.

AU - Privalle, L. S.

AU - Steiner, H. Y.

AU - Teshima, R.

AU - Van Ree, R.

AU - Woolhiser, M.

AU - Zawodny, J.

PY - 2004/4/1

Y1 - 2004/4/1

N2 - 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), β-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/μg 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.

AB - 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), β-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/μg 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.

KW - Biotechnology

KW - Digestive stability

KW - Genetically modified foods

KW - Pepsinolysis

KW - Protein allergenicity

UR - http://www.scopus.com/inward/record.url?scp=12144289196&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=12144289196&partnerID=8YFLogxK

U2 - 10.1016/j.yrtph.2003.11.003

DO - 10.1016/j.yrtph.2003.11.003

M3 - Article

VL - 39

SP - 87

EP - 98

JO - Regulatory Toxicology and Pharmacology

JF - Regulatory Toxicology and Pharmacology

SN - 0273-2300

IS - 2

ER -