TY - JOUR
T1 - Evaluation of spectral libraries and sample preparation for DIA-LC-MS analysis of host cell proteins
T2 - A case study of a bacterially expressed recombinant biopharmaceutical protein
AU - Heissel, Søren
AU - Bunkenborg, Jakob
AU - Kristiansen, Max Per
AU - Holmbjerg, Anne Fich
AU - Grimstrup, Marie
AU - Mørtz, Ejvind
AU - Kofoed, Thomas
AU - Højrup, Peter
N1 - Copyright © 2018. Published by Elsevier Inc.
PY - 2018
Y1 - 2018
N2 - Recombinantly expressed biopharmaceutical proteins often undergo a series of purification steps with the aim of removing contaminating material. Depending on the application of the protein, there are various requirements for the degree of purity, but host cell proteins (HCPs) will in general remain in small amounts. LC-MS has emerged as an orthogonal technique, capable of providing detailed information regarding the individual proteins. The aim of this case study was to characterize the HCPs associated with a biopharmaceutical protein, provided by Statens Serum Institut (DK), which is used in the field of tuberculosis and has not previously been studied by LC-MS. The developed method and acquired experiences served to develop a generalized strategy for HCP-characterization in our laboratory. We evaluated the use of different spectral libraries, recorded in data-dependent mode for obtaining the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced accurate quantification across several orders of magnitude, and the calculated purity was found to be in agreement with previously obtained ELISA data.
AB - Recombinantly expressed biopharmaceutical proteins often undergo a series of purification steps with the aim of removing contaminating material. Depending on the application of the protein, there are various requirements for the degree of purity, but host cell proteins (HCPs) will in general remain in small amounts. LC-MS has emerged as an orthogonal technique, capable of providing detailed information regarding the individual proteins. The aim of this case study was to characterize the HCPs associated with a biopharmaceutical protein, provided by Statens Serum Institut (DK), which is used in the field of tuberculosis and has not previously been studied by LC-MS. The developed method and acquired experiences served to develop a generalized strategy for HCP-characterization in our laboratory. We evaluated the use of different spectral libraries, recorded in data-dependent mode for obtaining the highest HCP coverage, combined with SWATH-based absolute quantification. The accuracy of two label-free absolute quantification strategies was evaluated using stable isotope peptides. Two different sample preparation workflows were evaluated for optimal HCP yield. . The label-free strategy produced accurate quantification across several orders of magnitude, and the calculated purity was found to be in agreement with previously obtained ELISA data.
U2 - 10.1016/j.pep.2018.03.002
DO - 10.1016/j.pep.2018.03.002
M3 - Journal article
C2 - 29526817
VL - 147
SP - 69
EP - 77
JO - Protein Expression and Purification
JF - Protein Expression and Purification
SN - 1046-5928
ER -