title: Assessment of stable isotope incorporation into recombinant proteins.
abstract: Stable isotope labeling combined with mass spectrometry has been widely used in a diverse set of applications in the biochemistry and biomedical fields. When stable isotope-labeled proteins are produced via metabolic labeling of cell culture, a comprehensive assessment of the labeling pattern is imperative. In this study, we present a set of mass spectrometry-based bioanalytical tools developed for quantitatively tracing the levels of the stable isotopes incorporated into the recombinant proteins (monoclonal antibodies and Fc fusion proteins expressed in different host systems) that include total mass analysis, peptide mapping analysis, and amino acid analysis. We show that these three mass spectrometry-based analytical methods have distinctive advantages and limitations and that they are mutually complementary in evaluating the quality of stable isotope-labeled proteins. In addition, we show that the analytical techniques developed here are powerful tools to provide valuable insights into studying cell metabolism and performing flux analysis during cell culture.
authors.affiliation: Analytical Sciences, Amgen, Thousand Oaks, CA 91320, USA. xinz@amgen.com
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.forename: Quanzhou
authors.initials: Q
authors.lastname: Luo
authors.forename: Izydor
authors.initials: I
authors.lastname: Apostol
authors.forename: Shun
authors.initials: S
authors.lastname: Luo
authors.forename: Matthew
authors.initials: M
authors.lastname: Jerums
authors.forename: Gang
authors.initials: G
authors.lastname: Huang
authors.forename: Xinzhao Grace
authors.initials: XG
authors.lastname: Jiang
authors.forename: Jessica
authors.initials: J
authors.lastname: Gastwirt
authors.forename: Nimesh
authors.initials: N
authors.lastname: Savjani
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Lewis
authors.forename: Ronald
authors.initials: R
authors.lastname: Keener
authors.forename: Jette
authors.initials: J
authors.lastname: Wypych
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D016466:CHO Cells; D018929:Cell Culture Techniques / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D007141:Immunoglobulin Fc Fragments / Q000096:biosynthesis*; D007553:Isotope Labeling / Q000379:methods*; D013058:Mass Spectrometry / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*
chemical_list: D000911:Antibodies, Monoclonal; D007141:Immunoglobulin Fc Fragments; D011993:Recombinant Fusion Proteins