title: N-glycan PK Profiling Using a High Sensitivity nanoLCMS Work-Flow with Heavy Stable Isotope Labeled Internal Standard and Application to a Preclinical Study of an IgG1 Biopharmaceutical.
abstract: OBJECTIVE
  In this study an innovative, highly sensitive work-flow is presented that allows the analysis of a possible influence of individual glyco-variants on pharmacokinetics already during pre-clinical development. Possible effects on the pharmacokinetics caused by glyco-variants have been subject of several studies with in part contradictory results which can be related to differences in the set-up.
  
  
  METHODS
  Using 96-well plate based affinity purification an IgG1 antibody was isolated from preclinical samples and glycans were analyzed individually by nanoLCMS. Prerequisite was a reference standard based on stable heavy isotope labeled glycans. The high sensitivity and low sample consumption enabled the integration into the preclinical development program.
  
  
  RESULTS
  The data of an IgG1 biopharmaceutical from a preclinical rabbit study showed that some N-glycoforms have a different PK profile compared with the average of all molecule variants as determined by ELISA. IgG1 high mannose glycoforms M5 and M6 were removed from circulation at a higher rate.
  
  
  CONCLUSIONS
  The results of the preclinical study demonstrated the applicability of the developed innovative workflow. The PK profile of glyco-variants could be determined individually. It was concluded that M6 was converted by mannosidases in circulation to M5 which in turn was selectively cleared by mannose receptor binding which is in-line with previously published results. Therefore the developed technology delivers reliable results and can be applied for PK profiling of other mAbs and other types of biopharmaceuticals.
authors.affiliation: Analytical Characterization, Sandoz Biopharmaceuticals, HEXAL AG, Keltenring 1+3, 82041, Oberhaching, Germany. fabian.higel@sandoz.com.
authors.forename: Fabian
authors.initials: F
authors.lastname: Higel
authors.affiliation: Analytical Characterization, Sandoz Biopharmaceuticals, HEXAL AG, Keltenring 1+3, 82041, Oberhaching, Germany. andreas.seidl@sandoz.com.
authors.forename: Andreas
authors.initials: A
authors.lastname: Seidl
authors.affiliation: Process Analytics, Sandoz Biopharmaceuticals, Schaftenau, Austria.
authors.forename: Uwe
authors.initials: U
authors.lastname: Demelbauer
authors.affiliation: Clinical Bioanalytics, Sandoz Biopharmaceuticals, HEXAL AG, Oberhaching, Germany.
authors.forename: Margot
authors.initials: M
authors.lastname: Viertlboeck-Schudy
authors.affiliation: Clinical Bioanalytics, Sandoz Biopharmaceuticals, HEXAL AG, Oberhaching, Germany.
authors.forename: Vera
authors.initials: V
authors.lastname: Koppenburg
authors.affiliation: Clinical R&D, Sandoz Biopharmaceuticals, HEXAL AG, Holzkirchen, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Kronthaler
authors.affiliation: IBMP - Institute for Biomedical and Pharmaceutical Research, Nürnberg-Heroldsberg, Germany.|Institute of Pharmacology, Faculty of Medicine, University Duisburg-Essen, Essen, Germany.
authors.forename: Fritz
authors.initials: F
authors.lastname: Sörgel
authors.affiliation: Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Ludwig Maximilians-Universität München, Munich, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Friess
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000097:blood* / Q000737:chemistry; D000915:Antibody Affinity; D001702:Biopharmaceutics / Q000379:methods*; D002247:Carbon Isotopes; D002853:Chromatography, Liquid / Q000379:methods; D004353:Drug Evaluation, Preclinical; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry; D007074:Immunoglobulin G / Q000097:blood* / Q000737:chemistry; D007279:Injections, Subcutaneous; D057230:Limit of Detection; D013058:Mass Spectrometry / Q000379:methods; D011134:Polysaccharides / Q000737:chemistry*; D011817:Rabbits; D012015:Reference Standards; D057188:Workflow
chemical_list: D000911:Antibodies, Monoclonal; D002247:Carbon Isotopes; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011134:Polysaccharides
keywords: N-glycosylation; high mannose; mass spectrometry; monoclonal antibody; pharmacokinetics