title: LC-MS/MS strategies for therapeutic antibodies and investigation into the quantitative impact of antidrug-antibodies.
abstract: OBJECTIVE
  We aimed to establish novel, high-throughput LC-MS/MS strategies for quantification of monoclonal antibodies in human serum and examine the potential impact of antidrug antibodies.
  
  
  METHODS
  We present two strategies using a thermally stable immobilized trypsin. The first strategy uses whole serum digestion and the second introduces Protein G enrichment to improve the selectivity. The impact of anti-trastuzumab antibodies on the methods was tested.
  
  
  CONCLUSIONS
  Whole serum digestion has been validated for trastuzumab (LLOQ 0.25 µg/ml). Protein G enrichment has been validated for trastuzumab (LLOQ 0.1 µg/ml), bevacizumab (LLOQ 0.1 µg/ml) and adalimumab (LLOQ 0.25 µg/ml). We have shown the potential for anti-drug antibodies to impact on the quantification and we have subsequently established a strategy to overcome this impact where total quantification is desired.
authors.affiliation: Bioanalytical Services, Covance Laboratories Ltd., Otley Road, Harrogate, HG31PY, UK.
authors.forename: Matthew
authors.initials: M
authors.lastname: Ewles
authors.affiliation: Bioanalytical Services, Covance Laboratories Ltd., Otley Road, Harrogate, HG31PY, UK.
authors.forename: Ranbir
authors.initials: R
authors.lastname: Mannu
authors.affiliation: Bioanalytical Services, Covance Laboratories Ltd., Otley Road, Harrogate, HG31PY, UK.
authors.forename: Chris
authors.initials: C
authors.lastname: Fox
authors.affiliation: Bioanalytical Services, Covance Laboratories Ltd., Otley Road, Harrogate, HG31PY, UK.
authors.forename: Johannes
authors.initials: J
authors.lastname: Stanta
authors.affiliation: Bioanalytical Services, Covance Laboratories Ltd., Otley Road, Harrogate, HG31PY, UK.
authors.forename: Graeme
authors.initials: G
authors.lastname: Evans
authors.affiliation: Bioanalytical Services, Covance Laboratories Ltd., Otley Road, Harrogate, HG31PY, UK.
authors.forename: Lee
authors.initials: L
authors.lastname: Goodwin
authors.affiliation: Biopharmaceutical CMC Solutions, Covance Laboratories Ltd, Otley Road, Harrogate, HG31PY, UK.
authors.forename: James
authors.initials: J
authors.lastname: Duffy
authors.affiliation: Biopharmaceutical CMC Solutions, Covance Laboratories Ltd, Otley Road, Harrogate, HG31PY, UK.
authors.forename: Len
authors.initials: L
authors.lastname: Bell
authors.affiliation: Biopharmaceutical CMC Solutions, Covance Laboratories Ltd, Otley Road, Harrogate, HG31PY, UK.
authors.forename: Sian
authors.initials: S
authors.lastname: Estdale
authors.affiliation: Biopharmaceutical CMC Solutions, Covance Laboratories Ltd, Otley Road, Harrogate, HG31PY, UK.
authors.forename: David
authors.initials: D
authors.lastname: Firth
mesh_terms: D000068879:Adalimumab / Q000097:blood / Q000276:immunology / Q000378:metabolism; D000888:Antibodies, Anti-Idiotypic / Q000737:chemistry* / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000097:blood* / Q000276:immunology / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D000068258:Bevacizumab / Q000097:blood / Q000276:immunology / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D057230:Limit of Detection; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D000068878:Trastuzumab / Q000097:blood / Q000276:immunology / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D000068879:Adalimumab; D000888:Antibodies, Anti-Idiotypic; D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D000068258:Bevacizumab; D010455:Peptides; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; D014357:Trypsin; C034284:IgG Fc-binding protein, Streptococcus; C508053:ERBB2 protein, human
keywords: ADA (antidrug antibodies); LC–MS/MS; adalimumab; antibody–drug conjugates; bevacizumab; digestion; monoclonal; trastuzumab