Development of an analytical reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry method for characterization of recombinant antibodies

Development of an analytical reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry method for characterization of recombinant antibodies

Analytical characterization of monoclonal antibodies has been hindered by the lack of appropriate chromatographic methods to be used in conjunction with high-resolution MS. Current methodologies for standard RP-HPLC are incompatible with antibodies due to irreproducibility, low recovery, short colum...

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Veröffentlicht in:Journal of Chromatography A 2004-10, Vol.1053 (1-2), p.299-305
Hauptverfasser: Dillon, Thomas M, Bondarenko, Pavel V, Speed Ricci, Margaret
Format: Artikel
Sprache:eng
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Antibodies - chemistry
Chromatography, Gel - methods
Chromatography, High Pressure Liquid - methods
Recombinant Proteins - chemistry
Spectrometry, Mass, Electrospray Ionization - methods
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container_title Journal of Chromatography A
container_volume 1053
creator Dillon, Thomas M
Bondarenko, Pavel V
Speed Ricci, Margaret
description Analytical characterization of monoclonal antibodies has been hindered by the lack of appropriate chromatographic methods to be used in conjunction with high-resolution MS. Current methodologies for standard RP-HPLC are incompatible with antibodies due to irreproducibility, low recovery, short column lifetimes, and poor resolution of degradation products. An analytical RP-HPLC-MS method was developed for monitoring and characterizing intact IgG1antibodies. Key parameters required for improved chromatographic resolution included long alkyl chains of the stationary phase (Zorbax SB300 C8), column temperatures elevated to 65-70 degrees C and combination of trifluoroacetic acid and heptafluorobutyric acid ion-pairing agents. RP chromatographic separation of degradation species and C-terminal lysine variants along with the characterization of glycosylation profile by mass spectrometry demonstrates the capability of this method for whole antibody analysis.
doi_str_mv 10.1016/s0021-9673(04)01410-4
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subjects Antibodies - chemistry
Chromatography, Gel - methods
Chromatography, High Pressure Liquid - methods
Recombinant Proteins - chemistry
Spectrometry, Mass, Electrospray Ionization - methods
title Development of an analytical reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry method for characterization of recombinant antibodies
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