TY - JOUR
T1 - An automated robotic platform for rapid profiling oligosaccharide analysis of monoclonal antibodies directly from cell culture
AU - Doherty, Margaret
AU - Bones, Jonathan
AU - McLoughlin, Niaobh
AU - Telford, Jayne E.
AU - Harmon, Bryan
AU - DeFelippis, Michael R.
AU - Rudd, Pauline M.
PY - 2013
Y1 - 2013
N2 - Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5- h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies.
AB - Oligosaccharides attached to Asn297 in each of the CH2 domains of monoclonal antibodies play an important role in antibody effector functions by modulating the affinity of interaction with Fc receptors displayed on cells of the innate immune system. Rapid, detailed, and quantitative N-glycan analysis is required at all stages of bioprocess development to ensure the safety and efficacy of the therapeutic. The high sample numbers generated during quality by design (QbD) and process analytical technology (PAT) create a demand for high-performance, high-throughput analytical technologies for comprehensive oligosaccharide analysis. We have developed an automated 96-well plate-based sample preparation platform for high-throughput N-glycan analysis using a liquid handling robotic system. Complete process automation includes monoclonal antibody (mAb) purification directly from bioreactor media, glycan release, fluorescent labeling, purification, and subsequent ultra-performance liquid chromatography (UPLC) analysis. The entire sample preparation and commencement of analysis is achieved within a 5- h timeframe. The automated sample preparation platform can easily be interfaced with other downstream analytical technologies, including mass spectrometry (MS) and capillary electrophoresis (CE), for rapid characterization of oligosaccharides present on therapeutic antibodies.
KW - Automation
KW - Cell culture media
KW - Glycomics
KW - Monoclonal antibodies
KW - Oligosaccharides
KW - Ultra-performance liquid chromatography
UR - http://www.scopus.com/inward/record.url?scp=84885636807&partnerID=8YFLogxK
U2 - 10.1016/j.ab.2013.07.005
DO - 10.1016/j.ab.2013.07.005
M3 - Article
C2 - 23872006
AN - SCOPUS:84885636807
SN - 0003-2697
VL - 442
SP - 10
EP - 18
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -