Development and application of a robust N-glycan profiling method for heightened characterization of monoclonal antibodies and related glycoproteins.
A highly robust hydrophilic interaction liquid chromatography (HILIC) method that involves both fluorescence and mass spectrometric detection was developed for profiling and characterizing enzymatically released and 2-aminobenzamide (2-AB)-derivatized mAb N-glycans. Online HILIC/mass spectrometry (MS) with a quadrupole time-of-flight mass spectrometer provides accurate mass identifications of the separated, 2-AB-labeled N-glycans. The method features a high-resolution, low-shedding HILIC column with acetonitrile and water-based mobile phases containing trifluoroacetic acid (TFA) as a modifier. This column and solvent system ensures the combination of robust chromatographic performance and full compatibility and sensitivity with online MS in addition to the baseline separation of all typical mAb N-glycans. The use of TFA provided distinct advantages over conventional ammonium formate as a mobile phase additive, such as, optimal elution order for sialylated N-glycans, reproducible chromatographic profiles, and matching total ion current chromatograms, as well as minimal signal splitting, analyte adduction, and fragmentation during HILIC/MS, maximizing sensitivity for trace-level species. The robustness and selectivity of HILIC for N-glycan analyses allowed for method qualification. The method is suitable for bioprocess development activities, heightened characterization, and clinical drug substance release. Application of this HILIC/MS method to the detailed characterization of a marketed therapeutic mAb, Rituxan(®), is described.
Analytical Research and Development, Biotherapeutics Pharmaceutical SciencesPfizer, Inc.AndoverMassachusetts 01810.
Tanya Q
TQ
Shang
Analytical Research and Development, Biotherapeutics Pharmaceutical SciencesPfizer, Inc.AndoverMassachusetts 01810.
Andrew
A
Saati
Genzyme, A Sanofi Company, FraminghamMassachusetts01701.
Kelly N
KN
Toler
Analytical R&D, Pfizer, Inc.St. LouisMissouri63017.
Jianming
J
Mo
Analytical R&D, Pfizer, Inc., Pearl RiverNew York10965.
Heyi
H
Li
Analytical R&D, Pfizer, Inc., Pearl RiverNew York10965.
Tonya
T
Matlosz
Analytical R&D, Pfizer, Inc., Pearl RiverNew York10965.
Xi
X
Lin
Analytical Research and Development, Biotherapeutics Pharmaceutical SciencesPfizer, Inc.AndoverMassachusetts 01810.
Jennifer
J
Schenk
Analytical Research and Development, Biotherapeutics Pharmaceutical SciencesPfizer, Inc.AndoverMassachusetts 01810.
Chee-Keng
CK
Ng
Analytical Research and Development, Biotherapeutics Pharmaceutical SciencesPfizer, Inc.AndoverMassachusetts 01810.
Toni
T
Duffy
Analytical Research and Development, Biotherapeutics Pharmaceutical SciencesPfizer, Inc.AndoverMassachusetts 01810.
Thomas J
TJ
Porter
Analytical Research and Development, Biotherapeutics Pharmaceutical SciencesPfizer, Inc.AndoverMassachusetts 01810. Electronic address: Jason.rouse@pfizer.com.
Jason C
JC
Rouse
D058846:Antibodies, Monoclonal, Murine-Derived / Q000737:chemistry*; D001702:Biopharmaceutics / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D055808:Drug Discovery / Q000295:instrumentation / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D011134:Polysaccharides / Q000737:chemistry*; D000069283:Rituximab; D021241:Spectrometry, Mass, Electrospray Ionization
D058846:Antibodies, Monoclonal, Murine-Derived; D006023:Glycoproteins; D011134:Polysaccharides; D000069283:Rituximab
HPLC; LC-MS; biopharmaceuticals characterization; glycosylation; liquid chromatography; mass spectrometry; monoclonal antibody; protein structure