title: Simple NMR methods for evaluating higher order structures of monoclonal antibody therapeutics with quinary structure.
abstract: Monoclonal antibody (mAb) drugs constitute the largest class of protein therapeutics currently on the market. Correctly folded protein higher order structure (HOS), including quinary structure, is crucial for mAb drug quality. The quinary structure is defined as the association of quaternary structures (e.g., oligomerized mAb). Here, several commonly available analytical methods, i.e., size-exclusion-chromatography (SEC) FPLC, multi-angle light scattering (MALS), circular dichroism (CD), NMR and multivariate analysis, were combined and modified to yield a complete profile of HOS and comparable metrics. Rituximab and infliximab were chosen for method evaluation because both IgG1 molecules are known to be homologous in sequence, superimposable in Fab crystal structure and identical in Fc structure. However, herein the two are identified to be significantly different in quinary structure in addition to minor secondary structure differences. All data collectively showed rituximab was mostly monomeric while infliximab was in mono-oligomer equilibrium driven by its Fab fragment. The quinary structure differences were qualitatively inferred from the less used but more reproducible dilution-injection-SEC-FPLC curve method. Quantitative principal component analysis (PCA) was performed on NMR spectra of either the intact or the in-situ enzymatic-digested mAb samples. The cleavage reactions happened directly in NMR tubes without further separation, which greatly enhanced NMR spectra quality and resulted in larger inter- and intra-lot variations based on PCA. The new in-situ enzymatic digestion method holds potential in identifying structural differences on larger therapeutic molecules using NMR.
authors.affiliation: Division of Pharmaceutical Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, 20993, United States. Electronic address: kang.chen@fda.hhs.gov.
authors.forename: Kang
authors.initials: K
authors.lastname: Chen
authors.affiliation: Division of Pharmaceutical Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, US Food and Drug Administration, St. Louis, MO, 63110, United States.
authors.forename: Dianna S
authors.initials: DS
authors.lastname: Long
authors.affiliation: Division II, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, 20993, United States.
authors.forename: Scott C
authors.initials: SC
authors.lastname: Lute
authors.affiliation: Division of Pharmaceutical Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, US Food and Drug Administration, St. Louis, MO, 63110, United States.
authors.forename: Michaella J
authors.initials: MJ
authors.lastname: Levy
authors.affiliation: Division II, Office of Biotechnology Products, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, US Food and Drug Administration, Silver Spring, MD, 20993, United States.
authors.forename: Kurt A
authors.initials: KA
authors.lastname: Brorson
authors.affiliation: Division of Pharmaceutical Analysis, Office of Testing and Research, Office of Pharmaceutical Quality, Center for Drug Evaluation and Research, US Food and Drug Administration, St. Louis, MO, 63110, United States.
authors.forename: David A
authors.initials: DA
authors.lastname: Keire
mesh_terms: D002623:Chemistry Techniques, Analytical; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry; D000069285:Infliximab / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D011487:Protein Conformation; D000069283:Rituximab / Q000737:chemistry*
chemical_list: D007140:Immunoglobulin Fab Fragments; D000069283:Rituximab; D000069285:Infliximab
keywords: Digestion; Dilution-injection; PCA; SEC-FPLC; in-situ