Enabling adoption of 2D-NMR for the higher order structure assessment of monoclonal antibody therapeutics

Abstract

The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.

An NMR-Based Similarity Metric for Higher Order Structure Quality Assessment Among U.S. Marketed Insulin Therapeutics

“…18 Subsequent work on monoclonal antibody HOS comparison reported even lower thresholds for the same protein across many laboratories. 31 However, most published studies on NMR spectral comparison for peptide or protein HOS of different brands of drugs lack quantification or a numerical similarity score and proposed threshold. Such a numerical score if reproducible and robust could be useful for evaluating the equivalence of generic or biosimilar drugs to the RLD.…”

NMR‐Studien an biologischen Makromolekülen (>100 kDa) ohne Notwendigkeit der Deuterierung: Das XL‐ALSOFAST Experiment mit verzögerter Entkopplung

“…Eine wichtige sich entwickelnde Anwendung von NMR in der pharmazeutischen Industrie ist die Qualitätskontrolle durch Messung von Methylspektren bei natürlicher Isotopenhäufigkeit von biologischen Therapeutika, wie monoklonalen Antikçrpern (150 kDa). [15][16][17]38] Die Mçglichkeit mit vollständig protonierten Systemen mit hohem Molekulargewicht zu Arbeiten und zur selben Zeit eine exzellente Artefaktunterdrückung zu bewerkstelligen macht das gs-XL-ALSOFAST-HMQC ideal für diese Anwendung. Wir haben die Leistung von verschiedenen Experimenten an einer Lçsung eines monoklonalen Antikçrpers (40 mg mL À1 ) bei 323 K getestet (Abbildung 5 D) und haben die Signal-Rausch-Verhältnisse für ausgewählte Signale dieser Fingerabdruck-Spektren ausgewertet.…”

“…mit einem einzigen Spektrum überprüft werden kann. [15][16][17][18] Auch hier kann die Messung auf einem Hochfeld-Spektrometer gut einen Tag in Anspruch nehmen.…”

Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

“…Producing crystalline mAbs is challenging, owing to the complexity of mAb HOS and the degree of mAb conformational heterogeneity. However, several complementing technologies have evolved in recent years to ascertain structure and interactions, including circular dichroism (CD), infrared (IR) and raman spectroscopy, cryogenic-electron microscopy, and nuclear magnetic resonance (NMR) spectroscopy [148,150,151,152,153,154,155,156,157]. Of the technologies available for HOS elucidation, 2D-NMR and X-Ray crystallography (followed by MS) provide the highest sensitivity and local specificity.…”