Interlaboratory Studies Using the NISTmAb to Advance Biopharmaceutical Structural Analytics

Abstract

Biopharmaceuticals such as monoclonal antibodies are required to be rigorously characterized using a wide range of analytical methods. Various material properties must be characterized and well controlled to assure that clinically relevant features and critical quality attributes are maintained. A thorough understanding of analytical method performance metrics, particularly emerging methods designed to address measurement gaps, is required to assure methods are appropriate for their intended use in assuring drug safety, stability, and functional activity. To this end, a series of interlaboratory studies have been conducted using NISTmAb, a biopharmaceutical-representative and publicly available monoclonal antibody test material, to report on state-of-the-art method performance, harmonize best practices, and inform on potential gaps in the analytical measurement infrastructure. Reported here is a summary of the study designs, results, and future perspectives revealed from these interlaboratory studies which focused on primary structure, post-translational modifications, and higher order structure measurements currently employed during biopharmaceutical development.

Keywords: biopharmaceutical; interlaboratory study; monoclonal antibody; nistmab; therapeutic protein.

FIGURE 1

Timeline of Global NISTmAb Interlaboratory Studies. (A) Representative timeline identifying the key milestones for an interlaboratory study. (B) Corresponding dates and key milestones for each NISTmAb interlaboratory study (MAM, glycosylation, NMR HOS, and HDX-MS).^a MAM New Peak Detection Publication (Mouchahoir et al., 2021).^b MAM Attribute Analytics Publication (In progress).^c Glycosylation Interagency Internal Report (DeLeoz et al., 2017). ^d Glycosylation Interlaboratory Publication (De Leoz et al., 2020).^e NMR HOS Interlaboratory Publication (Brinson et al., 2019).^f HDX-MS Interlaboratory Publication (Hudgens et al., 2019a)

FIGURE 2

Overview of MAM New Peak Detection Data Analysis. (A) A representation of new peak detection data is shown for a single charge state/isotope cluster for a new peak, changed peak, and unchanged peak (B) Peaks Reported as New, Missing, or Changed in Spike and Unknown Samples. New, missing, and changed peaks detected in the Spike (S) and Unknown (U) Samples were reported by each participant. For the Spike Sample, peaks that conformed to expectation are represented in blue: Spike Peptides, Modified Spike Peptides (e.g., Spike Peptide with a PTM) and Spike Peptide Impurities (e.g., Spike Peptide with additional residue, truncation, etc.); peaks that did not conform to expectation are represented in red: NISTmAb Peptides, Unidentified Peaks, Contaminants. Peaks detected in the Unknown Sample did not conform to expectation and are represented in red without further categorization. One participant self-reported peaks in the Unknown Sample as false positives (represented in yellow) and thus were counted as a conforming result. Each participant is represented by a unique symbol. This figure was adapted from Mouchahoir et al., 2021 (https://pubs.acs.org/doi/10.1021/jasms.0c00415), with permission from ACS Publications; further permissions related to this material should be directed to ACS.

FIGURE 3

Schematic description of the glycosylation interlaboratory study. Participating laboratories used the glycoanalytical and detection method(s) of their choice to determine relative abundance of glycans.

FIGURE 4

Representative Analysis of Data from NMR interlaboratory study. (A) simulated data package of many 1H, 13C methyl fingerprints; (B) PCA score plot of the interlaboratory NMR study; (C) Converted grayscale image of a 1H, 13C methyl fingerprint; (D) PCA score plot of 252 spectra used in the automated analysis of outliers. Panel 5B was reprinted from Brinson et al., 2019 (https://doi.org/10.1080/19420862.2018.1544454), with permission from Taylor and Francis Group, LLC. Please note the article was published under a creative commons open access license. Permission is granted subject to the terms of the License under which the work was published. Panels 5C and 5D were Reprinted from Sheen et al., 2020 (https://doi.org/10.1016/j.chemolab.2020.103973) with permission from Elsevier.

FIGURE 5

Schematic description of the HDX-MS interlaboratory study. Fab of NISTmAb test kits containing standardized solutions were overnight shipped to participating laboratories. Laboratories measured peptic peptide centroids and reported these results to NIST. NIST evaluated the accumulated data to determine interlaboratory precision.