Peptide mapping is a component of the analytical toolbox used within the biopharmaceutical industry to aid in the identity confirmation of a protein therapeutic and to monitor degradative events such as oxidation or deamidation. These methods offer the advantage of providing site-specific information regarding post-translational and chemical modifications that may arise during production, processing or storage. A number of such variations may also be induced by the sample preparation methods themselves which may confound the ability to accurately evaluate the true modification levels. One important focus when developing a peptide mapping method should therefore be the use of sample preparation conditions that will minimize the degree of artificial modifications induced. Unfortunately, the conditions that are amenable to effective reduction, alkylation and digestion are often the same conditions that promote unwanted modifications. Here we describe the optimization of a tryptic digestion protocol used for peptide mapping of the NISTmAb IgG1κ which addresses the challenge of balancing maximum digestion efficiency with minimum artificial modifications. The parameters on which we focused include buffer concentration, digestion time and temperature, as well as the source and type of trypsin (recombinant vs. pancreatic; bovine vs porcine) used. Using the optimized protocol we generated a peptide map of the NISTmAb which allowed us to confirm its identity at the level of primary structure. Graphical abstractPeptide map of the NISTmAb RM 8671 monoclonal antibody. Tryptic digestion was performed using an optimized protocol and followed by LC-UV-MS analysis. The trace represents the total ion chromatogram. Each peak was mapped to peptides identified using mass spectrometry data. Electronic supplementary materialThe online version of this article (10.1007/s00216-018-0848-6) contains supplementary material, which is available to authorized users.
The NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing. It must therefore embody the quality and characteristics of a typical biopharmaceutical product and be available long-term in a stable format with consistent product quality attributes. A stratified sampling and analysis plan using a series of qualified analytical and biophysical methods is described that assures RM 8671 meets these criteria. Results for the first three lots of RM 8671 highlight the consistency of material attributes with respect to size, charge, and identity. RM 8671 was verified to be homogeneous both within and between vialing lots, demonstrating the robustness of the lifecycle management plan. It was analyzed in concert with the in-house primary sample 8670 (PS 8670) to provide a historical link to this seminal material. RM 8671 was verified to be fit for its intended purpose as a technology innovation tool, external system suitability control, and cross-industry harmonization platform. Graphical abstractThe NISTmAb Reference Material (RM) 8671 is intended to be an industry standard monoclonal antibody for pre-competitive harmonization of best practices and designing next generation characterization technologies for identity, quality, and stability testing. Electronic supplementary materialThe online version of this article (10.1007/s00216-017-0800-1) contains supplementary material, which is available to authorized users.
The Multi-Attribute Method (MAM) Consortium was initially formed as a venue to harmonize best practices, share experiences, and generate innovative methodologies to facilitate widespread integration of the MAM platform, which is an emerging ultra-high-performance liquid chromatography–mass spectrometry application. Successful implementation of MAM as a purity-indicating assay requires new peak detection (NPD) of potential process- and/or product-related impurities. The NPD interlaboratory study described herein was carried out by the MAM Consortium to report on the industry-wide performance of NPD using predigested samples of the NISTmAb Reference Material 8671. Results from 28 participating laboratories show that the NPD parameters being utilized across the industry are representative of high-resolution MS performance capabilities. Certain elements of NPD, including common sources of variability in the number of new peaks detected, that are critical to the performance of the purity function of MAM were identified in this study and are reported here as a means to further refine the methodology and accelerate adoption into manufacturer-specific protein therapeutic product life cycles.
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