John E. Schiel

LC-MS/MS biopharmaceutical glycoanalysis: identification of desirable reference material characteristics

Glycosylation, the enzymatic addition of carbohydrates to a protein, is one of the most abundant post-translational modifications found in nature. There is variability in the number, location, and identity of glycans attached. As a result, a glycoprotein consists of a number of glycoforms with different combinations of glycans, potentially resulting in different stability, toxicity, and activity. This is especially important in the biopharmaceutical industry where product consistency and safety are vital. Glycoprotein analysis involves numerous mass spectrometry based techniques, each of which provides various aspects of characterization. The current paper describes two commonly used analytical techniques for glycoprotein characterization. In one experiment, nonspecific proteolysis is combined with a two-tiered mass spectrometry approach (MALDI-TOF and LC-MS/MS) to gain glycosylation site and glycan identity. In a second approach, glycans were enzymatically released, labeled with a fluorescent dye, and analyzed using LC-Fluorescence-MS/MS to give glycan identification and relative quantification. The type and degree of information yielded by each method is assessed in an effort to identify desired reference material characteristics for improving biopharmaceutical glycoanalysis.

Glycoprotein analysis using mass spectrometry: unraveling the layers of complexity

A glycoprotein exists as a heterogeneous mixture of forms due to differential glycosylation, each of which may confer different functionality and/or serve as a biochemical marker for disease. The complex structure of glycans make them a bioanalytical challenge requiring multiple mass spectrometry based approaches to gain different types of information. The following article will briefly describe recently utilized mass spectrometry methods to identify glycosylation sites and measure glycan composition, sequence, branching, and relative quantities. Potential metrological developments are discussed in light of current trends toward complete, reliable glycoanalytical characterization in a high-throughput manner.

Bioreactor Process Parameter Screening Utilizing a Plackett–Burman Design for a Model Monoclonal Antibody

Consistent high-quality antibody yield is a key goal for cell culture bioprocessing. This endpoint is typically achieved in commercial settings through product and process engineering of bioreactor parameters during development. When the process is complex and not optimized, small changes in composition and control may yield a finished product of less desirable quality. Therefore, changes proposed to currently validated processes usually require justification and are reported to the US FDA for approval. Recently, design-of-experiments-based approaches have been explored to rapidly and efficiently achieve this goal of optimized yield with a better understanding of product and process variables that affect a products critical quality attributes. Here, we present a laboratory-scale model culture where we apply a Plackett-Burman screening design to parallel cultures to study the main effects of 11 process variables. This exercise allowed us to determine the relative importance of these variables and identify the most important factors to be further optimized in order to control both desirable and undesirable glycan profiles. We found engineering changes relating to culture temperature and nonessential amino acid supplementation significantly impacted glycan profiles associated with fucosylation, β-galactosylation, and sialylation. All of these are important for monoclonal antibody product quality.

Universal proteolysis and MSn for N- and O- glycan branching analysis

The continually growing list of critical glycosylation-related processes has made analytical methodology for detailed glycan characterization an area of increasing interest. Glycosylation is a post translational modification of unsurpassed complexity due to the variety of compositions and linkages formed by these biopolymers. Structural characterization of glycan isomers has been achieved using ion trap mass spectrometry and MS(n) of released, permethylated glycans. However, N- and O-glycans require different sample preparation strategies; and release of the glycans may be hindered, result in degradation of the glycan, and/or produce limited yields of permethylated product. In the current report, we demonstrate universal proteolysis of both N- and O-linked glycoproteins to individual glycoamino acids. These samples were shown to be directly amenable to permethylation and MS(n) analysis for isomeric structural determination. Universal proteolysis and permethylation provides an identical sample preparation strategy for both classes of glycans that avoids potential pitfalls of commonly used release methods. This methodology should be applicable to all glycoproteins and serve as an alternative to glycan release for MS(n) branching analysis. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.

Comparison of Traditional 2-AB Fluorescence LC–MS/MS and Automated LC–MS for the Comparative Glycan Analysis of Monoclonal Antibodies

Monoclonal antibody therapeutics are a heterogeneous mixture of glycoforms. Multiple methods exist for defining the glycan composition and relative abundance of species present. In the current report, two MS-based methods were compared for their ability to both identify glycans and monitor differences in the glycoprofile. Gross changes in the glycoprofile can be identified either by visual inspection of fluorescence profiles and correlated to glycan identities when coupled with online MS/MS (LC-F-MS/MS) or through extracted ion chromatograms using LC-MS. In the present study, both an LC-F-MS/MS method and an automated LC-MS label free approach were able to identify minor differences in low abundance glycoforms, and data indicate a disparity in glycosylation between the analyzed batches of US and foreign-sourced mAb X. Thus, either method may be useful in characterizing monoclonal antibody therapeutics products and could serve as a potential screening test for understanding process, comparability, similarity, and possibly detecting counterfeit agents.

The NISTmAb Reference Material 8671 value assignment, homogeneity, and stability

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. 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.

The NISTmAb Reference Material 8671 lifecycle management and quality plan

AbstractComprehensive analysis of monoclonal antibody therapeutics involves an ever expanding cadre of technologies. Lifecycle-appropriate application of current and emerging techniques requires rigorous testing followed by discussion between industry and regulators in a pre-competitive space, an effort that may be facilitated by a widely available test metric. Biopharmaceutical quality materials, however, are often difficult to access and/or are protected by intellectual property rights. The NISTmAb, humanized IgG1κ Reference Material 8671 (RM 8671), has been established with the intent of filling that void. The NISTmAb embodies the quality and characteristics of a typical biopharmaceutical product, is widely available to the biopharmaceutical community, and is an open innovation tool for development and dissemination of results. The NISTmAb lifecyle management plan described herein provides a hierarchical strategy for maintenance of quality over time through rigorous method qualification detailed in additional submissions in the current publication series. The NISTmAb RM 8671 is a representative monoclonal antibody material and provides a means to continually evaluate current best practices, promote innovative approaches, and inform regulatory paradigms as technology advances. 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.

A simple and sensitive LC-ICP-MS method for the accurate determination of vitamin B12 in fortified breakfast cereals and multivitamin tablets

A sensitive liquid chromatographic (LC) method coupled with inductively coupled plasma mass spectrometry (ICP-MS) has been developed for the determination of vitamin B12. The method was based on efficient isocratic separation with a mobile phase consisting of 20 mmol L−1 ethylenediaminetetraacetic acid (EDTA) in 25/75 methanol–water mixture (volume fractions) operating at a flow rate of 0.2 mL min−1. After LC separation, ionic cobalt (Co), cyanocobalamin, methylcobalamin, and hydroxocobalamin were measured as 59Co by ICP-MS. For Co as cyanocobalamin, the analyte of interest of this work, the method has shown good repeatability with relative standard deviation (RSD) of 3% for ten measurements and excellent linearity between 0.1 ng g−1 and 100 ng g−1 (linear regression, r2 > 0.999). The limit of detection (LOD) for cyanocobalamin was found to be less than 1 ng g−1, which permits the method to be employed for the determination of ultra-trace concentrations of vitamin B12 in various types of dietary supplements and fortified food products. Cyanocobalamin in aqueous solution was found to decompose under the ambient light of the laboratory; therefore, dark room conditions are required for the determination of vitamin B12 in the form of cyanocobalamin to minimize the photon-induced decomposition. To determine total Co in a commercial high-purity cyanocobalamin using direct ICP-MS measurement as part of an effort to characterize the chemical for use as a calibrant, it was observed that quantitative measurement of Co was achieved only through a complete acid digestion. The method was applied to the determination of vitamin B12 in Standard Reference Material (SRM) 3233 Fortified Breakfast Cereal. SRM 3280 Multivitamin/Multielement Tablet was used for quality assurance of the cereal sample measurements. The vitamin B12 value of (0.187 ± 0.016) mg kg−1 found in SRM 3233 was comparable to (0.219 ± 0.066) mg kg−1 obtained by Grocery Manufacturers Associations Food Industry Analytical Chemists Committee (FIACC) using microbiological assay. The (4.38 ± 0.05) mg kg−1 of vitamin B12 found in quality assurance samples of SRM 3280 was in good agreement with the certified values of (4.8 ± 1.0) mg kg−1.

Qualification of NISTmAb charge heterogeneity control assays

AbstractThe NISTmAb is a monoclonal antibody Reference Material from the National Institute of Standards and Technology; it is a class-representative IgG1κ intended serve as a pre-competitive platform for harmonization and technology development in the biopharmaceutical industry. The publication series of which this paper is a part describes NIST’s overall control strategy to ensure NISTmAb quality and availability over its lifecycle. In this paper, the development and qualification of methods for monitoring NISTmAb charge heterogeneity are described. Capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) assays were optimized and evaluated as candidate assays for NISTmAb quality control. CIEF was found to be suitable as a structural characterization assay yielding information on the apparent pI of the NISTmAb. CZE was found to be better suited for routine monitoring of NISTmAb charge heterogeneity and was qualified for this purpose. This paper is intended to provide relevant details of NIST’s charge heterogeneity control strategy to facilitate implementation of the NISTmAb as a test molecule in the end user’s laboratory. Graphical AbstractRepresentative capillary zone electropherogram of the NIST monoclonal antibody (NISTmAb). The NISTmAb is a publicly available research tool intended to facilitate advancement of biopharmaceutical analytics.

Development of an LC-MS/MS peptide mapping protocol for the NISTmAb

AbstractPeptide 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.