Characterization of the Charge Heterogeneity of a Monoclonal Antibody That Binds to Both Cation Exchange and Anion Exchange Columns under the Same Binding Conditions.

Therapeutic antibodies play an important role in the public healthcare system to treat patients with a variety of diseases. Protein characterization using an array of analytical tools provides in-depth information for drug quality, safety, efficacy, and the further understanding of the molecule. A therapeutic antibody candidate MAB1 exhibits unique binding properties to both cation and anion exchange columns at neutral pH. This uniqueness disrupts standard purification processes and necessitates adjustments in manufacturing. This study identifies that the charge heterogeneity of MAB1 is primarily due to the N-terminal cyclization of glutamine to pyroglutamine and, to a lesser extent, succinimide intermediate, deamidation, and C-terminal lysine. Using three approaches, i.e., deferential chemical labeling, H/D exchange, and molecular modeling, the binding to anion exchange resins is attributed to negatively charged patches on the antibody's surface, involving specific carboxylic acid residues. The methodologies shown here can be extended to study protein binding orientation in column chromatography.

Correlating charge heterogeneity data generated by agarose gel isoelectric focusing and ion exchange chromatography methods.

An isoelectric focusing method (IEF) has been used to assess the charge heterogeneity profile of a monoclonal antibody during the early stages of product development. A more precise and sensitive ion exchange chromatography (IEC/CEX) method was developed and implemented as development progressed and was used concurrently with IEF for lot release and to monitor charge heterogeneity. Charge variants resolved by both methods (IEC and IEF) were purified and characterized. Tryptic peptide mapping and N- linked oligosaccharide profile analyses of the IEC and IEF fractions indicated a structural correlation between the charge variants separated by these two methods. The major sources of molecular heterogeneity were due to the variation in the sialyated carbohydrate structure and heavy chain C-terminal lysine truncation. By monitoring the rates of change in the charge heterogeneity profiles of the monoclonal antibody stored at elevated temperatures by the IEC and IEF methods, a positive correlation between the two methods was established. This approach enabled replacement of the IEF method with the more precise IEC method.

Bio-CoDa: A new analysis class to ensure accurate and precise monoclonal antibody CQA estimation and control.

Monoclonal antibody production processes control critical quality attributes (CQAs), which are the measures that provide proof of a product's identity and quality. Critical decisions rely on the accuracy and precision of these measures, as well as their appropriate statistical treatment. Many measures require special attention. For example, the charge heterogeneity CQA measured by ionic exchange chromatography reports proportions or percentages of the total integrated peak area of known species. Since proportions sum to a constant (1 or 100%), they fall into a special class of data called compositional data that have a unit sum constraint and therefore an inherent correlation. However, these measures are often analyzed assuming independence which is incorrect. Estimating statistics with incorrect assumptions can lead to inferential failures (e.g., shelf life failures), or can lead to missing important structural patterns in the data. Presented here is a new class of analysis methods for CQAs compositional data called Biologic Compositional Data Analysis (Bio-CoDa). The method is based on the elegant solution to analysis issues by Aitchison (1986). An introduction to the Bio-CoDa methods with rational is presented as well as examples demonstrating its strengths. Biotechnol. Bioeng. 2017;114: 2001-2010. © 2017 Wiley Periodicals, Inc.

Conformational characterization of the charge variants of a human IgG1 monoclonal antibody using H/D exchange mass spectrometry.

MAb1, a human IgG1 monoclonal antibody produced in a NS0 cell line, exhibits charge heterogeneity because of the presence of variants formed by processes such as N-terminal glutamate cyclization, C-terminal lysine truncation, deamidation, aspartate isomerization and sialylation in the carbohydrate moiety. Four major charge variants of MAb1 were isolated and the conformations of these charge variants were studied using hydrogen/deuterium exchange mass spectrometry, including the H/D exchange time course (HX-MS) and the stability of unpurified proteins from rates of H/D exchange (SUPREX) techniques. HX-MS was used to evaluate the conformation and solution dynamics of MAb1 charge variants by measuring their deuterium buildup over time at the peptide level. The SUPREX technique evaluated the unfolding profile and relative stability of the charge variants by measuring the exchange properties of globally protected amide protons in the presence of a chemical denaturant. The H/D exchange profiles from both techniques were compared among the four charge variants of MAb1. The two techniques together offered extensive understanding about the local and subglobal/global unfolding of the charge variants of MAb1. Our results demonstrated that all four charge variants of MAb1 were not significantly different in conformation, solution dynamics and chemical denaturant-induced unfolding profile and stability, which aids in understanding the biofunctions of the molecules. The analytical strategy used for conformational characterization may also be applicable to comparability studies done for antibody therapeutics.

An innovative approach for the characterization of the isoforms of a monoclonal antibody product.

Protein biopharmaceuticals, such as monoclonal antibodies (mAbs) are widely used for the prevention and treatment of various diseases. The complex and lengthy upstream and downstream production methods of the antibodies make them susceptible to physical and chemical modifications. Several IgG1 immunoglobulins are used as medical agents for the treatment of colon, breast, and head and neck cancers, and at least four to eight isoforms exist in the products. The regulatory agencies understand the complex nature of the antibody molecules and allow the manufactures to set their own specifications for lot release, provided the safety and efficacy of the products are established in animal models prior to clinical trials. During the manufacture of a mAb product, we observed lot-to-lot variability in the isoform content and, although the variability is within the set specifications for lot release, made attempts to gain mechanistic insight by isolating and characterizing the individual isoforms. Matrix-assisted laser desorption/ionization (MALDI) and liquid chromatography (LC)/mass spectrometry (MS)/MS analyses of the isolated isoforms indicate that this variability is caused by sialic acid content, as well as truncation of C-terminal lysine of the individual isoforms. Sialidase and carboxypeptidase treatment of the product confirm the observations made by MALDI and LC/MS/MS.