Large and light chain samples were prepared at 0

Large and light chain samples were prepared at 0.2 and 0.1mg/mL, respectively, in 8M urea with 1% (w/v, final) methyl cellulose, 4% 310 Pharmalytes, and pI markers (icIEF buffer). reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown. Keywords:monoclonal antibody, charge variants, isoelectric focusing, deamidation, stability 2D-PAGE, reduced, denatured chromatography == Abbreviations == two-dimensional polyacrylamide gel electrophoresis capillary isoelectric focusing full width half height heavy chain imaged capillary isoelectric focusing isoelectric focusing ion exchange chromatography light chain reduced and denatured size exclusion chromatography size exclusion chromatography == Introduction == With over 30 approved products worldwide and hundreds of on-going clinical trials, therapeutic antibody development is a well-established and vital segment of the pharmaceutical industry.1Moreover, advances in antibody engineering have resulted in novel targeted therapies and antibody formats such as antibody-drug conjugates and bispecific antibodies, which hold tremendous therapeutic potential.2,3Compared to non-proteinaceous drugs, therapeutic antibodies are several orders of magnitude larger and significantly more complex.4This complexity arises, in part, because therapeutic antibodies are manufactured in biological systems (e.g., Chinese hamster ovary cells), leading to differences in post-translational modifications (e.g., amino or carboxy terminal processing, glycosylation).5,6Additionally, various chemical alterations (e.g., deamidation, oxidation, fragmentation) may occur during purification and storage.7-9The combined effect of these modifications, which occur BPTES during both Rabbit Polyclonal to PLAGL1 up-stream and down-stream processes, is micro-heterogeneity in the physicochemical properties of the proteins that requires advanced analytical technologies to properly characterize. While micro-heterogeneity is a generally accepted property of both natural and recombinant proteins, methods to assess, control, and, if possible, reduce the occurrence in biotherapeutics intended for clinical use are needed.10A suite of orthogonal and complementary assays are typically employed to characterize the molecular properties of therapeutic antibodies such as size, glycosylation, and isoelectric point.8These quality attributes support drug safety and efficacy profiles, which are ultimately related to clinical performance.11Charge heterogeneity of a purified protein is manifest in the presence of minor populations with slightly different isoelectric points (either acidic or basic relative to the main species). These charge variants may BPTES stem from several distinct pathways, including deamidation, glycation, and differences in glycosylation.12Although evidence suggesting isolated charge variants have comparable safety and efficacy is beginning to emerge, the results cannot necessarily be extended to all molecules,a priori, and is likely to be highly dependent on the nature, location, and extent of the modifications.13Furthermore, charge heterogeneity must be monitored during development and production to ensure product consistency, and over the course of normal shelf-life to monitor stability.14Obtaining detailed quantitative analysis of the charge variant profile is an early requisite step toward uncovering the various pathways by which a therapeutic antibody acquires charge heterogeneity. This information has the potential to better inform structure/function/stability relationships, and to clarify any effect of charge heterogeneity on clinical safety and efficacy and shelf-life. Charge variant analysis of intact antibodies may BPTES be achieved using isoelectric focusing techniques (IEF) or ion exchange chromatography (IEX). Gel and capillary-IEF methods use a pH gradient and an electric field to separate charge variants based on the intrinsic charge of the molecule.14-16Although readily implemented without the need for sophisticated equipment, flatbed and vertical gel-IEF have been largely supplanted by capillary-based IEF (cIEF) methods because the latter provides rapid, quantitative characterization with greater resolution. Chromatographic methods are also relatively straightforward to implement and the different combinations of ion exchange resins, solution conditions, and elution gradients facilitate method optimization for each individual antibody.17In IEX, charge variants are resolved by disrupting the interactions between the stationary phase and the charged groups on the molecular.