Preparative scale methods such as OFFGEL, Rotofor, FFE and IEX offer alternative charge variant separations that are amenable to fraction collection and downstream characterization. However, fractionation and sample collection is impractical on these platforms. Capillary and microchip-based techniques afford very high separation efficiency even for very rapid separations (<15 min), while consuming very little sample. Charge sensitive analytical methods commonly employed for mAb charge heterogeneity characterization and monitoring include capillary isoelectric focusing (CIEF), imaged capillary isoelectric focusing (ICIEF), capillary zone electrophoresis (CZE), ion exchange chromatography (IEX), and free-flow electrophoresis (FFE). Routine monitoring of mAb charge heterogeneity is an integral part of any product quality and lifecycle management plan.
Determination of isoelectric point series#
This paper is part of a series of articles in this issue describing all aspects of the NISTmAb quality control strategy.Ĭharge heterogeneity of monoclonal antibodies may reflect a number of post-translational modifications and structural variants which affect product efficacy to varying degrees. Here, we describe the development and qualification of charge heterogeneity monitoring assays based on capillary electrophoresis, which form an integral part of the quality control strategy of the NISTmAb Reference Material. In support of this goal, NIST has an ongoing characterization and quality monitoring program using state-of-the-art analytical methods to contribute to a growing open-source knowledge base surrounding the NISTmAb.
The NISTmAb is intended to serve as a platform for open innovation and technology development through evaluation of established and novel analytical methods. With this need in mind, NIST developed Reference Material 8671 (“NISTmAb”), a class-representative humanized IgG1κ monoclonal antibody which has undergone extensive analytical and biophysical characterization by NIST and industry/academic/government stakeholders. Increased knowledge surrounding fundamental measurement principles of each measurement can lead to better, more streamlined product characterization and will facilitate new technology and product development. Selection of the appropriate testing strategy is guided by the attribute-specific information each method can provide regarding product quality. Biotherapeutic production in biological systems results in inherently heterogeneous drug products which cannot be fully characterized by any one analytical method, necessitating an analytical toolkit comprised of multiple orthogonal methods. Quality control mechanisms and their accompanying analytics, originally designed to support small molecule drug development, are still evolving to meet the increasingly sophisticated needs of the biotherapeutics market. 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.Ĭomplex biotherapeutics, in particular monoclonal antibodies, increasingly dominate the arena of new drugs submitted for regulatory approval. CZE was found to be better suited for routine monitoring of NISTmAb charge heterogeneity and was qualified for this purpose. CIEF was found to be suitable as a structural characterization assay yielding information on the apparent pI of the NISTmAb. Capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF) assays were optimized and evaluated as candidate assays for NISTmAb quality control. In this paper, the development and qualification of methods for monitoring NISTmAb charge heterogeneity are described. 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. The 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.
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