Comparison of two-dimensional gel electrophoresis patterns and MALDI-TOF MS analysis of therapeutic recombinant monoclonal antibodies trastuzumab and rituximab (original) (raw)
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International Journal of Molecular Sciences, 2014
Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.
2014
Abstract: Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has bee...
mAbs, 2015
Glycosylation affects the efficacy, safety and pharmacokinetics/pharmacodynamics properties of therapeutic monoclonal antibodies (mAbs), and glycoengineering is now being used to produce mAbs with improved efficacy. In this work, a glycoengineered version of rituximab was produced by chemoenzymatic modification to generate human-like N-glycosylation with a 2,6 linked sialic acid. This modified rituximab was comprehensively characterized by liquid chromatography-mass spectrometry and compared to commercially available rituximab. As anticipated, the majority of N-glycans were converted to a 2,6 linked sialic acid, in contrast to CHO-produced rituximab, which only contains a 2,3 linked sialic acid. Typical posttranslational modifications, such as pyro-glutamic acid formation at the N-terminus, oxidation at methionine, deamidation at asparagine, and disulfide linkages were also characterized in both the commercial and glycoengineered mAbs using multiple enzymatic digestion and mass spectrometric analysis. The comparative study reveals that the glycoengineering approach does not cause any additional posttranslational modifications in the antibody except the specific transformation of the glycoforms, demonstrating the mildness and efficiency of the chemoenzymatic approach for glycoengineering of therapeutic antibodies.
Analysis of recombinant monoclonal antibodies by capillary zone electrophoresis
Analytical platforms that characterize charge heterogeneity in therapeutic proteins, such as mAbs, are important tools that can be used to define quality attributes. CZE separates protein moieties close to their native state and is a valuable physicochemical analytical method that can be used in parallel with other orthogonal methods for characterization and comparability. In this study, custom conditions for the analysis of charge heterogene-ity of two mAbs were developed with regard to critical parameters in the BGE, running conditions, and sample treatment. The method application was tested for up to four mAbs and one mAb fragment. The electropherograms showed specific profiles and contrasting levels of basic and acidic isoforms with respect to the main isoform. Issues that surround this method, such as peak tailing and capillary lifetime, are summarized. Using this method, the identities of rituximab and trastuzumab were confirmed, based on the correspondence between the biosimilars and reference products, noninterference of the sample matrix, and the ability to separate spiked samples of related mAbs. The RSD of the isoform content and migration time for the method repeatability were less than 2 and 1%, respectively.
In this report, we present the characterization of a humanized monoclonal antibody specific for the human epidermal growth factor receptor (hEGFR). Direct analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of peptide mixtures and chromatographically isolated fractions allowed identification of 94.0% and 85.4% of the amino acid sequence of light and heavy chains, respectively. Microheterogeneity sources were identified in light and heavy chains and a previously unreported posttranslational modification for immunoglobulins was found. One N-glycosylation site was identified in the heavy chain with non-sialylated bianntenary fucosylated structures. This study is one of the first to assess the potential of MALDI-MS in combination with more conventional protein chemistry techniques for the characterization of monoclonal antibodies.
International Journal of Molecular Sciences, 2021
Different manufacturing processes and storage conditions of biotherapeutics can lead to a significant variability in drug products arising from chemical and enzymatic post-translational modifications (PTMs), resulting in the co-existence of a plethora of proteoforms with different physicochemical properties. To unravel the heterogeneity of these proteoforms, novel approaches employing strong cation-exchange (SCX) high-performance liquid chromatography (HPLC) hyphenated to mass spectrometry (MS) using a pH gradient of volatile salts have been developed in recent years. Here, we apply an established SCX-HPLC-MS method to characterize and compare two rituximab-based biotherapeutics, the originator MabThera® and its Indian copy product Reditux™. The study assessed molecular differences between the two drug products in terms of C-terminal lysine variants, glycosylation patterns, and other basic and acidic variants. Overall, MabThera® and Reditux™ displayed differences at the molecular le...
A general process for the development of peptide-based immunoassays for monoclonal antibodies
Cancer Chemotherapy and Pharmacology, 2010
Purpose Monoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations. Methods Alemtuzumab and rituximab are monoclonal approved for the treatment of B-cell malignancies and were used as a model system. Phage-displayed peptide libraries were screened for peptide sequences recognized by alemtuzumab (anti-CD52) or rituximab (anti-CD20). Synthetic biotinylated peptides were used in enzyme-linked immunosorbent assays (ELISA). Peptides directly synthesized on polymer resin beads were used in an immunoXuorescent-based assay. Results Peptide mimetope sequences were recovered for both mAb and conWrmed by competitive staining and kinetic measurements. A peptide-based ELISA method was developed for each. The assay for rituximab had a limit of detection of 4 g/ml, and the assay for alemtuzumab had a limit of detection of 1 g/ml. Antibody-speciWc staining of peptide conjugated beads could be seen in a dose-dependent manner. Conclusion Phage-displayed peptide libraries can be a source of highly speciWc mimetopes for therapeutic mAb. The biotinylated forms of those peptides are compatible with conventional ELISA methods with sensitivities comparable to other assay methods and suYcient for pharmacological studies of those mAb given at high dose. The process outlined here can be applied to any mAb to enable improved pharmacokinetic analysis during the development and clinical use of this class of therapies.
Analytical Biochemistry, 2015
Rapid and efficient structural analysis is key to the development of new monoclonal antibodies. We have developed a fast and easy process to obtain MS profiles of antibodies from culture supernatant. Treatment of the supernatant with IdeS generates 3 fragments of 25 kDa that can be analyzed by LC-MS TOF in one run (LC, Fd and Fc/2). This process gives rapid access to isoform and glycoform profiles. To specifically measure the fucosylation yield, we have included a one-pot treatment with EndoS that removes the distal glycan heterogeneity. Our process has been successfully compared to HPCE-LIF, currently considered as the gold standard method. Monoclonal antibodies (MAbs) have been developed as therapeutic agents in several disease areas; such as cancer, rheumatoid arthritis, and Alzheimer's disease. Between 2010 and 2014, of the 54 biopharmaceuticals approved in Europe and United States, 17 were MAb-based products [1]. The growing popularity of recombinant proteins has sparked a demand for advanced and cost-effective protein analysis techniques. In particular, the initial stages of MAb development, that include clone selection during the cell-line generation and optimization of production parameters, require a systematic characterization of the produced antibodies in a screening mode. This step consists of primary amino-acid sequence confirmation, identification and quantification of isoforms and lastly post-translational glycosylation profiling.
Characterization of Therapeutic Antibodies and Related Products
Analytical Chemistry, 2013
Therapeutic antibodies Liquid chromatography and electrophoresis separation methods Mass spectrometry Protein level analysis Peptide level analysis Compendium of IgG micro-variants identified by mass spectrometry Glyco-variants structure-function relationships Glyco-analytics at glycan level Glyco-analytics at protein and peptide levels Charge variants Cysteine-linked variants Oxidized variants Size and sequence variants Higher-order structure and mAb/Antigen complexes Native mass spectrometry Ion mobility mass spectrometry (IM-MS) Hydrogen/ Deuterium exchange mass spectrometry (HDX MS) Dimers and aggregates Monoclonal antibody quantification in biofluids MS identification and quantification of residual Host Cell Proteins Conclusions and future directions Author information