Monoclonal antibodies biosimilarity assessment using transient isotachophoresis capillary zone electrophoresis-tandem mass spectrometry (original) (raw)
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As research, development, and manufacturing of biosimilar protein therapeutics proliferates, there is great interest in the continued development of a portfolio of complementary analytical methods that can be used to efficiently and effectively characterize biosimilar candidate materials relative to the respective reference (i.e., originator) molecule. Liquid phase separation techniques such as liquid chromatography and capillary electrophoresis are powerful tools that can provide both qualitative and quantitative information about similarities and differences between reference and biosimilar materials, especially when coupled with mass spectrometry. However, the inherent complexity of these protein materials challenges even the most modern one-dimensional (1D) separation methods. Two-dimensional (2D) separations present a number of potential advantages over 1D methods, including increased peak capacity, 2D peak patterns that can facilitate unknown identification, and improvement in...
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The development and approval processes of biosimilar mAbs depend on their comparability to originators. Therefore, analytical comparisons are required to assess structural features and post-translational modifications (PTM) and thereby minimize the risk of being clinically meaningful differences between biosimilar and originator drug products. The glycosylation pattern of mAbs is considered to be an important critical quality attribute (CQA), and several analytical approaches have been proposed that facilitate characterizing and monitoring a glycosylation profile, albeit mainly at a glycan and glycopeptide level of analysis. In this study, we demonstrate the utility of hydrophilic interaction chromatography (HILIC) hyphenated with mass spectrometry (MS) for the qualitative profiling of glycosylation patterns at the protein level, by comparing originator and biosimilars mAbs (Remicade ® /Remsina ® /Inflectra ® , Herceptin ® /Trastuzumab B, and Erbitux ® /Cetuximab B) using a middle-up approach. We demonstrate the ability of HILIC to resolve hydrophilic variants of protein biopharmaceuticals at the middle-up level of analysis, its complementarity to reversed phase liquid chromatography (RPLC), and its hyphenation to MS. HILIC features combined to MS make a powerful analytical tool for the comparison of originator and biosimilar mAbs that could eventually be applied in routine analyses for quality control.
2020
Several of the 10 top selling drugs are antibody-based therapeutics that have lost or will lose patent protection soon.1 As highly complex glycoproteins, monoclonal antibodies (mAbs) have a wide range of micro-variants. Therefore, mAbs require extensive characterization through multiple methods for structure assessment, manufacturing control, and biosimilarity studies. Biosimilars contain a version of an active substance that is already an authorized original biological drug. Biosimilars require additional analytical and statistical characterizations for FDA approval.2 As drugs lose patent protection these biosimilars have started to enter the market.
mAbs
This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS(E)), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also use...
Journal of Pharmaceutical and Biomedical Analysis, 2013
Liquid chromatography-tandem mass spectrometry-based proteomics for peptide mapping and sequencing was used to characterize the marketed monoclonal antibody trastuzumab and compare it with two biosimilar products, mAb A containing D359E and L361M variations at the Fc site and mAb B without variants. Complete sequence coverage (100%) including disulfide linkages, glycosylations and other commonly occurring modifications (i.e., deamidation, oxidation, dehydration and K-clipping) were identified using maps generated from multi-enzyme digestions. In addition to the targeted comparison for the relative populations of targeted modification forms, a non-targeted approach was used to globally compare ion intensities in tryptic maps. The non-targeted comparison provided an extradimensional view to examine any possible differences related to variants or modifications. A peptide containing the two variants in mAb A, D359E and L361M, was revealed using the non-targeted comparison of the tryptic maps. In contrast, no significant differences were observed when trastuzumab was self-compared or compared with mAb B. These results were consistent with the data derived from peptide sequencing via collision induced dissociation/electron transfer dissociation. Thus, combined targeted and non-targeted approaches using powerful mass spectrometry-based proteomic tools hold great promise for the structural characterization of biosimilar products.
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.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences, 2016
Out of all categories, monoclonal antibodies (mAbs), biosimilar, antibody-drug conjugates (ADCs) and Fc-fusion proteins attract the most interest due to their strong therapeutic potency and specificity. Because of their intrinsic complexity due to a large number of micro-heterogeneities, there is a crucial need of analytical methods to provide comprehensive in-depth characterization of these molecules. CE presents some obvious benefits as high resolution separation and miniaturized format to be widely applied to the analysis of biopharmaceuticals. CE is an effective method for the separation of proteins at different levels. capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) have been particularly relevant for the characterization of size and charge variants of intact and reduced mAbs, while CE-MS appears to be a promising analytical tool to assess the primary structure of mAbs and related products. This review will be ...
Analytical Approach for Biosimilar Development: Special Focus on Monoclonal Antibody Biosimilars
International journal of biopharmaceutical sciences, 2018
Long-term safety and efficacy of biosimilars are approved by using abbreviated methods and clinical studies may not always adequate to ensure comparability of biosimilar mAbs comparing to its reference product. Therefore, the analytical strategy of the physicochemical comparison of a biosimilar to its reference product becomes an important data to indicate clinical similarity in safety and efficacy. FDA recommended that demonstration of biosimilarity between reference and biosimilar versions is based upon data derived from analytical studies to show "high similarity" to the reference product not withstanding minor differences in clinically inactive components. Therefore, the physicochemical analytical comparison between biosimilar and its reference product is the primary consideration during biosimilar development. In these review, the approach for physicochemical characterization, biological activity and impurities assessment were reviewed.
2017
Biopharmaceutical development and manufacturing of monoclonal antibody (mAb) therapies requires routine analyses and monitoring of various physiochemical properties. Here, we describe a unified capillary electrophoresis and electrospray ionization with mass spectrometry (CESI-MS) workflow for characterization of mAb samples. The workflow combines the unique separation capabilities of capillary zone electrophoresis (CZE) with the advantages of an MS-based detector to provide high resolution structural information and accurate molecular weight information. Small amounts (~10 ng) of samples containing intact (non reduced) or reduced mAbs can be analyzed to determine charge heterogeneity (including charge variants, glycoforms, and clipped species), purity, and molecular weight.
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.