Identification and quantification of product-related quality attributes in bio-therapeutic monoclonal antibody via a simple, and robust cation-exchange HPLC method compatible with direct online detection of UV and native ESI-QTOF-MS analysis

Sankaran, Praveen Kallamvalliillam; Kabadi, Pradeep; Honnappa, Chethan Gejjalagere; Subbarao, Malini; Pai, Harish V.; Adhikary, Laxmi; Palanivelu, Dinesh V.

doi:10.1016/j.jchromb.2018.10.019

Cited by 16 publications

(11 citation statements)

References 62 publications

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“…Within the 1D-LC analysis format, there has been an increase in the development of MS-compatible CEX methods and direct online coupling of CEX to MS using volatile salt-based pH gradients, giving rise to Native CEX-MS. This has been demonstrated by multiple groups with variations in the stationary phase (weak and strong ion exchangers) and MS platforms (ESI-MS, IM-MS, and orbitrap) for trastuzumab, adalimumab, infliximab, bevacizumab, and cetuximab (Bailey et al, 2018;Füssl et al, 2018;Sankaran et al, 2018;Jaag et al, 2021;Murisier et al, 2021) (Supplementary Table S7). Multidimensional platforms such as 2D-LC with CEX in first dimension followed by an MS-compatible second dimension, i.e., RP (desalting improves peak capacity and MS compatibility) have also been incorporated in biosimilarity (Alvarez et al, 2011;Stoll et al, 2015) (Supplementary Table S7).…”

Section: Charge Variantsmentioning

confidence: 92%

Section: Global Landscape On Biosimilar Approvalsmentioning

confidence: 92%

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Analytical Similarity Assessment of Biosimilars: Global Regulatory Landscape, Recent Studies and Major Advancements in Orthogonal Platforms

Nupur

Joshi

Gulliarme

et al. 2022

Front. Bioeng. Biotechnol.

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Biopharmaceuticals are one of the fastest-growing sectors in the biotechnology industry. Within the umbrella of biopharmaceuticals, the biosimilar segment is expanding with currently over 200 approved biosimilars, globally. The key step towards achieving a successful biosimilar approval is to establish analytical and clinical biosimilarity with the innovator. The objective of an analytical biosimilarity study is to demonstrate a highly similar profile with respect to variations in critical quality attributes (CQAs) of the biosimilar product, and these variations must lie within the range set by the innovator. This comprises a detailed comparative structural and functional characterization using appropriate, validated analytical methods to fingerprint the molecule and helps reduce the economic burden towards regulatory requirement of extensive preclinical/clinical similarity data, thus making biotechnological drugs more affordable. In the last decade, biosimilar manufacturing and associated regulations have become more established, leading to numerous approvals. Biosimilarity assessment exercises conducted towards approval are also published more frequently in the public domain. Consequently, some technical advancements in analytical sciences have also percolated to applications in analytical biosimilarity assessment. Keeping this in mind, this review aims at providing a holistic view of progresses in biosimilar analysis and approval. In this review, we have summarized the major developments in the global regulatory landscape with respect to biosimilar approvals and also catalogued biosimilarity assessment studies for recombinant DNA products available in the public domain. We have also covered recent advancements in analytical methods, orthogonal techniques, and platforms for biosimilar characterization, since 2015. The review specifically aims to serve as a comprehensive catalog for published biosimilarity assessment studies with details on analytical platform used and critical quality attributes (CQAs) covered for multiple biotherapeutic products. Through this compilation, the emergent evolution of techniques with respect to each CQA has also been charted and discussed. Lastly, the information resource of published biosimilarity assessment studies, created during literature search is anticipated to serve as a helpful reference for biopharmaceutical scientists and biosimilar developers.

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Section: Charge Variantsmentioning

confidence: 92%

Section: Global Landscape On Biosimilar Approvalsmentioning

confidence: 92%

Analytical Similarity Assessment of Biosimilars: Global Regulatory Landscape, Recent Studies and Major Advancements in Orthogonal Platforms

Nupur

Joshi

Gulliarme

et al. 2022

Front. Bioeng. Biotechnol.

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“…Acidic species are often related to PTM's like sialic acid or deamidation on asparagine, while basic variants are formed by aspartate isomerization, succinimide formation, variants of C terminal lysine and N terminal glutamine [49]. IEX is giving relative quantitative information about charge variants which can be important for the qualification of manufacturing batches [50].…”

Section: Separation Of Proteoforms Of Therapeutic Proteins 41 Separamentioning

confidence: 99%

Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

Hidayah¹,

Gaikwad²,

Heikaus³

et al. 2020

Proteoforms - Concept and Applications in Medical Sciences

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A characteristic of many proteoforms, derived from a single gene, is their similarity regarding the composition of atoms, making their analysis very challenging. Many overexpressed recombinant proteins are strongly associated with this problem, especially recombinant therapeutic glycoproteins from large-scale productions. In contrast to small molecule drugs, which consist of a single defined molecule, therapeutic protein preparations are heterogenous mixtures of dozens or even hundreds of very similar species. With mass spectrometry, currently highquality spectra of intact proteoforms can be obtained only, if the complexity of the mixture of individual proteoform-ions, entering the gas phase at the same time is low. Thus, prior to mass spectrometric analysis, an effective separation is required for getting fractions with a low number of individual proteoforms. This is especially true not only for recombinant therapeutic proteins, because of their huge heterogeneity, but also relevant for top-down proteomics. Purification of proteoforms is the bottleneck in analyzing intact proteoforms with mass spectrometry. This review is focusing on the current state of the art, especially of liquid chromatography for preparing proteoforms for mass spectrometric top-down analysis. The topic of therapeutic proteins has been chosen, because this group of proteins is most challenging regarding their proteoform analysis.

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“…7 As a conventional and nondenaturing technique, ionexchange chromatography (IEX) has been widely used to separate and isolate protein charge variants during protein purification and for subsequent characterization. [22][23][24] Upon the separation of charge variants by IEX, current strategies to determine the effects of modifications on specific charge variant peak involve isolating the peak of interest followed by various mass spectrometric analyses, such as intact mass analysis, peptide mapping, and glycan analysis. 10,25 In addition to being limited by time and resources, this two-step approach may overlook the minor species that do not exhibit distinctive UV peaks and introduce artifacts because of the lengthy sample preparation processes.…”

Section: Introductionmentioning

confidence: 99%

“…A strategy for the direct coupling of MS with IEX involved the application of a pH gradient using volatile salts. 23,24,[29][30][31] Online weak cation exchange (WCX)-MS has been reported for analyzing IgG2 mAbs by using an ammonium hydroxide-based pH gradient. 32 Characterization of digested mAbs and proteins with molecular weights below~30 kDa has also been described by utilizing an ammonium formate and ammonium acetate-based pH and salt gradient.…”

Section: Introductionmentioning

confidence: 99%

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

Shi

Xiao

Dillon

et al. 2020

mAbs

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2020) Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis, mAbs, 12:1, 1739825, ABSTRACT Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatographytop-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping. ARTICLE HISTORY

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Identification and quantification of product-related quality attributes in bio-therapeutic monoclonal antibody via a simple, and robust cation-exchange HPLC method compatible with direct online detection of UV and native ESI-QTOF-MS analysis

Cited by 16 publications

References 62 publications

Analytical Similarity Assessment of Biosimilars: Global Regulatory Landscape, Recent Studies and Major Advancements in Orthogonal Platforms

Analytical Similarity Assessment of Biosimilars: Global Regulatory Landscape, Recent Studies and Major Advancements in Orthogonal Platforms

Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

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