Comparative Analysis of Antibodies and Heavily Glycosylated Macromolecular Immune Complexes by Size-Exclusion Chromatography Multi-Angle Light Scattering, Native Charge Detection Mass Spectrometry, and Mass Photometry

Boer, Maurits A. den; Lai, Szu-Hsueh; Xue, Xiaoguang; Kampen, Muriel D. van; Bleijlevens, Boris; Heck, Albert J. R.

doi:10.1021/acs.analchem.1c03656

Cited by 35 publications

(30 citation statements)

References 88 publications

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“…Total IgA was captured from serum or milk from Donor 1 at collection 1 (C1), and subjected to mass analysis by mass photometry (MP) to assess the oligomeric state. MP is a label-free light scattering-based mass analysis technique, which allows for the determination of the mass of individual particles [ 34 – 36 ]. The resulting mass distribution histograms of purified serum and milk IgA are depicted in Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification of common and distinct origins of human serum and breastmilk IgA1 by mass spectrometry-based clonal profiling

et al. 2022

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The most abundant immunoglobulin present in the human body is IgA. It has the highest concentrations at the mucosal lining and in biofluids such as milk and is the second most abundant class of antibodies in serum. We assessed the structural diversity and clonal repertoire of IgA1-containing molecular assemblies longitudinally in human serum and milk from three donors using a mass spectrometry-based approach. IgA-containing molecules purified from serum or milk were assessed by the release and subsequent analysis of their Fab fragments. Our data revealed that serum IgA1 consists of two distinct structural populations, namely monomeric IgA1 (∼80%) and dimeric joining (J-) chain coupled IgA1 (∼20%). Also, we confirmed that IgA1 in milk is present solely as secretory (S)IgA, consisting of two (∼50%), three (∼33%) or four (∼17%) IgA1 molecules assembled with a J-chain and secretory component (SC). Interestingly, the serum and milk IgA1-Fab repertoires were distinct between monomeric, and J-chain coupled dimeric IgA1. The serum dimeric J-chain coupled IgA1 repertoire contained several abundant clones also observed in the milk IgA1 repertoire. The latter repertoire had little to no overlap with the serum monomeric IgA1 repertoire. This suggests that human IgA1s have (at least) two distinct origins; one of these produces dimeric J-chain coupled IgA1 molecules, shared in human serum and milk, and another produces monomeric IgA1 ending up exclusively in serum.

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Section: Resultsmentioning

confidence: 99%

Identification of common and distinct origins of human serum and breastmilk IgA1 by mass spectrometry-based clonal profiling

et al. 2022

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“…In addition, we use well-described methods. 13 , 29 , 34 , 35 Our multi-method approach helped explain differences in potency and characterize by-products formed during production. We were able to show by SDS-PAGE that there are multiple and non-covalent by-products in the HMW1 fraction.…”

Section: Discussionmentioning

confidence: 99%

“…Mass photometry is a handy and rapid tool to give insight into therapeutics and by-products under different conditions and is already finding its place in the analysis of therapeutics. 29 It allows the manipulation of the environment by introducing other buffers and reducing agents and ligands all within minutes per measurement. Another advantage is that this approach is not limited to only complex mAb formats.…”

Section: Discussionmentioning

confidence: 99%

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Characterization of high-molecular weight by-products in the production of a trivalent bispecific 2+1 heterodimeric antibody

Cramer

Franc

Heidenreich

et al. 2023

mAbs

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The development of increasingly complex antibody formats, such as bispecifics, can lead to the formation of increasingly complex high- and low-molecular-weight by-products. Here, we focus on the characterization of high molecular weight species (HMWs) representing the highest complexity of size variants. Standard methods used for product release, such as size exclusion chromatography (SEC), can separate HMW by-products from the main product, but cannot distinguish smaller changes in mass. Here, for the identification of the diverse and complex HMW variants of a trivalent bispecific CrossMAb antibody, offline fractionation, as well as production of HMW by-products combined with comprehensive analytical testing, was applied. Furthermore, HMW variants were analyzed regarding their chemical binding nature and tested in functional assays regarding changes in potency of the variants. Changes in potency were explained by detailed characterization using mass photometry, SDS-PAGE analysis, native mass spectrometry (MS) coupled to SEC and bottom-up proteomics. We identified a major portion of the HMW by-products to be non-covalently linked, leading to dissociation and changes in activity. We also identified and localized high heterogeneity of a by-product of concern and applied a CD3 affinity column coupled to native MS to annotate unexpected by-products. We present here a multi-method approach for the characterization of complex HMW by-products. A better understanding of these by-products is beneficial to guide analytical method development and proper specification setting for therapeutic bispecific antibodies to ensure constant efficacy and patient safety of the product through the assessment of by-products.

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“…Higher, but still acceptable variability is observed with SV-AUC and mass photometry. Mass photometry is a newer technology successfully applied in the immunological field [41]. In our studies on the formation of immune complexes, however, extensive adaptations were required.…”

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Pre-Clinical In-Vitro Studies on Parameters Governing Immune Complex Formation

et al. 2022

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The success of biotherapeutics is often challenged by the undesirable events of immunogenicity in patients, characterized by the formation of anti-drug antibodies (ADA). Under specific conditions, the ADAs recognizing the biotherapeutic can trigger the formation of immune complexes (ICs), followed by cascades of subsequent effects on various cell types. Hereby, the connection between the characteristics of ICs and their downstream impact is still not well understood. Factors governing the formation of ICs and the characteristics of these IC species were assessed systematically in vitro. Classic analytical methodologies such as SEC-MALS and SV-AUC, and the state-of-the-art technology mass photometry were applied for the characterization. The study demonstrates a clear interplay between (1) the absolute concentration of the involved components, (2) their molar ratios, (3) structural features of the biologic, (4) and of its endogenous target. This surrogate study design and the associated analytical tool-box is readily applicable to most biotherapeutics and provides valuable insights into mechanisms of IC formation prior to FIH studies. The applicability is versatile—from the detection of candidates with immunogenicity risks during developability assessment to evaluation of the impact of degraded or post-translationally modified biotherapeutics on the formation of ICs.

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Comparative Analysis of Antibodies and Heavily Glycosylated Macromolecular Immune Complexes by Size-Exclusion Chromatography Multi-Angle Light Scattering, Native Charge Detection Mass Spectrometry, and Mass Photometry

Cited by 35 publications

References 88 publications

Identification of common and distinct origins of human serum and breastmilk IgA1 by mass spectrometry-based clonal profiling

Identification of common and distinct origins of human serum and breastmilk IgA1 by mass spectrometry-based clonal profiling

Characterization of high-molecular weight by-products in the production of a trivalent bispecific 2+1 heterodimeric antibody

Pre-Clinical In-Vitro Studies on Parameters Governing Immune Complex Formation

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