Characterization of Therapeutic Antibodies and Related Products

Beck, Alain; Wagner‐Rousset, Elsa; Ayoub, Daniel; Dorsselaer, Alain Van; Sanglier-Cianférani, Sarah

doi:10.1021/ac3032355

Cited by 527 publications

(466 citation statements)

References 244 publications

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“…Structural characterization studies revealed that the size variants were due to either opened engineered scFv disulfide bonds with concomitant cysteine/glutathione capping on the engineered cysteines 33-35 or stable dimers formed by intermolecular disulfide bonds. We also describe a convenient approach to monitor and control these size variants with high-resolution size-exclusion chromatography (SEC).…”

Section: Introductionmentioning

confidence: 99%

Characterization and analysis of scFv-IgG bispecific antibody size variants

Cao¹,

Wang²,

Chung³

et al. 2018

mAbs

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Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.

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Section: Introductionmentioning

confidence: 99%

Characterization and analysis of scFv-IgG bispecific antibody size variants

Cao¹,

Wang²,

Chung³

et al. 2018

mAbs

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“…Therefore numerous works were published about the analysis of mAb and the characterization of the molecular heterogeneity [1]. The rituximab (Rtx) is a monoclonal antibody used to treat cancers of blood system such as B cells leukemia (non Hodgkin's lymphoma) and some autoimmune diseases (rheumatoid arthritis) [2].…”

Section: Introductionmentioning

confidence: 99%

Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

G¹

2014

J Chromatogr Sep Tech

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This paper focuses on the applicability of capillary zone electrophoresis (CZE) using uncoated fused silica capillaries for the determination of heterogeneity of rituximab (MabThera, Roche) and for the study of its solution and thermal stability. The best resolution for the charge variants of the main component was obtained with a buffer electrolyte containing 800 mM 6-amino caproic acid, 2 mM triethylene tetramine and 0.05% hydroxypropyl methylcellulose at pH = 5.2. It was found that the pH and the components of the buffer used for the electrophoretic separation and for the dilution of the sample prior to the analysis are important to the stability of the rituximab. We demonstrated the rituximab is stable in the pharmaceutical product MabThera due to the stabilizing additives, but the dilution of the MabThera caused a slow formation of acidic variants, while the amount of the basic variants did not change. After incubation of the diluted rituximab at higher temperature several charge variants could be determined by CZE.

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“…These PQAs can potentially include: product-related structural heterogeneity related to glycosylation profile, disulfide bond pattern, and higher order structure; product-related degradants and impurities, such as deamidation, oxidation, sequence variants; and process-related impurities and residuals, such as host cell protein (HCP), host cell DNA, and residual protein A [1]. Regulatory agencies have recently recommended a Quality by Design (QbD) approach for the manufacturing of therapeutic molecules [2][3][4][5], which requires in-depth understanding of these PQAs at the molecular level to ensure that the drug products meet the desired safety and efficacy profiles [6]. The QbD guidelines require development of a quality target product profile (QTPP) that identifies critical quality attributes (CQAs) and implementation of control strategies to ensure that the QTPP is achieved.…”

mentioning

confidence: 99%

“…The complexity of biologics attributes and the implementation of QbD strategies demand a multi-attribute method (MAM) that can monitor multiple biologics PQAs at the molecular level in a single assay. Coupling liquid chromatography (LC) to high resolution and high accuracy mass spectrometry (MS) techniques, LC-MS based peptide mapping has become a MAM approach that can identify and quantify multiple attributes of biologics simultaneously at the molecular level [6,7,11,12], as well as elucidate the mechanisms associated with degradation [7,13,14]. In a typical MAM workflow, therapeutic protein samples are denatured and reduced to break disulfide bonds between the cysteine residues, which are then alkylated by iodoacetamide.…”

mentioning

confidence: 99%

LC-MS multi-attribute method for characterization of biologics

Xu¹,

Qiu²,

Li³

2017

J Appl Bioanal

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Biologics, such as therapeutic monoclonal antibodies (mAbs), are complex protein molecules produced from mammalian tissue culture cells through recombinant DNA technology. As a result of naturally-occurring molecular heterogeneity as well as chemical and enzymatic modifications during manufacture, process, and storage, there are many product quality attributes (PQAs) presenting in therapeutic proteins. These PQAs can potentially include: product-related structural heterogeneity related to glycosylation profile, disulfide bond pattern, and higher order structure; product-related degradants and impurities, such as deamidation, oxidation, sequence variants; and process-related impurities and residuals, such as host cell protein (HCP), host cell DNA, and residual protein A [1]. Regulatory agencies have recently recommended a Quality by Design (QbD) approach for the manufacturing of therapeutic molecules [2][3][4][5], which requires in-depth understanding of these PQAs at the molecular level to ensure that the drug products meet the desired safety and efficacy profiles [6]. The QbD guidelines require development of a quality target product profile (QTPP) that identifies critical quality attributes (CQAs) and implementation of control strategies to ensure that the QTPP is achieved. QTPP is a prospective summary of the quality characteristics of a drug product to be achieved to ensure the desired quality, safety and efficacy [2]. QTPP describes the design criteria for the product and forms the basis for determination of the CQAs, critical process parameters (CPPs), and control strategy. A CQA is a physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality [2]. A CQA is identified based on the severity of harm to a patient resulting from failure to meet that quality attribute. Analytical methods to identify and quantify these PQAs, especially CQAs, are essential for the development of QTPP and implementation of control strategies. Conventionally, a panel of analytical techniques such as size-exclusion chromatography (SEC), ion-exchange chromatography (IEX), hydrophobic-interaction chromatography (HIC), or capillary electrophoresis (CE) is typically used to monitor product quality consistency as well as product variants and impurities at the intact protein level [7][8][9]. Although these chromatographic and electrophoretic methods widely are used as release assays for biologics [10], they cannot directly monitor biologically relevant PQAs at the molecular level, which does not align with QbD principles. The complexity of biologics attributes and the implementation of QbD strategies demand a multi-attribute method (MAM) that can monitor multiple biologics PQAs at the molecular level in a single assay. Coupling liquid chromatography (LC) to high resolution and high accuracy mass spectrometry (MS) techniques, LC-MS based peptide mapping has become a MAM approach that can identify and quantify multiple ...

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Characterization of Therapeutic Antibodies and Related Products

Cited by 527 publications

References 244 publications

Characterization and analysis of scFv-IgG bispecific antibody size variants

Characterization and analysis of scFv-IgG bispecific antibody size variants

Analysis of Rituximab, A Therapeutic Monoclonal Antibody by Capillary Zone Electrophoresis

LC-MS multi-attribute method for characterization of biologics

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