Facile quantitation of free thiols in a recombinant monoclonal antibody by reversed-phase high performance liquid chromatography with hydrophobicity-tailored thiol derivatization

Welch, Leslie; Dong, Xiao; Hewitt, Daniel; Irwin, Michelle; McCarty, Luke; Tsai, Christina; Baginski, Tomasz K.

doi:10.1016/j.jchromb.2018.05.039

Cited by 11 publications

(7 citation statements)

References 20 publications

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“…During technical development of mAb1 production process, the total free thiol content (exposed and buried free thiols) was routinely determined using an in-house developed reversed-phase high performance liquid chromatography (RP-HPLC) assay, with the free thiol derivatization under denaturing conditions and UV detection, capable of separating and quantitating free thiol-containing mAb1 variants [9]. Using this assay, a high variability of the total free thiol content (up to ~ 10-fold difference in % Free Thiol Peaks) was observed among various mAb1 drug substance (DS) batches produced at manufacturing-scale (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Enzymatic basis of the Fc-selective intra-chain disulfide reduction and free thiol content variability in an antibody produced in Escherichia coli

Baginski

Veeravalli

McKenna³

et al. 2022

Preprint

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Background: Escherichia coli (E. coli) is a promising host for production of recombinant proteins (including antibodies and antibody fragments) that don’t require complex post-translational modifications such as glycosylation. During manufacturing-scale production of a one-armed antibody in E. coli (periplasmic production), variability in the degree of reduction of the antibody’s disulfide bonds was observed. This resulted in variability in the free thiol content, a potential critical product quality attribute. This work was initiated to understand and prevent the variability in the total free thiol content during manufacturing. Results: In this study, we found that the reduction in antibody’s disulfide bonds was observed to occur during homogenization and the ensuing homogenate hold step where in the antibody is exposed to redox enzymes and small molecule reductants present in homogenate. Variability in the downstream processing time between the start of homogenization and end of the homogenate hold step resulted in variability in the degree of antibody disulfide bond reduction & free thiol content. The disulfide bond reduction in the homogenate is catalyzed by the enzyme disulfide bond isomerase C (DsbC) and is highly site-specific and occurred predominantly in the intra-chain disulfide bonds present in the Fc CH2 region. Our results also imply that lack of glycans in E. coli produced antibodies may facilitate DsbC accessibility to the disulfide bond in the Fc CH2 region, resulting in its reduction. Conclusions: During E. coli antibody manufacturing processes, downstream processing steps such as homogenization and subsequent processing of the homogenate can impact degree of disulfide bond reduction in the antibody and consequently product quality attributes such as total free thiol content. Duration of the homogenate hold step should be minimized as much as possible to prevent disulfide bond reduction & free thiol formation. Other approaches such as reducing homogenate temperature, adding flocculants prior to homogenization, using enzyme inhibitors, or modulating redox environments in the homogenate should be considered to prevent antibody disulfide bond reduction during homogenization and homogenate processing steps in E. coli antibody manufacturing processes.

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Section: Resultsmentioning

confidence: 99%

“…Free thiol analysis by RP-HPLC Total free thiol content (accessible and buried free thiols) was determined by RP-HPLC with free thiol derivatization and UV detection as described [9]. Brie y, mAb sample was diluted to 2 mg/mL with 20 mM sodium acetate buffer, pH 6.0.…”

Section: Analytical Measurementsmentioning

confidence: 99%

Enzymatic basis of the Fc-selective intra-chain disulfide reduction and free thiol content variability in an antibody produced in Escherichia coli

Baginski

Veeravalli

McKenna³

et al. 2022

Preprint

Self Cite

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“…An interesting development of maleimide labeling has been recently employed, where free-Cys in mAbs are labeled with N-tert-butylmaleimide (NtBM) ( Welch et al, 2018 ). When unlabeled and NtBM-labeled mAb are analyzed on C4 RP-HPLC, there is a retention time shift associated with NtBM-labeled mAb, allowing resolution of the two peaks and quantitation of free-Cys.…”

Section: Spectroscopic Methodsmentioning

confidence: 99%

A Review of Methodologies for the Detection, Quantitation, and Localization of Free Cysteine in Recombinant Proteins: A Focus on Therapeutic Monoclonal Antibodies

Metcalfe

2022

Front. Mol. Biosci.

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Free-cysteine residues in recombinant biotherapeutics such as monoclonal antibodies can arise from incorrect cellular processing of disulfide bonds during synthesis or by reduction of disulfide bonds during the harvest and purification stage of manufacture. Free cysteines can affect potency, induce aggregation, and decrease the stability of therapeutic proteins, and the levels and positions of free cysteines in proteins are closely monitored by both manufacturers and regulators to ensure safety and efficacy. This review summarizes the latest methodologies for the detection and quantification of free cysteines.

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“…Some examples of derivatization with this purpose are taken from the literature. For example, a facile quantitation of the free thiols in a recombinant monoclonal antibody by RP‐HPLC with hydrophobicity‐tailored thiol derivatization was reported by Welch et al (2018). For this purpose, the authors used maleimide‐based thiol‐reactive reagents with varying degrees of hydrophobicity, based on the addition reaction of thiol group to the C=C bond from the derivatization reagent (Winther & Thorpe, 2014).…”

Section: Hydrophobicity‐tailored Derivatizationmentioning

confidence: 99%

Derivatization procedures and their analytical performances for HPLC determination in bioanalysis

David

Moldoveanu

Galaon

2020

Biomedical Chromatography

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Derivatization, or chemical structure modification, is often used in bioanalysis performed by liquid chromatography technique in order to enhance detectability or to improve the chromatographic performance for the target analytes. The derivatization process is discussed according to the analytical procedure used to achieve the reaction between the reagent and the target compounds (containing hydroxyl, thiol, amino, carbonyl and carboxyl as the main functional groups involved in derivatization). Important procedures for derivatization used in bioanalysis are in situ or based on extraction processes (liquid-liquid, solid-phase and related techniques) applied to the biomatrix. In the review, chiral, isotope-labeling, hydrophobicity-tailored and post-column derivatizations are also included, based on representative publications in the literature during the last two decades. Examples of derivatization reagents and brief reaction conditions are included, together with some bioanalytical applications and performances (chromatographic conditions, detection limit, stability and sample biomatrix).

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Facile quantitation of free thiols in a recombinant monoclonal antibody by reversed-phase high performance liquid chromatography with hydrophobicity-tailored thiol derivatization

Cited by 11 publications

References 20 publications

Enzymatic basis of the Fc-selective intra-chain disulfide reduction and free thiol content variability in an antibody produced in Escherichia coli

Enzymatic basis of the Fc-selective intra-chain disulfide reduction and free thiol content variability in an antibody produced in Escherichia coli

A Review of Methodologies for the Detection, Quantitation, and Localization of Free Cysteine in Recombinant Proteins: A Focus on Therapeutic Monoclonal Antibodies

Derivatization procedures and their analytical performances for HPLC determination in bioanalysis

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