Optimization of a nano‐enzymatic reactor for on‐line tryptic digestion of polypeptide conjugates by capillary electrophoresis

Ladner, Yoann; Coussot, G.; Ebner, Stefanie; Ibrahim, Amal M.; Vidal, Laetitia; Perrin, Catherine

doi:10.1002/elps.201500349

Cited by 7 publications

(8 citation statements)

References 28 publications

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“…Optimization of an in‐capillary nano‐enzymatic reactor for on‐line tryptic digestion of therapeutic polypeptide conjugates was done by CE analysis of tryptic hydrolysates of poly‐L‐lysine (PLL, 15–30 kDa) and PLL‐conjugate with 5‐methoxytryptamine using the transverse diffusion of laminar flow profiles mixing methodology . The separation of oligolysines (Lys 2–Lys 12) in acidic BGEs in the pH range 2.5–5.0 was better in citrate BGEs than in phosphate BGEs.…”

Section: Applicationsmentioning

confidence: 99%

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

Kašička

2017

Electrophoresis

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The review brings a comprehensive overview of recent developments and applications of high performance capillary and microchip electroseparation methods (zone electrophoresis, isotachophoresis, isoelectric focusing, affinity electrophoresis, electrokinetic chromatography, and electrochromatography) to analysis, microscale isolation, purification, and physicochemical and biochemical characterization of peptides in the years 2015, 2016, and ca. up to the middle of 2017. Advances in the investigation of electromigration properties of peptides and in the methodology of their analysis (sample preseparation, preconcentration and derivatization, adsorption suppression and EOF control, and detection) are described. New developments in particular CE and CEC methods are presented and several types of their applications to peptide analysis are reported: qualitative and quantitative analysis, determination in complex (bio)matrices, monitoring of chemical and enzymatical reactions and physical changes, amino acid, sequence and chiral analysis, and peptide mapping of proteins. Some micropreparative peptide separations are shown and capabilities of CE and CEC methods to provide important physicochemical characteristics of peptides are demonstrated.

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Section: Applicationsmentioning

confidence: 99%

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

Kašička

2017

Electrophoresis

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“…Each plug was injected in the capillary with 4 psi during 5 s corresponding to 5.13 nL. The volume of proteolysis buffer (NH 4 HCO 3 ) was sufficient to maintain a pH of 8.0 for the reactants mixture during the enzymatic reaction and consequently, the formation of peptides occurred [36]. Figure 1 shows that the electrophoretic profiles of in‐line digested mAb with the three enzymes are different from the profile of intact mAb due to trypsin interferences.…”

Section: Resultsmentioning

confidence: 99%

“…Plugs of reactants are injected with high pressure for a short time to generate parabolic profiles of plugs. TDLFP was applied successfully for the tryptic digestion of polylysines [35,36] as well as intact mAbs [37] using a sandwich injection mode of the reactants. In-line tryptic digestion was directly implemented without prior pretreatment step (denaturation, reduction …).…”

Section: Introductionmentioning

confidence: 99%

Fast in‐line bottom‐up analysis of monoclonal antibodies: Toward an electrophoretic fingerprinting approach

et al. 2021

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For their characterization and quality control, monoclonal antibodies are frequently analyzed at the bottom‐up level to generate specific fingerprints that can be used to tackle post‐translational modifications or ensure production consistency between lots. To circumvent time‐consuming and labor‐intensive off‐line sample preparation steps, the implementation of integrated methodologies from sample preparation to separation and detection is highly valuable. In this perspective, capillary zone electrophoresis appears as a choice technique since the capillary can subsequently be used as a vessel for sample preparation and electrophoretic discrimination/detection of the reaction products. Here, a fast in‐line methodology for the routine quality control of mAbs at the bottom‐up level is reported. Simultaneous denaturation and reduction (pretreatment step) were conducted with RapiGest® surfactant and dithiothreitol before in‐line tryptic digestion. Reactant mixing was realized by transverse diffusion of laminar flow profile under controlled temperature. In‐line digestion was carried out with a resistant trypsin to autolysis. The main parameters affecting the digestion efficiency (trypsin concentration and incubation conditions) were optimized to generate mAb electrophoretic profiles free from trypsin interferences. An acidic MS‐compatible BGE was used to obtain high resolution separation of released peptides and in‐line surfactant cleavage. The whole methodology was performed in less than two hours with good repeatability of migration times (RSD = 0.91%, n = 5) and corrected peak areas (RSD = 9.6%, n = 5). CE‐fingerprints were successfully established for different mAbs and an antibody‐drug conjugate.

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“…Schematic representation of the electrophoretically mediated microanalysis (EMMA) methodology for digestion of protein substrate (S) by the proteolytic enzyme (E) in proteolysis buffer (PB) with subsequent CE separation of the digest products in the FS capillary filled with BGE. Reprinted with permission from [75]…”

Section: Sample Preparationmentioning

confidence: 99%

Peptide mapping of proteins by capillary electromigration methods

Kašička

2022

J of Separation Science

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This review article provides a wide overview of important developments and applications of capillary electromigration methods in the area of peptide mapping of proteins in the period 1997-mid-2022, including review articles on this topic. It deals with all major aspects of peptide mapping by capillary electromigration methods: i) precleavage sample preparation involving purification, preconcentration, denaturation, reduction and alkylation of protein(s) to be analyzed, ii) generation of peptide fragments by off-line or on-line enzymatic and/or chemical cleavage of protein(s), iii) postcleavage preparation of the generated peptide mixture for capillary electromigration separation, iv) separation of the complex peptide mixtures by one-, two-and multidimensional capillary electromigration methods coupled with mass spectrometry detection, and v) a large application of peptide mapping for variable purposes, such as qualitative analysis of monoclonal antibodies and other protein biopharmaceuticals, monitoring of posttranslational modifications, determination of primary structure and investigation of function of proteins in biochemical and clinical research, characterization of proteins of variable origin as well as for protein and peptide identification in proteomic and peptidomic studies.

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Optimization of a nano‐enzymatic reactor for on‐line tryptic digestion of polypeptide conjugates by capillary electrophoresis

Cited by 7 publications

References 28 publications

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

Recent developments in capillary and microchip electroseparations of peptides (2015–mid 2017)

Fast in‐line bottom‐up analysis of monoclonal antibodies: Toward an electrophoretic fingerprinting approach

Peptide mapping of proteins by capillary electromigration methods

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