Development of a multidimensional online method for the characterization and quantification of monoclonal antibodies using immobilized flow-through enzyme reactors

Reinders, Lars M H; Klassen, Martin D; Teutenberg, Thorsten; Jaeger, Martin; Schmidt, Torsten

doi:10.1007/s00216-021-03683-z

Cited by 4 publications

(3 citation statements)

References 27 publications

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“…Other advantages include full unattendance once the sequence starts and minimal new equipment purchase. When compared to other online type of approaches, , this technique offers a few important advantages, such as a retention of typical LC flow path that allows the use of the system for offline analysis, standard CDS usage with full compliance, and generally a simpler system that is easier to use, maintain, and troubleshoot. Additionally, it is possible to reduce the cost of consumables with the technique, as the trypsin column (Orachrom) and the custom-made desalting device are found to be extremely durable.…”

Section: Discussionmentioning

confidence: 99%

“…Others have also automated peptide mapping with HPLC equipment; however, this typically involves adding significant complexity to the system that subsequently prevents it from being used as a standard UHPLC to perform nonautomated analysis. − Many techniques are partially automated and require additional work before or after the digestion. − Liu et al automated peptide mapping using a sequential injection device that was fully integrated into a PAT workflow . That approach has several advantages over the liquid handler approaches in that each sample is digested prior to analysis and the system is fully online.…”

Section: Introductionmentioning

confidence: 99%

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Fully Unattended Online Protein Digestion and LC–MS Peptide Mapping

Richardson,

Zhang

2023

Anal. Chem.

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LC−MS based peptide mapping, i.e., proteolytic digestion followed by LC−MS/MS analysis, is the method of choice for protein primary structural characterization. Manual proteolytic digestion is usually a labor-intensive procedure. In this work, a novel method was developed for fully automated online protein digestion and LC−MS peptide mapping. The method generates LC−MS data from undigested protein samples without user intervention by utilizing the same HPLC system that performs the chromatographic separation with some additional modules. Each sample is rapidly digested immediately prior to its LC−MS analysis, minimizing artifacts that can grow over longer digestion times or digest storage times as in manual or automated offline digestion methods. In this report, we implemented the method on an Agilent 1290 Infinity II LC system equipped with a Multisampler. The system performs a complete digestion workflow including denaturation, disulfide reduction, cysteine alkylation, buffer exchange, and tryptic digestion. We demonstrated that the system is capable of digesting monoclonal antibodies and other proteins with excellent efficiency and is robust and reproducible and produces fewer artifacts than manually prepared digests. In addition, it consumes only a few micrograms of material as most of the digested sample protein is subjected to LC−MS analysis.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Fully Unattended Online Protein Digestion and LC–MS Peptide Mapping

Richardson,

Zhang

2023

Anal. Chem.

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“…During recent years, several authors have successfully demonstrated the application of two- or multi-dimensional LC coupled to MS (2D- or mD-LC–MS) methods for the qualitative and quantitative structural characterization of therapeutic antibodies and related protein formats. − The mD-LC–MS approach combines sample preparation and multi-level analysis of antibody attributes within an automated continuously connected setup (online) and removes the need for manual sample preparation procedures and sample handling (offline). , …”

mentioning

confidence: 99%

Identification of Hetero-aggregates in Antibody Co-formulations by Multi-dimensional Liquid Chromatography Coupled to Mass Spectrometry

et al. 2023

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Antibody combination therapies have become viable therapeutic treatment options for certain severe diseases such as cancer. The co-formulation production approach is intrinsically associated with more complex drug product variant profiles and creates more challenges for analytical control of drug product quality. In addition to various individual quality attributes, those arising from the interactions between the antibodies also potentially emerge through co-formulation. In this study, we describe the development of a widely applicable multi-dimensional liquid chromatography coupled to tandem mass spectrometry method for antibody homo-versus hetero-aggregate characterization. The co-formulation of trastuzumab and pertuzumab was used, a challenging model system, comprising two monoclonal antibodies with very similar physicochemical properties. The data presented demonstrate the high stability of the co-formulation, where only minor aggregate formation is observed upon product storage and accelerated temperature or light-stress conditions. The results also show that the homo-and hetero-aggregates, formed in low and comparable proportions, are only marginally impacted by the formulation and product storage conditions. No preferential formation of hetero-aggregates, in comparison to the already existing pertuzumab and trastuzumab homo-aggregates, was observed.

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