Recent mass spectrometry-based techniques and considerations for disulfide bond characterization in proteins

Abstract

Disulfide bonds are important structural moieties of proteins: they ensure proper folding, provide stability, and ensure proper function. With the increasing use of proteins for biotherapeutics, particularly monoclonal antibodies, which are highly disulfide bonded, it is now important to confirm the correct disulfide bond connectivity and to verify the presence, or absence, of disulfide bond variants in the protein therapeutics. These studies help to ensure safety and efficacy. Hence, disulfide bonds are among the critical quality attributes of proteins that have to be monitored closely during the development of biotherapeutics. However, disulfide bond analysis is challenging because of the complexity of the biomolecules. Mass spectrometry (MS) has been the go-to analytical tool for the characterization of such complex biomolecules, and several methods have been reported to meet the challenging task of mapping disulfide bonds in proteins. In this review, we describe the relevant, recent MS-based techniques and provide important considerations needed for efficient disulfide bond analysis in proteins. The review focuses on methods for proper sample preparation, fragmentation techniques for disulfide bond analysis, recent disulfide bond mapping methods based on the fragmentation techniques, and automated algorithms designed for rapid analysis of disulfide bonds from liquid chromatography-MS/MS data. Researchers involved in method development for protein characterization can use the information herein to facilitate development of new MS-based methods for protein disulfide bond analysis. In addition, individuals characterizing biotherapeutics, especially by disulfide bond mapping in antibodies, can use this review to choose the best strategies for disulfide bond assignment of their biologic products. Graphical Abstract This review, describing characterization methods for disulfide bonds in proteins, focuses on three critical components: sample preparation, mass spectrometry data, and software tools.

The impact of standard accelerated stability conditions on antibody higher order structure as assessed by mass spectrometry

“…Additional reports further support the conclusion that these breakages, along with any sequential bond formation, can affect the stability of mAbs. [65][66][67] However, given the absence of any disulfide mapping approach in our study, such as those previously reported in the literature, 68 we cannot conclusively support our hypothesis using CIU-IM-MS data alone.…”

Preparing Proteoforms of Therapeutic Proteins for Top-Down Mass Spectrometry

“…Du et al stated that during the manufacturing process, extensive reduction of antibodies has been observed after harvest operation or Protein A affinity chromatography and multiple process parameters correlate to the extent of the reduction [34]. The topic "disulfide bonds of therapeutic proteins" is in depth discussed by Lakbub et al [35].…”

Experimental Assignment of Disulfide‐Bonds in Purified Proteins

“…Therefore, it is useful and necessary (in cases where the MS/MS assignments are ambiguous) to confirm the disulfide bond assignments using the protocol described here. A recent comprehensive review of MS‐based techniques summarizes and compares most bottom‐up techniques and associated software for disulfide bond assignment (Lakbub, Shipman, & Desaire, ).…”