Research article
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Highlights
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Peptide mapping methods of semaglutide and liraglutide were developed. •
The sameness between synthetic and recombinant DNA peptides was demonstrated. •
High-resolution mass spectrometry was useful for peptide mapping analysis. •
Combining Glu-C and chymotrypsin peptide maps provided full sequence coverage.
Abstract
Semaglutide and liraglutide are long-acting glucagon-like peptide-1 receptor agonists used to treat type-2 diabetes and obesity. Recent advances in peptide synthesis and analytical technologies have enabled the development of synthetic generic peptide for reference listed drugs (RLD) originating from recombinant DNA (rDNA) technology. Since the original semaglutide and liraglutide were produced through rDNA technology, there has been great interest in developing their synthetic peptides as generic versions of the original drugs. Therefore, this study aimed to develop a peptide mapping method to describe the primary structure of semaglutide and liraglutide using ultra-performance liquid chromatography-high-resolution mass spectrometry (UPLC-HRMS), and to apply this method to demonstrate the sameness between synthetic peptides for generic drugs and rDNA peptides of the original drugs. Masses of the peptide fragments were identified using HRMS at an accurate level of mass error below 10 ppm, and their sequences were determined via MSE sequencing using in-source fragmentation, which was also useful for identifying the fatty acid chain modification site. Full sequence coverage of each semaglutide and liraglutide was accomplished by combining peptide maps generated using Glu-C and chymotrypsin. The proposed peptide mapping method using UPLC-HRMS was useful for determining active ingredient sameness between generic synthetic peptides and previously approved peptide drug products of rDNA origin.
Keywords
Glucagon-like peptide-1 receptor agonists
;
Semaglutide
;
Liraglutide
;
High-resolution mass spectrometry
;
Peptide mapping
Introduction
Peptide drugs have become an important class of pharmaceuticals with a steadily increasing number of approved drugs for treating a wide range of diseases, occupying a distinct market space between small organic molecule drugs and biologics [1], [2]. Since most peptides are rapidly cleared from the bloodstream, strategies to increase plasma half-life have been widely applied to injectable peptides [3], [4]. Additionally, several efforts have been made to develop oral delivery system of peptides [5], [6], [7]. Semaglutide and liraglutide are long-acting glucagon-like peptide-1 (GLP-1) receptor agonists developed using the lipidation technology [8]. They are used to treat type-2 diabetes and obesity, and their drug products have achieved a blockbuster status in the market [9].
Peptides can be produced by recombinant DNA (rDNA) technology or chemical synthesis. The representative rDNA peptides are glucagon, teriparatide, and teduglutide [10], [11], [12]. Semaglutide and liraglutide are made by a semi-recombinant process, where the peptide backbone is produced using rDNA technology, and fatty acid is subsequently conjugated to lysine (Lys) residue [8], [13]. In rDNA peptides, high-risk post-translational modifications, such as deamidation, oxidation, isomerization, and oligomerization, can be critical considerations, as the manufacturing environment and downstream processing conditions can promote their occurrence [14], [15]. Recently, several peptides have been chemically synthesized using solid-phase peptide synthesis, which has become the method of choice for producing peptides, making it possible to automate this process and produce peptides with sequences longer than 20 amino acids [16]. Typically, the drug substance of exenatide, composed of 39 amino acids, is manufactured via chemical synthesis [17].
Advances in peptide synthesis technology and the widespread use of synthetic peptide drugs have increased interest in the development of synthetic generic peptides through Abbreviated New Drug Applications (ANDAs) for reference listed drugs (RLD) originating from rDNA technology. Recently, the Food and Drug Administration (FDA) issued a guidance for industry titled “ANDAs for certain highly purified synthetic peptide drug products that refer to listed drugs of rDNA origin” in 2021 [18]. Following that, the European Medicines Agency (EMA) published a draft guideline titled “Guideline on the Development and Manufacture of Synthetic Peptides” in 2023 [19]. Both documents highlight the sameness of the active ingredient in a proposed generic with that of the RLD, recommending assessments of primary and secondary structures, oligomer state, physicochemical properties, and biological activities, as appropriate. Since the primary structure of a peptide is directly related to drug efficacy, it is important for the manufacturer to characterize the primary sequence of the active ingredient peptide by comparing it with the primary sequence of the RLD peptide.
Peptide mapping is an identity-testing method based on primary structure analysis and is recommended for peptide drugs with sequences longer than 20 amino acids [20]. In addition to primary structure analysis, peptide mapping is a powerful test to detect whether structural alterations have occurred and to demonstrate process consistency and chemical stability [21]. Peptide mapping is generally performed by chemical or enzymatic treatment of the test material to form small peptide fragments, followed by the separation and identification of the resultant fragments. The analysis of the fragmented peptides has generally been performed by high-performance liquid chromatography with UV detection (HPLC-UV) methods, by comparing the retention time of each fragment peak with the retention time of the fragments derived from a reference standard [22]. However, HPLC-UV methods may not be sufficient to achieve the full sequence coverage for peptides with complex structures because of the presence of unresolved or undetectable fragments. Mass spectrometry (MS) can be used to identify peptide fragments that cannot be detected by UV detector [23].
This study aimed to develop a peptide mapping method to describe the primary structure of the lipidated GLP-1 receptor agonists, semaglutide and liraglutide, using ultra-performance liquid chromatography-high-resolution MS (UPLC-HRMS), and to apply this method to demonstrate the sameness between synthetic peptides for semaglutide and liraglutide generic drugs and rDNA peptides of the original drugs (Ozempic® and Saxenda®). HRMS based on time-of-flight was employed to accurately determine the mass of each peptide fragment with four decimal places. The identity of each fragment was confirmed by comparison with in silico digestion results. In addition, amino acid sequence of each peptide fragment was determined by fragmentation pattern analysis using MSE. Two proteolytic enzymes, Glu-C and chymotrypsin, were used as peptide digestion agents for full sequence coverage of the target peptides.
Section snippets
Materials
Synthetic semaglutide and liraglutide were purchased from Bachem (Basel, Switzerland). Ozempic® and Saxenda® are commercial products containing semaglutide and liraglutide, respectively (Novo Nordisk, Bagsværd, Denmark). Sequencing-grade endoproteinase Glu-C and chymotrypsin were obtained from Promega (Madison, WI, USA) and Roche Diagnostics GmbH (Mannheim, Germany), respectively. LC-MS-grade water, acetonitrile, and formic acid were obtained from Thermo Fisher Scientific (Waltham, MA, USA).
Peptide mapping of semaglutide using Glu-C
Semaglutide contains three Glu and one Asp residues among 31 amino acids. Five fragments were expected after Glu-C digestion because Glu-C cleaves peptide chains at the carboxyl side of Asp and Glu (Fig. 1a). Fig. 1b shows the total ion chromatogram (TIC) of the synthetic semaglutide peptide digested with Glu-C. Six distinct peaks were observed and identified by comparing their masses with expected peptide masses obtained by in silico digestion (Table 1). The six peaks correspond to four
Conclusion
The comprehensive peptide mapping approach used in this study achieved full sequence coverage of semaglutide and liraglutide by combining peptide maps generated by Glu-C and chymotrypsin cleavage. The chymotrypsin-digested peptide map complemented the missing N-terminal sequence in the Glu-C-digested peptide map, whereas the Glu-C-digested peptide map included the missing C-terminal sequence in the chymotrypsin-digested peptide map. Highly accurate masses of the enzyme-digested fragments were
CRediT authorship contribution statement
Dong Hee Na: Writing review & editing, Supervision, Funding acquisition, Conceptualization. Soo Hyun Kim: Writing original draft, Methodology, Investigation. Sung Soo Kim: Writing original draft, Methodology, Investigation. Hyun Jun Kim: Methodology, Investigation, Formal analysis. Eun Ji Park: Data curation.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This research was supported by the Chung-Ang University Graduate Research Scholarship in 2023. This work was also supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-202400360020) and a grant (RS-202400397701) from Ministry of Food and Drug Safety in 2024. We would like to thank Editage (www.editage.co.kr) for English language editing.
1
These authors contributed equally to this work.
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