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... included Linzess ® (linaclotide) for the treatment of chronic constipation and Irritable Bowel Syndrome (IBS) with constipation in adults, Surfaxin ® (lucinactant) for treat- ment of Infant Respiratory Distress Syndrome (IRDS), Kyprolis ® (carfilzomib) a proteasome inhibitor to treat multiple myeloma, Gattex ® (teduglutide) to treat adults with short bowel syndrome (SBS), Bydureon ® (an extended-release version of Byetta, with the active ingredient exenatide) for patients with type 2 diabetes and Signifor ® (pasireotide) for the treatment of Cushing's disease [17,19]. A global overview of currently marketed peptide drugs is given in Table 2. ...
Context 2
... gen- eral lay-out for peptide drug monographs was proposed and was suggested to consist of appearance, solubility information, iden- tification by LC-UV supplemented by other techniques like mass spectrometry (MS), related peptides by LC-UV (including higher molecular weight derivatives), residual solvents (water, acetic acid, etc.), residual reagents, inorganic impurities (such as catalysts), Table 1 Overview of current peptide drug classes. Target Indication/activity Peptide example 26S proteasome Multiple myeloma Bortezomib ACTH r Diagnostic agent for cortisol disorder Cosyntropin Angiotensin II r Anti-hypertension Saralasin Bacterial cellwall synthesis Antibiotic (Gram-positive microbiological quality attributes and assay by LC-UV. Peptide- related substances in chemically synthesized peptide APIs are expected to adhere to thresholds of reporting (0.1%), identifica- tion (0.5%) and qualification (1.0%). ...
Citations
... (Luna et al. 2016) However, beyond its known toxicity and regulatory issues, piperidine is notable to induce the formation of aspartimide-based side products. (Amblard et al. 2006;Behrendt et al 2016;Coin et al 2007;D'Hondt et al. 2014;Flora et al. 2005;Jensen et al 2013;Mergler et al. 2003aMergler et al. , 2003bMergler and Dick 2005;Samson et al. 2019;Scala et al. 2018;Tam et al 1988;Toney et al. 1993;Wade et al. 2000) While it is well known that t-butyl esters (tBu) are unstable under acidic and basic conditions, and that the base-promoted aspartimide formation of Asp(OtBu) occurs during Fmoc removal or peptide coupling, (Neumann et al. 2020;Tam et al 1988) allyl esters have been less studied regarding their stability under Fmoc deprotection. (Delforge et al. 1996;Flora et al. 2005;Vigil-Cruz and Aldrich 1999). ...
Despite significant advancements in peptide chemistry, the persistent occurrence of aspartimide formation during peptide synthesis is a stumbling block, especially in stapled peptide through Lys-Asp lactamization. This obstacle not only results in low yields but also necessitates expensive purification processes and can render certain peptide sequences unattainable. In this context, the method proposed in this work utilizes morpholine to effectively address the issue and successfully achieve the synthesis of a challenging aspartimide-prone peptide sequence. The efficacy of morpholine in preventing aspartimide formation is emphasized, presenting a versatile solution applicable to peptides containing β-allyl ester aspartic acid for stapling peptide synthesis. Graphical Abstract
... The majority of the impurities are formed during the SPPS process, most notably, single amino acid deletions and additions, D-isomer formation and modification of the N-terminus. [6][7][8] As the peptide chain elongates, the impurities formed during each amino acid incorporation cycle accumulate and persist through the end of the synthesis. Some of these impurities may be difficult to reject in downstream crystallization, 9 chromatography, or membrane filtration and will remain in the active pharmaceutical ingredient. ...
The peptide coupling reaction is one of the most critical steps in the solid phase synthesis of therapeutic peptides/proteins. Improper reaction conditions can result in several common impurities such as single amino acid deletions, additions, N‐terminus modifications, and D‐isomers, all while potentially impacting the active pharmaceutical ingredient critical quality attributes. In this work, we developed a first‐principle mechanistic reaction kinetics model for the solid‐phase peptide/protein coupling reaction based on well‐established reaction mechanisms and experimental data from literature. Utilizing the reaction kinetics model, we present a systematic, quality by design approach for the coupling reaction control strategy. Critical process parameters are identified via univariate analysis and the design space is designated via multivariate risk assessment. The presented approach provides a novel solution for designing solid‐phase peptide/protein synthesis control strategies and identifying normal operating ranges for each process parameter, as well as the associated design space.
... Regarding artificial peptides, D-amino acids were reported to enhance the biostability of peptide drugs in the human body, since D-amino acids usually change the secondary structure of peptide drugs and interfere with the interaction between peptide drugs and their receptors [9,10]. On the contrary, DAACPs could also be accidentally synthesized and considered as undesired impurities during solid-phase peptide drug synthesis (SPPS) [11,12]. The inescapable probability of racemization during SPPS (approximately 0.4% or less per synthesis cycle) [13] indicated that the identification of DAACP impurities in peptide drug products is required and imposes an urgent quality control of biological therapeutics [14][15][16]. ...
D-amino acid-containing peptides (DAACPs) occur in biological and artificial environments. Since the importance of DAACPs has been recognized, various mass spectrometry-based analytical approaches have been developed. However, the capability of higher-energy collisional dissociation (HCD) fragmentation to characterize DAACP sites has not been evaluated. In this study, we compared the normalized spectra intensity under different conditions of HCD and used liraglutide along with its DAACPs as examples. Our results indicated that the difference in the intensity of y ions between DAACPs and all-L liraglutide could not only distinguish them but also localize the sites of D-amino acids in the DAACPs. Our data demonstrate the potential of using HCD for the site characterization of DAACPs, which may have great impact in biological studies and peptide drug development.
... Semaglutide impurities can include peptides of imperfect structure, resulting from the insertion of an undesired amino acid or deletion (the absence of one or more amino acid residues), oxidation or racemization of amino acids [49]. There are methods for obtaining semaglutide by solid-phase synthesis that can reduce the formation of racemic impurities, simplify the purification of the target product, its purity and yield, and also reduce costs [50,51]. ...
Semaglutide is a representative of analogues of the incretin hormone human glucagon-like peptide-1 (GLP-1) and is currently used in Russia for the treatment of type 2 diabetes mellitus (T2DM; in monotherapy and in combination therapy), including patients with obesity and overweight.
The aim of the work was to conduct a comparative assessment of the physicochemical properties, a biological activity, bioequivalence and safety, including tolerability and immunogenicity, of the drug Quincent® (semaglutide, 1.34 mg/ml, a solution for a subcutaneous administration, Promomed Rus LLC, Russia) and the drug Ozempic® (semaglutide, 1.34 mg/ml, a solution for a subcutaneous administration, Novo Nordisk A/S, Denmark) when administered to healthy volunteers.
Materials and methods. To assess the degree of similarity of the study drug Quincenta® (semaglutide, 1.34 mg/ml, a solution for a subcutaneous administration, Promomed Rus LLC, Russia) with a chemically synthesized active substance to the original (reference) drug Ozempic® (semaglutide, 1.34 mg/ml, a solution for a subcutaneous administration, Novo Nordisk A/S, Denmark), a comparative study of physicochemical properties and a biological activity was carried out. To assess the bioequivalence of the study drug and the reference drug, an open randomized parallel comparative study with the participation of healthy volunteers ( n =54), 54 participants of which had been included in the population, was conducted. The volunteers were randomized into 2 groups in a 1:1 ratio, and received a single dose subcutaneously either of the study drug (domestic semaglutide at a dose of 0.5 mg) or the reference drug (foreign semaglutide at a dose of 0.5 mg). The mode of administration was in the morning on an empty stomach. A semaglutide concentration was determined in serum samples using a previously validated enzyme-linked immunosorbent assay (ELISA) method. A quantitative determination of antibodies to semaglutide in the human serum by ELISA was carried out with a microplate photometer using ready-made kits pre-validated by the manufacturer. The conclusion about the bioequivalence of the compared drugs was made using an approach based on the assessment of 90% confidence intervals for the ratios of the geometric mean values of the parameters C max , AUC (0–t) of semaglutide in the measurement original units.
Results. The results of the comparative analysis of the study drug and the reference drug demonstrate the comparability of their physicochemical properties and biological activity. The results of the clinical study demonstrated the bioequivalence of the test drug and the reference drug. Thus, the pharmacokinetic parameters of the drugs were comparable to each other: the C max value for the study drug was 42.088±8.827 ng/ml, for the reference drug Ozempic® it was 42.2556±7.84. Herewith, the half-life for the study drug and the reference drug was 168.39±39.47 and 157.99±28.57 hours, respectively. The resulting 90% confidence intervals for the ratio of the C max and AUC 0–t values of the study drug and the reference drug were 90.89–109.15 and 91.66–111.27%, respectively. The tolerability of the drugs in the volunteers was notified as good. No adverse events were recorded during the study. No serious adverse events were reported throughout the study. According to the results of the immunogenicity analysis, no antibodies to Russian-made semaglutide were detected in the blood serum of the volunteers, which indicated the lack of Results. The results of a comparative analysis of the study drug and the reference drug demonstrate the comparability of physicochemical properties and biological activity. The results of the clinical study demonstrated the bioequivalence of the study drug and the reference drug. Thus, the pharmacokinetic parameters of the drugs were comparable to each other: the C max value for the study drug was 42.088±8.827 ng/ml, for the reference drug Ozempic® this figure was 42.2556±7.84. At the same time, the half-life for the study drug and the reference drug was 168.39±39.47 and 157.99±28.57 hours, respectively. The resulting 90% confidence intervals for the ratio of the C max and AUC 0–t values of the study drug and the reference drug were 90.89–109.15 and 91.66–111.27%, respectively. Tolerability of the drugs in volunteers was noted as good. No adverse events were recorded during the study. No serious adverse events were reported throughout the study. According to the results of the immunogenicity analysis, no antibodies to Russian-made semaglutide were detected in the blood serum of the volunteers, which indicated the lack of the drug immunogenicity.
Conclusion. In the course of the study, the comparability of the physicochemical properties and biological activity of the studied Russian drug with the chemically synthesized active substance Quincenta® to the reference drug Ozempic® was confirmed: the activity range of the studied drugs was within 80–120% in relation to the standard sample of semaglutide. The bioequivalence and a similar safety profile, including the immunogenicity and tolerability of the Russian drug Quincenta® (semaglutide 1.34 mg/ml, Promomed Rus LLC, Russia) were shown in comparison with the foreign drug Ozempic® (semaglutide 1.34 mg/ml, Novo Nordisk A/C, Denmark).
... Typically, solid-phase synthesis (SPSS) is used for their production. The main routes and causes of deviation from the intended amino acid sequence are well known for SPSS [11,12]. In such a setting, therefore, the number of contaminants to be taken into account may be small and manageable. ...
Quantitative analysis relies on pure-substance primary calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method for determining the mass fraction of host cell proteins (HCPs) in bioengineered proteins which are intended for use as protein calibration standards. In this study a purified hemoglobin-A2 (HbA 2 ) protein, obtained through its overexpression in E. coli, was used. Two different materials were produced: natural and U ¹⁵ N-labeled HbA 2 . For the quantification of impurities, precursor ion (MS1-) intensities were integrated over all E. coli proteins identified and divided by the intensities obtained for HbA 2 . This ratio was calibrated against the corresponding results for an E. coli cell lysate, which had been spiked at known mass ratios to pure HbA 2 . To demonstrate the universal applicability of LFQ, further proteomes (yeast and human K562) were then alternatively used for calibration and found to produce comparable results. Valid results were also obtained when the complexity of the calibrator was reduced to a mix of just nine proteins, and a minimum of five proteins was estimated to be sufficient to keep the sampling error below 15%. For the studied materials, HbA 2 mass fractions (or purities) of 923 and 928 mg(HbA 2 )/g(total protein) were found with expanded uncertainties ( U ) of 2.8 and 1.3%, resp. Value assignment by LFQ thus contributes up to about 3% of the overall uncertainty of HbA 2 quantification when these materials are used as calibrators. Further purification of the natural HbA 2 yielded a mass fraction of 999.1 mg/g, with a negligible uncertainty ( U = 0.02%), though at a significant loss of material. If an overall uncertainty of 5% is acceptable for protein quantification, working with the original materials would therefore definitely be viable, circumventing the need of further purification.
... Regulatory T-cell epitopes have now been identified in multiple human proteins and peptides, including the well-defined Treg epitopes found in immunoglobulin G (IgG), heat shock protein and Factor V. 30,53-55 Many (but not all) Treg epitopes can be identified using a combination of in silico tools (for identifying sequences resembling Treg epitopes) and in vitro validation methods (for validating their regulatory effect), as described in detail by van Herwijnen et al. and De Groot and colleagues. 55,56 Not surprisingly, T effector and Treg epitopes are present in peptide drugs that are derived from natural human genome sequences. The presence of Treg epitopes can reduce the risk of immunogenicity to the drug product ( Figure 1). ...
Peptide drugs play an important part in medicine owing to their many therapeutic applications. Of the 80 peptide drugs approved for use in humans, at least five are now off-patent and are consequently being developed as generic alternatives to the originator products. To accelerate access to generic products, the FDA has proposed new regulatory pathways that do not require direct comparisons of generics to originators in clinical trials. The 'Abbreviated New Drug Application' (ANDA) pathway recommends that sponsors provide information on any new impurities in the generic drug, compared with the originator product, because the impurities can have potential to elicit unwanted immune responses owing to the introduction of T-cell epitopes. This review describes how peptide drug impurities can elicit unexpected immunogenicity and describes a framework for performing immunogenicity risk assessment of all types of bioactive peptide products. Although this report primarily focuses on generic peptides and their impurities, the approach might also be of interest for developers of novel peptide drugs who are preparing their products for an initial regulatory review.
... Main channels of aberration from the intended amino acid sequence are well known for SPSS. 11,12 In such setting, therefore, the number of contaminants to be taken into account may be small and manageable. ...
Quantitative analysis depends on pure-substance primary
calibrators with known mass fractions of impurity. Here, label-free quantification (LFQ) is being evaluated as a readily available, reliable method to determine the mass fraction of host-cell proteins (HCPs) in bioengineered proteins. For example, hemoglobin-A2 (HbA2)
is being used, as obtained by overexpression in E.coli. Two different materials had been produced: natural, and U-15N-labeled HbA2. For quantification of impurity, precursor ion
(MS1-) intensities were integrated over all E.coli-proteins identified, and divided by the intensities obtained for HbA2. This ratio was calibrated against the corresponding results for E.coli-cell lysate, which had been spiked at known mass-ratios to pure HbA2. To demonstrate universal applicability of LFQ, further proteomes (yeast- and human K562) were then alternatively used for calibration, and confirmingly found to produce comparable results. Valid results could also be obtained on reduction of complexity of the calibrator down to a mix of nine proteins, and a minimum of five proteins is estimated to be sufficient to keep the sampling error less than 15%. For the studied materials,
HbA2-mass fractions were found of 916 +/- 15 mg/g and 922 +/-11 mg/g. Value assignment by LFQ contributes 1-2%, thus, to the overall uncertainty of HbA2-quantification,
if using these materials as calibrators. Further purification of the natural HbA2 yielded 999.1 +/- 0.15 mg/g, corresponding to approx 0.2% of uncertainty contribution, though at a significant loss of material. Accepting a target of 5% overall-uncertainty for protein-quantification, working with the original materials would absolutely have been viable, therefore.
... These official testing procedures often require calibration against a higher order physical standard (e.g., pharmacopeial reference standard) with an appropriately assigned value. Such reference standards are essential for assessing the quality attributes of therapeutic products such as peptides, as described in pharmacopeial methodologies and specifications [4,5]. Since the quality attributes (e.g., assay, purity) of the drug substance should be established against a well characterized reference standard, it is imperative that those attributes remain unchanged in the reference standard and that its original purity determination is valid under the conditions in which it will be used [6]. ...
Purpose
Peptides are an important class of therapeutics. Their quality is evaluated using a series of analytical tests, many of which depend on well-characterized reference standards to determine identity, purity, and strength.
Objective
Discuss approaches to producing peptide reference standards, including vialing, lyophilization, analytical testing and stability studies.
Methods
Case studies are used to illustrate analytical approaches to characterize reference standards, including methods for value assignment, content uniformity, and identity testing. Methods described include NMR, mass spectrometry, and chromatography techniques for identity testing and HPLC and GC methods for assessing peptide content and impurities.
Results
This report describes the analytical strategy used to establish peptide reference standard and illustrates how results from multiple labs are integrated to assign a value to the final lyophilized vial. A two-step process for value assignment is described, which uses a mass balance approach to assign a quantitative value to a bulk peptide material. The bulk material is then used as a standard to assign a final value to the vialed material. Testing to confirm peptide identity and to ensure consistency of the vialed material is also described. Considerations for addressing variability, identifying outliers, and implementing stability studies are also presented.
Conclusion
The methods and case studies described provide a benchmark for best practices in establishing the preparation, analytical testing, handling, and storage of peptide reference standards for the pharmaceutical industry. Some peptide features, such as chiral or isobaric amino acids, may require additional techniques to ensure a full characterization of the peptide reference standard.
... The inefficient deprotection of the amino acid side chain leads to unwanted peptide adducts. In SPPS, the oxidation of amino acid impurities was also observed [24]. The incomplete removal of solvent used in the peptide elongation step may lead to impurities such as substitution and addition products. ...
Obesity is a global health concern. Physical activities and eating nutrient-rich functional foods can prevent obesity. In this study, nano-liposomal encapsulated bioactive peptides (BPs) were developed to reduce cellular lipids. The peptide sequence NH2-PCGVPMLTVAEQAQ-CO2H was chemically synthesized. The limited membrane permeability of the BPs was improved by encapsulating the BPs with a nano-liposomal carrier, which was produced by thin-layer formation. The nano-liposomal BPs had a diameter of ~157 nm and were monodispersed in solution. The encapsulation capacity was 61.2 ± 3.2%. The nano-liposomal BPs had no significant cytotoxicity on the tested cells, keratinocytes, fibroblasts, and adipocytes. The in vitro hypolipidemic activity significantly promoted the breakdown of triglycerides (TGs). Lipid droplet staining was correlated with TG content. Proteomics analysis identified 2418 differentially expressed proteins. The nano-liposomal BPs affected various biochemical pathways beyond lipolysis. The nano-liposomal BP treatment decreased the fatty acid synthase expression by 17.41 ± 1.17%. HDOCK revealed that the BPs inhibited fatty acid synthase (FAS) at the thioesterase domain. The HDOCK score of the BPs was lower than that of orlistat, a known obesity drug, indicating stronger binding. Proteomics and molecular docking analyses confirmed that the nano-liposomal BPs were suitable for use in functional foods to prevent obesity.
... 21,22 Challenges with Peptide Purification, Separation, and Immunogenicity. Handling peptides with natural and noncanonical amino acids in the lab faces practical challenges: their synthesis often requires multiple steps resulting in impurities that are structurally very similar, 23 making purification difficult and time-consuming. 24,25 Reverse-phase HPLC is widely employed in peptide purification but is burdened by its own challenges related to strong retention of hydrophobic peptides, sustainability, and cost, particularly on a large scale. ...
The isoelectric point (pI) is a fundamental physicochemical property of peptides and proteins. It is widely used to steer design away from low solubility and aggregation and guide peptide separation and purification. Experimental measurements of pI can be replaced by calculations knowing the ionizable groups of peptides and their corresponding pKa values. Different pKa sets are published in the literature for natural amino acids, however, they are insufficient to describe synthetically modified peptides, complex peptides of natural origin, and peptides conjugated with structures of other modalities. Noncanonical modifications (nCAAs) are ignored in the conventional sequence-based pI calculations, therefore producing large errors in their pI predictions. In this work, we describe a pI calculation method that uses the chemical structure as an input, automatically identifies ionizable groups of nCAAs and other fragments, and performs pKa predictions for them. The method is validated on a curated set of experimental measures on 29 modified and 119093 natural peptides, providing an improvement of R2 from 0.74 to 0.95 and 0.96 against the conventional sequence-based approach for modified peptides for the two studied pKa prediction tools, ACDlabs and pKaMatcher, correspondingly. The method is available in the form of an open source Python library at https://github.com/AstraZeneca/peptide-tools, which can be integrated into other proprietary and free software packages. We anticipate that the pI calculation tool may facilitate optimization and purification activities across various application domains of peptides, including the development of biopharmaceuticals.
