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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
... 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.
... 08 (hPTH). It is entirely composed of natural amino acids and contains two methionine residues in positions 8 and 18. Oxidation of the methionine residues within a peptide is a common occurrence and is frequently identified in drug product impurities analysis (Grassi and Cabrele, 2019;D'Hondt et al., 2014). Traditional in silico immunogenicity prediction algorithms cannot accommodate sequences containing methionine sulfoxide (or sulfone) residues. ...
... The first phase of this effort focuses on generating preliminary data to ultimately develop correction factors for D-amino acids. D-amino acids are commonly encountered in synthetic peptide impurities (D'Hondt et al., 2014) or by design in novel peptide drug development to decrease the rate of proteolytic cleavage and therefore increase the half-life of the peptide drug candidate (Evans et al., 2020;Wang et al., 2022). Reported studies have demonstrated that D-amino acids incorporated into some positions in the core sequence of a known promiscuous HLA-DR binding peptide, Flu-HA (306-318), diminish HLA binding affinity compared to the L-amino acid counterpart (Azam et al., 2021). ...
The in silico prediction of T cell epitopes within any peptide or biologic drug candidate serves as an important first step for assessing immunogenicity. T cell epitopes bind human leukocyte antigen (HLA) by a well-characterized interaction of amino acid side chains and pockets in the HLA molecule binding groove. Immunoinformatics tools, such as the EpiMatrix algorithm, have been developed to screen natural amino acid sequences for peptides that will bind HLA. In addition to commonly occurring in synthetic peptide impurities, unnatural amino acids (UAA) are also often incorporated into novel peptide therapeutics to improve properties of the drug product. To date, the HLA binding properties of peptides containing UAA are not accurately estimated by most algorithms. Both scenarios warrant the need for enhanced predictive tools. The authors developed an in silico method for modeling the impact of a given UAA on a peptide’s likelihood of binding to HLA and, by extension, its immunogenic potential. In silico assessment of immunogenic potential allows for risk-based selection of best candidate peptides in further confirmatory in vitro, ex vivo, and in vivo assays, thereby reducing the overall cost of immunogenicity evaluation. Examples demonstrating in silico immunogenicity prediction for product impurities that are commonly found in formulations of the generic peptides teriparatide and semaglutide are provided. Next, this article discusses how HLA binding studies can be used to estimate the binding potentials of commonly encountered UAA and “correct” in silico estimates of binding based on their naturally occurring counterparts. As demonstrated here, these in vitro binding studies are usually performed with known ligands which have been modified to contain UAA in HLA anchor positions. An example using D-amino acids in relative binding position 1 (P1) of the PADRE peptide is presented. As more HLA binding data become available, new predictive models allowing for the direct estimation of HLA binding for peptides containing UAA can be established.
... In 2013, the US pharmacopeia convention (USP) formed a therapeutic peptide expert panel, and the panel recommended some guidelines for the quality of peptide synthesis and for considering peptides as active pharmaceutical ingredients. Table 9.3 lists some of the quality controls that are recommended for peptide API required to obtain regulatory approval Rastogi et al. 2019;Eggen et al. 2014;D'Addio et al. 2016;Zeng et al. 2019;D'Hondt et al. 2014a). ...
... Peptides can undergo degradation and aggregation when they are subjected to stress related to manufacturing (D'Hondt et al. 2014a;Stalmans et al. 2016), processing, and shipping. These stress factors include temperature fluctuations, shaking, pH changes, and surfaces of the containers. ...
Recent advances in the field of peptide therapeutics have led to the design and synthesis of many promising peptides. However, research and development to provide safe, stable, efficacious, and patient compliant formulation plays a vital role in bringing peptide therapeutics to market. The conventional parenteral route has made scientific advances to overcome multiple barriers, leading to the approval of many peptide-based products via parenteral route of administration in recent years. In parallel, oral, pulmonary, transdermal, and other delivery routes have been extensively investigated to deliver peptides with improved patient compliance. This includes the use of novel strategies for developing delivery systems that can offer various advantages over conventional dosage forms. This chapter focuses on fundamentals, formulations, and recent advances for successful peptide delivery.KeywordsPeptidesPharmaceutical marketFormulation developmentDelivery systemsParenteralOralPulmonaryTransdermalControlled release parenteralCarrier systems
... The advantages of peptide attract pharmaceutical company to develop it as a drug for their high activity and specificity. Small peptides as a drug are very specific in nature due to non-immunogenic, more stable, fast interaction and more-bioavailability [11]. It has been reported that compound which mimic the binding of the acetylated peptide substrate might be effective and selective inhibitor of SIRT2 [12]. ...
Inhibition of Sirtuin2 (SIRT2) protein rescues the α-synuclein toxicity in vitro and in vivo models of Parkinson’s disease (PD). Thioacetyl group can structurally mimic the acetyl group and restrain the deacetylating p53 reaction by SIRT2. This work evaluated the biological activity of designed pentapeptides inhibitor containing N-thioacetyl-lysine against SIRT2. Pentapeptide by introducing thioacetyl-lysine as an inhibitor of SIRT2 was screened by molecular docking and synthesized by solid phase method. The inhibition of pure recombinant SIRT2 as well as SIRT2 in serum of PD patients by peptide was done by fluorescent activity assay. The inhibition of SIRT2 was assessed in PC12 cell line by measuring acetylated α-tubulin level. The peptide YKK(ε-thioAc)AM and HRK(ε-thioAc)AM were found to be SIRT2 inhibitors by molecular docking. However, YKK(ε-thioAc)AM was more specific towards SIRT2 than SIRT1 (Sirtuin1). It inhibited recombinant SIRT2 by IC50 value of 0.15 µM and KD values 9.92 × 10⁻⁸/M. It also inhibited serum SIRT2 of PD. It increased the acetylation of α-tubulin in PC12 neuroblastoma cells which is essential for maintaining the microtubular cell functions of brain. It can be concluded that novel peptide YKK(ε-thioAc)AM may be a platform for therapeutic agent for Parkinson’s disease targeting SIRT2.
Graphic abstract
... Peptide related impurities may result from the amino acid residue insertion or deletion, or modification of peptide sequences. 6 In general, these impurities carry nominal safety or efficacy risks. However, in some cases, peptide-related impurities may affect the safety or effectiveness of a pharmaceutical drug product based on their levels. ...
Stress study of a drug substance or pharmaceutical drug product provides a vision into degradation pathways and degradation products of the active pharmaceutical ingredient and helps in interpretation of the chemical structure of the degradation impurities. In the current study, Ganirelix active ingredient presented in the Orgalutran V R was stressed with acidic and alkali hydrolysis, photolysis, thermal and oxidation conditions as per the guidelines of International Conference on Harmonization (ICH) Q1A (R2). Ganirelix was found to be labile under thermal and alkali hydrolytic stress conditions, while it was stable to acid hydrolytic, oxidative and photolytic stress. All degradation products were separated with a resolution > 1.5 on a C18 column (2.6 mm, 25 cmÂ4.6 mm) using a hydrophilic ion pair such as sodium perchlorate, at a concentration <0.04 M. In total, four major degradant impurities were found during stress study. These impurities were fractionated and desalted by flash chromatography for identification of chemical structures. LC-MS-QTOF analysis revealed that two degradation products are diastereomers of Ganirelix, one degradation product is a deamination compound and other degradation product result from the insertion of a new amino acid residue in the Ganirelix peptide sequence. The developed method is sensitive enough to quantify the related substances of Ganirelix at the 0.04% level with that of Ganirelix test concentration.
... AMP has a large-scale of actions and capabilities to operate in viral, bacterial, fungal infections, involved in mitogenic and signaling processes [20] and also shown anticancer activity. Sipuleucel-T with the trade name PROVENGE [21], [22] and Leuprolide with trade name LUPRON [1], [23], [24] are two examples from the list of FDA approved peptide-based cancer drugs for the treatment of prostate cancer. Furthermore, the HPRP-A1 peptide shown excellent antimicrobial and anticancer activity, according to the experimental study by Cuihua Hu et al. [6] and Wenjing Hao et. ...
Peptides, short-chained amino acids, have shown great potentials toward the investigation and evolution of novel medications for treatment or therapy. The wet-lab based discovery of potential therapeutic peptides and eventually drug development is a hard and time-consuming process. The computational prediction using machine learning (ML) methods can expedite and facilitate the discovery process of potential prospects with therapeutic effects. ML approaches have been practiced favorably and extensively within the area of proteins, DNA, and RNA to discover the hidden features and functional activities, moreover, recently been utilized for functional discovery of peptides for various therapeutics. In this paper, a systematic literature review (SLR) has been presented to recognize the data-sources, ML classifiers, and encoding schemes being utilized in the state-of-the-art computational models to predict therapeutic peptides. To conduct the SLR, fourty-one research articles have been selected carefully based on well-defined selection criteria. To the best of our knowledge, there is no such SLR available that provides a comprehensive review in this domain. In this article, we have proposed a taxonomy based on identified feature encodings, which may offer relational understandings to researchers. Similarly, the framework model for the computational prediction of the therapeutic peptides has been introduced to characterize the best practices and levels involved in the development of peptide prediction models. Lastly, common issues and challenges have been discussed to facilitate the researchers with encouraging future directions in the field of computational prediction of therapeutic peptides.
... The number of therapeutic peptide regulatory approvals is steadily increasing and hence the global peptide drug market is continuously growing [1][2][3] . The majority of peptide therapeutics on the market are of synthetic origin. ...
There is a huge, still increasing market for synthetic and therapeutic peptides. Their quality control is commonly based on a generic reversed-phase liquid chromatography (RPLC) method with C18 stationary phase and acetonitrile gradient with 0.1% trifluoroacetic acid in the mobile phase. It performs exceptionally well for a wide variety of impurities, yet structurally closely related impurities with similar sequences, not resolved in preparative RPLC, may easily coelute in the corresponding QC run as well. To address this problem an advanced generic 2D-LC impurity profiling method was developed in this work. It employs a selective comprehensive (high resolution sampling) RP×RP 2D-LC separation using a 100×2.1 mm ID column with the common acidic generic gradient in the first dimension, while RPLC under basic pH on a short 30×3 mm ID column is used in the second dimension. Recording data with a UV detector at 215 nm after ¹D separation provides the common generic 1D chromatogram. However, after the ²D separation a flow splitter enabled recording of the signals of complementary detectors comprising a diode array detector (DAD) in-line with a charged aerosol detector (CAD) and a quadrupole-time-of-flight (QTOF) mass spectrometer (MS) with an electrospray ionization (ESI) source. Generic conditions of this 2D-LC method have been established through optimization of ²D stationary and mobile phase considering different pH values and buffer concentrations. The orthogonal separation principle has been documented by a number of therapeutic peptides including Exenatide, Octreotide, Cyclosporine A and Oxytocin as well as some other proprietary synthetic peptides. The information density can be further enhanced by using the QTOF-MS detector by data independent acquisition with SWATH. Through this sequential window acquisition of all theoretical fragment ion mass spectra it became possible to collect MS/MS data comprehensively in the high-resolution sampling window, thus enabling the extraction of 2D-EICs from fragment ions and the generation of 2D-contour plots of all product ions. Using Oxytocin as an example for an important therapeutic peptide, the ability of this advanced generic sRP-UV×RP-DAD-CAD-ESI-QTOF-MS/MS method with SWATH for peptide quality control is discussed.
... In addition, impurities can arise during storage. A thorough review of impurities in peptide drugs, and where they occur in the synthesis process can be found in D'Hondt et al. "Related Impurities in Peptide Medicine (118). Analysis of these impurities can be performed with in silico tools and in vitro assays, similar to the process described below for biotherapeutics. ...
Immune responses to protein and peptide drugs can alter or reduce their efficacy and may be associated with adverse effects. While anti-drug antibodies (ADA) are a standard clinical measure of protein therapeutic immunogenicity, T cell epitopes in the primary sequences of these drugs are the key drivers or modulators of ADA response, depending on the type of T cell response that is stimulated (e.g., T helper or Regulatory T cells, respectively). In a previous publication on T cell-dependent immunogenicity of biotherapeutics, we addressed mitigation efforts such as identifying and reducing the presence of T cell epitopes or T cell response to protein therapeutics prior to further development of the protein therapeutic for clinical use. Over the past 5 years, greater insight into the role of regulatory T cell epitopes and the conservation of T cell epitopes with self (beyond germline) has improved the preclinical assessment of immunogenic potential. In addition, impurities contained in therapeutic drug formulations such as host cell proteins have also attracted attention and become the focus of novel risk assessment methods. Target effects have come into focus, given the emergence of protein and peptide drugs that target immune receptors in immuno-oncology applications. Lastly, new modalities are entering the clinic, leading to the need to revise certain aspects of the preclinical immunogenicity assessment pathway. In addition to drugs that have multiple antibody-derived domains or non-antibody scaffolds, therapeutic drugs may now be introduced via viral vectors, cell-based constructs, or nucleic acid based therapeutics that may, in addition to delivering drug, also prime the immune system, driving immune response to the delivery vehicle as well as the encoded therapeutic, adding to the complexity of assessing immunogenicity risk. While it is challenging to keep pace with emerging methods for the preclinical assessment of protein therapeutics and new biologic therapeutic modalities, this collective compendium provides a guide to current best practices and new concepts in the field.
