Next Article in Journal
Foot/Ankle Prostheses Design Approach Based on Scientometric and Patentometric Analyses
Previous Article in Journal
Scanning of Historical Clothes Using 3D Scanners: Comparison of Goals, Tools, and Methods
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:

Latest Advances on Synthesis, Purification, and Characterization of Peptides and Their Applications

Ayman El-Faham
Beatriz G. de la Torre
3,4,* and
Fernando Albericio
Department of Chemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia
Chemistry Department, Faculty of Science, Alexandria University, P.O. Box 426, Ibrahimia, Alexandria 12321, Egypt
KwaZulu-Natal Research Innovation and Sequencing Platform (KRISP), School of Laboratory Medicine and Medical Sciences, College of Health Sciences, University of KwaZulu-Natal, Durban 4041, South Africa
Peptide Science Laboratory, School of Chemistry and Physics, University of KwaZulu-Natal, Durban 4001, South Africa
Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), 08034 Barcelona, Spain
CIBER-BBN (Networking Centre on Bioengineering, Biomaterials and Nanomedicine) and Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain
Authors to whom correspondence should be addressed.
Appl. Sci. 2021, 11(12), 5593;
Submission received: 17 May 2021 / Revised: 12 June 2021 / Accepted: 15 June 2021 / Published: 17 June 2021
Peptides have a broad number of applications: from biochemical tools to diagnostic kits; from nanotechnology to new materials; and from vaccines to drugs. Increased interest in peptides in all these fields is exemplified by the growing number of peptide-based drugs available [1,2]. Table 1 shows the peptide drugs accepted by the US Food and Drug Administration (FDA) between Jan 2015 and May 2021 [3,4]. During these six and a half years, 21 peptide-based drugs have been launched onto the market. This number accounts for approximately 5% of the total number of drugs, including both new chemical entities and biologics, approved by the FDA in the same period. Importantly, although cancer and metabolism continue to be the main target, peptides are being used for a much wider spectrum of indications and they continue to gain applications with time (Table 1).
Only a few decades ago, it was unthinkable to consider producing an active pharmaceutical ingredient (API) of a chemical entity which requires more than seventy chemical steps—as in the case of Fuzeon® (enfurtivide, T20) [5]—fulfilling the requirements of the regulatory agencies, targeting both purity of the peptide API and identification of the impurities. This has been possible thanks to the breakthroughs in three fields: (i) chemical synthesis; (ii) purification; and (iii) characterization, which allowed consideration of peptides, and oligonucleotides too, as small molecules from purity and characterization points of view.
Coincidentally, at the same time, in the late fifties and early sixties, in the USA East Coast, Nobel Laureate R. Bruce Merrifield (Rockefeller University, New York) and the entrepreneur Jim Waters (Water Corporation, Milford, Massachusetts, USA) developed two techniques, for synthesis and purification, respectively, that over time proved to be crucial in making peptides and oligonucleotides (TIDES) an alternative to small molecules and biologics as drugs. Interestingly, the two techniques were based on a similar concept, namely the concourse of a solid support—a resin.
Merrifield first envisaged and then implemented a solid polymeric protecting group for the C-terminal carboxylic group, in the so-called solid-phase peptide synthesis (SPPS) methodology [6]. Thus, the growing peptide chain can be elongated with N-protected amino acids, while the peptide chain is in a pseudo-soluble state anchored to the solid support (resin). Thus, all reactions—coupling of the incoming residue and removal of the N-protecting group—can be carried out using excesses of reagents, thus ensuring excellent yields. Intermediates are not isolated or characterized, and the excesses of reagents and soluble side products are removed by filtration and extensive washing with solvents. Once the sequence is completed, the unprotected peptide is cleaved from the resin. In recent years, resins, linkers, protecting groups, coupling reagents, solvents, and finally green approaches have been described for fine tuning the SPPS approach.
Reversed-phase high-performance liquid chromatography (RP-HPLC), whose roots are found in the pioneering work of Waters [7], is based on the hydrophobic interaction between the molecules—peptides in this case—to be purified in a hydrophilic mobile phase, and the hydrophobic moieties (long alkyl chains and others), which are attached to the solid support, the stationary phase. Jean Rivier linked SPPS and RP-HPLC [8]. Thus, using an analog of LH-RH as a peptide model, he showed that the synthesis of a peptide at industrial scale can be carried out using SPPS techniques, rendering a crude product with sufficient quality to be purified by RH-HPLC.
Mass spectrometry (MS) is probably the technique that has evolved most over time [9]. In combination with HPLC (separation by HPLC and detection by MS), it has become a key tool through which to detect and, more importantly, identify the chemical structure of the impurities present after synthesis. These impurities often have subtle structural changes that hinder their separation and identification. In this regard, they can contain one amino acid less or one more, deletion or double hit incorporation, respectively; they can be an epimer (racemization of just one residue) or a β-peptide (isomerization of Ser- or Asp); and the residues of Cys, Met, and Trp are able to undergo oxidation, or those of Cys, Met, Trp, and Tyr alkylation too [10].
The list of peptides that have reached the market (Table 1) supports the strength and the importance of these compounds in the pharmaceutical arena. In this context, we have decided to publish a Special Issue in Applied Sciences, entitled “Latest Advances on Synthesis, Purification, and Characterization of Peptides and their Applications”, which provides excellent reviews and quality research articles covering the most recent developments in peptide synthesis, purification, and analysis. It is hoped that some of these new synthetic “tricks” will soon form part of the toolbox for the synthesis of peptides of therapeutic interest.

Author Contributions

A.E.-F., B.G.d.l.T. and F.A. equally contributed to writing the article. All authors have read and agreed to the published version of the manuscript.


The work in the laboratory of the authors was funded in part by the following: National Research Foundation (NRF) (Blue Skies Research Program # 120386).

Conflicts of Interest

The authors declare no conflict of interest.


  1. Muttenthaler, M.; King, G.F.; Adams, D.J.; Alewood, P.F. Trends in peptide drug discovery. Nat. Rew. Drug. Discov. 2021, 20, 309–325. [Google Scholar] [CrossRef] [PubMed]
  2. Al Musaimi, O.; Al Shaer, D.; Albericio, F.; de la Torre, B.G. 2020 FDA TIDES (Peptides and Oligonucleotides) Harvest. Pharmaceuticals 2021, 14, 145. [Google Scholar] [CrossRef] [PubMed]
  3. de la Torre, B.G.; Albericio, F. Peptide Therapeutics 2.0. Molecules 2020, 25, 2293. [Google Scholar] [CrossRef] [PubMed]
  4. FDA. Novel Drug Approvals for 2021. Available online: (accessed on 12 May 2021).
  5. Bray, B.L. Large-scale manufacture of peptide therapeutics by chemical synthesis. Nat. Rew. Drug. Discov. 2003, 2, 587–593. [Google Scholar] [CrossRef] [PubMed]
  6. Merrifield, R.B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149–2154. [Google Scholar] [CrossRef]
  7. McDonald, P.D. James Waters and His Liquid Chromatography People: A Personal Perspective. In The Chromatographic Society Golden Jubilee Book: Recognising Half a Century of Supporting Progress in Separation Science; ILM: St Albans, UK, 2006; Available online: (accessed on 15 May 2021).
  8. Hoeger, C.; Porter, J.; Boublik, J.; River, J. Preparative-scale synthesis and reversed-phase purification of a gonadotropin-releasing hormone antagonist. J. Chromatogr. 1989, 22, 307–318. [Google Scholar] [CrossRef]
  9. Wysocki, V.H.; Resing, K.A.; Zhang, Q.; Cheng, G. Mass spectrometry of peptides and proteins. Methods 2005, 35, 211–222. [Google Scholar] [CrossRef] [PubMed]
  10. Yang, Y. Side Reactions on Amino Groups in Peptide Synthesis. In Side Reactions in Peptide Synthesis; Academic Press: Oxford, UK, 2016; ISBN 978-0-12-801009-9. [Google Scholar]
Table 1. Peptide-based drugs approved by the FDA (2015–2021 (May)) [4,5].
Table 1. Peptide-based drugs approved by the FDA (2015–2021 (May)) [4,5].
YearActive Ingredient
Trade Name
2015Insulin degludec
DiabetesModified insulin with an aa deletion and a hexadecanedioic acid via γ-Glu at Lys (B29)
Multiple myelomaN-acylated, C-boronic acid dipeptide
Diabetes44 aa GLP-1 peptide with Lys6 at the C-terminal
2017Abaloparatide Tymlos®Osteoporosis34 aa analog of parathyroid hormone-related protein
2017Angiotensin II
HypotensionNatural octapeptide
2017Etelcalcetide Parsabiv®Hyperparathyroidism8 aa in two Cys chains: Ac-DCys-DAla-(DArg)3-DAla-DArg-NH2, L-Cys
2017Macimorelin Macrilen®Growth hormone deficiencyPseudotripeptide N-formylated
2017Plecanatide Trulance®Chronic idiopathic constipation16 aa with two disulfides
DiabetesGLP-1 peptide (31 aa in the chain) with hexadecanedioic acid via γ-Glu and mini PEG at Lys
2018177Lu DOTA-TATE
Neuroendocrine tumors, theranostics177Lu chelated by DOTA bound to Tyr3-octreotate
2019Afamelanotide Scenesse®Skin damage and pain13 aa linear peptide analog of α-MSH
2019Bremelanotide Vyleesi®Hypoactive sexual desire in women7 aa cyclic peptide analog of α-MSH
2019Enfortumab Vedotin-Ejfv PADCEV®Cancers expressing nectin-4ADC with a synthetic analog of the marine natural peptide dolastatin 10 (5 residues)
201968Ga DOTA-TOCNeuroendocrine tumors, diagnostics68Ga chelated by DOTA bound to Tyr3-octreotide
2019Polatuzumab Vedotin-Piiq Polivy®Diffuse large B-cell lymphomaADC with a synthetic analog of dolastatin 10 (5 residues)
Obesity8 aa, cyclic disulfide
Scintigraphic imaging64Cu chelated by DOTA bound to Tyr3-octreotate
202068Ga-PSMA-11Diagnosis of recurrent prostate carcinoma68Ga chelated by HBED-CC with a pending urea-based peptidomimetic
2020Belantamab mafodotin-blmf Blenrep®Relapsed or refractory multiple myelomaADC with a synthetic analog of the marine natural peptide dolastatin 10 (5 residues)
Lupus nephritisAnalog of cyclosporin, 11 aa cyclic peptide with several N-methyl aa
2021Dasiglucagon Zegalogue®Hypoglycemia in individuals with diabetes29 aa linear peptide
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

El-Faham, A.; de la Torre, B.G.; Albericio, F. Latest Advances on Synthesis, Purification, and Characterization of Peptides and Their Applications. Appl. Sci. 2021, 11, 5593.

AMA Style

El-Faham A, de la Torre BG, Albericio F. Latest Advances on Synthesis, Purification, and Characterization of Peptides and Their Applications. Applied Sciences. 2021; 11(12):5593.

Chicago/Turabian Style

El-Faham, Ayman, Beatriz G. de la Torre, and Fernando Albericio. 2021. "Latest Advances on Synthesis, Purification, and Characterization of Peptides and Their Applications" Applied Sciences 11, no. 12: 5593.

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop