Related impurities in peptide medicines

D׳Hondt, Matthias; Bracke, Nathalie; Taevernier, Lien; Gevaert, Bert; Verbeke, Frederick; Wynendaele, Evelien

doi:10.1016/j.jpba.2014.06.012

Cited by 105 publications

(76 citation statements)

References 148 publications

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“…Our results are supported by the original work by Singh and coworkers who clearly showed that the presence of TIS in the deprotection cocktail resulted in the removal of Acm. Other examples of the misunderstanding regarding the abilities of TIS to act as reducing agents are present in the literature …”

Section: Resultsmentioning

confidence: 99%

“…Our results are supported by the original work by Singh and coworkers who clearly showed that the presence of TIS in the deprotection cocktail resulted in the removal ties of TIS to act as reducing agents are present in the literature. 13,17 Since its introduction as a scavenger for protecting group cations in 1989, the role of TIS as a reductant has been overlooked, even though the original report cites the known role of trialkylsilanes as mild reductants in TFA. 1 For example, it has been shown that TIS can reduce the indole ring of tryptophan residues to indoline.…”

Section: As Demonstrated Inmentioning

confidence: 99%

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Reduction of cysteine‐S‐protecting groups by triisopropylsilane

Ste.Marie

Hondal

2018

Journal of Peptide Science

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Triisopropylsilane (TIS), a hindered hydrosilane, has long been utilized as a cation scavenger for the removal of amino acid protecting groups during peptide synthesis. However, its ability to actively remove S-protecting groups by serving as a reductant has largely been mischaracterized by the peptide community. Here, we provide strong evidence that TIS can act as a reducing agent to facilitate the removal of acetamidomethyl (Acm), 4-methoxybenzyl (Mob), and tert-butyl (But) protecting groups from cysteine (Cys) residues in the presence of trifluoroacetic acid (TFA) at 37 °C. The lability of the Cys protecting groups in TFA/TIS (98/2) in this study are in the order: Cys(Mob) > Cys(Acm) > Cys(But), with Cys(Mob) being especially labile. Unexpectedly, we found that TIS promoted disulfide formation in addition to aiding in the removal of the protecting group. Our results raise the possibility of using TIS in orthogonal deprotection strategies of Cys-protecting groups following peptide synthesis as TIS can be viewed as a potential deprotection agent instead of merely a scavenger in deprotection cocktails based on our results. We also tested other common scavengers under these reaction conditions and found that thioanisole and triethylsilane were similarly effective as TIS in enhancing deprotection and catalyzing disulfide formation. Our findings reported herein show that careful consideration should be given to the type of scavenger used when it is desirable to preserve the Cys-protecting group. Additional consideration should be given to the concentration of scavenger, temperature of the reaction, and reaction time.

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Section: Resultsmentioning

confidence: 99%

Section: As Demonstrated Inmentioning

confidence: 99%

Reduction of cysteine‐S‐protecting groups by triisopropylsilane

Ste.Marie

Hondal

2018

Journal of Peptide Science

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“…Concern about the purity and quality of commercial chemicals and biochemicals has previously been expressed by other researchers. For example, D’Hondt et al [42] highlighted the impact that contaminants may have in peptide medicines, not only with respect to their presence within the approved and clinically used products, but also during the initial drug discovery phase. During this initial phase, the presence of contaminants may greatly influence functionality studies thus resulting in conclusions that are incorrect.…”

Section: Discussionmentioning

confidence: 99%

Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies

et al. 2017

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The present study reports the perplexing results that came about because of seriously impure commercially available reagents. Commercial reagents and chemicals are routinely ordered by scientists and expected to have been rigorously assessed for their purity. Unfortunately, we found this assumption to be risky. Extensive work was carried out within our laboratory using commercially sourced preparations of the small leucine-rich proteoglycans (SLRPs), decorin and biglycan, to investigate their influence on nerve cell growth. Unusual results compelled us to analyse the composition and purity of both preparations of these proteoglycans (PGs) using both mass spectrometry (MS) and Western blotting, with and without various enzymatic deglycosylations. Commercial ‘decorin’ and ‘biglycan’ were found to contain a mixture of PGs including not only both decorin and biglycan but also fibromodulin and aggrecan. The unexpected effects of ‘decorin’ and ‘biglycan’ on nerve cell growth could be explained by these impurities. Decorin and biglycan contain either chondroitin or dermatan sulfate glycosaminoglycan (GAG) chains whereas fibromodulin only contains keratan sulfate and the large (>2500 kDa), highly glycosylated aggrecan contains both keratan and chondroitin sulfate. The different structure, molecular weight and composition of these impurities significantly affected our work and any conclusions that could be made. These findings beg the question as to whether scientists need to verify the purity of each commercially obtained reagent used in their experiments. The implications of these findings are vast, since the effects of these impurities may already have led to inaccurate conclusions and reports in the literature with concomitant loss of researchers’ funds and time.

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“…Ser, Thr, Cys, and cystine may undergo an acid-or basecatalyzed β-elimination reaction that results in dehydroalanine (∆Ala), β-methyl-∆Ala, and, in case of cystine, Scheme 10 Mechanisms of DKP formation a at positions 1 and 2 of a peptide chain, and b in the presence of Snn at position 2 also of thiocysteine (Scheme 12) ( Bar-Or et al 2008;D'Hondt et al 2014a). Due to the sp 2 hybridization of the α-carbon, the α,β-dehydro amino-acid residue has a planar geometry, which forces co-planarity of the two planar peptide units centered at the sp 2 α-carbon.…”

Section: β-Elimination Reactions Of β-Hydroxyland β-Sulfhydryl-amino mentioning

confidence: 99%

Susceptibility of protein therapeutics to spontaneous chemical modifications by oxidation, cyclization, and elimination reactions

Grassi

Cabrele

2019

Amino Acids

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Peptides and proteins are preponderantly emerging in the drug market, as shown by the increasing number of biopharmaceutics already approved or under development. Biomolecules like recombinant monoclonal antibodies have high therapeutic efficacy and offer a valuable alternative to small-molecule drugs. However, due to their complex three-dimensional structure and the presence of many functional groups, the occurrence of spontaneous conformational and chemical changes is much higher for peptides and proteins than for small molecules. The characterization of biotherapeutics with modern and sophisticated analytical methods has revealed the presence of contaminants that mainly arise from oxidation-and elimination-prone amino-acid side chains. This review focuses on protein chemical modifications that may take place during storage due to (1) oxidation (methionine, cysteine, histidine, tyrosine, tryptophan, and phenylalanine), (2) intra-and inter-residue cyclization (aspartic and glutamic acid, asparagine, glutamine, N-terminal dipeptidyl motifs), and (3) β-elimination (serine, threonine, cysteine, cystine) reactions. It also includes some examples of the impact of such modifications on protein structure and function.

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Related impurities in peptide medicines

Cited by 105 publications

References 148 publications

Reduction of cysteine‐S‐protecting groups by triisopropylsilane

Reduction of cysteine‐S‐protecting groups by triisopropylsilane

Contaminants in commercial preparations of ‘purified’ small leucine-rich proteoglycans may distort mechanistic studies

Susceptibility of protein therapeutics to spontaneous chemical modifications by oxidation, cyclization, and elimination reactions

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