Process Development of SPPS

Process Development of SPPS

Published on 21/09/2022

From mg synthesis of a peptide up to bulk quantities often makes a huge difference in the route of production. Read on for more information about Iris Biotech’s process development services.

During initial research work, yield during the solid phase peptide synthesis hardly matters as long as the desired product is obtained. Furthermore, modern chromatographic technologies, e.g. preparative HPLC, are so powerful that also only slight amounts of the desired product can rather easily be isolated out of complex mixtures even in the presence of several impurities and side-products.

In contrast, the industrial and particularly pharmaceutical production of peptides and peptide APIs requires high purity levels, and each impurity should ideally be identified and be present in less than 0.5%. Especially if one of the impurities has the same elution properties and retention time as the target peptide, the isolation of the desired sequences becomes challenging. As soon as the production becomes of commercial interest, all efforts are naturally cost dependent, the yield gained plays a major role accompanied by the efforts to isolate the desired product out of all present impurities.

Most common sources for peptide impurities are:

  • Raw materials; chiral compounds contain other stereoisomers, dipeptides, beta-alanine based dipeptides, contamination with other amino acids (e.g. leucine and isoleucine can contain significant amount of valine, if the wrong strain is being used during fermentative production), content of acetic acid, e.g. through transesterification of residual solvents like ethyl acetate;
  • Back-alkylation of protecting groups; t-butyl from t-butyl ethers and ester, as well as from t-butyloxycarbonyl protecting groups can alkylate electron rich aromatic rings as for example present in tryptophane;
  • Undesired linker fragmentation; both Wang- and Rink-linker can fragmentize under acidic cleavage conditions. Moieties like p-hydroxybenzyl or 2,4-dimethoxybenzyl can undergo an electrophilic alkylation;
  • Racemization; during each Fmoc-deprotection step the peptide is being treated with base, typically piperidine. Certain amino acids are particularly sensitive to racemization under prolonged contact with bases. The main candidates are cysteine, histidine, and serine, while certain sequences are prone to racemize;
  • Typically, SPPS synthesis is not carried out under inert atmosphere. Hence, oxygen will oxidize amino acids most sensitive to oxidation, such as methionine and tryptophan;
  • Cleavage is often carried out under strong acidic and dehydrating conditions. Wherever it is possible water can be removed, as it can be found in the case of serine, forming dehydroalanine;
  • In case of cyclic disulfide-bridged peptides, bridges might open, shift and reform in different manner.

Example of possible reactions creating undesired impurities, like oxidized methionine and alkylated tryptophan.


Our team has decades of experience in bringing peptides from research to commercial scale and will accompany you during this transition period with the following approach:


a) Repeating the synthesis with your given method; identification of all impurities formed and determination of yields at each step;

b) Systematic evaluation and optimization of each coupling step to maximize the yield of each individual step. This includes screening of different coupling reagents, solvents and additives, as well as reaction temperature;

c) Optimizing cleavage from the resin, including screening of different linkers, selection of different cleavage conditions, e.g. by variation of acids, reaction times, reaction temperature and addition of different scavengers;

d) Optimization of the final purification of the peptide by selection of appropriate chromatographic methods, i.e. ion chromatography or HPLC, as well as alternative methods liquid/liquid extraction methods or new state-of-the-art technologies, such as safety-catch technologies by PEC purification;

e) Technology transfer to the production of your choice.


Inquire for our Custom Synthesis services. Download our brochure for more information
Reach out to us for Process Development of your peptide.



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The impact of impurities in synthetic peptides on the outcome of t-cell stimulation assays; J. W. K. de Beukelaar, J. W Gratama, P. A. Smitt, G M. G. M. Verjans, J. Kraan, T. M. Luider, P. C. Burgers; Rapid Commun. Mass Spectrom. 2007; 21: 1282-1288.

T Cells Recognizing a Peptide Contaminant Undetectable by Mass Spectrometry; V. Brezar, S. Culina, T. Østerbye, F. Guillonneau, G. Chiappetta, Y. Verdier, J. Vinh, F. S. Wong, S. Buus, R. Mallone; Plos One 2011; 6: e28866.