Length of peptides to be synthesized:

Shorter peptides of length 2-50 amino acids (AAs) are easier to synthesize. Although peptides up to 108 AAs have also have been synthesized, the complexity of peptide synthesis increases with increase in length above 50 AAs. With solid phase synthesis in practice, it has been possible to synthesize peptides beyond 50 AA, but peptides longer than 50AA still pose a problem. Longer peptides can be synthezed by long peptides fragment synthesis, however, expertise with this technique is scarce.

The sequence of peptides to be synthesized:

Synthesis of peptides is easier when it is composed of natural or L- Amino Acids. Some of the AAs are very tricky, for example, if Proline is at 2nd position from C-terminal than it causes steric hindrances in the synthesis of the peptide. Similarly, hexapeptide containing all Glycines has found an extremely difficult situation in synthesis until now. Cost of peptide synthesis can be unprecedently high due to un-natural AAs or any modification at C-terminal of the peptide. It also requires a good peptide chemist with fairly good knowledge of chemistry.

Modifications at C-terminal in solid-phase peptide synthesis need very special types of resins and their cleavage method to unblock the desired chemical moiety at C-terminal. Tryptophan and Methionine containing peptides sometimes get oxidized during cleavage so special side-chain protections are used to prevent the oxidations, which increases the cost of synthesis. 

Many sequences contain Cysteine’s so that disulfide bonds can be established to make targeted cyclisation by inducing the oxidation reaction. Some peptides like conotoxins or venom peptides contain two or three disulfide bonds, which are highly tricky to synthesise and need differential side-chain protections to make two to three disulfide bridges.

Quantity of peptides:

Quantity of peptides required significantly affects the cost and here’s why. Even with current methodologies, it is costly to synthesise gram quantities, so larger quantities require special techniques like solid phase synthesis or more reactions. Quantity of peptide is determined based on the application. In case of research experiment like antibody production or NMR studies, 20-30mg peptide is sufficient. Applications such as drug testing and clinical trials require larger quantities. Solid Phase Peptide Synthesizers are available to produce batches of hundreds of gram or kilograms of crude peptides.

Purity of Peptides:

Peptide purity is another area, which determines the cost of peptides in a big way. Desalting by Gel filtration method normally provides a purity level; >70%. In this process, one gets rid of salts and scavengers from synthesized peptides. Using the HPLC method of purification one attains higher purity. In this purification process, the first run of HPLC gives a purity level >90%. However, to reach higher level of purities like >97% or >98% purity, more cycles of purification are required on HPLC. One can not control the purity level while running the HPLC, that is why quotations are normally given as >80%; >90% or >98% etc. Higher the purity, more is the loss of the desired peptide and also more is the cost. To attain the purity level of >97% to >98% cost of peptides is increased by 25% or even more. One should intelligently decide about the purity of peptide-based on the applications to keep the cost low. Recommended purities for Antibody production would be >80%, Structural studies >98%; Toxicity studies in Clinical Trials >90% and therapeutic peptides >98%.*

Since 1% TFA (Trifluoroacetic Acid) is used in all HPLC purifications so peptide achieved after purification is Trifluoroacetate salt of peptides. Therapeutic peptides are required to be converted to acetate salt of the peptdie. 

Characterization of Peptides:

Most of the times research-grade peptides are characterized for purity profile using Analytical HPLC and molecular weight by Mass Spectrometry. This is mostly provided free or as a part of the package while supplying synthetic peptides but in case of more physical characterizations like CD, NMR, Amino Acid Analysis, peptide content, water content, polarity and N-terminal Sequencing are performed on request and additional costs are incurred. A peptide supplier takes the guarantee of HPLC and Mass results but not for other physical characterizations.

Techncially speaking, no peptide supplier can take guarantee to performance of the custom synthesized peptide in biological experiments and assays. On the contrary, in the case of catalogue peptides the same can be guaranteed,

Another physical character of peptides where the confrontation between user and supplier may occur, is the solubility of custom peptides which can not be guaranteed by any supplier.

In last I wish to mention that all custom peptide sequences can not be synthesized with guarantee, sometimes steric hindrances do not let the reaction happen or the yield of peptide achieved is so low which can not be delivered.At times,  supplier need to repeat the synthesis twice or thrice,  to fulfil the supply obligation to the customer. Introduction of new reagents, coupling agents and technologies such as microwave synthesis applied in solid-phase peptide synthesis, is overcoming this problem and other racemization problems.

Written by Rakesh Arora, CEO, TechnoConcept India Pvt Ltd.

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