Peptide purity grades, salt exchange and modifications

The grade ladder from crude to 98 per cent and what each is used for, what a salt exchange adds to price and lead time, and the modifications a custom order can specify.

By Touchstone PeptidesSeptember 22, 20265 minute read

A custom peptide order is a specification, not a purchase of a molecule. The buyer chooses a purity grade, a salt form, a scale and any modifications, and each of those choices moves the price, the lead time and what the certificate will say. The grade that matters is the lowest one that answers the question being asked at the bench.

What "purity" means on a grade label

Purity on a peptide specification is area percent by reversed-phase HPLC: the proportion of the peptide material eluting as the target peak rather than as something else. GenScript defines peptide purity as the percentage of the target peptide compared to impurities, usually determined by RP-HPLC or UHPLC.

That is a comparison among peptide species. Salts, counter-ion and water are close to invisible at the wavelengths used, which is why a peptide can be 98 per cent pure and still be a minority of the mass in the vial. Purity is not potency works through the consequence, and net peptide content is the figure that closes the gap.

The grade ladder and what each grade is for

Grade	Typical purity	What suppliers list it for
Crude	Below the named thresholds	First-pass screening, mutation screening, sequence optimisation, protein-protein and receptor-ligand screening
Immuno grade	≥ 75%	ELISA, peptide arrays, antigens for polyclonal antibody production or affinity purification
Biochemistry grade	≥ 85%	In vitro bioassays, epitope mapping, polyclonal antibody production, phosphorylation and cell attachment work
High purity	≥ 95%	NMR, quantitative receptor-ligand work, quantitative blocking and competitive inhibition studies
Industrial grade	≥ 98%	Crystallography, structure-activity relationship work

Those categories and applications are GenScript's. Bachem describes its own ladder by application rather than by name, recommending greater than 95 per cent for NMR, crystallography, reference standards and enzyme-substrate or receptor-ligand studies, 90 to 95 per cent for antibody production and quantitative enzyme and receptor work, and greater than 80 per cent for non-quantitative western blotting. In both ladders, quantitative work needs a number you can trust to be the compound and screening work does not.

"Desalted" is a processing step rather than a purity claim. It removes residual salts and small-molecule by-products from crude material without a preparative purification, and it is what "crude, desalted" means on a quotation.

Scale, lead time and what is actually quoted

Bachem lists custom synthesis from 5 mg to 100 g, with purity levels from 80 to 97 per cent, and standard quantity brackets of 5 to 9 mg, 10 to 19 mg, 20 mg, 50 mg and 100 mg or custom amounts.
GenScript's regular service covers sequences up to 200 amino acids at milligram to kilogram scale from crude to 98 per cent, with an approximate two to three week turnaround, and an expedited service quoting as fast as five business days for 4 to 15 residues and seven business days for 16 to 25 residues, with a 4 mg minimum at 95 per cent or better.

Length drives everything. Each residue is another coupling and another deprotection, and the yield of the full-length chain falls with every one of them, which is the subject of solid phase synthesis and the price. A twenty-residue sequence at 98 per cent is a different manufacturing proposition from the same sequence at crude.

Ordering a salt exchange: price and lead time

Synthetic peptides usually arrive as trifluoroacetate salts, and a laboratory that needs acetate or chloride orders an exchange as a line on the quotation. LifeTein, which sells the service, notes that the exchanged salt forms are usually 20 to 30 per cent more expensive than regular TFA salt forms because of the peptide loss during the conversion. GenScript quotes its TFA exchange as a standard option reducing residual TFA below 10 per cent or a guaranteed option below 1 per cent, adding two to three business days. Why the counter-ion is there, the exchange routes compared by Roux and colleagues in 2008, and what the salt changes about the weighing arithmetic are covered in TFA salt or acetate salt.

The modifications a custom order can carry

Terminal and internal modifications are ordinary catalogue items at the custom houses. The common ones, with the purposes the suppliers give:

N-terminal acetylation. Removes the charge at the amino terminus, which suppliers describe as imitating the peptide's natural context within a protein, and slows exopeptidase degradation.
C-terminal amidation. Converts the terminal carboxyl group to a carboxamide, removing the corresponding charge, with the same rationale.
Biotin. Attached at either terminus, at the C-terminus usually through an added lysine side chain, for streptavidin capture and detection.
Fluorophores. FITC, FAM, TAMRA and cyanine dyes, commonly with an aminohexanoyl spacer between the dye and the chain to keep labelling efficient.
PEGylation. Polyethylene glycol chains attached covalently, which suppliers list as a way to raise the solubility of a difficult sequence.
Cyclization. Most often a disulfide bridge between two cysteines, or a macrocyclic linkage, which suppliers list for constraining conformation and stabilising the peptide.
Phosphorylation and methylation. Site-specific, for work on kinase and methyltransferase signalling.
Isotope labels. Stable-isotope residues for NMR and for quantitative mass spectrometry.
D-amino acids and unnatural residues. Substitutions that change protease susceptibility and conformation; several stocked catalogue peptides carry them.

JPT lists the same families, adding glycopeptides, sulfated peptides, internally quenched FRET substrates, peptide dimers and click-chemistry handles.

What to ask for on the quotation

The purity grade, stated as area percent by RP-HPLC, with the method.
The salt form, and the exchange if one is required.
Net peptide content, and water content, if the arithmetic matters.
Identity by mass spectrometry, reported with observed and calculated masses. Mass spectrometry identity sets out what that confirms and what it does not.
The quantity per vial and the number of vials, since a single large fill and several small ones are not the same product.
Any modification, written as it should appear in the sequence line.

Custom synthesis enquiries for this catalog go through the quote form.

Sources

For laboratory research use only. Not a drug, not a supplement, and nothing here is a claim about what any of this material does in a person or an animal.

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