Frequently Asked Questions PEC

The PurePep® EasyClean Kits enable you to purify peptides orthogonally using catch & release principles. The Kits contain the PEC-Linker, cartridges or multi-well plates, prefilled with activated filter material, and related consumables. You can choose between two different types of Kits:

• Starter Kit to purify eight peptides (individual or in parallel), either at 25 µmol or 100 µmol synthesis scale
• High-Throughput Kit to purify either 48 peptides at 20 µmol scale or 96 at 10 µmol scale (individually or in parallel)

• shaking device for fritted cartridges
• centrifuge (recommended) or filtration apparatus to collect peptide precipitates
• a vacuum manifold is required for the High-Throughput Kit
• optional for the Starter Kit: vacuum manifold for parallel set-up

Single purifications can be processed by hand, using the plunger and push-pull processing. Please make sure your peptide is always fully dissolved. We recommend a vacuum manifold or a vacuum flask with a septum for parallel processing.

Plate purifications require a vacuum manifold that is available upon request via peptides@gyrosproteintech.com

Our High-Throughput Kit enables purification of 96 peptides in a day. For higher throughput requirements, we offer Custom Process Developments to meet your demands.

No. The PEC-Technology can easily be adjusted in scale based on the requirement. For the range between 10 and 100 µmol, you can use the ready-to-use Starter Kits or High-Throughput Kits. We offer Custom Process Development service for scales up to kg.

PEC  Kits rely on catch & release principles for the orthogonal purification of chemically synthesized peptides in 7 simple steps.

Step 1: Coupling of a traceless cleavable linker, the PEC-RC+ Linker, at the N-terminus of the target peptide(s), synthesized via Solid-phase Peptide Synthesis (SPPS)

Step 2: Cleavage of the peptide from the SPPS resin and global deprotection with TFA-cocktails

Step 3: Dissolution of the peptide in solvents with high content of organic solvents (90% DMSO (Dimethyl sulfoxide) or pure HFIP

Step 4: Catch of the PEC-Linker RC+-tagged, target peptide through covalent capture on the activated filter material by oxime ligation

Step 5: Washing out of unbound substances such as truncated sequences and other organic synthetic dirt

Step 6: Reduction of the PEC-Linker at neutral conditions

Step 7: Safety release of the desired peptide by acid treatment

Read more details in the peer-reviewed publication R. Zitterbart, N. Berger, O. Reimann, G. Noble, S. Lüdtke, D. Sarma, O. Seitz, Traceless parallel peptide purification by a first-in-class reductively cleavable linker system featuring a safety-release, Chemical Science, 2021

In contrast to chromatography, which relies on physico-chemical differences of target peptide and peptidic impurities, PEC chemo-selectively isolates the target peptide from its crude mixture. Therefore, it reflects a truly orthogonal approach to HPLC to generate peptides with high quality.

Peptide purity is often critically affected by co-eluting side products that are hard to remove by chromatographic methods. These impurities lead to false-positive results during R&D activities and are problematic for the manufacturing of peptide APIs (Active Pharmaceutical Ingredient). Co-eluting side products become especially relevant when the time for single synthesis optimizations, such as peptide libraries or neoantigens, is not given.

The orthogonal PEC technology purifies peptides regardless of the impurity profile. The hydrophilic peptide Histone H3 (1-20), synthesized via SPPS, is a formidable example due to the co-eluting impurities. The chromatogram shown below, highlights very nicely how impurities can hide beneath an otherwise clean-looking main peak. Only mass analysis reveals the accumulation of truncations, which all represent a potential threat to your assay and are difficult to remove with chromatography. PEC efficiently removes these impurities regardless of their similarity to the target peptide.

You can use our PEC-grade Peptide Services and order custom peptides to test assay compatibility and conformity of PEC-grade with your target specifications.

Chromatogram of Histone peptide before and after PEC purification

Please consider the two rules for successful purification  with PEC Kits:

Purification with PEC Kits begins with an optimized SPPS

• Use an efficient, recommended capping protocol during SPPS to ensure that the PEC-linker modification happens selectively on the N-terminus of your target peptide
• Prevent side reactions using the correct amino acid building blocks and coupling conditions

Have the right chemicals with an adequate quality in place

• Use aldehyde and ketone-free solvents to prevent loss in yield during the process and analysis
• Use appropriate scavengers during global TFA deprotection to prevent the re-addition of protecting groups

Detailed instructions are provided in the PEC Kit manuals.

Reagents for coupling of the linker:

• ethyl-2-cyano-2-(hydroxyimino) acetate (Oxyma)
• scavengers for TFA (Trifluoroacetic acid) cleavage cocktail (g., TIS, EDT, thioanisol, phenol)

Salts for buffers:

• guanidinium chloride (GdmCl, in case of technical grade, filter the solution prior to use)
• sodium chloride (NaCl)

Acids / Bases:

• N,N-diisopropylethylamine (DIEA)
• trifluoroacetic acid (TFA)
• sodium carbonate (Na2CO3)

Solvents

• N,N-dimethylformamide (DMF), ≥ 99.5% (peptide synthesis grade)
• dimethyl sulfoxide (DMSO), ≥ 99.5% (synthesis grade)
• dichloromethylene (CH₂Cl₂), ≥ 99.5% (synthesis grade)
• acetonitrile (MeCN), ≥ 99.5% (synthesis grade & LC-MS grade)
• deionized water
• ethanol (EtOH), denatured
• Aldehyde and Ketone-free ether for peptide precipitation (g., Et₂O, MTBE, THF/heptane 1:1), ≥ 99.5% (synthesis grade)

The minimum shelf life of the kits is 6 month.

Yes, automated coupling on peptide synthesizers is possible. Detailed procedures are given in the manual.

If you have an alkylated N-terminus e.g., N-Me-Ala you can use PEC. If your N-terminus is acylated, you cannot use PEC, since the PEC-Linker is bound to the N-terminus of the peptide. Therefore, acylation of the N-terminus is not possible using the routine procedure. However, if you have a Lysine with an orthogonal protecting group, you can couple the linker on the backbone and modify your peptide on the N-terminus. We are happy to find a solution together with you during our PEC process and kit development services.

In principle, the final product after purification contains every compound coupled to the PEC-Linker. Potential impurities can already form during synthesis. These may be undesired sequences due to, e.g., amino acid contamination, incomplete Fmoc deprotection, or intra- or intermolecular side reactions (e.g., aspartimides) if these are present at the time of linker coupling.

Another essential synthesis-related source of contamination occurs when peptide acetylation is not (or only incompletely) performed after each amino acid coupling. As a result, the PEC-Linker may bind to truncated or undesired sequences, and those will then remain in the product as by-products.

Further peptide impurities may occur during the cleavage of the peptide from the synthesis resin. These are peptide sequences that already carry the PEC-Linker and then react with reactive species (e.g., cleaved protective groups or degradation products of the cleaved synthesis linker). These usually unwanted sequences are not removed and remain in the final product.

Respective measures are given in the manual to ensure a high quality product after PEC purification.

At all stages, before the peptide-PEC-Linker-conjugate is bound to the activated filter material, contaminations with aldehydes or ketones in the TFA-cleavage, ether- precipitation, storage, or analysis media can react with the oxyamine of the PEC-Linker. This side-reaction can reduce the yield and/or distort the analysis. Therefore, the purity of solvents used for PEC-Linker coupling or the ether used to precipitate the peptide is of vital importance. During chromatographic analysis, the purity of eluents and the column condition is significant.

No. Unwanted side-products formed in the target sequence during synthesis or purification, such as epimerization, cannot be removed by PEC (see “What affects purity?”). Yet, there are many methods available today to suppress these side-reaction early during synthesis. Our team of experts can help you optimize the conditions to obtain the best possible product quality.  

The possibility of using up to 90% solvents and chaotropic salts or HFIP helps to solubilize a variety of hydrophobic or aggregating peptides extending the range of addressable sequences.

The purification process is characterized by low consumption of organic solvents compared to chromatography because no continuous flow of eluents is required. All steps are simple solid-phase reactions, and therefore lesser amounts of solution are needed to wash off the excess of reagents.

Catch-and-release purification with our novel PEC-Linkers enables the purification of peptides in simple syringe reactors (cartridges). The protocol relies on covalent and discrete chemical reactions, which proceed independently. This allows the parallel processing of the reactors, for example with vacuum flask set-ups or more convenient vacuum manifolds. Having the right equipment in hand, you can therefore readily purify up to 8 peptides at the time with Starter Kits. The High-throughput Kit enables purification of 48 or 96 peptides in parallel using vacuum manifolds.

No. The breakdown species (residues) or the PEC-Linker will remain on the activated filter material, and reuse is not recommended.

If the chemistry fit, yes. The range of possible applications is broad and is not limited to peptide purification. Please reach out to our team of experts and engage with our New Modality Services.