Home / Methodology
Last reviewed: May 14, 2026 · Reviewed by Dr. Tshering Pedon
Every vial of research peptide that leaves Koi Peptides goes through eight defined stages: raw material sourcing, solid-phase peptide synthesis, cleavage, preparative HPLC purification, counter-ion handling, lyophilization, analytical quality control by FreedomDiagnostics, and packaging for shipment. This page documents each stage, the equipment and methods used, and the documentation generated. The intent is to give buyers a complete picture of where their material came from and how it was verified, so the analytical record on the Certificate of Analysis they receive carries the operational context behind it.
Koi operates as a research-use peptide supplier. Materials are not manufactured under cGMP and are not intended for human or veterinary use. The methodology described here is research-grade analytical chemistry, supported by U.S. Pharmacopeia methods and ICH guidelines where applicable, and verified per batch through an independent third-party laboratory.
The framework
Before going into each stage, here is the full chain at a glance:
Raw material sourcing
amino acid building blocks, resin, coupling reagents, solvents
Solid-phase peptide synthesis (SPPS)
assembly of the peptide chain on a solid support
Cleavage and deprotection
separation of the assembled peptide from the resin
Preparative HPLC purification
separation of the target peptide from synthesis impurities
Counter-ion handling
TFA exchange where the application requires it
Lyophilization and vialing
freeze-drying and packaging into sealed vials
Analytical QC by FreedomDiagnostics
identity, purity, content, contamination testing
Final packaging and shipment
protective packaging, transit handling, customs documentation
Each stage generates documentation that ties back to a single lot number, which is printed on the vial and indexed in the COA Library. Buyers can pull the full analytical record for any lot they receive by searching that lot number.
Stage 1
The quality of a finished peptide is bounded by the quality of the starting materials. Koi's raw material program covers four categories of inputs:
Sourced from established peptide chemistry suppliers with documented identity (HPLC, MS, optical rotation) and ≥99% purity by HPLC for standard residues. Non-standard residues (D-amino acids, N-methylated, labeled, or modified amino acids) carry the same identity-and-purity documentation from the supplier and are independently verified on receipt.
The Wang, Rink amide, or 2-chlorotrityl resin used for a given synthesis is selected based on the target peptide's C-terminus chemistry. Resin loading is measured per lot, recorded in the synthesis batch record, and used to calculate stoichiometry for the run.
HATU, HBTU, DIC, Oxyma, and DIPEA from suppliers with documented purity. Each lot is checked against expiration before use.
DMF, DCM, methanol, acetonitrile, and water at HPLC-grade or higher. Water for analytical work meets ASTM Type I or USP Purified Water specifications. Solvents are tested for residual moisture (Karl Fischer) when stability matters to the synthesis.
Every incoming raw material lot is logged against a supplier certificate of analysis. The supplier COA is retained for the operational life of any peptide synthesized from that material. This is the chain of custody that lets Koi trace a finished vial back to its starting materials if a quality question is ever raised.
Stage 2
Koi synthesizes peptides using solid-phase peptide synthesis (SPPS), the method developed by R. B. Merrifield in 1963 and recognized with the Nobel Prize in Chemistry in 1984 (Merrifield, Journal of the American Chemical Society, 1963). The chemistry is the standard for research peptides up to roughly 50 residues in length and produces material at the purity and scale our catalog requires.
The SPPS workflow follows the Fmoc/tBu protection strategy, which is the modern standard for the field:
The C-terminal amino acid is anchored to the resin.
Each subsequent residue is added through a deprotection step (piperidine in DMF removes the Fmoc protecting group) followed by a coupling step (HATU or HBTU activation in the presence of base couples the next Fmoc-amino acid to the growing chain).
Coupling efficiency is checked at intervals through ninhydrin (Kaiser) or chloranil tests, which detect free amine groups that indicate incomplete coupling.
Between every coupling and deprotection step, the resin is washed with DMF to remove excess reagents and byproducts.
Synthesis takes place on automated peptide synthesizers under written standard operating procedures. Each run generates a synthesis batch record that captures the date, the operator, the resin lot, the amino acid lots, the coupling reagent lots, and any in-process observations.
For longer or more challenging sequences, additional measures are applied. These can include double-coupling protocols where standard single-coupling efficiency falls below threshold, pseudoproline dipeptides to disrupt secondary structure formation during synthesis, or microwave-assisted coupling. The synthesis route selected for each compound is documented and reproducible across batches.
Stage 3
Once the full sequence is assembled on the resin, the peptide must be separated from the solid support and the side-chain protecting groups must be removed. This is done in a single chemical step using a cleavage cocktail based on trifluoroacetic acid (TFA), typically with scavengers such as triisopropylsilane (TIS), water, and 1,2-ethanedithiol (EDT) where cysteine or methionine residues require protection from oxidation during cleavage.
The cleavage cocktail composition is selected based on the residues in the peptide. The cocktail is removed under reduced pressure or by precipitation into cold diethyl ether, which produces a crude peptide as a precipitate that can be collected by filtration or centrifugation.
The crude peptide at this stage typically runs between 60% and 85% pure by HPLC, depending on the sequence length and the synthesis difficulty. Purification in the next stage brings the material to the catalog specification.
Stage 4
The crude peptide from cleavage carries the target sequence alongside truncated sequences (where coupling failed at one or more residues), deletion sequences (where a residue was skipped), oxidation products, and small amounts of cleavage byproducts. Preparative reversed-phase HPLC separates the target peptide from these impurities.
The standard method uses:
Fractions that meet the purity threshold are pooled. Fractions that fall outside the threshold are either set aside for a second purification pass or discarded. The pooled material is then concentrated by rotary evaporation to remove most of the acetonitrile, leaving an aqueous TFA solution of the purified peptide.
The output of this stage is purified peptide as a TFA salt, in solution, ready for counter-ion handling or direct lyophilization.
The purification yield depends on the sequence and the difficulty of separating closely related impurities, but well-behaved sequences typically come through preparative HPLC at 70 to 90% recovery of the target peptide loaded.
Stage 5
Peptides purified by TFA-buffered HPLC come off the column as TFA salts. The TFA counter-ion can account for 10 to 25% of the dry mass of a freshly purified peptide before any salt exchange, depending on the number of basic residues in the sequence (Roux et al., Journal of Peptide Science, 2008).
For most catalog peptides at Koi, the TFA salt form is retained. TFA is a stable, well-characterized counter-ion that does not interfere with most research applications, and retention of the TFA salt avoids the additional handling step that salt exchange introduces.
For peptides where the downstream application requires it, Koi performs TFA-to-acetate exchange through repeated lyophilization from dilute acetic acid, or TFA-to-chloride exchange through ion-exchange chromatography. The counter-ion form is documented on every COA so the buyer can calculate the correct net peptide content and dissolve the material in the right solvent.
Counter-ion choice matters most for cell culture work, where TFA can interfere with TLR4-related assays, and for any application where the dry mass calculation needs to be exact.
Stage 6
The purified peptide solution is frozen and then dried under reduced pressure (lyophilization, also called freeze-drying) to produce a stable, light, dry powder. Lyophilization is the standard preservation method for synthetic peptides because it removes water without exposing the peptide to heat or chemical stress.
The lyophilized cake is the form in which most Koi peptides ship. After drying, the material is portioned into vials at the specified fill weight, typically 5 mg or 10 mg per vial, depending on the catalog SKU, under controlled ambient conditions. Each vial is sealed, crimped, and labeled with:
The vial fill weight is the gross peptide mass. The net peptide content, which accounts for counter-ion, water, and any peptidic impurities, is reported separately on the COA. This is the figure that matters for any quantitative work. The mass balance approach behind it is the standard method described by USP and recognized in the peer-reviewed reference standards literature (Williamson et al., AAPS PharmSciTech, 2023).
Stage 7
Every batch is sampled at this stage and submitted to FreedomDiagnostics for independent analytical verification. FreedomDiagnostics is an independent third-party laboratory that issues the analytical results that appear on the Koi COA under its own letterhead, with a unique verification ID per submission.
The standard analytical panel covers:
Identity by electrospray ionization mass spectrometry (ESI-MS). Observed mass vs theoretical monoisotopic mass, with the mass spectrum included on the COA. Acceptance criterion: within ±1 Da on a quadrupole instrument.
Purity by analytical HPLC. Area percent of the target peak under specified method conditions (C18 reversed-phase, water/acetonitrile gradient with 0.1% TFA, UV detection at 210 or 214 nm). The chromatogram is included on the COA with retention time, method conditions, and integration. Acceptance criterion: ≥99% for the standard Koi catalog.
Net peptide content by UV detection. Absorbance at 205 or 280 nm against calibrated standards, reported separately from purity (Anthis & Clore, Protein Science, 2013).
Bacterial endotoxin by USP <85>. Numerical result in EU/mg, with the testing method specified. Acceptance criterion is set against the intended downstream application. For cell culture work the relevant published thresholds sit in the 0.1 to 1 EU/mg range (mAbs 2025, doi: 10.1080/19420862.2025.2458627).
Residual solvents by gas chromatography. Where the synthesis route required organic solvents that fall under ICH Q3C class 2 or class 3 controls, residuals are quantified and compared against the chapter's limits.
Heavy metals by ICP-MS. Run on routes that used palladium or other catalysts. Detection limits in the parts-per-billion range.
The COA
The COA issued by FreedomDiagnostics is the document that travels with the lot. It is published in the COA Library and searchable by the lot number printed on the vial. Any buyer can verify the FreedomDiagnostics COA ID directly against FreedomDiagnostics's own records, which is the verification step that distinguishes a third-party result from an unverifiable claim.
Stage 8
Lyophilized peptides are stable at ambient temperature for the duration of standard transit. This is a property of the lyophilized form itself: with water removed, the chemical and microbiological pathways that drive peptide degradation are essentially shut down, and the dry cake holds its analytical profile across normal shipping timeframes.
For standard domestic U.S. shipments, lyophilized vials ship in protective packaging without cold-chain handling. The peptide remains within specification on arrival, and the buyer's responsibility for cold storage begins at the receiving end.
For long-distance international shipments and high-summer routing in warm climates, Koi ships with thermal packaging at no charge. This is the conservative default for any route where peak transit temperatures could exceed comfortable ambient or where transit times extend beyond a standard window. The decision to add thermal packaging is made by the shipping team per order, not as a paid upgrade.
Every shipment includes:
Once a buyer receives the material, the recommended storage condition is -20°C for the lyophilized form, which gives the longest documented stability window. Once dissolved in a laboratory diluent or buffer, the peptide is stable for a shorter period and should be stored at 2 to 8°C.
Verifiable record
For every shipment, the documentation a buyer can pull is:
The batch-specific COA tied to the lot number on the vial
The HPLC chromatogram with method conditions, retention time, integration, and area percent
The mass spectrum with observed vs theoretical mass
The endotoxin test result by USP <85>, in EU/mg, with method specified
The FreedomDiagnostics verification ID that can be checked against FreedomDiagnostics's own records
The net peptide content figure, reported separately from purity
The counter-ion form and any residual solvent or heavy metal data where applicable
Historical COAs for any lot Koi has ever shipped remain accessible through the COA Library. This is the operational record that supports reproducibility on the buyer's end. If a researcher needs to reference the exact material used in an experiment six months or two years later, the COA they pulled at order time can be re-pulled at any point.
Scope and limits
Transparency works in both directions. Researchers sourcing peptides should know what a research-use vendor is and is not, and Koi states this directly:
Koi is a research-chemical supplier. Koi is not a pharmacy, compounding pharmacy, or outsourcing facility, and does not supply finished drug products.
Koi is not a cGMP pharmaceutical manufacturer. Koi's facility is not registered with the FDA as a drug manufacturer and Koi's analytical program is designed for research-grade material only. Pharmaceutical-grade peptides require full chain-of-custody documentation, sterile manufacturing environment, validated analytical methods, and ICH stability data that Koi does not provide.
Koi does not test for sterility. Lyophilized synthetic peptides produced through organic-solvent synthesis routes operate as low-bioburden processes, but Koi does not certify sterility under USP <71>, and Koi's materials are not represented as sterile.
Koi does not test for viral clearance, mycoplasma, or other contaminants relevant to biologically-derived materials. These tests apply to biologically-derived products, not to chemically-synthesized peptides, and they are outside the scope of the research-use analytical program.
Koi materials are for laboratory and research use only and are not for human consumption. Every product page carries the "For laboratory and research use only. Not for human consumption." statement. This is the operational basis on which the materials are sold.
The methodology described on this page is research-grade analytical chemistry, executed under written SOPs and verified through an independent third-party laboratory. It is sufficient for the laboratory research applications the materials are sold for. It is not sufficient for any application outside that scope, and Koi makes no claim that it is.
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• ≥99% purity target • Third-party tested • COA per batch • Fast US shipping
For research use only — not for human or veterinary use. Products are laboratory research chemicals, not drugs, supplements, or food.
