Storing Lyophilized Research Peptides: What Suppliers State

Lyophilization, or freeze-drying, removes water from a frozen peptide solution by sublimation and leaves a dry solid (see how research peptides are made). A review of protein lyophilization calls it the most commonly used method for preparing solid protein formulations, and reports that even after successful lyophilization with a stabilizer, proteins in the solid state may still have limited long-term storage stability ^[8]. That finding concerns proteins. A solid-state review that covers peptides as well describes chemical reactions that occur in dry solids ^[9].

The solid-state review lists the main reactions as deamidation, peptide-bond cleavage, oxidation, the Maillard reaction, beta-elimination and dimerization or aggregation, and names temperature, moisture content, excipients and amorphous versus crystalline state as influencing factors ^[9]. The reasoning behind cold, dry storage follows from the first two of those factors: temperature and moisture content both influence these reactions ^[9]. Supplier pages follow the same logic. They describe the dry powder as the format for long-term holding ^{[1, 2, 3, 4, 5, 6]}, and several state that peptides in solution are less stable than the dry powder ^{[3, 5, 6, 10]}.

Two caveats apply throughout. Supplier statements are general handling guidance for laboratory peptides, not guidance for any one compound. And the peer-reviewed work cited here is mostly in-vitro laboratory chemistry on proteins and model peptides, much of it from pharmaceutical-formulation research on other molecules, so it explains mechanisms without predicting how a particular peptide behaves as supplied. Apart from those papers, the sources are reviews and supplier technical pages. None concerns a compound covered on this site's research pages, and none is evidence about human use.

The table reports what each supplier's technical page states about the dry powder, unless a cell says otherwise. Suppliers are named only as sources of the cited handling pages. A supplier that does not appear in a row did not address that point for the dry powder on the pages reviewed, or the row notes where it did so only for solutions.

Sigma-Aldrich's page gives -80 °C for longer storage in one passage and -20 °C, or preferably -80 °C, in its closing summary ^[2], so the table gives both.

A vial taken from a freezer is colder than the surrounding air, so opening it straight away can allow moisture to condense on the cold solid and container. Ten of the eleven supplier pages reviewed state that the vial is allowed to reach room temperature first. Thermo Fisher says before dissolving, Biosyn says before use, and JPT's page does not mention it ^{[1, 2, 3, 4, 5, 6, 7, 10, 11, 12, 13]}. Bachem, Biosyn and both AAPPTEC pages name a desiccator for this step, and LifeTein and Thermo Fisher name one as preferable ^{[1, 4, 6, 7, 12, 13]}. This article cites no study that tests the condensation mechanism directly. It rests on the suppliers' statements and on how a cold surface behaves in humid air.

Bachem says peptides tend to be hygroscopic and LifeTein says they are often hygroscopic ^{[1, 6]}. GenScript names Asp, Glu, Lys, Arg and His residues as prone to absorbing moisture from the air (deliquescence), a list the other pages reviewed do not give ^[3].

The laboratory literature is more nuanced than "wetter is always worse". In freeze-dried powders of human insulin at 35 °C, the degradation rate increased with water content, including at hydration levels well below the suspected glass transition ^[16]. A review of deamidation in freeze-dried amorphous peptides and proteins reports two patterns of dependence on water: a steady increase in the rate constant with water, and a threshold pattern in which the rate is essentially independent of water at low water contents ^[17]. In model beta-turn cyclic peptides and their linear analogs in polyvinylpyrrolidone-based freeze-dried solids, the observed deamidation rate constants decreased dramatically, by four orders of magnitude, in glassy solids ^[18]. For one lyophilized Asn-containing hexapeptide, the pH of the starting solution had a significant effect on deamidation, while residual moisture and temperature were reported as less significant within the ranges tested ^[19]. Moisture is one variable among several.

Supplier pages differ on whether freeze-thaw matters for the dry powder. Sigma-Aldrich (for both lyophilized peptides and solutions), GenScript (in its lyophilized-peptide guidance) and JPT (for dry peptides) say to avoid repeated freeze-thaw cycles ^{[2, 3, 10]}. AAPPTEC's FAQ gives a general statement about freeze-thaw cycles that does not specify the form ^[13]. ChemPep, Thermo Fisher, Biomatik and AAPPTEC's resource page mention it only for solutions ^{[4, 5, 7, 11]}. Bachem, LifeTein and Biosyn do not mention it ^{[1, 6, 12]}.

For a sealed dry powder, the relevant variable is presumably cold-warm cycling, with a condensation risk each time a container is opened. That is a plausible mechanism, not one that any study cited here tests. JPT states that repeated freeze-thaw cycles can degrade dry peptides through microcondensation and says dividing the peptide into separate vials limits repeated exposure to ambient conditions; the page, as reviewed, cites no data for the mechanism ^[10].

Some pages describe limiting how often a container is opened. GenScript says to work out the amount needed for each experimental set in advance, to divide the peptide into separate vials accordingly, and not to open the stock vial repeatedly ^[3]. Thermo Fisher says to divide the material into single-use portions on receipt if necessary ^[4]. On handling time, Bachem and GenScript say to weigh the powder out quickly and reseal the container ^{[1, 3]}, LifeTein says to weigh rapidly and return unused peptide to cold storage promptly ^[6], and ChemPep says to reseal as soon as possible, preferably under dry nitrogen, and return the vial to cold storage ^[11]. LifeTein states that repeated exposure to room air, moisture and handling can reduce long-term stability ^[6].

GenScript states that each peptide's stability is unique and that certain residues can limit long-term stability even at the stated storage temperature ^[3]. Supplier residue lists overlap without matching. For the dry powder, Bachem names Asn, Gln, Met, Cys and Trp as having limited shelf lives ^[1], and LifeTein names Cys, Met, Trp, Asn, Gln and N-terminal Glu as sequences that may have a shorter shelf life ^[6]. Sigma-Aldrich, GenScript and Biomatik name Cys, Met and Trp as prone to oxidation ^{[2, 3, 5]}. Other residue lists appear in text about solutions. Biosyn and AAPPTEC's FAQ name oxidation of Cys, Met and Trp, deamidation of Gln and Asn, and Cys-Cys disulfide formation as possible degradation reactions in solution ^{[12, 13]}. GenScript (Cys, Met, Trp, Asp, Gln, N-terminal Glu) ^[3], Biomatik (Cys, Met, Trp, Asn, Gln, N-terminal Glu) ^[5] and Sigma-Aldrich (N, Q, C, M, W) ^[2] give residue lists for solutions.

Oxidation. A review of oxidation in proteins identifies Met, Cys, His, Trp and Tyr as the most susceptible residues, and states that oxidation during processing and storage can be induced by contaminating oxidants and by light, and catalyzed by transition metal ions ^[15]. In the two solid-state studies below, being dry did not prevent it. In a preliminary surface-sensitive (ToF-SIMS) study, the neuropeptide Met-enkephalin was oxidized to its sulfoxide at a faster rate under UV light than in laboratory air ^[20]. For a methionine in the protein IGF-I, light exposure, storage temperature and oxygen content each had a considerable effect on the oxidation rate, with no significant difference between aqueous solution and the solid state ^[21].

Deamidation. Deamidation of Asn is described as a major chemical degradation pathway of peptides and proteins ^[22]. In solution studies of model hexapeptides, it proceeds by direct hydrolysis in acid and, at neutral to alkaline pH, through a cyclic imide intermediate that gives both Asp and isoAsp products ^{[22, 23]}. The rate depends strongly on sequence. In model hexapeptides at neutral to alkaline pH, the size of the residue on the C-terminal side of Asn changed the rate significantly ^[23]. Replacing the Gly in a model Asn-Gly peptide with Leu or Pro slowed degradation 33- to 50-fold ^[24]. A mass-spectrometric method has been used to measure deamidation rates for 306 Asn-containing model peptide sequences at pH 7.4 and 37 °C ^[25].

These deamidation rate studies are solution-phase experiments. They show that some sequences are far more labile than others, not what rates to expect for a dry powder.

A certificate of analysis describes a lot at the time it was tested. It is not a record of the vial after shipping, storage, warming, opening and resealing. The storage statement and any test, retest or expiry date on the document are part of the context for the result: an HPLC purity figure and a mass measurement for identity describe that sample on that day.

Oxidation and deamidation are documented solid-state reactions of peptides and proteins, and moisture content and temperature influence them ^[9]. A COA result therefore describes the lot as tested. See how to read a COA, the companion guide on peptide COA red flags and the COA database.

Everything above concerns the dry powder. Several suppliers state that peptides in solution are less stable than the dry powder. GenScript says the shelf life in solution is much shorter ^[3], Biomatik says solutions are less stable than lyophilized powders ^[5], LifeTein says many peptides become more vulnerable to degradation, oxidation or aggregation in solution ^[6], and JPT describes solutions as much more prone to degradation ^[10]. Thermo Fisher describes peptide solutions as unstable ^[4], and Bachem says peptides may slowly degrade in solution ^[1]. Sequence-dependent degradation or shelf life in solution is stated by Bachem, Sigma-Aldrich, GenScript, Biomatik, ChemPep, Biosyn and AAPPTEC's FAQ ^{[1, 2, 3, 5, 11, 12, 13]}, and Sigma-Aldrich and Biomatik also tie solution stability to pH ^{[2, 5]}. Because solution stability is compound-specific and depends on conditions, this article gives no schedules or durations for solutions. The documentation supplied with the specific material applies.