How peptides degrade in storage: oxidation, deamidation, hydrolysis and how retest dates are set

Why a lot can change after its certificate was issued

A certificate of analysis describes a lot on the day it was tested. Peptides are chemically reactive molecules, and some sequences change measurably in storage. Bachem, a peptide manufacturer, states that most peptides containing Trp, Met, Cys, Asn or Gln have limited shelf life, and that adsorption of water may decrease stability and reduce the overall peptide content. [2] GenScript states that most lyophilized peptides are stable for several years at -20 C away from bright light, while noting that sequence affects this. [3]

So the useful question is not whether a peptide degrades but which pathways apply to this sequence, how fast under your storage conditions, and what evidence the supplier has for the date printed on the certificate.

The main pathways and what they look like on a re-run

Use the sequence on the certificate to see which rows apply. Mass shifts are what a mass spectrometer would see on the intact peptide; the HPLC column describes what tends to appear on a repeat chromatogram.

Oxidation and deamidation in more detail

A review of methionine chemistry in PNAS notes that a variety of oxidants react readily with methionine to form methionine sulfoxide, and that oxidant sources include hydrogen peroxide, metal-catalysed oxidation and irradiation. [5] Bachem advises special care for Trp, Met or Cys peptides to avoid oxidation, and GenScript recommends storing Cys, Met or Trp peptides under anaerobic conditions. [2] [3]

Asparagine deamidation proceeds through a succinimide intermediate to aspartic acid or isoaspartic acid, adding about 1 Da; aspartic acid can isomerise to isoaspartic acid by the same route. Liu and colleagues describe Asn-Gly as the sequence most prone to deamidation and report that rates depend on primary sequence and higher-order structure, with an inverse bell-shaped pH profile. [4] In their exenatide analysis, deamidated and intact forms were resolved by liquid chromatography and distinguished by mass spectrometry. [4]

A worked example: a 20-residue peptide with one Met and one Asn-Gly motif that shows 98.5 percent purity at release, and 96.8 percent with a new peak at +16 Da on a repeat run 18 months later, has a documented oxidation product. The same peptide with no new mass but a new shoulder near the main peak may have an isoAsp form that MS alone would not flag.

How ICH Q1A stability studies set retest dates

ICH Q1A(R2) is the stability guideline used for registered drug substances. It defines the re-test period as the time during which a drug substance is expected to remain within its specification when stored under the defined conditions, after which the batch should be re-tested before use; a batch can be re-tested multiple times as long as it continues to comply. [1] It also notes that for most labile biotechnological substances a shelf life is more appropriate than a re-test period. [1]

For the general case, long-term studies run at 25 C/60 percent RH or 30 C/65 percent RH and accelerated studies at 40 C/75 percent RH, with at least 12 and 6 months of data respectively at submission. For substances intended for freezer storage, the long-term condition is -20 C plus or minus 5 C for 12 months, the re-test period should be based on real-time data, and a single batch should be tested at an elevated temperature such as 5 C or 25 C to address short excursions during shipping or handling. [1] Long-term testing is normally every 3 months in the first year, every 6 months in the second, and annually after that, on at least three primary batches. [1] Significant change for a drug substance means failing its specification. [1]

Deciding whether an aged lot needs retesting

Research-use suppliers are not filing registration dossiers, so a retest date on their certificate may or may not rest on a Q1A-style study. Ask what it rests on. Then use the checks below before relying on old material for new work. This is a documentation decision, not advice on any use of the material.

• Is today past the retest date on the lot certificate? If so, the lot needs a new result before you rely on the original one. [1]
• Does the supplier have real-time data at the labelled storage temperature, or only an accelerated estimate? For freezer-stored material, Q1A expects real-time data. [1]
• Does the sequence contain Met, Cys, Trp, Asn-Gly, Asn, Gln or Asp? Those are the residues the table flags. [2] [4]
• Was the vial stored at the labelled condition throughout? Check your freezer log and any shipping excursion record.
• Has the vial been opened repeatedly, especially cold? Water uptake lowers net peptide content. [2] [3]
• If retesting, request both HPLC purity and MS, because isomerisation has no mass change while oxidation and deamidation do. [4]
• Compare the new chromatogram to the original peak by peak, not only the headline purity number.