Stability Testing, Retest Dates and Shelf Life

In plain English

Every material changes slowly in storage. A peptide powder can pick up water, oxidise, or break down into related peptides ^{[5, 8]}. Stability testing is the planned way of measuring that change.

Samples from several batches are stored in chambers held at set temperatures and humidities. At planned time points, samples are taken out and tested with the same methods used at release. The results show how fast quality drifts and how long the material stays within its specification ^[1].

From those data, a manufacturer sets a date. For an active ingredient this is usually a retest date: the point at which the material should be tested again before further use. For a finished medicine it is an expiry date: the end of its shelf life ^[1].

The short version

• Stability testing shows how quality changes with time, temperature, humidity and light ^[1].
• A retest period applies to a drug substance; a shelf life applies to a finished product ^[1].
• Peptides are usually studied refrigerated or frozen ^[5].
• Frozen materials get their retest period from real-time data only ^{[1, 2]}.
• In Australia, stability studies for registered medicines should reflect hot, humid Zone IV conditions ^[6].

Retest date, shelf life and expiry date: what’s the difference?

The ICH Q1A(R2) glossary defines the key terms ^[1]:

First, a batch of drug substance can be retested more than once, and a different portion used after each retest, as long as it still complies ^[1]. Second, for most biotechnological and biological substances that are known to be labile, Q1A says a shelf life is more appropriate than a retest period ^[1].

A retest date on a certificate of analysis is therefore not an expiry date. It is a promise that the material was expected to stay within specification until then, under the stated storage conditions. See Batch Numbers and Traceability.

What a formal stability study involves

Stress testing (forced degradation)

Stress testing comes first. It helps identify likely degradation products and pathways, and shows whether the analytical methods can detect them, which is what “stability-indicating” means ^[1]. For a drug substance, Q1A says stress testing is likely to be done on a single batch and should include ^[1]:

• temperatures in 10 °C steps above the accelerated condition (for example 50 °C, 60 °C);
• humidity of 75% relative humidity (RH) or more, where appropriate;
• oxidation and photolysis (light);
• hydrolysis across a wide pH range, in solution or suspension.

European guidance for synthetic peptides says forced degradation studies are expected, both to study how the peptide degrades and to test whether the methods detect that degradation ^[5]. Industry practice varies in the details: a 2020 benchmarking survey of 12 companies compared the conditions, time points and analytical techniques they used for forced degradation of biopharmaceuticals ^[13]. See How to Read an HPLC Chromatogram.

Formal studies

Formal stability data should come from at least three primary batches, made at least at pilot scale by the same synthetic route and a process that simulates the final one. Samples are stored in the same container closure system as the material itself, or one that simulates it ^[1]. Validated stability-indicating methods are used, and the tests cover attributes likely to change during storage ^[1].

For a proposed retest period of at least 12 months, long-term samples are normally tested every 3 months in the first year, every 6 months in the second, and yearly after that. The accelerated study needs at least three time points, such as 0, 3 and 6 months ^[1].

Photostability

ICH Q1B sets the light exposure for confirmatory photostability studies: an overall illumination of not less than 1.2 million lux hours and an integrated near-ultraviolet exposure of not less than 200 watt hours per square metre ^[3].

Standard ICH storage conditions

Q1A(R2) sets the conditions below for drug substances ^[1]:

For a drug substance, “significant change” means failure to meet its specification ^[1]. Refrigerated materials that change significantly at the accelerated condition get a retest period based only on the long-term data ^[1]. Frozen materials have no accelerated condition. Instead, a single batch is tested at an elevated temperature (for example 5 °C or 25 °C) to show the effect of short excursions during shipping or handling ^[1].

Peptides fall mostly into the refrigerated and frozen rows. European peptide guidance notes that peptides are generally stored at 5 °C ± 3 °C or −20 °C ± 5 °C to prevent or minimise degradation, with higher temperatures and humidities also studied to cover short excursions ^[5].

Q1A also says storage statements should avoid vague terms such as “ambient conditions” or “room temperature” ^[1].

What changes in a peptide during storage

European guidance asks that likely degradation pathways be discussed from the peptide’s amino acid composition and sequence: oxidation of cysteine and methionine, deamidation, hydrolysis and β-aspartate formation. It also asks for high-molecular-mass impurities (aggregates) to be investigated where relevant, and expects water content to be part of stability studies of hygroscopic powders ^[5].

Solid powders are not inert. A review of solid-state chemistry described deamidation, peptide bond cleavage, oxidation, the Maillard reaction, β-elimination, and dimerisation or aggregation in dried peptides and proteins. Temperature, moisture, excipients and whether the solid is amorphous or crystalline all influence these reactions ^[8].

Water deserves special attention. In freeze-dried amorphous peptides and proteins, reported deamidation rates either rise steadily with water content or stay almost flat until a threshold water content is passed, then accelerate (a “hockey stick” pattern) ^[9]. That is one reason a stability study tracks water alongside purity. See Water Content and Karl Fischer Titration and Peptide Storage and Stability.

For larger proteins and polypeptides, ICH Q5C notes that molecular conformation depends on non-covalent as well as covalent forces, and that such products are particularly sensitive to temperature changes, oxidation, light, ionic content and shear ^[4]. A 2010 review summarised chemical and physical instability, and stabilisation in solution and in the dried state ^[10].

From data to a retest period

The statistics

Q1A suggests one way to analyse an attribute that changes with time: find the time at which the one-sided 95% confidence limit for the mean degradation curve meets the acceptance criterion. Batches can be combined into one estimate only if statistical tests (with a significance level of 0.25) show their slopes and intercepts are similar; otherwise the retest period is based on the batch expected to fall out of specification first ^[1].

How far beyond the data a date can go

ICH Q1E limits extrapolation beyond the period covered by long-term data ^[2]:

Worked example: a refrigerated peptide

The figures below are illustrative. A lyophilised peptide has an HPLC purity specification of not less than 97.0%. Long-term data at 5 °C ± 3 °C:

1. Fit the trend. A straight-line fit gives a loss of about 0.057 percentage points per month.
2. Find where the mean line meets the limit. 98.6 − 0.057 × t = 97.0 gives t ≈ 28 months.
3. Apply the confidence limit. The lower one-sided 95% confidence limit for the mean crosses 97.0% earlier, at about 26 months ^[1].
4. Apply the extrapolation cap. With 12 months of data on refrigerated material, Q1E allows at most 1.5 × 12 = 18 months, and not more than 12 + 6 = 18 months ^[2].

The supportable retest period at this point is therefore 18 months, not 26 or 28. It can be extended later as more real-time data accumulate. A real study would also have to look at the other attributes, such as water content and individual impurities ^{[1, 5]}.

Release limits versus shelf-life limits

TGA guidance gives an example for finished medicines. If the lower assay limit is 95% at release and 90% at expiry, the shelf life allows a maximum fall of 5 percentage points. A batch released at 101% should therefore be dated from the time it falls to 96%, not 90%, so that a batch released at the 95% limit would still comply ^[6]. The same guidance says the maximum shelf life for chemically derived medicines is normally five years, and the maximum extrapolated shelf life is normally three years ^[6].

Can accelerated testing predict shelf life?

Sometimes. The Accelerated Stability Assessment Program (ASAP) uses a humidity-corrected Arrhenius equation and an “isoconversion” design, in which storage times at several temperature and humidity conditions are chosen to reach a fixed degradant level ^[11]. In the first published application to a peptide, bacitracin was stored at 50–80 °C and 0–63% RH for up to 21 days. The model’s predictions agreed with longer-term results measured at 30 °C/53% RH and 40 °C/75% RH ^[12].

The guidelines still put real-time data first. ICH Q5C says the primary data supporting a storage period for biotechnological products should come from long-term, real-time, real-condition studies ^[4]. Q1A and Q1E base frozen materials on long-term data only ^{[1, 2]}.

Temperature history during transport can be summarised by the mean kinetic temperature: a single temperature that gives the same thermal challenge as the varying temperatures actually experienced. It is higher than the simple average, because it takes account of the Arrhenius equation, which gives high temperatures extra influence ^[1]. Temperature excursions change the mean kinetic temperature and, through it, the expected stability ^[14].

Reading stability information for research material

A stability claim on a research peptide can be checked with a few questions:

1. Is the date a retest date or an expiry date? They mean different things ^[1].
2. Which storage condition does it assume? A date is only valid under the stated condition, such as 5 °C ± 3 °C or −20 °C ± 5 °C ^[1].
3. Is it based on data? Real-time data on the same material, in the same container, count for the most ^{[1, 4]}.
4. Which attributes were tracked? Purity alone can miss water uptake or aggregate formation ^{[5, 9]}.
5. Were the methods stability-indicating? A method that cannot separate degradation products cannot show they are absent ^{[1, 5]}.

What this means in Australia

• Adopted guidance. The TGA has adopted ICH Q1A(R2) with no Australian annotations ^[7], and its stability guidance lists the European versions of Q1E and Q5C among the guidelines it has adopted ^[6].
• Australian climate. Major Australian population centres have hot, humid summers and are classified as climatic Zone IV. The TGA asks for stability studies on medicines registered in Australia to be performed under conditions representative of Zone IV ^[6].
• Research-use labels. The TGA has stated that a “research use only” disclaimer does not make supply lawful, change a product’s regulatory status, permit importation, or remove advertising or supply obligations ^[15]. A retest date describes one batch’s expected quality under stated storage; it does not change any of this.

This is general information, not legal advice. Laws change; check the current Poisons Standard and TGA guidance.

Frequently asked questions

What is the difference between a retest date and an expiry date?

A retest date tells when a drug substance should be tested again to confirm it still meets specification. An expiry date marks the end of a finished product’s shelf life, after which it must not be used ^[1].

What are the ICH accelerated stability conditions?

For the general case, 40 °C ± 2 °C at 75% RH ± 5% RH for 6 months. For refrigerated materials, 25 °C ± 2 °C at 60% RH ± 5% RH ^[1].

How far can a shelf life be extrapolated?

For room-temperature storage with little change, up to twice the long-term data period but not more than 12 months beyond it. Refrigerated materials are limited to 1.5 times, and not more than 6 months beyond ^[2].

Why are peptides stored refrigerated or frozen?

To prevent or minimise degradation such as oxidation, deamidation and hydrolysis ^[5].

How many batches does a formal stability study need?

At least three primary batches, made at least at pilot scale ^[1].

References

1. International Council for Harmonisation. ICH Q1A(R2): Stability testing of new drug substances and products. Step 4, 6 February 2003. database.ich.org. Accessed 25 October 2026.
2. International Council for Harmonisation. ICH Q1E: Evaluation of stability data. 2003. database.ich.org. Accessed 25 October 2026.
3. International Council for Harmonisation. ICH Q1B: Stability testing: photostability testing of new drug substances and products. 1996. database.ich.org. Accessed 25 October 2026.
4. International Council for Harmonisation. ICH Q5C: Quality of biotechnological products: stability testing of biotechnological/biological products. Step 4, 30 November 1995. database.ich.org. Accessed 25 October 2026.
5. European Medicines Agency. Guideline on the development and manufacture of synthetic peptides. EMA/CHMP/CVMP/QWP/367182/2025. Adopted 4 December 2025; effective 1 June 2026. ema.europa.eu. Accessed 25 October 2026.
6. Therapeutic Goods Administration. Stability testing for prescription medicines. Version 1.1, March 2017. tga.gov.au. Accessed 25 October 2026.
7. Therapeutic Goods Administration. ICH Topic Q 1 A (R2): Stability testing of new drug substances and products. International scientific guidelines adopted in Australia. tga.gov.au. Accessed 25 October 2026.
8. Lai MC, Topp EM. Solid-state chemical stability of proteins and peptides. J Pharm Sci. 1999;88(5):489–500. doi:10.1021/js980374e. PMID: 10229638
9. Ohtake S, Feng S, Shalaev E. Effect of water on the chemical stability of amorphous pharmaceuticals: 2. Deamidation of peptides and proteins. J Pharm Sci. 2018;107(1):42–56. doi:10.1016/j.xphs.2017.09.003. PMID: 28923320
10. Manning MC, Chou DK, Murphy BM, et al. Stability of protein pharmaceuticals: an update. Pharm Res. 2010;27(4):544–575. doi:10.1007/s11095-009-0045-6. PMID: 20143256
11. Waterman KC. The application of the Accelerated Stability Assessment Program (ASAP) to quality by design (QbD) for drug product stability. AAPS PharmSciTech. 2011;12(3):932–937. doi:10.1208/s12249-011-9657-3. PMID: 21748541
12. Waterman R, Lewis J, Waterman KC. Accelerated stability modeling for peptides: a case study with bacitracin. AAPS PharmSciTech. 2017;18(5):1692–1698. doi:10.1208/s12249-016-0635-7. PMID: 27714699
13. Halley J, Chou YR, Cicchino C, et al. An industry perspective on forced degradation studies of biopharmaceuticals: survey outcome and recommendations. J Pharm Sci. 2020;109(1):6–21. doi:10.1016/j.xphs.2019.09.018. PMID: 31563512
14. Kommanaboyina B, Rhodes CT. Effects of temperature excursions on mean kinetic temperature and shelf life. Drug Dev Ind Pharm. 1999;25(12):1301–1306. doi:10.1081/ddc-100102303. PMID: 10612028
15. Therapeutic Goods Administration. Understanding your responsibilities when importing, compounding and supplying unapproved peptide products. Safety advisory, 13 April 2026. tga.gov.au. Accessed 25 October 2026.