1How to calculate F0.
F0 is the lethality of a saturated steam process: “the equivalent time in minutes at a temperature of 121 °C delivered by the process to the load in its container with reference to micro-organisms possessing a theoretical Z-value of 10”.¹ Each reading becomes a lethal rate, the minutes at 121 °C that one minute at that temperature is worth, and F0 sums them:
F0 = Δt × Σ 10^((T − 121)/10)
T is the temperature in °C inside the slowest-to-heat container and Δt the reading interval in minutes. The D-value is the time that cuts a population of organisms to 10%, and the z-value the temperature change that alters the D-value tenfold.¹ The slowest-heating unit receives the least heat, so its probe sets the cycle’s F0, not the chamber drain or control sensor.²¹
| Temperature | Lethal rate per minute |
|---|---|
| 110 °C | 0.079 |
| 115 °C | 0.251 |
| 118 °C | 0.501 |
| 120 °C | 0.794 |
| 121 °C | 1.000 |
| 122 °C | 1.259 |
In a spreadsheet, put the time of each reading in column A from row 2, the probe temperature in column B and the reading interval in minutes in G1 (0.5 for 30-second readings). In C2 enter =IF(B2>=110,10^((B2-
Validation systems may differ. Kaye’s software accumulates lethality by “averaging the current Ileth and previous Ileth values”, the instantaneous lethal rates.³ To reproduce that number, enter =(C2+C3)/2*$G$1 in D3, fill it down and sum column D.
Two more choices change the number, so the protocol fixes both. The Japanese Pharmacopoeia’s 121.1 °C reference gives a result 2.3% lower than EMA’s 121 °C.⁴ And EMA says: “If temperatures below 110 °C are included (during heat-up and cool-down) in the determination of F0, this should be justified.”¹
2Example: F0 at the cold spot of a PQ run.
The site, product and data are invented. Run PQ-AC05-02 is a performance qualification (PQ) run of cycle FL-121 on autoclave AC-05, with the maximum load of 2,400 vials of a 10 ml aqueous solution. The chamber drain probe reached 121.0 °C at minute 5. Probe TC-07, in a vial at the cold spot found in the heat distribution studies, reached 121 °C at minute 8, and the 15-minute exposure was timed from then, not from minute 5: “the whole of the load should reach the required temperature before measurement of the sterilising time-period starts” (Annex 1, 8.52).⁵
| Minutes | TC-07 reading | Lethal rate | F0 added |
|---|---|---|---|
| 1–2 | 104.0, 108.5 °C | Below 110 °C | 0 |
| 3–7 | 111.2 to 120.3 °C | 0.105, 0.200, 0.363, 0.589, 0.851 | 2.11 |
| 8–22 | 121.4 °C each | 1.096 each | 16.45 |
| 23–25 | 119.6 to 112.4 °C | 0.724, 0.331, 0.138 | 1.19 |
| 26–27 | 108.1, 103.5 °C | Below 110 °C | 0 |
| Total | 19.75 |
- F0 at TC-07 is 19.75 minutes; at TC-02, the fastest-heating vial, 23.85. Both meet the protocol’s limits: at least 15 at every load probe, at most 30 at any, the ceiling set by the product’s heat degradation data.
- Averaging also gives 19.75, because the readings either side of the counted period are zero. Counting from 100 °C gives 19.89 by simple sum and 19.87 by averaging; a 121.1 °C reference gives 19.30.
- The bioburden limit is 100 CFU/100 ml, so a vial holds at most 10 CFU. With a biological indicator D121 of 1.8 minutes, 19.75 ÷ 1.8 = 10.97 log reductions, so the sterility assurance level (SAL) is 10 × 10^−10.97 = 1.1 × 10⁻¹⁰, better than the 10⁻⁶ required. EMA prefers this calculation, “from the maximum bioburden per container and the D-value of the biological indicator used in the validation”.¹
- At F0 8 the same sum gives 10 × 10^−4.44 = 3.6 × 10⁻⁴, which fails, so a cycle near F0 8 depends on knowing the bioburden.
- A weaker indicator would flatter the result: with a low-D indicator “the SAL demonstrated becomes numerically higher, but does not provide as high a safety margin”, so EMA expects a D121 of at least 1.5 minutes, or additional data.¹
3Heat penetration summary: template.
FDA accepts a data summary of each loading study.⁶ This template adds the fields investigators ask about and the signatures that make it a record, filled in for the example run.
| Field | Run PQ-AC05-02 | Source |
|---|---|---|
| Autoclave | AC-05, with manufacturer and model | FDA II.A.3, II.B.3 |
| Cycle and load | FL-121; maximum load, 2,400 × 10 ml vials | FDA II.B.3; CP question 20 |
| Protocol | VP-AC05-01, approved by QA before the first run | PI 006-4 4.1.6, 4.3.3.2 |
| Run date and time | 14 October 2026, 09:40 | FDA II.B.3 |
| Run in series | 2 of 3 consecutive | FDA II.B.1 |
| Executed by | Site validation engineer or qualified contractor, signed | PI 006-4 4.3.3.1 |
| Equilibration time | Not applicable to a fluid load; required for porous cycles | Annex 1 8.59 |
| Dwell time | 15 minutes, from TC-07 at 121 °C | FDA II.B.3; Annex 1 8.52 |
| Dwell range | 121.4 to 121.9 °C, average 121.6 °C; spread 0.5 °C, limit 2.0 °C | FDA II.B.3; Annex 1 8.59 |
| Minimum F0 | 19.75 at TC-07; limit 15 | FDA II.A.3, II.B.3 |
| Maximum F0 | 23.85 at TC-02; limit 30 | FDA II.A.3, II.B.3 |
| Probe calibration | Passed before and after the run | CP question 22; GUI-0074 |
| Indicators | All BIs negative; positive control grew | Annex 1 8.42; GUI-0074 |
| Deviations | None; any would also enter the deviation system | PI 006-4 3.4.3(l) |
| Data integrity | Confirmed by a QA reviewer who did not run it | PI 006-4 3.4.3(m) |
| Report approval | QA, once the third run’s indicators are read | FDA PV IV.C.1; PI 006-4 3.4.3(b) |
4Who runs autoclave PQ, and how long it takes.
The validation master plan names who writes, runs and approves each protocol and report; the template’s signature rows show who signs what.⁸ For equipment, FDA’s process validation guidance says “The quality control unit must review and approve the qualification plan and report”.⁹
| Days | Step | What it involves | Record |
|---|---|---|---|
| 1–5 | IQ and OQ | Control and recording instruments calibrated | IQ and OQ reports |
| 6–7 | Empty chamber | Heat distribution within the protocol’s uniformity limits | Chamber profile |
| 8–10 | Loaded chamber | Maximum and minimum loads, probes between units, repeated to see if the cold spot moves | Certified profile per load |
| 11–16 | Heat penetration | Probes in containers filled to maximum with the slowest-heating solution, BIs alongside; three consecutive runs per load, one a day | A summary per run, as above |
| 17–23 | Incubation | The last run’s BIs read after 7 days | BI results, positive controls |
| 24–30 | Report | Data integrity check, deviations, QA approval | Routine parameters and loading patterns |
One run a day leaves time to calibrate the probes before and after each run, which investigators check.⁷ Indicators set the end date: FDA’s guidance for indicator makers says “Generally, FDA recommends 7 days as the conventional incubation time”, and a shorter time has to be validated.¹⁰ Each further loading pattern repeats days 8 to 30, a little over three weeks.
5What autoclave validation must show.
21 CFR 211.113(b) requires “validation of all aseptic and sterilization processes”.¹¹ Annex 1 asks for sterilising conditions “in all parts of each type of load to be processed”, shown by physical measurements and, where appropriate, biological indicators (8.36).⁵ Beyond the heat studies above, the validation covers these checks.
| Check | What to show | When |
|---|---|---|
| Steam quality | Non-condensable gases, dryness, superheat; condensate meets the WFI monograph | PQ at every user point, then periodically |
| Leak test | Chamber holds vacuum | Normally weekly for vacuum cycles |
| Air removal | Air removal test cycle or an air detector | Normally daily |
| Indicators | Each new lot verified; positive controls grow | Every PQ run |
| Porous loads | Items dry on removal | Every cycle |
| Routine cycle | Unique cycle ID; record reviewed for batch release | Every cycle |
Annex 1 and PIC/S name the steam quality tests but not their limits. HTM 01-01 Part C, NHS guidance for hospital sterilisers, gives the European standard values: non-condensable gases not exceeding 3.5%, superheat not exceeding 25 °C, and a dryness value of at least 0.9, or 0.95 for metal loads.¹²
5.1Where FDA, the EU and Health Canada differ.
- FDA: its 1994 guidance sets no minimum F0 and no fixed requalification interval. The application states the minimum and maximum F0 and describes “routine and unscheduled requalification of production autoclaves, including frequency”; more bioburden information “may be needed for bioburden-based autoclave processes than for overkill processes”.⁶
- EU: Annex 1, in operation since 25 August 2023, requires revalidation “at least annually for load patterns that are considered worst case”, other patterns at a frequency justified in the contamination control strategy, and investigation of any cycle that deviated, such as one with a longer heating phase (8.39, 8.41).⁵
- Health Canada sets the number of runs by risk management and makes physical and microbial requalification at least once a year a “must”. “Sterilization has not been achieved if there is a growth of any biological challenge organisms”, and if no processing error explains the growth, the process is unacceptable.²¹³
- UK and PIC/S: MHRA points manufacturers to Volume 4 of the EU rules, which contains Annex 1.¹⁴ Annex 1 was revised jointly with PIC/S.⁵
6Which steam cycle to choose.
EMA lists its cycles “with decreasing lethality when read from top to bottom, thus the first feasible process should be selected”: start at the top and stop at the first cycle the product survives.¹
| Cycle | Lethality | Hold | Bioburden | Dossier |
|---|---|---|---|---|
| Ph. Eur. reference | ≥ 15 min | ≥ 121 °C | 100 CFU/100 ml, not routine | Confirmation of validation only |
| Overkill | F0 > 12 | ≥ 121 °C | 100 CFU/100 ml, not routine | Validation summary |
| F0 > 8, above 115 °C | F0 > 8 | > 115 °C | Routine; characterised if BI D121 < 1.5 | Summary, plus more data with a low-D BI |
| F0 > 8, above 110 °C | F0 > 8 | > 110 °C | Routine and characterised | As above |
| Post-aseptic heat | F0 < 8 | > 110 °C | 0 CFU/100 ml after aseptic filtration | Summary plus additional data |
If even the last full cycle degrades the product, filter and fill aseptically. A heat treatment with F0 below 8 can add assurance, but “should not compensate for poor aseptic manufacturing practice”.¹
7What inspectors ask and cite.
FDA’s sterile drug compliance programme lists moist heat questions for investigators. Two tie routine cycles back to the validation:⁷
“Is the slowest to heat spot (‘cold spot’) in each load monitored during each autoclave cycle?”
“Has the firm determined lag times for all container sizes/mass, product viscosities, etc. and adjusted their cycles accordingly?”
Annex 1 adds that “BI results in isolation should not be used to override other critical parameters and process design elements” (8.42).⁵
FDA’s warning letter of 29 October 2019 to Cadila Healthcare, for its plant at Moraiya, Ahmedabad, shows where a failed requalification leads. In June 2018 a periodic requalification did not reach the required F0; the firm named a root cause, judged other products unaffected and did not extend the CAPA. In March 2019 the same equipment failed again:¹⁵
“Again, several sensors did not achieve the required F0, and one did not reach the (b)(4) temperature. In addition, at the end of the incubation, the biological indicators at multiple locations in the (b)(4) showed microbiological growth.”
A batch was recalled. FDA found the investigation “did not sufficiently address why your originally validated cycle parameters were not met and why the process fell out of a state of control”, noted seven requalification deviations in two years, and asked for a review of every affected US batch still in expiry, including the F-value, z-value and indicator D-value data behind it.¹⁵
8What changed from 2024 to 2026.
- ISO 17665:2024, published in March 2024, replaced ISO 17665-1:2006 and ISO/TS 17665-2 and 17665-3.¹⁶ EMA’s guideline still cites Parts 1 and 2.¹
- Health Canada’s GUI-0074, published on 12 November 2024, replaced its separate moist heat, gaseous and irradiation guides (GUI-0010, GUI-0007 and GUI-0009).¹⁷
- PIC/S PI 006-4 came into force on 1 October 2026, the first revision since PI 006-3 of September 2007.⁸
9How it runs in Seal.
In Seal, a validated load pattern becomes the template each autoclave load is scanned against, and the cycle’s phases, lethality, probe traces and indicators stay attached to that load and its items. A wet pack is bounded to the positions and items it affects, and load release is a quality decision, not the controller’s cycle-complete flag.
Released items carry their sterile hold into the aseptic batches that use them, so a later failed indicator or calibration finding is bounded to the affected items and the batches that consumed them.
References
- 1Guideline on the sterilisation of the medicinal product, active substance, excipient and primary container, EMA/
CHMP/ CVMP/ QWP/ 850374/ 2015, European Medicines Agency, effective 1 October 2019, section 4.1.1, Table 1 and definitions. https:/ / www. ema. europa. eu/ en/ documents/ scientific- guideline/ guideline- sterilisation- medicinal- product- active- substance- excipient- and- primary- container_ en. pdf (read 8 November 2026) - 2Guide to validation of terminal sterilization process of drugs (GUI-0074), Process validation, sterilization by moist heat, Health Canada, 12 November 2024. https:/
/ www. canada. ca/ en/ health- canada/ services/ drugs- health- products/ compliance- enforcement/ good- manufacturing- practices/ guidance- documents/ guide- validation- terminal- sterilization- process- drugs- gui- 0074/ process- validation- sterilization- moist- heat. html (read 8 November 2026) - 3Understanding Lethality Calculation: Minutes or Seconds?, Kaye (Amphenol), 23 June 2023, the validation system maker’s description of its own calculation. https:/
/ www. kayeinstruments. com/ en/ news/ blog- post/ understanding- lethality- calculation- minutes- or- seconds (read 8 November 2026) - 4The Japanese Pharmacopoeia, 18th edition, English version, General Information G4-10-162, Sterilization and Sterilization Indicators, Ministry of Health, Labour and Welfare. https:/
/ www. mhlw. go. jp/ content/ 11120000/ 000904454. pdf (read 8 November 2026) - 5EudraLex Volume 4, Annex 1, Manufacture of Sterile Medicinal Products, European Commission, 22 August 2022, document history and sections 6.17 and 8.36 to 8.61. https:/
/ health. ec. europa. eu/ system/ files/ 2022- 08/ 20220825_ gmp- an1_ en_ 0. pdf (read 8 November 2026) - 6Guidance for Industry for the Submission Documentation for Sterilization Process Validation in Applications for Human and Veterinary Drug Products, FDA, November 1994, sections II.A to II.C. https:/
/ www. fda. gov/ media/ 71442/ download (read 8 November 2026) - 7Compliance Program 7356.002A, Sterile Drug Process Inspections, section 3.4 and Attachment A, moist heat questions 8 to 24, FDA, implemented 11 September 2015. https:/
/ www. fda. gov/ media/ 75174/ download (read 8 November 2026) - 8PI 006-4, Recommendations on Qualification and Validation, PIC/S, in force 1 October 2026, document history and sections 3.4.3, 4.1.6, 4.3.3, 6.3.9 and 6.3.11. https:/
/ picscheme. org/ docview/ 11277 (read 8 November 2026) - 9Process Validation: General Principles and Practices, guidance for industry, FDA, January 2011, section IV.C.1. https:/
/ www. fda. gov/ media/ 71021/ download (read 8 November 2026) - 10Biological Indicator (BI) Premarket Notification [510(k)] Submissions, guidance for industry and FDA staff, FDA, 4 October 2007, section E. https:/
/ www. fda. gov/ media/ 71134/ download (read 8 November 2026) - 1121 CFR 211.113, Control of microbiological contamination, eCFR. https:/
/ www. ecfr. gov/ current/ title- 21/ chapter- I/ subchapter- C/ part- 211/ subpart- F/ section- 211. 113 (read 8 November 2026) - 12Health Technical Memorandum 01-01, Management and decontamination of surgical instruments, Part C, Steam sterilization, Department of Health, 2016, paragraphs 2.85, 2.86, 3.20 and 3.30 and Table 5. https:/
/ www. england. nhs. uk/ wp- content/ uploads/ 2021/ 05/ HTM0101PartC. pdf (read 8 November 2026) - 13Guide to validation of terminal sterilization process of drugs (GUI-0074), Phase 3, ongoing process verification, Health Canada, 12 November 2024. https:/
/ www. canada. ca/ en/ health- canada/ services/ drugs- health- products/ compliance- enforcement/ good- manufacturing- practices/ guidance- documents/ guide- validation- terminal- sterilization- process- drugs- gui- 0074/ phase- 3- ongoing- process- verification. html (read 8 November 2026) - 14Medicines: good manufacturing practice and good distribution practice, Medicines and Healthcare products Regulatory Agency, last updated 13 May 2024. https:/
/ www. gov. uk/ guidance/ good- manufacturing- practice- and- good- distribution- practice (read 8 November 2026) - 15Warning letter 320-20-05 to Cadila Healthcare Limited, Moraiya, FDA, 29 October 2019, observation 2. https:/
/ www. fda. gov/ inspections- compliance- enforcement- and- criminal- investigations/ warning- letters/ cadila- healthcare- limited- 584856- 10292019 (read 8 November 2026) - 16ISO 17665:2024, Sterilization of health care products, Moist heat, ISO, March 2024. https:/
/ www. iso. org/ standard/ 80271. html (read 8 November 2026) - 17Overview: Guide to validation of terminal sterilization process of drugs (GUI-0074), Health Canada, published 12 November 2024. https:/
/ www. canada. ca/ en/ health- canada/ services/ drugs- health- products/ compliance- enforcement/ good- manufacturing- practices/ guidance- documents/ guide- validation- terminal- sterilization- process- drugs- gui- 0074. html (read 8 November 2026)
AQuestions and answers
What is the difference between overkill and bioburden-based sterilisation?
An overkill cycle delivers so much lethality that the bioburden hardly matters: EMA lists it as a cycle with F0 above 12 minutes, and the Japanese Pharmacopoeia as a 12 log reduction, also referred to as F0 12, regardless of bioburden. A bioburden-based cycle delivers less heat, so it depends on knowing how many organisms each container holds and how heat-resistant they are. FDA says more bioburden information may be needed for those cycles, and EMA requires routine bioburden testing for them.
Does one positive biological indicator fail a validation run?
Health Canada says sterilisation has not been achieved if any biological challenge organism grows, and the process must be treated as unacceptable unless a processing error explains it. Annex 1 adds that indicator results alone should not override the physical data, so a positive indicator in a run with good temperatures still needs investigating, starting with its positive control, handling and lot.
Does each fill volume need its own heat penetration study?
Not automatically, but it needs to be covered. FDA says more studies may be needed when different fill volumes run in the same container line, and its investigators ask whether lag times were determined for every container size, mass and viscosity. A worst-case approach, such as validating the largest fill of the slowest-heating solution, has to be shown to cover the rest.
Is F0 used for dry heat sterilisation?
No. EMA and the Japanese Pharmacopoeia define F0 for moist heat, with a z-value of 10 °C. The Japanese Pharmacopoeia defines a separate FH for dry heat: equivalent minutes at 160 °C for organisms with a z-value of 20 °C. For dry heat, EMA asks for the cycle’s time and temperature and a bioburden limit to be stated, with the Ph. Eur. reference of at least 160 °C for at least 2 hours, and cycles below it validated physically and biologically to an SAL of 10⁻⁶.