This is a ready-to-use protocol. Replace every <<FILL: ...>> placeholder with your own specifics, execute per your validation program, and route through your normal review and approval. A worked filled specimen follows the template. Verify each cited regulation against the current source before you rely on it.
Approval page
| Field | Entry |
|---|---|
| Protocol title | Dirty Hold Time and Clean Hold Time Study for <<FILL: equipment / train>> |
| Protocol number | <<FILL>> |
| Parent cleaning validation protocol | <<FILL: protocol number>> |
| Version | <<FILL>> |
| Effective date | <<FILL>> |
| Role | Name | Signature | Date |
|---|---|---|---|
| Author | <<FILL>> | ||
| Microbiology reviewer | <<FILL>> | ||
| QA approver | <<FILL>> |
1. Objective
To establish, with documented evidence, the maximum dirty hold time (DHT), the maximum interval between end of manufacturing and start of cleaning during which the cleaning procedure remains proven effective, and the maximum clean hold time (CHT), the maximum interval cleaned equipment may be stored before use while remaining within chemical and microbial limits, for <<FILL: equipment / train>>.
2. Scope
This protocol covers <<FILL: equipment / train, products represented>>. Both DHT and CHT are validated against worst-case conditions: the most difficult-to-clean residue represented, the longest realistic delay the operation could impose, and the storage condition (temperature, humidity, covered/uncovered, port status) actually used in practice.
3. Justification of worst-case target times
| Study | Proposed maximum time | Justification |
|---|---|---|
| DHT | <<FILL: e.g. up to 9 days>> | <<FILL: state the real operational driver, e.g. longest campaign length between cleans, weekend/holiday scheduling gaps>> |
| CHT | <<FILL: e.g. up to 14 days>> | <<FILL: state the real operational driver, e.g. longest realistic interval between cleaning and next scheduled use>> |
Target times must reflect real operational practice, not a convenient round number. A DHT validated at 48 hours while campaign manufacturing routinely soils equipment for nine days is not a validated state; it is a gap waiting to be found.
4. Roles
| Role | Responsibility |
|---|---|
| Manufacturing / equipment SME | Confirms realistic soiling and storage conditions; executes the hold and the subsequent standard clean |
| Microbiology | Performs bioburden (and endotoxin, where applicable) sampling and testing |
| QC analytical | Performs chemical residue sampling and testing at the DHT endpoint per the parent protocol’s method |
| Validation | Designs the study, reviews results against acceptance criteria |
| QA | Approves the protocol and the final report |
5. Method: Dirty hold time
- Soil the equipment (or a representative coupon/section) using the actual product and process, or a scientifically justified surrogate, to represent real end-of-manufacturing residue.
- Hold for the target maximum DHT plus a margin of
<<FILL: e.g. 10%>>, under<<FILL: the warmest/most humid realistic condition that promotes drying and bioburden growth>>. - At the end of the hold, execute the standard cleaning procedure exactly as written, with no adjustment for the extended soil time.
- Sample per the parent protocol’s worst-case locations and methods (chemical residue, cleaning agent, and bioburden/endotoxin where applicable).
- Compare results to the same acceptance limits used in the parent cleaning validation protocol.
6. Method: Clean hold time
- Clean the equipment per the standard, validated cleaning procedure.
- Store under the actual representative storage condition:
<<FILL: covered/uncovered, ports capped or open, storage area, temperature/humidity>>. - Sample at intermediate and target maximum CHT timepoints:
<<FILL: e.g. time zero, 50% of target, 100% of target>>. - At each timepoint, sample for bioburden (and endotoxin for sterile/injectable applications), and for chemical residue where airborne or rinse-water carryover is a credible risk.
- Compare results to the microbial and (where applicable) chemical acceptance limits.
7. Acceptance criteria
| Study | Criterion |
|---|---|
| DHT | Chemical residue (recovery-corrected) at or below the per-swab/per-rinse limit after the maximum DHT; bioburden at or below the defined limit |
| CHT | Bioburden (and endotoxin, where applicable) at or below the defined limit at the target maximum CHT; any chemical carryover criterion (if applicable) met |
Any single result outside limits at any timepoint fails that study; the study does not average across timepoints or locations to mask a single failing result.
8. Deviation handling
Any out-of-limit result is investigated per <<FILL: SOP-ID for deviations>> before the DHT or CHT is finalized. A failing DHT or CHT study means the proposed maximum time is not validated; either shorten the proposed time and retest, or investigate and improve the cleaning or storage process.
9. Summary and conclusion
<<FILL: state the validated maximum DHT and CHT, the conditions they were validated under, and confirm operations cannot exceed either without triggering a deviation>>.
10. Maintaining the validated hold times
Once established, DHT and CHT are maximum limits, not targets. Operations exceeding either validated time is a deviation per <<FILL: SOP-ID for deviations>>, not a routine occurrence to be tolerated. Re-validate if the process, product mix, or storage conditions change materially. See change control for validated systems.
References
EU GMP Annex 15 (Qualification and Validation), 2015 revision. FDA Guide to Inspections Validation of Cleaning Processes (1993).
Confirm the current version and clause numbers of each reference before issue.
Filled specimen
Objective (excerpt): Establish DHT and CHT for the 500 L mixing vessel train, representing the worst-case product identified in the parent protocol CV-2026-014.
| Study | Proposed maximum time | Justification |
|---|---|---|
| DHT | 9 days | Longest campaign length between scheduled cleans, per production scheduling data for the prior 12 months |
| CHT | 14 days | Longest realistic gap between cleaning completion and next scheduled batch, accounting for changeover and campaign planning |
DHT result (excerpt): Equipment soiled with worst-case product residue, held 10 days (9-day target plus 10% margin) at ambient warehouse temperature and humidity representative of the worst summer condition on file. Standard cleaning executed with no adjustment. Swab results at all four worst-case locations: recovery-corrected residue below the 43.4 ug/swab limit (highest result 28.1 ug/swab). Bioburden below the 25 CFU/25cm2 action limit at all locations (highest result 6 CFU/25cm2). DHT of 9 days validated.
CHT result (excerpt): Equipment cleaned and stored capped, in the designated clean-equipment storage area, sampled at time zero, day 7, and day 14. Bioburden at day 14: 3 CFU/25cm2, within the 25 CFU/25cm2 limit. CHT of 14 days validated.
Summary and conclusion (excerpt): DHT of 9 days and CHT of 14 days are validated for the 500 L mixing vessel train under the conditions described. Production scheduling must not exceed either time without triggering a deviation and re-cleaning or re-qualification.
Common inspection findings this protocol prevents
- A DHT validated at a short, convenient duration while real campaign manufacturing routinely exceeds it.
- CHT sampled only at time zero or under ideal conditions, never challenged at the real maximum storage duration.
- Hold times treated as numbers written into an SOP with no underlying study behind them.
How to adapt this protocol
- Set the proposed maximum times in section 3 from your actual operational data, not a default assumption.
- Add endotoxin sampling to sections 5 and 6 where the equipment supports sterile or injectable manufacturing.
- Pair this protocol with the MACO and acceptance limit worksheet so the DHT chemical acceptance criterion is the same recovery-corrected limit used in the parent protocol.