This is a ready-to-use calculation worksheet for the endotoxin log-reduction step of a depyrogenation qualification or requalification. It turns the raw assay values into a defensible reduction figure and the pass/fail decision. Replace every <<FILL: ...>> placeholder, attach it to the parent protocol, and have a second person verify the arithmetic. A filled specimen follows. This is educational reference content, not regulatory advice.
Header
| Field | Entry |
|---|---|
| Worksheet number | <<FILL: FORM-ID>> |
| Parent protocol / report | <<FILL: PQ number>> |
| Equipment / cycle | <<FILL>> |
| EI lot and certified potency | <<FILL>> |
| Assay method and compendial chapter | <<FILL: e.g. kinetic chromogenic LAL per USP <85>, or recombinant Factor C per USP <86>>> |
| Assay detection limit used | <<FILL: e.g. 0.05 EU/mL>> |
| Recovery volume per indicator | <<FILL: e.g. 10 mL>> |
| Date of assay | <<FILL>> |
The rule (read before filling)
- t=0 is the measured positive control, not the label claim. Positive controls are unprocessed EIs from the same lot, extracted and assayed the same day by the same method. Their mean recovered EU is the starting value.
- Log reduction = log10(mean positive control EU) minus log10(residual EU on the processed indicator).
- If a processed indicator reads below the detection limit, bound the residual at the detection limit converted to EU per indicator (detection limit in EU/mL multiplied by the recovery volume) and report a “greater than or equal to” reduction.
- Acceptance: every worst-case position, every cycle, at least 3.0 log10.
Step 1: Positive control recovery (t=0)
| PC replicate | Recovered EU per indicator |
|---|---|
| PC-1 | <<FILL>> |
| PC-2 | <<FILL>> |
| PC-3 | <<FILL>> |
| Mean PC EU (t=0) | <<FILL>> |
| log10(mean PC EU) | <<FILL>> |
| Check: mean PC EU > 1000? | Yes / No (if No, challenge inadequate, stop) |
Step 2: Residual on processed indicators and reduction
For each processed EI, enter the assayed residual (or ”< DL” and the bounded value). Reduction = log10(mean PC EU) minus log10(residual EU).
| Position | Cycle | Processed EI residual (EU) | Bounded value if < DL | log10(residual) | Log reduction | >= 3.0? |
|---|---|---|---|---|---|---|
<<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> | Yes / No |
Bounded value if below detection limit = <<FILL: DL in EU/mL>> x <<FILL: recovery volume mL>> = <<FILL: EU>> per indicator.
Step 3: Decision
| Item | Result |
|---|---|
| All positions and cycles >= 3.0 log10 | Yes / No |
| Any position below 3.0 (list) | <<FILL: none, or detail>> |
| Deviation raised (if any failure) | <<FILL: number or N/A>> |
| Overall | Pass / Fail |
Sign-off
| Role | Name | Signature | Date |
|---|---|---|---|
| Calculated by | <<FILL>> | ||
| Verified by (second person) | <<FILL>> | ||
| QA reviewed | <<FILL>> |
Filled specimen
Example for tunnel DPT-02, EI lot certified 6,800 EU, kinetic chromogenic assay, detection limit 0.05 EU/mL, recovery volume 10 mL (so the below-DL bound is 0.05 x 10 = 0.5 EU per indicator).
Step 1 positive controls: PC-1 6,200; PC-2 6,400; PC-3 6,300. Mean = 6,300 EU. log10(6300) = 3.80. Mean above 1000, challenge adequate.
Step 2:
| Position | Cycle | Residual (EU) | Bounded | log10(residual) | Log reduction | >= 3.0? |
|---|---|---|---|---|---|---|
| P1 left edge | 1 | < DL | 0.5 | -0.30 | 4.10 | Yes |
| P2 right edge | 1 | < DL | 0.5 | -0.30 | 4.10 | Yes |
| P3 leading | 1 | 1.2 | n/a | 0.08 | 3.72 | Yes |
Step 3: All positions and cycles at or above 3.0 log10. No deviation. Overall: Pass. Calculated by A. Patel, verified by S. Rao, QA reviewed by R. Gomez.
Note how P3, the only position with a measurable residual, still cleared 3 logs because the recovered t=0 was comfortably above 1000 EU. Had the positive controls recovered only 600 EU (log10 = 2.78), no position could have demonstrated 3 logs even with a perfect cycle, which is why Step 1 gates the whole worksheet.
Common inspection findings this worksheet prevents
- The nominal inoculation figure used as t=0 instead of the measured positive control.
- A below-detection result recorded as “zero residual,” overstating the reduction.
- Positive controls assayed on a different day or by a different method than the processed indicators.
- No second-person verification of the arithmetic behind a release-relevant number.
How to adapt this worksheet
- Set your assay detection limit and recovery volume in the header; they drive the bounding value.
- Add rows for every position and cycle in your study.
- Keep this worksheet with the raw assay records and the parent protocol.
- Require an independent verifier signature before the result feeds a disposition.