This is a ready-to-use calculation worksheet for viral clearance data generated under a spiking study protocol. It is a calculation aid, not a validated tool; if you build it into a spreadsheet used for GxP decisions, validate that spreadsheet per your infrastructure qualification program. Replace every <<FILL: ...>> placeholder with your own values. A worked filled specimen follows. Confirm every input and threshold against your approved study protocol.
Section 1: per-step LRV calculation
For each spiked step, LRV is calculated from total virus (titer multiplied by volume), not titer alone.
| Field | Load (pre-step) | Product pool (post-step) |
|---|---|---|
| Infectious titer (log10 TCID50/mL or PFU/mL) | <<FILL>> | <<FILL>> |
| Volume (mL) | <<FILL>> | <<FILL>> |
| Total virus (log10 titer + log10 volume) | <<FILL: A>> | <<FILL: B>> |
| LRV = A minus B | <<FILL>> |
If the post-step result is below the assay’s limit of detection, report the LRV as a “greater than” value bounded by the limit of detection and the sample volume assayed, not as an absolute number: <<FILL: e.g. LRV greater than 5.3, limited by 10 mL assayed at LOD 1.5 log10>>.
Section 2: inactivation kinetics (for time-dependent steps, e.g. low-pH hold)
| Time point (min) | Infectious titer (log10) | LRV from t=0 |
|---|---|---|
| 0 | <<FILL>> | 0.0 |
<<FILL>> | <<FILL>> | <<FILL>> |
<<FILL>> | <<FILL>> | <<FILL>> |
| Process hold time | <<FILL>> | <<FILL>> |
Confirm the kill curve reaches a plateau (essentially complete inactivation) before the process hold time. A single endpoint with no intermediate time points does not demonstrate kinetics.
Section 3: orthogonality check before summing
| Step | Mechanism | Orthogonal to which other step(s)? | Include in cumulative sum? |
|---|---|---|---|
<<FILL>> | <<FILL: inactivation / size-exclusion removal / charge-based removal>> | <<FILL>> | <<FILL: Yes/No + rationale>> |
<<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> |
<<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> |
Only steps with genuinely different clearance mechanisms may be summed independently. Two steps sharing the same mechanism are not both counted at full value without documented justification.
Section 4: cumulative process clearance per virus
| Virus | Step 1 LRV | Step 2 LRV | Step 3 LRV | Cumulative LRV |
|---|---|---|---|---|
<<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL: sum>> |
<<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> | <<FILL>> |
Section 5: safety margin statement
| Input | Value |
|---|---|
| Estimated viral burden of unprocessed bulk (e.g., retrovirus-like particles by TEM, particles/L or particles/dose) | <<FILL>> |
| Cumulative process LRV for the relevant virus | <<FILL>> |
| Estimated particles remaining after clearance (burden minus 10^LRV) | <<FILL>> |
| Safety margin statement | <<FILL: e.g. "cumulative clearance exceeds the estimated load by more than N log10">> |
Section 6: sign-off
| Role | Name | Signature | Date |
|---|---|---|---|
| Calculated by | <<FILL>> | ||
| Reviewed by (Validation) | <<FILL>> | ||
| Approved by (QA) | <<FILL>> |
Filled specimen
Illustrative low-pH inactivation step for X-MuLV. Numbers are illustrative.
Section 1:
| Field | Load | Product pool |
|---|---|---|
| Infectious titer (log10 TCID50/mL) | 6.8 | less than 1.5 (below detection) |
| Volume (mL) | 500 | 480 |
| Total virus (log10) | 9.5 | less than 4.2 |
| LRV | greater than 5.3 |
Section 2 (kinetics):
| Time (min) | Titer (log10) | LRV from t=0 |
|---|---|---|
| 0 | 6.8 | 0.0 |
| 15 | 3.1 | 3.7 |
| 30 | less than 1.5 | greater than 5.3 |
| 60 (process hold) | less than 1.5 | greater than 5.3 |
Plateau reached by 30 minutes, well before the 60-minute process hold; kinetics support the claim.
Section 4 (cumulative, illustrative):
| Virus | Low-pH LRV | AEX LRV | Filtration LRV | Cumulative |
|---|---|---|---|---|
| X-MuLV | greater than 5.3 | greater than 4.5 | greater than 4.2 | greater than 14.0 |
Section 5: cumulative clearance of X-MuLV (greater than 14 log10) exceeds the estimated retrovirus-like particle burden of the unprocessed bulk by a wide margin, consistent with ICH Q5A(R2) expectations for the relevant virus in a rodent cell line.
Common inspection findings this worksheet prevents
- LRV computed from titer alone, ignoring volume, which silently inflates or deflates the true total-virus reduction.
- A below-detection result presented as an exact LRV number instead of a bounded “greater than” value.
- Non-orthogonal steps summed without a documented mechanism justification.
- A safety margin asserted without stating the estimated viral burden it is being compared against.
How to adapt this worksheet
- Populate Section 1 for every studied step from the contract lab’s raw data report.
- Complete Section 2 for any inactivation (time-dependent) step; skip it for pure removal steps.
- Work through the orthogonality check in Section 3 before summing anything in Section 4.
- State the safety margin in Section 5 against your own estimated viral burden data (e.g., TEM particle counts on unprocessed bulk).
- If this worksheet becomes a standing spreadsheet tool, validate it and control its formulas per change control.