0 item(s)
View cart
You have no items in your shopping cart.
27
Oct
A cell bank qualification lab recently asked us a practical question: the PERT protocol calls for ultracentrifugation, but they don't have an ultracentrifuge. Can they still test their Master and Working Cell Banks?
It's a common situation, so we've summarized our answer here: what the options are, what you trade off, and what to validate before you rely on the data.
Article Table of Contents
Why PERT for cell bank testing
Production cell lines such as CHO, HEK293, Vero and hybridomas can carry endogenous or adventitious retroviruses. ICH Q5A expects cell banks to be characterized for them. Sequence-specific PCR only finds viruses you already know to look for. PERT (Product-Enhanced Reverse Transcriptase) measures the reverse transcriptase enzyme that every retrovirus carries, so it picks up retroviral particles regardless of their sequence.
In a one-step real-time PERT assay, any RT in the sample copies a supplied RNA template into cDNA, which is then amplified and detected by qPCR in the same tube. A dilution series of a known RT (MMLV) gives a standard curve, so results come out as RT activity, not just positive or negative.
The standard method: concentrated supernatant
Retroviral particles in culture supernatant are usually too dilute to test directly. The reference workflow concentrates them first:
- Centrifuge the supernatant at 11,000 × g for 10 minutes to remove cells and debris.
- Filter through a 0.45 µm membrane.
- Ultracentrifuge at 100,000 × g for 60 minutes to pellet the virus.
- Resuspend the pellet in PERT Sample Diluent, keep on ice, and test 1–5 µL per reaction.
That ultracentrifugation step is what makes low viral loads detectable. It is also the step many smaller QC labs can't run in-house.
No ultracentrifuge? Your options
| Approach | How it works | Our view |
|---|---|---|
| Cell lysate | Lyse 1 × 10⁶ cells in PERT Sample Diluent and test 0.1–1 µg total protein | Recommended alternative. Described in the kit protocol; no special equipment. |
| Ultrafiltration | Concentrate supernatant in 100 kDa MWCO centrifugal filters; test the retentate | Widely used in the field, but not validated by Biofargo. Recovery must be confirmed in your matrix. |
| Unconcentrated supernatant | Dilute raw supernatant into PERT Sample Diluent and test directly | Not recommended. Viral levels may fall below the detection limit, and only a small volume can be processed. |
Step by step: the cell lysate method
- Harvest 1 × 10⁶ cells from the MCB or WCB vial or culture.
- Pellet the cells using your lab's standard gentle conditions for that cell line and remove the medium completely.
- Resuspend the pellet in 200 µL PERT Sample Diluent.
- Incubate on ice for 20 minutes to lyse.
- Measure total protein with any total protein assay.
- Add 0.1–1 µg total protein per 20 µL PERT reaction, alongside the MMLV RT standard curve, an NTC and an NSC.
Whichever preparation you use, every sample must be taken up in PERT Sample Diluent before it goes into the RT-qPCR step.
What you give up in sensitivity
Dropping ultracentrifugation typically costs about 2–3 Ct cycles, roughly a 4- to 8-fold loss in signal. For a cell bank with a high retroviral load that may not matter. For a low-level contaminant, it can be the difference between a weak positive and a false negative.
The assay itself is very sensitive (several dozen RT molecules, with at least six logs of linear range), so the limiting factor is almost always how much virus reaches the tube, not the chemistry.
What to validate for MCB/WCB work
The Biofargo PERT kit is Research Use Only and is not a GMP/GLP-grade kit. If the data will support an IND or BLA, the method needs your own analytical validation. At a minimum:
- Recovery: spike a known amount of RT or a model retrovirus and measure how much your preparation method recovers.
- Matrix interference: confirm your culture medium and lysate do not inhibit RT activity or PCR.
- Controls in every run: NTC, NSC and the MMLV RT standard curve. A run is invalid if NTC or NSC amplifies with Ct < 35, or if the standards fail.
- Side-by-side comparison: where possible, compare your alternative preparation with ultracentrifuged samples (an outside lab can run these) to document the sensitivity difference.
- Limit of detection: establish LoD for your chosen preparation; it has not been defined for the cell lysate method.
FAQ
Can I skip ultracentrifugation and test supernatant directly?
We don't recommend it. Use the cell lysate method or ultrafiltration instead.
How many cells do I need for the lysate method?
1 × 10⁶ cells lysed in 200 µL PERT Sample Diluent gives enough material for testing at 0.1–1 µg total protein per reaction.
How much sample goes into each reaction?
1–5 µL of concentrated supernatant or retentate, or 0.1–1 µg total protein of lysate, in a 20 µL reaction.
Will it run on my qPCR instrument?
Any real-time PCR system with a FAM channel works, including the 7500 Fast.
Shop the Products in This Article
|
Reverse Transcriptase Assay (PERT), One-Step Real-Time qPCR Kit Cat. No. 221044 · 50 reactions |
Shop Now |
Explore the PERT Reverse Transcriptase Assay →
Planning PERT testing for your cell banks? Email contact@biofargo.com and our team will help you choose a sample preparation approach.
