Table 1.
Quality-control assays for clinical-grade retroviral and lentiviral vectors
| Testing | Example assays | Criteria | |
|---|---|---|---|
| Purity | Lentiviral vector | ||
| Total proteins (ng mL−1) | ELISA | Report results | |
| Bovine serum albumin (ng mL−1) | ELISA | Report results | |
| Benzonase (ng mL−1) | ELISA | Report results or <100 ng mL−1 | |
| Plasmid DNA (copies per 100 ng) | VSVg qPCR optional or serial washes | Below detection or decrease over time | |
| Host cell specific DNA (copies per 100 ng) | qPCR | Report results | |
| SV40 LTA and E1A qPCR | qPCR | Below detection | |
| Safety | Retroviral and lentiviral vector | ||
| Sterility | USP, No growth for 14 days culture on Vero indicator, PTC | No growth for 14 days | |
| Mycoplasma | Negative | ||
| Endotoxin/pyrogen | LAL | <10 EU mL−1 | |
| In vitro Adventitious agents | Assay on MRC5, Vero and A549 cells | Negative | |
| Retroviral vector | |||
| RCR | Marker-rescue cell culture assay | No RCR detected | |
| General safety (first lot) | Current USP | Absence of adverse agents | |
| Lentiviral vector | |||
| RCL | Co-culture on C8166 cells with amplification and indicator phases | No RCL detected | |
| Potency | Retroviral and lentiviral vector | ||
| Infectious viral particles | Gene transfer/expression assay in cell line of choice | Report results | |
| Retroviral vector | |||
| Total viral particles | EM | Only type C retroviral like particles detected | |
| Lentiviral vector | |||
| Total viral particles | p24 ELISA, qRT-PCR | Report value | |
| Others | Retroviral and lentiviral vector | ||
| Physicochemical characteristics | pH (optional) | 6.9–7.8 | |
| Appearance | Opaque |
Abbreviations: ELISA, enzyme-linked immunosorbent assay; LAL, limulus amebocyte lysate; qPCR, quantitative PCR; qRT-PCR, quantitative reverse transcription PCR; RCL, replication competent lentivirus; RCR, replication competent retrovirus; USP, U.S. pharmacopeial convention.