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Lentiviral Packaging Plasmids Features and Benefits
Recombinant lentiviruses are derived from HIV-1, so certain precautions must be taken to ensure its safe use in gene delivery. The biggest concern is the possibility of generating replication-competent lentivirus through crossover events between elements in the viral vector and the packaging cell line. To improve safety, viral accessory genes are deleted and essential genes are supplied in trans by the packaging and envelope plasmids. To produce lentivirus particles, the transfer plasmid containing the gene of interest (GOI), the envelope plasmid, and the packaging plasmid are co-transfected into the packaging cell line (usually HEK293T cells). For our lentivirus packaging services, the lentivirus particles are purified using sucrose gradient ultracentrifugation to ensure safe use in vivo. Viral titer is then determined using RT-qPCR with primers to the long-inverted terminal repeats (LTRs) present in the viral genome. The viral particles are then shipped overnight and can be used immediately for in vitro or in vivo research.
- Immediate, reliable, and phase appropriate supply – Lentiviral packaging plasmids are available in three different grades: Research Grade (RG), High Quality (HQ) and GMP
- Fit-for-purpose, consistent quality – Reliable production process, material, and personnel controls aligned with custom plasmid orders
- Kanamycin antibiotic resistance
- Animal Component-free production
- Proven track record in LVV production
Plasmid DNA from Concept to Cure
Exploring the challenges and considerations throughout CGT development, start with the end in mind and download this resource to inform your critical starting materials strategy..
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Research Grade Plasmids
Our portfolio of off-the-shelf plasmid products consists of lentiviral Rev, Gag-Pol, and VSV-G in research grade (RG), high quality (HQ), and GMP grades.
| Quality Level | Product | Pack Size |
|---|---|---|
| Research Grade | RG-pLV-GAG-Kan | 2 mg 10 mg Custom Inquiry |
| RG-pLV-REV-Kan | ||
| RG-pLV-VSVG-Kan |
Pricing may be reflected as a custom request, based on current off-the-shelf stock availability and the amount of material requested.
These lentiviral vector plasmids are based on GMP master cell banks and are all subjected to a standard testing panel. In addition, pipeline products include antibiotic-free ORT plasmids and generic backbone plasmids for linearization to be used for mRNA templates.
| Quality Level | Product | Pack Size |
|---|---|---|
| High Quality | HQ-pLV-GAG-Kan | Please Inquire |
| HQ-pLV-REV-Kan | ||
| HQ-pLV-VSVG-Kan | ||
| GMP | GMP-pLV-GAG-Kan | Please Inquire |
| GMP-pLV-REV-Kan | ||
| GMP-pLV-VSVG-Kan |
Catalog number: RG/HQ/GMP pLV-GAG-Kan
Description: RG/HQ/GMP grade packaging plasmid for third generation lentiviral vector production
| Format | Plasmid DNA |
| E. coli Selection | Kanamycin |
| Length (bp) | 8138 |
Catalog number: RG/HQ/GMP pLV-REV-Kan
Description: RG/HQ/GMP grade packaging plasmid for third generation lentiviral vector production
| Format | Plasmid DNA |
| E. coli Selection | Kanamycin |
| Length (bp) | 4266 |
Catalog number: RG/HQ/GMP pLV-VSVG-Kan
Description: RG/HQ/GMP grade envelope plasmid for third generation lenti viral vector production
| Format | Plasmid DNA |
| E. coli Selection | Kanamycin |
| Length (bp) | 6456 |
Click here to view lentivirus packaging protocols.
Production Process & Release Testing
GMP plasmids are manufactured to the highest standards under fully compliant GMP conditions at our licensed plasmid DNA center of excellence. High Quality plasmids maintain full traceability on materials, aligned processing and testing, and are manufactured in segregated processing suites at our purpose-built HQ plasmid manufacturing facility. Research grade plasmids represent a cost-effective solution for early research and preclinical requirements while still maintaining appropriate levels of documentation and quality control to provide confidence and ease of transition to higher quality standards when required for later stage clinic and commercial vector manufacture.
*Sterility and Mycoplasma testing are not performed for R&D grade plasmid
| Assay | Method | R&D / HQ acceptance Criteria | GMP acceptance Criteria |
|---|---|---|---|
| Identity | Sequencing | Confirm full plasmid sequence | Confirm full plasmid sequence |
| Quantity – nucleic acid concentration | UV A260 | 2.0±0.2mg/mL | 2.0±0.2mg/mL |
| Identity by restriction digest | Restriction digest | Plasmid Identity confirmed | Plasmid Identity confirmed |
| Total plasmid purity | Gel electrophoresis | ≥95% | ≥95% |
| Supercoiled plasmid | Capillary electrophoresis | ≥85% | ≥85% |
| Residual host cell RNA | RP-HPLC | ≤5% | ≤1% |
| Residual genomic DNA | qPCR | ≤5% | ≤2% |
| Residual host cell protein | ELISA | ≤2% | ≤1% |
| Residual Kanamycin | Colorimetric analysis | ≤5 ppm | ≤5 ppm |
| Bioburden | USP <61>, Ph. Eur. 2.6.12, 2.6.13 | ≤1 cfu/10mL | ≤1 cfu/10mL |
| Sterility* | USP <1071> | No growth | No growth |
| Mycoplasma* | EP/USP | Negative | Negative |
| Endotoxin | USP <85>, Ph. Eur. 2.6.14 | ≤10 EU/mg | ≤10 EU/mg |
| Appearance | Visual inspection | Clear, colorless solution | Clear, colorless solution |
| pH | USP <791>, Ph. Eur. 2.2.3 | 8.0 ± 0.5 | 8.0 ± 0.5 |
| 260/280 ratio | UV spectrophotometry | 1.8-2.0 | 1.8-2.0 |
Need Custom Plasmid Production?
With plasmid DNA also manufactured on request, if you do not see the AAV plasmid product you require, our CDMO team offers a comprehensive range of solutions for applications ranging from preclinical research studies to clinical trials and commercial scale.
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Lentiviral vector packaging plasmid maps
Charles River Laboratories lentiviral packaging plasmids have been developed to deliver reliable vector production with high infectious titer compared to other commercially available products.
Figure 1. Infectious titer data for LVV production in HEK293T cells using Charles River’s packaging plasmids compared to commercially available packaging plasmids. Vector material was purified and concentrated to equivalent volumes before infectious titers were determined.
Frequently Asked Questions (FAQs) About Off-the-Shelf Lentiviral Vector Packaging Plasmids
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What is the difference between second generation and third generation lentivirus packaging systems?
Third generation lentivirus packaging systems were developed to further segregate viral packaging components. Moving to a four-plasmid system, vs. three-plasmids used in second generation systems, supports reduced likelihood of generating replication-competent lentivirus. Third generation systems are therefore considered safer than second generation and established protocols are now in place to address potential yield impacts that were historically observed vs. second generation approaches.
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Do packaging plasmids affect titer levels?
The short answer is yes. However, packaging plasmid design is one part of a complex range of factors that can influence transfection and subsequent viral vector titers. Plasmids used for production of AAV (Helper and rep/cap plasmids) and LVV (envelope and packaging plasmids) are relatively well established, with proven track records in clinic. However, depending on the cell line, transfection method, specific gene of interest used in the transfer plasmid, and the onward manufacturing and purification processes, optimization is advised to deliver the highest possible yields while maintaining product quality. Reach out to our expert team to discuss your requirements.