Lentiviral Vector Manufacturing

BioInnovatise Viral Vector Team

Updated June 17, 2023

To deliver your genetic payload via viral vector to infect a host cell, you will require some form of viral vector production. Lentivirus is an excellent viral vector for gene delivery across therapeutic applications such as cell and gene therapy, immunology, oncology, regenerative medicine, and vaccine development.

Several terms are used interchangeably when discussing lentiviral vector production: lentivirus packaging and lentiviral manufacturing. Although both involve lentivirus, there are actually differences in these two phrases when it comes to the production process and production volumes.

Lentivirus Packaging

Lentivirus packaging refers to the production of lentiviral particles by transfecting producer cells with the necessary genetic components. The general process for lentivirus packaging is as follows:

  1. Plasmid Transfection: Producer cells (our lentivirus packaging productions utilize HEK293T cells for high transfection efficiency) are transfected with several plasmids.
    • Packaging Plasmid: Contains genes necessary for virus assembly (example: gag, pol). 2nd generation lentivirus packaging and 3rd generation lentivirus packaging have different amounts and types of packaging plasmids. Read more about lentivirus packaging plasmids and lentivirus packaging generations.
    • Envelope Plasmid: Encodes the viral envelope protein (example: VSV-G) that determines the tropism of the virus.
    • Transfer Plasmid: Contains the gene of interest flanked by long terminal repeats (LTRs) and other elements necessary for integration into the host genome.
  2. Production of Viral Particles: The transfected cells produce and release lentiviral particles containing the gene of interest into the culture medium.
  3. Harvesting: The lentiviral particles are collected from the culture medium.
Lentivirus Packaging Transfection Process Overview Lentivirus Packaging Protocol

The above diagram illustrates the general lentivirus packaging process at BioInnovatise and every production is completed according to the client’s specific lentivirus packaging protocol

Lentiviral Vector Manufacturing

The lentiviral vector manufacturing process encompasses the entire end-to-end process of producing lentiviral vectors at a larger scale, suitable for pre-clinical, clinical, or commercial use. This process includes:

  1. Upstream Processing:
    • Cell Culture Expansion: Growing a sufficient number of producer cells.
    • Transfection: Large-scale transfection of producer cells with the necessary plasmids, similar to the packaging step but on a much larger scale.
  2. Harvesting: Collecting the lentiviral particles from the culture medium.
  3. Downstream Processing:
    • Purification: Using techniques such as ultracentrifugation, chromatography, or filtration to purify the lentiviral particles from the culture medium and remove contaminants.
    • Concentration: Increasing the concentration of viral particles to the desired level for use.
  4. Quality Control: Testing the final product for potency, purity, sterility, and other quality attributes to ensure it meets regulatory standards.
  5. Formulation and Storage: Preparing the lentiviral vector for storage and distribution, often involving the addition of stabilizing agents and storage at appropriate temperatures.

3 Key Differences Between Lentivirus Packaging and Manufacturing

  • Scale: Lentivirus packaging is typically done on a small scale, suitable for research purposes, whereas lentivirus manufacturing is done on a larger scale, suitable for clinical or commercial use. Smaller scales have less purity requirements than larger scale productions that should be suitable for larger research models and humans. See more about production scales below.
  • Processes Involved: Lentivirus packaging focuses primarily on the initial production and harvesting of lentiviral particles. Lentiviral vector manufacturing includes additional steps such as large-scale production, purification, quality control, and formulation.
  • Regulatory Considerations: The lentiviral vector manufacturing process involves stringent regulatory compliance and quality control measures to ensure the safety and efficacy of the lentiviral vectors for therapeutic use.

Lentivirus Packaging and Lentiviral Vector Manufacturing Production Scales

Research Scale | Small Scale Production | 250 ul to 1 L

Typically involves producing lentiviral vectors in small volumes, such as milliliters to a few liters, for laboratory research purposes. This scale is often sufficient for in cell culture vitro studies and small animal experiments such as mice.

Preclinical Scale | Medium Scale Production | 5 L to 50 L

Involves producing lentiviral vectors in larger volumes, such as tens of liters, to support preclinical studies, including larger animal models such as beagles or non-human primates. This scale ensures enough material to evaluate safety, efficacy, and biodistribution before progressing to human trials.

Clinical Scale | Large Scale Production | 20 L – 500 L

Required for clinical trials and therapeutic applications. This involves producing lentiviral vectors in volumes ranging from tens to hundreds of liters. The manufacturing process must adhere to Current Good Manufacturing Practice (CGMP) standards to ensure product consistency, safety, and regulatory compliance for safe application in humans.

  • Phase I/II Clinical Trials – 20 L to 100 L
  • Phase III Clinical Trials and Commercial Production – 100 L to 500 L. Require large volumes to support widespread clinical use and eventual commercial distribution. This scale involves rigorous quality control and documentation to meet regulatory requirements for therapeutic products.
Commercial Scale | Very Large Scale Production | 500 L to 2000 L or More

For approved therapeutic products, lentiviral vector manufacturing must support consistent supply for ongoing clinical use. This involves the production of lentiviral vectors in very large volumes, potentially scaling up to thousands of liters. The process needs to be highly automated and optimized for efficiency and scalability. For ongoing clinical use and commercial distribution, volumes can range from hundreds to thousands of liters.

Determining the production scale that best fits your application is important to making sure your research timeline stays on track and does not take on excessive costs.

If you’re unsure which lentivirus packaging production volume is right for your application, contact our team

Learn about our quick turnaround lentivirus packaging services.

Want to learn more about the latest in lentivirus based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News continuously collect and publish the latest information on lentivirus-based research.

Real-time polymerase chain reaction validation equipment used for lentivirus titering RT-qPCR