art rendering of lentiviral vector gene therapy

Lentiviral vector manufacturing solutions for cell and gene therapy applications

The increased interest in cell and gene therapy has led to the need for more cost-effective and scalable lentiviral vector (LV vector) manufacturing platforms. Lentiviral vector gene therapy is now widely used because of its use in advanced clinical trials and regulatory approved therapies in combination with technological improvements in characterization and safety profiling.

As a world leader in serving science, the innovative solutions from Thermo Fisher Scientific are designed to accelerate the development of gene-modified cell therapies and gene therapies. With solutions that span the entire lentivirus workflow, we offer superior products, services, and expertise to help companies develop breakthrough lentivirus gene therapies.

Request information

Thermo Fisher Scientific offers solutions and expertise for every phase of your LV workflow

Our scientists have experience in developing systems that work well together. Explore how we provide solutions no matter what stage or scale your lentivirus gene therapy.

Lentivirus production workflow

Lentivirus production workflow. While there are many ways to produce Lentiviral vectors and no “standard” production process exists, a generic RUO production workflow might look like this. Complete CDMO support from LV cell line development to cGMP vector manufacturing. We offer CDMO support from LV cell line development to cGMP vector manufacturing.

Upstream production

Central to lentiviral expression is large-scale plasmid production and lentiviral purification, followed by an upstream process of cell culture and transfection or infection, respectively, depending on whether mammalian or insect cell lines are used. Either adherent or suspension cell lines can be used such as HEK293. Plasmid triple transfection is used for HEK293 cells. An alternative option for custom lentivirus production is the development of a stable producer cell line.

Plasmid development and production

The Invitrogen GeneArt Gene Synthesis service offers chemical synthesis, cloning, and sequence verification of virtually any desired genetic sequence. You will receive a bacterial stab and/or purified plasmid containing your synthesized gene, ready for downstream applications. In addition, subcloning and lentiviral plasmid services are offered to subclone your sequence into any vector.

Large amounts of plasmid DNA are needed to transfect the host cells. Before lentiviral transfection can take place, the plasmid DNA must be harvested and purified from the fermentation culture. For clinical use, multiple quality control tests are conducted to make sure the plasmid DNA is free from any process- or product-related impurities. We supply upstream additives including mammalian, animal and animal origin-free peptones.

From lentiviral expression to large scale production and purification, we’ve got you covered.

Cell expansion

To begin custom lentivirus production, a suitable mammalian or insect cell line is selected and expanded. HEK293 cells are the predominant mammalian platform for viral vector production chosen for their ability to grow at high cell density and can be suspension-adapted.

LV-MAX transfection reagent—a high-efficiency, cationic, lipid-based transfection reagent uniquely designed for co-transfecting multiple plasmids into high-density viral production cell cultures with high transfection efficiency and low toxicity.

Once the mammalian host cells have been transfected with the appropriate lentiviral plasmids, they are moved into shaker flasks, rocker systems, a cell factory or bioreactor—depending on production scale—for viral production. We supply cell culture media, and serum to reduce time to market.

Automation

In addition to standard cell lines, Thermo Fisher Scientific also offers custom cell line engineering services in our Brammer Bio CDMO service group. 

Plasmid transfection and viral vector production

In this phase of the lentivirus vector production workflow, multiple plasmids are used to introduce the gene of interest and the lentiviral vector coding genes into the host cell. The transfected host cells then generate lentiviral vectors.

LV-MAX transfection reagent—a high-efficiency, cationic, lipid-based transfection reagent uniquely designed for co-transfecting multiple plasmids into high-density Viral Production Cell cultures with high transfection efficiency and low toxicity.

The CTS LV-MAX Transfection Kit is an essential component of the CTS LV-MAX lentiviral production system. Based on extensive Design of Experiment (DOE) methodologies, the cationic lipid-based transfection reagent, lentiviral production supplement, and LV enhancer are specifically screened for maximal delivery of multiple DNA plasmids to achieve the highest possible lentiviral titer when used in conjunction with CTS Viral Production Cells cultured in CTS LV-MAX Production Medium.

Lipofectamine 2000 Transfection Reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results in a broad spectrum of hard-to-transfect cell lines and lentiviral vector gene therapy.

We can subclone and package your synthetic gene into any vector you send us. Get your gene ready to use in your downstream applications with our GeneArt Subcloning & Express Cloning Service.

In addition, we also offer the following transfection reagents:

Downstream processing

The second half of the lentivirus production workflow requires the clarification and purification of the viral particles, and the final fill and finish of the clinical product. Downstream processing can be a laborious and costly process, so effective methods of generating high purity and high yield of lentivirus particles is important.

Purification

We aim to deliver products that are a platform solution for your commercially viable custom lentivirus production. To scale your research, we have developed chromatography resins for lentiviral purification and created the mixers, buffers, and single-use solutions for robust vector production.

Fill and finish

From equipment to VVS, we offer the fill and finish services to get your lentivirus gene therapy from research to commercialization. Our cGMP-grade equipment is prepared for comprehensively filling and finishing large-scale lentivirus expression.

Learn more about our sterile bioprocessing consumables and equipment
Learn more about Nalgene bottles and carboys

Viral production analytical tests

Analytics are employed after each stage of the lentiviral vector production workflow and before final lot release. During the analytics steps, the gene therapy product/process is characterized and analyzed to ensure product quality and safety. Critical Quality Attributes (CQAs) and product specifications are tested as well as specific assays for sterility, identity, purity and potency.

Per regulatory requirements, cell-culture based therapies must be free of mycoplasma. Manufacturers have traditionally outsourced testing to labs that specialize in the 28-day culture-based test method. For manufacturers of gene and cell therapy products, as well as other low-dose and short-shelf life therapeutics, it is not feasible to wait 28 days for test results. Real-time PCR-based assays provide a viable alternative to the culture-based method and provide results in hours while meeting the required sensitivity.

Quantification of residual host cell DNA from the producing cell line is part of the purity tests for gene therapy products. Regulatory agencies provide guidance on the maximum acceptable level of DNA per dose of vector and plasmid DNA. Therefore, rapid and accurate quantification is crucial to manufacturers.

The nature of gene therapy products and other low-dose, short-shelf life therapies are not suitable for testing that requires a long time to receive results and uses large sample volumes. Real-time PCR based assays provide accurate, actionable results in hours, while meeting sensitivity requirements.

Learn more about our mycoplasma end-to-end real-time PCR solution that provides timely results that you can trust and have been proven to meet regulatory requirements.

Learn more about our integrated residual host cell DNA quantitation system that provides high-quality, reproducible results that meet regulatory requirements.

Learn more about real-time PCR assays

Viral vector contract development and manufacturing

In May of 2019, Thermo Fisher Scientific acquired Brammer Bio, a leading viral vector CDMO for companies developing gene-modified cell therapies and in vivo gene therapies. As part of Thermo Fisher Scientific, Brammer Bio will continue to provide process and analytical development, clinical and commercial supply of viral vector drug substance and drug product, as well as regulatory support, enabling our clients to bring novel medicines to market. Our team is flexible to support every client’s unique product needs, timelines and regulatory pathways. Through recent investments in our world-class facilities and planned expansion, Thermo Fisher Scientific’s Viral Vector CDMO Services are strategically positioned to support your immediate and long-term viral vector manufacturing needs.

In addition to product release, stability testing, process and analytical qualification, we provide global quality compliance and regulatory support.

Get more information about our CDMO services 

 

Resources

Support

Intended use of the products mentioned on this page vary. For specific intended use statements please refer to the product label.