In previous posts on lentiviral vector (LVV) manufacturing, we provided a general overview of the process with a focus on the scale-up of upstream and downstream processing. Here, we introduce the concept of analytics for LVV manufacture with a discussion of the basic assays used to determine the quantity of virus that is being produced.
Quality Control Assays for LVV Manufacture
Viral titre measures the amount of virus in a given volume and is a critical quality attribute of LVV production. The two primary testing methods used in a manufacturing setting are infectious titre and physical tire:
- Infectious Titre: a functional assay to determine the infectivity of viral particles. This assay is performed by transducing HEK293T cells with serially diluted virus. Infectivity of the virus is measured by quantifying the number of HEK293T target cells that have the viral genes incorporated into their genome. During process development activities, a fluorescent proxy (GFP or any other fluorescent reporter) for the gene-of-interest (GOI) may be used to facilitate timelines. However, when transitioning to GMP manufacturing, the GOI must be specifically measured. This may be performed by counting cells that are positive for GOI-specific antibodies using flow cytometry or droplet digital polymerase chain reaction (ddPCR) to quantify the amount of GOI integrated into the cells. The results are reported in transducing units (TU) per mL.
- Physical titre: an assay to quantify the number of viral particles in a given volume performed by measuring the amount of p24 protein present in a test sample, using an ELISA kit. This test does not provide any information about the functional properties of the virus and tends to overestimate the amount of viral titre because the assay measures the total amount of p24 protein in the medium and does not differentiate between infective and non-infective viral particles.
Measurement of process residuals:
- Host Cell Protein: downstream processing of LVV requires measuring host cell protein content to determine the purity of the LVV sample. Host cell proteins are process residuals and need to be removed through downstream processing. The quantity of host cell proteins is also an indication that the cell was expending its energy in the production of host cell proteins rather than lentiviral proteins. Host cell protein content can be measured using commercially available ELISA kits.
- Host Cell DNA: similar to host cell protein, host cell DNA is another process residual and it is important to measure its content and remove it during downstream processing. Host cell DNA content is also measured to determine viral production efficiency. Host cell DNA content can be measured using commercially available kits.
These assays are best used in combination to accurately assess viral titre. Assessment of viral titre is an important quality check during upstream manufacturing, which is carried out by testing small samples of media from virus-producing cultures. At CCRM, we have performed several optimization studies to adapt the infectious and physical titre assays to be used with LVV produced from suspension cultures. These improvements will ensure that these assays, originally developed for adherent cells, can be seamlessly integrated into large-scale, suspension culture-based manufacturing protocols.
These assays can also play a role during process development where they are used to compare the ability of different cell lines to produce virus. Our team has used this approach to compare virus production efficiency in different stable producer lines and transient cell lines.
Improving Assays for LVV Production
Using existing assays for viral titre is a relatively established and straight-forward step during upstream and downstream processing. However, when working with a contract development and manufacturing organization (CDMO), bottlenecks can occur during technology transfer that may require a different approach to assessing infectious titre.
Future analytics development could be aimed at automating assays for viral titre using an automated liquid handling robotic platform to help speed up processing of potentially hundreds of samples required for the evaluation of multiple replicates. Working with an experienced process development team can help you to improve your LVV manufacturing process by incorporating quality control assays that ensure important critical quality attributes for your LVV product.
Contact us to learn how our team can help you improve your LVV manufacturing process.