Lentiviral Vector Bioprocessing

Abstract

Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.

Keywords: Lentiviral vectors; bioprocessing; cell and gene therapy; lentivirus; manufacturing; pseudotyping.

Figure 1

Illustrative examples of chromatography resins in use for lentiviral vector purification with increasing convective mass transfer from left to right.

Figure 2

Bioprocess options in the production of lentiviral vectors. ${}^{\left(1\right)}$ Some studies have shown sequence of membrane filtration of different pore sizes or inclusion of low-speed centrifugation prior [145,183,189]. Sequence of filtration processes would be an option depending on scale and cell density and product and impurity profile. ${}^{\left(2\right)}$ These are examples of sequences of operations used in pre-clinical and clinical investigations [145,268,269]. ${}^{\left(3\right)}$ Benzonase may be added at a variety of steps within the downstream process.