Mujahid, Noor; (2025) Characterisation of Membrane Filtration Processes in the Manufacture of Lentiviral Vectors for Cell and Gene Therapy. Doctoral thesis (Ph.D), UCL (University College London).

Text NM_Thesis_Final.pdf - Submitted Version Access restricted to UCL open access staff until 1 September 2027. Download (23MB)

Abstract

Lentiviral Vectors (LV) can be used in Cell and Gene Therapies (CGT) to deliver therapeutic genetic material to host cells. These therapies are moving from early development to wider use, creating a need for scalable and efficient manufacturing processes to address the challenges of high cost and low recovery. The production of lentiviral vectors involves two main stages: upstream (generation of the vector) and downstream (purification and production of a concentrated, high-quality functional vector in a stable and sterile form). Downstream processing commonly involves membrane processing, including normal flow filtration (NFF) for clarification and sterile filtration, as well as Tangential Flow Filtration (TFF) for vector concentration or formulation. This thesis explores how a better understanding of the environment and the interaction between LV and its process conditions can improve LV recovery (up to 3-fold) during clarification, concentration/diafiltration using TFF, and anion exchange chromatography, while also considering stability and robustness. To improve the yield during clarification, the role of membrane chemistry in reducing LV loss was investigated. One key innovation is the novel application of membrane surface zeta potential techniques to understand the impact of membrane ionic charges on the rate and extent of fouling of different LV feeds. Experiments revealed an association between membrane material, in-process conditions, and recovery. For instance, during the filtration of crude LV harvest, Polyethersulfone (PES) showed a significantly higher recovery rate than Nylon at two different load challenges. This correlated with Nylon’s positive surface charge compared to the negative LV crude feed, which could result in a higher rate of adsorption and interaction with the membrane surface, resulting in the loss of functional vector particles. The foulants responsible for the fouling were further investigated by visualising the membrane surfaces using Confocal (CLSM) and Scanning Electron Microscopy (SEM). In studies of TFF, the focus was on understanding how the order of processing steps, the mode of operation, and the properties of the membrane affect the yield, productivity, and quality of lentiviral vectors (LV). The results showed that the sequence of TFF unit operations is crucial for achieving high recovery, both in small-scale and large-scale systems (up to a 50-fold membrane area ratio). Data from TFF fouling studies indicated that filtering LV requires understanding the interplay between operating Transmembrane Pressure (TMP) and shear. The high-shear operation was found to improve LV recovery by promoting increased permeate flux and membrane cleaning up to a point. Ultra Scale-Down (USD) devices were used to further investigate the fouling behaviour and process limitations. After preparing a range of upstream conditions, they were used in additional TFF experiments to examine the effects of downstream recovery and buffer conditions during TFF and subsequent anion exchange chromatography on aggregation, stability, and recovery. The results of these studies show that selecting the appropriate upstream and downstream conditions can enhance LV recovery and product profile. To the best of my knowledge, this will be the first time that a whole bioprocess analysis of LV recovery has been studied from LV generation through plasmid transfection to anion-exchange chromatography (AEX) in the context of membrane processing and LV robustness.

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