Development of Large-Scale Downstream Processing for Lentiviral Vectors

Abstract

The interest in lentiviral vectors (LVs) has increased prominently for gene therapy applications, but few have reached the later stages of clinical trials. The main challenge has remained in scaling up the manufacturing process for the fragile vector to obtain high titers for in vivo usage. We have previously scaled up the LV production to iCELLis 500, being able to produce up to 180 L of harvest material in one run with perfusion. The following challenge considers the purification and concentration of the product to meet titer and purity requirements for clinical use. We have developed a downstream process, beginning with clarification, buffer exchange, and concentration, by tangential flow filtration. This is followed by a purification step using single membrane-based anion exchange chromatography and final formulation with tangential flow filtration. Different materials and conditions were compared to optimize the process, especially for the chromatography step that has been the bottleneck in lentiviral vector purification scale-up. The final infectious titer of the lentiviral vector product manufactured using the optimized scale-up process was determined to be 1.97 Â 10 9 transducing units (TU)/mL, which can be considered as a high titer for lentiviral vectors.

Advances in Lentivirus Purification

“…recently reported functional recoveries close to 100% using a depth filter for clarification of large supernatant volumes containing VSV‐G pseudotyped LV. [ 21 ] However, the success of this operation can be highly dependent on the pseudotype. Using a GaLV‐TR pseudotyped LV, Boudeffa et al.…”

“…Many times, nuclease step immediately precedes [ 21,68 ] or succeeds clarification. [ 24,69 ] In other processes, it is included in the USP [ 26,55 ] or later DSP phases after some volume reduction.…”

“…[ 56,99 ] Efforts in standardization have been made resulting in a standard for LV analytics [ 100 ] and an LV reference material that will be released soon. [ 21,25 ] Each lot must be tested for identity , purity , potency , and safety . [ 1,4 ] Some examples of assays used for LV release are depicted in Table 3 .…”

“…The success of these clinical trials requires improved methods for purification of LV both quantitively, increasing process capacity, and qualitatively replacing traditional methods used at small scales that are not economically and/or technically viable for larger scales. [ 20,21 ]…”

Lentiviral Vector Bioprocessing

“…(2) These are examples of sequences of operations used in pre-clinical and clinical investigations [145,268,269]. (3) Benzonase may be added at a variety of steps within the downstream process.…”