Aspects of process development for virus vector production to improve quality and quantity The use of viral vectors as gene delivery and vaccine vehicles has developed rapidly during the last two decades owing to several viral properties. Viruses can infect cells efficiently, often have a broad tissue tropism and can achieve very high levels of either stable or transient transgene expression. Furthermore, their intrinsic immune-stimulatory properties can have adjuvant effects during the treatment of cancer or infectious disease and, importantly for manufacturing scale-up, some viruses can be grown to very high titre (Ͼ1012 particles/mL). The development of robust production procedures is essential to move therapeutics that utilize viral vectors into clinical trials, and to make them cost effective for market supply. Here, we describe some of the aspects of production that must be considered and optimized when producing virus vectors on an intermediate or large scale. By drawing examples from our experience of vector production, we show that upstream and downstream processes must be designed hand-in-hand to maximize the quantity and quality of any virus product. iral vectors have increasingly become the delivery that can circumvent these issues. In addition, for Matthew Reece-Ford Vmechanism of choice for gene-based therapeutics viruses that are to be used as gene delivery systems, Anthony G. Hitchcock because of their superior efficiency/toxicity profile. the ratio of total virus particles to infectious virus Kai S. Lipinski Adenoviral and retroviral vectors are the most particles (P:I ratio) needs to be considered and commonly used; approximately 1000 trials have been minimized, as this determines the therapeutic window performed worldwide and about half of these make between beneficial effect and toxicity and, therefore, use of these two vector types.1 Developing optimized the dose of product that can be delivered to a patient. large-scale production, harvest and purification Figure 1 provides an overview of upstream and strategies is vital if regulatory requirements regarding downstream factors that together determine the the purity and traceability of the product and the quality and quantity of the virus production process. quality control of equipment and facilities are to be met, and to move these innovative medicines through Upstream production strategies clinical evaluation to market approval.2,3 Downstream The upstream production process for generating viral and upstream processes need to optimize the quality vectors involves infecting cells with a virus, culturing (mainly purity and biological activity) and quantity of the cells to allow the virus to replicate and then the virus produced, and also be designed to minimize harvesting the virus from the cells. Each of these production costs.4,5 stages must be carefully considered when designing a Virus preparation methods on a small scale regularly cGMP production process to ensure that the maximum include procedures that are difficult to scale-up, are yields and quality of viral vector are generated. time and labour consuming, and can cause residual Infection. The ratio of the number of infectious contamination problems (for collection of protocols agents, in this case virus particles, to the number of regarding adenoviral vector generation see targets (i.e., cells) is known as the multiplicity of reference 6). In this article, we will discuss methods infection or MOI. As master and working virus seed I 1 stocks (MVSS and WVSS) are costly, of thumb, the POI is about 50% of the culture, the most commonly used cGMP manufacturing processes maximum possible cell density as this system is the stirred tank bioreactor. should use the lowest MOI that can allows a window for additional cell This system allows the fine-tuning of still produce maximum virus yield. growth. For most suspension cultures growth parameters such as dissolved There are no standard MOIs for any grown in batch-mode, cell density is oxygen, pH, temperature and timing of combination of cell and virus so these around 1.0–1.5ϫ106 viable cells/mL. media replenishment for optimal cell must be determined at the Culture systems. Cell lines can behave growth and increased productivity. optimization stage for each new differently when scaled up from Manufacture of lytic viruses is process. laboratory to large culture systems. typically performed in a batch mode The minimum MOI that can be used Therefore, scale-up requires process where cells are grown and infected for successful virus production will optimization and process development in a closed system without depend on the starting material. If to transfer virus production processes replenishment of the media. However, crude lysate of a virus is used or the to the clean room. if virus manufacture occurs during a virus has a high P:I ratio, a relatively For cGMP manufacture, a more extended duration or if cell low MOI (Ͼ1.0) is usually more suspension culture system is often densities increase beyond the level appropriate as, at higher ratios, toxicity the favoured option because the normally supported by the growth resulting from noninfectious particles scale-up issues are much simpler, but media, the media must be replenished and excess cellular viral toxic proteins there are several viruses including using either a fed-batch system can decrease virus production (data adeno-associated virus (AAV) and (glucose and amino acid feed) or a not shown). Cell toxicity is less of a Lentivirus that are commonly perfusion system (complete media problem if purified virus material is produced in adherent cell lines. change). used and/or the virus has got a low Of the two main systems for Harvesting. The standard method for intrinsic P:I ratio, meaning that a higher adherent cultures, the most lysing cells within the manufacturing MOI can be chosen (data not shown). commonly used is the cell factory environment has been multiple Toxicity is further minimized if the (CF) (Nunc or Corning), which relies rounds of freeze/thaw using liquid purification process achieves on flat surfaces in the form of Nitrogen (Ϫ196 °C) and a water-bath the separation of infectious and multilayers. At commercial-scale, the set at ϩ37 °C. However, there noninfectious virus particles. CF can be purchased in a 40-layer are several issues that make this For the production of a lytic human format that requires specialized method problematic for harvesting, adenoviral vector carrying a nontoxic robotic systems to inoculate, feed especially within a clean room. As transgene, the MOI is typically 1–20 and harvest cells. The alternative the freeze-thaw process requires infectious units per cell.4 Lower MOIs system uses microcarriers a number of open manipulations, often result in slow and inefficient maintained in suspension. These can maintaining containment of the virus virus production. Other viruses can be divided into two major classes; may not be feasible at large-scale establish effective virus amplification nonporous carriers allow cells to production. Aerosol formation can using very low MOIs (e.g., 0.01–0.1). grow only on their surface, while occur if standard polypropylene The optimal MOI for virus macroporous carriers contain tubes are used, as positive pressure production is also linked to the time channels that cells can grow inside.10 is generated when the liquid freezes after infection that is best for virus For large-scale suspension cell and expands. Upon thawing, a harvest.7–9 Some viruses (e.g., replicating Reovirus and HSV) will successfully replicate during a prolonged period, starting from low Figure 1 Schematic illustration of upstream and downstream process parameters (USP, DSP) MOI (data not shown); other viruses, that influence quantity and quality of virus production. [MOI: multiplicity of infection; POI: such as human adenovirus-based point of infection; POH: point of harvest]. vectors, show optimal virus Choice of Type of production vessel: production if infection time is kept Cell growth production T-flask, cell factory, roller bottle, MOI POI POH to 48–72 h.4,5 conditions cell line tank bioreactor, wave-reactor Cell proliferation tends to decline after virus infection. The rate of this slow-down mainly depends on the MOI used, as the metabolic burden on the USP USP cells increases with the relative amount Optimized virus production of virus present.8 The density of the cell DSP DSP culture once cells are infected with virus particles, the point of infection (POI), must be carefully calculated to enable optimum virus production. Final polishing: To calculate the optimum POI, it is Harvest DNase Capture step: Clarification Tangential gel filtration method treatment ion-exchange essential to draw a growth curve that flow filtration HIC affinity TFF shows the maximum cell density before viability decreases significantly. As rule I 2 negative pressure can be generated, virus release are being investigated pressure and a ‘Z’ chamber to induce potentially introducing contaminants to solve these issues. shear into the fluid path and break to the sample. Furthermore, the One option is using a Microfluidizer open cells.11 Several different process is both time and labour HC5000 CE (MFIC Corporation; pressures (data not shown) and intensive. Alternative methods for Newton, MA, USA), which uses high numbers of passes were tested (Figure 2a) and the results show that this alternative method generally leads to better recovery of viral Figure 2 Comparative virus yield data using different virus recovery particles than the standard methods. (A) Graphical representation of genomic (red) and infectious freeze/thaw cycles. Production particle (peach) concentration of the recombinant adenovirus harvested of 1.6 L batches of clinical grade by varying numbers of passes through a microfluidizer at an optimized virus by freeze/thaw cycles takes ϫ pressure of 2000 psi (or using the standard method of 3 freeze/thaw approximately 6 h, but only 2 h using ϫ [3 F/T]).