Improving Yield For Gene Therapy Through Transfection Optimization

By Evan Riley, M.Res, MBA, LSSBB, Senior Process Engineer, Kymanox Corporation

This article was first published in Cell & Gene Therapy

This article discusses gene therapy and the challenges in manufacturing gene therapy products at commercial scale. It covers the differences between stable and transient transfection methods used to insert therapeutic genes into host cells. The focus is on techniques to improve transient transfection yields during upstream fermentation by replicating small-scale, high-transfection conditions. Approaches like microfluidics and optimizing bioreactor design are described as potential solutions to increase transfection rates and make gene therapies more affordable and accessible.

Gene therapy is a modern therapeutic technique that utilizes viruses to deliver corrective genes to treat a targeted disease [1]. While still a burgeoning field, gene therapy has gained popularity as biomarkers that reliably correlate with specific diseases are being discovered and create therapeutic targets. Researchers can correct diseases by inserting a correct version of a particular gene into a patient’s genome [2]. This idea appears straightforward on the surface but is actually a complicated and inefficient process, compared to conventional biomanufactured active pharmaceutical ingredients (APIs) [1], [3]. The complications in manufacturing lead to high drug prices and thus limited patient use. A recent study found that the average price for a gene therapy treatment is approximately $1 million dollars [4]. This high price tag limits gene therapy to those patients who have health insurance to cover the cost [4].

Currently, only 27 cell and gene therapies have been approved by the Food and Drug Administration (FDA) as of January 2023 [5]. One of the limiting factors to developing more cell and gene therapies is the small number of commercial-scale facilities and available expertise to leverage for this specialty. The standard biomanufacturing process can be divided into three distinct departments—fermentation (upstream), purification (downstream), and formulation and fill (drug product). This article will focus on the translatable methods that can be extrapolated from the standard fermentation portion of the process to the emerging technologies that can be used for improving production of cell and gene therapies.

A typical fermentation process starts with a working cell bank of host cells that are grown in progressively larger vessels in order to increase the size of the cell culture and ultimately the number of available cells for API production. A key step for the successful transfection of the host cells during the upstream manufacturing [6] is the insertion of foreign nucleic acid into a host cell, which will then be indiscriminately copied to produce the API coded in the nucleic acid [1]. This process creates an API which can be harvested and transferred to the purification portion of the process. Fermentation typically starts with a cell culture that has already been transfected or the host cells are transfected once the culture has been grown to an appropriate size. Transfection can be divided into two types: stable and transient transfection [1].

Stable transfection is the insertion of a foreign gene into the host cell’s DNA. This form of transfection can be a costly, time-consuming investment, but has many benefits [1], [3], [7]. Stable transfection allows for the continuation of the transfected nucleic acid throughout cell replications and can be combined with other gene coding methods to ensure all of the cells in the upstream fermentation contain the gene responsible for producing the API [1], [6], [7]. However, stable transfection must be weighed against the business strategies of the company. To successfully implement stabilized transfected cells, the cell line must be developed, the API must be characterized, cells must be banked, and testing must be carried out, all of which can take months or possibly years to meet company and regulatory demands [1], [7]. Clinical trial requirements, patent time constraints, and pressure to be the first to market must also be considered by the companies. For these reasons, companies may decide not to risk the time or financial investment to pursue stable transfection and defer instead to the less efficient transient transfection.

Transient transfection is a much quicker and cheaper transfection method that relies on the insertion of a plasmid into host cells for a temporary introduction of foreign nucleic acid [1], [6]. In this process, host cells copy the circular pieces of foreign DNA to produce therapeutic viral vectors. These plasmids enter the chemically induced pores on the cell membrane of the host cell, where they will reside in the cytoplasm in the same fashion as host cell nucleic acids [1]. Despite the temporary nature of transient transfection, the plasmids last long enough to produce enough of the APIs which can then be harvested and purified via downstream purification methods [1]. The short time investment incentivizes companies to pursue this route in order to be first to market, allowing the pharmaceutical company to patent protect their significant financial investments in the pursuit of a new therapeutic API.

Most commonly biomanufactured pharmaceuticals, such as monoclonal antibodies, DNA vaccines, and mRNA vaccines only require a single plasmid for API production [8]. Gene therapy is more complicated as cells must successfully uptake three separate plasmids: one plasmid coding for the viral capsid, one coding for the therapeutic gene to be inserted into the patient, and a final plasmid to produce proteins necessary to assemble the therapeutic virus within the host [1], [6], [9], [10]. If any of the three plasmids are missing, then the API will be incorrectly assembled resulting in reduced yield. Many decisions during the early process development can affect cellular growth and its interactions with plasmids [1], [6], [9], [10]. Some of these decisions include the timing to introduce the plasmids in the cellular lifecycle, media characterization, pore inducer, and the plasmid ratio, which can either encourage or hinder the uptake of plasmids and ultimately determine the success or failure of a batch. Production yields for gene therapy based viral titers have seen ranges from single digits up to &GT90% yield depending on the process specifics chosen to pursue this type of API [11]. Larger scale processes have less control over the heterogeneity of parameters throughout the bioreactors which is reflected in reduced yield after scaling up [9]. The goal of any company should be to replicate the success and high transfection rates found throughout small scale facilities and academia.

Producers can replicate small scale conditions through techniques like roller bottles or T-flasks to achieve higher titer production. A roller bottle is designed to hold 0.1 L to 0.3 L of suspended media and functions by slowly rotating allowing the cells to be exposed to the gasses in the headspace of the bottle [3]. This culture technique performs exceptionally well for both adhering cells as well as suspended cell cultures, but these conditions become increasingly burdensome as the scale of production increases [4]. As manufacturers move from pilot scale to large scale, the roller bottle equivalent of a 2,000 L suspended tank becomes approximately 10,000 roller bottles. This number is unsustainable from both an environmental and business standpoint as the bottles are both wasteful and extremely costly. The question then is how to avoid a loss of yield when scaling up the fermentation process. Relying on the passive diffusion of polyethylenimine (PEI) attached plasmids has seen limited success especially in larger vessels. There are a few techniques currently utilized in the field to mimic the conditions found in small scale facilities. The plasmids and cells need to amalgamate by being forced into close proximity [1], [2], [6], [7], [9].

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