Animal component‐free Agrobacterium tumefaciens cultivation media for better GMP‐compliance increases biomass yield and pharmaceutical protein expression in Nicotiana benthamiana

Abstract

Transient expression systems allow the rapid production of recombinant proteins in plants. Such systems can be scaled up to several hundred kilograms of biomass, making them suitable for the production of pharmaceutical proteins required at short notice, such as emergency vaccines. However, large‐scale transient expression requires the production of recombinant Agrobacterium tumefaciens strains with the capacity for efficient gene transfer to plant cells. The complex media often used for the cultivation of this species typically include animal‐derived ingredients that can contain human pathogens, thus conflicting with the requirements of good manufacturing practice (GMP). We replaced all the animal‐derived components in yeast extract broth (YEB) cultivation medium with soybean peptone, and then used a design‐of‐experiments approach to optimize the medium composition, increasing the biomass yield while maintaining high levels of transient expression in subsequent infiltration experiments. The resulting plant peptone Agrobacterium medium (PAM) achieved a two‐fold increase in OD600 compared to YEB medium during a 4‐L batch fermentation lasting 18 h. Furthermore, the yields of the monoclonal antibody 2G12 and the fluorescent protein DsRed were maintained when the cells were cultivated in PAM rather than YEB. We have thus demonstrated a simple, efficient and scalable method for medium optimization that reduces process time and costs. The final optimized medium for the cultivation of A. tumefaciens completely lacks animal‐derived components, thus facilitating the GMP‐compliant large‐scale transient expression of recombinant proteins in plants.

Comparison of microbial and transient expression (tobacco plants and plant‐cell packs) for the production and purification of the anticancer mistletoe lectin viscumin

“…A. tumefaciens strain GV3101:pMP90RK was used for transient expression in N. benthamiana leaves and N. tabacum BY‐2 PCPs. A. tumefaciens cells were cultured in liquid peptone agrobacterium medium PAM4 (Houdelet et al, ) supplemented with 50 mg/L carbenicillin and 25 mg/L kanamycin at 28°C.…”

Ready‐to‐Use Stocks of Agrobacterium tumefaciens Can Simplify Process Development for the Production of Recombinant Proteins by Transient Expression in Plants

“…The laboratory‐scale preparation of cryo‐stocks from A. tumefaciens fermentation broth was straightforward using standard centrifuges, with a hands‐on time of <60 min for buffer preparation, cell pelleting, resuspension, and filling, yielding up to 0.5 L of 10× (OD 600 nm = 10) ready‐to‐use aliquots. At the manufacturing scale, shake flasks are replaced with stirred‐tank bioreactors for the cultivation of A. tumefaciens and an OD 600 nm of ≈20 can be achieved . The reactor volume will thus be ≈350 L for a single batch of transient expression, given that an OD 600 nm of 1.0 is typically used during infiltration when silencing inhibitors such as p19 are not included, and up to 7000 L of infiltration solution can be required per 1000 kg plant batch .…”

“…Furthermore, the number of staff required to operate the process at a specific time can be reduced because the workload can be distributed more evenly over the duration of a production campaign. For example, at an intermediate 1500 L (≈200 kg plant biomass) scale, instead of individual A. tumefaciens fermentations before every plant infiltration, one 350 L bioreactor with an OD 600 nm of 20 could supply bacteria for five infiltrations with an OD 600 nm of 0.85. At an even larger 7000 L scale (≈1000 kg plant biomass), bacterial fermentation can be completed weeks before the plants are infiltrated.…”

“…We analyzed the cost structure for 36 production scenarios with a weekly batch size of 20–1500 kg plant biomass; a campaign size of 5, 25, or 50 batches; the need for one, two, or five A. tumefaciens strains during (co‐)infiltration; and the use of conventional or cryo‐stock strategies to prepare the infiltration suspension. We used a 1000 kg plant biomass batch as a base case, assumed that an OD 600 nm of 20 would be achieved for all bacteria, and also assumed that the infiltration OD 600 nm for the principal bacterial strain (encoding the major recombinant protein) would be 0.85, whereas that for any additional protein products would be 0.3 and that for accessory proteins such as p19 would be 0.2. All bioreactor volumes were scaled to match the demand of the base case using the conventional bacterial preparation (a 350 L reactor for the principal strain with an ≈17% safety margin, and 150 L or 100 L reactors for additional product proteins and accessory proteins, respectively).…”

“…Transient expression is therefore ideal for the rapid and large‐scale production of plant‐made pharmaceuticals (PMPs) such as antibodies, vaccines, or enzymes, under conditions compatible with a good manufacturing practice. However, the importance of A. tumefaciens fermentation and preinfiltration handling has not been widely discussed . Both are highly relevant for large‐scale manufacturing because plant growth and bacterial cultivation must be synchronized to facilitate a seamless infiltration process.…”