Predictive models for transient protein expression in tobacco (Nicotiana tabacum L.) can optimize process time, yield, and downstream costs

Abstract

The transient expression of recombinant biopharmaceutical proteins in plants can suffer inter-batch variation, which is considered a major drawback under the strict regulatory demands imposed by current good manufacturing practice (cGMP). However, we have achieved transient expression of the monoclonal antibody 2G12 and the fluorescent marker protein DsRed in tobacco leaves with ∼ 15% intra-batch coefficients of variation, which is within the range reported for transgenic plants. We developed models for the transient expression of both proteins that predicted quantitative expression levels based on five parameters: The OD(600 nm) of Agrobacterium tumefaciens (from 0.13 to 2.00), post-inoculation incubation temperature (15-30°C), plant age (harvest at 40 or 47 days after seeding), leaf age, and position within the leaf. The expression models were combined with a model of plant biomass distribution and extraction, generating a yield model for each target protein that could predict the amount of protein in specific leaf parts, individual leaves, groups of leaves, and whole plants. When the yield model was combined with a cost function for the production process, we were able to perform calculations to optimize process time, yield, or downstream costs. We illustrate this procedure by transferring the cost function from a production process using transgenic plants to a hypothetical process for the transient expression of 2G12. Our models allow the economic evaluation of new plant-based production processes and provide greater insight into the parameters that affect transient protein expression in plants.

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

“…Seeds were germinated on mineral wool blocks (Cultilène, Tilburg, Netherlands) and the plants were cultivated in a greenhouse with a 25/22°C day/night temperature and a 16-h photoperiod (≈200 µmol s -1 m -2 ; λ = 400-700 nm) at 70% relative humidity, as previously described [12]. The plants were irrigated with 0.1% m/v Ferty 2 Mega (Kammlott GmbH, Erfurt, Germany) and were grown for about two months (prior to flowering) before the infiltration of tobacco leaf discs (Nicotiana tabacum cv.…”

“…Furthermore, plant-derived peptones cost ≈50−150 € kg -1 , which is 80% lower than animalderived components such as beef extract (≈700 € kg -1 ). The cultivation medium can account for more than 5% of the total costs associated with infiltration [12] so cost savings can be achieved even if greater quantities of plant peptones are required compared to animal-derived ingredients. Overall, we reduced the costs by more than 50% when using PAM rather than YEB (Table 2).…”

“…The presence of animal-derived products in these media means that additional processing steps are necessary to avoid the propagation of pathogens. Bacterial growth in shake flasks containing YEB is typically limited to an OD 600 of ≈5.0, corresponding to ≈7.5 × 10 9 cells per mL [11], whereas typical infiltration media require an OD 600 of ≈1.0 [12]. Therefore, the fermentation volume must be equivalent to at least 20% of the bacterial suspension volume used during infiltration.…”

Membrane stability and methaemoglobin content of human erythrocytes incubated in aqueous leaf extract of Nicotiana tabacum product

“…Accordingly, there are reports that researchers and bioengineering entrepreneurs of Developed Countries have already begun the use N. tabacum as hosts for bioengineering processes. [9][10][11][12][13] Likewise, the prospect of utilizing the plant product as biological pesticides in the form of nicotine tartrate, [14][15][16][17] and medicinal preparations/decoction have shown promising prospects. [18,19] Although there are concerns about the use of N. tabacum extracts as therapeutic agents because of the addictive tendency and harmful effects of nicotine content, reports have shown that salt extract of N. tabacum leaves are devoid of nicotine.…”

Effect of nitrate concentration in nutrient solution on hemagglutinin content of Nicotiana benthamiana leaves in a viral vector-mediated transient gene expression system

“…Recombinant proteins are biosynthesized from substances derived from the plant itself; therefore, plant growth conditions, which exert significant effects on plant metabolism and nutritional status, likely influence the level of recombinant protein accumulation. In fact, recent studies have shown that environmental factors, such as light intensity and temperature during plant growth, significantly influence recombinant protein production (Buyel and Fischer 2012;Matsuda et al 2012). Therefore, we propose that optimizing plant growth conditions should be a fundamental approach to increasing the accumulation of PMP proteins.…”