Patent Application
20250171726
The present invention relates to growing biomass, in particular with the aim of producing molecules of interest (like for example recombinant proteins and/or secondary metabolites), in an optimized bioreactor.
Hairy roots from plants have been widely studied and used for the production of specialized/secondary metabolites of industrial and pharmaceutical interest. Since the 1990s, the production of recombinant proteins has been considered as another promising application of hairy root cultures. This system presents numerous similarities with the one used to produce recombinant proteins from mammalian cell lines (such as, e.g., Chinese hamster ovary (CHO) cells, human cell lines, bacteria), among which the fact that the whole process is maintained under sterile conditions in a confined environment.
This plant-based technology offers relevant and advantageous differences compared to the mammalian expression systems. The selected hairy root clones are easily grown in tailor-made optimal conditions in a simple culture medium which has a better safety profile than culture media containing human- or animal-derived constituents. This process allows the large-scale production of compounds (often referred to as molecules of interest) such as recombinant proteins or secondary metabolites in an axenic environment in bioreactors. Moreover, contrary to the common mammalian cells-based expression systems, the hairy-root expression system allows a straightforward recovery of the molecules of interest that does not require an additional extraction phase.
However, as in every production method, it is important to obtain the highest possible amount of molecules of interest (like proteins or metabolites) in a same bioreactor in order to preserve an optimal reproducibility and decrease the number of bioreactors used in parallel in an attempt to reduce the carbon footprint.
This invention thus relates to a bioreactor for the production of biomass, the bioreactor being configured to contain at least 15 liters of a liquid comprising:
Thus, this solution achieves the above objective. In particular, it makes it possible to obtain a significant improvement in the productivity of molecules of interest while maintaining a high biomass growth by using bubbles of at least 5 mm of diameter.
The bioreactor according to the invention may include one or more of the following characteristics, taken in isolation from one another or in combination with one another:
A further object of the present invention is a system for the production of biomass, said system comprising a bioreactor according to any one of the here-above listed technical features and a gas injection device, the gas injection device being put in fluidic communication with the sparger.
A third object of the present invention is a method for growing biomass in a culture medium, said culture medium being put in motion by a bioreactor according to any one of the here-above listed technical features, said method comprising the following steps:
The invention will be better understood, and other aims, details, characteristics and advantages thereof will emerge more clearly on reading the detailed explanatory description which follows, including embodiments of the invention given by way of illustration and purely illustrative and non-limiting examples, with reference to the accompanying drawings:
The invention thus relates to a bioreactor 10 for the production of biomass, such as hairy roots, in particular with the aim of producing molecules of interest. The bioreactor is a column like reactor extending along a longitudinal axis X with a bottom extremity 10A and an upper extremity 10B.
In the present invention, the terminology “biomass” refers to any living organisms that may be cultured (or grown) in vitro. In some embodiments, the biomass refers to a plant (i.e., plant biomass), in particular to plant roots (i.e., root biomass). Example of plant roots that may be grown in the bioreactor include hairy roots, rhizocals, and adventitious roots. Thus, in some embodiments, the biomass is adventitious roots, hairy roots, rhizocals, or any combination thereof. In some embodiments, the biomass is hairy roots (i.e., hairy root biomass).
The terminology “adventitious roots” refers to root emergences which appear during the physiological development of the plant and hence occurs naturally. The terminology “hairy roots” refers to root emergences which appear after the infection of a plant by Rhizobium rhizogenes (previously referred to as Agrobacterium rhizogenes) bacteria or by Rhizobium radiobacter (also known as Agrobacterium Tumefaciens) bacteria harboring rol genes for example. The terminology “rhizocal” or “rhizocallus” refers to a conic-shaped structure connected to the roots (in particular to the hairy roots), also termed lateral root emergence, which develops alongside of the roots in a solidarized way. In practice, rhizocals (or “rhizocalli”) may be induced in a culture of roots by the addition of auxin in the culture medium.
The terminology “molecule of interest” refers to molecules (or compounds), such as recombinant proteins and/or secondary metabolites, which may be produced (and likely secreted) by the biomass, in particular by roots such as hairy roots.
The bioreactor 10, more precisely the internal tank 14 of the bioreactor 10, is configured to contain at least 15 liters of a liquid. In the present invention, the terminology “a liquid” or “liquid” refers to at least one liquid. In the other words, in the present invention, the terminology “a liquid” or “liquid” may include one or several liquids. In some embodiment, the liquid is a culture medium.
Roots, and in particular hairy roots are commonly cultivated (or grown) on solid medium or in liquid medium. Hairy roots may be cultivated in a standard culture medium (such as Gamborg B5, MS, etc.) or using an optimized culture medium where each nutrient concentration is specifically defined to allow better growth and/or molecule productivity. In a preferred embodiment, the internal tank 14 of the bioreactor 10 is configured to contain a volume ranging from 200 to 350 L of liquid. In some embodiments, the internal tank 14 of the bioreactor 10 can contain up to 1000 L of liquid, and even up to 5000 L. The internal tank 14 is classically put together by welding several pieces together.
In these regards, as can be seen on the embodiment depicted on
The bioreactor 10 according to the present invention might be part of a system for the production of biomass, said system comprising a bioreactor 10 and a gas injection device put in fluidic communication with the sparger 16.
The external support structure 12 is designed to hold the internal tank 14 as a sheath, as can be seen on
As can also be seen on
In a preferred embodiment, the internal tank 14 is disposable. This makes it easier to grow the biomass in sterile conditions.
More precisely, the internal tank 14 is made of five strips welded together. This makes it possible, once the internal tank 14 is filled with liquid, to form a reservoir much closer to a cylinder than if the internal tank 14 had been made from one or two strips. This allows the pressure forces exerted by the liquid filling the internal tank 14 to be distributed more evenly over the entire internal surface of the internal tank and along the different welding sites.
The internal tank 14 displays at least one closed extremity 20. In some embodiments, said closed extremity 20 can display a conical shape, the cone being centered around the revolution axis X. The conical shape of the closed extremity 20 of the internal tank 14 of the bioreactor 10 makes it possible both to reduce dead volumes and also to facilitate recirculation of the liquid. This closed extremity 20 is configured to be the bottom extremity 10A of the bioreactor 10. In some embodiments, in which the biomass has to been grown in a sterile environment, the upper extremity of the internal tank 14 (being the upper extremity 10B of the bioreactor 10) is also closed and comprises an air outlet 11. Said upper extremity may further comprise several inlet conducts 130 to add liquid and nutrients, one first outlet conduct 131 to collect sample and one second outlet conduct 132 to collect trash.
Depending on the embodiments, the internal tank 14 presents a height ranging from 1000 to 5000 mm and a width ranging from 300 to 1000 mm.
The internal volume 22 of the internal tank 14 is free of any internal structure, in particular any internal support structure. This means that the internal volume 22 of the internal tank 14 is one single continuous space: no internal support structure like a transverse or crossing conduct, an internal strand or an internal wall is to be found inside the internal tank 14 in order to separate this unique internal volume 22 into several internal spaces.
In some embodiments, the internal tank 14 comprises at least one loop 24 secured to its wall. The at least one loop 24 is configured to hold at least one fluidic conduct and to maintain said fluidic conduct in contact with the wall. The fluidic conduct might be used to transport some water or fresh culture medium. In some embodiments, the loop 24 is secured on the external face of the wall of the internal tank 14 and in some alternative embodiments, the loop 24 is secured on the internal face of the wall of the internal tank 14. However, even if the loop 24 is secured on the internal face of the wall, said loop and the associated fluidic conduct being maintained in contact with the wall, they do not separate the unique internal volume 22 into several internal spaces. Neither the loop 24 nor the fluidic conducts can be considered as internal support structures.
The design of the internal tank 14 of the bioreactor 10 includes different factors among which the geometric ratio (rG), which is defined, in the present application, as being the ratio between the diameter d and the useful height H (H being the height of the liquid column inside the internal tank 14) of the internal tank 14:
This ratio defines the size of the internal tank 14 and thus the space available for the biomass (for example hairy roots) to grow. Different ratios can be used regarding the present invention depending on the volume of the internal tank 14. For a 25 L scale, rG ranges between 0.1 and 0.2, for a 200 L scale, rG ranges between 0.3 and 0.5 and for a 350 L scale, rG ranges between 0.4 and 0.6. Empirical knowledge indicates that these ratios rG intervals have no impact on the hairy roots growth.
The sparger 16 and the internal tank 14 are arranged in a way that the generated bubbles 26 form a bubble column once the internal tank 14 is filled with liquid and the sparger 16 is fed with gas. In a preferred embodiment, it is one single bubble column. The bubble 26 column enables the liquid to be put in motion.
As can be seen on
The walls of the sparger can be made of inox, metal or plastic, for example.
An internal conduct 17 presenting a diameter ranging from 2 to 5 mm extends downstream from the opening O and enables gas to enter the sparger 16 through the opening O. The sparger 16 further comprises, on its external wall, at least one radial aperture 170 put radially in fluidic communication with the internal conduct 17. In some embodiments, the sparger 16 comprises several radial apertures 170. The opening O is thus put in fluidic communication with each radial aperture 170 by means of the internal conduct 17.
The at least radial aperture 170 presents a diameter ranging from 0.5 to 1.5 mm, preferably 1 mm. In the embodiment depicted on
The sparger 16 is therefore preferably arranged and secured in the center of the closed extremity 20 of the internal tank 14. The sparger 16 is centered with regard to the revolution axis X, thus enabling the formation of a central air column.
This makes the bioreactor 10 according to the present invention of the “bubble column” type, meaning that the agitation of the liquid is generated by the introduction of gas at the bottom of the bioreactor. Moreover, in the bioreactor 10 of the present invention being an air-lift system, the air flow of the bubble column directly influences the agitation and the type of flow in the internal tank 14 of the bioreactor 10. The higher the air of the bubble column flows, the more the liquid flow is increased. Some examples of experimental results will be discussed further below.
In order to feed the sparger 16 with gas, preferably air, the sparger 16 is put in fluidic communication with a gas injection device 28, preferably an air injection device. The gas injection device 28 is designed to generate a gas flow rate (preferably air flow rate) ranging from 0.5 L/min to 10 L/min, preferably 1 L/min.
When the sparger is fed with gas while the internal tank 14 is filled with liquid, the internal volume 22 of the internal tank 14 is separated into a gassed and an ungassed region. The bubbles generate a vertically circulating flow between the gassed and ungassed regions, as can be seen on
The control system 18 allows the acquisition and/or the control of several monitoring signals inside the liquid like for example pH, conductivity, dissolved oxygen. The control system comprises for example a control box like BioFlo® 120 Eppendorf or Biostat® B Sartorius. This control system 18 thus allows a precise monitoring of the growing process of the biomass.
The bioreactor 10 according to the present invention enables the implementation of a method for growing biomass in a culture medium, said culture medium being put in motion by air bubbles 26, said method comprising the following steps:
The objective of the herein presented experiments was to compare two bioreactors with bubbling systems based on two sparger technologies. For that, the bubble size distribution in function of the water column height was measured by picture acquisition and image analysis. Furthermore, the effect of the bubble size on the growth of the biomass and on the production of a recombinant protein was assessed.
A single-use bioreactor containing 25 L of distilled water was used for the bubble size measurement acquisition. In order to obtain clear and workable pictures, the air flow rate was maintained at 1 L/min during all the experiment and for both spargers. The spargers were placed at the bottom of the bioreactor.
Pictures were taken using a camera (Canon®) mounted on a tripod with adjustable height. The distance between the camera and the bioreactor was set at 185 mm during all the experiment in order to have exactly the same capture conditions. Pictures were taken from the bottom of the bioreactor to the top with regular height steps.
Image analysis was done using the software ImageJ. The “Ellipse” tool of the software was used to encircle each bubble on the pictures acquired. The “Measurement Analysis” tool was then used to calculate the area of the encircled bubbles. As this data was given by the software in pixel units, a calibration was needed to know the equivalence between pixels and distance in mm. For that, a control consisting in a sphere of 50 mm diameter was used for pixels calibration. A picture of the sphere was acquired in exactly the same conditions as the bubbles and the picture analysis has allowed to establish the equivalence between millimeters and pixels. This calibration was used to calculate the bubble area in square millimeters. Bubble areas were then averaged for each height step in order to represent the distribution of the average area in function of the height of the bioreactor, for both spargers.
Hairy roots of Brassica rapa rapa producing the protein IDUA (alpha-L-iduronidase) were cultivated in a bioreactor in standard conditions. At the end of the hairy root culture, the roots were collected, washed in protein recovery solution, rinsed with distilled water and dried at 70° C. during 48 h. Dry weight (in gDW) was then measured and reported to the culture volume (in L) to calculate the biomass concentration (in gDW/L).
To assess the amount of IDUA protein produced by the hairy root culture, the enzymatic activity of the IDUA protein was measured in the collected protein recovery solution using a fluorimetric enzymatic assay based on the fluorogenic substrate sodium 4-methylumbelliferyl-α-L-Iduronide (4MU-I; Santa Cruz Biotechnology). The 4MUI substrate was diluted to a working solution of 400 μM 4MU-I with the reaction buffer 0.4 M sodium formate, pH 3.5. Twenty-five μL of sample to analyze were added to 25 μL of 400 μM 4MU-I substrate. The mixture was incubated at 37° C. for 30 min and 200 μL glycine carbonate buffer (pH 9.8) was added to quench the reaction. 4-Methylumbelliferone (4MU) (Sigma) was used to prepare the standard calibration curve. Fluorescence was measured using a plate reader (TECAN Infinite M1000™ Männedorf, Switzerland) with excitation at 355 nm and emission at 460 nm. IDUA activity in measured in enzymatic unit per liter of sample (U/L). IDUA productivity (in mU/gDW/d) was then calculated by dividing the IDUA units (in U) by the dry weight (gDW) and by the production duration (in days).
The conclusion that can be drawn from those results is that the generated bubbles are twice smaller when using a microbubble sparger than when using a large bubble sparger like in the present invention.
It appears quite clearly that the biomass concentration obtained after 39 days of culture is about two times greater when using a large bubble sparger according to the present invention than when using a commercially available wave bioreactor.
It also appears quite clearly that the amount of protein produced after 39 days of culture is about three times greater when using a large bubble sparger according to the present invention than when using a wave bioreactor.
No significant impact on biomass production can be detected.
Despite the similar biomass concentration observed in
Therefore, using a bioreactor with a large bubble sparger like in the present invention allows to significantly increase the protein production by a hairy root culture, notably in comparison to the protein production by a hairy root culture in a bioreactor with a microbubble sparger or in a wave bioreactor.
| Number | Date | Country | Kind |
|---|---|---|---|
| 22305436.2 | Apr 2022 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2023/058535 | 3/31/2023 | WO |
