WO2016092073A1

WO2016092073A1 - Coupled systems of aeration, agitation and heat exchange for the culture of microorganisms in single use bioreactors

Info

Publication number: WO2016092073A1
Application number: PCT/EP2015/079423
Authority: WO
Inventors: Jordi Joan Cairó Badillo; Antoni Casablancas Mira; Martí Lecina Veciana; Ivan Martinez Monge; Carlos PAREDES MUÑOZ
Original assignee: Universitat Autonoma De Barcelona
Priority date: 2014-12-12
Filing date: 2015-12-11
Publication date: 2016-06-16

Abstract

The present invention describes a device for improving oxygen and heat transfer in single use bioreactors. Oxygen transfer is enhanced by an agitation-aeration system generating a flow of microbubbles with pore sizes ranging from 0,5 microns up to 1 mm. The system for the generation of microbubbles might be coupled to an agitation shaft, be independent of it or might be a combination of both simultaneously. The invention also includes a set of baffles harbouring a heat exchanger inside. This system increases the bioreactor's surface for heat exchange. This internal heat exchanger may be used in combination with an additional external heat exchanger located in an external recirculation loop of culture broth. The recirculation loop increases both the agitation of the bioreactor and the heat exchange capacity. This invention enables the use of 2-2000 liters single use plastic bioreactors (made of biocompatible plastic) for the culture of different biological specimens (bacteria, microalgae, yeasts, fungi, animal and plant cells) using a single bioreactor type.

Description

Coupled systems of aeration, agitation and heat exchange for the culture of microorganisms in single use bioreactors

Introduction

The use of single use bioreactors (SUB) for animal cell culture at scales between

2 and 2000 litres has been already described [1]. The applications of the molecules produced with animal cell technology spans the fields of biomedicine, biotechnology, food industry and environment. However, there are equally relevant substances produced with other organisms such as bacteria, yeasts, fungi (mainly aerobic, but also facultative and anaerobic) and microalgae. Unfortunately, currently available SUBs technology is not suitable for such microorganism and cannot meet commercial productivity levels. The main limitation encountered is in regard to the poor oxygen transfer rate between the gas and the liquid phase, as well as to the poor chilling capacity. The aeration and chilling needs increase proportionally to cell density, as oxygen is a key substrate and heat is a product of the aerobic catabolism of the main carbon source. When the heat generated cannot be removed fast enough, the process temperature increases resulting in poor cell growth and low product amount and/or quality.

From a productive standpoint, SUB technology has the following advantages over stainless steel bioreactors: lower fixed investments in main and auxiliary equipment, reduced needs of continuous cycles of cleaning and sterilization and their verification in compliance with GMP standards and, as a consequence, reduced manpower requirements [2]. Moreover, the easy handling of SUB allows concatenating different batches, or even switching processes, thanks to the reduced turnaround time. The rapid transition between processes increases the overall plant/facilities yearly productivity.

This invention extends the application of single use bioreactors to bioprocesses based on bacteria, yeasts, fungi and microalgae culture by improving the oxygen (or other gases) transfer to the system, as well as, by increasing the removal of the heat generated as a consequence of cell metabolism and growth. These improvements are based on the increase of K_La (oxygen mass transfer coefficient), and thus the gas transfer rate, by using a micro-bubble generating system, and by increasing the heat exchanging area. The bubble-generation device may be coupled to the agitation shaft, independent of it or both simultaneously. The heat transfer (removal) capacity of the proposed SUB is achieved by adding an internal recirculation loop harboured into the baffles located in the bioreactor's shell.

This invention enables the application of single use bioreactors for the culture of aerobic bacteria, yeasts, fungi, microalgae, animal cells and vegetal cells in batch processes as well as in high cell density processes.

References:

[1 ] Fenge C, Lijllau E. Cell Culture Biorreactors for Pharmaceutical and Cell Based Therapies. Ozturk S, HU WS, Eds. Taylor and Francis Group: Oxford, UK. 2006; 155- 224.

[2] Sinclaire A, Monge M. Quantitative Economic Evaluation of Single-use Disposables in Bioprocessing. Pharmaceutical Engineering, 2002:20-34. Description of the Invention

As stated previously, the low oxygen transfer and heat transfer capability of SUBS limits its widespread use. In bioreactors, the oxygen transfer rate is directly related to the volumetric oxygen mass transfer coefficient (K_La) that can be viewed as the inverse of the resistance to oxygen transfer. Moreover, the K_La is the combination of two terms: the specific mass transfer coefficient (K_L) and the specific phase exchange area (a). For a given system, the calculation of the K_La can be performed using a complex formula which depends on many parameters such as the agitation rate, the gas flow and the viscosity of the media.

The proposed approach consists on improving two parameters strongly linked to k_La: a) Increasing the exchange area between both phases (gas-liquid) by reducing the size of the microbubbles.

b) Optimizing the agitation system configuration to achieve higher agitation rate.

The values of K_La obtained with an experimental prototype of the proposed system are even higher than those achieved with conventional bioreactors working under identical operation parameters. As it can be seen in the Figure 1 , K_La values increase with the stirring rate, reaching up to 75% of improvement at 1000 rpm of stirring rate, 2 vvm of aeration and 37°C of temperature, compared to conventional glass bioreactor In one embodiment the microbubbles generator system is made of sintered metal, plastic or nylon. In one embodiment the microbubbles generator system is made of compressed ceramic or glass particles.

Fig. 2 shows the growth profile of an Escherichia coli strain in a conventional bioreactor and in our SUB prototype. The later reached a cell density 31 % higher than the conventional bioreactor at the onset of oxygen transfer limitation, i.e. when the p0₂ set point could not be maintained anymore once the maximum oxygen transfer is reached.

The single use bioreactor developed is made of a biocompatible plastic material, and provides a controlled environment comparable to that of stainless steel bioreactors. The bioreactor is heat sealed and includes both an agitation shaft and the microbubble generation system.

In one embodiment, the coupling of the agitation shaft is done through a mechanical lock located on the top section of the SUB, whereas in some other embodiments the mechanical lock is located in the bottom section of the SUB.

In some embodiments, the microbubble-generator is integral to the agitation shaft, which has an internal channel for the gas inflow. In some other embodiments the microbubble-generator is located on the bottom section of the SUB being apart of the agitation shaft. Yet in some other embodiments both systems are included in the SUB design.

In one embodiment, heat removal transfer is achieved thanks to an internal recirculation loop of chilling water harboured into the baffles on the bioreactor's walls, increasing the heat exchange area and therefore the heat transfer rate. In one embodiment this chilling system may work in conjunction with an additional external heat exchanger located on a culture broth recirculation loop.

The bioreactor as a whole will consist in a gamma-sterilized disposable item and will be presented in a sealed envelope or bag. All required manipulations needed for operating the bioreactor such as sterile filling, inoculation, connection to/from ports or filters, and monitoring elements coupling will not require specific tools. For the final user or SUB operator, the bioreactor will be a consumable item. Brief Description of the Drawings

For a better understanding of what has been disclosed, various embodiments of the present invention will now be discussed with reference to the presented drawings. The drawings are understood to be diagrammatic and/or schematic and are not necessarily drawn to scale nor are they to be limiting of the spirit and scope of the present invention.

Fig. 1 comparison of K_La values obtained with a conventional 5-litre bioreactor and the 5-litre SUB prototype. Stirring rates of 400, 700 and 1000 rpm were assessed. Values are the mean of 3 replicates and the error bar corresponds to the standard deviation.

Fig. 2 growth profile of an Escherichia coli strain obtained with a conventional 5- litre bioreactor (A) and the 5-litre SUB prototype (B).

Fig. 3 is a vertical section of an embodiment of the bioreactor according to the invention. Vertical section of an embodiment with an agitator shaft, driven by an electric motor, entering from the top of the bioreactor. This shaft has an internal channel for the gas inflow. Microbubbles are generated through sintered metal, glass, plastic or ceramic cylinders which are part of the shaft. This embodiment also shows a plate at the bottom of the reactor to create microbubbles.

1 agitation shaft

2 stirring paddle

3 a bottom microbubble plate diffuser

3 b agitation shaft microbubble diffuser

4 mechanical lock and top port for gas inlet

5 bottom port for gas inlet

6 a-b-c-d gas inlets

7 bioreactor baffles

8 external heat exchange device

9 cooling-heating inlet (a) and outlet (b)

10a, 1 1 a, 12a, 13a ports for the introduction, withdrawal and return of liquids

10b, 1 1 b, 12b, 13b peristaltic pumps 14 a-b-c gas exhaustion outlet and condenser

15a-b peristaltic pump and piping to external heat exchange device

16 (a-g) optical and invasive sensor ports

17 temperature sensor port

18 port for sampling

19 harvesting port

20 plastic bag bioreactor

In one embodiment, the gases inflow is done at the bottom of the bioreactor through a porous sintered metal, glass, ceramic or plastic polymer particles plate as can be seen in Figs. 4, 5A and 5C.

Fig. 4 detail of the sintered, glass, ceramic or plastic particles plate located at the bottom of the single use bioreactor. The plate constitutes one of the microbubbles generator systems proposed. In the embodiment in which the gas inlet flow is performed through the agitation shaft, the mechanic lock is replaced by a board supported on rotating shafts, as is shown in Figs. 5B and 5C.

Fig. 5 vertical sectional views of the aeration system coupled or not to the stirring shaft through the top part of the SUB. The three embodiments are as follows: 5A: Agitation shaft without aeration and sintered particles plate at the bottom of the reactor to generate microbubbles.

1 agitation shaft

2 stirring paddle

3 bottom microbubble plate difuser

4 mechanical lock and top port for gas inlet

5 bottom gas inflow port

6 a-b gas inlet pipe

5B: Shaft with an internal channel for gas inflow, microbubbles are generated through sintered metal, glass, ceramic or plastic polymer cylinders which are part of the shaft. 1 agitation shaft

2 stirring paddle

3 agitation shaft microbubble diffuser

4 mechanical lock and top port for gas inlet

5 a-b gas inlet pipe

5C: Embodiment in which the aeration systems detailed in Figs. 5A and 5B are combined together in the same SUB.

1 agitation shaft

2 stirring paddle

3 a bottom microbubble plate difuser

3 b agitation shaft microbubble diffuser

4 mechanical lock and top port for gas inlet

5 bottom gas inflow port

6 a-b-c-d gas inlet pipe

According to the above embodiment, the agitation shaft and the electrical motor may be coupled at the top of the bioreactor through a mechanical seal or lock. This mechanical lock is different depending of the entrance of gas for this part.

In one embodiment the stirring shaft is supported on a radial-axial bearing and mechanical lock supported on a metallic axis or shaft.

In one embodiment the stirring shaft is supported on a radial-axial bearing and mechanical seal supported on a plastic polymer axis or shaft.

The detail of the heat exchanger for temperature control of the bioreactor is shown in Fig. 6.

In one embodiment, the heat exchanger is composed by coiled pipes located into the bioreactor shell and baffles. In one embodiment, the heat exchanger is a bioreactor jacket that includes the baffles inner volume as a part of the bioreactor jacket.

In one embodiment the chilling fluid or agent is refrigerated by a cryostat platform and is pumped back to the bioreactor using an external pump. In one embodiment the chilling agent is tap water directly connected to the heat exchanger. Fig. 6 detail of the heat exchanger harboured into the bioreactor shell and into the baffles.

In one embodiment, any of both heat exchanger systems located into the shell (jacket or pipes), may be complemented with an external heat exchanger located on the recirculation loop. The external heat exchanger may have any of the following structures: double tube heat exchanger (Fig. 7a), shell and tube heat exchanger. (Fig. 7b) or plate and frame exchanger (Fig. 7c).

Fig. 7a, 7b and 7c show vertical section views of the external heat exchanger that may complement the heat exchanger located into the bioreactor jacket and baffles.

Fig. 7a detail of the double tube heat exchanger.

1 a Recirculating loop inflow

1 b Recirculating loop outflow

2a Chilling agent Inflow

2b Chilling agent outflow

4 cooling-heating piping

5 baffles

Fig. 7b detail of the shell and tube heat exchanger.

1 a Recirculating loop inflow

1 b Recirculating loop outflow

2a Chilling agent Inflow

2b Chilling agent outflow 3 shell

4 internal tubes

5 baffles

Fig. 7c. detail of the plate and frame heat exchanger.

1 a Recirculating loop inflow

1 b Recirculating loop outflow

2a Chilling agent Inflow

2b Chilling agent outflow

3 shell 4 plates

Preferred description of the invention:

The following describes in more detail the different parts of the apparatus and its operation.

Description of the bioreactor and the elements for optical ports and external connection systems.

In brief, Figure 3 describes:

The bioreactor is composed by a closed cylindrical vessel made of biocompatible plastic material following the single use bioreactor concept. Once the bioreactor is filled in with the culture medium and it is inoculated (using the sterile port connection system), it can be handled and transported to a non-sterile environment were the culture follows its course until ready for harvesting.

The single use bioreactor is equipped with several optical ports located on the bottom section, ensuring that the ports are always submerged in the culture broth. Different optical sensors and probes, based on fluorimetry or light scattering/transmission, can be plugged into the optical ports . The sensors may be (but are not limited to) pH, p02, turbidity, NIR, etc.

The bioreactor also has inlet and outlet gas ports, liquid filling ports, inoculation port, sampling port, harvesting port, and other liquid inlet ports for buffers, acid or base punctual additions.

Figure 3 details the location of the heat exchanger through an external recirculation loop, power to the agitation shaft, as well as port for sensors (optical or not) and microsensors.

The bioreactor will also have gas inlet and outlet ports to provide adequate partial pressure of oxygen to the culture, as well as other gases that may be necessary in certain applications. Sterility of the gaseous streams is ensured by the use of 0,22 micron filter sterilization connected to these ports. The filling, inoculation and sampling of the bioreactor is carried out through one or more dedicated ports. For continuous or fed-batch operations, one or more of these ports may be used for the addition or removal of liquids as needed.

Measurements of the culture parameters will be conducted externally, without interfering with the sterile barrier of the bioreactor. Possible measurement methods to be used are:

- Optical access to the contents of each bioreactor, from transparent walls or parts thereof.

- Access to the external pins of sensors or microsensors located on the inside of the bioreactors.

- A system of access points (ports) for devices that respects the sterile barrier, enables the automatic monitoring and control of cultivation parameters.

The monitoring elements will be connected to a Digital Control Unit (DCU) allowing the monitoring of bioprocess main parameters, the control of operational variables of the system and also the automation of the bioprocess.

In a preferred embodiment the single-use bioreactor has a volume between 5 and 2000 litres.

Description of the agitation-aeration system

The agitation/aeration system according to one of the embodiments presented in Figures 4 and 5 comprises:

An agitation-aeration system, shown in Figures 5A, 5B and 5C consisting of a mechanical agitator element, driven by an electric motor. The agitator shaft may be held from the top section of the bioreactor or from the bottom part of it. The shaft will have an internal channel that may be used as inlet gas flow for the aeration of the bioreactor. Sintered metal cylinders whit microspores will be coupled at the end of the perforated shaft, in order to generate the microbubbles needed for the efficient mas transfer from the gas to the liquid phase. This bubble generation system may be combined or alternatively substituted by a sintered metal, ceramic, glass or polymeric plastic plate at the bioreactor base. Each device comprises a single central agitation shaft to homogenize its content. The stirring assembly is powered by an electric motor.

The bioreactor will also have an outlet port for exhaust air equipped with sterilizing 0,22 micron filter to ensure the sterility of the system. Alternatively, the bioreactor has an agitation shaft without the internal gas conduit, is driven by an electric motor and there is a sintered metal plate at the base of the reactor to create microbubbles (Figure 5) with pore sizes ranging from 0,5 microns up to 1 mm.

Description Of The internal Recirculation heat exchanger

The culture temperature will be controlled by a heat exchanger system harboured into the bioreactor's shell. In one embodiment the heat exchanger is composed by a coiled pipe. In one embodiment, the heat exchanger will be composed by a bioreactor's jacket. In both cases, the set of baffles will be part of the heat exchanger. In one embodiment, any of those heat transfer systems may be combined with an additional external heat exchanger located on an external recirculation loop. The culture broth will be pumped through the external recirculation loop being the external heat exchanger part of it. An external recirculation loop containing a heat exchanger (using tubes or plates) is depicted in Figures 6, 7a, 7b and 7c. This system is mainly made of stainless steel or with plastic material.

Claims

1 . Device for improving the oxygen and heat transfer suitable for a single-use bioreactor (SUB) characterized in that it comprises: a) Aeration system,

b) heat exchange system, and

c) baffles system as part of the SUB shell that adapt the shape of the flexible single-use bioreactor.

2. The device of claim 1 , wherein heat exchange system is coupled to an external recirculation loop.

3. The device of claim 1 , wherein heat exchange system comprises a device harbored into baffles and shell of SUB that acts as heat exchanger and, at the same time, as a holder of the vessel.

4. The device of any of the previous claims, wherein aeration system comprises: a) Microbubble generation system, and

b) Agitation means.

5. The device of claim 4, wherein the agitation means is a mechanical shaft.

6. The device of claim 4, wherein the microbubble generation system is integrated in the shaft

7. The device of claim 4, wherein the microbubble generation system is a disc with pores.

8. The device of claim 4, wherein the microbubble generation system is integrated in the shaft and a disc with pores.

9. The device of any of the claims 4-8, wherein the pore sizes of the microbubble generation system range from 0,5 microns up to 1 mm.

10. The device of any of the claims 4-9, wherein the construction material of any of the microbubble generation system comprises sintered metal, glass, ceramic or plastic particles.

1 1. The device of any of the claims 3-10, wherein the heat exchange system comprises a bioreactor jacket that has baffles as a part of it.

12. The device of any of the claims 3-10, wherein the heat exchange system comprises a coiled pipe located into the jacket that has the baffles as a part of it.

13. The device of any of the claims 3-10, wherein the heat exchange system comprises a double tube, tubes and shell or plate and frame heat exchangers.

14. The device of any of the previous claims, wherein the heat exchange system is made at least of stainless steel, metal or plastic material.

15. Single use bioreactor characterized in that it comprises a device according any of the previous claims.