Tunable disruption of eukaryotic protoplasts that release intact organelles

Abstract

The present invention relates to an apparatus for disrupting cell walls-free cells without disrupting a biologically active compartment contained therein surrounded by lipid bilayers and a method of using said apparatus, wherein shear force generating means (S) generate a shear force region suitable for disrupting said cells. Optionally, a pump (P) for pumping an aqueous medium containing the cells at an adjustable flow rate or flow rate, preferably separately adjusting residence time and shear force, and a separating device (C) for separating the at least one released bioactive compartment and / or the bioactive liquid phase around it from the debris are combined. The equipment is an integral part of a stand-alone system or production line. The apparatus and methods are capable of producing products comprising high yields of bioactive compartments that exhibit high biological activity, particularly in energy regeneration and / or protein synthesis.

Description

Technical Field

[0001] The present invention relates to an apparatus for breaking cell-wall-free cells without breaking the biologically active compartments surrounded by lipid bilayers contained therein and a method for using the apparatus, wherein a shear force generating device (S) generates a shear force region suitable for breaking the cells. Optionally, a pump (P) and a separation device (C) are combined, the pump (P) being used to pump an aqueous medium containing the cells at an adjustable flow rate and / or flow rate, and the separation device (C) being used to separate the broken cells and their fragments from at least one released biologically active compartment and / or the biologically active liquid phase surrounding it. The apparatus is an integral part of an independent system or production line. The apparatus and method are capable of producing a composition containing a high yield of biologically active compartments, which exhibit high biological activity, particularly in energy regeneration and / or protein synthesis. Background Art

[0002] In the technical field of cell culture, cell lysates are produced from living cells for cell-free production and processing, such as cell-free production and processing of recombinant proteins, using different methods to produce lysates from cells. In particular, for protein biosynthesis based on natural systems, for various reasons, it is desirable to use cell-free systems to avoid: subsequent difficulty in extracting protein from living cells, (long) separate cell line development for each desired target protein, long culture time, low protein productivity. Therefore, it is desirable to extract desired mechanisms from the cells, such as protein mechanisms, and use them for recombinant protein biosynthesis. For cell-free protein synthesis, it is necessary to extract microsomal vesicles derived from endoplasmic reticulum and Golgi apparatus, promote the formation of disulfide bonds, glycosylation and co-translation integration of membrane proteins. In addition, it is also desirable to extract mitochondria that provide energy for the biosynthesis of proteins. In order to achieve high yield and rapid biosynthesis of proteins, high concentrations of the above-mentioned biologically active compartments are advantageous. In the prior art, particularly homogenization methods (such as using Dounce homogenizer (Braun, Melsungen, Germany)) are used to break cell-free cell walls. Thereafter, the unbroken cells, nuclei and cell membrane fragments are removed to obtain the desired lysate for protein synthesis. However, this device is not suitable for serial production and is not scalable, so the lysate yield obtained is low. In addition, valve homogenization produces particles in the range of 0.5-1 μm (Zhang et al., Table 1, Figure 3), which is expected to damage the desired microsomes and undesired nuclei. In particular, the presence of plant DNA affects the biosynthesis of the recombinant protein, because the plant DNA will also be targeted and will produce natural plant proteins instead of the recombinant protein target. WO9712959A1 discloses a method for breaking cultured cells lacking cell walls, which comprises passing cells suspended in a culture medium through a low-pressure impact jet device, with the purpose of separating naturally occurring products (such as proteins, polysaccharides, recombinant proteins and viruses). However, the method and the device cannot separate biologically active compartments and are significantly different from the technical solution of the present invention. Summary of the invention

[0003] Therefore, an object of the present invention is to provide a method and apparatus for adjustably breaking eukaryotic cells, particularly eukaryotic (micro) protoplasts to release complete organelles (e.g., mitochondria and / or microsomes). An object is to provide an apparatus and method that allows to break (micro) protoplasts in an efficient, scalable and tandem manner without destroying organelles. Another object is to provide an apparatus and method that allows to adjust and regulate shear forces and residence time independently of each other on the relevant magnitude. Another object of the present invention is to provide sterilizable equipment and a method that can carry out aseptic closed processing.

[0004] Therefore, the present invention provides a technical solution as described in detail below, which has the advantage that it realizes mutually adjustable / adjustable shear force and residence time by rotor speed and / or flow rate and / or flow rate, so that it is in the corresponding magnitude. According to the device and method described in the present invention, it is also possible to realize an expandable closed and sterile system. Therefore, there is no available device and method so far to allow the scale of cell disruption and ensure the release of biologically active compartments. In addition, the devices for particle processing, sonication, homogenizers or mills known so far, or other cell disruption devices in series, can not provide adjustable shear force of the correct magnitude. Continuous shear force generating devices, such as rotor-stator-mixers, are generally helpful for pumping fluids, such as liquid media containing cells described in the present invention, and can be used without external feed pumps. The device described in the present invention provides independent control of shear and residence time for the first time, realizing a reasonable balance between pumping speed and shear force. Thereby, gentle disruption of cells (especially cell cultures) is provided, and the gentle disruption of the cells provides complete organelles, such as biologically active compartments. The advantages of the present invention are that the lysate obtained by carrying out the method by the apparatus is improved and faster protein production is achieved with a higher protein yield.

[0005] A first aspect of the invention relates to a device for disrupting cell wall-less cells (preferably (micro)protoplasts and / or spheroplasts) without disrupting the biologically active compartments contained therein surrounded by a lipid bilayer, the device comprising:

[0006] - a shear force generating device (S) for generating a shear force zone suitable for disrupting said cells,

[0007] - a tank A for providing the cell, preferably a plant cell, more preferably a cell of a plant of the genus Nicotiana of the family Solanaceae, most preferably the plant is Nicotiana tabacum, most preferably a BY-2 cell line from Nicotiana,

[0008] - optionally, a pump (P), said pump (P) being located upstream of (S) or downstream of (S) for pumping the aqueous medium comprising said cells at an adjustable flow rate and / or flow rate,

[0009] - at least one conduit connecting tank A to the shear force generating device (S), and / or at least one conduit connecting (S) to tank B,

[0010] - a tank B for collecting the product from the previous step, said product comprising at least one released biologically active compartment, and

[0011] - separation means (C) for separating the disrupted cells and their fragments from the at least one released biologically active compartment and / or the biologically active liquid phase surrounding it.

[0012] In an embodiment of the device of the present invention, the device comprises a shear force generating device (S) and a pump (P) located upstream of (S) or downstream of (S), wherein the pump (P) is used to pump the aqueous medium (S) containing the cell wall-free cells at an adjustable flow rate and / or flow rate. Figure 1 ). Figure 1 Some possible embodiments of the combination of the device (S) according to the invention with an external pump (P) are shown. However, the device is suitable for the method according to the invention and the product according to the invention can be obtained without the external pump (P), because the residence time as defined herein is adjustable using only the device (S). In this embodiment, the residence time (VZ) is the quotient of the internal volume Vi of the shear generating device (S) and the flow rate through the shear generating device (S). All embodiments relating to the residence time, either alone or in combination with the preferred shear force generating device (S) (centrifugal pump or rotor-stator system), apply accordingly to the device of the invention without an external pump. A person skilled in the art following the teachings of the invention will be able to adjust the setting of (S) accordingly to a suitable residence time.

[0013] In another embodiment of the apparatus according to the invention, the shear force generating device (S) is particularly suitable and designed to generate shear by stirring the aqueous medium. Fig. 9 As shown, different devices (S) for generating shear are known. However, only those devices that mediate shearing through a fluid (preferably an aqueous medium containing the cell wall-free cells according to the present invention, and in particular by stirring the fluid) fall within the meaning of the present invention.

[0014] In another embodiment of the apparatus according to the invention, the shear force generating device (S) is a centrifugal pump, a high shear force mixer or a rotor-stator mixer with a toothed design or with a bladed design.

[0015] In an embodiment, a device for disrupting cell wall-less cells (preferably (micro)protoplasts and / or spheroplasts) without disrupting the biologically active compartments contained therein surrounded by a lipid bilayer, the device comprising:

[0016] - a shear force generating device (S), preferably a shear force generating device (S) adapted and designed to generate shear by stirring an aqueous medium, preferably a rotor-stator system, more preferably an IKA magic lab or an IKA UTL25,

[0017] - a tank A for providing the cell, preferably a plant cell, more preferably a cell of a plant of the genus Nicotiana of the family Solanaceae, most preferably the plant is tobacco, most preferably a BY-2 cell line from tobacco,

[0018] a pump (P), located upstream of (S) or downstream of (S), for pumping the aqueous medium containing the cells at an adjustable flow rate and / or flow rate,

[0019] - at least one conduit connecting tank A to the shear force generating device (S), and / or at least one conduit connecting (S) to tank B,

[0020] - a tank B for collecting the product from the previous step, said product comprising at least one released biologically active compartment, and

[0021] - separation means for separating the disrupted cells and their fragments from the at least one released biologically active compartment and / or the biologically active liquid phase surrounding it.

[0022] The device is suitable for adjusting different residence times defined herein. More preferably, the above-mentioned rotor-stator system can be adjusted to a residence time of less than 75 seconds, less than 74 seconds, less than 73 seconds, less than 72 seconds, less than 71 seconds, less than 70 seconds, less than 65 seconds, less than 60 seconds, less than 55 seconds, less than 50 seconds, less than 45 seconds, less than 40 seconds, less than 30 seconds and less than 25 seconds, less than 20 seconds, less than 10 seconds. In this example and for specific equipment design and method, the residence time is less than 10 seconds, less than 9, 8, 7, 6, 5, 4, 2, 1 seconds.

[0023] In an embodiment, a device for disrupting cell wall-less cells (preferably (micro)protoplasts and / or spheroplasts) without disrupting the biologically active compartments contained therein surrounded by a lipid bilayer, the device comprising:

[0024] - a shear force generating device (S), preferably a shear force generating device (S) adapted and designed to generate shear by stirring an aqueous medium, preferably a centrifugal pump, more preferably a PuraLev i100SU,

[0025] - a tank A for providing the cell, preferably a plant cell, more preferably a cell of a plant of the genus Nicotiana of the family Solanaceae, most preferably the plant is tobacco, most preferably a BY-2 cell line from tobacco,

[0026] a pump (P), located upstream of (S) or downstream of (S), for pumping the aqueous medium containing the cells at an adjustable flow rate and / or flow rate,

[0027] - at least one conduit connecting tank A to the shear force generating device (S), and / or at least one conduit connecting (S) to tank B,

[0028] - a tank B for collecting the product from the previous step, said product comprising at least one released biologically active compartment, and

[0029] - separation means (C) for separating the disrupted cells and their fragments from the at least one released biologically active compartment and / or the biologically active liquid phase surrounding it.

[0030] The device is suitable for adjusting the different residence times defined herein. More preferably, the above-mentioned centrifugal pump can be adjusted to a residence time of less than 75 seconds, less than 74 seconds, less than 73 seconds, less than 72 seconds, less than 71 seconds, less than 70 seconds, less than 65 seconds, less than 60 seconds, less than 55 seconds, less than 50 seconds, less than 45 seconds, less than 40 seconds, less than 30 seconds and less than 25 seconds, less than 20 seconds, less than 10 seconds. In this example and for specific equipment design and method, the residence time is less than 10 seconds, less than 9, 8, 7, 6, 5, 4, 2, 1 seconds.

[0031] In an embodiment of the device according to the invention, the shear force generating means (S) (synonym, shear generating means (S)) is adapted to and controllably generate shear forces (synonym, shear) sufficient to disrupt cells without damaging the lipid bilayer of the at least one biologically active compartment contained therein. Preferably, the shear force generating means (S) is a shear force generating means (S) that mediates the shear force through a fluid (particularly an aqueous medium). This is different from other devices that can mediate shear forces through solids ( Fig. 9 ). More preferably, the shear force generating device (S) of the device according to the invention is suitable and designed to generate shear forces by stirring an aqueous medium (= liquid) containing cells without cell walls. Such a device comprising said device (S) is also suitable for the method according to the invention, wherein said shear forces are applied by stirring the aqueous medium containing said cells, preferably by means of a centrifugal pump, a high shear mixer or a rotor-stator-mixer with a toothed design or with a blade design.

[0032] Preferably, the shear force used does not damage the "biologically active content". Preferably, (S) is a centrifugal pump, a high shear mixer or a rotor-stator mixer with a toothed design or with a bladed design ( Figure 2 ). A rotor-stator system with a rotor having a toothed design consists of a disk with one or more rings of teeth mounted on a rotating shaft. One or more sets of concentric tooth rings together with the stator constitute the rotor-stator system ( Figure 2 B) However, the shear force generating components of the shear force generating device (S) may be designed differently.

[0033] In one embodiment, the shear force generating device (S) is a rotor-stator system ( 2018). Such rotor-stator systems are also suitable for stand-alone systems. Suitable rotor-stator systems are commercially available, for example IKA magic LAB and IKA UTL 25inline, which are adjustable in the speed range [min-1] of 3,000-26,000, 10000-24000, 3000-20,000 depending on the design, and can be adjusted to a throughput of 2-20 l / min, 5-15 l / min, 5-12 l / min, 11.6 l / min up to the following in [l / h] Each: 1-1500 [l / h], 1-100 [l / h], 10-1000 [l / h], 10-200 [l / h], 30-200 [l / h], 50-200 [l / h], 1-20 [l / h], up to 1500, 1-20 [kg / h], and / or adjustable to a peripheral speed [m / s] of 5-40, 5-30, 5-23 and 6-16. Within the meaning of the present invention, the speed of the rotor-stator system is preferably at least 3000 rpm, at least 4000 rpm, at least 4500 rpm, 5000 rpm, at least 6000 rpm, at least 7000 rpm, at least 8000 rpm, at least 9000 rpm, at least 10000 rpm or more for an aqueous medium, or any other speed between the above ranges. In particular, for the rotor-stator system, preferably, the rotor tip speed is in the range of at least 4 m / sec to equal to or less than 32 m / sec, and can be adjusted in combination with a speed of at least 6000 rpm to obtain a residence time of less than 25 seconds (Example 9).

[0034] magic Used for mixing and dispersing, especially in the chemical and food industries, and therefore a fluid-mediated shear force generating device ( Fig. 9 ). magic Suitable for developing new processes and scaling up the same methods to large-scale production, because magic Many parameters are the same as those of the IKA production machine series 2000. It is ideally suited for batch applications, but is also available in different versions for tandem applications. The basic unit of the tandem machine is equipped with a single-stage dispersing module ULTRA- UTL. A wide range of process parameters can be determined, such as speed, shear rate, temperature, pressure and time. Due to the high rotor tip speed of up to 40 m / s and the high energy input during the rotation of the medium into the product, optimal mixing, dispersing and grinding results can be achieved. During the mixing process, and due to the energy input into the medium containing the cells, the cells contained therein are disintegrated.

[0035] Said speed depends on the scale of the crushing process according to the invention and on the device used, a stand-alone system or preferably a cascaded embodiment of the device according to the invention. Thus, the present invention encompasses any adjusted speed, optionally or preferably in combination with any adjusted speed of the pump, in order to obtain the desired directly obtained "processed product" as defined herein and performed by a device capable of realizing a scaled crushing process.

[0036] The above-mentioned rotor-stator system, preferably IKA magic LAB and IKA UTL 25inline, can be adjusted to different residence times as defined herein. More preferably, the above-mentioned rotor-stator system, preferably IKA magic LAB and IKA UTL25inline, can be adjusted to such a residence time, wherein the residence time is the duration for which the cells in the aqueous medium are retained in the region of shear forces and / or turbulence, which is sufficient to disrupt the cells without disrupting the at least one biologically active compartment surrounded by a lipid bilayer contained therein. Preferably, the residence time for the above-mentioned rotor-stator system, preferably IKA magic LAB and IKA UTL 25inline, is less than 25 seconds, less than 20 seconds, less than 10 seconds, less than 9, 8, 7, 6, 5, 4, 2, 1 seconds.

[0037] Preferably, the residence is reduced as much as possible, which can be achieved by changing the design of (S) in terms of geometry and volume in combination with the design of the pump, respectively. Therefore, it is clear to those skilled in the art that in order to scale up, the device and its components must be adjusted to maintain the desired residence time. In Example 9, it is shown that for an IKAmagic lab with 1x teeth, a residence time of 12 seconds is achieved by a flow rate of 150 mL / min and a rotor speed of 31.4 m / s, which is sufficient to effectively disrupt cells and provide active lysate for high protein yield production.

[0038] The "processed product obtained directly" by the method according to the invention (preferably performed using the apparatus according to the invention) is an aqueous medium downstream of the shear force generating device after at least one disruption cycle according to the invention and comprises disrupted cell wall-free cells, preferably disrupted microprotoplasts, protoplasts and / or spheroplasts. The directly obtained processed product preferably comprises at least one biologically active compartment and its surrounding biologically active liquid (= liquid medium). The liquid and / or at least one compartment realize at least ATP synthesis and / or energy regeneration and / or at least one protein biosynthesis-related process (expression, transcription, translation, translocation, protein folding and / or protein modification). The "biologically active content" comprises: biologically active molecules such as enzymes, soluble proteins and / or small molecules derived from the cytosol (= surrounding biologically active liquid) and / or mitochondria, microsomes, nuclei, Golgi apparatus and / or endoplasmic reticulum (ER) of the cell wall-free cells. Therefore, a "functional product" is defined by its ability to perform or allow cell-free protein biosynthesis of recombinant proteins. This can be demonstrated by using the method described herein. Preferably, the functional product comprises at least one released biologically active compartment (part of the "biologically active content") capable of ATP synthesis, energy regeneration, capable of at least one protein biosynthesis-related process (expression, transcription, translation, translocation, protein folding and / or protein modification). More preferably, the "functional product" is separated from the debris (nucleus, cell membrane debris and (micro) protoplast debris) and exhibits the ability of cell-free protein biosynthesis of recombinant proteins. Preferably, the production capacity is more than 10 ml / day to 100 L / day, up to 1000 L / day. Preferably, the device (alone or as an integral component of production) is automatically operated.

[0039] In another embodiment, the shear generating device is a centrifugal pump, e.g. puraLevi100 SU (e.g. PLD-i100SU.1, PLD-i100SU.5, PLD-i100SU.2, PLD-i100SU.3). The pump is based on the principle of magnetic levitation, the pump impeller is suspended, contactless, inside a sealed housing and driven by the magnetic field of the electric motor. The pump head is disposable, sterile, or sterilizable before use, and suitable for biological materials. The flow rate is adjusted by the rotor speed and also allows the pressure to be controlled. The maximum flow rate is 17L / min, the maximum pressure difference is 2bar / 29psi, and the maximum medium viscosity is <20cP. The flow rate of the centrifugal pump (preferably for aqueous media) is preferably adjustable within the following ranges: 0.00-50 mL / min, 0.0 to 40 L / min, 0.0 to 30 L / min, 0.0 to 20 L / min, at least 2.5 L / min up to 50 L / min to 20 L / min, at least 5.0 L / min up to 50 L / min to 20 L / min, at least 7.5 L / min up to 50 L / min to 20 L / min, at least 10 L / min up to 50 L / min to 20 L / min, at least 12.5 L / min up to 50 L / min to 20 L / min, at least 15 L / min to 20 L / min, at least 17.5 L / min to 20 L / min. This range of flow rates can be achieved with rotor speeds in the following ranges: 3000 rpm to 10000 rpm, 3000 rpm to 9000 rpm, 4000 rpm to 9000 rpm, 4000 rpm to 8000 rpm, 4000 rpm to 7000 rpm, 4000 rpm to 6000 rpm, and 4000 rpm to 5000 rpm.

[0040] In particular, in another embodiment, the rotor speed of the centrifugal pump as the shear generating device (S) is in the range of 500rpm to 3000rpm, 500rpm to 2500rpm, 500rpm to 2000rpm, 500rpm to 1500rpm and 500rpm to 1000rpm.

[0041] As disclosed herein, the centrifugal pump is a shear generating device (S) according to the present invention, which is suitable for adjusting the rotor tip speed from equal to or greater than 2 m / s to 50 m / s. The rotor speed range is 3000 rpm to 9000 rpm, resulting in a rotor tip speed of 6.8 m / sec to 20.3 m / sec (Example 9). In particular, the rotor speed is adjusted to 500 rpm, resulting in a rotor tip speed of 1.1 m / sec (m / sec is synonymous with m / s), which is combined with a residence time in the range of 25 seconds to less than 75 seconds to obtain an effective lysate in the method (Example 9).

[0042] The above centrifugal pumps, such as puraLev i100 SU (e.g. PLD-i100SU.1, PLD-i100SU.5, PLD-i100SU.2, PLD-i100SU.3) and the centrifugal pump of preferred embodiment 9 can be adjusted to different residence times as defined herein. More preferably, the above centrifugal pump, e.g. puraLev i100 SU (e.g. PLD-i100SU.1, PLD-i100SU.5, PLD-i100SU.2, PLD-i100SU.3) and preferably the centrifugal pump of embodiment 9 can be adjusted to such a residence time, which is the duration of time that the cells in the aqueous medium are retained in the region of shear force and / or turbulence, which is sufficient to disrupt the cells without disrupting the at least one biologically active compartment surrounded by a lipid bilayer contained therein. Preferably, for the above-mentioned centrifugal pump (e.g. The residence time of the centrifugal pump of puraLev i100SU (e.g. PLD-i100SU.1, PLD-i100SU.5, PLD-i100SU.2, PLD-i100SU.3)) and preferably embodiment 9 is less than 75 seconds, less than 74 seconds, less than 73 seconds, less than 72 seconds, less than 71 seconds, less than 70 seconds, less than 65 seconds, less than 60 seconds, less than 55 seconds, less than 50 seconds, less than 45 seconds, less than 40 seconds, less than 30 seconds and less than 25 seconds. Preferably, the residence time is reduced as much as possible, which can be achieved by changing the design of (S) in terms of geometry and volume in combination with the design of the pump, respectively. However, as described above, the present invention provides a device and a technical solution with the following advantages: it achieves independently adjustable / adjustable shear force and residence time by means of rotor speed and / or flow rate and / or flow velocity, so that they are at corresponding orders of magnitude. The embodiment of the apparatus according to the invention comprising a centrifugal pump with an external feed pump enables for the first time independent control of shear and residence time and a reasonable balance between pumping rate and shear force.

[0043] The device is adjusted to a residence time of less than about 25 seconds (Example 9, Table 3) by a centrifugal pump (e.g., PuraLev i100SU, with drive: IPD-100.3-03-02, and pump head: DCP-200.3, rotation speed of 500 rpm, flow rate of 56.4 mL / min, combined with a rotor tip speed of 1.1 m / s), sufficient to effectively disrupt the cell wall-free cells (preferably from the BY-2 cell line) contained in the aqueous medium, and sufficient to provide such an active lysate, which is capable of performing at least one process related to protein biosynthesis (including expression, transcription, translation, translocation, protein folding and / or protein modification), and capable of ATP synthesis and / or energy regeneration, as shown in detection number 1 in Table 3 ( Fig.10 , 11 and 12). Due to the fewer broken cells without cell walls ( Fig.13 , Column C, No. 1), the resulting functional product contains a relatively low content of released bioactive compartments (part of the "bioactive content") that are capable of ATP synthesis, energy regeneration and / or at least one protein biosynthesis-related process, as defined herein. However, it is feasible to repeat the "cycle run" defined herein to increase the amount of released bioactive components and thereby increase the activity of the lysate. However, the same device is adjusted to a residence time of less than 71 seconds or to about 25 seconds (Example 9, Table 3), a rotation speed of 4500 rpm or 9000 rpm, a flow rate of 20 mL / min or 56.4 mL / min, and a rotor tip speed of 10.1 or 20.3 m / s, respectively, which is sufficient to effectively break the cell wall-free cells (preferably from the BY-2 cell line) contained in the aqueous medium and provide an active lysate for high protein yield production (Table 3, Fig.10 , 11 1 and 12 in test numbers 2-5). Therefore, it is clear to those skilled in the art that, in order to scale up, the device and its components must be adjusted to maintain the desired residence time as shown in this example.

[0044] The i100 disposable pump is designed for demanding fluid applications in bioprocessing, where extremely low shear, pulsation-free and continuously controlled flow ensures the highest product yield. Based on the principle of magnetic levitation, the pump impeller is suspended, contactless, inside a sealed housing, driven by the magnetic field of the electric motor, producing a continuous and smooth flow, and is therefore a fluid-mediated shear force generating device (S) ( Fig. 9 ). The pump head is disposable and can be replaced in seconds. Fluid flow and pressure are precisely controlled by electronically regulating the impeller speed. The motor and pump controller are integrated into the drive housing, significantly reducing the footprint.

[0045] (2018) provide guidance for the scalability of rotor-stator systems and the calculation of batch and cascade forces (pp. 8-10), and compare the rotor-stator systems to centrifugal pumps. Zhang et al. disclose different embodiments of high shear mixers (HSMs) that are suitable as shear generating devices according to the present invention. In particular, Zhang et al. Figure 1 , which represents different geometrical variations of commercial high shear mixers, such as toothed tandem units (Ytron-Quadro z), blade screen tandem units (Silverson 150 / 250MS), radial discharge units (left-SilversonL4R, right-VMI Rayneri) and axial discharge units (Greerco 1.5HR). These HSMs can be used in tandem production or as stand-alone systems (batch units), as described by Espinoza et al.

[0046] Preferably, the production line has a production capacity of at least 10 ml / day, wherein "day" is the production day, which is defined as the time from the end of cell culture until the "functional product" is obtained.

[0047] In another embodiment of the device according to the present invention, the pump is suitable for adjusting the residence time of the aqueous medium containing cells in (S) independently of the shear force adjusted and generated by (S), wherein the residence time is the duration of the shear force applied to the aqueous medium containing the cells. The "residence time" can be controlled or modulated by adjusting the flow rate and / or flow rate of the pump (P). In combination with the desired geometry of the shear force generating device (preferably a rotor-stator-mixer), the residence time can be controlled or modulated by adjusting the flow rate and / or flow rate of the pump, without relying on the adjustment of the shear force of (S). Therefore, the flow rate and / or flow rate are used to manipulate the residence time. As shown in Table 2 in Example 2, the residence time in the rotor-stator system is adjusted to 45 seconds by the flow rate of the pump.

[0048] Example 9 shows that a shear force generating device (S) (e.g. a rotor-stator-mixer or a centrifugal pump with an external feed pump) enables independent control of shear and residence time and allows a reasonable balance between pumping speed and shear force. Thus, gentle disruption of cell wall-free cells (preferably from the BY-2 cell line) is provided. Figure 10-13), in particular gentle disruption of cell cultures, providing intact organelles (e.g., biologically active compartments), most preferably gentle disruption of BY-2 cell lines. The invention has the advantage that the lysate obtained by performing the method with the device is improved and allows faster protein production with higher protein yields.

[0049] Flow rate specifies the rate at which fluid is transported through a system, which is the amount of fluid moved per unit time (e.g., mL / min). Flow rate specifies the speed at which a fluid moves through a system, measured as distance per unit time (e.g., m / s). Both flow rate and flow rate are related to the rotor-stator system. Flow rate, combined with the geometry of the rotor-stator shear region (particularly the volume of the shear region), determines the residence time in the shear region. Similarly, flow rate, combined with the geometry of the rotor-stator shear region (particularly the path length through the shear region), determines the residence time in the shear region.

[0050] Preferably, the residence time is the duration of applying the shear force to the aqueous medium containing the cells, which is sufficient to break the cells without breaking the at least one biologically active compartment surrounded by a lipid bilayer contained therein. More preferably, the residence time is the duration of time that the cells are retained in the shear force and / or turbulent region. In order to accelerate the fragmentation and the entire production process, the residence is as short as possible. In particular, the residence time is as short as possible so as to accelerate the fragmentation and the entire production process by using the device according to the present invention. In Example 2 (Table 2), for the rotor-stator system, the residence time (VZ) has been adjusted to 45 seconds, wherein the residence time (VZ) is the quotient of the internal volume Vi of the shear generating device and the flow rate (R) of the pump (see Example 9).

[0051] Preferably, the residence time is less than 25 seconds, less than 20 seconds, less than 10 seconds. In the present example, and for a specific equipment design and processing, the residence time is less than 10 seconds, less than 9, 8, 7, 6, 5, 4, 2, 1 seconds. In particular, in the present embodiment 9, and for the specific equipment and processing design of the centrifugal pump (S), the residence time is greater than 70 seconds and less than 71 seconds, about 70.5 seconds and about 25 seconds, and for the specific equipment and processing design of the rotor-stator system (S), the residence time is approximately less than 25 seconds. Preferably, the residence is shortened as much as possible, which can be achieved by changing the design of (S) in terms of geometry and volume in combination with the design of the pump, respectively. Therefore, it is clear to those skilled in the art that in order to scale up, the equipment and its components must be adjusted to maintain the same residence time as shown in the present embodiment.

[0052] The residence time is the duration of applying the shear force to the aqueous medium containing the cells, particularly during the method according to the present invention, which is sufficient to break the cells without breaking the at least one biologically active compartment surrounded by a lipid bilayer contained therein. At the same time, under the conditions, an active lysate for high protein yield production is provided (see Examples 2 and 9).

[0053] "As short as possible" should be understood in the context of the geometric shapes of the various shear force generating devices (S) according to the present invention and considering the directly obtained processed products as defined herein. In addition, "as short as possible" considers various devices including a separate shear force generating device (S) or a shear force generating device (S) combined with an external feed pump. Since each device defines the limits of the adjustable values, it is clear to those skilled in the art that "as short as possible" means depending on the final device settings. In particular, the residence time is less than 75 seconds, less than 74 seconds, less than 73 seconds, less than 72 seconds, less than 71 seconds, less than 70 seconds, less than 65 seconds, less than 60 seconds, less than 55 seconds, less than 50 seconds, less than 45 seconds, less than 40 seconds, less than 35 seconds, less than 30 seconds and less than 25 seconds, less than 20, less than 10 seconds, less than 9, 8, 7, 6, 5, 4, 2, 1 seconds.

[0054] The pump is preferably a self-priming and / or self-sealing anti-backflow, positive displacement (e.g. peristaltic) pump with an adjustable pump speed. Preferably, the pump controls the flow rate and / or flow rate so that an isotonic or hypertonic aqueous medium is transported into S, particularly into the space between the rotor and the stator. One embodiment includes a peristaltic pump, for example, using a Watson Marlow 120u / dv, whose pump speed is adjustable in the range of 0 to 200rpm (maximum). In Example 9, a peristaltic pump Hei-Flow ultimate 120 with a SPquick 1.6 head was used, whose speed was adjustable in the range of 5 to 120rpm and whose flow rate was adjustable in the range of 0.83-861ml / min. However, the pump speed (rpm) of the pump is at least 10 rpm, at least 15 rpm, at least 20 rpm, at least 50 rpm, at least 100 rpm, 150 rpm, at least 200 rpm, at least 250 rpm, at least 300 rpm, at least 500 rpm or any integer between these values. Peristaltic pumps do not contain valves, seals or glands that cause clogging or corrosion, and are therefore the simplest pumps. The aqueous medium is in contact only with the inner wall and the inner space of the conduit. This ensures the sanitation of the process, and there is no risk of the pump contaminating the aqueous medium or the aqueous medium contaminating the pump. Peristaltic pumps can operate at a temperature of at least -10°C to 50°C and are allowed to operate at a controlled temperature. The pump is suitable for viscous, shear-sensitive aqueous media, even containing solid components. According to the present invention, the aqueous medium includes biological materials containing cells and cell wall-free cells, which are not considered solid. According to the manufacturer's information, the pump head can be adjusted to adapt to 1.6 mm wall tubes with a size of 0.5 mm inner diameter to 4.8 mm inner diameter. It is clear to those skilled in the art that the flow rate may vary over time due to changes in the aqueous medium, the viscosity of the aqueous medium, the processing temperature, the inlet and outlet pressures, the system configuration and / or the pipeline properties. In general, the flow rate is between 0.001 and 170 ml / min. Examples of flow rates for different pipeline settings are shown below:

[0055]

[0056] In this embodiment 9, the flow rate is adjusted to a range of 20 mL / min to 150 mL / min in the method of the present invention. Therefore, preferably, the flow rate is adjusted to the following range in the method of the present invention: greater than or equal to 20 mL / min to less than or equal to 150 mL / min, preferably greater than or equal to 20 mL / min to less than or equal to 120 mL / min, greater than or equal to 20 mL / min to less than or equal to 100 mL / min, greater than or equal to 20 mL / min to less than or equal to 80 mL / min, and more preferably greater than or equal to 20 mL / min to less than or equal to 60 mL / min.

[0057] However, it is recommended to determine the flow rates under the operating conditions of the method and apparatus according to the invention before implementing them. If combined with another pump, the pump speed can be adjusted accordingly. In particular, in the case of scale-up of the crushing method according to the invention, and depending on the respective equipment used, a stand-alone system or preferably a series embodiment of the equipment, it will be necessary to adjust the pump size, process parameters and flow rates. Therefore, the present invention covers any adjusted pump speed, preferably in combination with any adjusted speed of the shear force generating device, in order to obtain the desired directly obtained "processed product" as defined herein, and performed by an apparatus that allows the scale-up of the crushing method.

[0058] Predicting turbulence and shear in shear force generating devices of different scales is difficult. In liquid-liquid methods (which are proposed to be applied because cells in culture and due to their high water content can be considered as "liquids"), highly localized intense turbulence and shear in the shear force generating devices (particularly rotor-stators and HSMs) lead to multiple disruption mechanisms, resulting in the destruction of droplets (Zhang et al., citing Kolmogoroff cited therein). According to the present invention, the method can be transferred to cells without cell walls.

[0059] In another embodiment of the device according to the present invention, the device is a monolithic device of a production line (preferably a closed and sterile production line, more preferably a production line for a plant cell line, particularly a plant cell-free lysate production line). In another embodiment of the device according to the present invention, the device is a stand-alone system, preferably a closed and sterile stand-alone system, more preferably a system for crushing plant cells. Preferably, the plant cell is a plant cell line of tobacco, most preferably the BY-2 cell line from tobacco.

[0060] Thus, the device may be a stand-alone system, such as Figure 1As shown. In the independent system, tank A provides the starting material. The starting material is an aqueous medium containing cells without cell walls, more preferably a cell culture of animal cells, plant or fungal (micro) protoplasts and bacterial spheroplasts, and is suitable for large-scale culture. The cell culture is preferably broken as defined herein after pretreatment, more preferably after evacuation of the plant cell line. Tank A is connected to the shear force generating device (S) through a conduit, and the downstream conduit connects the shear force generating device (S) to tank B. The pump (P) is optionally integrated into the independent system and can be placed upstream or downstream of the shear force generating device (S). In the independent system, the separation device (C), preferably a centrifuge, is located downstream of the shear generating device (S), in particular downstream of the pump (if the pump is integrated). The independent system is capable of allowing the cyclic operation of the crushing. "Cyclic operation" refers to a predetermined condition of repeating the speed of the shear generating device (S) and optionally a combination of the speed of the pump. Applying force more than once to the aqueous medium containing the biological material. According to the present invention, the aqueous medium passes through the shear force area more than once, at least twice, three times, four times or more if necessary. The disruption according to the present invention may include the following steps at least once, at least twice, at least three times, at least four times or more: providing an aqueous medium containing cells without cell walls (preferably in tank A), moving the aqueous medium into a shear force generating device (S), applying shear force to the aqueous medium containing the cells, and continuing such a residence time, the residence time is sufficient to disrupt the cells without disrupting the at least one biologically active compartment when the aqueous medium passes through S, and disrupting the cells without disrupting the lipid bilayer of the at least one biologically active compartment.

[0061] As already described herein, when a shear force generating device (S) without an external pump (P) is used, the device is also suitable for the present invention, and is also suitable for the method described herein and for obtaining the product described herein. The residence time as defined herein can also be adjusted by the device (S) alone. All embodiments involving the residence time alone or in combination with a preferred shear force generating device (S) (preferably a centrifugal pump or a rotor-stator system) are applicable to the device of the present invention without an external pump (P). By applying the technical teachings of the present invention, a person skilled in the art will be able to adjust the setting of (S) to a suitable residence time accordingly. One method of adjusting the same residence time setting is to repeat the cyclic operation of the crushing defined above. Therefore, under predetermined conditions of the speed of the shear force generating device (S), the "cyclic operation" is repeated to the aqueous medium containing the biological material. Therefore, the aqueous medium passes through the shear force zone more than once, at least twice, three times, four times or more times if necessary.

[0062] In an embodiment of the device according to the present invention, the device is an integral device of a production line ( Figure 1 Correspondingly applicable, wherein tank A, tank B and / or (C) are coupled to downstream or upstream components), it is possible to separate upstream tanks, assemblies and / or devices for upstream. Any type of pre-treatment known to the technician in the cell line production system field of cell bioreactor, biotechnology bioreactor and any species can be considered, such as microbial cell line, human cell line, yeast, fungi and plant. Each production line is preferably closed and aseptic production line, more preferably automated operation and control. More preferably, it is a production line for plant cell line, i.e. the plant cell line of tobacco, most preferably the BY-2 cell line from tobacco.

[0063] Preferably, the production line has a production capacity of at least 10ml / day, wherein "day" is the number of production days, which is defined as the time from the end of cell culture until the "functional product" is obtained. "Functional product" is defined by its ability to perform or allow the cell-free protein biosynthesis of recombinant protein. Preferably, the functional product comprises at least one released biologically active compartment, which can carry out ATP synthesis, energy regeneration, and / or at least one protein biosynthesis-related process (expression, transcription, translation, translocation, protein folding and / or protein modification). More preferably, "functional product" is separated from debris (cell membrane debris and (micro) protoplast debris), and shows the ability of cell-free protein biosynthesis of recombinant protein. Preferably, the production capacity is more than 10ml / day to 100L / day, up to 1000L / day. Preferably, the equipment (alone or as an integral component of production) is automatically operated.

[0064] The stand-alone system has a capacity of at least 100 ml, the capacity in the tank (A) is at least 1 L, at least 1 L, at least 5 L, at least 10 L, at least 100 L, at least 1000 L, and can be adjusted to any desired processing volume. In particular, the scale-up is adjustable depending on the predetermined volume of the tank (A), the volume that can be processed simultaneously by the shear generating device (S) and the volume that can be processed simultaneously by the optional pump (P).

[0065] Another aspect of the invention is a production line comprising an apparatus according to the invention, wherein tank (A) is placed downstream of at least one component for pre-treating biological material and the conduit connects tank (A) to (S), and wherein tank (B) is placed downstream of (S) and optionally, pump (P) is placed downstream or upstream of (S). The "pre-treatment" described herein includes any other treatment of the biological material defined herein in a production protocol applied in a production line or in a stand-alone system. The pre-treatment component may include a cell wall digestion process, supply of buffers, reagents, etc. Preferably, the at least one component is a device for de-vacuolization of a plant cell line or a tank for providing de-vacuolized plant material.

[0066] In one embodiment, when it is desired to repeat more than one crushing cycle, tank (B) and tank (A) are identical. This embodiment is applicable to the independent system as well as the production line. In the latter, the production line comprises the circulation crushing device with a suitable conduit and a mode for opening the circulation crushing device to continue downstream processing. After crushing is completed, the functional product defined herein is moved to a separation device (C), preferably a centrifuge. In a preferred embodiment, tank (B) is a device for separation, preferably a centrifuge. The centrifuge is suitable for separating at least one released (especially, complete) bioactive compartment surrounded by a lipid bilayer. Preferably, the lipid bilayer of the separated compartment is complete. Therefore, separation will not damage or break the released bioactive compartment.

[0067] Another aspect of the invention is a method for disrupting cell wall-less cells (preferably (micro)protoplasts and / or spheroplasts) comprising at least one biologically active compartment surrounded by a lipid bilayer (preferably, the cells range from equal to or greater than 0.5 μm to 200 μm, more preferably equal to or greater than 5 μm to 100 μm, 10 μm to 100 μm, more preferably equal to or greater than 10 μm to 70 μm, more preferably equal to or greater than 20 μm to 70 μm), the method comprising the following steps, preferably performed by using any device according to the invention,

[0068] - providing an aqueous medium comprising said cell-wall-free cells, preferably in tank A, preferably an aqueous medium comprising a biological material comprising cell-wall-free cells and preferably having a viscosity of less than 20 mPa s (at 21° C.) (less than 15 mPa s, less than 10 mPa s, in the range of 0.5 to 10 mPa s, preferably in the range of 0.5 to 5 mPa s), preferably provided in tank (A) of the apparatus according to the invention,

[0069] - moving said aqueous medium into the shear force generating device (S) as described herein, preferably at a predetermined speed as described herein for (S) and / or (P),

[0070] - optionally, regulating the flow rate and / or the flow rate of the aqueous medium by means of a pump (P) as described herein, preferably by means of a pump speed as described herein,

[0071] - generating a shear region, preferably a shear and / or turbulence region, within (S), more preferably in a centrifugal pump or a rotor-stator system,

[0072] - applying shear forces to the aqueous medium containing said cells for a residence time sufficient to disrupt said cells without disrupting said at least one biologically active compartment, preferably applying shear forces and / or centrifugal forces within said shear forces and / or turbulence zone,

[0073] - when said aqueous medium passes through (S), said cells are disrupted without disrupting the lipid bilayer of said at least one biologically active compartment,

[0074] - release of at least one intact biologically active compartment and its surrounding biologically active fluid, preferably into an aqueous medium,

[0075] - optionally, collecting at least one released intact biologically active compartment and its surrounding biologically active fluid, preferably in tank (B),

[0076] - separating the at least one released intact biologically active compartment and the biologically active liquid surrounding it, preferably by means of a separation device (C) according to the invention, preferably wherein at least the fraction of debris ((micro)protoplasts and / or spheroplast lipid bilayers) is removed,

[0077] - preferably obtaining a released fraction of the intact bioactive compartment and its surrounding bioactive fluid, and

[0078] - Optionally, isolating said at least one released intact biologically active compartment.

[0079] The method according to the invention is suitable for being carried out only by the described stand-alone system or production line. All embodiments of the described apparatus are correspondingly applicable to the method for crushing.

[0080] By "disrupting" cells is meant that the lipid bilayer of a cell without a cell wall (in particular, a protoplast and / or a microprotoplast or a spheroplast) is "broken" or "the cell is opened" so as to be able to release its biological contents, as defined herein. "Disruption" in the meaning of the present invention refers to a fluid-mechanical process by shear forces, which is significantly different from sonication, where acoustic energy is applied at different frequencies, such as ultrasonic frequencies (>20 kHz), to reduce the particle size in a sample. It is also significantly different from a homogenization process, where two (immiscible) liquids are mixed and treated to obtain a homogenous mixture, such as to obtain uniformly dispersed particles. By homogenization, the biologically active compartments are also disrupted ( Fig. 9 ).

[0081] Preferably, the range of the cell wall-free cells is equal to or greater than 5 μm to 200 μm, more preferably equal to or greater than 5 μm to 100 μm, 10 μm to 100 μm, more preferably equal to or greater than 10 μm to 70 μm, and more preferably equal to or greater than 20 μm to 70 μm. Preferably, the cells contain protoplasts and / or microprotoplasts or spheroplasts or other comparable biologically active structures of another species. Preferably, the aqueous medium containing the cell wall-free cells is substantially free of cellulose, substantially free of chitin, and / or substantially free of pectin. Preferably, the cell wall-free cells are derived from plant cells, fungal yeasts and / or eukaryotic cell lines, such as CHO. The complete biologically active compartments and the biologically active liquid surrounding them together form the functional product. "Biologically active liquid" comprises the cytosol of the cell wall-free cells (preferably (micro) protoplasts and / or spheroplasts) and the contents of the aqueous medium. The cytosol contains cell line-specific, species-specific components, such as enzymes, soluble proteins and / or small molecules. The aqueous medium may contain nutrients and other process-specific components. The separation of the functional product is carried out by the separation device (C) described in the present invention, preferably by centrifugation. The separation can be carried out once or multiple times. In addition, the separation step can be carried out before, after or in place of the separation. Preferably, the separation is carried out after the separation, more preferably by filtering and / or centrifuging the separated functional product. The difference between separation and separation is that the purpose of separation is to remove debris (cell membrane debris and protoplast debris) from the functional product, while the purpose of separation is to separate and potentially concentrate the desired fraction of the biologically active compartment from the functional product. The desired "fraction" is defined by the function and / or particle size of the biologically active compartment according to the present invention. Preferably, separation is carried out to obtain the "fraction" of biologically active microsomes and / or biologically active mitochondria.

[0082] Washing steps can also be integrated into the method according to the present invention. Washing can be carried out before or after at least one pretreatment (e.g., de-vacuolization), before or after the disruption according to the present invention, before or after another disruption cycle, before or after separation, and / or before or after at least one separation. Therefore, when appropriate, washing steps can be integrated between any steps according to the present invention. Whether washing is needed or desired depends on the volume obtained by the upstream steps and the downstream devices / tanks and / or steps or the volume desired for the final preparation. It may be necessary to perform at least one washing step in order to separate at least one released intact biologically active compartment from the separated functional product. Washing is performed using a suitable buffer / solution with lipid bilayer stabilization properties to stabilize the separated compartments, preferably microsomes and / or mitochondria. However, washing is not mandatory in order to obtain a "functional product" of good quality.

[0083] Functional products obtained by method according to the present invention, any directly obtained processed products or intermediate products and separated functional products can be through further processing steps.Therefore, depend on optional separation and / or washing and / or sterilization, obtain functional products of different purity (functional products with desired purity).After reaching desired purity, further processing can be followed, and comprise that the product obtained is washed and / or mixed and / or supplemented and / or sterilized and / or frozen, dried, gas dried and / or lyophilized respectively.But, subsequent steps (such as freezing, drying, gas dried and / or lyophilized) can also be the step of production line according to the present invention.In this embodiment, final product is defined as dried and / or frozen lysate (final lysate) with desired purity.

[0084] "Separation" refers to centrifuging and / or filtering the product (preferably a functional product) in the above steps to obtain a higher content of at least one biologically active compartment, and preferably has a higher purity than the purity of the intermediate product obtained from the previous step. By separation, the content of the surrounding biologically active liquid is reduced, and the content of the biologically active compartment is increased. Preferably, by separation, the "fraction" of the desired biologically active compartment is separated and / or concentrated, for example, the fraction of the biologically active compartment capable of ATP synthesis and / or energy regeneration, or the fraction of the biologically active compartment capable of at least one protein biosynthesis-related process (including expression, transcription, translation, translocation, protein folding and / or protein modification). Preferably, the ATP synthesis and / or energy regeneration activity fraction contains biologically active compartments within the range of 0.5-2 μm. The protein biosynthesis-related process activity fraction contains biologically active compartments within the range of 0.5-30 μm, and is capable of at least one protein biosynthesis-related process, including expression, transcription, translation, translocation, protein folding and / or protein modification. In the methods described herein, separation provides the desired fraction having the desired biological activity.

[0085] In another embodiment of the method according to the invention, a predetermined rotor tip speed of equal to or greater than 2 m / s to 50 m / s is applied by (S), preferably in the range of at least 4 m / sec to equal to or less than 32 m / sec. An appropriate range is adjusted to the scale of the device according to the invention, which is used for the method according to the invention, as effectively shown in Example 9. The rotor tip speed is preferably in the following range: at least 2.5 m / sec to equal to or less than 38 m / sec, 3 m / sec to 38 m / sec, 3.5 m / sec to 38 m / sec, 3.5 m / sec to 36 m / sec, 3.5 m / sec to 34 m / sec, more preferably in the range of at least 4 m / sec to equal to or less than 32 m / sec (Example 9). The above rotor tip speeds can be converted to similar uses for alternative shear generating means (S) for the device according to the invention, the alternative shear generating means (S) comprising a centrifugal pump, a high shear mixer (e.g., ultra turaxx) or a rotor-stator-mixer with a toothed design or with a bladed design (Example 9). In particular, preferably, in combination with the arrangement of the various embodiments of the apparatus according to the invention as described herein, preferably in Example 9, a rotor tip speed in the range of at least 4 m / sec to equal to or less than 32 m / sec is applied. Thus, in one embodiment of the method according to the invention, the residence time in the case of cell disruption without disruption of at least one biologically active compartment is equal to or less than 75 seconds; in a preferred embodiment of the method according to the invention, the shear force generating means (S) is a rotor-stator-mixer with a toothed design or with a blade design, and the residence time for cell disruption without disruption of at least one biologically active compartment is less than 25 seconds. In another preferred embodiment of the method according to the invention, the shear force generating means (S) is a centrifugal pump, and the residence time for cell disruption without disruption of at least one biologically active compartment is less than 75 seconds.

[0086] In an embodiment of the method according to the invention, the shear force is generated by stirring the aqueous medium containing the cells by a shear force generating device (S). In another embodiment of the method according to the invention, the shear force generating device S is a centrifugal pump, a high shear force mixer or a rotor-stator-mixer with a toothed design or with a bladed design, more preferably a rotor-stator-mixer with a toothed design or with a bladed design ( Figure 2 ). Embodiments with different centrifugal pumps, rotor-stator-mixers are described herein and are accordingly adapted to the method, as shown in Example 9. In the method, the shear force generating device (S) is used in combination with a pump (P) or without a pump (P).

[0087] Preferably, the rotation of the rotor is adjusted so as to generate an acceleration force causing radial movement of the medium and axial suction of the medium. The aqueous medium is thereby forced towards the shear gap and the cells are moved by the shear forces and thrusts between the rotor and the stator and impact the cells at the shear gap. The cells are thereby disrupted, in particular the lipid bilayer of the cells without disrupting the lipid bilayer of at least one biologically active compartment contained therein.

[0088] In another embodiment of the method according to the present invention, the biologically active compartment (preferably the released biologically active compartment) is capable of ATP synthesis, energy regeneration, at least one protein biosynthesis-related process (expression, transcription, translation, translocation, protein folding and / or protein modification). It is preferably released into the aqueous medium ("directly obtained processed product", "functional product"). Different fractions can be separated and / or isolated therefrom. Preferably, one fraction of the separated biologically active compartment according to the present invention is mainly capable of ATP synthesis and / or energy regeneration, and another fraction of the separated biologically active compartment according to the present invention is mainly capable of at least one process related to protein biosynthesis: expression, transcription, translation, translocation, protein folding and / or protein modification. The latter fraction, in particular, is capable of coupled transcription and co-translational translocation of at least one complex protein (e.g., GOx) and / or expressed transmembrane protein (TP) (e.g., ACE2) that requires post-translational modification PTM to at least one biologically active compartment (preferably the biologically active compartment is a microsome, more preferably an endogenous microsome).

[0089] According to another embodiment of the method of the present invention, the at least one biologically active compartment has an average particle size in the range of at least 0.5 μm to less than 30 μm. More preferably, the compartment can be divided into at least two fractions: a fraction comprising a main biologically active compartment in the range of 0.5-2 μm and a fraction of a main biologically active compartment in the range of 0.5-30 μm. The fraction of the main biologically active compartment in the range of 0.5-2 μm is mainly capable of ATP synthesis and / or energy regeneration, while the fraction of the main biologically active compartment in the range of 0.5-30 μm is mainly capable of at least one process related to protein biosynthesis: expression, transcription, translation, translocation, protein folding and / or protein modification, more preferably, mainly capable of translocating and co-translationally translocating at least one complex protein (e.g., GOx) requiring post-translational modification PTM and / or expressed transmembrane proteins into at least one biologically active compartment (preferably the biologically active compartment is a microsome, more preferably an endogenous microsome).

[0090] Preferably, a combination of two different biologically active compartments is obtained (fractions as described herein), wherein one fraction comprises a biologically active compartment in the range of 0.5-2 μm and is at least capable of ATP synthesis and / or energy regeneration, and the other fraction comprises a biologically active compartment in the range of 0.5-30 μm and is capable of at least one process associated with protein biosynthesis, including expression, transcription, translation, translocation, protein folding and / or protein modification.

[0091] In another embodiment of the method according to the invention the disruption efficiency is defined as the amount of disrupted cells compared to the amount of undisrupted cells, preferably or alternatively, as the amount of released biologically active compartments compared to the amount of undisrupted cell wall-free cells provided.

[0092] Each amount is determined by counting the provided cell wall-free cells. Preferably, each amount of the biologically active compartment is counted by microscopic analysis, determination of markers / components of the biologically active compartment (e.g., known and detectable transmembrane proteins) or by particle flow measurement. The relationship of one amount to another is expressed as a percentage.

[0093] The method, wherein the disruption efficiency is the product of the shear gradient and the residence time of the aqueous medium in the shear force and / or turbulence region and is defined as being strong enough to disrupt or break or break apart the cell wall-less cells ((micro)protoplasts) (preferably the membranes of (micro)protoplasts) to release biologically active compartments, preferably with a diameter of less than 70 μm. Preferably, the biologically active compartments are intact organelles from plant (micro)protoplasts containing mitochondria, microsomes, nuclei, endoplasmic reticulum and / or Golgi bodies. Preferably, the separated fractions contain released intact microsomes and / or mitochondria from plant (micro)protoplasts but without nuclear DNA.

[0094] Preferably, the compartments are mitochondria and / or microsomes and most preferably, the released biologically active compartments do not contain cell nuclei and / or nuclear (karyological) DNA, preferably, the separated fractions do not contain cell membranes or fragments thereof, cell nuclei and / or nuclear DNA.

[0095] In another embodiment of method according to the present invention, described method is the monolithic method (as described in the context of equipment according to the present invention) of production and processing, preferably the monolithic method of cell lysate production and processing.Described production and processing is cell line production and processing, and its purpose is preferably to produce cell lysate and / or obtain desired fraction from described cell line.Described desired fraction is certain soluble protein, transmembrane protein in the lipid bilayer embedded in bioactive compartment of the present invention and / or another kind of component, main product or by-product of described production and processing.More preferably, described production and processing is plant cell lysate production and processing, most preferably is the plant cell lysate production and processing of tobacco BY2, for the manufacture of the lysate for cell-free protein synthesis.

[0096] Another aspect of the invention is a composition comprising at least one biologically active compartment and a biologically active liquid surrounding it, wherein said liquid and / or compartment is capable of carrying out at least one step of protein biosynthesis, which is preferably obtained by the method according to the invention. More preferably, it is obtained by the method according to the invention, which is performed independently or as part of a tandem production by using an apparatus according to the invention. All embodiments of at least one biologically active compartment, fraction and / or a biologically active liquid surrounding it are applicable to the composition according to the invention.

[0097] Another aspect of the present invention is such an isolated biologically active compartment, which is surrounded by a lipid bilayer and exhibits at least the ability of ATP synthesis and / or energy regeneration and / or at least the ability in at least one protein synthesis process (preferably, including transcription, translation, post-translational modification, protein folding and / or translocation). Preferably, the isolated biologically active compartment surrounded by a lipid bilayer is obtained by the method according to the present invention and / or preferably by using the device according to the present invention. All embodiments of at least one biologically active compartment, fraction and / or the biologically active liquid surrounding it are applicable to the isolated biologically active compartment surrounded by a lipid bilayer according to the present invention.

[0098] In another embodiment of the present invention, the isolated biologically active compartment surrounded by a lipid bilayer or a composition comprising at least one biologically active compartment comprises at least one transmembrane protein, at least one inner membrane and / or outer membrane associated protein, and / or at least one soluble protein within its internal space, respectively.

[0099] Preferably, the biologically active compartment is obtained by the method described in the present invention, and preferably by using the device described in the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0100] Figure 1: shows a schematic arrangement of an apparatus according to the present invention, wherein (S) is a shear generating device, (P) is a pump, tank A is used to provide an aqueous medium, tank B is used to collect downstream processed products, and (C) is a separation device (preferably a centrifuge).

[0101] Figure 2 : Schematically shows A) the blade design and B) the tooth design of the rotor-stator-mixer of the shear generating device (S) according to the invention.

[0102] Figure 3 : The workflow (experimental protocol) of the method according to an embodiment of the present invention, wherein it is integrated into the production line described herein. Different starting materials ("starting materials comprising a cell line of interest"), optional "pretreatments" and starting materials for the method according to the present invention ("starting materials comprising biological materials") are shown. The products defined herein are associated with the corresponding steps described herein. Optional washing and / or sterilization ("washing / sterilization") steps can be integrated between any steps. Sterilization can be applied to components, devices and / or parts supplemented or installed by the device according to the present invention.

[0103] Figure 4: eYFP yields from cell lysates containing bioactive compartments (e.g., mitochondria and microsomes) obtained by different disruption methods. In order to demonstrate that efficient cell disruption while ensuring bioactive lysates, protein synthesis methods are feasible methods. Disruption efficiency as a function of protein productivity. A: Disruption was performed using a high-pressure homogenizer (Microfluidizer: Model M-110L; LM-10). B: Disruption efficiency of N2 disruption (N2), freeze-thaw (-80 / -20°C), ultrasonic treatment (Ultrasonic), and sonication (Sonoplus). C: Disruption efficiency of IKA magic lab under different conditions (Table 2) as a function of eYFP productivity (10 ng / μL (black bars), 20 ng / μL (grey bars), 40 ng / μL (dashed bars)). D: Disruption efficiency of IKA magic lab under different conditions as a function of GOx productivity (5 ng / μL (black bars), 10 ng / μL (grey bars), 20 ng / μL (dashed bars)). E: Determination of the fragmentation efficiency of the method according to the invention as a function of the productivity of a functional transmembrane protein (TP), here angiotensin-converting enzyme ACE2, via binding to its ligand, a commercially available receptor binding domain RBD.

[0104] Figure 5: Microscopic evaluation (40x) of lysates of the BY-2 cell line; A: pumping speed: 40 (30 mL / min), ultraTurrax speed 2, 4 or 6; B: pumping speed: 30 (22.5 mL / min), ultraTurrax speed 2, 4 or 6; C: pumping speed: 20 (15 mL / min), ultraTurrax speed 2, 4 or 6; for A, B and C at Ultra turrax speed 2, three distinct fractions are visible; for A, B and C at Ultra turrax speed 4, three distinct fractions are visible, but not as clearly as at Ultra turrax speed 2; for A, B and C at Ultra turrax speed 4, only two distinct fractions are visible.

[0105] Figure 6 : Macroscopic evaluation of lysates derived from BY-2 cell line after disruption and centrifugation at 1,500×g for 15 min; A: pumping speed: 40 (30 mL / min), ultra Turrax speed of 2, 4 or 6, B: pumping speed: 30 (22.5 mL / min), ultra Turrax speed of 2, 4 or 6, C: pumping speed: 20 (15 mL / min), ultra Turrax speed of 2, 4 or 6; for A, B and C, three distinct fractions are visible at Ultra turrax speed of 2. The bottom fraction contains the unbroken cell wall-less cells (micro) protoplasts; the fraction above contains the undesired fraction containing the nuclei and debris (lipid bilayer debris and (micro) protoplast debris); the upper fraction contains the desired fraction (lysate) containing the bioactive compartments and surrounding bioactive fluid (ribosomes, mitochondria, microsomes, etc.). For A, B and C at Ultra turrax speed 4, three fractions are visible, but not as clear as at Ultra turrax speed 2; for A, B and C at Ultra turrax speed 4, only two different fractions are visible.

[0106] Figure 7 : Microscopic evaluation after disruption in one pass using IKA magic LAB (single rotor / stator at 10000 rpm and 37 mL / min feed rate).

[0107] Figure 8: Microscopic evaluation after disruption using IKA magic LAB (with a feed rate of 40 mL / min, using a triple or single rotor / stator, respectively). A) triple rotor / stator, 6000 rpm after two passes, B) single rotor / stator, 6000 rpm after two passes, C) triple rotor / stator, 6000 rpm after one pass, D) feeding, E) triple rotor / stator, 9000 rpm after one pass.

[0108] Fig. 9 : Overview and classification of well-known mechanical shear generating or shear force generating devices (S). Some are suitable and arranged to mediate shear forces through fluids, while others, which are different therefrom, are suitable and arranged to mediate shear forces through solids. For each group of "fluids" or "solids", different forces generate shear through the "fluids" or "solids". The shear force generating device (S) described in the present invention is characterized in that the individual components in (S) are subjected to a stirring or stirring motion, wherein the aqueous medium containing the cells is stirred / mixed under shear forces in (S). In contrast, other "fluid" prior art devices are characterized by pressure or ultrasound as the shear generating force. These are not part of the present invention.

[0109] Fig.10 : Oxygen consumption of lysate, expressed as the change in dissolved oxygen concentration (%) in the lysate over time (h). Lysate reaction conditions: reaction volume = 300 μL, oscillation frequency = 700 rpm, 48-well round plate, BioLector, plasmid pLB0077, plasmid concentration 10 ng / μL, target protein = GOx. Lysate numbers correspond to the detection numbers in Table 3. The lysate LYCEDL110CR(1) obtained by disruption using the shear force generating device PuraLev i100SU at 500 rpm (rotor tip speed 1.1 m / s; residence time 25 sec) and flow rate 56.4 mL / min showed oxygen consumption equivalent to a decrease in dissolved oxygen concentration of about 15%; the lysate LYCEDL180CR(8) obtained by disruption using the IKA UTL25 with blades at 6000 rpm (rotor tip speed 9.4 m / s; residence time 24 sec) and flow rate 20 mL / min showed an increase in oxygen consumption equivalent to a decrease in dissolved oxygen concentration of about 30%; while the lysate LYCEDL180CR(8) obtained by disruption using the PuraLev i100SU at 500 rpm (rotor tip speed 1.1 m / s; residence time 25 sec) and flow rate 56.4 mL / min showed an increase in oxygen consumption equivalent to a decrease in dissolved oxygen concentration of about 15%; The lysate LYCEDL120CR-160CR (2-6) obtained by the i100SU at 4500rpm or 9000rpm (rotor tip speed 10.1 or 20.3m / s; residence time 25 or 70.5 seconds) and flow rate 56.4 or 20mL / min showed further increased oxygen consumption, equivalent to a decrease in dissolved oxygen concentration of about 55-65%. A dissolved oxygen concentration of 100% means that no oxygen is consumed.

[0110] Fig.11 : Expression of eYFP protein using lysates 1, 2, 3, 4, 5, 6, 7 and 8. Reaction conditions: reaction volume = 25 μL, shaking frequency = 500 rpm, 384-well plate, plasmid pLB0001, plasmid concentration = 10 ng / μL, target protein = eYFP. Data are shown as the mean + / - standard deviation of two independent protein expression reactions. Lysate numbers correspond to the assay numbers in Table 3.

[0111] Fig.12 : Expression of Gox protein using lysates 1, 2, 3, 4, 5, 6, 7 and 8. Reaction conditions: reaction volume = 50 μL, shaking frequency = 500 rpm, 96 half-well plates, plasmid pLB0077, plasmid concentration = 10 ng / μL, target protein = Gox. Data are shown as the mean +- standard deviation of two independent protein expression reactions. The lysate numbers correspond to the assay numbers in Table 3.

[0112] Fig.13 : Microscopic and macroscopic evaluation of lysates from BY-2 cell line after disruption. The lysate numbers correspond to the assay numbers in Table 3. Lysates 1-5: PuraLev i100SU, at different pumping speeds at 500rpm, 4500rpm or 9000rpm (see Table 3); Lysate 6: IKA magic lab, with 1x gear ring, at 20000rpm; and Lysate 8: IKAUTL25, with blades, at 6000rpm (see Table 3). Lysate "0" represents untreated material (no disruption). Column A shows lysates after disruption at 40x magnification, and column B shows lysates after disruption at 100x magnification. Column C shows photos of the lysates in the tubes after disruption followed by centrifugation (500×g, 10min); Lysate 1 (A / B / C) still shows intact microprotoplasts (A / B), and after centrifugation (C) shows three different fractions. The fraction at the bottom comprises the (micro) protoplastid without cell wall cells that are not broken; The fraction above it comprises such undesirable fraction that is separated, and the described undesirable fraction that is separated comprises nucleus and debris (lipid double layer debris, nucleus and (micro) protoplastid debris); The fraction at the top again comprises such desired fraction (lysate), and it comprises biologically active compartment and surrounding biologically active liquid (ribosome, mitochondria, microsome etc.). Lysate 2 and 8 have shown the complete microprotoplastid (A / B) and the less bottom fraction (C) of the amount that reduces. Lysate 3-6 hardly shows complete microprotoplastid (A / B) and lacks the described bottom fraction (C).

[0113] References

[0114] Buntru et al. (2014) – M. Buntru, S. Vogel, H. Spiegel and S. Schillberg; Tobacco BY-2 cell-free lysate: an alternative and highly-productive plant-based in vitro translation system. BMC Biotechnology 2014, 14:37. https: / / doi.org / 10.1186 / 1472-6750-14-37

[0115] Buntru et al. (2015) – M. Buntru, S. Vogel, K. Stoff, H. Spiegel, S. Schillberg; A Versatile Coupled Cell-Free Transcription–Translation System Based on Tobacco BY-2 Cell Lysates. Biotechnology and Bioengineering, Vol. 112, No. 5, May, 2015. DOI 10.1002 / bit.25502 Espinoza et al. - C.J.U. Espinoza, F. Alberini, O. Mihailova, A.J. Kowalski, M.J.H. Simmons; Flow, turbulence and potential droplet break up mechanisms in an in-line Silverson 150 / 250 high shear mixer. Chemical Engineering Science: X 6(2020) 100055. https: / / doi.org / 10.1016 / j.cesx.2020.100055

[0116] -Andreas Rotor-Stator Mixers:From Batch toContinuous Mode of Operation—A Review.Processes 2018,6,32;doi:10.3390 / pr6040032John et al.-GTJohn,I.Klimant,C.Wittmann,E.Heinzle:IntegratedOptical Sensing of Dissolved Oxygen in Microtiter Plates:A Novel Tool for Microbial Cultivation.Biotechnol Bioeng81:829–836,2003.DOI:10.1002 / bit.10534

[0117] Zhang et al.-Jinli Zhang*, Shuangqing Xu, Wei Li; High shear mixers: A review of typical applications and studies on power draw, flow pattern, energy dissipation and transfer properties. Chemical Engineering and Processing 57–58(2012)25–41; DOI: 10.1016 / j.cep.2012.04.004. Example

[0118] Example 1 - Proof of Concept

[0119] 1.1 Experimental Setup

[0120] A peristaltic pump (here a Watson Marlow 120 U / DV pump with an inner tube diameter of 3.175 mm) was used in conjunction with an Ultra Turrax (IKA ULTRA- T 10) combination. Three different pumping speeds and three different ultra turrax speeds (about 10 ml each) were used. The starting material was a pretreated aqueous medium, which contained devacuolated cell wall-free BY-2 cells derived from tobacco suspension cell culture (Buntru et al., 2014, 2015) and was supplemented with 1.5 times (v / v) TR buffer. During the experiment, the starting material was kept at low temperature (less than 10°C).

[0121] For the aqueous medium, a TR buffer containing 30 mM HEPES KOH buffer (pH 7.6), 40 mM potassium glutamate, 0.5 mM magnesium glutamate and 2 mM DTT was used. After completion of the method, the directly obtained processing product (as defined herein) comprises the TR buffer, the released bioactive compartment and / or its surroundings.

[0122] Table 1: Applied shear force and flow rate

[0123]

[0124] 1.2 Results

[0125] Microscopic evaluation ( Figure 5 ) shows the processed product obtained directly after crushing with the ultra turrax and pump as shown in Table 1. Figure 5 It is shown that both pump speed and / or flow rate and Ultra turrax speed have an impact on fragmentation, with higher ultraturrax speeds (top to bottom in the set of figures) and lower pump speeds and / or flow rates (left to right in the set of figures) each individually leading to a more fragmented process product (lower content of visible microstructure). Figure 5 C / 6 shows the most extreme settings of both parameters, where essentially all BY-2 (micro)protoplasts were disrupted. Macroscopic evaluation of BY-2 cell line lysate after disruption and centrifugation at 1,500×g for 15 min ( Figure 6 ) show that all (micro)protoplasts were successfully disrupted when the pumping speed was 30, 22.5 or 15 mL / min and the ultraTurrax speed was 6. Only two fractions were visible, one containing the bioactive compartments (mitochondria and microsomes) and the surrounding bioactive liquid, and the other containing the nuclei and / or debris of the (micro)protoplasts. Pumping speeds of 30, 22.5 and 15 mL / min and ultraTurrax speeds of 2 and 4 were not sufficient to disrupt the entire biomaterial.

[0126] Example 2 - IKA magic LAB test

[0127] 2.1 Experimental Setup

[0128] Equipment for crushing

[0129] A peristaltic pump (here a Watson Marlow 120u / dv pump) was used in combination with an IKAmagic LAB as a shear generator. Unless otherwise specified, the same setup was used ( Figure 5 , 6 , 7 and 8).

[0130] IKA Module Single rotor / stator or triple rotor / stator Feeding speed 37mL / min Number of passes 1 Rotor speed 10,000rpm

[0131] For the following biosynthesis assays (2.2.1 and 2.2.2), the lysates were generated by disruption using IKA magic lab and the experimental setup was as follows.

[0132] Table 2: Applied shear force and flow rate

[0133]

[0134] The same starting materials as above were used and the temperature was kept low (less than 10°C) during the experiment.

[0135] 2.2 Verification of crushing effect:

[0136] Through vision ( Figure 6 ),microscope( Figure 5 , 7 and 8 ) and protein synthesis to assess the fragmentation effect.

[0137] 2.2.1 Validation of the assay for cytoplasmic protein eYFP expression as described by Buntru et al. (page 9)

[0138] Enhanced yellow fluorescent protein (eYFP) is used as a marker protein. It is expressed in lysates outside the microsomes and does not require microsome-mediated post-translational modifications (PTMs). However, active protein synthesis and energy regeneration mechanisms (ribosomes, mitochondria, etc.) are required. The higher the eYFP protein expression (yield), the higher the measurable fluorescence. Alternative model proteins known in this context are firefly luciferase (FFLuc) and Renilla luciferase (Buntru et al., 2014).

[0139] 2.2.2 Validation of microsomal expression assays with post-translational modifications and folding (GOx)

[0140] In order to verify that the released microsomes are active and can be post-translationally modified and folded, the expression of multi-domain glycoprotein (a homodimer composed of 80kDa monomers covalently linked by disulfide bonds, and each monomer has 8 N-glycosylation sites) glucose oxidase (GOx) from Aspergillus niger was tested. For expression, the template encoding protein GOx was cloned into plasmid pALiCE02. The lysate samples after incubation were treated with 0.5% DDM (5% DDM storage solution from PBS) at room temperature for 10min. GOx standards from Aspergillus niger (Sigma Aldrich) were used to draw a calibration curve within the range of 0-500 μg / ml. Samples and standards were diluted in a transparent 96-well plate at 1:2500 in an assay buffer consisting of 0.33M glucose, 0.67mM ABTS and 1.67U / ml HRP (SigmaAldrich) and 0.1M potassium phosphate buffer (pH 6). The absorbance at 420 nm was measured over time in an infinite PRO Tecan plate reader for 15 min. The linear absorbance increase over time of the calibration samples was used to calculate the sample GOx activity.

[0141] 2.2.3 Validation of the assay for transmembrane protein expression embedded in the microsomal lipid bilayer ( Figure 4E ).

[0142] To verify that intact microsomes contain transmembrane proteins that are correctly folded and embedded in the lipid bilayer, an alternative approach is to use the expression of a model transmembrane protein (e.g. angiotensin converting enzyme ACE2) using the corresponding lysates after disruption. Since the so-called Covid pandemic, it has been known that ACE2 is expressed on alveolar epithelial cells and capillary endothelial cells, while actin is the cellular doorway for SARS-CoV-2, allowing infection of the cells. Commercially available ligands (RBD) are well known. For the assay, the template encoding ACE2 was cloned into the vector pALiCE02 (LenioBio GmbH) and Strep- II is fused to the C-terminus of the DNA template for protein orientation and subsequent capture on a microtiter plate. The melittin signal peptide sequence (MSP) is fused at the N-terminus for targeting microsomes. ACE2 is expressed and translocated into intact microsomes, wherein the N-terminal binding site of the ACE2 is located inside the microsome, while the C-terminus remains outside to allow capture. PBS containing 1% DDM is added to each well, and the plate is incubated at room temperature for 15 min. The microtiter plate is washed (washed 3 times with PBS-0.05% Tween (v / v) (PBST)) and blocked with 1x blocking buffer (ab126587, Abcam) to prevent nonspecific binding (1h, RT), and washed again. In order to capture ACE2 embedded in microsomes and perform protein binding analysis, the commercially available RBD SARS-CoV Spike / RBD protein (RBD, His Tag) (40150-V08B2, SinoBiological) is diluted to 2.5 μg / ml in blocking buffer. The plate was incubated at room temperature for 1 h, followed by a washing step. In order to be able to bind to ACE2, the microsomes were treated with 1% DDM to break up the captured microsomes. For protein detection, a detection antibody (HRP labeled) that binds to the protein was added. For ACE2-commercial RBD binding detection, a His Tag horseradish peroxidase-coupled antibody (MAB050H, Bio-Techne) diluted 1:4000 in blocking buffer and StrepMAB-Classic-HRP (2-1509-001, IBA-lifesciences) diluted 1:15,000 were added. The plate was incubated (1 h, RT), then washed, and then an indirect ELISA (by measuring sample absorbance) was performed to determine the structure and binding efficiency of the protein. Detection of absorbance (e.g. (650 nm) TECAN Infinite M1000 Pro machine, Tecan i-Control2.0 software) demonstrates the expression of correctly folded ACE2 because it effectively binds to the ligand RBD of SARS-CoV-1. High absorbance values ​​demonstrate that high ACE2 expression, post-translational modification, folding and embedding into the microsomal lipid bilayer are prerequisites for RBD ligand binding. Therefore, this assay is suitable for verifying the release of the bioactive compartment according to the present invention, such as Figure 4E shown.

[0143] 2.3 Results

[0144] 2.3.1 Homogenization, sonication, etc. versus shear disruption

[0145] The disruption method using a microfluidic disruptor: Model M-110L; LM-10 was to evaluate whether homogenization (as used in the prior art in other technical fields) can achieve lysate production with high yields of bioactive components. However, only low yields of expressed protein (less than 0.01 mg eYFP / ml) were observed. Figure 4A ). Therefore, only low levels of bioactive components were achieved in the lysate produced by homogenization. Other prior art methods, such as sonication, etc., were tested accordingly. Figure 4B As shown in Figure 2, the yield of expressed eYFP is very low, which is due to the low content of biologically active components in the lysate. puraLev i100 SU and Ultra turrax (IKA ULTRA- The products obtained by the method performed in T 10) will be analyzed using the same method. Figure 4C The eYFP yields from lysates after different disruption methods are shown.

[0146] The crushing method of the present invention utilizes a shear generating device (S) (here IKA magic lab), such as Figure 4C and Figure 4D As shown. Figure 4A Compared to the results shown in B, the protein yields obtained ranged from 1 mg / mL to 2.8 mg / mL eYFP, depending on the disruption conditions and the plasmid concentration used ( Figure 4C , listed from left to right as 10, 20, 40 ng / μL). The data show that the various disruption settings of IKAmagic lab resulted in bioactive compartments that were able to achieve high target protein titers in lysate reactions. In addition, the assay using GOx showed that the GOx titers varied depending on the disruption conditions and the plasmid concentration used ( Figure 4D , 5, 10, 20 ng / μL from left to right), the obtained lysates were also able to produce complex GOx proteins with post-translational modifications mediated by intact microsomes, producing 40-150 au (arbitrary units) GOx ( Figure 4D ).

[0147] 2.3.2 Microscopic evaluation Figure 7 )

[0148] Figure 7 Indicates the starting material before crushing ( Figure 7 A) and after crushing at 6,000 rpm in one pass ( Figure 7B) Microscopic evaluation (x100). The picture shows that under these settings, most of the cells without cell walls have been broken. Since not all cells without cell walls will be broken under these settings, it also shows that the force applied is low enough so as not to destroy the cell compartment but to release the cell compartment with biological activity.

[0149] 2.3.3 Results Figure 8 )

[0150] Pass number / Other Feeding speed Rotor speed IKA Module Figure 8 2 60mL / min 6,000rpm Triple rotor / stator A 2 60mL / min 6,000rpm Single rotor / stator B 1 60mL / min 3,000rpm Triple rotor / stator C 1 / Feeding tube and IKA are filled with TR buffer 60mL / min 9,000rpm Triple rotor / stator E 0 / Feed na 0rpm na D

[0151] Figure 8 Represents a microscopic assessment of the effect of starting material as well as disruption settings on the amount of disrupted cell wall-free cells. Figure 8 D shows cells without cell walls before disruption. Figure 8 C shows that after a single pass at a rotor speed of 3,000 rpm, most of the cell wall-free cells remained intact. In contrast, Figure 8 A, B, and E show broken cells without cell walls. Figure 8 Comparison of A and 8B shows the influence of the rotor-stator configuration. It shows that, using the same setup, except for the triple rotor / stator versus a single rotor / stator module, different shear forces can be achieved to finely tune the appropriate force to disrupt cell wall-free cells.

[0152] 3. Summary

[0153] At present, the expression of eYFP and GOx shows that the functional lysate is obtained by the crushing method of the present invention. Not only is the protein biosynthesis machinery intact and able to express soluble proteins (eYEP), but post-translational modification of the multi-domain protein GOx can also occur. Therefore, the crushing method and equipment according to the present invention allow the production of functional lysates for the biosynthesis of recombinant proteins on a variety of scales. At present, the successful scale-up of the key steps in the production of the lysate has been successfully demonstrated, and the improvement of production efficiency has been ensured and improved while the quality of the directly obtained processed products has been improved.

[0154] Example 3 - Blade and tooth design experiments

[0155] Series production with IKA UTL 25Inline showing the influence of the blade design of the rotor compared to the toothed design ( Figure 2 ).

[0156] Experimental setup

[0157] Peristaltic pumps, such as Watson Marlow 120u / dv pumps, are combined with shear generating devices having a) blade design and b) tooth design, respectively. The starting materials are the same as those described in Examples 1 and 2. The rotor speed is adjusted at 3,000-15,000 rpm, such as 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000 and 15000, respectively. The pump flow rate is adjusted within the range of 10-100 mL / min, respectively:

[0158]

[0159] The precise adjustment is shown in Example 9 and Table 3.

[0160] Example 4 - Centrifugal Pump Test

[0161] Experimental setup

[0162] Centrifugal pumps, e.g. puraLev i100 SU, replacing the rotor-stator-mixer. The starting materials are the same as described in Examples 1 and 2. The centrifugal speed (in rpm) will be higher and the peristaltic pump feed rate will be lower because the centrifugal pump will exert a lower force on the cell wall-free cells than the rotor-stator-mixer. 4,500-9,000 rpm centrifugal speeds will be used with peristaltic pump speeds of 1-50 mL / min. In addition, multiple passes through the centrifugal pump will also be used. The precise adjustments are shown in Example 9 and Table 3.

[0163] Example 5 - Scale-up trials

[0164] In order to achieve the residence time of the cell wall-free cells in the shear field as a scale-up parameter, an Ultra turrax (IKA ULTRA- Parallel experiments were performed on IKA T 10), IKA UTL 25inline and IKA magic LAB to confirm the residence time together with the shear force in the shear field as a criterion for scale-up.

[0165] Example 6 - Viscosity Determination

[0166] The viscosity of different starting materials that can be disrupted using the method and using an embodiment of the device according to the present invention is determined and adjusted. The viscosities of the shear generating device will be tested. Viscosities of about 0.5 cP to 5 cP are aqueous media and are feasible for each embodiment of the device and method of the present invention. However, viscosities greater than 5 to less than 15 mPa s may represent cell culture media containing higher concentrations of biological material to be disrupted, for example after centrifugation.

[0167] Example 7 - Separation experiment followed by determination of the amount of compartments, the amount of each cell wall-less cell and the pass status.

[0168] The biologically active compartments were fractionated by centrifugation.

[0169] This is accomplished by measuring markers for the components and determining the amount of nuclear DNA depending on the disruption setup.

[0170] Example 8 - Disruption of Yeast Protoplasts, CHO and Other Species

[0171] The most common cell lines used in the field of cell-free protein synthesis (e.g., as described in Buntru et al., 2015 and Buntru et al., 2014, page 2) were compared and disrupted as described herein to release and separate bioactive compartments and bioactive fluids suitable for cell-free biosynthesis of recombinant proteins.

[0172] Example 9

[0173] 9.1. Experimental setup and adjustments according to the invention

[0174] The components of the device according to the invention are listed in Table 3. The centrifugal pump PuraLev i100SU used here is equipped with a "driver" IPD-100.3-03-02 (epoxy, 0.3m PVC, IC915, item number: 100-10105) and a "SU pump head" DCP-200.3 (PP, Barb 1 / 2", item number: 100-90792). The driver drives the pump head (magnetically in this case) and uses this to adjust the rpm. Other versions of the "driver" and pump head are also applicable accordingly. The rotor-stator system (S) used here is of toothed ring design, such as Figure 2 B as shown in the left figure.

[0175] Table 3: Experimental overview Equipment used for all experiments Pump: Hei-Flow ultimate 120 peristaltic pump with SP quick 1.6 head.

[0176]

[0177] *Driver: IPD-100.3-03-02 driver, DCP-200.3 pump head

[0178] Calculation of residence time

[0179] The residence time (VZ) for each device can be calculated, provided that the internal volume of the device used is known or can be calculated by, for example, metering (volume measurement). Based on the known flow rate (R) (in mL / min) and the internal volume (Vi) (in mL), the following residence time VZ=Vi / R results can be obtained, for example,

[0180] a) For IKA magic lab (Vi = 30 mL), the flow rate is 150 mL / min (Table 3) or 40 mL / min (Table 2)

[0181] VZ = 30 mL / 150 mL / min = 12 seconds (see Table 3) bzw,

[0182] VZ = 30 mL / min / 40 mL / min = 45 seconds (see Table 2).

[0183] b) For UTL25 (Vi = 8 mL), the flow rate is 10 mL / min:

[0184] VZ = 8 mL / 10 mL / min = 75 seconds.

[0185] c) For a centrifugal pump with pump head DCP-200.3 (Vi = 23.5 mL), the flow rate is 20 mL / min or 56.4 mL / min

[0186] VZ = 23.5 mL / 20 mL / min = 70.5 seconds,

[0187] VZ = 23.5 mL / 56.5 mL / min = 25 seconds.

[0188] 9.2 Method for determining oxygen consumption

[0189] One way to demonstrate that the cell lysate (the directly obtained processed product) is active and functional in the sense of the present invention is to demonstrate the functionality of the mitochondria, since they provide the energy for the biosynthesis of proteins. To visualize the function of the mitochondria after disruption, the BioLector Pro (Beckman Coulter GmbH, Aachen, Germany) was used. With this device, oxygen transfer / consumption can be measured with an oxygen-sensitive fluorescent dye located at the bottom of the wells of a microtiter plate (John et al., 2001). The measurement principle is described in the literature of John et al. Figure 1 The fluorescence of the dye is measured by an optical fiber and correlated to the oxygen concentration in the liquid surrounding the dye. If oxygen is consumed, the oxygen consumption is measured by the dissolved oxygen (DO) concentration level in the well. As John et al. Figure 2If no oxygen is consumed, the measured oxygen concentration is 100%. In the case of lower DO levels, the oxygen consumption in the liquid is displayed.

[0190] 9.3 Results

[0191] 9.3.1 Oxygen consumption

[0192] Oxygen consumption demonstrated the presence of active / functional mitochondria in the lysates produced by the apparatus in Table 3 and according to the method with modifications described in Table 3. All produced lysates 1, 2, 3, 4, 5, 6 and 8 consumed oxygen ( Fig.10 ).

[0193] This example shows that, using a corresponding device, the desired settings of shear force and residence time can be set independently of one another and that the desired directly obtained processed product is therefore active in each case in the sense of the invention. The consumption of oxygen demonstrates that the directly obtained processed product contains biologically active compartments and is capable of ATP synthesis and energy regeneration. As shown below, the protein yield further demonstrates that the same directly obtained processed product is also capable of processes associated with protein biosynthesis: expression, transcription, translation, translocation, protein folding and / or protein modification.

[0194] 9.3.2 Expression of eYFP

[0195] The expression of eYFP has been analyzed as described above in 2.2.1 and demonstrated the ability of lysates 1, 2, 3, 4, 5, 6, 7 and 8 to produce cytoplasmic protein (here eYFP). This indirectly demonstrated that the key components of cytoplasmic protein expression in the lysates (transcription, translation, mitochondrial energy regeneration and protein folding) are functional.

[0196] 9.3.3 Expression of GOx

[0197] The expression of GOx was analyzed as described above in 2.2.2 and demonstrated the ability of the lysates to couple transcription and co-translational translocation of GOx, a more complex protein requiring post-translational modifications (PTMs). Fig.12 It was shown that lysates 1, 2, 3, 4, 5, 6 and 8 were able to produce GOx. This indirectly proves that the key components of microsomal protein expression (such as transcription, translation, mitochondrial energy regeneration, transport, protein folding and post-translational modification) in the lysates produced by the devices and methods described in Table 3 are functional.

[0198] Therefore, both oxygen consumption and protein synthesized indicate that directly accessible processing products can be obtained by different devices and processes (Table 3).

Claims

1. A device for disrupting cells without cell walls without disrupting the biologically active compartments contained therein surrounded by lipid bilayers, the device comprising: - a shear force generating device (S) for generating a shear force zone suitable for disrupting said cells, - a tank A for providing said cells, - optionally, a pump (P), said pump (P) being located upstream of (S) or downstream of (S) for pumping the aqueous medium comprising said cells at an adjustable flow rate and / or flow rate, - at least one conduit connecting tank A to the shear force generating device (S), and / or at least one conduit connecting (S) to tank B, - a tank B for collecting the product from the previous step, said product comprising at least one released biologically active compartment, and - separation means (C) for separating the disrupted cells and their fragments from at least one released biologically active compartment and / or its surrounding biologically active liquid phase.

2. Apparatus according to claim 1, wherein the shear force generating means (S) is adapted and controllably capable of generating shear forces sufficient to disrupt cells without damaging the lipid bilayer of at least one biologically active compartment contained therein.

3. An apparatus according to claim 1 or 2, wherein the pump is suitable for adjusting the residence time of the aqueous medium containing the cells within (S) independently of the shear force adjusted and generated by (S), wherein the residence time is the duration during which the shear force is applied to the aqueous medium containing the cells.

4. The device according to any one of claims 1 to 3, wherein the device comprises a shear force generating device (S) and a pump (P) located upstream of (S) or downstream of (S), wherein the pump (P) is used to pump the aqueous medium containing the cells at an adjustable flow rate and / or flow rate.

5. Apparatus according to any one of claims 1 to 4, wherein the shear force generating means (S) is adapted and designed to generate shear by stirring the aqueous medium.

6. Apparatus according to any one of claims 1 to 5, wherein the shear force generating device (S) is a centrifugal pump, a high shear force mixer or a rotor-stator-mixer with a toothed design or with a bladed design.

7. The device according to any one of claims 1 to 6, wherein the device is an integral device of a production line, preferably an integral device of a closed and sterile production line.

8. A production line comprising an apparatus according to any one of claims 1 to 7, wherein tank A is located downstream of at least one component for pre-treating biological material and the conduit connects tank A to S, and wherein tank B is located downstream of S and, optionally, pump P is located downstream or upstream of S.

9. A method for disrupting cells without cell walls comprising at least one biologically active compartment surrounded by a lipid bilayer, the method comprising the following steps: - providing an aqueous medium comprising said cell wall-free cells, preferably in a tank A, - moving the aqueous medium into a shear force generating device (S) according to claim 1 or 2, - optionally, regulating the flow rate and / or the flow rate of the aqueous medium by means of a pump (P) according to claim 3, - a shear force region is generated within (S), - applying shear forces to the aqueous medium containing the cells for a residence time sufficient to disrupt the cells but not the at least one biologically active compartment, - when said aqueous medium passes through (S), said cells are disrupted without disrupting the lipid bilayer of said at least one biologically active compartment, - release of at least one complete biologically active compartment and its surrounding biologically active fluid, - optionally, collecting at least one released intact biologically active compartment and the biologically active fluid surrounding it, preferably in tank B, - separating the at least one released intact biologically active compartment and its surrounding biologically active fluid, and - Optionally, isolating said at least one released intact biologically active compartment.

10. The method according to claim 9, wherein the shear force is generated by stirring the aqueous medium containing the cells using the shear force generating device (S).

11. The method according to claim 9 or 10, wherein the shear force generating device (S) is a centrifugal pump, a high shear force mixer or a rotor-stator mixer with a toothed design or with a blade design.

12. The method according to any one of claims 9 to 11, wherein a predetermined rotor tip speed equal to or greater than 2 m / s up to 50 m / s is applied by (S).

13. The method according to any one of claims 9 to 12, wherein the residence time for disrupting the cells without disrupting the at least one biologically active compartment is equal to or less than 75 seconds.

14. The method according to any one of claims 9 to 12, wherein the shear force generating device (S) is a rotor-stator-mixer with a toothed design or with a blade design, and the residence time for disrupting the cells without disrupting the at least one biologically active compartment is less than 25 seconds.

15. The method according to any one of claims 9 to 14, wherein the shear force generating means (S) is a centrifugal pump and the residence time for disrupting the cells without disrupting the at least one biologically active compartment is less than 75 seconds.

16. The method according to any one of claims 9 to 15, wherein the biologically active compartment is capable of ATP synthesis, energy regeneration, at least one protein biosynthesis-related process, expression, transcription, translation, translocation, protein folding and / or protein modification.

17. The method of any one of claims 9 to 16, wherein the at least one biologically active compartment has an average particle size ranging from at least 0.5 μm to less than 30 μm.

18. The method according to any one of claims 9 to 17, wherein the disruption efficiency is defined as the amount of disrupted cells compared to the amount of undisrupted cells, preferably or alternatively, as the amount of released biologically active compartments compared to the amount of undisrupted cell wall-free cells provided.

19. The method according to any one of claims 9 to 18, wherein the method is an integrated method of a production process, preferably an integrated method of a cell lysate production process.

20. A composition comprising at least one biologically active compartment and a biologically active fluid surrounding it, wherein said fluid and / or said compartment is capable of carrying out at least one step of protein biosynthesis.

21. An isolated biologically active compartment surrounded by a lipid bilayer, wherein the biologically active compartment exhibits at least one ability for ATP synthesis and / or energy regeneration, and / or at least one ability for at least one protein synthesis process, wherein the protein synthesis process preferably includes transcription, translation, post-translational modification, protein folding and / or translocation.

22. The biologically active compartment of claim 20 or 21, wherein the biologically active compartment comprises at least one transmembrane protein, at least one inner membrane and / or outer membrane associated protein, and / or at least one soluble protein within the interior space.

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