Regulatable disruption of eukaryotic protoplasts for release of intact organelles

Abstract

The present invention relates to an apparatus for destroying a cell without a cell wall containing a biologically active compartment surrounded by a lipid bilayer and a method of using the apparatus, wherein a shear force generating device (S) generates a region of shear force suitable for destroying the cell. Optionally, a pump (P) for pumping an aqueous medium containing the cells at an adjustable flow rate or flow velocity, preferably adjusting the residence time and shear force separately, and a separation device (C) for separating at least one released biologically active compartment and / or the biologically active liquid phase surrounding it from debris are combined. The apparatus is an independent system or an integrated part of a production line. The apparatus and method enable the production of products containing high yields of biologically active compartments exhibiting high biological activity, particularly in energy regeneration and / or protein synthesis.

Description

Technical Field

[0001] The present invention relates to an apparatus for destroying a cell without a cell wall containing a biologically active compartment surrounded by a lipid bilayer and a method of using the apparatus, wherein a shear force generating device (S) generates a region of shear force suitable for destroying the cell. Optionally, a pump (P) for pumping an aqueous medium containing the cells at an adjustable flow rate and / or flow velocity, and a separation device (C) for separating the disrupted cells and their fragments from at least one released biologically active compartment and / or the biologically active liquid phase surrounding it are combined. The apparatus is a stand-alone system or an integrated part of a production line. The apparatus and method enable the production of a composition containing a high yield of biologically active compartments exhibiting high biological activity, particularly in energy regeneration and / or protein synthesis.

[0002] In the technical field of cell culture, in particular, for the production of cell lysates from living cells, for example for cell-free production processes of recombinant proteins, various methods are used to produce lysates from cells. In particular, with regard to protein biosynthesis based on natural systems, in order to avoid the extraction of proteins from living cells, which later becomes difficult, the development of individual cell lines (over a long period) for each desired target protein, long culture times, and low protein production rates, it is desirable to use cell-free systems for several reasons. Therefore, it is desirable to extract a desired mechanism, such as a protein mechanism, from cells and use it for recombinant protein biosynthesis. For cell-free protein synthesis, the extraction of microsomal vesicles derived from the endoplasmic reticulum and Golgi, which promote the formation of disulfide bonds, glycosylation, and co-translational integration of membrane proteins, is necessary. Furthermore, it is also desirable to extract mitochondria, which provide energy for protein biosynthesis. To achieve high yields of protein and rapid biosynthesis, high concentrations of the aforementioned biologically active compartments are advantageous. In the prior art, in particular, homogenization methods, such as a Dounce homogenizer (Braun, Melsungen, Germany), are used to disrupt cells without cell walls. Subsequently, intact cells, nuclei, and cell membrane fragments are removed to obtain the desired lysate for protein synthesis. However, this device is not suitable for in-line production and is not scalable, and thus the yield of lysate is low. Furthermore, valve homogenization results in particles in the range of 0.5 to 1 μm, which are expected to damage the desired microsomes and undesired nuclei (Zhang et al., Table 1, Figure 3). In particular, plant DNA is also targeted, and the presence of plant DNA affects the biosynthesis of recombinant proteins because native plant proteins are produced instead of the recombinant protein target. WO 97 / 12959 A1 discloses a method for disrupting cultured cells lacking cell walls, including passing cells suspended in a culture fluid through a low-pressure impact jet device for the purpose of isolating naturally occurring products such as proteins, polysaccharides, recombinant proteins, and viruses. However, the method and device are unable to separate biologically active compartments and are significantly different from the solution of the present invention.

[0003] Accordingly, one object of the present invention is to provide a method and an apparatus for the controllable disruption of eukaryotic cells, in particular eukaryotic (mini) protoplasts, for releasing intact organelles such as mitochondria and / or microsomes. It is an object to provide an apparatus and a method that enable efficient, scalable, and inline disruption of (mini) protoplasts without destroying the organelles. Another object is to provide an apparatus and a method that enable adjustment and regulation of shear force and residence time independently of each other to a relevant degree of magnitude. Yet another object of the present invention is to provide a sterilizable apparatus and a method that enables a closed sterilization process.

[0004] Accordingly, the present invention has the advantage of enabling shear force and residence time that are adjustable / regulatable to a relevant degree of magnitude via rotor speed and / or flow rate and / or flow velocity, independently of each other, and provides a solution as described in detail below. The apparatus and method according to the present invention further enable an expandable closed sterile system. Thus, there has hitherto been no available device and method that enable scaling of cell disruption to ensure release of biologically active compartments. Furthermore, none of the hitherto known devices for particle treatment, sonication, homogenizer or grinder, or other inline cell disruption devices provide a shear force that is adjustable to the correct degree of magnitude. Continuous shear force generating devices, such as rotor-stator mixers, usually contribute to the pumping of fluids, such as liquid media containing the cells of the present invention, and are used without an external supply pump. The apparatus according to the present invention proposes for the first time an independent control of shear and residence time and enables a reasonable balance between pumping speed and shear force. Thereby, gentle disruption of cells, in particular cell cultures, is provided that provides intact organelles such as biologically active compartments. The advantage of the present invention is that the lysate obtained by the described method implemented by the described apparatus is improved and enables faster protein production with a higher protein yield.

[0005] A first aspect of the invention in an apparatus for disrupting a cell without cell wall containing a biologically active compartment surrounded by a lipid bilayer, preferably a (mini) protoplast and / or spheroplast, is that the apparatus comprises: - a shear force generating device (S) for generating a region of shear force suitable for disrupting the cell; - a tank A for providing a cell, preferably a plant cell, more preferably a plant of the genus Nicotiana of the Solanaceae family, most preferably the plant is N. tabacum, and most preferably a BY-2 cell line from N. tabacum; - optionally, a pump (P) arranged upstream or downstream of (S) for pumping an aqueous medium containing the cell at an adjustable flow rate and / or flow velocity; - at least one conduit connecting tank A and the shear force generating device S and / or at least one conduit connecting (S) and tank B; - a tank B for recovering the product from the previous step containing at least one released biologically active compartment; - a separation device (C) for separating the disrupted cells and their fragments from at least one released biologically active compartment and / or the biologically active liquid phase surrounding it. The apparatus is provided with the above components.

[0006] In certain embodiments of the device of the present invention, the device comprises a shear force generating device (S) and a pump (P) arranged upstream or downstream of (S) for pumping an aqueous medium containing cells without cell walls at an adjustable flow rate and / or flow velocity (Figure 1). Figure 1 shows some possible embodiments of the combination of the device (S) according to the present invention and an external pump (P). However, since the residence time can be adjusted using only the device (S) as defined herein, it is suitable for the method according to the present invention and is suitable for achieving the product of the present invention without an external pump (P). 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 regarding the residence time are applied as appropriate to the device of the present invention without an external pump, either alone or in combination with a preferred shear force generating device (S) (centrifugal pump or rotor-stator system). A person skilled in the art following the teachings of the present invention has the ability to adjust the settings of (S) according to the appropriate residence time.

[0007] In a further embodiment of the device of the present invention, the shear force generating device (S) is particularly suitable for generating shear by agitation of the aqueous medium and is designed accordingly. As shown in Figure 9, different devices (S) for generating shear are known. However, only those that mediate shear by a fluid, preferably an aqueous medium containing cells without cell walls of the present invention, particularly by agitation of the fluid, are within the scope of the meaning of the present invention.

[0008] In another embodiment of the device of the present invention, the shear force generating device (S) is a centrifugal pump, a high shear mixer, or a tooth-type or blade-type rotor-stator mixer.

[0009] In one form, a device for disrupting cells without cell walls containing it, preferably (mini) protoplasts and / or spheroplasts, without disrupting the biologically active compartment surrounded by the lipid bilayer, - A shearing force generating device (S), preferably suitable for generating shear by stirring an aqueous medium, a shearing force generating device (S) so designed, preferably a rotor-stator system, more preferably an IKA magic lab or IKA UTL25, and, - Cells, preferably plant cells, more preferably plants of the genus Nicotiana of the Solanaceae family, most preferably the plant is N. tabacum, and a tank A for providing a BY-2 cell line from N. tabacum, - A pump (P) arranged upstream or downstream of (S) for pumping an aqueous medium containing the cells at an adjustable flow rate and / or flow velocity, - At least one conduit connecting tank A and the shearing force generating device S and / or at least one conduit connecting (S) and tank B, - A tank B for recovering the product from the previous step, containing at least one released biologically active fraction, - A separation device for separating disrupted cells and their fragments from at least one released biologically active fraction and / or the biologically active liquid phase surrounding it An apparatus comprising.

[0010] This apparatus is suitable for adjusting different residence times as defined herein. More preferably, the aforementioned rotor-stator system is adjustable to residence times of less than 75, less than 74, less than 73, less than 72, less than 71, less than 70, less than 65, 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, less than 10 seconds. In this example and for specific apparatus designs and processes, the residence is less than 10 seconds, 9, 8, 7, 6, 5, 4, 2, 1 second.

[0011] In one embodiment, an apparatus for disrupting cells without a cell wall containing a biologically active fraction surrounded by a lipid bilayer, preferably (mini) protoplasts and / or spheroplasts, without disrupting the biologically active fraction, - A shearing force generating device (S), preferably for stirring an aqueous medium, preferably a centrifugal pump, more preferably a PuraLev i100SU, suitable for generating shear and so designed, and - Cells, preferably plant cells, more preferably plants of the Solanaceae tobacco genus, most preferably the plant is N. tabacum, and most preferably a tank A for providing BY-2 cell lines from N. tabacum, and - A pump (P) arranged upstream or downstream of (S) for pumping an aqueous medium containing the cells at an adjustable flow rate and / or flow velocity, and - At least one conduit connecting tank A and the shearing force generating device S and / or at least one conduit connecting (S) and tank B, and - A tank B for recovering the product from the previous step, containing at least one released biologically active fraction, and - A separation device (C) for separating the disrupted cells and their fragments from at least one released biologically active fraction and / or the surrounding biologically active liquid phase Comprising an apparatus.

[0012] This apparatus is suitable for adjusting different residence times as defined herein. More preferably, the aforementioned centrifugal pump is adjustable to residence times of less than 75, less than 74, less than 73, less than 72, less than 71, less than 70, less than 65, 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. In this example and for specific apparatus designs and processes, the residence is less than 10 seconds, 9, 8, 7, 6, 5, 4, 2, 1 second.

[0013] In certain embodiments of the device according to the invention, a shear force generating device (S) (synonym, shear generating device (S)) is suitable for generating shear forces (synonym, shear) sufficient to disrupt cells without damaging at least one lipid bilayer containing a biologically active compartment and is controllable. Preferably, the shear force generating device (S) is a shear force generating device (S) that mediates the shear force through a fluid, particularly an aqueous medium. This is distinguished from other devices that can mediate shear forces through solids (Figure 9). More preferably, the shear force generating device (S) of the device according to the invention is suitable for generating shear forces by agitation of an aqueous medium (= liquid) containing cells without cell walls and is designed accordingly. Such a device containing the device (S) is also suitable for the process according to the invention, and the shear force is exerted by agitating the aqueous medium containing the cells, preferably by a centrifugal pump, a high shear mixer, or a tooth-type or blade-type rotor-stator mixer.

[0014] Preferably, the applied shear force does not damage the "biologically active content". Preferably, (S) is a centrifugal pump, a high shear mixer, or a tooth-type or blade-type rotor-stator mixer (Figure 2). A rotor-stator system containing a tooth-type rotor consists of a circular plate attached to a rotating shaft having a circle of one or more teeth. Together with the stator, one or more sets of concentric circles of teeth constitute the rotor-stator system (Figure 2B). However, different designs of the shear force generating components of the shear force generating device (S) are possible.

[0015] In one embodiment, the shear force generating device (S) is a rotor-stator system which is a suitable inline production embodiment of the present invention (Hakansson, 2018). Such rotor-stator systems are equally suitable for standalone systems. Suitable rotor-stator systems, for example, IKA MagicLab and IKA UTL 25 inline are commercially available and, depending on the design, the rotor-stator system can be adjusted within speed ranges of 3,000 - 26,000, 10,000 - 24,000, 3,000 - 20,000 [min⁻¹], throughput of 2 - 20 l / min, 5 - 15 l / min, 5 - 12 l / min, 11.6 l / min, maximum [l / h] of 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], and / or circumferential speeds of 5 - 40, 5 - 30, 5 - 23 and 6 - 16 [m / s]. Within the scope of the present invention, preferably for an aqueous medium, the speed of the rotor-stator system is at least 3000 rpm, at least 4000 rpm, at least 4500 rpm, 5000 rpm, at least 6000 rpm, at least 7000, at least 8000 rpm, at least 9000, at least 10000 rpm or more, or any other speed in between the aforementioned ranges. In particular, preferably in the case of a rotor-stator system, the rotor tip speed is within a range of at least 4 m / s and 32 m / s or less, can be adjusted in combination with a speed of at least 6000 rpm, and a residence time of less than 25 seconds can be obtained (Example 9).

[0016] MAGIC LAB (registered trademark) is used for mixing and dispersing, especially in the chemical and food industries, and is therefore a fluid-mediated shear force generating device (Figure 9). MAGIC LAB (registered trademark) is suitable for the development of new processes and the scaling-up of the same methods to large-scale production because many parameters of MAGIC LAB (registered trademark) and IKA production machines of the 2000 series are the same. It is ideally suited for batch applications, but different versions are also available for in-line applications. The basic unit of the in-line machine is equipped with a single-stage dispersion module ULTRA-TURRAX (registered trademark) UTL. Various process parameters such as speed, shear rate, temperature, pressure, and time can be determined. Optimal mixing, dispersion, and grinding results are achieved due to a rotor tip speed of up to 40 m / s and a high energy input into the product resulting from the rotation of the medium. During the mixing process and due to the energy input into the medium containing cells, the cells contained therein are disrupted.

[0017] The speed depends on the scaling of the disruption method according to the present invention and on the device used, a stand-alone system, or preferably an in-line embodiment of the device according to the present invention. Thus, the present invention includes any adjusted speed, optionally or preferably in combination with any adjusted speed of a pump, to obtain the desired directly obtained "process product" implemented by a device that defines and enables a scaled disruption method as defined herein.

[0018] The foregoing rotor - stator system, preferably the IKA Magic Lab and the IKA UTL 25 Inline, is adjustable to different residence times as defined herein. More preferably, the foregoing rotor - stator system, preferably the IKA Magic Lab and the IKA UTL 25 Inline, is adjustable to a residence time that is the period of time in which cells in an aqueous medium are held in a region of shear force and / or turbulent flow sufficient to disrupt the cells without destroying at least one biologically active compartment enclosed by a lipid bilayer. Preferably, the residence time of the foregoing rotor - stator system, preferably the IKA Magic Lab and the IKA UTL 25 Inline, is less than 25 seconds, less than 20, less than 10 seconds, less than 9, 8, 7, 6, 5, 4, 2, 1 second.

[0019] It is preferred to reduce the achievable residence as low as possible by adapting the design of the shape and volume of (S) in combination with the design of the pump, respectively. Thus, it is obvious to a person skilled in the art that when scaling up the device and its components, it is necessary to adjust to maintain the desired residence time. In Example 9, it is shown that a residence time of 12 seconds for the IKA Magic Lab with 1x teeth 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 the cells and provide an active lysate for high protein yield production.

[0020] The "directly obtained process product", which is carried out by the method according to the invention, preferably by the use of the device 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 contains cells without disrupted cell walls, preferably disrupted miniprotoplasts, protoplasts and / or spheroplasts. This directly obtained process product preferably contains at least one biologically active compartment and the surrounding biologically active liquid (= liquid medium). The liquid and / or at least one compartment enable 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" includes biologically active molecules such as enzymes, soluble proteins and / or small molecules derived from the cytosol of cells without cell walls (= the surrounding biologically active liquid), and / or mitochondria, microsomes, nuclei, Golgi and / or ER. Therefore, the "functional product" is defined by its ability to carry out or enable cell-free protein biosynthesis of recombinant proteins. It is demonstrated using the methods presented herein. Preferably, the functional product contains at least one released biologically active compartment (part of the "biologically active content") capable of ATP synthesis, energy regeneration, 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 debris (nuclei, cell membrane debris and (mini) protoplast debris) and exhibits the ability of cell-free protein biosynthesis of recombinant proteins. Preferably, the production capacity is greater than 10 ml / day up to a maximum of 100 L / day, up to a maximum of 1000 L / day. Preferably, the device operates automatically, either alone or as an essential component of production.

[0021] In another embodiment, the shear generating device is a centrifugal pump, such as the Levitronix® puraLev i100 SU (e.g., PLD-i100SU.1, PLD-i100SU.5, PLD-i100SU.2, PLD-i100SU.3). This pump is based on the principle of magnetic levitation, and the impeller of the pump is suspended non - contact in a sealed casing and driven by the magnetic field of the motor. The pump head is sterilizable before disposal, sterilization or use and is suitable for biological substances. The flow rate is adjusted by the rotor speed, and pressure control is also possible. The maximum flow rate is 17 L / min, the maximum Diff pressure is 2 bar / 29 psi and the maximum viscosity of the medium is <20 cP. Preferably, the flow rate of the centrifugal pump for an aqueous medium is preferably adjustable in the range of 0.00 - 50 mL / min, 0.0 to a maximum of 40 L / min, 0.0 to a maximum of 30 L / min, 0.0 to a maximum of 20 L / min, at least 2.5 L / min to a maximum of 50 L / min - 20 L / min, at least 5.0 L / min to a maximum of 50 L / min - 20 L / min, at least 7.5 L / min to a maximum of 50 L / min - 20 L / min, at least 10 L / min to a maximum of 50 L / min - 20 L / min, at least 12.5 L / min to a maximum of 50 L / min - 20 L / min, at least 15 L / min to a maximum of 20 L / min, at least 17.5 L / min to a maximum of 20 L / min. This flow rate range can be achieved by rotor speeds in the range of 3000 rpm to a maximum of 10000 rpm, 3000 rpm to a maximum of 9000 rpm, 4000 rpm to a maximum of 9000 rpm, 4000 rpm to a maximum of 8000 rpm, 4000 rpm to a maximum of 7000 rpm, 4000 rpm to a maximum of 6000 rpm and 4000 rpm to a maximum of 5000 rpm.

[0022] In particular, in another embodiment, the rotor speed of the centrifugal pump as the shear generating device (S) is in the range of 500 rpm to a maximum of 3000 rpm, 500 rpm to a maximum of 2500 rpm, 500 rpm to a maximum of 2000 rpm, 500 rpm to a maximum of 1500 rpm, and 500 rpm to a maximum of 1000 rpm.

[0023] As disclosed herein, the centrifugal pump is a shear generating device (S) according to the present invention suitable for adjusting the rotor tip speed from 2 m / sec or more up to a maximum of 50 m / sec. A rotor speed range from 3000 rpm up to a maximum of 9000 rpm results in a rotor tip speed from 6.8 m / sec to 20.3 m / sec (Example 9). In particular, by adjusting the rotor speed to 500 rpm, the rotor tip speed becomes 1.1 m / sec (m / sec is synonymous with m / s), which in combination with a residence time in the range of less than 25 - 75 seconds results in effective dissolution in the method (Example 9).

[0024] The aforementioned centrifugal pumps, such as Levitronix® puraLev i100 SU (e.g., PLD-i100SU.1, PLD-i100SU.5, PLD-i100SU.2, PLD-i100SU.3), and preferably the centrifugal pump of Example 9, are adjustable to different residence times as defined herein. More preferably, the aforementioned centrifugal pumps, such as Levitronix® puraLev i100 SU (e.g., PLD-i100SU.1, PLD-i100SU.5, PLD-i100SU.2, PLD-i100SU.3), and preferably the centrifugal pump of Example 9, are adjustable to a residence time that is a period during which cells in an aqueous medium are held in a region of shear force and / or turbulent flow sufficient to disrupt the cells without destroying at least one biologically active compartment enclosed by a lipid bilayer. Preferably, the residence time for the aforementioned centrifugal pumps, such as Levitronix® puraLev i100 SU (e.g., PLD-i100SU.1, PLD-i100SU.5, PLD-i100SU.2, PLD-i100SU.3), preferably the centrifugal pump of Example 9, is less than 75 seconds, less than 74, less than 73, less than 72, less than 71, less than 70, less than 65, less than 60 seconds, less than 55 seconds, less than 50 seconds, less than 45 seconds, less than 40 seconds, less than 30 and less than 25 seconds. It is preferred to minimize the achievable residence time as much as possible by adapting the design of the shape and volume of (S) in combination with the pump design, respectively. However, as described above, the present invention provides a device and solution that has the advantage of enabling adjustable / regulatable shear force and residence time to a related degree of magnitude via rotor speed and / or flow rate and / or flow velocity that are independent of each other. Embodiments of the device according to the present invention comprising a centrifugal pump together with an external supply pump achieve for the first time independent control of shear and residence time and enable a reasonable balance between pumping speed and shear force.

[0025] A centrifugal pump, for example, equipped with a driver IPD-100.3-03-02 and a pump head DCP-200.3, in combination with a rotor tip speed of 1.1 m / s, by a PuraLev i100SU having a speed of 500 rpm and a flow rate of 56.4 mL / min, adjusting the device to a residence time of less than approximately 25 seconds (Example 9, Table 3) is sufficient to effectively disrupt cells without cell walls contained in an aqueous medium, preferably from the BY-2 cell line, and as shown in Test No. 1, Table 3 (Figures 10, 11, 12), at least one protein biosynthesis-related process including expression, transcription, translation, translocation, protein folding and / or protein modification is possible, and it is sufficient to provide an active lysate capable of ATP synthesis and / or energy regeneration. Since there are few cells without disrupted cell walls (Figure 13, Column C, No. 1), the functional product achieved has a low content of the released biologically active compartment (part of the "biologically active content") capable of ATP synthesis, energy regeneration and / or at least one protein biosynthesis-related process as defined herein. However, in order to increase the amount of the released biologically active components and as a result increase the activity of the lysate, it is feasible to repeat the "circular run" as defined herein. However, in combination with rotor tip speeds of 10.1 or 20.3 m / s respectively, at speeds of 4500 rpm or 9000 rpm, and flow rates of 20 mL / min or 56.4 mL / min, by adjusting the same device to a residence time of less than 71 seconds or approximately 25 seconds (Example 9, Table 3), it is sufficient to effectively disrupt cells without cell walls contained in an aqueous medium, preferably from the BY-2 cell line, and provide an active lysate for the production of a high protein yield (Test Nos. 2 - 5, Table 3, Figures 10, 11 and 12). Therefore, it will be apparent to those skilled in the art that when scaling up, the devices and their components need to be adjusted to maintain the desired residence time as shown in this example.

[0026] The PuraLev® i100 disposable pump is designed for the demanding fluid applications in bioprocesses where extremely low shear, pulseless, and continuously controlled flow ensures the highest product yields. Based on the principle of magnetic levitation, the pump impeller is suspended non - contact within a sealed casing and driven by the magnetic field of the motor to provide a continuous smooth flow, thus being a fluid - mediated shear force generating device (S) (Figure 9). The pump head is disposable and can be exchanged in a few seconds. The flow rate and pressure of the fluid are accurately controlled by electronically regulating the impeller speed. The motor and pump controller are integrated into the driver's housing, resulting in a significant reduction in the installation area.

[0027] Hakansson, 2018 gives guidance for scaling of the rotor - stator system and for calculating forces in batch and in - line (pages 8 - 10), and compares the rotor - stator system with centrifugal pumps. Various embodiments of high - shear mixers (HSMs) suitable as shear - generating devices according to the present invention are disclosed by Zhang et al. In particular, Figure 1 of Zhang et al. represents different geometric variations of commercially available high - shear mixers, such as the teeth in - line unit (Ytron - Quadro z), the blade screen in - line unit (Silverson 150 / 250 MS), the radial discharge unit (left - Silverson L4R, right - VMI Rayneri) and the axial discharge unit (Greerco 1.5 HR). These HSMs can be used in in - line production and as stand - alone systems (batch units), as described by Espinoza et al.

[0028] Preferably, the production line has a production capacity of at least 10 ml / day, where "day" is defined as the production day from the end of cell culture until the "functional product" is obtained.

[0029] In another embodiment of the device according to the invention, the pump is suitable for adjusting the residence time of the aqueous medium containing the cells in (S), independently of the shear force adjusted and generated by (S), where the residence time is the period during which the shear force acts on the aqueous medium containing the cells. The "residence time" can be controlled or regulated by adjusting the flow rate and / or the flow velocity of the pump (P). In combination with the desired shape of the shear force generating device, preferably a rotor - stator mixer, the residence time can be controlled or regulated by adjusting the flow rate and / or the flow velocity of the pump, independently of the adjustment of the shear force in (S). Thus, the flow rate and / or the flow velocity are used to manipulate the residence time. As shown in Example 2, Table 2, a residence time of 45 seconds is adjusted within the rotor - stator system by the flow rate of the pump.

[0030] In Example 9, it has been shown that a shear force generating device (S), for example, a rotor - stator mixer or a centrifugal pump with an external supply pump, enables independent control of shear and residence time, allowing a reasonable balance between the pumping rate and the shear force. Thereby, gentle disruption of the cell - wall - free cells, preferably from the BY - 2 cell line, is provided (Figs. 10 - 13), especially of the cell culture, thereby providing intact cell organelles, for example, biologically active compartments, most preferably of the BY - 2 cell line. The advantage of the present invention is that the lysate obtained by the described method implemented by the described device is improved, enabling faster protein production with a higher protein yield.

[0031] Flow rate specifies the rate of fluid transport through a system and is the amount of fluid moved per unit time (e.g., mL / min). Flow velocity specifies the velocity at which a fluid moves through a system, measured as distance per unit time (e.g., m / sec). Both flow rate and flow velocity are related to the rotor-stator system. Flow rate, in combination with the shape of the rotor-stator shear force region (specifically, the volume of the shear force region), determines the residence time in the shear force region. Similarly, flow velocity, in combination with the shape of the rotor-stator shear force region (specifically, the path length through the shear force region), determines the residence time in the shear force region.

[0032] Preferably, the residence time is the period during which shear forces sufficient to disrupt cells are exerted on an aqueous medium containing cells without disrupting at least one biologically active compartment enclosed by a lipid bilayer. More preferably, the residence time is the period during which cells are held in a region of shear force and / or turbulent flow. Residence is as short as possible to accelerate the overall disruption and production processes. In particular, the residence time is as short as possible to accelerate the overall disruption and production processes by use of the apparatus according to the invention. In Example 2 (Table 2), a residence time (VZ) of 45 seconds was adjusted for the rotor-stator system, where the residence time (VZ) is the quotient of the internal volume Vi of the shear generation device and the pump flow rate (R) (see Example 9).

[0033] Preferably, the residence time is less than 25 seconds, less than 20, and less than 10 seconds. In this example and with certain device designs and processes, the residence is less than 10 seconds, 9, 8, 7, 6, 5, 4, 2, 1 second. In particular, in Example 9 and with a specific device and process design of the centrifugal pump (S), the residence time is more than 70 seconds and less than 71 seconds, approximately 70.5 seconds, and approximately 25 seconds, and with a specific device and process design of the rotor - stator system (S), the residence time is less than approximately 25 seconds. It is preferred to reduce the residence as much as possible by adapting the design of the shape and volume of (S) in combination with the pump design respectively. Therefore, when scaling up, it is obvious to those skilled in the art that the devices and their components need to be adjusted to maintain the same residence time as shown in this example.

[0034] The residence time is the period during which, particularly during the method of the present invention, sufficient shear force to disrupt the cells is exerted on the aqueous medium containing the cells without disrupting at least one biologically active compartment surrounded by a lipid bilayer. Together with these conditions, an active lysate for the production of a high protein yield is provided (see Examples 2 and 9).

[0035] "As short as possible" should be understood in the context of the shape of each shear - force - generating device (S) according to the present invention and considering the process product obtained directly as defined herein. Further, "as short as possible" considers each device comprising the shear - force - generating device (S) alone or in combination with an external supply pump. Since each device defines the limits of adjustable values, it is obvious to those skilled in the art that "as short as possible" means depending on the final device setup. In particular, the residence time is less than 75 seconds, less than 74, less than 73, less than 72, less than 71, less than 70, less than 65, less than 60, less than 55, less than 50, less than 45, less than 40, less than 35, less than 30 and less than 25 seconds, less than 20, less than 10 seconds, 9, 8, 7, 6, 5, 4, 2, 1 second.

[0036] The pump is preferably a self-priming and / or self-sealing positive displacement (e.g., peristaltic) pump with an adjustable pump speed. Preferably, the pump controls the flow rate and / or flow velocity at which an isotonic or hypertonic aqueous medium is transported to S, particularly the space between the rotor and the stator. One embodiment includes a peristaltic pump, for example, Watson marlow 120u / dv with an adjustable pump speed in the range of up to 0 - 200 rpm was used. In Example 9, a peristaltic pump Hei-Flow ultimate 120 with an SP quick 1.6 head was used, and the speed can be adjusted from 5 - 120 rpm and the flow rate from 0.83 - 861 ml / min. However, the pump speed (rpm) of this 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. The peristaltic pump does not include valves, seals or glands that clog or corrode and is thus the simplest pump. The aqueous medium only contacts the inner wall and the internal space of the conduit. This ensures hygienic handling and there is no risk of the pump contaminating the aqueous medium or the aqueous medium contaminating the pump. The peristaltic pump can operate at temperatures from at least -10°C to a maximum of 50°C, enabling operation under controlled temperature. This pump is suitable for viscous shear-sensitive aqueous media even if it contains solid components. According to the present invention, the aqueous medium contains biological substances including cells and cell-free cells that are not considered solids. According to the manufacturer's information, the pump head can be sized from an inner diameter of 0.5 mm to an inner diameter of 4.8 mm and adjusted to accommodate a 1.6 mm wall tube. It is obvious to those skilled in the art that the flow rate can vary due to changes in the aqueous medium, the viscosity of the aqueous medium, the processing temperature, the inlet and discharge pressures, the system configuration and / or the piping performance over time. Generally, the flow rate is from 0.001 - 170 ml / min. Some examples of the flow rate for different piping settings are shown below.

Table 1

[0037] In Example 9 of the present invention, the flow rate of the method according to the present invention was adjusted in the range of 20 mL / min to 150 mL / min. Therefore, it is preferable to adjust the flow rate in the method according to the present invention to be 20 mL / min or more and 150 mL / min or less, preferably 20 mL / min or more and 120 mL / min or less, 20 mL / min or more and 100 mL / min or less, 20 mL / min or more and 80 mL / min or less, and more preferably 20 mL / min or more and 60 mL / min or less.

[0038] However, it is recommended to determine the flow rate under operating conditions before implementing the method and apparatus according to the present invention. The pump speed can be adjusted according to the situation when another pump is combined. In particular, in the case of scaling the disruption method according to the present invention, adjustments to the pump size, processing parameters, and flow rate will be required depending on the apparatus used, the stand-alone system, or preferably the in-line embodiment of the apparatus accordingly. Therefore, the present invention preferably includes any adjusted pump speed in combination with any adjusted speed of the shear force generating device in order to obtain the desired directly obtained "process product" implemented by an apparatus that defines and enables a scaled disruption method herein.

[0039] It is difficult to predict the turbulent flow and shear in shear force generating devices of different scales. In the liquid-liquid approach, it has been proposed to apply this approach due to the cells in the culture and the high water content of the cells that can be regarded as "liquid", and the highly localized intense turbulent flow and shear in the shear force generating device, particularly the rotor-stator and HSM, cause multiple disruption mechanisms that result in the disruption of droplets (see Zhang et al., with Kolmogoroff cited therein). This approach can be diverted to cells without cell walls according to the present invention.

[0040] In another embodiment of the device according to the invention, the device is an integrated device for a production line, preferably a closed aseptic production line, more preferably a production line for a plant cell line, in particular a lysate production line without plant cells. In another embodiment of the device according to the invention, the device is a stand-alone system, preferably a closed aseptic stand-alone system, more preferably a system for disrupting plant cells. Preferably, the plant cells are a plant cell line of N. tabacum, most preferably the BY-2 cell line from N. tabacum.

[0041] Accordingly, the device may be a stand-alone system as schematically shown in FIG. 1. In a stand-alone system, tank A provides the starting material. The starting material includes cells without cell walls, more preferably animal cells, plant cell cultures or fungal (mini) protoplasts and bacterial spheroplasts, and is an aqueous medium suitable for large-scale culture. As defined herein, the cell culture, preferably after pretreatment, more preferably after vacuolation of the plant cell line, is disrupted. Tank A is connected via a conduit to a shear force generating device (S), and the downstream conduit connects the shear generating device (S) to tank B. A pump (P) may optionally be incorporated into the stand-alone system and may be placed upstream or downstream of the shear generating device (S). Within the stand-alone system, a separation device (C), preferably a centrifuge, is placed downstream of the shear generating device (S), particularly downstream of the pump if the pump is incorporated. The stand-alone system allows for a cyclic operation of disruption. "Cyclic operation" means that a predetermined condition, in combination with the speed of the shear generating device (S) and optionally the speed of the pump, is repeated. The force is exerted multiple times on the aqueous medium containing the biological material over a longer period of time. According to the present invention, the aqueous medium passes through the shear force region two or more times, at least two times, three times, four times or more as needed. The disruption according to the present invention includes at least the steps of providing an aqueous medium containing cells without cell walls (preferably in tank A), moving the aqueous medium into the shear force generating device (S), exerting a shear force on the aqueous medium containing the cells for a residence time sufficient to disrupt the cells without disrupting at least one biologically active compartment, and disrupting the cells without disrupting the lipid bilayer of at least one biologically active compartment while the aqueous medium is passing through S, and may include at least one pass, at least two passes, at least three passes, at least four passes or more of the steps of disrupting the cells.

[0042] As already described herein, the device according to the invention is also suitable for obtaining the method and the product of the invention when the shear force generating device (S) is used without an external pump (P). The residence time as defined herein is also adjustable with the device (S) alone. All embodiments described herein in relation to the residence time are applicable to the device of the invention without an external pump (P), either alone or in combination with a preferred shear force generating device (S) (preferably a centrifugal pump or a rotor-stator system). By applying the technical teachings of the invention, a person skilled in the art can adjust the settings of (S) according to the appropriate residence time. One way to adjust the settings for the same residence time is to repeat the cycle operation of the disruption defined above. Thus, the "circulation operation" is repeated on the aqueous medium containing the biological material at the speed of the shear force generating device (S) under predetermined conditions. Thus, the aqueous medium passes through the shear force region two or more times, at least two times, three times, four times or more as necessary.

[0043] In an embodiment of the device according to the invention, the device is an integrated device of the production line (applying FIG. 1 according to the situation, and tank A, tank B and / or (C) are coupled to downstream or upstream units), and for the upstream process, upstream tanks, components and / or devices are possible. Any kind of pretreatment known to those skilled in the art in the field of production systems of cell bioreactors, biotechnology bioreactors, and any kind of cell line, such as microbial, human cell line, yeast, fungal and plant cell lines, is conceivable. For each of the production lines, it is preferred that it is a closed aseptic production line, more preferably that it operates and is controlled automatically. More preferably, it is a production line for a plant cell line, a plant cell line of N. tabacum, most preferably the BY-2 cell line from N. tabacum.

[0044] Preferably, the production line has a production capacity of at least 10 ml / day, where "day" is the production day defined as the period from the end of cell culture to obtaining the "functional product". The "functional product" is defined by its ability to perform or enable cell-free protein biosynthesis of recombinant proteins. Preferably, the functional product includes at least one released biologically active compartment capable of 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, the "functional product" is separated from debris (cell membrane debris and (mini) protoplast debris) and exhibits the ability of cell-free protein biosynthesis of recombinant proteins. Preferably, the production capacity is more than 10 ml / day up to a maximum of 100 L / day, a maximum of 1000 L / day. Preferably, the device operates automatically, either alone or as an essential component of production.

[0045] The standalone system has a volume of at least 100 ml, at least 1 L, at least 1 L, at least 5 L, at least 10 L, at least 100 L, at least 1000 L in tank (A) and can be adjusted to any desired process volume. In particular, the scaling can be adjusted according to the predetermined volume of tank (A), the volume that the shear generation device (S) can process simultaneously, and the volume that the optional pump (P) can process simultaneously.

[0046] Another aspect of the present invention is a production line comprising the device according to the present invention, wherein the tank (A) is arranged downstream of at least one unit for the pretreatment of the biological material, the conduit connects the tank (A) and (S), the tank (B) is arranged downstream of (S), and optionally the pump (P) is arranged downstream or upstream of (S). As described therein, "pretreatment" includes any other treatment of the biological material as defined herein in the production protocol applied within the production line or in a stand-alone system. The unit for pretreatment may include a cell wall digestion process, a buffer solution, a supply of drugs, etc. Preferably, at least one unit is a device for the vacuolization of a plant cell line or a tank providing vacuolized plant material.

[0047] In one embodiment, the tank (B) and the tank (A) are identical if it is desired to repeat the disruption two or more cycles. This embodiment is applicable to both stand-alone systems and production lines. In the latter case, the production line includes a circulation disruption device with means for opening the circulation disruption device for continuing the appropriate conduits and downstream processes. After the disruption is completed, the functional product as defined herein is transferred to a separation device (C), preferably a centrifuge. In a preferred embodiment, the tank (B) is a device for separation, preferably a centrifuge. The centrifuge is suitable for separating at least one released, particularly intact, biologically active compartment surrounded by a lipid bilayer. Preferably, the lipid bilayer of the separated compartment is intact. Thus, the separation does not damage and disrupt the released biologically active compartment.

[0048] Another aspect of the invention is a method for the disruption of cell wall - free cells, preferably (mini) protoplasts and / or spheroplasts, comprising at least one biologically active compartment enclosed by a lipid bilayer, preferably carried out using any device according to the invention, wherein preferably the cells are in the range from 0.5 μm or more up to a maximum of 200 μm, more preferably from 5 μm or more up to a maximum of 100 μm, from 10 μm up to a maximum of 100 μm, more preferably from 10 μm or more up to a maximum of 70 μm, more preferably from 20 μm or more up to a maximum of 70 μm. - Providing an aqueous medium containing cell wall - free cells, preferably in tank (A), preferably the aqueous medium contains biological material containing cell wall - free cells and preferably has a viscosity of less than 20 mPa·s (at 21 °C), in the range of less than 15 mPa·s, less than 10 mPa·s, from 0.5 to a maximum of 10 mPa·s, preferably from 0.5 to a maximum of 5 mPa·s, preferably provided in tank (A) of the device according to the invention. - Moving the aqueous medium into the shear - force generating device (S) according to the invention, preferably at the predetermined rate described herein for (S) and / or (P). - Optionally, adjusting the flow rate and / or flow volume of the aqueous medium by means of the pump (P) according to the invention, preferably at the pump rate described herein. - Generating a region of shear force, preferably a region of shear force and / or turbulent flow, more preferably in a centrifugal pump or a rotor - stator system, within (S). - Applying shear force over a residence time sufficient to disrupt the cells, without disrupting at least one biologically active compartment, preferably applying shear force and / or centrifugal force within the region of shear force and / or turbulent flow, to the aqueous medium containing the cells. - Disrupting the cells while the aqueous medium passes through (S), without disrupting the lipid bilayer of at least one biologically active compartment. - Releasing at least one intact biologically active compartment and the biologically active liquid surrounding it, preferably into the aqueous medium. - Optionally, recovering at least one released intact biologically active fraction, together with its surrounding biologically active liquid, preferably in tank (B). - A separation step of at least one released intact biologically active fraction and its surrounding biologically active liquid, preferably by the separation device (C) according to the invention, wherein preferably at least part of the residue ((mini) protoplasts and / or spheroplast lipid bilayers) is removed. - Optionally, obtaining a fraction released from the intact biologically active fraction, preferably together with its surrounding biologically active liquid, and - Optionally, isolating at least one released intact biologically active fraction The method comprises the steps of

[0049] The method according to the invention is suitable for implementation by stand-alone systems or production lines described separately. All embodiments of the device are applied to the method for disruption, depending on the situation.

[0050] "Disrupting" a cell means that the lipid bilayer of a cell without a cell wall, in particular a protoplast and / or a mini-protoplast or a spheroplast, is "broken down" or "the cell is opened" in order to enable the release of its biological contents as defined herein. "Disrupting" in the sense of the present invention is a fluid mechanical process by shear forces significantly different from sonication, which applies acoustic energy at different frequencies (e.g., ultrasonic frequencies (>20 kHz)) to reduce the particle size in a sample. This is also significantly different from the process of homogenization, which mixes and processes two (immiscible) liquids to achieve a homogeneous mixture, e.g., a process of homogenization that results in uniformly dispersed particles. By homogenization, biologically active fractions are also disrupted (Figure 9).

[0051] Preferably, the cell wall-free cells range from 5 μm or more up to a maximum of 200 μm, more preferably from 5 μm or more up to a maximum of 100 μm, from 10 μm up to a maximum of 100 μm, more preferably from 10 μm or more up to a maximum of 70 μm, and even more preferably from 20 μm or more up to a maximum of 70 μm. Preferably, the cells comprise protoplasts and / or miniprotoplasts or spheroplasts, or other equivalent biologically active structures of other species. Preferably, the aqueous medium containing the cell wall-free cells is essentially free of cellulose, essentially free of chitin, and / or essentially 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 intact biologically active compartment and the biologically active liquid surrounding it together form a functional product. The "biologically active liquid" includes the cytosol of the cell wall-free cells, preferably (mini)protoplasts and / or spheroplasts, as well as 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) according to the invention, preferably by centrifugation. The separation may be carried out one or several times. Furthermore, a purification step may be carried out before, after or instead of the separation. The purification is preferably carried out after the separation, more preferably by filtration and / or centrifugation of the separated functional product. The difference between separation and purification is that the purpose of separation is to remove debris (cell membrane debris and protoplast debris) from the functional product, whereas the purpose of purification is to isolate 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 invention. Preferably, the purification is carried out to obtain a "fraction" of biologically active microsomes and / or biologically active mitochondria.

[0052] The washing step may be further incorporated into the method according to the invention. Washing may be carried out before or after at least one pre-treatment (e.g., vacuolation), before or after disruption according to the invention, before or after a further disruption cycle, before or after isolation and / or before or after at least one separation. Thus, where appropriate, the washing step may be incorporated between any steps according to the invention. Whether washing is necessary or desirable depends on the volume obtained from the upstream steps, as well as the volume required for the downstream device / tank and / or process or final formulation. It may be desirable to carry out at least one washing step in order to isolate at least one released intact biologically active compartment from the separated functional product. Washing is carried out using a suitable buffer / solution having the stabilizing properties of the lipid bilayer in order to stabilize the isolated compartment, preferably microsomes and / or mitochondria. However, washing is not essential for obtaining a good quality "functional product".

[0053] The functional product obtained by the method according to the invention, any directly obtained process product or intermediate product, as well as the separated functional product may be subjected to further processing steps. Thus, depending on the optional isolation and / or washing and / or sterilization, functional products of different degrees of purity (functional products having the desired purity) are obtained. After the desired degree of purity has been achieved, further processing may include, following the washing and / or mixing and / or supplementation and / or sterilization and / or freezing, drying, gas drying and / or lyophilization of the respective obtained product. However, subsequent steps such as freezing, drying, gas drying and / or lyophilization may also be steps in the production line according to the invention. In this embodiment, the final product is defined as a dried and / or frozen lysate (final lysate) having the desired degree of purity.

[0054] "Isolation" means that the product from a previous step, preferably a functional product, is centrifuged and / or filtered in order to achieve a higher degree of purity compared to the higher content of at least one biologically active fraction, preferably the purity of the intermediate product obtained from the previous step. Isolation reduces the content of the surrounding biologically active liquid and increases the content of the biologically active fraction. Preferably, isolation results in the isolation and / or concentration of the desired "fraction" of the biologically active fraction, for example, a fraction capable of ATP synthesis and / or energy regeneration, or a fraction capable of at least one protein biosynthesis-related process including expression, transcription, translation, translocation, protein folding and / or protein modification. Preferably, the active fraction of ATP synthesis and / or energy regeneration contains biologically active fractions in the range of 0.5 to 2 μm. The active fraction of the protein biosynthesis-related process contains biologically active fractions in the range of 0.5 to 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 method according to the invention, isolation provides the desired fraction having the desired biological activity.

[0055] In another embodiment of the method according to the invention, a predetermined rotor tip speed from 2 m / s or more up to a maximum of 50 m / s is applied by (S), preferably in the range from at least 4 m / s to 32 m / s or less. The appropriate range is adjusted to the scale of the device according to the invention and used for the method according to the invention, as effectively shown in Example 9. The rotor tip speed is preferably in the range from at least 2.5 m / s to 38 m / s or less, 3 m / s to 38 m / s, 3.5 m / s to 38 m / s, 3.5 m / s to 36 m / s, 3.5 m / s to 34 m / s, more preferably in the range from at least 4 m / s to 32 m / s or less (Example 9). The aforementioned rotor tip speed can be converted for similar use of an alternative shear generating device (S) by the device of the invention comprising a centrifugal pump, a high shear mixer (e.g., Ultra Turrax), or a tooth or blade type rotor-stator mixer (Example 9). In particular, in combination with different embodiments of the current device according to the present specification, preferably the settings of Example 9, it is preferred to apply a rotor tip speed in the range from at least 4 m / s to 32 m / s or less. Thus, in one embodiment of the method according to the invention, the residence time for cell disruption is 75 seconds or less without disrupting at least one biologically active compartment. In a preferred embodiment of the method according to the invention, the shear force generating device (S) is a tooth or blade type rotor-stator mixer, and the residence time for cell disruption is less than 25 seconds without disrupting at least one biologically active compartment. 15. In another preferred embodiment of the method according to the invention, the shear force generating device (S) is a centrifugal pump, and the residence time for cell disruption is less than 75 seconds without disrupting at least one biologically active compartment.

[0056] In certain embodiments of the method according to the invention, the shear force is generated by agitation of 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 mixer, or a tooth or blade type rotor-stator mixer, more preferably a tooth or blade type rotor-stator mixer (Figure 2). Embodiments using different centrifugal pumps, rotor-stator mixers are described herein and, as shown in Example 9, are applied to the method depending on the situation. In this method, the shear force generating device (S) is combined with a pump (P) or used without a pump (P).

[0057] Preferably, the rotation of the rotor is adjusted, thereby generating accelerating forces that cause radial movement of the medium and axial suction of the medium. Thereby, the aqueous medium is forced towards the shear gap and the cells are moved by the shear and thrust between the rotor and the stator and the shear gap affects the cells. Thereby, the cells are disrupted and in particular the lipid bilayer of the cells is disrupted without disrupting at least one lipid bilayer containing biologically active compartments.

[0058] In another embodiment of the method according to the 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. Preferably, it is released into an aqueous medium ("directly obtained process product", "functional product"). From there, different fractions can be separated and / or isolated. Preferably, one fraction of the isolated biologically active compartment according to the invention is capable of mainly ATP synthesis and / or energy regeneration, and another fraction of the isolated biologically active compartment according to the invention is capable of mainly at least one protein biosynthesis-related process, expression, transcription, translation, translocation, protein folding and / or protein modification. Later fractions are capable of the coupled transcription and cotranslational translocation of at least one complex protein that requires post-translational modification PTM (e.g., GOx) and / or expressed transmembrane protein (TP) (e.g., ACE2) to at least one biologically active compartment, preferably microsomes, more preferably endogenous microsomes.

[0059] In another embodiment of the method according to the invention, 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 is distinguishable into at least two fractions comprising a fraction of mainly biologically active compartments in the range of 0.5 to 2 μm and a fraction of mainly biologically active compartments in the range of 0.5 to 30 μm. The fraction of mainly biologically active compartments in the range of 0.5 to 2 μm is mainly capable of ATP synthesis and / or energy regeneration, and the fraction of mainly biologically active compartments in the range of 0.5 to 30 μm is mainly capable of at least one protein biosynthesis-related process, expression, transcription, translation, translocation, protein folding and / or protein modification, more preferably mainly of the coupled transcription and cotranslational translocation of at least one complex protein requiring post-translational modification PTM (e.g., GOx) and / or expressed transmembrane protein to at least one biologically active compartment, preferably microsomes, more preferably endogenous microsomes.

[0060] Preferably, a combination of two different biologically active compartments is obtained (the fractions described herein), one fraction comprising biologically active compartments in the range of 0.5 to 2 μm and being capable of at least ATP synthesis and / or energy regeneration, and the other fraction comprising biologically active compartments in the range of 0.5 to 30 μm and being capable of at least one protein biosynthesis-related process including expression, transcription, translation, translocation, protein folding and / or protein modification.

[0061] In another embodiment of the method according to the invention, the disruption efficiency is defined as the amount of disrupted cells relative to the amount of non-disrupted cells, preferably or alternatively as the amount of released biologically active compartments relative to the amount of cells provided without an intact cell wall.

[0062] Each amount is determined by counting the provided cell wall-free cells. Each amount of the biologically active fraction is preferably counted by microscopic analysis, assay for a marker / component of the biologically active fraction (e.g., a known detectable transmembrane protein), or by particle flow measurement. The relationship between one amount and another is expressed as a percentage.

[0063] A method in which the disruption efficiency is the product of the shear force and / or the shear gradient in the turbulent region and the residence time of the aqueous medium, and which is defined as being strong enough to disrupt or crack or break up the membrane of cell wall-free cells ((mini) protoplasts), preferably (mini) protoplasts, preferably to release biologically active fractions with a diameter of less than 70 μm. Preferably, the biologically active fraction is an intact organelle containing mitochondria, microsomes, nuclei, ER and / or Golgi bodies from plant (mini) protoplasts. Preferably, the isolated fraction contains intact microsomes and / or mitochondria released from plant (mini) protoplasts but does not contain nuclear DNA.

[0064] Preferably, the fraction is mitochondria and / or microsomes, most preferably the released biologically active fraction does not contain nuclei and / or nuclear (nucleolar) DNA, and preferably the isolated fraction does not contain cell membranes and their fragments, nuclei and / or nuclear DNA.

[0065] In another embodiment of the method according to the invention, the method is an essential method of a production process (a production process as described in the context of the device according to the invention), preferably a production process of a cell lysate. The production process is a production process of a cell line and preferably aims to produce a cell lysate and / or obtain a desired fraction from the cell line. The desired fraction is a certain specific soluble protein, a transmembrane protein embedded in the lipid bilayer of the biologically active fraction according to the invention, and / or another component that is a main or by-product of the production process. More preferably, the production process is a production process of a plant cell lysate of Nicotiana tabacum BY2, most preferably for producing a lysate for use in cell-free protein synthesis.

[0066] Another aspect of the invention is a composition comprising at least one biologically active fraction and the biologically active liquid surrounding it, wherein the liquid and / or the fraction preferably enables at least one step of protein biosynthesis obtained by the method according to the invention. More preferably, it is obtained by the method according to the invention implemented by the use of the device according to the invention (stand-alone or as part of an in-line production). All embodiments of at least one biologically active fraction, fraction and / or the biologically active liquid surrounding it are applied to the composition according to the invention as appropriate to the situation.

[0067] Another aspect of the invention is an isolated biologically active fraction surrounded by a lipid bilayer and showing the ability of at least one protein synthesis process, preferably including at least ATP synthesis and / or energy regeneration ability and / or at least transcription, translation, post-translational modification, protein folding and / or translocation. Preferably, the isolated biologically active fraction surrounded by the lipid bilayer is obtained by the method according to the invention, preferably by the use of the device according to the invention. All embodiments of at least one biologically active fraction, fraction and / or the biologically active liquid surrounding it are applied to the isolated biologically active fraction surrounded by the lipid bilayer according to the invention as appropriate to the situation.

[0068] In another embodiment of the invention, a composition comprising an isolated biologically active compartment or at least one biologically active compartment enclosed by a lipid bilayer each comprises at least one transmembrane protein, at least one inner and / or outer membrane-associated protein, and / or at least one soluble protein in the internal space.

[0069] Preferably, the biologically active compartment is obtained by the method according to the invention, preferably by using the device according to the invention.

Brief Description of the Drawings

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[0071] References 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 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 Hakansson - Andreas Hakansson; Rotor-Stator Mixers: From Batch to Continuous Mode of Operation - A Review. Processes 2018, 6, 32; doi:10.3390 / pr6040032 John et al. - G.T.J. John, I. Klimant, C. Wittmann, E. Heinzle: Integrated Optical Sensing of Dissolved Oxygen in Microtiter Plates: A Novel Tool for Microbial Cultivation. Biotechnol Bioeng 81:829 - 836, 2003. DOI:10.1002 / bit.10534 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

[0072] Examples Example 1 - Proof of Concept 1.1 Experimental Setup A peristaltic pump, here a watson marlow 120u / dv pump with an inner tube diameter of 3.175 mm, was combined with an Ultra - Turrax (IKA ULTRA - TURRAX® T 10) as the shear - generating device. Three different pumping speeds and three different Ultra - Turrax speeds were applied (each approximately 10 mL). The starting material was a pretreated aqueous medium containing vacuolated, cell - wall - free BY - 2 cells derived from a suspension cell culture of N. tabacum (Buntru et al., 2014, 2015) supplemented with 1.5 - fold (v / v) TR buffer. During the experiment, the starting material was kept at a low temperature (less than 10 °C).

[0073] 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 the completed process, the directly obtained process product (as defined herein) contains the TR buffer, the released biologically active fraction and / or its surroundings. [Table 2]

[0074] 1.2 Results Microscopic evaluation (Figure 5) shows the process products obtained directly after disruption using Ultra-Turrax and a pump, as represented in Table 1. Figure 5 shows that both pump speed and / or flow rate and Ultra-Turrax speed have an impact on disruption, and that both higher Ultra-Turrax speeds (from the top to the bottom of the panels in the figure) and lower pump speeds and / or flow rates (from the left to the right of the panels in the figure) individually result in more disrupted process products (with less content of visible microstructure). The most extreme settings of both parameters are shown in Figure 5C / 6, where essentially all (mini) protoplasts of BY-2 were disrupted. Macroscopic evaluation (Figure 6) of the lysates of the BY-2 cell line after disruption and centrifugation at 1,500 x g for 15 minutes shows that all (mini) protoplasts were successfully disrupted at pumping speeds of 30, 22.5 or 15 mL / min and an Ultra-Turrax speed of 6, respectively. Only two fractions are visible: a fraction containing biologically active compartments (mitochondria and microsomes) and the surrounding biologically active liquid, and a fraction containing the debris of nuclei and / or (mini) protoplasts. Pumping speeds of 30, 22.5 and 15 mL / min at Ultra-Turrax speeds 2 and 4 were not sufficient to disrupt the entire biological material.

[0075] Example 2 - Trials with an IKA MagicLab 2.1 Experimental setup Apparatus for disruption A peristaltic pump, here a watson marlow 120u / dv pump, was used in combination with an IKA MagicLab as a shear generating device. The same setup was used unless otherwise stated (Figures 5, 6, 7 and 8). [Table 3]

[0076] For the subsequent biosynthesis assays (2.2.1 and 2.2.2), lysates were produced by performing disruption with an IKA MagicLab in the following experimental setup. [Table 4]

[0077] The same starting material was used as described above and kept at low temperature (below 10°C) during the experiment.

[0078] 2.2 Proof of destructive efficacy: The destructive efficacy is evaluated visually (Figure 6), by microscopy (Figures 5, 7, and 8), and by protein synthesis.

[0079] 2.2.1 Assay for demonstrating the expression of cytosolic protein, eYFP, described by Buntru et al. (page 9) Enhanced yellow fluorescent protein (eYFP) is used as a marker protein. This is expressed in the lysate outside the microsomes and does not require post-translational modification (PTM) mediated by the microsomes. However, active protein synthesis and energy regeneration mechanisms (ribosomes, mitochondria, etc.) are required. The higher the protein expression (yield) of eYFP, the greater the measurable fluorescence. In this context area, alternative model proteins, firefly luciferase (FFLuc) and Renilla reniformis luciferase (Buntru et al., 2014) are known.

[0080] 2.2.2 Assay for demonstrating microsomal expression using post-translational modification and folding (GOx) To demonstrate that the released microsomes are active and capable of folding the translation - post - modification and expression of a multi - domain glycoprotein (a homodimer containing 80 kDa monomers covalently linked by disulfide bonds and each having eight N - glycosylation sites), glucose oxidase (GOx) from Aspergillus niger was tested. For expression, the template encoding the protein GOx was cloned into plasmid pALiCE02. After incubation, the lysate samples were treated with 0.5% DDM from a 5% DDM stock in PBS for 10 minutes at room temperature. A calibration curve was prepared in the range of 0 - 500 μg / ml using a GOx standard from Aspergillus niger (Sigma Aldrich). Samples and standards were diluted 1:2500 in an assay buffer consisting of 0.33 M glucose, 0.67 mM ABTS and 1.67 U / ml HRP (Sigma Aldrich) in 0.1 M potassium phosphate buffer at pH 6 in a transparent 96 - well plate. The absorbance over time at 420 nm was measured for 15 minutes using an infinite PRO Tecan plate reader. The sample GOx activity was calculated using the linear increase in absorbance over time of the calibration samples.

[0081] 2.2.3 Assay to demonstrate the expression of transmembrane proteins embedded in the lipid bilayer of microsomes (Figure 4E). Another way to demonstrate intact microsomes containing correctly folded membrane - spanning proteins embedded in a lipid bilayer is the expression of a model membrane - spanning protein, such as angiotensin - converting enzyme ACE2, using the respective lysates after disruption. The so - called Covid pandemic ACE2 is expressed in alveolar epithelial cells and capillary endothelial cells, and actin is known as the cellular entry and exit point for SARS - CoV - 2 that enables cell infection. Commercially available ligands (RBD) are well - known. For the assay, a template encoding ACE2 was cloned into the vector pALiCE02 (LenioBio GmbH), and a Strep - Tag® II was fused to the DNA template at the C - terminus for protein orientation, and then captured on a microtiter plate. For microsome targeting, a melittin signal peptide sequence (MSP) was fused to the N - terminus. ACE2 was expressed and translocated into intact microsomes, where the N - terminal binding site of ACE2 was inside the microsome and the C - terminus remained outside, enabling capture. 1% DDM in PBS was added to each well, and the plate was incubated at room temperature for 15 minutes. The microtiter plate was washed (three times with PBS - 0.05% Tween (v / v) (PBST)), blocked by adding 1× blocking buffer (ab126587, Abcam) to prevent non - specific binding (1 hour, room temperature), and washed again. For the capture of ACE2 embedded in microsomes and protein - binding analysis, a commercially available RBD SARS - CoV spike / RBD protein (RBD, His - tag) (40150 - V08B2, SinoBiological) was diluted to 2.5 μg / ml in the blocking buffer. The plate was incubated at room temperature for 1 hour, followed by a washing step. To enable binding to ACE2, the microsomes were treated with 1% DDM to disrupt the captured microsomes. For protein detection, a detection antibody (HRP - labeled) that binds to the protein was added.For the detection of ACE2-commercial RBD binding, antibodies conjugated with His-tagged horseradish peroxidase in blocking buffer (MAB050H, Bio-Techne) 1:4000 and StrepMAB-Classic-HRP (2-1509-001, IBA-lifesciences) 1:15,000 were added. The plates were incubated (1 hour, room temperature), then washed, and indirect ELISA (by measuring the sample absorbance) was performed to determine the protein structure and binding efficiency. Detection of absorbance (e.g., (650 nm) TECAN Infinite M1000 Pro machine, Tecan i-Control 2.0 software) demonstrates the expression of correctly folded ACE2 for its effective binding to the RBD ligand of SARS-CoV-1. High absorbance values demonstrate a high yield of ACE2 expression, post-translational modification, folding, and embedding into the lipid bilayer of microsomes as a prerequisite for RBD ligand binding. Thus, this assay is suitable to demonstrate the release of the biologically active fraction according to the invention, as shown in Figure 4E.

[0082] 2.3 Results 2.3.1 Homogenization, sonication, etc. against shear force disruption Microfluidizer: The disruption method using models M-110L and LM-10 was carried out to evaluate whether homogenization, as used in prior art in other technical fields, could enable the production of lysates with high yields of biologically active components. However, only low yields of the expressed protein, less than 0.01 mg of eYFP / ml, were observed (Figure 4A). As a result, only low levels of biologically active components were obtained in the lysates produced by homogenization. Other prior art methods such as sonication were tested as appropriate. The yields of the expressed eYFP were shown to be very low (Figure 4B), which is due to the low content of biologically active components in the lysates. The same assay will be applied to / applied to the products obtained by the methods carried out using IKA Magic Lab, IKA UTL 25, Levitronix® puraLev i100 SU, and Ultra-Turrax (IKA ULTRA-TURRAX® T 10). Figure 4C shows the eYFP yields produced by lysates after different disruption methods.

[0083] The shearing generation device (S), here the disruption method according to the present invention using IKA Magic Lab, is shown in Figures 4C and 4D. Compared with the results shown in Figures 4A and B, the achieved protein yields were eYFP of 1 mg / mL to 2.8 mg / mL depending on the disruption conditions and the plasmid concentrations used (from left to right columns in Figure 4C, 10, 20, 40 ng / μL). The data indicate that different disruption settings of IKA Magic Lab result in biologically active fractions capable of reaching high target protein titers in the lysate reaction. Furthermore, the assay using GOx indicates that the obtained lysates were able to produce complex GOx proteins with post-translational modifications mediated by intact microsomes with 40 - 150 a.u. (arbitrary units) GOx depending on the disruption conditions and the plasmid concentrations used (from left to right columns in Figure 4D, 5, 10, 20 ng / μL) (Figure 4D).

[0084] 2.3.2 Microscopic evaluation (Figure 7) Figure 7 represents the microscopic evaluation (100x) of the starting material before disruption (Figure 7A) and after disruption at 1 pass and 6,000 rpm (Figure 7B). The photographs show that cells without most of their cell walls are disrupted with these settings. Since not all cells without cell walls are disrupted with these settings, it also shows that the force exerted is low enough not to disrupt the cell compartments but sufficient to release biologically active material.

[0085] 2.3.3 Results (Figure 8)

Table 5

[0086] Figure 8 represents the microscopic evaluation of the starting material and the effect of disruption settings on the amount of cells without disrupted cell walls. Figure 8D shows cells without cell walls before disruption. Figure 8C shows that most cells without cell walls remain intact even after a single pass at a rotor speed of 3,000 rpm. In contrast, Figures 8A, B, and E show that cells without cell walls are disrupted. The comparison between Figures 8A and 8B shows the influence of the rotor - stator configuration. This indicates that different shear forces can be achieved using the same settings except for the triple vs single rotor / stator module, allowing for fine - tuning of the appropriate force to disrupt cells without cell walls.

[0087] 3. Summary Here, it has been shown that by the expression of eyFP and GOx, functional lysates can be obtained by the disruption method according to the present invention. The protein biosynthesis machinery is complete and not only is it possible to express soluble proteins (eYEP), but also post - translational modification of the multi - domain protein GOx occurs. As a result, the disruption method and apparatus according to the present invention enable the production of functional lysates at various scales for the use in recombinant protein biosynthesis. Here, the successful scaling of an important step in lysate production and the improvement of production efficiency while ensuring and improving the quality of the directly obtained process product are well demonstrated.

[0088] Example 3 - Blade and Tooth Type Trials Inline production using an IKA UTL 25 inline to show the influence of the blade type compared to the tooth type of the rotor (Figure 2)

[0089] Experimental Setup Peristaltic pumps, e.g., a watson marlow 120u / dv pump combined with a shear generating device of a) blade type and b) tooth type respectively. The same starting materials as described in Examples 1 and 2. Adjustment of the rotor speed from 3,000 to 15,000 rpm, e.g., 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000 and 15000 respectively. Adjustment of the pump in the range of 10 - 100 mL / min respectively: [Table 6] Accurate adjustments are shown in Example 9 and Table 3.

[0090] Example 4 - Centrifugal Pump Trials Experimental Setup Instead of a rotor - stator mixer, a centrifugal pump, e.g., Levitronix® puraLev i100 SU. The same starting materials as described in Examples 1 and 2. Since the force exerted on the cell without a cell wall is smaller for the centrifugal pump compared to the rotor - stator mixer, the centrifugal speed (rpm) is higher and the supply speed of the peristaltic pump is lower. A centrifugal speed of 4,500 - 9,000 rpm is used together with a peristaltic pump speed of 1 - 50 mL / min. Additionally, multiple passes through the centrifugal pump are used. Accurate adjustments are shown in Example 9 and Table 3.

[0091] Example 5 - Scale - up Trials As a scale-up parameter, to implement the residence time of cells without cell walls in a shear force field, Ultra-Turrax (IKA ULTRA-TURRAX® T 10), IKA UTL 25 in-line, and IKA Magic Lab were used in parallel experiments to confirm the residence time as a scale-up criterion along with the shear force in the shear force field.

[0092] Example 6 - Viscosity determination Viscosity determination and adjustment of different starting materials that can be disrupted by the methods described and the use of one embodiment of the device according to the present invention. Test the viscosity that can be performed using a shear generating device. Viscosities from approximately 0.5 cP to a maximum of 5 cP are aqueous media and are feasible using each embodiment of the device and method of the present invention. However, viscosities from greater than 5 to less than 15 mPa·s can correspond to more concentrated (e.g., after centrifugation) cell culture media containing the biological material to be disrupted.

[0093] Example 7 - Isolation experiment, followed by determination of the amount of the fraction, amount per cell without cell wall, and qualification. Fractionation of biologically active fractions by centrifugation. Performed by assay of component markers and determination of the amount of nuclear DNA according to the disruption settings

[0094] Example 8 - Disruption of yeast protoplasts, CHO, and other species Comparison of the most common cell lines used in the field of cell-free protein synthesis (e.g., described on page 2 of Buntru et al. 2015 and Buntru et al. 2014) are disrupted as described herein to release and separate biologically active fractions and biologically active liquids suitable for cell-free biosynthesis of recombinant proteins.

[0095] Example 9 9.1. Experimental setup and adjustment according to the present invention The components of the device according to the invention are listed in Table 3. The centrifugal pump PuraLev i100SU used here comprises a "driver" IPD-100.3-03-02 (epoxy, 0.3 m PVC, IC915, product number: 100-10105) and an "SU pump head" DCP-200.3 (PP, Barb 1 / 2'', product number: 100-90792). The driver drives the pump head, - magnetically in this case -, whereby the rpm is adjusted. Other versions of the "driver" as well as pump heads are suitable depending on the situation. The rotor - stator system (S) used here is designed with a toothed ring as shown on the left side of Figure 2B.

[0096]

Table 7

[0097] Calculation of the residence time The residence time (VZ) can be calculated for each device as long as the internal volume of the device used is known, or can be calculated, for example, by metering (volume measurement). Based on a known flow rate (R) (unit: mL / min) and internal volume (Vi) (unit: mL), for example, the following residence time VZ = Vi / R is obtained. a) In the case of an IKA Magic Lab (Vi = 30 mL) with a flow rate of 150 mL / min (Table 3) or 40 mL / min (Table 2) VZ = 30 mL / 150 mL / min = 12 s (see Table 3) bzw. VZ = 30 mL / min / 40 mL / min = 45 s (see Table 2) b) In the case of a UTL25 (Vi = 8 mL) with a flow rate of 10 mL / min: VZ = 8 mL / 10 mL / min = 75 s c) In the case of a centrifugal pump with a pump head DCP-200.3 (Vi = 23.5 ml) at a flow rate of 20 mL / min or 56.4 mL / min VZ = 23.5 mL / 20 mL / min = 70.5 s, VZ = 23.5 mL / 56.5 mL / min = 25 s.

[0098] 9.2 Method for determining oxygen consumption One way to demonstrate that the cell lysate (a directly obtained process product) is active and functional in the sense of the present invention is to demonstrate the functionality of the mitochondria, since the mitochondria provide the energy for protein biosynthesis. To show the functionality of the mitochondria after disruption, BioLector Pro (Beckman Coulter GmbH, Aachen, Germany) was used. With this equipment, the transfer / consumption of oxygen can be measured using an oxygen-sensitive fluorescent dye (John et al. 2001) placed at the bottom of the microtiter plate well. The measurement principle is shown graphically in Figure 1 of John et al. and is explained on pages 2 - 3, which is hereby incorporated by reference. The fluorescence of the dye is measured by an optical fiber and correlated with the oxygen concentration in the liquid surrounding the dye. When oxygen is consumed, the oxygen consumption is measured by the level of the dissolved oxygen (DO) concentration in the well. As shown by John et al. in Figure 2, when oxygen is not consumed, the measured oxygen concentration is 100%. In the case of lower DO levels, it indicates the consumption of oxygen in the liquid.

[0099] 9.3 Results 9.3.1 Oxygen consumption The consumption of oxygen demonstrates that active / functional mitochondria are present in the lysates produced by the devices of Table 3 and by the methods with the adjustments described in Table 3. All produced lysates 1, 2, 3, 4, 5, 6 and 8 consume oxygen (Figure 10).

[0100] In this example, each device can set the desired settings of shear force and residence time independently of each other. Therefore, in any case, it is shown that the directly obtained process product is active in the sense of the present invention. Oxygen consumption proves that the directly obtained process product contains a biologically active compartment and is capable of ATP synthesis and energy regeneration. As shown below, the fact that the same directly obtained process product can also perform protein biosynthesis-related processes, expression, transcription, translation, translocation, protein folding and / or protein modification is further proven by the protein yield.

[0101] 9.3.2 Expression of eYFP The expression of eYFP was analyzed as described above in 2.2.1, and for lysates 1, 2, 3, 4, 5, 6, 7 and 8, cytosolic proteins, the ability to produce eYFP herein was demonstrated. This indirectly demonstrates that the important components of cytosolic protein expression in the lysate (transcription, translation, energy regeneration by mitochondria and protein folding) are functional.

[0102] 9.3.3 Expression of GOx The expression of GOx was analyzed as described above in 2.2.2, demonstrating the ability of the lysate for the coupled transcription and cotranslational translocation of GOx, a more complex protein that requires post-translational modification PTM. Figure 12 shows that lysates 1, 2, 3, 4, 5, 6 and 8 are capable of producing GOx. This indirectly demonstrates that the important components of microsomal protein expression in the lysate, such as transcription, translation, energy regeneration by mitochondria, translocation, protein folding and post-translational modification, produced by the devices and methods described in Table 3 are functional.

[0103] Therefore, it is shown that process products (Table 3) directly obtained by different devices and processes are obtained by both oxygen consumption and the synthesized protein.

Claims

1. A device for destroying cell walls without a cell wall, without destroying the biologically active compartment surrounded by a lipid bilayer, - A shear force generating device (S) for generating a region of shear force suitable for destroying the cells, - Tank A for providing the aforementioned cells, -Optionally, a pump (P) located upstream of (S) or downstream of (S) to pump the aqueous medium containing the cells at an adjustable flow rate and / or flow rate, - At least one conduit connecting the tank A and the shear force generating device (S) and / or at least one conduit connecting (S) and tank B, - Tank B for recovering products from the previous process, which contains at least one released biologically active compartment, - Separation device (C) for separating the destroyed cells and their fragments from the at least one released biologically active compartment and / or the biologically active liquid phase surrounding it. A device equipped with the following features.

2. The apparatus according to claim 1, wherein the shear force generating device (S) is suitable for generating a shear force sufficient to destroy the cells without damaging at least one lipid bilayer containing a biologically active compartment, and is controllable.

3. The apparatus according to claim 1, wherein the pump is suitable for adjusting the residence time of the aqueous medium containing the cells in (S), independently of the shear force adjusted and generated by (S), the residence time being the period of time the shear force is applied to the aqueous medium containing the cells.

4. The apparatus according to claim 1, comprising a shear force generating device (S) and a pump (P) positioned upstream of (S) or downstream of (S) for pumping an aqueous medium containing the cells at an adjustable flow rate and / or flow velocity.

5. The apparatus according to claim 1, wherein the shear force generating device (S) is suitable for generating shear by stirring the aqueous medium and is designed to do so.

6. The apparatus according to claim 1, wherein the shear force generating device (S) is a centrifugal pump, a high shear force mixer, or a tooth-type or blade-type rotor-stator mixer.

7. The apparatus according to claim 1, which is an integrated apparatus of a production line, preferably a closed sterile production line.

8. A production line comprising the apparatus according to claim 1, wherein tank A is located downstream of at least one unit for pretreatment of biological material, the conduit connects tank A and S, tank B is located downstream of S, and optionally pump P is located downstream or upstream of S.

9. A method for disrupting a cell wall-less cell containing at least one biologically active compartment surrounded by a lipid bilayer, - A step of providing an aqueous medium containing the cells without cell walls, preferably in tank A. - A step of moving the aqueous medium into the shear force generating device (S) described in claim 1, -Optionally, a step of adjusting the flow rate and / or flow rate of the aqueous medium using the pump (P) described in claim 3. - A process to generate a region of shear force within (S), - A step of applying a shearing force to the aqueous medium containing the cells for a residence time sufficient to destroy the cells without destroying the at least one biologically active compartment. - A step of destroying the cells without destroying the lipid bilayer of the at least one biologically active compartment while the aqueous medium is passing through (S), - A process of releasing at least one intact biologically active compartment and the surrounding biologically active fluid, -Optionally, a step of recovering the at least one released intact biologically active compartment together with the surrounding biologically active liquid, preferably in tank B. - A step of separating the at least one released intact biologically active compartment from the biologically active surrounding fluid, and - Optionally, the isolation step of the at least one released intact biologically active compartment. Methods that include...

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

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

12. The method according to claim 9, wherein a predetermined rotor tip speed of 2 m / s or more and up to 50 m / s is applied by (S).

13. The method according to claim 9, wherein the residence time for destroying the cells without destroying the at least one biologically active compartment is 75 seconds or less.

14. The method according to claim 9, wherein the shear force generating device (S) is a tooth-type or blade-type rotor-stator mixer, and the residence time for destroying the cells without destroying the at least one biologically active compartment is less than 25 seconds.

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

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

17. The method according to claim 9, wherein 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.

18. The method according to claim 9, wherein the disruption efficiency is defined as the amount of disrupted cells relative to the amount of undisrupted cells, preferably or alternatively, as the amount of biologically active compartments released relative to the amount of provided but undisrupted cell wall-less cells.

19. The method according to claim 9, which is an essential method for a production process, preferably a cell lysate production process.

20. A composition comprising at least one biologically active compartment and a biologically active liquid surrounding it, wherein the liquid and / or the compartment enable at least one step of protein biosynthesis.

21. An isolated, biologically active compartment surrounded by a lipid bilayer, exhibiting the ability to perform at least ATP synthesis and / or energy regeneration, and / or at least one protein synthesis process, preferably including at least transcription, translation, post-translational modification, protein folding, and / or rearrangement.

22. A biologically active compartment according to claim 20 or 21, comprising at least one transmembrane protein, at least one inner membrane and / or outer membrane-related protein, and / or at least one soluble protein within the internal space.

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