A new process for protein extraction

Abstract

A method for extracting cytoplasmic or periplasmic proteins, the method comprising providing a first cell suspension containing cells, the cells containing cytoplasmic or periplasmic proteins to be extracted, the first cell suspension having a first temperature, and heating the first cell suspension at an operating temperature, at which at least a fraction of the cells are subject to heat-induced lysis and at least a fraction of the cytoplasmic or periplasmic proteins to be extracted are not subject to irreversible denaturation. The step of heating the first cell suspension comprises providing an aqueous solution, the aqueous solution having a second temperature higher than the first temperature, and the heating step comprises mixing the first cell suspension and the aqueous solution to thereby obtain a second cell suspension, the second cell suspension having a third temperature higher than the first temperature. A system for extracting cytoplasmic or periplasmic proteins, the system comprising a static mixer.

Description

[Technical Field]

[0001] The present application relates to a method for extracting cytoplasmic or periplasmic proteins, said method comprising the step of heating a cell suspension at a temperature at which heat-induced lysis of cells containing the cytoplasmic or periplasmic proteins to be extracted occurs and at which irreversible denaturation of the cytoplasmic or periplasmic proteins to be extracted does not occur. The present application also relates to a system for extracting cytoplasmic or periplasmic proteins. [Background technology]

[0002] Large-scale production of recombinant proteins for medical and biotechnological applications requires process development and optimization to meet the demands of a cost-effective, reproducible production process that yields a high-quality final product. A process that works well in small-scale production may not be feasible for large-scale production due to technical or economic reasons. When targeting industrial production, process development under conditions that mimic the potential large-scale process, followed by scale-up of the developed production process, is essential.

[0003] In the production of recombinant proteins expressed by cells, efficient cell lysis and recovery of the desired product are crucial. Several methods for lysing cells have been developed and described. These include mechanical homogenization, ultrasonic homogenization, pressure homogenization, heat treatment, freeze / thaw cycles, and osmotic and chemical lysis. The method selected depends on various factors, such as the characteristics of the protein of interest to be extracted from the cells, the intracellular location of the protein, the volume involved, and the required throughput.

[0004] For the extraction of thermostable proteins, heat lysis can be advantageous because it may result in precipitation of unwanted host cell-derived proteins and / or removal of insoluble aggregates, thus potentially aiding in subsequent purification. Furthermore, when used in screening steps, heat lysis favors thermostable mutants, which are more likely to result in greater yields of correctly folded protein.

[0005] However, for large-scale production, the time required to heat and cool the cell suspension can affect the overall yield because the protein of interest may begin to precipitate or degrade. The time required to heat and cool the cell suspension can further cause problems with the generation of product-related impurities. Therefore, lysis by heat treatment is not optimal for large-scale production of proteins by cell expression because heating and cooling in larger bioreactors or vessels requires long periods of time, thereby making the results of the extraction process less controllable. Therefore, improved extraction procedures are needed to improve process efficiency and protein yield in large-scale cell expression processes. Summary of the Invention

[0006] It is an object of the present invention to provide efficient extraction of cytoplasmic or periplasmic proteins of interest, i.e., to provide high yields and / or high quality of said proteins, particularly in large-scale production. Another object of the present invention is to provide extraction of cytoplasmic or periplasmic proteins of interest while avoiding or reducing precipitation or degradation of the extracted protein of interest. A further object of the present invention is to provide extraction of cytoplasmic or periplasmic proteins of interest while avoiding or reducing the generation of product-related impurities.

[0007] These objects, as well as other objects of the present invention, which should be apparent to those skilled in the art after considering the following description, are achieved in one aspect of the present invention by a method for extracting cytoplasmic or periplasmic proteins, said method comprising: - providing a first cell suspension comprising cells, said cells comprising cytoplasmic or periplasmic proteins to be extracted, said first cell suspension having a first temperature; and - heating the first cell suspension at an operating temperature, at which at least a fraction of the cells are subject to heat-induced lysis and at least a fraction of the cytoplasmic or periplasmic proteins to be extracted are not subject to irreversible denaturation; wherein heating the first cell suspension comprises: - providing an aqueous solution, said aqueous solution having a second temperature higher than said first temperature; and - mixing the first cell suspension with an aqueous solution, thereby obtaining a second cell suspension, said second cell suspension having a third temperature higher than the first temperature; Includes.

[0008] Therefore, the method for extracting cytoplasmic or periplasmic proteins can be summarized as follows: - providing a first cell suspension comprising cells, said cells comprising cytoplasmic or periplasmic proteins to be extracted, said first cell suspension having a first temperature; and - heating said first cell suspension at an operating temperature at which heat-induced lysis of cells occurs and which does not cause irreversible denaturation of cytoplasmic or periplasmic proteins to be extracted; wherein heating the first cell suspension comprises: - providing an aqueous solution, said aqueous solution having a second temperature higher than said first temperature; and - mixing the first cell suspension with an aqueous solution, thereby obtaining a second cell suspension, said second cell suspension having a third temperature higher than the first temperature; Includes.

[0009] Heating the first cell suspension to the operating temperature thus results in the liberation of the cytoplasmic or periplasmic protein of interest, thereby providing the liberated cytoplasmic or periplasmic protein of interest. Mixing the first cell suspension containing lysed cells with a warm solution allows for rapid heating of the cell suspension to the lysis temperature, thereby increasing the recovery rate of the extracted protein. The first cell suspension can be mixed with the aqueous solution in a batch or continuous manner, preferably in a continuous manner. The mixing ratio between the first cell suspension and the aqueous solution can be in the range of 1:0.1 to 1:14, preferably in the range of 1:1 to 1:14, more preferably in the range of 1:2 to 1:12, and more preferably in the range of 1:3 to 1:10. It is contemplated that the dilution achieved by mixing the first cell suspension with the aqueous solution reduces viscosity and the risk of the extracted protein of interest adhering to and / or coprecipitating with other proteins.

[0010] Using the method of the present invention, cells can be heated for 10 minutes or less, for example, in the range of 0.1 seconds to 10 minutes, preferably 5 minutes or less, for example, in the range of 0.1 seconds to 5 minutes, more preferably 1 minute or less, for example, in the range of 0.1 seconds to 1 minute, more preferably 10 seconds or less, for example, in the range of 0.1 seconds to 10 seconds, and most preferably 1 second or less, for example, in the range of 0.1 seconds to 1 second, to reach the third temperature.

[0011] As used herein, extraction of a cytoplasmic or periplasmic protein of interest refers to the liberation of the protein from the cytoplasm or periplasm of cells in which the protein of interest is expressed. As used herein, the operating temperature at which heat-induced lysis of cells occurs and irreversible denaturation of the cytoplasmic or periplasmic protein to be extracted does not occur refers to a temperature at which at least a minority fraction, preferably a major fraction, or essentially all of the cells are subject to lysis, and at least a minority fraction, preferably a major fraction, or essentially all of the protein molecules of the cytoplasmic or periplasmic protein to be extracted are not subject to irreversible denaturation. As used herein, denaturation refers to a process in which proteins partially or completely lose the quaternary, tertiary, and / or secondary structure present in their native state.

[0012] The aqueous solution is typically a buffer solution, as is commonly used in cell and protein processing. In other words, the aqueous solution has a composition suitable for use in the present method. Therefore, one skilled in the art can adapt the composition of the aqueous solution to optimize the recovery of the protein to be extracted. Adapting the composition of the aqueous solution typically involves selecting appropriate buffer components, appropriate salt concentrations, conductivity and / or pH, and / or optional additives. The additives may include those that protect the protein of interest from modification or degradation, or those that enhance the precipitation of undesirable cellular components.

[0013] Thus, the aqueous solution may have a pH and / or conductivity and / or may contain additives that enhance extraction of the protein of interest. Furthermore, the aqueous solution may have a pH and / or conductivity and / or may contain additives that enhance protection of the protein of interest against modification, degradation, misfolding, or precipitation. Furthermore, the aqueous solution may have a pH and / or conductivity and / or may contain additives that enhance denaturation or precipitation of undesirable cellular components, such as host cell-derived proteins, DNA, RNA, endotoxins, or other cellular components. Furthermore, the aqueous solution may have a pH and / or conductivity and / or may contain additives that affect, preferably lower, the temperature at which cell lysis occurs.

[0014] The first cell suspension is typically provided by subjecting a cell culture medium to centrifugation or filtration, or may be provided by mixing a frozen cell pellet, preferably obtained from a cell culture by centrifugation or filtration, with a warm buffer solution.

[0015] The first temperature, i.e., the temperature at which the provided cell suspension containing cells containing the protein of interest to be extracted is provided, may be in the range of 0° C. to 37° C., preferably in the range of 2° C. to 37° C., more preferably in the range of 8° C. to 30° C., more preferably in the range of 18° C. to 25° C. The first temperature may alternatively be below 0° C., when the cell suspension includes an antifreeze agent such as glycerol.

[0016] The operating temperature, i.e., the temperature at which cell lysis occurs, may be 90°C or less, for example, in the range of 20°C to 90°C, preferably in the range of 40°C to 90°C, more preferably in the range of 50°C to 90°C, more preferably in the range of 60°C to 90°C, more preferably in the range of 70°C to 90°C, more preferably in the range of 70°C to 85°C, and most preferably in the range of 75°C to 85°C.

[0017] The second temperature, i.e., the temperature of the provided aqueous solution, may be 110°C or less, for example, in the range of 40 to 110°C, preferably in the range of 50 to 110°C, more preferably in the range of 60 to 99°C, more preferably in the range of 70 to 99°C, more preferably in the range of 80 to 99°C, more preferably in the range of 90 to 99°C, and most preferably in the range of 90 to 95°C.

[0018] The third temperature, i.e., the temperature of the second cell suspension obtained by mixing the first cell suspension and the aqueous solution, may be 90°C or lower, for example, in the range of 40 to 90°C, preferably in the range of 50 to 90°C, more preferably in the range of 60 to 85°C, more preferably in the range of 65 to 85°C, more preferably in the range of 65 to 80°C, more preferably in the range of 65 to 78°C, and most preferably in the range of 68 to 78°C. The third temperature may alternatively be in the range of 70 to 80°C. At the third temperature, at least a fraction of the cytoplasmic or periplasmic proteins to be extracted is preferably not subject to irreversible denaturation.

[0019] Heating the first cell suspension may further include heating the second cell suspension from the third temperature to the operating temperature. Thus, heating the first cell suspension by mixing with a warmer solution may be followed by further heating toward the lysis temperature. Such further heating may be appropriate when the operating temperature cannot be reached simply by mixing the first cell suspension with a warmer solution, which may be the case when limitations apply to the mixing ratio or temperature of the mixing fluids. Furthermore, such further heating allows for precise control of the temperature of the second cell suspension. Heating the second cell suspension from the third temperature to the operating temperature is preferably achieved by indirect heat exchange, such as a tubular heat exchanger or a plate heat exchanger.

[0020] The third temperature is preferably 10°C or less, and preferably 5°C or less, below the operating temperature. It is desirable to heat the first cell suspension extensively toward the lysis temperature, primarily by mixing with the warmer solution. Therefore, it is advantageous if the third temperature is close to the operating temperature. A remaining temperature difference of 5 or 10°C will allow for fine-tuning of the temperature with further heating.

[0021] Alternatively, heating the first cell suspension may further include cooling the second cell suspension from the third temperature to the operating temperature. Thus, heating the first cell suspension by mixing with a warmer solution may be followed by cooling. This cooling is appropriate when a temperature higher than the desired operating temperature is reached by mixing the first cell suspension with a warmer aqueous solution. Furthermore, this cooling allows for precise control of the temperature of the second cell suspension. Cooling the second cell suspension from the third temperature to the operating temperature is preferably achieved by indirect heat exchange, such as a tubular or plate heat exchanger. The third temperature is preferably at least 10°C above the operating temperature, preferably at least 5°C above it. As noted above, a remaining temperature difference of 5 or 10°C may allow for fine adjustment of the temperature by cooling.

[0022] Alternatively, the third temperature may be the operating temperature, and if, with adequate precision, the operating temperature can be reached directly by mixing the first cell suspension with a warmer solution, further heating to the lysis temperature may be omitted.

[0023] The method may further include maintaining the second cell suspension at an operating temperature. While maintaining the cell suspension at elevated temperatures for extended periods, such as during heating and subsequent cooling of the suspension, can adversely affect the recovery of the protein being extracted, simply reaching the lysis temperature may not be sufficient for optimal extraction. Therefore, it is advantageous for the extraction method to include maintaining the second cell suspension at the operating temperature for a period of time. As used herein, "maintaining at the operating temperature" refers to maintaining at a substantially operating temperature, i.e., at a lower temperature that may result from any undesired heat loss from the second cell suspension. The second cell suspension is maintained at the operating temperature for a period of time ranging from 1 second to 20 minutes, or 10 seconds to 20 minutes, preferably from 1 second to 10 minutes, or 10 seconds to 10 minutes, more preferably from 1 second to 5 minutes, or 10 seconds to 5 minutes, and most preferably from 10 seconds to 4 minutes, e.g., from 10 seconds to 30 seconds, or from 1 minute to 4 minutes.

[0024] The method may further comprise the step of cooling the second cell suspension from the operating temperature to a fourth temperature, preferably at which at least a fraction of the reversibly denatured cytoplasmic or periplasmic proteins to be extracted undergo renaturation. In other words, the method may further comprise the step of cooling the second cell suspension from the operating temperature to a fourth temperature, preferably at which the regeneration of the reversibly denatured cytoplasmic or periplasmic proteins to be extracted occurs. As already mentioned, maintaining the cell suspension at a high temperature for a long period of time adversely affects the recovery of the proteins to be extracted. Therefore, it is advantageous to include in the extraction method the cooling of the second cell suspension from the operating temperature to the fourth temperature. For the same reason, it is advantageous to cool the second cell suspension before any subsequent steps for separating the proteins to be extracted from cellular debris and / or native host cell proteins. In order to ultimately recover the proteins to be extracted in their native form, the fourth temperature is advantageously a temperature at which the regeneration of the reversibly denatured cytoplasmic or periplasmic proteins to be extracted occurs. As used herein, this temperature refers to the temperature at which renaturation of reversibly denatured cytoplasmic or periplasmic proteins to be extracted occurs, and refers to the temperature at which at least a minor fraction, preferably a major fraction or essentially all, of any reversibly denatured protein molecules of the proteins to be extracted undergo renaturation to their native state. The fourth temperature may be in the range of 2 to 37°C, preferably in the range of 8 to 30°C, or in the range of 25 to 37°C, more preferably in the range of 18 to 25°C.

[0025] The residence time at a temperature higher than both the first temperature and the fourth temperature is preferably 20 minutes or less, for example, in the range of 1 second to 20 minutes, or in the range of 10 seconds to 20 minutes, and more preferably 10 minutes or less, for example, in the range of 1 second to 10 minutes, or in the range of 10 seconds to 10 minutes, and more preferably 5 minutes or less, for example, in the range of 1 second to 5 minutes, or in the range of 10 seconds to 5 minutes.

[0026] After extraction of the cytoplasmic or periplasmic protein of interest and, if possible, cooling of the cell suspension, the method may further comprise separating the cytoplasmic or periplasmic protein of interest from cellular debris and / or native host cell-derived proteins. Such separation methods are well known to those skilled in the art and typically involve precipitation, filtration, centrifugation, and / or one or more forms of chromatography.

[0027] The cell can be a prokaryotic cell, such as an E. coli cell, or a eukaryotic cell.

[0028] The cytoplasmic or periplasmic protein to be extracted may comprise a three-helix bundle protein domain of a bacterial receptor protein, or a variant thereof. In certain embodiments, the three-helix bundle protein domain is selected from domains of bacterial receptor proteins. Non-limiting examples of such domains are i) five different three-helical domains, such as domain B, of protein A from Staphylococcus aureus, and derivatives thereof. In some embodiments, the three-helix bundle protein domain is a variant of protein Z, derived from domain B of staphylococcal protein A (Wahlberg E et al., 2003, PNAS 100(6):3185-3190), and ii) the albumin-binding domain (ABD) of streptococcal protein G (Kraulis et al., FEBS Lett 378:190, 1996), or a derivative thereof.

[0029] The step of mixing the first cell suspension with the aqueous solution can be carried out in a static mixer or a stirred vessel, preferably in a static mixer. As is conventional in the art, a static mixer can be a pipe equipped with a series of fixed blades, typically helical blades, or bars, typically a grid of interlocking and / or connecting bars. Preferably, the mixing is carried out in a continuous mode in a static mixer. When the flow of the first cell suspension intersects with the flow of the aqueous solution in the static mixer, a second cell suspension having a higher temperature than the first cell suspension provided is obtained substantially instantaneously.

[0030] The above object is achieved in another aspect by a system for extracting a cytoplasmic or periplasmic protein of interest, said system comprising: - a cell suspension supply conduit having an inlet and an outlet, the inlet being connectable to a cell suspension container; an aqueous solution supply conduit having an inlet and an outlet, the inlet being connectable to an aqueous solution container; a static mixer having at least one inlet and one outlet, wherein the at least one inlet is in fluid communication with the outlet of the cell suspension supply conduit and the outlet of the aqueous solution supply conduit; a first heat exchanger having an inlet for the cell suspension to be heated and an outlet for the heated cell suspension, the inlet being in fluid communication with the outlet of the static mixer; a second heat exchanger having an inlet for the cell suspension to be cooled and an outlet for the cooled cell suspension, the second heat exchanger having an inlet in fluid communication with the outlet of the first heat exchanger; a discharge conduit having an inlet and an outlet, the inlet being in fluid communication with the outlet of said second heat exchanger, the outlet being connectable to a protein suspension vessel or a protein suspension processing system; Equipped with.

[0031] The system is suitable for carrying out the method disclosed above. The static mixer provides rapid heating of the cell suspension to the lysis temperature, allowing for increased recovery of the protein to be extracted. As is conventional in the art, the static mixer can be a pipe equipped with a series of fixed blades, typically helical blades, or bars, typically a grid of interlocking and / or connecting bars. The static mixer can have one inlet, which is in fluid communication with both the outlet of the cell suspension supply conduit and the outlet of the aqueous solution supply conduit, or it can have two inlets, one in fluid communication with the outlet of the cell suspension supply conduit and the other in fluid communication with the outlet of the aqueous solution supply conduit.

[0032] The first and second heat exchangers may independently be tube heat exchangers, plate heat exchangers, or conduits surrounded by a jacket or vessel.

[0033] The system may further include a holding conduit providing fluid communication between the outlet of the first heat exchanger and the inlet of the second heat exchanger, the holding conduit preferably being secured by a jacket or container, or The holding unit is surrounded by insulation or a heating blanket, such as an electric heating blanket. The holding unit provides an opportunity to maintain the temperature of the cell suspension substantially at the temperature achieved by the first heat exchanger for a certain period of time. The residence time in the holding unit can be in the range of 1 second to 20 minutes, or 10 seconds to 20 minutes, preferably in the range of 1 second to 10 minutes, or 10 seconds to 10 minutes, and more preferably in the range of 1 second to 5 minutes, or 10 seconds to 5 minutes. As a general practice, the desired residence time can be obtained by selecting an appropriate volume for the holding unit relative to the flow rate of the cell suspension, or vice versa.

[0034] The cell suspension supply conduit may include a pump that drives the cell suspension toward the outlet of the cell suspension supply conduit. The aqueous solution supply conduit may include a pump that drives the aqueous solution toward the outlet of the aqueous solution supply conduit. One or both of these pumps may be a positive displacement pump, such as a peristaltic pump.

[0035] The system may further include at least one heating unit that provides a heat medium to the first heat exchanger and / or the jacket or container of the holding unit.

[0036] The system may be adapted to operate at temperatures and / or flows as disclosed elsewhere herein. The flow through the static mixer, first heat exchanger, holding unit and second heat exchanger, and the conduits connecting them, is preferably turbulent to reduce stagnation of, for example, cell debris or proteins, in the pipes and equipment. [Brief explanation of the drawings]

[0037] [Figure 1] FIG. 1 is a schematic diagram of the system described in this invention.

[0038] [Figure 2] FIG. 2 shows the SDS-PAGE analysis of Example 3.

[0039] [Figure 3] FIG. 3 shows the SDS-PAGE analysis of Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0040] 1 shows a system 100 for extracting cytoplasmic or periplasmic proteins. The system 100 comprises a cell suspension supply conduit 102 and an aqueous solution supply conduit 104, both of which are connected to a static mixer 106. The system 100 further comprises a first heat exchanger 108, a holding unit 110, and a second heat exchanger 112, which are connected in series. The static mixer 106 is connected to the first heat exchanger 108. The system further comprises an exhaust conduit 114. The second heat exchanger 112 is connected to the exhaust conduit 114.

[0041] The cell suspension supply conduit 102 is connected to a cell suspension container 120 and is equipped with a peristaltic pump 122. The aqueous solution supply conduit 104 is connected to an aqueous solution container 124 and is equipped with a peristaltic pump 126. The discharge conduit 114 is connected to a protein suspension container 128. The holding unit 110 is equipped with a jacket 130.

[0042] During system operation, cell suspension container 120 provides a first cell suspension at room temperature, while aqueous solution container 124 provides a buffer solution at 95°C. Pumps 122, 126 pump the first cell suspension and buffer solution from containers 120, 124 to static mixer 106, where the cell suspension and aqueous solution are mixed in a 1 / 5 ratio, resulting in a second cell suspension at approximately 70°C. The second cell suspension is passed to first heat exchanger 108, where the temperature of the second cell suspension is increased to 75°C. From first heat exchanger 108, the second cell suspension is passed via retention unit 110 to second heat exchanger 112, where the temperature of the second cell suspension is reduced to 25°C. The second cell suspension has a residence time of 5 minutes in retention unit 110. From second heat exchanger 112, the second cell suspension is passed via discharge conduit 114 to protein suspension container 128, from which it may be collected for further processing.

[0043] System 100 further includes a heating unit 140. Tap water is provided to the heating unit via conduit 142. Heating unit 140 heats the tap water and provides a heat transfer medium to first heat exchanger 108 and jacket 130 via conduits 144 and 146, respectively. The heat transfer medium is returned to heating unit 140 via conduits 148 and 150, respectively. The tap water in conduit 142 also provides a refrigerant medium to second heat exchanger 112. The refrigerant medium is discharged via conduit 152. [Example]

[0044] Example 1, Heat-Induced Extraction of BPEP01 The description in this example refers to the cultivation of two replicate production batches of a ∼19 kDa polypeptide called BPEP01, which contains two copies of the Z variant (Z01) and the albumin-binding domain from GA3 of streptococcal protein G, heat-induced extraction involving the use of a static mixer, and subsequent analysis, including comparison with heat treatment using a fermentor.

[0045] Materials and Methods

[0046] Culture: Culture scale was either 6 L or 20 L. E. coli T7E3 cells (GeneBridge) were transformed with a plasmid containing the product gene fragment. A research cell bank (RCB) was created using Vegtone LB medium (Sigma-Aldrich) containing 50 mg / L kanamycin. When the culture reached an OD600 of 0.94, glycerol was added to a final concentration of 15%, and the culture was aliquoted into vials (1 ml / vial) and frozen at -80°C.

[0047] Shake flask medium (6.7 g / l yeast nitrogen base (Becton Dickinson), 5.5 g / l glucose monohydrate, 7 g / l dipotassium monohydrogen phosphate, 1 g / l trisodium citrate dihydrate, 50 mg / ml kanamycin) was inoculated with 200 μl / l of thawed RCB vials. After incubation at 30 °C to an OD of >4, the fermenter containing the medium (ammonium sulfate 3.75 g / L, dipotassium monohydrogen phosphate 3.3 g / L, monopotassium dihydrogen phosphate 4.95 g / L, trisodium citrate dihydrate 1.88 g / L, antifoam 204 (Sigma-Aldrich) 1 ml / L, magnesium sulfate 6.1 mol / L, kanamycin 50 mg / L, glucose 1.2 g / L, iron(III) chloride hexahydrate 74 mg / L, zinc sulfate 24 mg / L heptahydrate, copper(II) sulfate pentahydrate 4 mg / L, manganese(II) sulfate monohydrate 16 mg / L, calcium chloride dihydrate 10 mg / L) was inoculated into shake flasks at an OD of 0.05–0.1. Cultivation was typically carried out at 37 °C with stirring and under overpressure (<0.5 bar), with the dissolved oxygen level controlled at >30%. The pH was controlled at pH 7, and glucose feeding was initiated 3 hours after inoculation. The temperature was lowered to 33°C after 17.5 hours, and 0.6 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added 18 hours later to induce protein expression. The culture was terminated after 27–30 hours.

[0048] Cell concentration: Cultures were harvested by either centrifugation or tangential flow filtration. Centrifugation was performed at 9,800 × g for 15 minutes at 23°C, and the supernatant was discarded. To mimic the large-scale separation device, the cell pellet was resuspended to a cell slurry of approximately 700 g / kg using 10 mM sodium phosphate, pH 7.0. Two 0.5 m plates of regenerated cellulose (P2C01MV05, Merck-Millipore) were used. 2 Tangential flow filtration was performed on a 1000 kDa filter where the culture was concentrated by a factor of three, followed by diafiltration with three diafiltration volumes of 50 mM sodium acetate buffer, pH 6.0.

[0049] Heat-induced extraction using a static mixer system: 10 mM sodium phosphate, 2 mM EDTA, pH 7 (expected to reach pH 6.5 during heat treatment), [heat release buffer 1], was heated to 91–95°C in a medium preparation tank of a multi-fermentation system (System Greta, Belach Bioteknik). In two separate heat treatment runs, a peristaltic pump was used to direct the 23°C cell concentrate (~1.6 L and ~4.3 L, respectively) and the heated heat release buffer 1 into a static mixer (PMS3, ESSKA.se Industriteknik) at flow rates of 30 ml / min and 137 ml / min, respectively, resulting in a 5.6-fold dilution of the cell concentrate. After mixing, the cell suspension was transferred to a holding unit (Pumpsil® 6.4 × 1.6 mm tubing with a volume of 56 cm) placed in a water bath set at 76–78°C. 3 The cell suspension was then introduced into a cooling coil (S30, Bryggbolaget, Watson Marlow). The resulting device resulted in heating of the cell suspension to approximately 76 °C (operating temperature) with a 20-second hold time. After heating and holding, the cell suspension was introduced into a cooling coil (S30, Bryggbolaget) placed in a bucket of ice water. Ice was repeatedly added to the water to maintain the temperature of the heat-treated cell suspension at ~25 °C.

[0050] Heat-induced extraction using a fermentor: The cell concentrate was mixed with 50 mM sodium acetate buffer, pH 6.0, followed by the addition of EDTA to a final concentration of 2 mM and pH adjustment to pH 6.5 with 0.5 M disodium hydrogen phosphate, resulting in a 3.8-fold dilution of the cell concentrate. The cell suspension was heated to 76°C for 3 min using the heating system of a jacketed fermentor BR20 (Belach Bioteknik). The total time for heating, holding, and cooling to 25°C was approximately 1 h, simulating a large-scale heat treatment in a 200 L fermentor.

[0051] Protein analysis: Quantitation was achieved by small-scale affinity chromatography purification of small fractions, followed by Abs280 measurement of the purified eluate.

[0052] result

[0053] Product quantitation in heat-treated cell suspensions using a static mixer system showed an average recovery of 100% in two representative runs. In comparison, use of a fermentor heat treatment procedure resulted in an 87% recovery. The resulting process improvement using the static mixer was 15% in terms of product recovery.

[0054] Example 2, Heat-Induced Extraction of BPEP02 This example describes the cultivation of two different Z variants (Z02a and Z02b) and a ∼19 kDa polypeptide called BPEP02, which contains the albumin-binding domain from GA3 of streptococcal protein G, and its heat-induced extraction, including the use of a static mixer, and subsequent analysis. Comparison with heat treatment using a fermentor is included.

[0055] Materials and Methods

[0056] Cultivation: The culture size was either 2 L or 20 L. Cultivation was essentially as described in Example 1, except that the final OD600 at the time of RCB preparation was 0.80 and the temperature was reduced to 31°C after 17.5 hours of cultivation.

[0057] Cell concentration: Cultures were harvested by centrifugation or tangential flow filtration. The cells were centrifuged at 15,900 × g for 25 min at 4 °C, and the supernatant was discarded. Cells were then concentrated by centrifugation using 2 × 0.5 m of regenerated cellulose (P2C01MV05, Merck-Millipore). 2 Tangential flow filtration was performed on a 1000 kDa filter where the culture was concentrated by a factor of three, followed by diafiltration with three diafiltration volumes of 10 mM phosphate buffer, pH 8.

[0058] Heat-induced extraction using a static mixer system: Cells were frozen prior to heat treatment. 25 mM sodium phosphate, 2 mM EDTA, pH 8 (expected to reach pH 7.3 during heat treatment), [heat release buffer 2], was heated to 91–95°C in a medium preparation tank of a multi-fermentation system (System Greta, Belach Bioteknik). Using two peristaltic pumps, the 23°C cell concentrate (~6 L) and the heated heat release buffer 2 were introduced into a static mixer (PMS3, ESSKA.se Industriteknik) at flow rates of 25 ml / min and 142 ml / min, respectively, resulting in a 6.7-fold dilution of the cell concentrate. After mixing, the cell suspension was transferred to a holding unit (S30, Bryggbolaget, estimated volume 500 cm) placed in a water bath set at 76°C. 3 The resulting device resulted in heating of the cell suspension at approximately 76 °C (operating temperature) for 3 min. After heating, the cell suspension was introduced into a cooling coil (S30, Bryggbolaget) placed in a bucket of ice water. Ice was repeatedly added to the water to maintain the temperature of the heat-treated cell suspension at ~25 °C.

[0059] Heat-induced extraction using a fermenter: Cells were frozen before heat treatment. The cell concentrate was mixed with 179 mM phosphate, 11 mM citrate buffer, followed by the addition of EDTA to a final concentration of 2 mM, resulting in a 5-fold dilution of the cell concentrate. The pH of the resulting cell suspension was 7.3. The cell suspension was heated to 76°C for 3 minutes using the heating system of the fermenter BR20 (Belach Bioteknik). The total time for heating, holding, and cooling to 25°C was 75 minutes.

[0060] Protein analysis: Quantitation was achieved by small-scale affinity chromatography purification of small fractions, followed by Abs280 measurement of the purified eluate.

[0061] result

[0062] Product determination in heat-treated cell suspensions using the static mixer system showed a recovery of 69%. In comparison, using the fermentor heat treatment procedure, the recovery was 46%. The resulting process improvement using the static mixer was 50% in terms of product recovery.

[0063] Example 3. Heat-induced extraction of BPEP03 This example describes the cultivation of a ∼19 kDa polypeptide, designated BPEP03, containing two copies of the Z variant (Z03) and the albumin-binding domain from streptococcal protein G, GA3, and subsequent analysis using a heat-induced extraction process involving the use of a static mixer, as well as comparison with heat treatment using a fermentor.

[0064] Materials and Methods

[0065] Cultivation: The culture volume was 1 L. The culture was essentially carried out as described in Example 1, except that the temperature was lowered to 31° C. after 17.5 hours of cultivation.

[0066] Cell concentration: Cultures were harvested by centrifugation at 9,800 × g for 15 minutes at 23 °C, and the supernatant was discarded. To mimic the large-scale separation device, the cell pellet was resuspended in 10 mM phosphate buffer, pH 7.4, to obtain a cell slurry of ~700 g / kg.

[0067] Heat-induced extraction using a static mixer system: 25 mM phosphate, 2 mM EDTA, pH 8.5 [heat release buffer 3] was heated to 91–95 °C in a media preparation tank (System Greta, Belach Bioteknik) of a multi-fermentation system. Using two peristaltic pumps, the 23 °C cell concentrate (~0.15 L) and the heated heat release buffer 3 were introduced into a static mixer (PMS3, ESSKA.se Industriteknik) at flow rates of 25 ml / min and 114 ml / min, respectively, resulting in a 5.6-fold dilution of the cell concentrate. After mixing, the cell suspension was introduced into a holding unit (S30, Matrevolution, estimated 417 ml retention volume) placed in a water bath set at 77.6 °C. The resulting device resulted in instantaneous heating of the cell suspension to 75 °C, pH ~7.4, with a 3-minute holding time. After heating and holding the suspension at operating temperature, the cell suspension was introduced into a cooling coil (S30, Bryggbolaget) placed in a bucket of ice water. Ice was repeatedly added to the water to maintain the temperature of the heat-treated cell suspension at ∼25 °C.

[0068] Heat-induced extraction using a fermenter: The cell concentrate was mixed with 25 mM phosphate, 2 mM EDTA, pH 8.5, to obtain the same proportions of cell concentrate and buffer as described for the static mixer procedure in the section above. Heating of the cell suspension was performed to simulate a large-scale heat treatment in a >200 L fermenter using the Fermenter BR20 heating system (Belach Bioteknik). Therefore, a heating profile was set in the fermenter to heat from 25 °C to 75 °C for approximately 50 min, followed by a 3 min hold at 75 °C, and finally a 30 min cool down at 25 °C. The total heating, hold, and cool down time was approximately 83 min.

[0069] Protein analysis: Quantitation was achieved by small-scale affinity chromatography purification of small fractions, followed by Abs280 measurement of the purified eluate. Additionally, SDS-PAGE analysis of purified fractions was performed to assess product-related impurities, such as dimerization and degradation.

[0070] result

[0071] Product determination in the heat-treated cell suspension from heat treatment in a static mixer showed a recovery of 71%, while heat treatment in a fermentor resulted in a recovery of 33%. Thus, the resulting process improvement using the static mixer was 115% in terms of product recovery.

[0072] During the comparison, significant quality advantages were also detected for static mixer heat-treated samples compared to fermentor heat-treated samples, as demonstrated by SDS-PAGE analysis. Figure 2 shows SDS-PAGE analysis of affinity-purified lysates containing BPEP03 after heat treatment in a static mixer (lane 2) and a fermentor (lane 3), each loaded at 8 μg on the gel. Novex™ Sharp Protein Standards (MW: 260, 160, 110, 80, 60, 50, 40, 30, 20, 15, 10, and 3.5 kDa) were loaded in lane 1. The fermentor heat-treated samples showed both more degradation and dimerization. In addition to the improved recovery and better sample profile when using a static mixer, the process of using a static mixer for heat treatment enables industrial production that is not feasible with fermentor-based heat treatment.

[0073] Example 4, Heat-Induced Extraction of BPEP04 The description in this example refers to the cultivation, heat-induced extraction, including the use of a static mixer, and subsequent analysis of an approximately 14 kDa albumin-binding protein called BPEP04, which contains two albumin-binding domains of streptococcal protein G, GA2 and GA3, and a C-terminal cysteine ​​residue, and a comparison with heat treatment using a fermentor.

[0074] Materials and Methods

[0075] Cultivation (1 L scale), lab-scale static mixer system, and heat-induced extraction using fermentors, as well as protein analysis, were carried out essentially as described in Example 3.

[0076] result

[0077] Quantification of product in heat-treated cell suspensions from either static mixer or fermentor heat treatments showed 100% recovery. However, the comparison demonstrated an advantage in quality of the static mixer heat-treated samples compared to the fermentor heat-treated samples, as shown by SDS-PAGE analysis. Figure 3 shows SDS-PAGE analysis of affinity-purified lysates containing BPEP04 after static mixer heat treatment (lane 2) and fermentor heat treatment (lane 3), each loaded at 8 μg on the gel. Novex™ Sharp Protein Standard was loaded in lane 1. The fermentor heat-treated sample exhibited both more degradation and a higher fraction of multimeric forms (dimers, trimers, and tetramers).

[0078] Example 5. Heat-induced extraction of BPEP05 The description in this example refers to the cultivation of a ∼6.7 kDa polypeptide, designated BPEP05, containing one copy of the Z variant (Z04) and a C-terminal cysteine ​​residue, followed by heat-induced extraction using a static mixer and subsequent analysis, including comparison with heat treatment using a fermentor.

[0079] Materials and Methods

[0080] Cultivation (1 L scale) and heat-induced extraction using a static mixer system and a fermenter were essentially performed as described in Example 3, except that instead of using an RCB, a shake flask starter culture was inoculated with a flow-through culture of TSB+YE medium and incubated at 30°C for 5 hours. Quantitation of the products was performed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS).

[0081] result

[0082] UPLC-MS quantification of products in heat-treated cell suspensions from static mixer and fermentor heat treatments each showed 43% better recovery when using a static mixer compared to fermentor heat treatment.

[0083] Example 6: Large-scale heat dissipation of BPEP01 We demonstrated the production of BPEP01 using the heat treatment described in this invention in a large-scale process. Cultivation and harvesting were performed essentially as described in Example 1, but at a 100 L culture scale using a disc stack centrifuge (GEA Westfalia) for cell concentration. Heat treatment was performed in a static mixer with the same proportions of cell suspension and [heat release buffer 1] as in Example 1, and was performed using a heat treatment system designated S175 with a retention volume of 13.6 L and a total flow rate of 6.8 L / min, at an operating temperature of 76 ± 1°C and a 2-minute retention time. The recovery rate from the large-scale run was 100%, which corresponded well to the recovery rate obtained in the small-scale run described in Example 1. Therefore, the results of this experiment confirmed the scalability and industrial applicability of this method.

[0084] Example 7: Large-scale heat dissipation of BPEP02 Three batches, two of which were conducted under GMP (Good Manufacturing Practice), were successfully performed using the heat treatment described in this invention in a large-scale process for the production of BPEP02. Cultivation and harvesting were performed essentially as described in Example 2, at a 300 L cultivation scale. Heat treatment was performed in a static mixer with the same proportions of cell suspension and [heat dissipation buffer 2] as in Example 2, using a heat treatment system designated S163 with a 26 L retention volume and a total flow rate of 8.67 L / min, at an operating temperature of 80–84°C, and a 3-minute retention time. Recoveries from the three large-scale runs were 73–85%, corresponding well to those obtained in the small-scale runs described in Example 2. Therefore, the results of this experiment confirmed the scalability and industrial applicability of this method.

Claims

1. A method for extracting cytoplasmic or periplasmic proteins, - providing a first cell suspension comprising cells, said cells comprising cytoplasmic or periplasmic proteins to be extracted, said first cell suspension having a first temperature; and - heating the first cell suspension at an operating temperature, at which at least a fraction of the cells are subject to heat-induced lysis and at least a fraction of the cytoplasmic or periplasmic proteins to be extracted are not subject to irreversible denaturation; wherein heating the first cell suspension comprises: - providing an aqueous solution, said aqueous solution having a second temperature higher than said first temperature; and - mixing the first cell suspension with an aqueous solution, thereby obtaining a second cell suspension, said second cell suspension having a third temperature higher than the first temperature, - said first temperature is in the range of 0 to 37°C; and / or - the operating temperature is in the range of 50 to 90°C; and / or - the second temperature is less than or equal to 110°C; and / or - the third temperature is in the range of 50 to 90°C; the steps of: wherein the cells are E. coli cells.

2. heating the first cell suspension; - heating the second cell suspension from the third temperature to the operating temperature; The method of claim 1 further comprising:

3. 3. The method of claim 1, wherein the third temperature is no more than 10° C. below the operating temperature.

4. The method of any one of claims 1 to 3, wherein the third temperature is no more than 5°C lower than the operating temperature.

5. The method of claim 1 , wherein the third temperature is the operating temperature.

6. - maintaining the second cell suspension at said operating temperature for a period ranging from 1 second to 20 minutes; The method of any one of claims 1 to 5, further comprising:

7. - cooling the second cell suspension from said operating temperature to a fourth temperature, said fourth temperature being a temperature at which at least a fraction of the reversibly denatured cytoplasmic or periplasmic proteins to be extracted are subject to renaturation. The method of any one of claims 1 to 6, further comprising:

8. - separating the cytoplasmic or periplasmic proteins to be extracted from cell debris and / or native host cell proteins; The method of any one of claims 1 to 7, further comprising:

9. 9. The method of claim 7 or 8, wherein the fourth temperature is in the range of 2 to 37°C.

10. 10. The method according to claim 7, wherein the residence time at a temperature higher than both the first temperature and the fourth temperature is 20 minutes or less.

11. The method of any one of claims 1 to 10, wherein the cytoplasmic or periplasmic protein to be extracted comprises a three-helix bundle protein domain of a bacterial receptor protein, or a mutant thereof.

12. 12. The method according to any one of claims 1 to 11, wherein the mixing of the first cell suspension and the aqueous solution is carried out in a static mixer or in a stirred vessel.

13. A system for extracting cytoplasmic or periplasmic proteins for carrying out the method according to any one of claims 1 to 12, - a cell suspension supply conduit having an inlet and an outlet, the inlet of the cell suspension supply conduit being connectable to a cell suspension container; an aqueous solution supply conduit having an inlet and an outlet, the inlet of said aqueous solution supply conduit being connectable to an aqueous solution container; a static mixer having at least one inlet and one outlet, wherein at least one inlet of said static mixer is in fluid communication with the outlet of said cell suspension supply conduit and the outlet of said aqueous solution supply conduit; a first heat exchanger having an inlet for the cell suspension to be heated and an outlet for the heated cell suspension, the inlet of the first heat exchanger being in fluid communication with the outlet of the static mixer; a second heat exchanger having an inlet for the cell suspension to be cooled and an outlet for the cooled cell suspension, the inlet of the second heat exchanger being in liquid communication with the outlet of the first heat exchanger; a discharge conduit having an inlet and an outlet, the inlet being in fluid communication with the outlet of said second heat exchanger, the outlet being connectable to a protein suspension vessel or a protein suspension processing system; A system with.

14. The system of claim 13 further comprising a retention conduit providing fluid communication between the outlet of the first heat exchanger and the inlet of the second heat exchanger.

15. 15. The system of claim 14, wherein the holding conduit is surrounded by a jacket or container, by insulation, or by a heating blanket.

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