NEW PROCESS OF EXTRACTING PROTEIN
20220411743 · 2022-12-29
Inventors
Cpc classification
C12M47/06
CHEMISTRY; METALLURGY
International classification
Abstract
A method of extracting a cytoplasmic or periplasmic protein. The method comprises provision of a first cell suspension comprising cells, the cells containing a cytoplasmic or periplasmic protein of interest to be extracted, the first cell suspension having a first temperature, and heating of the first cell suspension to an operating temperature, at which operating temperature at least a fraction of the cells is subject to heat-induced lysis and at least a fraction of the cytoplasmic or periplasmic protein of interest to be extracted is not subject to irreversible denaturation. The heating of the first cell suspension comprises provision of an aqueous solution, the aqueous solution having a second temperature that is higher than the first temperature, and mixing of the first cell suspension with the aqueous solution, thereby obtaining a second cell suspension, the second cell suspension having a third temperature that is higher than the first temperature. A system for extracting a cytoplasmic or periplasmic protein. The system comprises, i.a., a static mixer.
Claims
1. A method of extracting a cytoplasmic or periplasmic protein, the method comprising: providing of a first cell suspension comprising cells, the cells containing a cytoplasmic or periplasmic protein of interest to be extracted, the first cell suspension having a first temperature, and heating of the first cell suspension to an operating temperature, wherein at least a fraction of the cells is subject to heat-induced lysis and at least a fraction of the cytoplasmic or periplasmic protein of interest to be extracted is not subject to irreversible denaturation, and wherein the heating of the first cell suspension comprises: providing an aqueous solution, the aqueous solution having a second temperature that is higher than the first temperature, and mixing of the first cell suspension with the aqueous solution, thereby obtaining a second cell suspension, the second cell suspension having a third temperature that is higher than the first temperature.)
2. The method according to claim 1, wherein the heating of the first cell suspension further comprises: heating of the second cell suspension from the third temperature to the operating temperature.
3. The method according to claim 1, wherein the third temperature is no more than 10° C. lower than the operating temperature.
4. The method according to claim 1, wherein the third temperature is the operating temperature.
5. The method according to claim 1, wherein the method further comprises: maintaining of the second cell suspension at the operating temperature for a time period of about 1 s to 20 min, about 10 s to 20 min, about 1 s to 10 min, about 10 s to 10 min, about 1 s to 5 min, about 10 s to 5 min, about 10 s to 4 min, about 10 s to 30 or about 1 min to 4 min.
6. The method according to claim 1, wherein the method further comprises: cooling of the second cell suspension from the operating temperature to a fourth temperature, wherein the fourth temperature is a temperature at which at least a fraction of reversibly denatured cytoplasmic or periplasmic protein of interest to be extracted is subject to renaturation.
7. The method according to claim 1, wherein the method further comprises: separating the cytoplasmic or periplasmic protein of interest from cell debris and/or native host cell proteins.
8. The method according to claim 1, wherein the first temperature is about in the range of 0 to 37° C., preferably in the range about 2 to 37° C., more preferably in the range about 8 to 30° C., more preferably in the range or about 18 to 25° C.
9. The method according to claim 1, wherein the fourth temperature is about 2 to 37° C., about 8 to 30° C., about 25 to 37° C., or about 18 to 25° C.
10. The method according to claim 6, wherein the time at a temperature that is higher than both the first temperature and the fourth temperature is no more than 20 min, about 1 s to 20 min, about 10 s to 20 min, about 10 min, about 1 s to 10 min, about 10 s to 10 min, about 5 min, about 1 s to 5 min, about 10 s to 5 min.
11. The method according to claim 1, wherein the cells are prokaryotic cells or eukaryotic cells.
12. The method according to claim 1, wherein the cytoplasmic or periplasmic protein of interest to be extracted comprises a three-helix bundle protein domain of a bacterial receptor protein, or a variant thereof.
13. The method according to claim 1, wherein the mixing of the first cell suspension with the aqueous solution is performed in a static mixer or in an agitated vessel.
14. A system for extracting a cytoplasmic or periplasmic protein, the system comprising: 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 the aqueous solution supply conduit being connectable to an aqueous solution container; a static mixer having at least one inlet and an outlet, the at least one inlet of the static mixer being in liquid communication with the outlet of the cell suspension supply conduit and with the outlet of the aqueous solution supply conduit; a first heat exchanger having an inlet for cell suspension to be heated and an outlet for heated cell suspension, the inlet of the first heat exchanger being in liquid communication with the outlet of the static mixer; a second heat exchanger having an inlet for cell suspension to be cooled and an outlet for 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 of the discharge conduit being in liquid communication with the outlet of the second heat exchanger and the outlet of the discharge conduit being connectable to a protein suspension container or to a protein suspension treatment system.
15. The system of claim 14, further comprising a holding conduit providing liquid communication between the outlet of the first heat exchanger and the inlet of the second heat exchanger, the holding conduit being surrounded by a jacket, a vessel, a heat insulating material, a heating blanket, or an electrical heating blanket.
16. The method according claim 1, wherein the third temperature is no more than 5° C. lower than the operating temperature.
17. The method of claim 1, wherein the operating temperature is below 90° C., about 20 to 90° C., about 40 to 90° C., about 50 to 90° C., about 60 to 90° C., about 70 to 90° C., about of 70 to 85° C., or about 75 to 85° C.
18. The method of claim 1, wherein the second temperature is below 110° C., about 40 to 110° C., about 50 to 110° C., about 60 to 99° C., about 70 to 99° C., about 80 to 99° C., about 90 to 99° C., about 90 to 95° C.
19. The method according to claim 1, wherein the third temperature is below 90° C., about 40 to 90° C., about 50 to 90° C., about 60 to 85° C. about 65 to 85° C., about 65 to 80° C., about 65 to 78° C., or about 68 to 78° C.
20. The method according to claim 11, wherein the prokaryotic cells are E. coli.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0051]
[0052]
[0053]
DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
[0054]
[0055] The cell suspension supply conduit 102 is connected to a cell suspension container 120 and comprises a peristaltic pump 122. The aqueous solution supply conduit 104 is connected to an aqueous solution container 124 and comprises a peristaltic pump 126. The discharge conduit 114 is connected to protein suspension container 128. The holding unit 110 is provided with a jacket 130.
[0056] During operation of the system, the cell suspension container 120 provides a first cell suspension at room temperature whereas the aqueous solution container 124 provides a buffer solution at 95° C. The pumps 122, 126 transport the first cell suspension and the buffer solution from the containers 120, 124 to the static mixer 106, where the cell suspension and the aqueous solution are mixed in a ratio of 1:5, resulting in a second cell suspension at approx. 70° C. The second cell suspension is passed to the first heat exchanger 108, where the temperature of the second cell suspension is raised to 75° C. The second cell suspension is passed from the first heat exchanger 108, via the holding unit 110 to the second heat exchanger 112, where the temperature of the second cell suspension is lowered to 25° C. The second cell suspension has a residence time in the holding unit 110 of 5 min. The second cell suspension is passed from the second heat exchanger 112 via the discharge conduit 114 to the protein suspension container 128, from where it may be collected for further treatment.
[0057] The system 100 further comprises a heating unit 140. The heating unit is provided with tap water via a conduit 142. The heating unit 140 heats the tap water and provides the first heat exchanger 108 and the jacket 130 with heating medium via conduits 144 and 146, respectively. The heating medium is returned to the heating unit 130 via conduits 148 and 150, respectively. The tap water in conduit 142 also provides the second heat exchanger 112 with cooling medium. The cooling medium is discharged via a conduit 152.
Examples
Example 1, Heat-Induced Extraction of BPEP01
[0058] The description in this Example refers to cultivation, heat-induced extraction involving use of a static mixer, and subsequent analysis, of two repeated production batches of an approximately 19 kDa polypeptide, referred to as BPEP01, comprising two copies of a Z variant (Z01) and an albumin binding domain derived from GA3 of Streptococcal protein G. A comparison with heat treatment using a fermenter is included.
Materials and Methods
[0059] Cultivation: The scale of the cultivations was either 6 L or 20 L. E. coli T7E2 cells (GeneBridges) were transformed with plasmids containing the gene fragment of the product. A Research Cell Bank (RCB) was generated using Vegitone LB-medium (Sigma-Aldrich) containing 50 mg/l kanamycin. When the culture had reached OD600=0.94, glycerol was added to a final concentration of 15%, and the culture was aliquoted into vials (1 ml/vial), which were frozen at −80° C.
[0060] 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 a thawed RCB vial. After incubation at 30° C. to an OD600>4, a fermenter containing medium (3.75 g/l ammonium sulphate, 3.3 g/l dipotassium monohydrogen phosphate, 4.95 g/l monopotassium dihydrogen phosphate, 1.88 g/l trisodium citrate dihydrate, 1 ml/l antifoam 204 (Sigma-Aldrich), 6.1 mol/l magnesium sulphate, 50 mg/l kanamycin, 1.2 g/l glucose, 74 mg/l iron (III) chloride hexahydrate, 24 mg/l zinc sulfate heptahydrate, 4 mg/l copper (II) sulfate pentahydrate, 16 mg/l manganese (II) sulfate monohydrate, 10 mg/l calcium chloride dihydrate) was inoculated with the shake flask culture to an OD600 of 0.05-0.1. The cultivations were generally run at 37° C. under stirring and overpressure 0.5 bar) to control the dissolved oxygen level at 30%. The pH was controlled at pH=7 and a glucose feed was initiated 3 h after inoculation. After 17.5 h the temperature was lowered to 33° C., and after 18 h 0.6 mM of Isopropyl-β-D-thiogalactopyranoside (IPTG) was added for induction of protein production. The cultivations were stopped after 27-30 h.
[0061] Cell concentration: The cultivations were harvested either by centrifugation or tangential flow filtration. Centrifugation was performed at 9,800×g for 15 min at 23° C. and the supernatant was discarded. To reflect a large-scale separator, the cell pellet was resuspended giving about 700 g/kg cell slurry using 10 mM sodium phosphate, pH 7.0. Tangential flow filtration was run on 2×0.5 m.sup.2 1000 kDa-filters of regenerated cellulose (P2C01 MV05, Merck-Millipore), where the cultivation was concentrated to one third, followed by diafiltration with three diafiltration volumes of 50 mM sodium acetate buffer, pH 6.0.
[0062] Heat-induced extraction using a static mixer system: 10 mM sodium phosphate, 2 mM EDTA, pH 7 (expected to result in a pH of 6.5 during the heat treatment), [Heat releasing buffer 1], was heated to 91-95° C. in the media preparation tank of a multifermenter system (System Greta, Belach Bioteknik). In two separate heat treatment runs, peristaltic pumps were used to lead the 23° C. cell concentrate (−1.6 L and −4.3 L, respectively) and the heated Heat releasing buffer 1, respectively, to the static mixer (PMS3, ESSKA.se Industriteknik) with a flow rate of 30 ml/min and 137 ml/min, respectively, resulting in a 5.6 times dilution of the cell concentrate. After mixing, the cell suspension was led to the holding unit (Pumpsil® 6.4×1.6 mm tubing with a volume of 56 cm.sup.3, Watson Marlow) placed in a water bath set at 76-78° C. The resulting set-up led to a heating of the cell suspension to approximately 76° C. (operating temperature) with a holding time of 20 s. After heating and holding, the cell suspension was led to a cooling coil (S30, Bryggbolaget) placed in a bucket with ice water. Ice was added repeatedly to the water in order to keep the temperature at −25° C., in the heat treated cell suspension.
[0063] Heat-induced extraction using a fermenter: The cell concentrate was mixed with 50 mM sodium acetate buffer pH 6.0 followed by addition of EDTA to a final concentration of 2 mM and pH adjustment to pH 6.5 using 0.5 M disodium hydrogen phosphate, resulting in a 3.8 times dilution of the cell concentrate. Heating of the cell suspension was performed at 76° C. for 3 min using the heating system of the jacketed fermenter 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 fermenter.
[0064] Protein analysis: Quantification was made by small-scale affinity chromatography purification of a minor fraction, followed by Abs280 measurements of the purified eluate.
Results
[0065] Quantification of the product in the heat treated cell suspension using a static mixer system showed in average 100% recovery in two representative runs. As a comparison, using the fermenter heat treatment procedure resulted in a recovery of 87%. The resulting process improvement, in terms of recovery of product, using the static mixer thus was 15%.
Example 2, Heat-Induced Extraction of BPEP02
[0066] The description in this Example refers to cultivation, heat-induced extraction involving use of a static mixer and subsequent analysis of an approximately 19 kDa polypeptide, referred to as BPEP02, comprising two different Z variants (Z02a and Z02b) and an albumin binding domain derived from GA3 of Streptococcal protein G. A comparison with heat treatment using a fermenter is included.
Materials and Methods
[0067] Cultivation: The scale of the cultivations was either 2 L or 20 L. The cultivation was performed essentially as described in Example 1, with the exceptions that the final OD600 during RCB preparation was 0.80, the temperature was lowered to 31° C. after 17.5 h of cultivation.
[0068] Cell concentration: The cultivation was harvested by centrifugation or tangential flow filtration. Centrifugation was performed at 15,900×g for 25 min at 4° C. and the supernatant was discarded. Tangential flow filtration was run on 2×0.5 m.sup.2 1000 kDa-filters of regenerated cellulose (P2C01 MV05, Merck-Millipore), where the cultivation was concentrated to one third, followed by diafiltration with three diafiltration volumes of 10 mM phosphate buffer, pH 8.
[0069] Heat-induced extraction using a static mixer system: The cells were frozen before subjected to heat treatment. 25 mM sodium phosphate, 2 mM EDTA, pH 8 (expected to result in a pH of 7.3 during the heat treatment), [Heat releasing buffer 2], was heated to 91-95° C. in the media preparation tank of a multifermenter system (System Greta, Belach Bioteknik). Two peristaltic pumps were used to lead the 23° C. cell concentrate (˜6 L) and the heated Heat releasing buffer 2, respectively, to the static mixer (PMS3, ESSKA.se Industriteknik) with a flow rate of 25 ml/min and 142 ml/min, respectively resulting in a 6.7 times dilution. After mixing, the cell suspension was led to the holding unit (S30, Bryggbolaget, with an estimated volume of 500 cm.sup.3) placed in a water bath set at 76° C. The resulting set-up led to a heating of the cell suspension to approximately 76° C. (operating temperature) for 3 min. After heating, the cell suspension was led to a cooling coil (S30, Bryggbolaget) placed in a bucket with ice water. Ice was added repeatedly in order to keep the final temperature at ˜25 ° C., in the heat treated cell suspension.
[0070] Heat release using a fermenter: The cells were frozen before they were subjected to heat treatment. The cell concentrate was mixed with 179 mM phosphate 11 mM citrate buffer followed by addition of EDTA to a final concentration of 2 mM, resulting in a 5 times dilution of the cells. Resulting pH of the cell suspension was 7.3. Heating of the cell suspension was performed at 76° C. for 3 min using the heating system of the fermenter BR20 (Belach Bioteknik). The total time for heating, holding and cooling to 25° C. was 75 min.
[0071] Protein analysis: Quantification was made by small-scale affinity chromatography purification of a minor fraction, followed by Abs280 measurements of the purified eluate.
Results
[0072] Quantification of the product in the heat treated cell suspension using a static mixer system showed 69% recovery. As a comparison, using the fermenter heat treatment procedure, the recovery was 46%. The resulting process improvement, in terms of recovery of product, using the static mixer thus was 50%.
Example 3, Heat-Induced Extraction of BPEP03
[0073] The description in this Example refers to cultivation, heat-induced extraction involving use of a static mixer, and subsequent analysis of an approximately 19 kDa polypeptide, referred to as BPEP03, comprising two copies of a Z variant (Z03) and an albumin binding domain derived from GA3 of Streptococcal protein G. A comparison with heat treatment using a fermenter is included.
Materials and Methods
[0074] Cultivation: The scale of the cultivation was 1 L. The cultivation was performed essentially as described in Example 1, with the exceptions that the temperature was lowered to 31° C. after 17.5 h hours of cultivation.
[0075] Cell concentration: The cultivation was harvested by centrifugation. Centrifugation was performed at 9,800×g for 15 min, 23° C. and the supernatant was discarded. To reflect a large-scale separator, the cell pellet was resuspended in a 10 mM phosphate buffer, pH 7.4, yielding a −700 g/kg cell slurry.
[0076] Heat-induced extraction using a static mixer system: 25 mM phosphate, 2 mM EDTA, pH 8.5, [Heat releasing buffer 3], was heated to 91-95° C. in the media preparation tank of a multifermenter system (System Greta, Belach Bioteknik). Two peristaltic pumps were used to lead the 23° C. cell concentrate (˜0.15 L) and the heated Heat releasing buffer 3, respectively, to the static mixer (PMS3, ESSKA.se Industriteknik) with a flow rate of 25 ml/min and 114 ml/min, respectively resulting in a 5.6 times dilution of the cell concentrate. After mixing, the cell suspension was led to the holding unit (S30, Matrevolution, estimated to a holding volume of 417 ml) placed in a water bath set at 77.6° C. The resulting set-up led to an instantaneous heating of the cell suspension to 75° C., pH ˜7.4, with a 3 min holding time. After heating, and holding the suspension at the operating temperature, the cell suspension was led to a cooling coil (S30, Matrevolution) placed in a bucket with ice water. Ice was added repeatedly in order to keep the final temperature at ˜25° C., in the heat treated cell suspension.
[0077] Heat-induced extraction using a fermenter: The cell concentrate was mixed with 25 mM phosphate, 2 mM EDTA, pH 8.5 giving 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 heating system of the fermenter BR20 (Belach Bioteknik). Thus, a heating profile was set in the fermenter, where heating from 25° C. to 75° C. was set to take approximately 50 min followed by a holding step at 75° C. for 3 min and finally cooling to 25° C. in 30 min. The total time for heating, holding and cooling was approximately 83 min.
[0078] Protein analysis: Quantification was made by small-scale affinity chromatography purification of a minor fraction, followed by Abs280 measurements of the purified eluate. Furthermore, SDS-PAGE analysis of the purified fraction was performed to assess product related impurities, such as dimerization and degradation.
Results
[0079] Quantification of the product in the heat treated cell suspension from heat treatment with a static mixer showed 71% recovery, whereas heat treatment in a fermenter resulted in a recovery of 33%. Thus, the resulting process improvement, in terms of recovery of product, using the static mixer was 115%.
[0080] During the comparison also a major advantage in terms of product quality was detected for the static mixer heat treated sample compared to fermenter heat treated as depicted in the SDS-PAGE analysis.
Example 4, Heat-Induced Extraction of BPEP04
[0081] The description in this Example refers to cultivation, heat-induced extraction involving use of a static mixer, and subsequent analysis of an approximately 14 kDa albumin binding protein referred to as BPEP04 comprising the two albumin binding domains GA2 and GA3 of Streptococcal protein G, and a C-terminal cysteine residue. A comparison with heat treatment using a fermenter is included.
Materials and Methods
[0082] Cultivation (1 L scale), heat induced extraction using the lab-scale static mixer system and a fermenter, respectively, as well as protein analysis were performed essentially as described in Example 3.
Results
[0083] Quantification of the product in the heat treated cell suspension from heat treatment with static mixer or heat treatment with fermenter, both showed 100% recovery. However, the comparison showed an advantage in terms of product quality for the static mixer heat treated sample compared to fermenter heat treated as depicted in the SDS-PAGE analysis.
Example 5, Heat-Induced Extraction of BPEP05
[0084] The description in this Example refers to cultivation, heat-induced extraction involving use of a static mixer, and subsequent analysis of an approximately 6.7 kDa polypeptide, referred to as BPEP05, comprising one copy of a Z variant (Z04) and a C-terminal cysteine residue. A comparison with heat treatment using a fermenter is included.
Materials and Methods
[0085] Cultivation (1 L scale) and heat induced extraction using the static mixer system and a fermenter, respectively, were performed essentially as described in Example 3, with the exception that instead of establishing an RCB, the shake flask starter culture was inoculated with a culture run with TSB+YE-medium, which had been incubated for 5 h at 30° C. Quantification of the product was carried out by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS).
Results
[0086] UPLC-MS quantification of the product in the heat treated cell suspension from heat treatment with static mixer and fermenter, respectively, showed 43% better recovery when using static mixer compared to heat treatment in fermenter.
Example 6, Large-Scale Heat Release of BPEP01
[0087] Production of BPEP01 using a heat treatment according to the present invention in a large scale process has successfully been demonstrated. Cultivation and harvest were performed essentially as described in Example 1, but in a 100 L cultivation scale and 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 releasing buffer 1] as in Example 1 and using a heat treatment system denoted S175, with a holding unit volume of 13.6 L and a total flow rate of 6.8 L/min, giving 2 min hold time at an operating temperature of 76±1° C. The recovery from the large-scale run was 100% and corresponds well to the recovery obtained in small scale run, described in Example 1. Thus, the result of this experiment confirms scalability and industrial applicability.
Example 7, Large-Scale Heat Release of BPEP02
[0088] Three batches, of which two were performed under GMP (Good Manufacturing Practice), have successfully been run using a heat treatment according to the present invention in a large-scale process, for the production of BPEP02. Cultivation and harvest were performed essentially as described in Example 2, in a 300 L cultivation scale. Heat treatment was performed in a static mixer with the same proportions of cell suspension and [Heat releasing buffer 2] as in Example 2 and using a heat treatment system denoted S163, with a holding unit volume of 26 L and a total flow rate of 8.67 L/min, giving 3 min hold time at an operating temperature of 80-84° C. The recoveries from the three large-scale runs were 73-85% and correspond well to the recovery obtained in small scale run described in Example 2. Thus, the result of this experiment confirms scalability and industrial applicability.