Automated periplasmic extraction

Abstract

The invention relates to an automated process for extracting a peptide of interest located in periplasmic space and / or comprising an acidification step.

Description

[0001] AUTOMATED PERIPLASMIC EXTRACTION

[0002] Technical field

[0003] The present invention relates to the automated extraction of a peptide of interest whose production has been induced in the periplasmic space.

[0004] Prior art

[0005] The production of a protein of interest in the periplasmic space is known. This has the advantage of establishing a first barrier with cytosolic contaminants including genomic DNA.

[0006] In practice, the outer membrane of a Gram-negative bacterium, for example Escherichia coli, or Pseudomonas sp., is selectively ruptured, but not the inner membrane, which allows the contents of the periplasmic space to be released into the medium. Then, a simple centrifugation allows the cells to be removed with all the contaminants present. Osmotic shock, where the cells are conditioned in a hypertonic medium (e.g. sucrose 20-25% by weight, at a neutral or alkaline pH), then suddenly placed in contact with a large volume of water, is commonly used for this purpose, but always at neutral or alkaline pH.

[0007] This technique, however, has several drawbacks and limitations. The first difficulty is that the inner membrane must not be ruptured, which imposes limits on the solutions that can be used, the treatment times, and the mechanical constraints that can be applied to these weakened cells. The second, reciprocal difficulty, is that the outer membrane must be sufficiently ruptured, which, on the contrary, imposes somewhat severe conditions. In addition, peptides that are absorbed into the membranes, or even inserted into them, may not be well recovered, or could even weaken the inner membrane, which has been reported as problematic in the case of acidification when diphtheria toxin is produced in the periplasmic space of Escherichia coli (O'Keefe and Collier, PNAS Vol. 86, pp. 343-346, 1989).

[0008] Another limitation is the difficulty of scaling-up: indeed, some peptides must be mass-produced, which requires the ability to process large quantities of cells while ensuring substantially identical processing conditions for all cells: inhomogeneity or variable processing times are problematic. Thus, the use of larger containers promotes local inhomogeneities, which directly affects (i) the recovery yields of the peptide of interest, due, for example, to poor rupture of the outer membrane (osmotic shock too weak), and (ii) the purity, due to the unwanted rupture of the inner membrane (e.g. osmotic shock too strong). In addition, the means of mixing and mechanical agitation are limited, otherwise there is a risk of breaking the internal membranes or even shearing the genomic DNA, which would render the batch completely unusable.

[0009] Among the improvements, some have proposed the addition of salts or chaotropic molecules such as, among others, alkali or alkaline earth chlorides or sulfates, sodium phosphate, ammonium acetate, ammonium chloride, various detergents (sodium deoxycholate, Octyl-glucoside, Tween®80, Triton X-100), guanidinium chloride or urea, so as to further weaken the outer membrane. However, as mentioned above, these molecules also risk further weakening the inner membrane, especially in the context of solutions that are difficult to homogenize. WO 2008 / 088757 proposed a continuous extraction of the periplasmic content, where, as in batch mode, the cells are equilibrated in a hypertonic solution. This suspension is then pumped and, using a T-system, water is added in large quantities via a second pump, so as to cause an osmotic shock, before centrifuging the mixture.The mixing of the two streams is done exclusively by static means, and the pH of the solutions is 7.2. A yield of approximately 70% is reported. However, the time the cells are in contact with water is not described, nor is the purity of the protein of interest.

[0010] Brief summary of the invention

[0011] The present invention relates firstly to a method for the automated extraction of a peptide of interest artificially secreted into the periplasmic space of a Gram-negative bacterium comprising the successive steps of:

[0012] - placing said bacteria in suspension in a hypertonic solution,

[0013] - pumping said suspension into a first mixing chamber in a hypertonic medium, at a constant and calibrated flow rate Q1;

[0014] - simultaneously pumping at a constant and calibrated flow rate Q2 a hypotonic solution into said mixing chamber, so that the mixture containing said cells, the hypertonic solution and the hypotonic solution remains at least 10 seconds in said mixing chamber, so as to ensure rupture of the external membrane of said bacteria by osmotic shock while preserving the integrity of the internal membrane of said bacteria;

[0015] - pumping said mixture to withdraw it from said mixing chamber to a pipe, at a constant and calibrated flow rate Q3, so that the volume in said first mixing chamber allows the desired residence time to be ensured in said first mixing chamber, said pipe having a length and a diameter so as to ensure a contact time of at least 10 seconds additional to the contact time in said first mixing chamber;

[0016] - collect the said mixture, then

[0017] - centrifuging said collected mixture so as to recover the clarified supernatant, comprising said peptide of interest.

[0018] Brief description of the drawings

[0019] Figure 1 shows the automated extraction process in its simplest version.

[0020] Figure 2 shows the automated extraction process including an acidification step via a T-connection.

[0021] Figure 3 shows the automated extraction process including an acidification step via a second mixing chamber.

[0022] Figure 4 shows two examples of purification of two different proteins expressed in the periplasmic space, each having a size of approximately 15 kDa. Detailed description of one embodiment of the invention

[0023] A first aspect of the present invention relates to a method for the automated extraction of a peptide of interest artificially secreted into the periplasmic space of a Gram-negative bacterium comprising the successive steps of: - placing the bacteria in suspension in a hypertonic solution, - pumping 2 into a first mixing chamber 1 the suspension in a hypertonic medium, at a constant and calibrated flow rate Q1; - simultaneously pumping at a constant and calibrated flow rate Q2 a hypotonic solution 3 into the mixing chamber 1, so that the mixture containing the cells, the hypertonic solution and the hypotonic solution remains at least 10 seconds in the mixing chamber 1, so as to ensure rupture of the external membrane of the bacteria by osmotic shock while preserving the integrity of the internal membrane of the bacteria;- pumping the mixture to withdraw it from the mixing chamber 1 to a pipe 4, at a constant and calibrated flow rate Q3, so that the volume of the mixture in the mixing chamber 1 allows the desired residence time to be ensured in the mixing chamber 1, the pipe 4 having a length and a diameter so as to ensure a contact time of at least 10 seconds additional to the contact time in the mixing chamber 1; - collecting the mixture 7, then - centrifuging the collected mixture so as to recover the clarified supernatant, comprising the peptide of interest. This makes it possible to treat large quantities of cells while ensuring the integrity of the internal membrane and ensuring a purity at least equivalent to that obtained by a batch process, even practiced on a much more modest scale.;

[0024] The residence time in the mixing chamber 1 is preferably between 10 seconds and 10 minutes, advantageously between 30 seconds and 5 minutes, for example between 1 and 3 minutes, such as approximately 2 minutes.

[0025] Similarly, the residence time in the pipe 4 is preferably between 10 seconds and 10 minutes, advantageously between 30 seconds and 5 minutes, for example between 1 and 3 minutes, such as approximately 2 minutes.

[0026] Preferably, this method further comprises the step of adding an acidic solution 5 to the solution comprising the cell suspension, the hypertonic solution and the hypotonic solution (before the centrifugation step), while retaining (substantially) the solubility of the peptide of interest.

[0027] Indeed, although the usual processes are commonly carried out at neutral or alkaline pH (in any case rarely at pH < 6.5, or even <6), which are compatible with those of the downstream chromatographic steps, which is considered advantageous, the inventors have noticed that acidification makes it possible to easily increase the level of purity of the peptide to be isolated, which is very advantageous.

[0028] This step is only advantageous for peptides remaining (substantially) soluble in an acidic medium. Thus, the method preferably comprises a preliminary step of determining the solubility of the peptide in an acidic medium. This makes it possible to (substantially) preserve the peptide of interest in solution while applying the most marked acidification possible, which allows the precipitation of as many contaminants as possible. In the context of the present invention, "(substantially) preserve the peptide of interest in solution" preferably means that at least 50% (by weight), at least 60% by weight, at least 65%, at least 70, 75, 80, 85, 90, or even 95, 96, 97, 98, 99 or substantially all of the peptide of interest is preserved (maintained) in solution. An advantageous way to perform the measurement is via acrylamide gel electrophoresis followed by specific staining compatible with quantification.Thus, preferably, the acid solution is added so as to obtain a final pH of between 3.0 and 6.0, preferably between 4.0 and 5.0, even more preferably between 4.2 and 4.8 (depending on the pH which is compatible with the solubility of the peptide of interest to be purified, a pH as acidic as possible being preferred). The pH, preferably, is continuously monitored by a probe, and is adjusted to the desired value by modulating the flow rate Q4.

[0029] According to an alternative (Figure 2), the step of acidifying the solution is carried out directly at the level of the pipe 4 receiving the flow Q3 (or a periplasmic extract obtained differently, or even a cell lysate, see below) via the continuous addition to said flow Q3 of a flow Q4 of an acid solution 5, preferably so as to obtain a final pH of between 3.0 and 6.0, preferably between 4.0 and 5.0, even more preferably between 4.2 and 4.8. Thus the flow Q4 is not necessarily constant, but can (slightly) vary; in other words, the fine control of the pH is, according to the invention, more important than a slight variation of the final volume 7.

[0030] According to the other alternative (Figure 3), the step of adding the acid solution 5 is carried out in a second mixing chamber 6, the second mixing chamber 6 receiving the flow rate Q3 and a flow rate Q4 of the acid solution 5, so as to produce a homogeneous mixture, this mixture being drawn from the second mixing chamber 6 at a constant flow rate, so as to ensure a volume allowing the desired residence time in the second mixing chamber 6.

[0031] As in the first alternative, preferably the acidic solution has a pH between 3.0 and 6.0, such as between 4.0 and 5.0, even more preferably between 4.2 and 4.8.

[0032] Preferably, the acidifying solution comprises (consists essentially of) an acid, preferably acetic acid, preferably at a concentration of between 0.2 M and 3.0 M, such as about 0.5 M. A concentrated solution is advantageous because it avoids excessively increasing volumes. However, the pH is more difficult to adjust in this case and the viscosity of the solution increases, complicating mixing. Other acids may be advantageously used, for example hydrochloric acid, citric acid or phosphoric acid.

[0033] Preferably, the solutions are added and withdrawn from the bottom of the first and / or second mixing chamber.

[0034] Advantageously, the flows Q1 and Q2 are mixed via the turbulence induced by the arrival of the flow Q1 and / or Q2 in the mixing chamber 1.

[0035] Preferably, the flows Q1 and Q2 are mixed via mechanical agitation 9 in the first mixing chamber 1, this mechanical agitation being calibrated so as not to cause the rupture of the internal membranes of the bacteria.

[0036] Alternatively, or in addition, the streams Q3 and Q4 are mixed via mechanical stirring 8 in the second mixing chamber 6, this mechanical stirring 8 being calibrated. When the second mixing chamber is not used, the streams Q3 (or a periplasmic extract obtained differently) and Q4 are mixed by the turbulence induced at the junction. This advantageously allows rapid acidification.

[0037] Preferably, and in particular in connection with the addition of an acidifying solution, the hypertonic solution and / or the hypotonic solution of the process are at a pH between 3.0 and 5.0, preferably between 3.5 and 4.0.

[0038] If the peptide to be purified is compatible with such acidification, the use of solutions causing acid osmotic shock will simplify the downstream acidification effort.

[0039] Furthermore, surprisingly, the inventors noticed that the internal membranes of the bacteria were better preserved in acidic, even highly acidic, conditions, while the external membranes remained just as sensitive to osmotic shock, regardless of the pH tested.

[0040] Advantageously, the pH of the hypertonic solution and / or the pH of the hypotonic solution is set by a buffer with a concentration between 10 and 100 mM, preferably between 15 and 50 mM, advantageously around 20 mM, for example a 20 mM acetic acid (acetate) buffer.

[0041] Thus, this buffer is relatively weak and the pH of the medium, after rupture of the external membrane of the bacteria, may be higher, but this may be corrected and the desired pH may advantageously be finely fixed during subsequent acidification, if it takes place. A related aspect of the present invention is a method for purifying peptides artificially secreted into the periplasmic space of Gram-negative bacteria comprising the successive steps of: - applying an osmotic shock to said bacteria, so as to specifically release the periplasmic space, - applying an acidification of said released periplasmic space, so as to specifically precipitate the bacterial contaminants, but not the peptide of interest and - obtaining the purified peptide by sedimentation and / or by centrifugation.

[0042] As mentioned above, this method advantageously comprises the preliminary step of determining the solubility of the peptide of interest in different acidic solutions and using a solution sufficiently acidic to precipitate the bacterial contaminants while maintaining the peptide of interest in solution. In practice, this method is also advantageous even if a minor proportion of the peptide of interest is no longer in solution due to the acidification, provided that a larger proportion of contaminating proteins is removed by this acidification. Thus, in the context of the present invention, "maintaining the peptide of interest in solution" preferably means that at least 50% (by weight), at least 60% by weight, at least 65%, at least 70, 75, 80, 85, 90, or even 95, 96, 97, 98, 99 or substantially all of the peptide of interest is maintained in solution.An advantageous way to perform the measurement is via acrylamide gel electrophoresis followed by specific staining compatible with quantification. Preferably, the pH of the acidification solution (or the solution after addition of the acidification solution) in this method is between 3.0 and 6.0, preferably between 4.0 and 5.0 (the pH is as acidic as possible while ensuring the solubility of the peptide of interest).

[0043] Preferably, the acid solution added in this method comprises an acid, preferably selected from acetic acid, citric acid, hydrochloric acid and phosphoric acid at a concentration preferably between 0.2 M and 3 M, preferably between 0.3 M and 1.5 M, even more preferably between 0.4 M and 1.0 M.

[0044] Preferably, in this method, the acid solution is added continuously and / or the homogenization of the solution comprising the periplasmic extract with the acid solution is rapid. For example, the mixing of the two solutions is done continuously via "T" or "Y" connections, which makes it possible to cause local turbulence, conducive to rapid homogenization.

[0045] Alternatively, the acid solution 5 can advantageously be added via a (the second) mixing chamber 6. This mixing chamber 6 receives the flow rate Q3 (which is, in this alternative, a periplasmic extract not necessarily obtained from the first mixing chamber 1, such as a periplasmic extract obtained by a batch process or via a continuous process) and a flow rate Q4 of the acid solution 5, so as to produce a homogeneous mixture, this mixture being withdrawn from the (second) mixing chamber 6 at a constant flow rate, so as to ensure a volume allowing the desired residence time in the (second) mixing chamber 6. Calibrated mechanical stirring can advantageously be applied so as to accelerate homogenization.The inventors noticed that using an acid, rather than a buffer mixture (the acid and a base, so as to better fix a precise pH), was advantageous because it limited variations in the conductivity of the composition after mixing.

[0046] In synergy with the above, preferably the hypertonic and / or hypotonic solutions used for the osmotic shock of this method are acidic, preferably at a pH between 3.0 and 5.0, more preferably between 3.5 and 4.0.

[0047] Advantageously, the pH of the hypertonic solution and / or the pH of the hypotonic solution is set by a buffer with a concentration between 10 and 100 mM, preferably between 15 and 50 mM, advantageously around 20 mM, for example a 20 mM acetic acid (acetate) buffer.

[0048] Alternatively, this acidification process is performed directly on a lysate of Gram-negative bacteria expressing a recombinant peptide (of interest), rather than on the periplasmic extract.

[0049] Both of the above methods, and the variant of the second method, have been successfully applied to CRM 197, as well as to other peptides remaining soluble under acidic conditions.

[0050] For example, the peptide of interest (or recombinant, or expressed in the periplasmic space) is advantageously chosen from an enzyme, an antibody fragment (preferably a "single domain antibody" and / or "immunoglobulin single variable domain"), a coagulation factor and an epitope and / or, advantageously, this peptide has a molecular weight of between 5 and 200 kDa, preferably between 10 and 50 kDa, preferably between 12 and 20 kDa. Other characteristics and advantages of the present invention will be drawn from the non-limiting description which follows, and with reference to the drawings and examples.

[0051] Examples.-

[0052] It is understood that the present invention is in no way limited to the embodiments described above and that many modifications may be made thereto without departing from the scope of the appended claims.

[0053] Comparative example:

[0054] The inventors used 2 x 30g of frozen Escherichia coli cell paste expressing a protein of interest (CRM197; SEQ ID NO:1) in the periplasmic space. The paste was thawed at room temperature and then resuspended in two flasks with each time 60 ml of a hypertonic buffer (20 mM sodium acetate, 5 mM EDTA; 20% (w:w) sucrose; pH 3.6) and gently mixed (manually) for approximately 10 minutes, until the aggregates disappeared.

[0055] In each vial containing the suspension, it was then diluted with 210 mL of a cold hypotonic buffer (5 mM MgCL2, 20 mM sodium acetate, pH 3.6) and gently mixed (manually) for 30 seconds. Then, in one of the conditions, the pH of the suspension was adjusted by the addition of an acetic acid solution, so as to obtain a final pH of 4.5. Both conditions were then centrifuged for 20 minutes at 8000 rpm at 4°C in an Avanti centrifuge.

[0056] The supernatant, which contains the periplasmic extract, is recovered and filtered at 0.2 μm. The inventors observed to their surprise that this brief osmotic shock time was sufficient and that acidification increased the purity of the protein of interest (from 50% to 65%) by inducing the precipitation of contaminating proteins from E. coli. The protein of interest did not precipitate at this pH.

[0057] Example 1

[0058] Automated periplasmic extraction. Another batch of Escherichia coli cell paste expressing a protein of interest (CRM197; SEQ ID NO:1) in the periplasmic space was used, this time for automated periplasmic extraction.

[0059] Thawed dough (450 g) at room temperature was incubated for 15 minutes in 900 ml of a hypertonic solution (20 mM sodium acetate, 5 mM EDTA; 20% (w:w) sucrose; pH 3.6) with gentle mechanical stirring.

[0060] The sample is then pumped into a mixing chamber at a fixed and constant flow rate Q1 and, at the same time, the cold hypotonic solution is added at another fixed and constant flow rate Q2, so that the entire hypotonic solution (3.1 L) is pumped during the same time as the hypertonic suspension comprising the cells. The hypotonic solution is MgCL25mM, sodium acetate 20 mM, pH 3.6. Then, when a volume allowing a desired contact time is reached in the mixing chamber, the mixture is pumped at a constant flow rate Q3 which is the sum of flows Q1 and Q2 (the volume in the mixing chamber therefore remains substantially constant, until the mixing chamber is emptied) and this mixture passes into a pipe of determined diameter and length, so as to ensure a residence time of 30 seconds.The piping leads to a second mixing chamber where an acetic acid solution (0.5M HAc; pH=4.5) is pumped at a suitable flow rate to ensure a pH of 4.5. The pH is monitored continuously. The residence time of the mixture in the acidification chamber is 30 seconds and, once the volume allowing this residence time is reached, a pump extracts the mixture containing the acidified sample at a flow rate Q5 (Q5=Q3+Q4). Then, the mixture is centrifuged and the supernatant, which contains the clarified periplasmic extract, is retained. A purity as good as in the manual process with acidification, 65%, was obtained. However, the present method allows the processing of significantly larger quantities, 15 times more in this example, while ensuring process control, which guarantees its reproducibility, even when very large quantities are processed.

[0061] The inventors also tested the same periplasmic extraction system, but without the addition of acetic acid after osmotic shock. The purity was significantly lower, less than 50%. However, again, this automated process allows for the processing of large quantities. The protein of interest can, if necessary, be purified via one or more chromatographies.

[0062] Example 2

[0063] Alternatively, the second mixing chamber is omitted, and the acidification solution (0.5 M HAc) is added directly to the end of the tubing containing the mixture of hypertonic and hypotonic solutions through which flow Q3 passes, so as to obtain a final pH of 4.5; this is done via a "T" fitting and the homogeneity of the mixture is ensured by adapting the diameter of the pipes, the contact time being ensured by further adapting the length of the pipework. Example 3 - automation and generalization

[0064] The inventors then applied the technology for the purification of two peptides fermented and then exported into the periplasmic space of E. coli. These two peptides are quite similar in size (15.5 and 12.6 kDa, Figures 4A and 4B), but were less well produced and / or exported into the periplasmic space than the protein in the examples above, which has a direct effect on purity.

[0065] Figure 4 (A): column 1, molecular weight markers; column 2: old batch (control); column 3: periplasmic extract according to the invention; Columns 4-7, periplasmic extract after acidification (successive dilutions 1x, 2x, 4x and 8x). Column 8-10: bovine serum albumin (BSA) reference; 1 μg; 0.5 μg and 0.25 μg).

[0066] Figure 4 (B): column 1, molecular weight markers; column 2: old batch (control); column 3: periplasmic extract according to the invention; Columns 4-6, periplasmic extract after acidification (successive dilutions 1x, 2x and 4x). Column 7-10: previously purified protein reference (4 μg; 2 μg; 1 μg and 0.5 μg).

[0067] The first peptide retains almost complete solubility under acidic conditions.

[0068] This peptide represents 6.9% of the periplasmic space (column 3). When acid precipitation was performed (column 4), this peptide now represents 18.4%.

[0069] The second peptide has reduced solubility under acidic conditions.

[0070] This peptide represents 15% of the periplasmic space. When acid precipitation has been performed, this peptide now represents 28% of the total, although approximately 35% of the peptide has precipitated and is therefore lost. However, since the method is simple to implement, and does not involve expensive components, the inventors consider that the acid precipitation step remains advantageous, even for this peptide.

Claims

DEMANDS 1. Automated extraction process for a peptide of interest artificially secreted into the periplasmic space of a Gram-negative bacterium comprising the successive steps of: - place the said bacteria in suspension in a hypertonic solution, - pump the said suspension into a first mixing chamber in a hypertonic medium, at a constant and calibrated flow rate Q1; - simultaneously pump a hypotonic solution into said mixing chamber at a constant and calibrated flow rate Q2, so that the mixture containing said cells, the hypertonic solution and the hypotonic solution remains for at least 10 seconds in said mixing chamber, so as to ensure rupture of the external membrane of said bacteria by osmotic shock while preserving the integrity of the internal membrane of said bacteria; - pump said mixture to draw it from said mixing chamber into a pipe, at a constant and calibrated flow rate Q3, so that the volume in said first mixing chamber allows the required residence time to be ensured in said first mixing chamber, said pipe having a length and diameter so as to ensure a contact time of at least 10 seconds additional to the contact time in said first mixing chamber; - collect said mixture, then - centrifuge said collected mixture so as to recover the clarified supernatant, including said peptide of interest.

2. The method according to claim 1 further comprising the step of adding an acidic solution to the solution comprising the cell suspension, the hypertonic solution and the hypotonic solution while maintaining the solubility of the peptide of interest, preferably in which the solubility of the peptide in acidic medium has been predetermined.

3. The process according to claim 2 wherein the step of acidifying the solution is carried out directly at the level of the piping receiving the flow Q3 via the continuous addition to said flow Q3 of a flow Q4 of an acidic solution, preferably so as to obtain a final pH between 3.0 and 6.0, preferably between 4.0 and 5.0, even more preferably between 4.2 and 4.

8.

4. The process according to claim 2 wherein the step of adding an acid solution is carried out in a second mixing chamber, said second mixing chamber (6) receiving the flow rate Q3 and a flow rate Q4 of said acid solution, so as to achieve a homogeneous mixture, said mixture being withdrawn from said second mixing chamber (6) at a constant flow rate, so as to ensure a volume allowing the required residence time in said second mixing chamber (6), preferably said acid solution having a pH between 3.0 and 6.0, such as between 4.0 and 5.0, more preferably between 4.2 and 4.

8.

5. The process according to any one of claims 2 to 4 wherein the acid solution is acetic acid, preferably at a concentration between 0.2 M and 3.0 M, such that about 0.5 M.

6. The method according to any one of the preceding claims in which the solutions are added and withdrawn from the bottom of the first (1) and / or the second (6) mixing chamber.

7. The method according to any one of the preceding claims 1 to 6 in which the flows Q1 and Q2 are mixed via the turbulence induced by the arrival of the flow Q1 and / or Q2 in the mixing chamber.

8. The method according to any one of the preceding claims 1 to 7 wherein the first (1) and / or the second (2) mixing chamber comprises a mechanical agitator (8, 9), said mechanical agitator (8, 9) being calibrated so as not to cause rupture of the internal membranes of the bacteria.

9. The process according to any one of the preceding claims wherein the hypertonic solution and / or the hypotonic solution are at a pH between 3.0 and 5.0, preferably between 3.5 and 4.

0.

10. The process according to claim 9, wherein the pH of the hypertonic solution and / or the pH of the hypotonic solution is fixed by a buffer of concentration between 10 and 100 mM, preferably between 15 and 50 mM, advantageously around 20 mM. 1 1. The process according to claim 9 or 10 wherein the hypertonic and / or hypotonic solution comprises a 20 mM acetic acid (acetate) buffer.

12. A process for purifying peptides artificially secreted into the periplasmic space of Gram-negative bacteria, comprising the successive steps of: - to perform an osmotic shock on said bacteria, so as to specifically release the periplasmic space, - apply acidification to said released periplasmic space, so as to specifically precipitate bacterial contaminants, but not the peptide of interest and - obtain the purified peptide by sedimentation and / or centrifugation.

13. A process for purifying recombinant peptides expressed in Gram-negative bacteria comprising the successive steps of: - to perform a lysis of said bacteria, - apply acidification of said bacterial lysate comprising said recombinant peptide, so as to specifically precipitate bacterial contaminants, but not said recombinant peptide and - obtain the purified peptide by sedimentation and / or centrifugation.

14. A method according to claim 12 or 13 comprising the preliminary step of determining the solubility of the peptide of interest in different acidic solutions and of using a solution sufficiently acidic to precipitate bacterial contaminants while keeping the peptide of interest in solution.

15. A method according to any one of claims 12 to 14 wherein the pH of the acidification solution is between 3.0 and 6.0, preferably between 4.0 and 5.

0.

16. A process according to any one of claims 12 to 15, wherein the acid solution comprises an acid, preferably selected from acetic acid, citric acid, hydrochloric acid and phosphoric acid, at a concentration preferably between 0.2 M and 3 M, preferably between 0.3 M and 1.5 M, even more preferably between 0.4 M and 1.0 M.

17. A method according to any one of claims 12 and 13 to 16 wherein the acid solution is added continuously to the periplasmic extract, wherein a stream comprising the periplasmic extract is continuously mixed with a stream comprising the acid solution.

18. A method according to any one of claims 12 and 13 to 16, wherein the acid solution (5) is added via a mixing chamber (6), preferably under calibrated mechanical stirring (8).

19. A method according to any one of the preceding claims 12 and 14 to 18, wherein the hypertonic and / or hypotonic solutions used for osmotic shock are acidic, preferably at a pH between 3.0 and 5.0, more preferably between 3.5 and 4.0, and / or preferably via an acid buffer with a concentration between 10 and 100 mM, preferably between 15 and 50 mM.

20. A method according to any one of the preceding claims wherein the peptide is selected from an enzyme, an antibody fragment, a coagulation factor, an epitope and CRM197 and / or wherein said peptide has a molecular weight between 5 and 200 kDa, preferably between 10 and 50 kDa, preferably between 12 and 20 kDa.

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