Process for purifying cationic protein fractions and the fraction thus obtained

Abstract

The present invention relates to a method for purifying cationic protein fractions by removing endotoxins; it also relates to the purified fractions thus obtained.

Description

Title of the invention: Process for purifying cationic protein fraction and fraction thus obtained

[0001] The present invention relates to a process for purifying cationic protein fractions by removing endotoxins; it also relates to the purified fractions thus obtained.

[0002] Endotoxins are components of the cell wall of Gram-negative bacteria. Released during the lysis or destruction of these bacteria, they are responsible for systemic inflammatory manifestations, such as septic shock, during infections by this type of bacteria. For this reason, an endotoxin content limit is defined for injectable drugs or products (such as water) by authorities such as the FDA (https: / / www.fda.gov / inspection-compliance-enforcement-and-criminal-investigation / inspection-technical-guides / bacterial-endotoxinspyrogens). Even outside of injectable uses, uses such as in the cosmetic field, medical devices, or even nutrition increasingly require reducing the endotoxin content of the ingredients used.

[0003] Endotoxins, also called lipopolysaccharides (LPS), consist of a lipid (lipid A) to which a glycan chain is attached. The glycan portion is composed of two parts, one part called the central oligosaccharide (core oligosaccharide) and the other part called the O-side chain polysaccharide (O-antigen); [Fig.l] illustrates their schematic representation (Maeshima & Fernandez, 2013). Each LPS molecule has multiple negative charges originating from the phosphate and acid groups of lipid A and the core oligosaccharide. Endotoxins are known to be thermally and chemically stable. The endotoxin content is expressed in IU (international unit) which is equivalent to one EU (endotoxin unit) (3.4 Test for bacterial endotoxins, The International Pharmacopoeia - 9th edition); As a guide, 1 ng of LPS corresponds to approximately 10 EU (WHO International Standard, 3rd IS for endotoxin), a value which may vary depending on the strains of bacteria..

[0004] In the context of the development of drugs based on proteins derived from biotechnological processes and using biological material, various methods of protein purification have been developed in order to eliminate endotoxins; these methods are, for example, solvent extraction, affinity chromatography (such as polymyxin B grafted resins), membrane techniques (such as ultrafiltration), ion exchange chromatography, hydrophobic interaction chromatography (Petsch, D., 2000. Endotoxin removal from protein solutions. Journal of Biotechnology 76, 97-119; Ongkudon, CM, Chew, JH, Liu, B., Danquah, MK, 2012. Chromatography Removal of Endotoxins: A Bioprocess Engineer's Perspective. ISRN Chromatography 2012, 1-9).

[0005] However, it is known that the removal of endotoxins from a cationic protein fraction is difficult to achieve because most cationic proteins such as lysozyme (Petsch, D., Deckwer, W.-D., Anspach, FB, 1998. Proteinase K Digestion of Proteins Improves Detection of Bacterial Endotoxins by the LimulusAmebocyte Lysate Assay: Application for Endotoxin Removal from Cationic Proteins. Analytical Biochemistry 259, 42-47), ribonuclease A and lactoferrin (Elass-Rochard, E., Roseanu, A., Legrand, D., Trif, M., Salmon, V., Motas, C., Montreuil, J., Spik, G., 1995. Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide. Biochem J 312, 839-845) have strong interactions with LPS molecules that possess several negative charges.

[0006] A method for removing endotoxins bound to a cationic protein, in particular lactoferrin, has been proposed in WO 2009 / 009706 (Glanbia Nutritionals); this method comprises the steps of a) binding the protein to a cation exchange resin; b) eluting the endotoxin using a low ionic strength solution in the absence of added surfactant; c) eluting the protein with a high ionic strength solution. This method makes it possible to obtain a lactoferrin isolate containing less than 1 IU / mg of endotoxins.

[0007] A similar method for removing lactoferrin-bound endotoxins has been proposed in WO 2010 / 112988 (Jean-Paul Perraudin); this method makes it possible to obtain a lactoferrin isolate containing less than 50 pg / mg (i.e. approximately 0.5 IU / mg) of endotoxin.

[0008] These two methods show that starting from a cationic protein such as lactoferrin having a high affinity with cation exchange resins, after having fixed this cationic protein on these resins, the endotoxins bound to this cationic protein can be dissociated and eliminated with a solution of low to medium ionic strength (0.25-0.5 M NaCl solution) without detaching this cationic protein from the cation exchange resins. This affinity with cation exchange resins is dependent on the positive charges (their magnitude and their location) originating from the constituent cationic amino acids (lysine, arginine and histidine) of the cationic proteins.However, these methods do not effectively remove endotoxins present in or bound to a cationic protein or a fraction of cationic proteins having low to medium affinity with cation exchange resins, since these proteins would then be eluted together with the endotoxins.

[0009] The present invention provides a method for effectively removing endotoxins present in a cationic protein fraction or bound to a cationic protein or a cationic protein fraction regardless of the magnitude and location of their positive charges.

[0010] The present invention thus relates to a method for purifying a fraction of cationic proteins comprising the following steps a) to d):

[0011] a) obtaining a solution:

[0012] - pH between 6.5 and 7.5;

[0013] - comprising cationic proteins all having a higher isoelectric point at 7.5 and acidic proteins having an isoelectric point lower than 6.5 in a content of less than 1% by weight relative to the weight of the total proteins;

[0014] - of conductivity greater than 45 mS / cm, preferably greater than 50 mS / cm or 60 mS / cm;

[0015] By way of illustration and without limitation, such a solution may be chosen from: • a solution containing lactoferrin from cow's milk eluted with 100 mS / cm of NaCl solution from cation exchange resins (e.g. SP Sepharose Big Beads, Cytiva Life Sciences); • a solution containing a total fraction of cationic proteins from cow's milk eluted with 10% NaCl from cation exchange resins (e.g. SPEC 70 SLS, Sartorius); • a solution containing a total fraction of cationic proteins from cow's milk (containing lactoperoxidase, ribonucleases, lactoferrin) with 40 mS / cm of NaCl solution from cation exchange resins (e.g. SP Sepharose Big Beads, Cytiva Life Sciences), supplemented with the saturated NaCl solution up to the conductivity of 60 mS / cm; • a concentrated solution of bovine lactoferrin by ultrafiltration from an eluate at 100 mS / cm; • a solution of microfiltered cationic whey protein isolate (protein / dry matter >90%), supplemented with saturated NaCl solution up to a conductivity of 60 mS / cm; • a powder of caprine lactoferrin reconstituted with a 5% saline solution; • egg white lysozyme powder reconstituted with 0.5 M NaCl solution.

[0016] b) diafiltration of said cationic protein solution with endotoxin-free water, preferably ultrafiltered reverse osmosis water, using an ultrafiltration membrane with a cut-off threshold of 5 to 50 kDa, the cut-off threshold value is chosen according to the molecular weight of cationic proteins in the solution, until a conductivity of less than or equal to 10 mS / cm is obtained, preferably less than or equal to 5 mS / cm, more preferably less than or equal to 1 mS / cm; during this diafiltration, which lowers the conductivity of the cationic protein solution from greater than 45 mS / cm to less than 10 mS / cm, this solution continuously passes through an anion exchange support, preferably membrane (e.g. Sartobind Q, Sartorus Stedium Biotech) or monolithic (e.g. CIMmultus QA, BIA Separations), the support being such that it has little steric exclusion effect, in order to adsorb and eliminate almost all of the endotoxins present in the solution;

[0017] c) optionally, microfiltration with a membrane with a cut-off threshold between 0.2 and 1.4 pm in order to reduce the microbial load;

[0018] d) optionally, spray drying or freeze drying of the solution to obtain a powdered cationic protein isolate.

[0019] [Fig. 2] illustrates a schematic representation of the method according to the invention. Figures 3 and 4 illustrate the examples of diagrams allowing the implementation of step b) of the method according to the invention.

[0020] Alternatively, the present invention relates to a method for purifying a cationic protein isolate comprising the following steps a) to d):

[0021] a) obtaining a solution:

[0022] - pH between 6.5 and 7.5;

[0023] - comprising cationic proteins all having a higher isoelectric point at 7.5 and acidic proteins having an isoelectric point lower than 6.5 in a content of less than 1% by weight relative to the weight of the total proteins;

[0024] - conductivity less than 1 mS / cm;

[0025] b) passing said solution through an anion exchange support, preferably membrane (e.g. Sartobind Q, Sartorus Stedium Biotech) or monolithic (e.g. CIMmultus QA, BIA Separtions), the support being such that it has little steric exclusion effect, in order to adsorb and eliminate almost all endotoxins present in the solution; the solution is preferably passed several times through an anion exchange support, preferably at least 3 times;

[0026] c) optionally, microfiltration with a membrane having a cut-off threshold between 0.2 and 1.4 pm in order to reduce the microbial load to an acceptable level for the conformity of the finished product;

[0027] d) optionally, spray drying or freeze drying of the solution to obtain a powdered cationic protein isolate.

[0028] [Fig.5] illustrates a schematic representation of a device allowing the implementation of the alternative method according to the invention.

[0029] The present invention also relates to a cationic protein fraction capable of being obtained or as it is obtained by the methods according to the invention and as it has an endotoxin content of less than 5 IU / mg of proteins, preferably less than 1 IU / mg of proteins, more preferably less than 0.1 IU / mg of proteins and most preferably less than 0.01 IU / mg of proteins.

[0030] According to one embodiment, the cationic proteins of the fraction are derived from milk; they can then comprise mainly lactoferrin or mainly lactoperoxidase or mainly ribonucleases or even contain TGF-[3 in a content greater than 20 pg / g, preferably greater than 50 pg / g, most preferably between 100 and 200 pg / g of proteins. By mainly constituted, we mean a fraction comprising at least 50%, but also greater than 90% or 95% by weight of proteins relative to the weight of the dry matter. The fractions according to the invention can also contain mainly a mixture of cationic proteins from milk or whey. Figures

[0031] [Fig.l]: schematic representation of an LPS (Maeshima & Fernandez 2013);

[0032] [Fig.2]: Schematic representation of the purification process of cationic protein isolate;

[0033] [Fig.3]: Examples of flowchart of the purification process of cationic protein isolate; process combining diafiltration and anion exchange support in parallel; and

[0034] [Fig.4]: process combining diafiltration and anion exchange support in series;

[0035] [Fig.5]: Schematic representation of the alternative process for purifying cationic protein isolate. EXAMPLES

[0036] Example 1: Benchtop test with a liquid concentrate of bovine lactoferrin

[0037] 1) A liquid concentrate of lactoferrin from cow's milk was obtained according to a industrial cation exchange chromatography process, SP Sepharose Big Beads (Cytiva Sweden) in a radial flow column (Albert Handtmann Armaturenfabrick GmbH) by passing pasteurized skimmed cow's milk, then successively eluting with a NaCl solution at 36 mS / cm and that at 110 mS / cm, finally the 2nd eluate was concentrated on an ultrafiltration of MWCO 20 kDa.

[0038] The protein concentration is 13 mg / mL, the purity of bovine lactoferrin on protein is 95%, the conductivity of this solution is 65 mS / cm, and the pH is 6.8 (“LF 1 Concentrate”).

[0039] 2) 75 mL of this liquid concentrate of bovine lactoferrin at 65 mS / cm was diafiltered at benchtop scale using ÀKTA flux s (Cytiva Sweden) with a Start AXH hollow fiber ultrafiltration module (MWCO of 10 kDa). Diafiltration was performed with ultrapure deionized water prepared by Milli-Q (Millipore) discontinuously up to 5 mS / cm; when the retentate volume reached 50% of the initial volume, ultrapure water was added to the initial volume, which was repeated 5 times. Thus, the deionized bovine lactoferrin concentrate was obtained (“LF 2 Concentrate”),

[0040] The same diafiltration procedure described above was carried out, but this time the concentrate (retentate) was passed in parallel through a Q-type (quaternary ammonium) anion exchange membrane cartridge, Sartobind® Q nano 3 mL (Sartorius Stedim) at a flow rate of 15 mL / min in recirculation for 80 minutes during a gradual decrease in conductivity from 65 to 5 mS / cm by this diafiltration. Thus, the bovine lactoferrin concentrate demineralized by diafiltration and treated by anion exchange membrane was obtained ("Concentrate LF 3").

[0041] 3) The concentration of endotoxins in each demineralized concentrate of Lactoferrin obtained by diafiltration was measured by the LAL (limulus amoebocyte lysate) test in Lonza kinetic chromogenics. In parallel, the bovine lactoferrin concentration in each concentrate was measured by the HPLC PI method (C18 300 Å column, 0.1% TFA / CH3CN gradient, detection at 280 nm). The results are expressed as IU / mg of bovine lactoferrin.

[0042] [Tables 1] LF concentrate 1 LF concentrate 2 LF concentrate 3 Endotoxins (Ul / mg LF) 55.8 50.9 0.084

[0043] Table 1 - Endotoxins present in bovine lactoferrin concentrates

[0044] Example 2: Benchtop test with a liquid isolate of bovine lactoferrin

[0045] 1) A liquid microfiltrate of lactoferrin from cow's milk was obtained according to an industrial cation exchange chromatography process, SP Sepharose Big Beads (Cytiva Sweden) in a radial flow column (Albert Handtmann Armaturenfabrick GmbH) by passing pasteurized skimmed cow's milk concentrated by reverse osmosis to 130 g / L of dry matter, then successively eluting with a 38 mS / cm NaCl solution and that at 10%, then the 2nd eluate was concentrated on an ultrafiltration of MWCO 20 kDa, then diafiltered on an ultrafiltration of MWCO 10 kDa with the osmosis water up to 1 mS / cm, finally the diafiltered retentate was microfiltered on a ceramic membrane at 0.8 pm in double layer (Membrarox®, Pall Corporation).

[0046] 2) This liquid isolate of bovine lactoferrin was diluted with demineralized water prepared by Milli-Q (Millipore) to have the protein concentration is 16 mg / mg (w / v) and the purity of bovine lactoferrin on protein is 95%, the conductivity of this solution is 0.15 mS / cm, and the pH is 6.9 (“LF 1 Isolate”). 75 mL of this liquid lactoferrin isolate was passed through a Sartobind® Q nano 3 mL cartridge (Sartorius Stedim) at a flow rate of 13 mL / min in recirculation for 90 minutes. (“LF 2 Isolate”)

[0047] 3) The concentration of endotoxins in each lactoferrin isolate was measured by the LAL (limulus amoebocyte lysate) test in Lonza kinetic chromogenics. In parallel, the bovine lactoferrin concentration in each concentrate was measured by the HPLC PI method (Cl8 300 Å column, 0.1% TFA / CH3CN gradient, detection at 280 nm). The results are expressed in IU / mg of bovine lactoferrin.

[0048] [Tables2] LF Isolate 1 LF Isolate 2 Endotoxins (Ul / mg LF) 62.7 <0.05

[0049] Table 2 - Endotoxins present in bovine lactoferrin isolates

[0050] Example 3: Benchtop test with a liquid isolate of bovine lactoferrin

[0051] 1) A liquid microfiltrate of lactoferrin from cow's milk was obtained according to an industrial cation exchange chromatography process, SP Sepharose Big Beads (Cytiva Sweden) in a radial flow column (Albert Handtmann Armaturenfabrick GmbH) by passing pasteurized skimmed cow's milk, then successively eluting with a NaCl solution at 36 mS / cm and that at 110 mS / cm, then the 2nd eluate was concentrated on an ultrafiltration of MWCO 20 kDa, then diafiltered on an ultrafiltration of MWCO 10 kDa with osmosis water up to 1 mS / cm, finally the diafiltered retentate was microfiltered on a ceramic membrane at 1.4 pm in double layer (Membrarox®, Pall Corporation).

[0052] The protein concentration is 147 mg / mL, the purity of bovine lactoferrin on protein is 95%, the conductivity of this solution is 0.8 mS / cm, and the pH is 6.8 (“LF 3 Isolate”).

[0053] 2) 70 mL of this liquid lactoferrin isolate was passed through a cartridge of Sartobind® Q nano 3 mL (Sartorius Stedim) at a flow rate of 6 mL / min (“Isolat LF 4”).

[0054] The recovered liquid lactoferrin isolate (65 mL) was passed through the Sartobind® Q nano 3 mL cartridge, and then again the recovered liquid lactoferrin isolate was passed through the Sartobind® Q nano 3 mL cartridge, in all 3 passes (“LF 5 Isolate”).

[0055] The recovered lactoferrin liquid isolate (60 mL) was passed through the Sartobind® Q nano 3 mL recirculating cartridge for 30 minutes at a flow rate of 6 mL / min in total 6 equivalent passes (“LF 6 Isolate”).

[0056] The recovered lactoferrin liquid isolate (55 mL) was passed through the Sartobind® Q nano 3 mL recirculating cartridge for 37 minutes at a flow rate of 6 mL / min in total 10 equivalent passes (“LF 7 Isolate”).

[0057] 3) The concentration of endotoxins in each lactoferrin isolate was measured by the LAL (limulus amoebocyte lysate) test in Lonza kinetic chromogenics. The results are expressed in IU / mg of protein.

[0058] [Tables3] Isolate LF 3 Isolate LF 4 Isolate LF 5 Isolate LF 6 Isolate LF 7 Endotoxins (Ul / mg proteins) 59.8 7.2 4.6 2.2 1.5

[0059] Table 3 - Endotoxins present in protein isolates

Claims

Claims

1. Process for purifying a fraction of cationic proteins comprising the following steps: a) obtaining a solution: - of pH between 6.5 and 7.5; - comprising cationic proteins all having an isoelectric point greater than 7.5 and acidic proteins having an isoelectric point less than 6.5 in a content of less than 1% by weight relative to the weight of the total proteins; b) continuous passage of said cationic solution with a conductivity greater than 45 mS / cm through an anion exchange support, preferably membrane or monolithic, by diafiltering said solution with endotoxin-free water, preferably ultrafiltered osmosis water, using an ultrafiltration membrane with a cut-off threshold of 5 to 50 kDa until a conductivity less than or equal to 10 mS / cm, preferably less than or equal to 5 mS / cm, more preferably less than or equal to 1 mS / cm is obtained;or passage of said solution whose conductivity is less than 1 mS / cm through an anion exchange support, preferably membrane or monolithic; the solution is preferably passed several times through an anion exchange support, preferably at least 3 times; c) optionally, microfiltration with a membrane with a cut-off threshold of between 0.2 and 1.4 pm; d) optionally, drying by atomization or by lyophilization of the solution, in order to obtain a fraction of cationic proteins.;

2. Cationic protein fraction obtained by the process according to claim 1, characterized in that it has an endotoxin content of less than 0.1 IU / mg of protein.

3. Cationic protein fraction according to claim 2, characterized in that the cationic proteins of the fraction are derived from milk.

4. Cationic protein fraction according to any one of claims 2 to 3, characterized in that the cationic proteins mainly comprise lactoferrin.

5. Cationic protein fraction according to any one of claims 2 to 3, characterized in that the cationic proteins mainly comprise lactoperoxidase.

6. Cationic protein fraction according to any one of claims 2 to 3, characterized in that the cationic proteins mainly comprise ribonuclease s.

7. Cationic protein fraction according to any one of claims 2 to 3, characterized in that the cationic proteins contain TGF-[3 at a content greater than 20 pg / g of protein.

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