Method for purifying cationic protein fractions and fractions obtained thereby

Abstract

This invention provides a method for purifying a cationic protein fraction and the resulting purified fraction. [Solution] A method for purifying a cationic protein fraction is provided, comprising the following steps: a) Having a pH of 6.5 to 7.5, It contains cationic proteins with an isoelectric point greater than -7.5 and acidic proteins with an isoelectric point less than 6.5 in amounts of less than 1% by weight relative to the total weight of the protein. Obtain a solution having a conductivity greater than -45 mS / cm. b) The cationic protein solution is dialyzed using an ultrafiltration membrane having a cutoff threshold of 5 to 50 kDa with endotoxin-free water, preferably ultrafiltration permeated water, until an conductivity of 10 mS / cm or less is obtained, and during this dialyzed filtration, the solution is continuously passed through an anion exchange medium, preferably a membrane or monolithic medium.

Description

Technical Field

[0001] The present invention relates to a method for purifying a cationic protein fraction by removing endotoxin, and also relates to the purified fraction obtained in this manner.

Background Art

[0002] Endotoxin is a component of the cell wall of Gram-negative bacteria. When released upon lysis or destruction of these bacteria, endotoxin causes systemic inflammatory conditions such as septic shock in infectious diseases caused by this type of bacteria. For this reason, regulatory authorities such as the FDA (https: / / www.fda.gov / inspections-compliance-enforcement-and-criminal-investigations / inspection-technical-guides / bacterial-endotoxinspyrogens) have set upper limits for endotoxin content in pharmaceuticals and injections (such as water). Furthermore, in fields other than injections, such as cosmetics, medical devices, and nutritional supplements, there is an increasing demand for reducing the endotoxin content of the ingredients used.

[0003] Endotoxins, also known as lipopolysaccharides (LPS), consist of lipids (lipid A) with sugar chains attached. The sugar chain portion is composed of two parts: a core oligosaccharide and an O-side-chain polysaccharide (O-antigen). A schematic diagram is shown in Figure 1 (Maeshima & Fernandez 2013). Each LPS molecule carries multiple negative charges from the phosphate and acid groups of lipid A, as well as from the core oligosaccharide. Endotoxins are known to be thermally and chemically stable. Endotoxin content is expressed in IU (International Units), equivalent to 1 EU (endotoxin unit) (3.4 Test for bacterial endotoxins, The International Pharmacopoeia - 9th edition). As a guideline, 1 ng of LPS corresponds to approximately 10 EU (WHO International Standard, 3rd IS for endotoxin), but this may vary depending on the origin of the bacterial strain.

[0004] In the development of protein-based pharmaceuticals derived from biotechnology techniques and the implementation of biological materials, various methods for purifying proteins have been developed to remove endotoxins. These methods include, for example, solvent extraction, affinity chromatography (such as polymyxin B graft resin, although this process is not permitted for food preparation), membrane technology (such as ultrafiltration), ion exchange chromatography, and 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. Chromatographic Removal of Endotoxins: A Bioprocess Engineer's Perspective. ISRN Chromatography 2012, 1-9).

[0005] However, lysozyme (Petsch, D., Deckwer, W.-D., Anspach, FB, 1998. Proteinase K Digestion of Proteins Improves Detection of Bacterial Endotoxins by the Limulus Amebocyte 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) Many cationic proteins, such as the region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide (Lactoferrin-Lipopolysaccharide Interaction: Involvement of the 28-34 loop region of human lactoferrin in high-affinity binding to Escherichia coli 055B5 lipopolysaccharide. Biochem J 312, 839-845), have strong interactions with several negatively charged LPS molecules, making it difficult to remove endotoxins from cationic protein fractions.

[0006] A method for removing endotoxins bound to cationic proteins, particularly lactoferrin, is proposed in WO2009 / 009706 (Glanbia Nutritionals); this method comprises the steps of a) binding the protein to a cation exchange resin; b) eluting the endotoxin with a low ionic strength solution without the addition of a surfactant; and c) eluting the protein with a high ionic strength solution. This method can yield lactoferrin isolates containing less than 1 IU / mg of endotoxin.

[0007] A similar method for removing endotoxin bound to lactoferrin is proposed in WO2010 / 112988 (Jean-Paul Perraudin). This method can yield lactoferrin isolates containing less than 50 pg / mg (i.e., approximately 0.5 IU / mg) of endotoxin.

[0008] These two methods demonstrate that, from fractions containing cationic proteins with high affinity to cation exchange resins, such as lactoferrin, endotoxins bound to these cationic proteins can be dissociated and removed using a low-to-medium ionic strength solution (0.25-0.5 M NaCl solution) without detaching the cationic proteins from the cation exchange resins. This affinity to the cation exchange resin depends on the positive charge (its magnitude and position) derived from the cationic amino acids (lysine, arginine, histidine) that constitute the cationic protein. However, these methods cannot effectively remove endotoxins present in or bound to cationic proteins or fractions of cationic proteins that have low-to-medium affinity to the cation exchange resins, because these proteins elute along with the endotoxins. [Overview of the Initiative]

[0009] The present invention provides a method that enables the efficient removal of endotoxins, cationic proteins, or proteins bound to cationic protein fractions, regardless of the magnitude or location of their positive charge. [Modes for carrying out the invention]

[0010] Accordingly, the present invention relates to a method for purifying a cationic protein fraction comprising the following steps a) to d): a) - Having a pH of 6.5 to 7, - Contains cationic proteins with an isoelectric point greater than 7.5 and acidic proteins with an isoelectric point less than 6.5 in amounts of less than 1% by weight relative to the total weight of protein. - Having an electrical conductivity greater than 45 mS / cm, preferably greater than 50 mS / cm or 60 mS / cm, Obtain a solution; Such solutions may be selected from the following, illustratively and without limitation: - A solution containing milk-derived lactoferrin eluted from a cation exchange resin (e.g., SP Sepharose Big Beads, Cytiva Life Sciences) with a 100 mS / cm NaCl solution; - A solution containing the entire fraction of milk-derived cationic proteins eluted from a cation exchange resin (e.g., SPEC70SLS, Sartorius) with 10% NaCl; - A solution containing the entire fraction of milk-derived cationic proteins (including lactoperoxidase, ribonuclease, and lactoferrin) eluted from a cation exchange resin (e.g., SP Sepharose Big Beads, Cytiva Life Sciences) with a 40 mS / cm NaCl solution, supplemented with a saturated NaCl solution to a conductivity of 60 mS / cm; - Concentrated bovine lactoferrin solution obtained by ultrafiltration from eluate at 100 mS / cm; - A solution of microfiltered whey cationic protein isolate (protein / dry matter >90%), supplemented with saturated NaCl solution to a conductivity of 60 mS / cm; - Powdered goat lactoferrin reconstituted with 5% physiological saline; - Lysozyme powder of egg white reconstituted with a 0.5 M NaCl solution. b) The cationic protein solution is dialyzed using an ultrafiltration membrane having a cutoff threshold of 5-50 kDa, with endotoxin-free water, preferably ultrafiltration permeable water, wherein the cutoff threshold is selected as a function of the molecular weight of the cationic protein in the solution, and is generally 5-50 kDa, but may be 20 kDa or less, or 1-20 kDa, or 5-20 kDa, or 20 kDa, 10 kDa or less, or 1-10 kDa, or 5- The conductivity may be 10 kDa, or 10 kDa, 5 kDa or less, or 1 to 5 kDa, or 5 kDa, and the diafiltration is carried out until a conductivity of 10 mS / cm or less, preferably 5 mS / cm or less, and more preferably 1 mS / cm or less is obtained, during which the conductivity of the cationic protein solution is reduced from more than 45 mS / cm to less than 10 mS / cm, and this solution is passed continuously through an anion exchange medium, preferably a membrane medium (e.g., Sartobind Q, Sartorus Stedium Biotech) or a monolithic medium (e.g., CIMmultus QA, BIA Separations), which has little steric exclusion effect in order to adsorb and remove substantially all endotoxins present in the solution; c) Optionally, microfiltration (microfiltration) is performed using a membrane with a cutoff threshold of 0.2–1.4 μm to reduce the microbial load; d) Optionally, the solution is spray-dried or freeze-dried to obtain a powdered cationic protein isolate.

[0011] Figure 2 is a schematic diagram of the method according to the present invention. Figures 3A and 3B show examples of diagrams that enable the implementation of step b) of the method according to the present invention.

[0012] Alternatively, the present invention relates to a method for purifying cationic protein isolates comprising the following steps a) to d): a) - Having a pH of 6.5 to 7.5 - Contains cationic proteins with an isoelectric point greater than 7.5 and acidic proteins with an isoelectric point less than 6.5 in amounts of less than 1% by weight relative to the total weight of protein. - Having an electrical conductivity of less than 1 mS / cm, Obtain a solution; According to a particular embodiment, this second method is applied to an isolate obtained by the previous method. b) The solution is passed through an anion exchange medium, preferably a membrane medium (e.g., Sartobind Q, Sartorus Stedium Biotech) or a monolithic medium (e.g., CIMmultus QA, BIA Separtions), the medium having little steric exclusion effect in order to substantially adsorb and remove all endotoxins present in the solution; the solution is preferably passed through the cation exchange medium several times, preferably at least three times; c) Optionally, microfiltration (microfiltration) is performed using a membrane with a cutoff threshold of 0.2–1.4 μm to reduce the microbial load to an acceptable level for the suitability of the finished product; d) Optionally, the solution is spray-dried or freeze-dried to obtain a powdered cationic protein isolate.

[0013] Figure 4 shows a diagram of the apparatus that enables the implementation of the alternative method according to the present invention.

[0014] The present invention also relates to cationic protein fractions that can or may be obtained by the methods according to the present invention, having an endotoxin content of less than 5 IU / mg protein, preferably less than 1 IU / mg protein, and more preferably less than 0.1 IU / mg protein.

[0015] According to one embodiment, the cationic proteins of the fraction are derived from milk, and then consist predominantly of lactoferrin, or consist predominantly of lactoperoxidase, or consist predominantly of ribonuclease, or may contain TGF-β in an amount of more than 20 μg / g, preferably more than 50 μg / g, most preferably 100 to 200 μg / g, of protein. The phrase "consist predominantly of" means a fraction that contains at least 50% by weight, further 90% by weight, or more than 95% by weight of said protein relative to the dry weight of the dry matter. The fraction according to the present invention may also predominantly comprise a mixture of cationic proteins from milk or whey. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] [Figure 1] Schematic diagram of LPS (Maeshima & Fernandez 2013). [Figure 2] Schematic diagram of a method for purifying a cationic protein isolate. [Figure 3] Example of a schematic diagram of a method for purifying a cationic protein isolate. A: a method in which diafiltration and an anion exchange medium are combined in parallel; B: a method in which diafiltration and an anion exchange medium are combined in series. [Figure 4] It is a schematic diagram of an alternative method for purifying a cationic protein isolate. EXAMPLES

[0017] Example 1: Bench test using liquid bovine lactoferrin concentrate 1) In a radial flow column (Albert Handtmann Armaturenfabrick GmbH), industrial cation exchange chromatography using SP Sepharose Big Beads (Cytiva Sweden) was performed: pasteurized skim milk was passed through the column, then eluted sequentially with a 36 mS / cm NaCl solution and the same solution at 110 mS / cm, and finally the second eluate was concentrated by 20 kDa MWCO ultrafiltration, to obtain a liquid concentrate of milk-derived lactoferrin.

[0018] The protein concentration is 13 mg / mL, the purity of bovine lactoferrin relative to the protein is 95%, the conductivity of this solution is 65 mS / cm, and the pH is 6.8 ("Concentrate LF1").

[0019] 2) 75 mL of this liquid bovine lactoferrin concentrate at 65 mS / cm was dialyzed on a bench scale using an AKTA flux s (Cytiva Sweden) equipped with a Start AXH ultrafiltration hollow fiber module (MWCO 10 kDa). Diafiltration was performed discontinuously with ultrapure desalinated water prepared in Milli-Q (Millipore) to 5 mS / cm, and when the volume of the retainate reached 50% of the initial volume, ultrapure water was added to the initial volume, and this was repeated 5 times. Thus, a desalted bovine lactoferrin concentrate was obtained ("Concentrate LF2").

[0020] The same dialysis filtration procedure as described above was performed, but while the conductivity gradually decreased from 65 to 5 mS / cm due to this dialysis filtration, the concentrate (holding solution) was then recirculated 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 for 80 minutes. In this way, a bovine lactoferrin concentrate that had been desalted by dialysis filtration and treated with an anion exchange membrane was obtained ("Concentrate LF3").

[0021] 3) The endotoxin concentration in each desalted lactoferrin concentrate obtained by diafiltration was measured using Lonza's kinetic chromogenic LAL (horseshoe crab slime cell lysate) assay. In parallel, the bovine lactoferrin concentration in each concentrate was measured by HPLC PI (column C18 300Å, 0.1% TFA / CH3CN gradient, detection at 280nm). The results are expressed as IU / mg bovine lactoferrin.

[0022] [Table 1]

[0023] Example 2: Bench test using liquid isolate of bovine lactoferrin 1) Using a radial flow column (Albert Handtmann Armaturenfabrick GmbH), industrial cation exchange chromatography was performed using SP Sepharose Big Beads (Cytiva Sweden). Pasteurized skimmed milk concentrated to a dry weight of 130 g / L was passed through a reverse osmosis membrane, and then continuously eluted at 10% with a 38 mS / cm NaCl solution. The two eluates were then concentrated by 20 kDa MWCO ultrafiltration, followed by dialysfiltration using permeable water to 1 mS / cm by 10 kDa MWCO ultrafiltration. Finally, the retained solution after dialysfiltration was microfiltered (microfiltered) using a 0.8 μm double-layer ceramic membrane (Membrarox®, Pall Corporation) to obtain a liquid microfiltrate of lactoferrin derived from milk.

[0024] 2) This liquid bovine lactoferrin isolate was diluted with ultra-high purity deionized water prepared in Milli-Q (Millipore) to a protein concentration of 16 mg / mg (w / v), a purity of bovine lactoferrin relative to the protein of 95%, a conductivity of 0.15 mS / cm, and a pH of 6.9 ("Isolate LF1"). 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 and recirculated for 90 minutes (hereinafter referred to as "Isolate LF2").

[0025] 3) The endotoxin concentration in each lactoferrin isolate was measured using Lonza's kinetic chromogenic LAL (horseshoe crab slime cell lysate) assay. In parallel, the bovine lactoferrin concentration in each concentrate was measured by HPLC PI (column C18 300 Å, 0.1% TFA / CH3CN gradient, detection at 280 nm). The results are expressed as bovine lactoferrin IU / mg.

[0026] [Table 2]

[0027] Example 3: Bench test using liquid isolate of bovine lactoferrin 1) Using an industrial cation exchange chromatography method with a radial flow column (Albert Handtmann Armaturenfabrick GmbH) and SP Sepharose Big Beads (Cytiva Sweden), pasteurized skim milk was passed through the column, followed by successive elution with a 36 mS / cm NaCl solution and a 110 mS / cm NaCl solution. The second eluate was then concentrated by 20 kDa MWCO ultrafiltration, followed by dialysfiltration with permeable water down to 1 mS / cm using a 10 kDa MWCO ultrafiltration. Finally, the retained solution obtained by dialysfiltration was microfiltration through a 1.4 μm double-layer ceramic membrane (Membrarox®, Pall Corporation) to obtain a liquid microfiltrate of lactoferrin derived from milk.

[0028] The protein concentration is 147 mg / mL, the purity of bovine lactoferrin relative to the protein is 95%, the conductivity of this solution is 0.8 mS / cm, and the pH is 6.8 ("Isolate LF3").

[0029] 2) 70 mL of this liquid lactoferrin isolate was passed through a Sartobind® Q nano 3 mL cartridge (Sartorius Stedim) at a flow rate of 6 mL / min ("Isolate LF4"). The recovered lactoferrin liquid isolate (65 mL) was passed through a Sartobind® Q nano 3 mL cartridge, and then passed through the Sartobind® Q nano 3 mL cartridge again for a total of three times ("Isolate LF5"). The recovered lactoferrin liquid isolate (60 mL) was recirculated in a Sartobind® Q nano 3 mL cartridge at a flow rate of 6 mL / min for 30 minutes, allowing for a total of six equal passes ("Isolate LF6"). The recovered lactoferrin liquid isolate (55 mL) was circulated through a Sartobind® Q nano 3 mL cartridge at a flow rate of 6 mL / min for 37 minutes, allowing for a total of 10 equal passes ("Isolate LF7").

[0030] 3) Endotoxin concentrations in each lactoferrin isolate were measured using Lonza's kinetic chromogenic LAL (horseshoe crab dysmorphic cell lysate) assay. Results are expressed as IU / mg protein.

[0031] [Table 3]

[0032] Example 4: Bench test using liquid isolate of TGF-β containing milk-derived cationic protein 1) A TGF-β-containing cationic protein fraction derived from milk was obtained according to the method described in Example 1 of Japanese Patent EP1912513. The TGF-β2 content, analyzed using an ELISA kit (Quntikine TGF-2, R&D Systems) in the microfiltrate obtained before spray drying, was 115 μg / g protein.

[0033] 2) This liquid cationic protein isolate containing TGF-β was diluted with ultra-high purity deionized water prepared with Milli-Q (Millipore) to a protein concentration of 2.6 mg / mg (w / v), a conductivity of 0.89 mS / cm, and a pH of 7.1 ("Milk Cationic Protein Isolate 1"). 50 mL of this liquid milk cationic protein isolate was recirculated for 70 minutes at a flow rate of 5 mL / min through a Sartobind® Q nano 3 mL cartridge (Sartorius Stedim) ("Milk Cationic Protein Isolate 2").

[0034] 3) The endotoxin concentration in each liquid milk cationic protein isolate was measured using Lonza's kinetic chromogenic LAL (horseshoe crab dysmorphic cell lysate) assay. The results are expressed as IU / mg of protein.

[0035] [Table 4]

Claims

1. A method for purifying cationic protein fractions, including the following steps: a) - Having a pH of 6.5 to 7.5, - Contains cationic proteins with an isoelectric point greater than 7.5 and acidic proteins with an isoelectric point less than 6.5 in amounts of less than 1% by weight relative to the total weight of protein. - Having an conductivity greater than 45 mS / cm, Obtain a solution; b) The cationic protein solution is dialyzed using an ultrafiltration membrane having a cutoff threshold of 5 to 50 kDa with endotoxin-free water, preferably ultrafiltration permeated water, until an conductivity of 10 mS / cm or less, preferably 5 mS / cm or less, and more preferably 1 mS / cm or less is obtained, and during this dialyz filtration, the solution is continuously passed through an anion exchange medium, preferably a membrane or monolithic medium; c) Optionally, perform microfiltration using a membrane with a cutoff threshold of 0.2 to 1.4 μm; d) Optionally, spray-dry or freeze-dry this solution.

2. A method for purifying cationic protein isolates, including the following steps: a) - Having a pH of 6.5 to 7.5, - Contains cationic proteins with an isoelectric point greater than 7.5 and acidic proteins with an isoelectric point less than 6.5 in amounts of less than 1% by weight relative to the total weight of protein. - Having an electrical conductivity of less than 1 mS / cm, Obtain a solution; b) The solution is passed through an anion exchange medium, preferably a membrane or monolithic medium; the solution is preferably passed through the anion exchange medium several times, preferably at least three times; c) Optionally, perform microfiltration using a membrane with a cutoff threshold of 0.2 to 1.4 μm; d) Optionally, the solution is spray-dried or freeze-dried to obtain a powdered cationic protein isolate.

3. A cationic protein fraction that can be obtained by the method of claim 1 or claim 2, characterized in that the endotoxin content is less than 0.1 IU / mg protein.

4. A cationic protein fraction obtained by the method of claim 1 or claim 2, characterized by having an endotoxin content of less than 5 IU / mg protein, preferably less than 1 IU / mg protein, and more preferably less than 0.1 IU / mg protein.

5. The cationic protein fraction according to claim 3 or claim 4, characterized in that the cationic protein in the fraction is derived from milk.

6. A cationic protein fraction according to any one of claims 3 to 5, characterized in that the cationic protein mainly consists of lactoferrin.

7. A cationic protein fraction according to any one of claims 3 to 5, characterized in that the cationic protein mainly consists of lactoperoxidase.

8. A cationic protein fraction according to any one of claims 3 to 5, characterized in that the cationic protein mainly consists of ribonuclease.

9. The cationic protein fraction according to any one of claims 3 to 5, characterized in that the cationic protein contains TGF-β in an amount greater than 20 μg / g protein.

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