Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6437—Coagulation factor VIIa (3.4.21.21)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
  • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
  • A23J1/20—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from milk, e.g. casein; from whey
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
  • A61D19/00—Instruments or methods for reproduction or fertilisation
  • A61D19/04—Instruments or methods for reproduction or fertilisation for embryo transplantation
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/32—Bonded phase chromatography
  • B01D15/325—Reversed phase
  • B01D15/327—Reversed phase with hydrophobic interaction
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01D—SEPARATION
  • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
  • B01D15/08—Selective adsorption, e.g. chromatography
  • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
  • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
  • B01D15/3847—Multimodal interactions
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/745—Blood coagulation or fibrinolysis factors

Definitions

• the present invention relates to a method for extracting one or more proteins, in particular globular proteins whose tertiary structure induces a hydrophobic pocket, present in milk, by means of a support to which a ligand having both a hydrophobic character and an ionic character.
• proteins are an essential part of molecules carrying biological information. This is particularly the case of a large number of hormones, growth factors, blood coagulation factors or antibodies.
• proteins are polymers based on amino acids, most often high molecular weight that can not be obtained at reasonable cost by chemical synthesis.
• Such therapeutically useful proteins are usually isolated and purified from, for example, living organisms, tissues or human or animal blood. This is particularly the case of insulin extracted from the pig pancreas, coagulation factors, such as factor VIII or factor IX, extracted from blood plasma, or immunoglobulins.
• coagulation factors such as factor VIII or factor IX
• the above protein preparation methods are widely used today, they have disadvantages.
• the low level of certain proteins extracted from platelets from the blood does not allow to isolate them in sufficient quantities to satisfy the ever increasing therapeutic needs.
• the presence of viruses, prions or other pathogens in the plasma requires additional steps of viral inactivation and / or viral elimination to be included in plasma protein production such products that can be used for therapeutic purposes.
• Bacterial systems for example E. CoIi, are widely used and effective. They allow the production of recombinant proteins at low cost. However, such systems are limited to the preparation of simple, non-glycosylated proteins that do not require elaborate folding procedures.
• Fungal systems are also used for the production of secreted proteins.
• the disadvantage of these fungal systems lies in the fact that they are at the source of post-translational modifications, consisting, for example, in a grafting of glycan units and sulfate groups, which strongly affect the pharmacokinetic properties of the proteins produced, in particular by the addition of various groups of mannose derivatives.
• Systems using baculoviruses can produce a variety of proteins, such as vaccine proteins or growth hormone, but their application on an industrial scale is not optimized.
• Mammalian cell culture is also used for the preparation of complex recombinant proteins, such as monoclonal antibodies.
• Cell expression systems lead to correctly folded and modified recombinant proteins.
• the low yield compared to the cost of production is a major drawback.
• transgenic plants to obtain proteins in large quantities.
• These systems generate post-translational modifications specific to plants, in particular by adding to the proteins produced highly immunogenic xylose residues, thus limiting their use for therapeutic purposes.
• transgenic animals for the production of recombinant vaccines or complex therapeutic proteins.
• the proteins thus obtained have a glycosylation close to that of the human being and are correctly folded.
• These complex proteins do not consist solely of a simple polypeptide chain, such as, for example, growth hormone, but they are modified in various ways after the assembly of the amino acids, in particular by specific cleavages, glycosylations and carboxymethylations. In the vast majority of cases, such modifications can not be made by bacterial or yeast cells.
• transgenic animals combining both the expression levels found in bacterial cell systems and the post-translational modifications obtained through cell cultures, while generating lower production costs than by implementing the systems of cell expression.
• milk has been the subject of work leading to consider it a source of very satisfactory secretion of recombinant proteins.
• the recombinant proteins, produced from milk of transgenic animals, can be easily obtained by grafting the gene coding for the protein of interest on the regulatory region of one of the milk protein synthesis genes that will direct it. specifically in the mammary gland, then its secretion into the milk.
• patent application EP 0 527 063 which describes the production of a protein of interest in the milk of a transgenic mammal, the expression of the gene coding for the protein of interest being controlled by a promoter of a whey protein.
• Other patent applications or patents describe the preparation of antibodies (EP 0 741 515), collagen (WO 96/03051), human factor IX (US 6 046 380) and factor VIII / von Willebrand factor complexes ( EP 0 807 170) in the milk of transgenic mammals.
• milk is a mixture of 90% water comprising various constituents that can be grouped into three categories.
• the first category called whey (or whey), consists of carbohydrates, soluble proteins, minerals and water-soluble vitamins.
• the second category called lipid phase (or cream), contains fat in the form of emulsion.
• the third category called the protein phase, consists of about 80% of caseins, which form a set of precipitable proteins at a pH of 4.6 or under the action of rennet, enzymatic coagulant, in the presence of calcium.
• the different caseins form a colloidal micellar complex, capable of reaching diameters of approximately 0.5 ⁇ m, with phosphocalcic salts, for example in the form of aggregates ("clusters") of tricalcium phosphate or Cag (PO 4 O-Such micelles are formed from casein subunits consisting of a hydrophilic ⁇ -casein-surrounding layer surrounding a hydrophobic core, the phosphocalcic salts being electrostatically bonded to the hydrophilic layer. These phosphocalcic salts can also be present in the internal volume of the micelle without being bound to the casein.
• This protein phase also contains soluble proteins, such as lactalbumin and lactoglobulins, as well as albumins and immunoglobulins from the blood.
• the recombinant protein secreted in the milk of transgenic animals may be present in the whey or in the protein phase, or both.
• the richness and complexity of each category of milk constituents makes more difficult to carry out an extraction of this protein, in particular that trapped in the micelles of caseins.
• Another difficulty lies in the fact that the majority presence of this protein in one of the two phases is not predictable with certainty.
• a recombinant protein may also have affinities for milk calcium ions present in the form of either salts and / or various soluble complexes or phosphocalcic salts of casein micelles. These affinities translate into electrostatic bonds between the protein and divalent calcium cations.
• the protein / calcium ion affinities make it possible to define the affinity constants which, depending on their value, determine the binding force.
• most of the proteins having an affinity for calcium ions are related to phosphocalcic salts of micelles. Its extraction requires the implementation of complex steps, which poses problems of implementation and performance.
• US Patent 4,519,945 describes a method of extracting a recombinant protein by preparing a precipitate of caseins and whey from milk, implementing acidification and heating steps, as mentioned above. This process generates a significant loss of the activity of the protein in question and a low extraction yield.
• US 6,984,772 discloses a method of purifying recombinant fibrinogen from milk of a transgenic mammal. This process comprises a step of separating the whey from the casein pellet and the protein phase by successive centrifugations. The whey is isolated and then stored for the rest of the process leading to a purified fibrinogen solution.
• Patent application WO 2004/076695 describes a method for filtering recombinant proteins from milk of transgenic animals.
• This method comprises a first milk clarification step, that is to say a step of removing the milk components so as to obtain a solution that can be filtered through a filter membrane whose pores have a diameter. 0.2 ⁇ m.
• a first milk clarification step that is to say a step of removing the milk components so as to obtain a solution that can be filtered through a filter membrane whose pores have a diameter. 0.2 ⁇ m.
• Such a step results in the elimination of casein micelles. Therefore, the implementation This step may be unacceptable, in terms of yield, if the casein micelles are likely to contain a protein of interest trapped within their structure.
• US Pat. No. 6,183,803 describes a process for isolating proteins naturally present in milk, such as lactalbumin, and recombinant proteins, for example human albumin or ⁇ 1-antitrypsin, from milk.
• This method comprises an initial step of contacting the milk comprising a protein of interest with a chelating agent. This results in the destructuring of casein micelles, which leads to a clarified milk serum comprising caseins, whey proteins and the protein of interest. The method then comprises a step of restructuring the casein micelles by adding to the liquid medium
• the coagulation proteins and especially those known to be synthesized under the influence of vitamin K, are in this category.
• the Applicant has set itself the objective of providing a method for extracting, from milk, milk proteins, natural or not, such as the factor VII (FVII), recombinant factor VIII and factor IX, in particular having an affinity for ionic forms of milk calcium, simplified implementation, further leading to a satisfactory production yield, while maintaining the biological activity of the protein.
• FVII factor VII
• recombinant factor VIII recombinant factor VIII and factor IX
• the Applicant has developed a method for extracting a protein present in milk, having at least one hydrophobic bag and a negative charge at the natural pH of the milk, comprising the following steps: a) Skimming and delipidation of said milk, b) Passage of the delipidated and skimmed fraction containing said protein on a chromatographic support on which is grafted a ligand having both a hydrophobic character and an ionic character, under pH conditions. allowing said protein
• the method of the invention is advantageous in that it is very easy to implement, since it comprises only a few steps on the one hand, and on the other hand that it does not necessarily require the setting in implementation of a step of clarifying the milk before implementing the first step of retaining the protein of interest on the support on which the ligand is grafted.
• the process of the invention can be applied to fresh milk or frozen milk.
• the milk can be milk. Any mammalian female containing a protein of interest, such as the cow, the ewe, the goat, the rabbit, the mouse, the rat, the sow, this list not being limiting.
• the fluidification step can be carried out by adding an aqueous solvent to the raw milk.
• the aqueous solvent is a solution based on phosphate salt with a concentration of less than 100 .mu.M, with a pH of between 7.5 and 8.5, preferably between 8.0 and 8.3, as a 30 mM sodium phosphate pH 8.0 solution, this list not being limiting.
• Such an aqueous solvent may also contain sodium chloride whose maximum concentration is about 40 mM.
• Such solutions maintain the stable micellar structure of the milk (casein micelles in suspension).
• “Skimming” means the separation of fat from milk to give two fractions: skimmed milk and cream. Skimming is a technique well known to those skilled in the art, and may be carried out by example by the use of a cream separator, organic solvents such as trichloroacetic acid, this list is not limiting. In a particular embodiment, skimming of the milk is carried out by filtration on glass fiber filters with positive Zeta potential. As an example of such a filter, mention may be made of Ultipor® GF-plus filters as well as Supradisk HP series filters or AKS active carbon series (PaIl Life science), GF filters (Whatman), Millistack + series filters DE (Millipore), Zetaplus VR or Delipid filters (Cuno 3M).
• the Ultipor® GF Plus filter is used at the threshold of 1 ⁇ m (PaIl) and the depth filter VR02 or VR04 (PaIl).
• This filtration step also makes it possible to delipidate the fraction, that is to say to remove the lipids, such as fatty acids, glycerides and sterols.
• This delipidation can be carried out by frontal filtration of the milk on the Ultipor® GF plus filter after having allowed the diluted milk to rest for 30 minutes (thus the fat floats on the surface, which allows an optimization of the cream / milk separation).
• This skimming and delipidation step is indispensable because the ligand grafted onto the support, which has both a hydrophobic character and an ionic character, adsorbs the lipids.
• the lipids must be removed before step b), otherwise the protein of interest can not, or little, be retained on the ligand. This would result in a loss of yield of the extraction process of the protein of interest.
• step b) the fraction resulting from step a), simply skimmed and delipidated, is directly adapted to the implementation of step b) which very advantageously represents a purification step by chromatography. 'affinity.
• Step b) should not be performed at a pH lower than the isoelectric point (pi) of caseins, which is between 4.6 and 5. Indeed, if step b) is implemented at a pH less than the pi of the caseins, these precipitate, which may create significant damage to the chromatographic medium.
• step b) is carried out at a pH of between 5 and 8.5.
• the skimmed and denatured milk resulting from step a) is therefore applied to the chromatographic support of step b) at a pH of between 5 and 8.5.
• this pH is between 5.5 and 8, or between 6 and 7.5.
• this pH is between 6.5 and 6.8.
• this pH is the natural pH of the milk.
• the proteins having natural sites of interaction for example proteins having an affinity antigens / antibodies, enzymes / substrates, enzymes / inhibitors, pseudoaffinity, have, by nature, a position relative charges and hydrophobic areas and they vary little between pH 5 and pH 8.5.
• the proteins are, at this pH, negatively charged.
• the interactions between the ligand and the protein are essentially hydrophobic interactions.
• the chromatography technique advantageously used in the method of the invention allows the retention of the delipidated and skimmed fraction containing the protein of interest on a support on which is grafted a ligand having both a hydrophobic character and an ionic character.
• a ligand having both a hydrophobic character and an ionic character.
• Such a ligand makes it possible to bind the proteins of interest which, by their structure, have a hydrophobic pocket, and to allow the impurities to pass, even the casein micelles.
• the hydrophobic internal zones of the proteins will be able to bind to this type of ligand which will allow interactions with the protein of interest, which guarantees a high affinity for the protein of interest, and an increased selectivity for the proteins to be purified.
• the non-retained proteins are essentially the major part of the caseins, Whey acid protein (WAP), transferrin, lactoglobumin, lactalbumin and serum albumin.
• WAP Whey acid protein
• transferrin lactoglobumin
• lactalbumin serum albumin
• this process is suitable for the extraction of any protein which has the indicated characteristics, ie at the natural pH of milk, in the range of about 6.5-6.8 the protein has hydrophobic regions and carries negative charges, at least the charge balance is very advantageously in favor of negative charges.
• the pH conditions allow the protein to be retained, or in general, to bind to the ligand of the chromatographic medium, either by interactions hydrophobic, either by electrostatic interaction or by hydrophobic interaction.
• Such conditions depend to a large extent on the isoelectric point of the protein to be purified and thus on the pH of the process. It is possible to choose a chromatographic support whose ligand will be positively charged (of the anion exchange type) and to adjust the pH so that the protein is globally negatively charged.
• a negatively charged ligand can be used when working at the pH advantageously used for the implementation of the invention, ie between 5 and 8.5.
• the functional terminal group of this ligand will for example be a sulphonyl or carboxyl group, and set the pH, especially at a value greater than 6, so that the protein has a positive overall charge.
• This embodiment is applicable when the isoelectric point of the protein to be purified is such that at basic pH, especially greater than 6, the protein is positively charged. It must also be ensured that the pH is compatible with the implementation of the process so as not to degrade in particular the proteins to be extracted and the milk.
• the pH of step b) is from 5 to 8.5, and is chosen such that the interactions between the protein and the ligand are essentially hydrophobic in nature.
• the chromatographic support is preferably equilibrated with a solution (equilibration buffer) based on phosphate salt with a concentration of less than 100 mM, with a pH of between 7.5 and 8. , 5, preferably between
• Such a solution may also contain sodium chloride whose maximum concentration is about 100 mM, preferably in the range of 20-60 inM.
• Such a solution may also be based on citrate salt, in particular trisodium citrate 0.20-0.30 M, preferably 0.25 M, pH 7.5-8.5, conductivity between 30 and 40 mS / cm, in particular 35 mS / cm.
• washing step just after step b) with, advantageously, a buffer identical to the equilibration buffer. It is ensured that this washing step is effective by measuring the optical density (OD) at a determined length, for example at 280 nm, which must be reduced to the value of zero or to the value of the baseline. The fraction thus obtained can be harvested according to the case.
• OD optical density
• the completion of the elution step c) may be carried out using any eluent known to those skilled in the art, allowing the protein to no longer be retained, for example by virtue of the effects of ionic repulsions but also of chaotropic effects.
• the elution is carried out thanks to the effects of ion repulsion.
• the structural form of the proteins and the charge of the proteins can be modified by varying the pH of the elution buffer or the chosen elution buffer.
• a mixture of urea in the case where the ligand grafted onto the chromatographic support is positively charged (of the anion exchange type), a mixture of urea, with a concentration of between 1.2 and 8M, and glycine, • concentration of between 25 and 50 mM, said concentrations being the final concentrations in 'the mixture.
• concentrations being the final concentrations in 'the mixture.
• eluents are aqueous solutions of acidic pH between pH 4 and 6, the aqueous mixtures comprising the components, preferably two or three, selected from the group consisting of sodium phosphate with a concentration of between 5 and 50 mM, preferably 30 mM, and ethylene glycol, sodium citrate, concentration of between 5 and 50 mM, preferably 30 mM, and ethylene glycol, sodium phosphate, of concentration between 5 and 50 mM, preferably 30 mM, and ethylene glycol, Tris / NaCl and a calcium salt of concentration between 1 and 10 mM, preferably 5 mM, and ethylene glycol, sodium caprylate, concentration between 10 and 100 mM, preferably 30 mM, and ethylene glycol.
• sodium phosphate with a concentration of between 5 and 50 mM, preferably 30 mM, and ethylene glycol, sodium citrate, concentration of between 5 and 50 mM, preferably 30 mM, and ethylene glycol, sodium
• aqueous mixture whose conductivity, linked to the presence of components, is less than 3 mS / cm, such as a 30 mM sodium phosphate solution.
• concentrations are the final concentrations in the mixture.
• ethylene glycol can be replaced by propylene glycol, which is less toxic, or any other solvent.
• urea in the presence of amino acids as eluent (final concentrations ranging from 1.2 to 8 M for urea and from 25 to 50 mM for glycine or any other amino acid), this solution permitting thanks to its chaotropic power to lift the ligand interaction for adsorbed proteins.
• the pH of these aqueous mixtures is very advantageously between 7 and 8.5, too much pH acids being more denaturing for the target protein and may lead to insoluble proteins.
• aqueous mixtures may further comprise a nonionic detergent, preferably Triton® X100, at levels of between 0.5 and
• water and preferably PPI water (bi-distilled water for injection).
• the elution can be carried out by reducing the pH to a value lower than the pKa of the ligand if the it is lower than the isoelectric point of the protein, or by lowering the pH to a value lower than the isoelectric point of the protein if it is lower than the pKa of the ligand.
• a purification step is still necessary in order to eliminate the contaminating proteins of the milk, such as lactoferrin, lactalbumin, transferin, albumin, immunoglobulins.
• Such purifying means are well known to those skilled in the art.
• affinity chromatography, hydrophobic chromatography, cation or anion exchange chromatography, exclusion / diffusion chromatography this list not being limiting.
• This step d) also advantageously allows a good renaturation of the target proteins, that is to say a correct folding, conferring to the protein a biological activity equivalent to that of the native protein.
• this renaturation can also be carried out by simple dialysis or diafiltration in order to eliminate the denaturing agent.
• the different chromatographic steps are carried out by any conventional chromatographic apparatus, comprising in particular a pumping device and a detection system, in particular by UV-visible absorption.
• the means for recovering the fraction containing the protein of interest are well known to those skilled in the art. As such, mention may be made of affinity chromatography, hydrophobic chromatography, cation or anion exchange chromatography, exclusion / diffusion chromatography, using conventionally used eluants.
• a milk clarification step is preferably carried out after the skimming and defatting step (step a) and before step b).
• clarification of the milk it is meant a step of removing the micelles, by destructuring them, which leads to a clarified milk serum comprising caseins, whey proteins and the protein of interest.
• This embodiment makes it possible to obtain better yields, since the proteins of interest associated with casein micelles are in this case capable of enriching the purified fraction, which is not the case in the embodiment without clarification step, in which the micelles are evacuated, resulting in the proteins of interest associated with them at the same time.
• This embodiment also makes it possible to perform a submicrobial filtration that allows the elimination of microbial or cellular agents and cellular debris present in the milk (bacteria, epithelial cells, milk lymphocytes.
• the milk clarification step is carried out by adding a chelating agent at a concentration such that, after mixing with the milk, the micellar structure of the milk disappears, resulting in clarified milk (casein in solution or whey) .
• a chelating agent such as trisodium citrate or EDTA.
• a final concentration of 0.25 M sodium citrate at pH 8.0 allows a perfect clarification of the milk.
• step a after the skimming and delipidation step (step a), and before step b), the casein subunit aggregates are eliminated, in particular by filtration or centrifugation. according to usual implementations.
• This step allows the destabilization, by the precipitation, of the colloidal state in which the milk is.
• the proteins of interest are released or dissociated from micelles or casein subunits, thereby recovering these micelle-associated proteins.
• the ligand having both a hydrophobic character and an ionic character is 4-Mercapto-Ethyl-Pyridine.
• An example of a support comprising this ligand is the HyperCel® MEP gel (Ciphergen®).
• conditions permitting adsorption of the protein of interest on such a support may be a pH of at least 0.5 pH unit above the isoelectric point of the protein (negative charge of the protein). and at least 1 pH unit below the pI of the ligand (positive gel charge).
• the pH chosen may be a pH of 8.
• the adsorption of the protein on the ligand is done under pH conditions such as the interaction between the protein and the protein.
• ligand is an essentially hydrophobic interaction.
• this pH is between 5 and 8.5.
• the Applicant has observed that the elution thereof can be carried out by lowering the pH to a pH lower than the pKa of the ligand, which is less than at 4.8.
• the elution step c) can be carried out by the aqueous solutions and mixtures defined above, at a pH higher than the pKa of this ligand, it is that is greater than 4.8, in particular at pH greater than 6 and less than 9 and very advantageously between 7.0 and 8.5, and also with water, and preferably water PPI (water bi-distilled for injection).
• the protein in this case is, for example, factor VII.
• the Applicant has found, surprisingly, that the MEP Hypercel® gel, having a 4-Mercapto-Ethyl-Pyridine type ligand or the Hitrap IgY gel, having a 2-Mercaptopyridine type ligand exhibits a selectivity towards the FVII whose structure is "in conformity" with the reference molecule, that is to say with the activatable molecule.
• the Applicant observed in the non-adsorbed forms of FVII on this gel the existence of a systematic gap between the activatable forms (amidolytic FVII assay) and the total forms (FVII antigen assay) of FVII.
• the amidolytic FVII assay is equivalent to an in vitro activation of the antigens, with 1 antigen unit of the plasma giving, by definition, 1 amidolytic unit, therefore an activity ratio equal to 1.
• the protein may undergo physicochemical or biochemical denaturations. This ratio is considered to be "normal” when it is between 0.8 and 1.2, and advantageously equal to 1.
• the lactic secretion of factor VII is not homogeneous at 100%. it is known to those skilled in the art to see differences in post-translational transformations, in particular glycosylation and folding of the three-dimensional structure of proteins.
• the mammary cells thus manufacture FVII after transgenesis, said FVII is glycosylated and folded before being secreted by the mammary gland into the milk. There is no guarantee that 100% of the molecules manufactured will be functional. It is likely that naturally occurring control mechanisms are altered in transgenic cells and particular differences in the processing of glycoforms in transgenic proteins compared to natural proteins are observed.
• MEP hypercel® having a 4-Mercapto-Ethyl-Pyridine ligand, or the Hitrap IgY gel, having a 2-mercaptopyridine type ligand, have a ratio of 0.4 to
• 0.5 and the eluted forms have a ratio of 1.0 to 1.4.
• the MEP Hypercel® gel selects by adsorption the antigenic forms closest to the natural forms, and it may be thought that the non-adsorbed forms, on the other hand, may have manufacturing defects by the transgenic animal.
• This is a a definite advantage because the goal is to extract human injectable FVII milk from humans without significant side effects, and the FVII must be as close as possible to natural forms.
• the ratio amidolytic activity / antigen is an indicative tool of this state.
• the ligand having both a hydrophobic character and an ionic character is Mercapto-Benzimidazole-Sulfonic acid.
• a support comprising this ligand is the HyperCel® MBI gel (Ciphergen®) or the Capto-MMC (GE Healthcare).
• this kind of support it is necessary to implement slightly more acidic retention conditions (step b)) between pH 5 and 6. At such pH, the solubility of the caseins is low and there may even be a beginning precipitation.
• salts such as 1M NaCl makes it possible to maintain the casein solubility between pH 5 and pH 6.
• the elution is carried out with the eluents, in particular the aqueous solutions and mixtures, indicated above.
• step d) is carried out by anion exchange chromatography, in particular by the use of an anion exchange medium of strong base type, that is to say with quaternary ammonium groups of -NR 3 + type, R being an alkyl group such as methyl or ethyl.
• an anion exchange medium of strong base type that is to say with quaternary ammonium groups of -NR 3 + type, R being an alkyl group such as methyl or ethyl.
• R being an alkyl group such as methyl or ethyl.
• the ionic strength being advantageously low, and the pH make the anion exchange step, which makes it possible to concentrate the molecule of interest, in particular FVII, to transform it into activated factor VII and to purify it, make the anion exchange step particularly suitable.
• a conformational elution change of form bound specifically to the calcium fixation which induces a charge change in the N-terminal part (gla-domains) which induces a charge change in the N-terminal part (gla-domains)
• the charge of the protein becomes negative after saturation with calcium).
• step d) preferably carried out by anion-exchange chromatography
• the elution of the protein is carried out using a solution of calcium ions with a concentration of between 1 and 50 mM, preferably between 2 and 25 mM, preferably between 3 and 12.5 mM or between 4 and 6 mM, the source of calcium ions being for example provided by calcium chloride.
• step d) the elution of the protein is carried out using a solution of 5mM calcium ions.
• the solution used for elution may be based on copper, zinc or manganese salts.
• the method of the invention is capable of being used for the extraction of a recombinant protein or naturally present in the milk of the mammal under consideration.
• the protein may be a protein naturally present in milk, and represents, for example, ⁇ -lactoglobulin, lactoferrin, ⁇ -lactalbumin, or proteoses peptones, or their mixture.
• the protein may also be a protein not naturally present in milk.
• the protein is a recombinant protein
• the milk containing it is a transgenic milk.
• proteins not naturally present in milk can be synthesized by nonhuman transgenic mammals, thanks to recombinant DNA techniques and transgenesis.
• transgenic animal is intended to mean any non-human animal having incorporated in its genome an exogenous DNA fragment, in particular coding for a protein of interest, this animal expressing the protein encoded by the exogenous DNA, and capable of transmitting the DNA exogenous to its descendant.
• any non-human mammal is suitable for the production of such a milk.
• the rabbit, the ewe, the goat, the cow, the sow and the mouse can be used, this list not being limiting.
• the secretion by the mammary glands of the protein of interest involves the control of the expression of the recombinant protein in a tissue-dependent manner.
• control methods are well known to those skilled in the art.
• the control of the expression is carried out by means of sequences allowing the expression of the protein towards a particular tissue of the animal. These sequences are in particular the promoter sequences, as well as the signal peptide sequences. Examples of promoters well known to those skilled in the art are the WAP (whey acidic protein) promoter, the casein promoter and the ⁇ -lactoglobulin promoter.
• WAP whey acidic protein
• a method for producing a recombinant protein in the milk of a transgenic animal may comprise the following steps: a synthetic DNA molecule comprising a gene coding for a protein of interest, this gene being under the control of a promoter of a protein secreted naturally in milk, is integrated into an embryo of a non-human mammal. The embryo is then placed in a female mammal of the same species which gives birth to a transgenic animal. Once this subject has developed sufficiently, the lactation of the mammal is induced, then the milk collected. The milk then contains the recombinant protein of interest.
• a plasmid containing the WAP promoter is manufactured by introducing a sequence comprising the promoter of the WAP gene, this plasmid being made in such a way as to be able to receive a foreign gene placed under the control of the WAP promoter.
• the gene coding for a protein of interest is integrated and placed under the control of the WAP promoter.
• the plasmid containing the promoter and the gene coding for the protein of interest are used to obtain transgenic animals, for example rabbits, by microinjection into the male pronucleus of rabbit embryos. The embryos are then transferred into the oviduct of hormonally prepared females. The presence of the transgenes is revealed by the Southern technique from The DNA extracted from the transgenic rabbits obtained. Concentrations in animal milk are evaluated using specific radioimmunoassays.
• the protein is a coagulation protein.
• the protein of the invention is chosen from factor II (FII), factor VII (FVII), factor IX (FIX) and the factor
• the protein of the invention is FVII or activated FVII (FVIIa).
• FVII or FVIIa can be produced according to the teaching of EP 0 527 063, the summary of which is given above.
• a DNA fragment whose sequence is that of human FVII is then placed under the control of the promoter
• the FVII of the invention is activated.
• FVIIa results, in vivo, from cleavage of the zymogen by different proteases (FIXa, FXa, FVIIa) into two chains joined by a disulfide bridge.
• FVIIa alone has very little enzymatic activity, but complexed with its cofactor, tissue factor (FT), it triggers the coagulation process by activating FX and FIX.
• FVIIa has a coagulant activity 25 to 100 times higher than that of FVII when interacting with tissue factor (FT).
• the protein is factor VII (factor VII).
• FVII can be activated in vitro by the factors Xa, VIIa, 11a, IXa and XIIa.
• the FVII of the invention may also be activated during its purification process.
• Another object of the invention is the use of a positive Zeta potential fiberglass filter for skimming and simultaneous delipidation of a mammalian milk.
• a positive Zeta potential fiberglass filter for skimming and simultaneous delipidation of a mammalian milk.
• Another object of the invention is the use of a support on which is grafted a ligand having both a hydrophobic character and an ionic character, for the extraction of a protein present in skimmed milk and delipidated.
• a pi plasmid is first prepared by introducing the Bam HI-Hind III sequence (6.3 Kb fragment) of the WAP gene (described in Devinoy et al., Nucleic Acids Research, Vol 16, No. 16, August 25
• the vector p1 is therefore a plasmid capable of receiving a foreign gene placed under the control of the WAP 6.3 kb promoter.
• the introduction of the foreign gene can be done, for example, in the SaI I site of the poly linker.
• the inserts containing the entire promoter and foreign genes can be isolated from the plasmid after cleavage at the two Not 1 sites which are at the ends of the poly linker of the plasmid p-poly III-I.
• Plasmid p2, obtained from plasmid p1 contains the rabbit WAP gene promoter (6.3 Kb) and the human FVIII gene. The fragment used to obtain the transgenic rabbits is between the two NotI sites.
• a Hind III site was introduced into the leader (leader) gene sequence by site-directed mutagenesis to serve as a cloning site.
• Transgenic rabbits were obtained by the conventional microinjection technique (Brinster et al., Proc Natl Acad Sci USA (1985) 82, 4438-4442).
• 1-2 ⁇ l containing 500 copies of the gene were injected into the male pronucleus of mouse embryos.
• the constructs were made in the p-poly vector IH-I (Lathe et al., Gene (1987) 57, 193-201).
• the Not 1 - Not 1 fragments of this vector containing the recombinant genes were microinjected.
• the embryos were then transferred to the oviduct of hormonally prepared adoptive females. About 10% of the embryos handled gave birth to rabbits and 2-5% of the embryos handled to transgenic rabbits. The presence of transgenes has been revealed by the Southern blot technique from DNA extracted from the tails of rabbits. FVII concentrations in the blood and milk of animals were evaluated using specific radioimunmunological tests.
• FVII The biological activity of FVII was evaluated by adding milk to the culture medium of rabbit mammary cells or exbrants.
• the raw material is raw rabbit milk that is unskinned and frozen, containing about 150 grams per liter of protein and as much fat (15% cream), it is first of all necessary to "thin and degrease" the medium to make it suitable for chromatographic conditions.
• a volume of thawed raw milk is mixed with at least 2 or at most 9 volumes of aqueous solvent
• Protocol A This gives a perfectly fluid raw material and sufficiently delipidated for chromatographic techniques. Protocol A:
• the aqueous solvent is a solution of low ionic phosphate (less than 100 mM), with or without the addition of sodium chloride.
• the solvent contains a chelating agent such as trisodium citrate or EDTA at a concentration such that after mixing with the milk, the micellar structure of the milk disappears resulting in "clarified" milk (casein in solution or whey).
• a chelating agent such as trisodium citrate or EDTA at a concentration such that after mixing with the milk, the micellar structure of the milk disappears resulting in "clarified" milk (casein in solution or whey).
• a final concentration of 0.25M sodium citrate at pH 8.0 allows a perfect clarification of the milk.
• the gel Before injection of the biological material, the gel is equilibrated by passing a solution of 25 mM sodium phosphate containing 40 ⁇ M sodium chloride at pH 8.2 and having a conductivity of 8 mS / cm at 25 ° C. ( balancing buffer). The flow rate of the pump is adjusted to 1.5 ml / min, ie a contact time through the gel estimated at 1 minutes I / O (input / output). The column is connected to a UV lamp detector at 280 nm and the optical density signal is continuously recorded on paper. 40 ml of biological material is injected, a "departure MEP" sample was set aside for analysis.
• Solution B giving 17 ml of eluate 2-MEP -> 50% equilibration buffer + 50% ethylene glycol.
• Solution C giving 9 ml untested - Balancing buffer adjusted to pH 3 with glacial acetic acid.
• the gel is then regenerated by passage of sodium hydroxide (NaOH) 1M, then is kept in an IM sodium chloride medium supplemented with ethanol qs 20% (v / v).
• NaOH sodium hydroxide
• IM sodium chloride medium supplemented with ethanol qs 20% (v / v).
• the volume ratio of milk / volume of MEP gel increases from 3 to 20 - fractionation per 100 ml.
• nonionic detergent Triton X100
• a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH a basic pH. It is also planned to replace ethylene glycol (CH 2 OH-CH 2 OH) with less toxic propylene glycol (CH 2 OH-CH 2 -CH 2 OH) and also a test with urea (NH 2 -CO- NH 2 ) denaturing and renaturing agent at 6M.
• Triton X100 Triton X100
• FVIIrAg Antigenic assay of FVII in an ELISA system (specific antibody detection).
• 1 IU / ml of FVII is equivalent to 0.5 ⁇ g / ml of pure FVII protein.
• FVII proenzyme
• FVIIa enzyme which activates FX in FXa which causes plasma coagulation (thrombin generation which acts on fibrinogen).
• the FVII: am / FVII: Ag ratio (expressed in%) reflects the functional state of the FVII molecule during purification
• FVII is divided into 2 forms on Q-Sepharose FF ion exchangers, an elution of almost pure FVII in 5 mM Calcium (di-te "Ca 2+ 5mM fraction") and an elution of low purity FVII in Calcium 50 mM (called “Ca 2+ fraction 50mM”).
• 1 volume of gel QSFF 10 ml Treatment of the MEP eluate ( 1st pass after treatment with protocol A)
• the FVII resulting from the sequence MEP + QSFF gives a very purified FVII whose activation is carried out at about 50%. However, this activation continues naturally and slowly in the medium, at room temperature.

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