Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/02—Bacterial antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
  • B01J13/0004—Preparation of sols
  • B01J13/0039—Post treatment
• • 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
  • C07K1/30—Extraction; Separation; Purification by precipitation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
  • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
• • 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
  • C07K14/75—Fibrinogen
• • 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
  • C07K14/76—Albumins
  • C07K14/765—Serum albumin, e.g. HSA
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
  • C07K16/065—Purification, fragmentation
• • 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
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/814—Enzyme separation or purification
  • Y10S435/816—Enzyme separation or purification by solubility
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/82—Proteins from microorganisms
  • Y10S530/825—Bacteria

Definitions

• the present invention relates to the fractionation of mixtures of proteinaceous substances such as plasma proteins, toxins, antitoxins, enzymes, desoxyribonuclease and viruses.
• Liquid-liquid partitions require large volumes of phases to ensure suflicient dilution for complete solubility. The separation must then be followed by the additional step of recovering the fractions from the liquids.
• the present invention is based on the discovery of outstanding advantages attainable when polyethylene glycol is used to depress the dispersibility of proteinaceous substances of a mixture in the presence of a single liquid, more particularly aqueous, phase.
• Some of the most important advantages are the following (in addition to the advantages discussed further above in the context of the alcohol fractionation method)
• the precipitation (or its reversal) is substantially independent of the concentration of the particular protein and is substantially only dependent on the concentration of the polyethylene glycol.
• the amount of substrate precipitated is not stoichiometrically dependent on the amount of precipitant.
• the process allows the easy isolation of such substances as fibrinogen, which cannot be isolated satisfactorily by any liquid-liquid partition method.
• the PEG has a molecular weight of at least 600 with an upper limit of 20,000.
• the best results were attainable with PEG having a molecular weight in the range 1,500 to 20,000, say
• polyethylene glycol acts as a stabiliser of protein solutions, a factor which is of particular importance in dealing with dilute solutions of protein, e.g., purified bacterial toxins which tend to denature on standing for prolonged periods.
• the process may be carried out by fractional precipitation, for example at least two fractions are precipitated successively and selectively by increasing the concentration of the precipitant in the solution.
• the reverse procedure comprises the steps of fractionally extracting a solid mixture of the two or more proteinaceous compounds with an aqueous dispersion of PEG capable of selectively dispersing one of the said compounds while leaving the other compound at least partly undispersed.
• This may also be done by precipitating a mixture of at least two substances to be separated from one another and selectively eluting precipitated substance from the precipitate with at least one dispersion of PEG having a lower PEG concentration than the PEG concentration necessary to achieve complete precipitation.
• Consecutive precipitations may be carried out under different conditions, e.g., with regard to the molecular weight of the PEG or by varying other factors as will be explained further below. It is also possible to combine the process with other fractionating processes, e.g., electrodecantation and alcohol precipitation, particularly in cases where any one method alone produces inadequate separations.
• a protein fraction is more readily precipitated from solution when the pH of the solution is close to the isoelectric point of the protein components to be removed.
• protein fractions are more easily precipitated when the ionic concentration of the medium in which they are dispersed is lowered.
• the temperature may be varied to increase the separation between two protein fractions. The above-mentioned variations of pH, ionic strength and temperature have to some extent been found to have the same effect when applied to the present process.
• Polyethylene glycol used in the process can be removed by extraction with a solvent, e.g., chloroform, provided such solvent does not denature the particular precipitate.
• a solvent e.g., chloroform
• Eleetrodecantation may also be employed to remove small amounts of PEG from protein fractions. In other cases the removal of the PEG is unnecessary, in some cases the presence of the precipitant may even be desirable because of its stabilizing effect.
• Other possible methods of removing PEG from contaminated fractions involve the use of anion and cation exchangers, acetone or sometimes ammonium sulphate, alternatively, the protein or like fraction precipitated can be redispersed and then reprecipitated by salting-out procedures and with ethanol. However, it must be stressed once again for some applications the retention of minor amounts of PEG in the final product is not objectionable.
• the interaction between the precipitant and the solution is brought about at a temperature essentially above 0 C., advantageously at a temperature at least as high as ordinary room temperature.
• the process differs in an important and very advantageous manner from the cold alcohol fractionation process. Another important difference is to be found in the feature that the best precipitates are obtained if the predetermined ration of PEG and solution of substance to be precipitated is brought into admixture within a period of minutes. For example, to obtain the best results the precipitant is admixed at a rate of at least 4 kg. per minute per 50 litres of solution. The precipitant may also be added to the solution cocurrently.
• the cold alcohol precipitation method requires the alcohol concentration to be raised to the required level over a much longer period. This difference is due to the fact that in the present process the interaction between PEG and substance being precipitated is usually completely reversible.
• the precipitates formed by the process are on the whole particularly easy to handle, particularly if the solution after the admixture of the precipitating agent is maintained under conditions of gentle agitation before separating the precipitate from the solutions, say for a period of half an hour.
• Some embodiments of the process applied to the fractionation of a mixture of compounds comprising the said protein type structure comprise carrying out the fractionation with at least two different precipitating agents in combination, of which at least one is an inorganic precipitant, capable as such of precipitating protein type substances from aqueous solution.
• said inorganic precipitant is ammonium sulphate.
• a particular application of the process to the separation of gamma-globulin from blood plasma comprises precipitating an impure gamma-globulin from plasma by the addition thereto of ammonium sulphate, dissolving the precipitate in an aqueous medium and mixing the solution with polyethylene glycol to recover a more purified precipitate of gamma-globulin.
• the last-mentioned precipitate is preferably redissolved in an aqueous medium, contaminants of the gamma-globulin are precipitated by mixing the solution with a predetermined amount of polyethylene glycol, the precipitate is separated from the remaining solution and the concentration of polyethylene glycol in the solution is incretased to precipitate gammaglobulin.
• the removal of impurities at the lower concentration of polyethylene glycol takes place essentially at a pH of 4.4-4.8 whereas the last-mentioned precipitation of the gamma-globulin takes place at a pH between 6 and neutral.
• the final precipitate of gamma-globulin resulting from precipitation with polyethylene glycol may be freed of polyethylene glycol contamination by redissolving the precipitate and precipitating the gammaglobulin from the solution with a different precipitant, e.g. cold ethanol in a manner known per se.
• Another embodiment is applied to the separation of both fibrinogen and gamma-globulin and comprises mixing the plasma with ammonium sulphate up to a concentration at which most of the fibrinogen present precipitates, leaving the bulk of the gamma-globulin and some fibrinogen in solution, separating the precipitate from the solution, increasing the concentration of ammonium sulphate to precipitate at least the bulk of the gamma globulin and redissolving both precipitates separately, mixing the fibrinogemenriched solution with polyethylene glycol to precipitate an essentially pure fibrinogen fraction, while maintaining essentially neutral conditions and mix ingthe gamma-globulin-enriched solution with polyethylene glycol to precipitate therefrom gamma-globulin in a more highly purified form than before.
• the lastmentioned precipitate of gamma-globulin is preferably redissolved in an aqueous medium and further processed which was suitable for the in the manner described in the previous embodiment for the purification of gamma-
• Example 1 Fractionation of human blood plasma Human plasma was diluted with water in a ratio of 1:1 to result in a solution having a protein concentration of 3.8 gms. per ml. water. 5 ml. portions of this solution were added to 5 ml. portions of PEG (molecular weight 6,000) of increasing concentration dissolved in 4 M phosphate buffer, pH 7.0. The mixtures were left in a water bath at 21 C. for 30 minutes and then spun down at this temperature at 14,000 rpm. for 15 minutes. Protein determinations were made of the supernatant fluids and these were plotted against the PEG concentrations.
• PEG molecular weight 6,000
• the protein fraction which was precipitated at 3% was shown to be a fibrinogen concentrate as it clotted on addition of thrombin.
• the gamma-globulin fraction was recovered and between 21% and 26% PEG concentration the albumin fraction, contaminated with a trace of beta-globulin, was separated.
• Example 2 Purification of Clostridium novyi toxin 0.925 gm. of dried toxin was dissolved in 100 ml. of M phosphate buffer pH 7.0. Fractionation with PEG (molecular weight 6,000) in phosphate buffer was carried out exactly as with plasma at 21 C. and the fractions were spun down at 12,000 rpm. for 15 minutes. The bulk of the toxin was recovered at PEG concentrations between 7% and 9%.
• the gamma component was precipitated at 7% PEG concentration.
• the supernatant fluid after removal of the gamma component at 7% concentration was raised to 12% PEG concentration when the T component with approximately of gamma-globulin was recovered. This component contained 90% of the total activity of the original serum.
• Example 5 Fractionation of pancreatic desoxyribonuelease Frozen bovine pancrease derived from vitender longhorn cattle is minced in a meat grinder and extracted for 24 hours with 0.25 N H 80 in a cold room. The extract is clarified and ammonium sulphate added to a concentration of and the precipitate discarded. The supernatant fluid is raised to concentration and the precipitate formed is redissolved in water and ammonium sulphate added to a concentration of 30%. 2 gms. of this precipitate are dialysed salt-free against very dilute H 80 pH 3.0, and diluted to ml. with H O.
• Example 7 Preparation of homogeneous gammaglobulin from blood plasma In the following preparation it is only desired to prepare gamma-globulin, and all other components of the original mixture (blood plasma) are to be discarded.
• the precipitate is then redissolved in phosphate buffer (0.02 M, pH 7.0) to result in a solution having a 3% protein concentration (9,000 cc.).
• This solution is now fractionated at 18 C. by rapidly pouring into the solution polyethylene glycol (molecular weight 6,000) up to a total concentration of 14% PEG. Pouring rapidly in this context means as rapidly as is practical without entraining appreciable amounts of air bubbles. It is found that the total addition can be achieved within one minute. The mixture is left standing for approximately half an hour with gentle stirring (the optimum time may vary from batch to batch).
• the precipitate is separated by centrifugation and contains gamma-globulin in admixture with fibrinogen and traces of alpha-Z-globulin and beta-globulin.
• the solution which contains ammonium sulphate, some albumin and alpha-globulin is discarded.
• the precipitate is redissolved in acetate buffer (pH 4.6, 0.02 M) to result in a protein concentration of 2.0% (9,000 cc.).
• This solution is fracionated at room temperature by the addition of polyethylene glycol in the same manner as above, except that the polyethylene glycol concentration is brought to 12% only and that the polyethylene glycol used is buffered with acetate buffer (pH 4.6, 0.02 M).
• acetate buffer pH 4.6, 0.02 M
• the pH of the solution is adjusted, if necessary, with glacial acetic acid to pH 4.6. This time the precipitate, after having been removed by centrifugation is discarded.
• To the residual solution more polyethylene glycol (molecular weight 6,000) is added to raise the PEG content to 14%.
• the pH is also raised from 4.6 to between 6.3 and 6.5. This results in the precipitation of homogeneous gamma-globulin, which is separated by centrifugation.
• the precipitate is dissolved in phosphate buffer (pH 7.0, 0.02 M) to result in a protein concentration of 3% (3,500 cc.), which solution is clarified by filtration. From this clarified solution the gamma-globulin may then be precipitated in a conventional manner at -8 C. with 25% ethanol. The precipitate is removed by centrifugation and vacuum dried. The dried gamma-globulin may then be dissolved in an 0.8% saline solution (using pyrogen-free Water) to result in a solution containing 16% of gamma-globulin. This solution is clarified, filtered and filled under sterile conditions into glass vials. A yield of 60% was obtainable.
• the purity of the gamma-globulin prepared by the above-described method was compared by free electrophoresis with a sample of commercial gammaglobulin prepared by cold ethanol fractionation. The same substances were also compared by ultracentrifugation.
• the gamma-globulin product prepared in accordance with the invention showed a single component only in both tests, Whereas the commercial gamma-globulin was found to be contaminated with approximately 3% of a substance having properties corresponding to those of normal human albumin and only contained 55% of substance having the actual properties of human gammaglobulin.
• mice were injected intraperitoneally with 0.5 ml. amounts of 16% gamma-globulin prepared by the abovedescribed method. No side effects could be observed.
• Example 8 Preparation of pure fibrinogen and pure gamma-globulin from blood plasma 11 litres of human blood plasma (diluted with aqueous citric acid to a protein concentration of approximately 7%) are subjected to a preliminary fractionation at 10 C. by the addition of ammonium sulphate only to result in a concentration of 1.4 M ammonium sulphate. This results in a crude fibrinogen precipitate which is removed from the solution. The ammonium sulphate content of the remaining liquid is raised to 1.9 M, 330 gms. of sodium chloride are added and the temperature is raised to 18 C.
• the crude gamma-globulin precipitate is re-dissolved in phosphate bulfer (pH 7.0, 0.02 M) to result in a protein concentration of 2.5% (8,500 cc.).
• Polyethylene glycol (M. wt. 6,000) is added in the manner described in Example 7, while maintaining a temperature of 1 8 C. to give a polyethylene glycol concentration in the solution of 14%.
• the precipitate containing mainly gamma-globulin, some fibrinogen and some other impurities is now dissolved in acetate buffer (pH 4.6, 0.02 M) to result in a protein concentration of 2.4% (9,000 cc.) and polyethylene glycol is again added to the solution (at room temperature) to produce a concentration of 12.8% PEG.
• the PEG used is also buffered.
• the further procedure to obtain pure gamma-globulin follows the procedure described in Example 7.
• the crude fibrinogen fraction obtained in the first precipitation step is dissolved in a citrate/saline buffer to result in a solution containing 1% protein (4,500 cc.) and having a pH of 7.0.
• Polyethylene glycol buffered with phosphate buffer pH 7.0, 0.02 'M
• the PEG is added in the same manner as described in Example 7 for the preparation of pure gamma-globulin.
• the precipitate is separated by centrifugation and found to be homogeneous fibrinogen containing some polyethylene glycol.
• the fibrinogen precipitate is redissolved in citrate/saline buffer to result in a protein concentration of 1% (4,500 cc.) and centrifuged to remove denatured and insoluble material. From the clear solution pure fibrinogen is precipitated by adding ethanol to a concentration of 8% at -3 C. in a manner known as such. The precipitate is vacuum dried and may then be stored under sterile conditions in a manner known per se.
• Fibrinogen prepared by the above-mentioned methods was found to be homogeneous apart from approximately 1% of macroglobulin which compares very favourably with a macroglobulin content of approximately 10% of macrog-lobulin found to be present in a comparative material produced by the known alcohol fractionation method.
• Example 9 Purification of fibrinogen by extraction
• the crude fibrinogen precipitate obtained by the first fractionation step of Example 8 is mixed with kieselguhr and filled into a glass column.
• a solution of 4.5% PEG in water buffered with phosphate buffer (pH 7.0, 0.02 M) is prepared and slowly percolated through the column at room temperature.
• the impurities are selectively extracted from the column, leaving a purified fibrinogen behind on the column.
• Example l0.--Determination of the precipitation index As a measure of the comparative efllciency of a particular precipitant to precipitate a particular substance from a colloidal solution thereof, an arbitrary index, herein referred to as the precipitation index, may be conveniently used. This precipitation index is defined as the concentration of precipitant required to precipitate 50% of the substance to be precipitated from a solution containing 1 gm. per 100 ml. of the substance at a predetermined pH and temperature. In the present example it is assumed that the precipitation indices of polyethylene glycol samples of different molecular weight for gammaglobulin are to be determined.
• a series of standard test solutions of the dilferent polymer samples are prepared in phosphate buffer of molarity of 0.066 and pH 7.2 to contain 2, 4, 6 n 2 gms. per 100 ml. of solution (n being any integer from 1 to 50).
• ml. amounts of a 2% solution of gamma-globulin are placed in a series of tubes of a preparative ultracentrifuge. This is followed by the addition of 5 ml. of polymer solution of increasing concentration to the tubes. After thorough mixing the tubes are transferred to the rotor of the centrifuge, the temperature of which is maintained at 21 C. After allowing 30 minutes for the tubes to reach temperature equilibrium, the precipitates which form are removed by centrifugation at 12,000 rpm. for 15 minutes. The supernatant fluids are then carefully removed and their concentration determined by a standard biuret procedure.
• Example 11Effect of precipitant on denaturisation of protein precipitate In some cases a change of the pH at which precipication is carried out helps to avoid denaturisation. Thus fibrinogen was observed to be denatured when precipi tated with PEG at pH 4.6 but not when precipitated at pH 6.3 to 7.0.
• a process for fractionating a mixture of proteinaceous substances which comprises:
• polyethylene glycol used is selected with a molecular weight in the range of 1,500 to 20,000.
• a process as claimed in claim 3 in which the concentration of polyethylene glycol in admixture with the mixture being separated is successively increased thereby to precipitate successive fractions of different composition.
• a process as claimed in claim 3 which comprises co-currently in -a predetermined ratio, bringing into intimate and uniform contact with one another two material streams, one being the mixture to be separated dispersed in water;
• a process as claimed in claim 3 which comprises carrying out the fractionation with at least two different precipitating agents in combination, of which at least one is an inorganic precipitant, capable as such of precipitating protein type substances from aqueous dispersion and one other is said polyethylene glycol.
• a process as claimed in claim 8 applied to the separation of gamma-globulin from blood plasma which comprises precipitating ran impure gamma-globulin from plasma by the addition thereto of ammonium sulphate, dissolving the precipitate in an aqueous medium and mixing the solution with polyethylene glycol to recover a more purified precipitate of gamma-globulin.
• a process as claimed in claim 9 in which the lastmentioned precipitate is re-dissolved in an aqueous medium, contaminants of the gamma-globulin are precipitated by mixing the solution with a predetermined amount of polyethylene glycol, the precipitate is separated from the remaining solution and the concentration of polyethylene glycol in the solution is increased to precipitate gammaglobulin.
• a process as claimed in claim 7 applied to the separation of both fibrinogen and gamma-globulin from blood plasma which comprises mixing the plasma with ammonium sulphate up to a concentration :at which most of the fibrinogen present precipitates, leaving the bulk of the gamma-globulin and some fibrinogen in solution, separating the precipitate from the solution, increasing the concentration of ammonium sulphate to precipitate at least the bulk of the gamma-globulin and re-dissolving both precipitates separately, mixing the redissolved first precipitate with polyethylene glycol to now precipitate an essentially pure fibrinogen fraction while maintaining essentially neutral conditions, and mixing the redissolved second precipitate with polyethylene glycol to now precipitate therefrom gamma-globulin in a more highly purified form than before.
• a process as claimed in claim 1 which comprises:

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Cited By (51)

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  • NL NL286954D patent/NL286954A/xx unknown
• 1962
  • 1962-12-19 GB GB48051/62A patent/GB1006258A/en not_active Expired
• 1967
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