Patent GB-669461-A (GB)
Title: Improvements in or relating to the fractionation of proteins and the products thereof
IPC: A61K35/16
Filed: 1945-10-17
Abstract: The mixed proteins of human blood plasma are fractionated by a selective precipitation which comprises bringing together directly in aqueous solution the mixed proteins, an electrolyte and a water-miscible organic precipitant for protein, maintaining said solution at a temperature at least low enough to prevent denaturation of the proteins to be recovered, while maintaining the pH of the solution as near to the point of minimum solubility in said solution of the protein or protein fraction to be precipitated as is consistent with maintaining solubility in said solution of the protein or proteins to remain dissolved, and utilizing a concentration of precipitant and ionic strength such that the solubility of the protein or protein fraction to be precipitated is reduced as far as possible without rendering insoluble those proteins which are to remain in solution, and recovering the precipitated protein or protein fraction insoluble under the conditions of pH, temperature, precipitant concentration and ionic strength utilized. Alternatively, the fractionation is effected by a selective extraction from a mixture of the proteins in the solid state comprising contacting the solid mixture with an aqueous solution of a water-miscible organic precipitant for protein in the presence of an electrolyte at a temperature at least low enough to prevent denaturation of the proteins to be recovered, while maintaining the pH of the solution as near to the point of minimum solubility in said solution of the protein or protein fraction to remain undissolved as is consistent with p  maintaining solubility in said solution of the protein or protein fraction to be dissolved, and utilizing a concentration of precipitant and ionic strength such that the solubility of the protein or protein fraction to remain undissolved is reduced as far as possible without rendering insoluble those proteins to be dissolved and recovering the extracted protein or protein fraction soluble under such conditions of temperature, pH, precipitant concentration and ionic strength utilized.  By varying one or more of the conditions after a first precipitation or extraction, successive precipitations or extractions of further different fractions may be accomplished.  Specific organic precipitants are methanol, ethanol, acetone, various glycols and dioxane.  The blood plasma may be obtained by separating the corpuscles from blood by centrifuging or sedimentation, citrates being added to prevent clotting of fibrinogen.  In general, in carrying out the extractions and precipitations the variables are controlled within the ranges of temperature between 0 DEG  C. and the freezing point of the solution, pH between 4.4 and 7.8, ionic strength between 0.01 and 0.2, concentration of organic precipitant between 10 and 40 per cent by volume of the solution. In a fractionation by selective precipitation, fibrinogen is precipitated from plasma with ethanol at 10 per cent concentration at close to -3 DEG  C. and an ionic strength of 0.13 provided by the salts naturally present in the plasma.  The gamma-globulins are then precipitated by lowering the temperature to -5 DEG  C. and increasing the ethanol concentration to 25 per cent.  Next, the alpha- and beta-globulins are precipitated by bringing the pH to 5.5-6.0 and the alcohol concentration to 40 per cent, the temperature remaining at -5 DEG  C. Finally, albumin is precipitated by lowering the temperature to -15 DEG  C. or the pH to 4.4-4.8.  In the penultimate step, the use of an ionic strength of 0.05 ensures that the solubility of the albumin is increased sufficiently to allow satisfactory separation of the globulins therefrom; the temperature being left unchanged, the albumin may be precipitated by adding an acetate buffer of pH 4.2 whereby the pH is changed to 4.7 and the ionic strength to 0.06. Reference is made to the precipitation of a mixed protein fraction obtained when the alcohol concentration is 15 per cent and the temperature is lowered from 0 DEG  to -5 DEG  C. and a further precipitate obtained when the alcohol concentration is then raised to 20-22 per cent; euglobulin and pseudo globulin occur in the resulting fraction. Methods for bringing the alcohol and protein solutions together include spraying one into the other, forcing alcohol or alcohol-water mixtures through a tube of small diameter (e.g. a capillary tube) or a sintered glass plate into the protein solution with stirring or by feeding the alcohol and protein solution into a T-piece, mixing occurring during outflow through the stem. Precipitated proteins may be dried by freezing solid the wet mass and then subjecting it to a vacuum.  In the extraction procedure, albumin for example may be extracted from a solid mixture of plasma proteins under conditions such that the albumins only are soluble and the others are insoluble and not denatured and may also be subsequently selectively extracted.  In detailed illustrations: (1) fibrinogen and gamma globulins are precipitated from blood plasma at -5 DEG  C., ionic strength 0.09 and alcohol concentration 40 per cent and the solid precipitate is then immersed in aqueous alcohol at 10 per cent concentration, the temperature being between 0 DEG  and -3 DEG  C. and ionic strength (due to occluded salts in the precipitate) is 0.13 whereby the globulins are extracted from the insoluble fibrinogen; (2) a mixture of fibrinogen and gamma-, alpha- and betaglobulins are precipitated from plasma by a variation of the conditions of (1), and under conditions given, the solid precipitate is extracted with aqueous alcohol removing first the alpha- and beta-globulins and then the gamma-globulins.  An impure albumin fraction obtained by either of the above methods may be converted into a crystalline pure form by dissolving it in an aqueous solution of ethanol containing 15-40 per cent alcohol at a pH of about 5.5, an ionic strength of 0.01 to 0.2 and a temperature of 0 DEG  to -5 DEG  C. and maintaining the solution within that temperature range until crystallization occurs. Reference is made to the therapeutic use of the albumin and fibrinogen fractions.  It is stated that fibrinogen forms thermosetting resins and that a protein fraction containing fibrinogen and gamma-globulins may be used in the manufacture of plastic compositions.  Specification 669,460 is referred to.