Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/14—Blood; Artificial blood
  • A61K35/16—Blood plasma; Blood serum
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
  • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
  • A61L2/0011—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using physical methods

Definitions

• This invention relates to lipoprotein-free, stable serum and plasma and more particularly to stable hepatitis-free serum and plasma and to a process for making the same.
• Stabilized plasma protein solutions are generally prepared by fractionation. Adsorption on bentonite and treatment with monochloroacetate are also known (Donald Dawson et al. lSR Volume II, No. 1, January, 1962, 31). However, fractionation yields only plasma protein solutions which contain no therapeutically important fractions. With the other processes, it is not possible to achieve any stability.
• lt isstill another object to protect sera and plasma from deterioration caused by lipoproteins present therein without the use of chemical additives and without subsequent denaturation of the serum protein.
• colloidal silicic acid is capable under certain reaction conditions quantitatively to absorb not only alpha alpha and fi-lipoproteins but in addition the fibrinogen present in serum and plasma.
• serum and plasma are treated by intimately contacting the same with colloidal silicic acid, protein solutions are obtained which still contain all the therapeutically important constituents and which are substantially stable to storage due to the complete removal of fibrinogen which is unstable in solution as well as the complete removal of the lipoproteins, so that at a constant refrigeration temperature 4 to 6C) storage is possible for up to 2 years without any appearance of cloudiness and without any loss of therapeutic activity.
• the present invention is directed to a process for the preparation of lipoprotein-free, stable and sterile serum, which is characterized in that blood serum or blood plasma is intimately mixed with 250 to 500 mg. of colloidal silicic acid per gram of total protein and is thereafter purified, following removal of the silicic acid, by a combined treatment with UV irradiation (UV ultraviolet) and sterile filtration.
• blood serum or blood plasma is intimately mixed with 250 to 500 mg. of colloidal silicic acid per gram of total protein and is thereafter purified, following removal of the silicic acid, by a combined treatment with UV irradiation (UV ultraviolet) and sterile filtration.
• the sterile filtration is carried out first and UV-irradiation thereafter, although the sequence is not critical.
• the treatment of the plasma and serum according to the invention is preferably carried out at temperatures of up to 50C, in particular-between 20 and 50C.
• the preferred pH range during the processing lies between 6.5 and 8.
• the duration of the mixing process depends on the type of apparatus used for mixing.
• Mixing times as used in practice consist for example, of l to 8 hours treatment using mixers rotating at a suitable speed.
• the UV-irradiation is preferably carried out at an intensity of 1 mW/cm minute.
• viruses analogous to the hepatitis virus are largely removed from the plasma or serum. It has already been indicated above thatwith the present state of the art, it is not possible to carry out experiments with the hepatitis virus on the animal. (in vivo) or in vitro. Therefore, it is necessary to rely on tests carried out with analogous viruses, the presence of which can be scientifically proved.
• the coli-phage has been described by LoGrippo as a particularly suitable analog for the hepatitis virus (lnvestigations of the Use of ,B-Propiolactones in Virus Inactivation; G.A.
• preserved plasma or serum is obtained which is stable in storage and free from hepatitis virus.
• Quantitative removal of viruses may be achieved especially if a filtration step using layer filters of a suitable nature and pore size, and preferably if asbestoscontaining filters impervious to bacteria, are employed in a filtration step carried out between the adsorption operation and UV-irradiation.
• the model viruses used were coli-T-phages.
• the adsorbed lipoproteins can be eluted from the silicic acid precipitate'which is obtained by the process of the invention, largely without denaturation, by utilizing concentrated salt solutions in the alkaline pH range. Concentration of the lipoproteins thus recovered can be effected by a preceding elution of other adsorbed proteins by means of salt solutions of low concentratlon.
• the lipoprotein solution is suitable for use as a biochemical and immunochemical reagent, in particular for medical diagnosis.
• the storage stable lipoprotein-free sera and plasma canbe used as infusion liquids as for any of the other conventional uses for which such materials are conventionally employed.
• the colloidal silicic acid used in the practice of the invention can be any of those commercially available, especially those sold as Aerosil silica by DEGUSSA of Germany or as Cab-O-Sil by Cabot in the United States. Since the products are ultimately intended for human administration, the only requirement is that the adsorbent not contain any interfering contaminants. Thus, for example, the whole Aerosil silica line is suitable except for those which contain substantial amounts of starch, e. g. Aerosil compositum, or hydrophobic-imparting radicals, e.g. Aerosil R. 972, and their Cab-O- Sil counterparts.
• the surface area and particle size of the colloidal silicic acid may vary widely embracing, for example, the entire spread of commercial products which have primary particle sizes of about 3 to 50 m,u. and densities ranging from about 2.5 up to as high as 7.8 pounds per cubic foot, achieved by compression.
• the standard products are known as Aerosil 200 or Cab-O-Sil M-5 which have medium range surface areas of about 200 square meters per gram. Because of their higher surfaces Cab-O-Sil H-S or Aerosil 300, having 300 square meters of surface per gram, are even more effective on a weight basis; the extra high surface products such as Cab-O-Sil EH-S and Aerosil 380 have 380 square meters of surface per gram and are most effective.
• the surface area could be as low as square meters per gram, e.g Aerosil MOX 80, or even as low as 50 square meters per gram, e.g. Aerosil OX 50, although somewhat larger quantities would be required for a given result.
• the surfaces are all so large, however, that the indicated range of about 250 to 500 mg. of colloidal silicic acid per gram of total protein covers the practical operations; if materials of lower surface area are used, the upper part of the weight range will be required and/or possibly longer contact times, higher surface area materials performing their role in lesser amounts and/0r shorter times.
• EXAMPLE 1 A 1 liter flask was filled with 20 g of colloidal silicic acid. (The exact product employed was the silicic acid marketed by the firm DEGUSSA under the Trade Name Aerosil.) Thereafter 200g of glass beads (0.4 cm diameter), were introduced into the flask and it was closed with muslin and sterilized in hot air for 2 hours at l80C. The serum which was to be treated (pH 7.5) was then introduced into the flask under sterile conditions. After the flask had been sealed, it was rotated for 4 hours at a rate of 40 to 50 revolutions per minute overhead.
• the adsorption efiect is in the main directly dependent on the speed of rotation.
• the contents of the flask were then heated for 4 hours at 45C in a water bath without stirring or shaking, and thereafter centrifuged after cooling for 30 minutes at 5,000 revolutions per minute.
• the flask contents were then subjected to UV irradiation in a Dill apparatus.
• the liquid was then filtered off under sterile conditions. After 4 weeks storage at +5C, the liquid if necessary could be filtered off from the slight precipitate which sometimes forms.
• the silicic acid precipitate was worked up as follows:
• the voluminous precipitate was frozen at 20C, thawed at room temperature, and centrifuged for 15 minutes at 5,000 revolutions per minute.
• the upper layer which demonstrated on paper electrophoresis and immunoelectrophoresis substantially the same composition as the main portion of adsorbed serum or plasma, was filtered under sterile conditions after UV irradiation, and thereafter filtered again, if necessary, after 4 weeks storage at 5C.
• Sera prepared in this manner were found to be both acutely and chronically toxicologically compatible and entirely free from pyrogens.
• EXAMPLE 2 1 liter of human serum and 20g of colloidal silicic acid sterilized using hot air were thoroughly stirred at pH 7.5 (physiological pH) for 4 hours at 45C, and after cooling, centrifuged for 30 minutes at 5,000 revolutions per minute and irradiated with UV light. Thereafter the contents were filtered under sterile conditions. After 4 weeks of storage at 5C, any precipitate which had formed was filtered off.
• EXAMPLE 3 1 liter of human ACD plasma was adjusted to a 5.5 percent protein content, and at a pH of 6.5 thoroughly stirred for 4 hours at 45C with 30g of colloidal silicic acid which had been sterilized with hot air. The stirred mixture was then centrifuged, irradiated with UV light and filtered under sterile conditions. After 4 weeks of storage at 5C, the liquid was filtered to remove any precipitate which had formed.
• EXAMPLE 4 A 1 liter flask was filled with 20g of colloidal silicic acid, preferably the silicic acid product sold by the firm DEGUSSA under the Trade Name Aerosil was used. 200g of glass beads having a diameter of 0.4 cm were introduced and the flask closed with muslin and sterilized with hot air for 2 hours at 180C. The serum to be treated (having a pH of 7.5) was introduced into the flask under sterile conditions. After the flask had been sealed, it was rotated for 4 hours at 40 to 50 revolutions per minute overhead. The adsorption as previously noted directly depends on the speed of rotation used.
• the contents of the flask were then heated for 4 hours at 45C in a water bath without stirring or shaking, and after cooling they were centrifuged for 30 minutes at 5,000 revolutions per minute following filtration using filter layers of suitable nature and pore size, the filtrate was irradiated with UV light using therefor a DILL apparatus. After 4 weeks of storage at +5C, the precipitate, if any had formed, was filtered off.
• the silicic acid precipitate remaining was worked up as follows: The voluminous precipitate was frozen at 20C, thawed at room temperature, and centrifuged for minutes at 5,000 revolutions per minute. The upper layer, which was analyzed by paper electrophoresis and immunoelectrophoresis had substantially the same composition as the main portion of adsorbed serum of plasma, was filtered, prior to UV irradiation, using therefor suitable filter layers and preferably asbestoscontaining filters which are impervious to bacteria.
• Sera prepared in this manner were both acutely and chronically toxicologically compatible and free from pyrogens.
• EXAMPLE 6 1 liter of human serum was thoroughly stirred at pH 7.5 (physiological pH) in admixture with g of hot-air sterilized colloidal silicic acid for 4 hours at 45C. After cooling, the contents were centrifuged for minutes at 5,000 revolutions per minute and were then filtered through asbestoscontaining filters constructed so they were impervious to bacteria. The filtrate was then irradiated with UV light. After 4 weeks of storage at 5C, the precipitate, if any had formed, was filtered off.
• the product thereby obtained had the same desirable properties as the product described in Example 5.
• EXAMPLE 7 1 liter of human ACD plasma was adjusted so as to have a 5 .5 percent protein content, and at a pH of 6.5 thoroughly stirred for 4 hours at 45C together with 30g of colloidal silicic acid which had been sterilized by hot air. The plasma-silicic acid batch was then centrifuged, filtered through asbestos-containing filters impervious to bacteria, and irradiated with UV light. After 4 hours of storage at 5C, the precipitate, if any had formed, was filtered off.
• the serum obtained was both acutely and chronically toxicologically compatible and entirely free from pyrogen.
• the recoveredprecipitate was suspended in 1 liter of H 0, and 450g of solid NaCl were then added thereto. The mixture was then stirred for 1 hour at pH 9.0, the pH being kept constant by the addition of NaOH. After centrifuging, the top layer was dialyzed for 24 hours against 20 liters of 0.7% NaCl solution at 5C, followed by dialysis against 1 liter of 10 percent polyvinyl pyrrolidone K 90. It was thereby concentrated to about 2 percent protein.
• the solution was then filtered under sterile conditions, the filter comprising multiple layers of filter material of pore sizes smaller than the solids to be removed.
• the colloidal silicic acid was the standard Aerosil material, i.e. that identified either as Aerosil or Aerosil 200 and having 200 square meters of surface per gram and a primary particle size of about 16 mp.
• Aerosil material i.e. that identified either as Aerosil or Aerosil 200 and having 200 square meters of surface per gram and a primary particle size of about 16 mp.
• a set of side-by-side runs were made with the same silicic acid as well as with others, as follows:
• Adsorbent In an identical run using montmorillonite, obtained from Carl Roth OHG, Düsseldorf, Germany, of undefined particle size and surface area and of 80 percent I purity, less than 5 percent of the lipoproteins were adsorbed.
• EXAMPLE 1 1 and examples are set forth by way of illustration and not limitation and that various modifications and changes may be made without departing from the spirit and scope of the present invention.
• a process for the preparation of lipoprotein-free, stable and sterile serum or plasma which comprises intimately admixing, at temperatures of up to about 50C, a blood member selected from the group consisting of serum and plasma with 250 to 500 mg of colloidal silicic acid per gram of total protein, whereinthe colloidal silicic acid has a primary particle size of about 3 to 50 m, a bulk density of about 2.5 to 7.8 pounds per cubic foot and 'a surface area of about 50 to 380 square meters per gram, and thereafter separating the silicic acid having the adsorbed lipoproteins from the blood member thereon.

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Citations (2)

• 1967
  • 1967-02-24 NL NL6702923A patent/NL6702923A/xx unknown
  • 1967-03-07 GB GB10621/66D patent/GB1115849A/en not_active Expired
  • 1967-03-17 AT AT258467A patent/AT270868B/de active
  • 1967-03-23 BE BE696020D patent/BE696020A/xx unknown
  • 1967-03-31 SE SE04492/67A patent/SE328089B/xx unknown
  • 1967-04-05 FR FR101581A patent/FR7395M/fr not_active Expired
  • 1967-09-29 SU SU1186878A patent/SU477580A3/ru active
• 1970
  • 1970-10-07 US US78949A patent/US3686395A/en not_active Expired - Lifetime

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