WO2004014942A1 - Methode de preparation d'une lipoproteine a partir d'une source de sang - Google Patents

Methode de preparation d'une lipoproteine a partir d'une source de sang Download PDF

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Publication number
WO2004014942A1
WO2004014942A1 PCT/AU2003/001013 AU0301013W WO2004014942A1 WO 2004014942 A1 WO2004014942 A1 WO 2004014942A1 AU 0301013 W AU0301013 W AU 0301013W WO 2004014942 A1 WO2004014942 A1 WO 2004014942A1
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WIPO (PCT)
Prior art keywords
lipoprotein
blood component
divalent metal
metal cation
precipitate
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PCT/AU2003/001013
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English (en)
Inventor
Sandra Smith
Paul Bernal
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Selborne Biological Services (Australia) Pty Limited
Meat & Livestock Australia Limited
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Priority claimed from AU2002950726A external-priority patent/AU2002950726A0/en
Priority claimed from AU2002950730A external-priority patent/AU2002950730A0/xx
Application filed by Selborne Biological Services (Australia) Pty Limited, Meat & Livestock Australia Limited filed Critical Selborne Biological Services (Australia) Pty Limited
Priority to AU2003257228A priority Critical patent/AU2003257228A1/en
Publication of WO2004014942A1 publication Critical patent/WO2004014942A1/fr

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    • 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
    • C07K1/32—Extraction; Separation; Purification by precipitation as complexes
    • 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
    • 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

Definitions

  • the present invention relates to the preparation of lipoprotein from a blood source involving the precipitation of the lipoprotein from the blood source utilising a divalent metal cation in combination with a polyanion precipitating agent.
  • Serum cholesterol levels are commonly monitored for the purpose of indicating general health and well being as elevated levels are associated with a range of conditions including arthrosclerosis, coronary artery diseases, metabolic dysfunction, thyroid related diseases, liver disease and diabetes mellitus. Cholesterol levels are typically determined by comparison to standard values obtained using known amounts of cholesterol. Cholesterol is also commonly used as a component in culture medium for the growth and maintenance of micro-organisms such as bacterial and mycoplasma species.
  • US Patent No. 4,290,774 describes a method for purification of lipoprotein cholesterol that involves absorbing the lipoprotein from blood plasma or serum onto silica, eluting the absorbed lipoprotein and further processing the eluted material by adjusting pH and salt concentration prior to heat treatment. An alkaline carbonate and alkaline earth salt is then added to the heat treated material to obtain a precipitate containing denatured proteins which is discarded during the recovery of the remaining cholesterol. Similar processes are described in US Patent No.5,409,840 and US Patent No. 4,762,792.
  • the lipoprotein II precipitate obtained using the higher dextran sulfate and CaCl 2 concentrations contained 66% of the bovine serum cholesterol.
  • the lipoprotein II fraction has been found to become turbid making it unsuitable for subsequent use.
  • Total protein in bovine serum including protein in lipoprotein itself is typically in a range of from 72 to 79 g/1 while the amount of albumin in the serum typically ranges from 24g/ litre to 38 g/litre.
  • a human serum sample analysed in the Proksch and Bonderman (1976) article contained 36.75 g/litre of albumin and a total protein content of 56 g/litre.
  • the present invention stems from the finding that the amount of albumin and other contaminating proteins that precipitate with lipoprotein from a source such as plasma or serum using a divalent metal cation and polyanion precipitating agent can be substantially decreased by increasing the amount of the divalent cation with respect to the concentration of the polyanion used. This is highly surprising as it would be expected that by increasing the concentration of the divalent metal cation more contaminating protein and in particular, more albumin would be precipitated resulting in a less pure lipoprotein fraction. Accordingly, by manipulating the amount of the divalent metal cation used relative to the selected polyanion precipitating agent, the amount of the albumin and other proteins in the precipitate can be manipulated and it is this observation which has led to the present invention.
  • a method for preparing lipoprotein from a liquid blood component containing albumin comprising: adding a divalent metal cation and a polyanion precipitating agent to the blood component; mixing the divalent metal cation and the precipitating agent with the blood component to obtain a precipitate containing lipoprotein; and collecting the precipitate from the blood component for further purification of the lipoprotein; wherein the divalent metal cation is added to the blood component in an amount relative to the precipitating agent such that less than 10% of the albumin in the blood component is precipitated with the lipoprotein, and the precipitate obtained has a ratio of albumin to total protein below 1:3 by weight and contains greater than 20% of the lipoprotein associated cholesterol present in the blood component.
  • the collecting of the precipitate will comprise adding a filter aid to the blood component following the precipitation of the precipitate to provide a solution of the filter aid and the precipitate, and recovering the precipitate and the filter aid from the solution.
  • the filter aid will generally comprise diatomaceous earth.
  • the purification of the lipoprotein from the precipitate collected from the blood component will comprise:
  • the dissolving solution will usually comprise one or more agents for inhibiting the undissolved matter dissolving into the solution with the lipoprotein.
  • the cholesterol may also be separated from the precipitated lipoprotein and collected.
  • the invention further extends to a method of preparing cholesterol from the precipitated lipoprotein.
  • a liquid blood component containing albumin comprising:
  • the lipoprotein precipitated in a method of the invention will be obtained in a single stage precipitation process.
  • single stage precipitation process is meant that the lipoprotein is obtained from the blood component without preparing the blood component for the precipitation of the lipoprotein by firstly precipitating one or more initial fractions from the blood component.
  • the amount of the divalent metal cation needed to be added to the liquid blood component in order to attain a reduced albumin or other conterminating protein level in the precipitate in combination with the polyanion precipitating agent may be determined by varying the amount of the divalent metal cation relative to the amount of the polyanion precipitating agent and evaluating the albumin and/ or total protein content in the resulting precipitate.
  • a method for determining an amount of a divalent metal cation for preparing lipoprotein from a liquid blood component containing albumin in combination with a polyanion precipitating agent comprising: (a) adding an initial amount of the divalent metal cation and an amount of the polyanion precipitating agent to a sample of the blood component;
  • step (c) repeating steps (a) and (b) one or more times using a further sample of the liquid blood component and varying the amount of the divalent metal cation relative to the amount to the polyanion precipitating agent in the further blood component each time, respectively; and (d) determining the amount of the divalent metal cation relative to the polyanion precipitating agent required to obtain a precipitate containing less than 10% of the albumin in the blood component and having a ratio of albumin to total protein in the precipitate below 1:3 by weight.
  • the amount of the precipitating agent used in step (c) will be held substantially constant and the amount of the divalent metal cation varied each time.
  • the amount of the divalent metal cation determined in step (d) will be greater than the initial amount of the divalent metal cation used in step (a).
  • liquid blood component' is to be taken to mean blood plasma, serum or a fraction thereof.
  • the plasma, serum or fraction thereof may be used neat or diluted with a suitable buffer.
  • serum or plasma will be utilised in a method of the invention. Most preferably, serum will be used.
  • the polyanion precipitating agent may be any such agent that is capable of precipitating the lipoprotein from the blood component in combination with the divalent metal cation.
  • the polyanion precipitating agent will be selected from the group consisting of sulfated polymers, sulfated polysaccharides, and mixtures thereof.
  • the precipitating agent used will comprise a single compound as distinct from a mixture.
  • the precipitating agent will be dextran sulfate.
  • the divalent metal cation used in a method of the invention will be a divalent metal cation of a salt and the method will further comprise selecting the salt for addition to the liquid blood component.
  • the salt will be a Group HA metal salt.
  • a method for preparing lipoprotein from a liquid blood component comprising:
  • the divalent metal cation and the dextran sulfate are added to the blood component in a ratio of moles of the divalent metal cation to dextran sulfate in grams per litre of the blood component of 1:9.5 or greater, and the precipitate contains less than 10% of the albumin in the blood component and greater than 20% of the lipoprotein associated cholesterol present in the blood component.
  • lipoprotein prepared by a method of the invention.
  • cholesterol purified from lipoprotein prepared by a method of the invention is provided.
  • Lipoprotein concentrate prepared in accordance with one or more embodiments of the present invention may contain a reduced amount of albumin and other contaminating protein(s) compared to levels obtained by prior art precipitation methods, making the concentrate suitable for subsequent heat inactivation treatment with the clarity of the concentrate being substantially unaffected by the heat treatment.
  • Being able to heat treat the lipoprotein is highly advantageous as heat treatment is a simple and cost effective process which denatures deleterious enzymes and other proteins that may be present in the lipoprotein, and inactivates or kills any contaminating microorganisms such as viruses, without any further separation or treatment steps being required.
  • Figure 1 Graphs showing total protein in lipoprotein preparations prepared by a method of the invention as a percentage of the total protein in the starting bovine serum against CaCl 2 concentration;
  • Figure 2 Graphs showing the ratio of total protein to lipoprotein associated cholesterol by weight in lipoprotein preparations against CaCl 2 concentration
  • Figure 3 Graphs showing the percentage lipoprotein associated cholesterol recovered from bovine serum against CaCl 2 concentration.
  • bovine serum will be utilised in methods of the invention and may be readily obtained by separating the serum from clotted blood collected freshly at abattoirs.
  • human serum and serum from other mammalian species including those of the porcine, ovine, equine families may be utilised.
  • plasma from any such species may also be readily obtained by collecting the blood into an anti-coagulant such as heparin, and centrifuging the blood to remove blood cells and other cellular material.
  • separation of the plasma may be achieved by density gradient centrifugation using Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) or other suitable density gradient.
  • Particularly preferred fractions of plasma or serum containing lipoprotein that may be used as the starting material in methods of the invention include fibrinogen-poor fractions, and the like.
  • the plasma, serum or fraction thereof may be dialysed against a suitable buffer or diluted by a suitable buffer prior to precipitation of the lipoprotein.
  • the sulfated polymer for precipitating the lipoprotein in combination with the divalent metal cation may be selected from the group consisting of sulfated polysaccharides, dextran sulfate, sulfated polygalacturonic acid methyl ester, polyanetholsulfonate, sulfated amylopectin, polyvinyl sulfate, and mixtures thereof.
  • the polyanion precipitating agent will be dextran sulfate having a molecular weight in a range of from about 5kDa to about 1000 kDa. Most preferably, the dextran sulfate will have a molecular weight in a range of from 250 kDa to about 750 kDa. Typically, the dextran sulfate will be added to the blood component in an amount of 2 g/1 or greater and preferably, in an amount in a range of from 2.6 g/1 to 3.0 g/1 of the blood component.
  • the invention is not limited to dextran sulfate and other commercially available physiologically acceptable sulfated polysaccharide polyanion precipitating agents such as heparin may be utilised.
  • the amount of polyanion precipitating agent required for precipitating the lipoprotein can be determined by adding an amount of the selected precipitating agent to the blood component in the presence of an amount of the selected divalent metal cation, and determining the degree of precipitation of the lipoprotein. This can be repeated a number of times varying the amount of polyanion precipitating agent each time while maintaining the concentration of the divalent metal cation constant, until the desired level of precipitation of the lipoprotein is achieved. This process is then again repeated keeping the determined amount of precipitating agent constant but varying the amount of the divalent metal cation relative to the precipitating agent added, until the amount of albumin or contaminating protein precipitating with the lipoprotein has been minimised or otherwise reduced to the required level.
  • the divalent metal cation will usually be selected from Ca 2+ and Mg 2+ However, other divalent metal cations may also be utilised including Be + , Ba 2+ , and Mn 2+ .
  • the divalent metal cation will be a Group IIA divalent metal cation.
  • the selected divalent metal cation will generally be utilised in the form of a salt.
  • the salt will generally comprise halogen, sulfate or phosphate anion(s).
  • the salt will be a chloride or sulfate salt of the divalent metal cation and most preferably, CaCl 2 .
  • Increasing the concentration of CaCl 2 above 27.75 g/1 when used in combination with dextran sulfate with a molecular weight of 500 kDa at a concentration of 2.85 g/1 to precipitate lipoprotein from bovine serum for instance, can result in a significant decrease in the level of precipitation of albumin and other contaminating proteins with the lipoprotein.
  • the CaCl 2 will be added to the blood component in an amount greater than about 30 g/1, more preferably, in a range of from about 30 g/1 to about 55 g/1 or greater and preferably, in a range of from about 35 g/1 to about 50 g/1.
  • a concentration of 30g/l corresponds to a concentration of 0.3M. Accordingly, the use of a concentration of 0.3M CaCl 2 or greater and 2.85g/l of dextran sulfate equates to a ratio of moles of Ca 2+ to dextran sulfate in grams per litre of the blood component of 1:10.55 or greater.
  • the polyanion precipitating agent and the salt may be dissolved in a minimum amount of a suitable solvent such as distilled or deionised water for being added to the serum.
  • a suitable solvent such as distilled or deionised water
  • the salt will be added directly to the serum undissolved and preferably, before the polyanion precipitating agent is added.
  • the temperature of the serum or plasma during the precipitation of the lipoprotein will be maintained at a temperature of less than 10° C and typically at 4 - 5° C, but the precipitation step may also be carried out at higher temperatures including room temperature.
  • the pH of the blood component used will generally not be altered from its natural pH level for the precipitation of the lipoprotein. However, altering the pH of the blood component is not excluded. Typically, the pH of the blood component for precipitation of the lipoprotein will be in range of from about 6.0 - 8.0 and usually, in a range of from about 7.2 - 7.6.
  • the precipitate is collected and preferably washed a number of times to remove residual contaminants from the precipitate.
  • the precipitate is then typically dissolved in a suitable dissolving solution such as a solution of potassium chloride and potassium oxalate, and filtered through an appropriate size filter prior to the filtrate containing the dissolved lipoprotein being concentrated by ultrafiltration.
  • the filtrate may be concentrated by any other conventional method known in the art such as by precipitation using ammonium sulfate or other salts and /or precipitating agent, and adsorption or desorption methods, and subsequently dialysed against a suitable buffer.
  • the filtrate will typically be concentrated to obtain a cholesterol concentration in a range of from about 50 to 3000 mg/ dl and more preferably, in a range of from about 1000 to 1500 mg/dl.
  • the concentrate may be subjected to heat treatment. This also eliminates the risk of any contaminating bacteria or other micro-organisms that may be present at this stage.
  • the clarity of the concentrate remains substantially unaffected by the heat treatment. Turbidity of the heat treated concentrate can be determined by measuring percentage transmission of the heat treated concentrate at a wavelength of 650 run. Typically, the transmission of the heat treated concentrate will be greater than 70% and usually, will be in a range of from 80 - 86%.
  • the cholesterol may be separated from the protein in the isolated lipoprotein fraction using any method known in the art. Suitable methods include affinity chromatography.
  • the degree of precipitation of albumin and lipoprotein in the precipitate can be readily evaluated by subtracting the level of the albumin and the lipoprotein in the plasma or serum remaining after precipitation from the original levels prior to precipitation.
  • the level of the albumin or lipoprotein can be readily determined using conventional techniques known in the art such as for example, electrophoresis by SDS PAGE or immunodiffusion methods involving comparison with standard samples having a known albumin or lipoprotein content.
  • the amount of total protein in the precipitate can be determined by measuring total protein in the plasma or serum before and after precipitation of the lipoprotein. Methods for measuring total protein include colormetric methods (see for example: Ohnishi, S.T., Barr, J.K., Anal. Biochem.
  • the precipitate will contain greater than about 30% or 40% of the lipoprotein associated cholesterol present in the serum or plasma. Most preferably, the precipitate will contain a majority of the cholesterol and most preferably, 60 or 70% of the cholesterol or more.
  • the lipoprotein in the precipitate will generally be primarily high density lipoprotein (HDL) with the remainder of the lipoprotein being low density lipoprotein (LDL).
  • the precipitated lipoprotein will comprise greater than 70% HDL and more usually, about 76% to about 86% of the lipoprotein.
  • Suitable assays for determining the level of HDL in a sample include enzymatic colorimetric tests (eg. Sugiuchi, H. et al. Direct measurement of high density lipoprotein cholesterol in serum with polyethylene glycol-modified enzymes and sulfated -cyclodextran. Clin. Chem. 41:717-724 (1995)).
  • the divalent metal cation will be added in an amount to maintain precipitation of albumin relative to the amount of total protein precipitated below a ratio of albumin to total protein of 1:4 or 1:5 by weight or more preferably, below a ratio of 1:9, 1:19 or 1:49. Most preferably, the ratio will be below 1:99, 1:199, or 1:499 by weight or even lower.
  • the albumin in the precipitate will be less than about 5% of the total albumin in the serum or plasma, and more preferably, less than about 2% of the albumin. Most preferably, the precipitate will be substantially free of albumin.
  • the precipitate will contain a total protein to total cholesterol ratio of 2.9 by weight or less, preferably a ratio of 2.8 or 2.6 or less, more preferably a ratio of 2.5 or 2.4 or less or most preferably, a ratio of 2.0 by weight or less.
  • a volume of 1000 litres of pooled bovine serum was transferred to a stainless steel tank and mixed to ensure homogeneity of the serum.
  • An amount of 44 kg of CaCl 2 was then added undissolved to the serum to obtain a serum concentration of the CaCl 2 of 44 g/1.
  • the CaCl 2 was added in small amounts over the entire surface of the serum and the serum stirred for a minimum of 90 minutes.
  • a filter aid solution was prepared by adding 30 kg of diatomaceous earth (Celite 503, Celite Corporation, USA) in 1000 litres of RO water. The pH of the filter aid solution was checked to ensure it was not above 7.5. If necessary, the pH was adjusted to pH 7.5 or just below this value prior to the filter aid being added to the precipitated serum. The precipitated serum was then passed through a filter press (Edwards & Jones 630 mm x 630 mm single screw electromechanical semi- mechanised recessed chamber filter press). The filtrate was checked to ensure it was clear, and was recirculated as necessary through the filter press until clear prior to the filtrate being discarded. To ensure all precipitate was collected, 200 litres of CaCl 2 solution (0.734 kg CaCl 2 / 100 litres RO water) was subsequently passed through the filter press.
  • CaCl 2 solution 0.734 kg CaCl 2 / 100 litres RO water
  • the precipitate was then collected and washed by adding the precipitate to 500 litres of CaCl 2 solution (0.734 kg of CaCl 2 /100 litres of RO water), and the resulting solution was stirred for a minimum of 60 minutes. Following stirring, the solution was passed through the filter press as described above and the precipitate recollected. A further 200 litres of CaCl 2 solution (0.734 kg CaCl 2 / 100 litres RO water) was pumped through the filter press to ensure all precipitate was collected. A second CaCl 2 wash was then performed as described above.
  • the washed precipitate was then added to 500 litres of RO water in a stainless steel tank and stirred well for a further minimum period of 60 minutes prior to the washed precipitate being collected using the filter press.
  • a volume of 300 litres of dissolving solution was prepared by dissolving potassium chloride (7.455 kg/ 100 litres) and potassium oxalate (1.842 kg/ 100 litres) in RO water.
  • the pH of the dissolving solution was checked and adjusted to 6.5 if necessary.
  • Potassium oxalate removes calcium as an insoluble calcium oxalate and dissociates the dextran sulfate - lipoprotein complex.
  • the ionic strength of the potassium chloride and the pH of the solution allow the lipoprotein to dissolve while maintaining the dextran sulfate insoluble.
  • the washed lipoprotein precipitate was added to the dissolving solution and stirred for a minimum period of 90 minutes to fully dissolve the lipoprotein in the precipitate.
  • the dissolved lipoprotein was then filtered to remove the filter aid and precipitated dextran sulfate, by passing the solution through the filter press and then through a 0.2 micron membrane filter (Cat No. AB2NAZ7PH4, Pall Biopharmaceuticals). The filtrate was retained for further processing.
  • Example 1.2 Concentration and diafiltration of dissolved lipoprotein The filtrate retained from Example 1.2 was recirculated through an ultrafiltration unit (Pall Filtron Centrasette 10 fitted with Alpha Open Channel 30K polyethersulfone (PES) membrane cassettes) under the following conditions: a retentate flow rate of 6 litres /min/ 0.5 m 2 (cassette area); an inlet pressure of 1.7 bar; a retentate pressure of 1.2 bar; and a filtrate pressure of 0.
  • PES polyethersulfone
  • the filtrate hose was placed to drain to concentrate the solution to obtain the required volume for a cholesterol concentration of 12 g/litre.
  • the required concentrate volume can be readily determined by measuring the volume of the filtrate retained following filtration of the dissolved lipoprotein and its cholesterol concentration. Any conventional method suitable for measuring cholesterol may be utilised such as by colorimetric methods utilising commercially available kits (eg. Sigma-Aldrich, Cat. No. 401-25P).
  • the concentrate was subjected to diafiltration following the addition of an equal volume of RO water to the concentrate. Diafiltration was continued until the volume of the concentrate was returned to that prior to the addition of RO water. Addition of further RO water and diafiltration was repeated as necessary until the osmolality of the lipoprotein concentrate was less than or equal to 5.
  • the pH of the lipoprotein solution resulting from Example 1.3 above was measured and adjusted to 8.0 ( ⁇ 0.1) if necessary.
  • the lipoprotein solution was subsequently transferred to a heating tank and heated to a temperature in a range of from 80°C to 82°C, and maintained within that temperature range for three hours. At the end of this time period, the temperature of the lipoprotein solution was decreased to a temperature in a range of from 60°C to 62°C. Overall, the lipoprotein solution was at or above that temperature for a total period of 10 hours including the 3 hour period at 80°C to 82°C.
  • the cholesterol level of the lipoprotein solution was adjusted to 10.7 g/litre with injection grade water prior to adjusting the pH of the lipoprotein solution to 8.0 ( ⁇ 0.1) if required.
  • the resulting concentrate was clear.
  • the prepared lipoprotein concentrate was found to contain a ratio of total protein to lipoprotein associated cholesterol of between 2.0 to 3.0 by weight and more typically, a ratio of from 2.39 to 2.86.
  • the total protein in the concentrates ranged from 21.7 g/litre to 34.30 g/litre.
  • the concentrates contained from 2.43% to 3.11% of the total protein in the bovine serum.
  • the amount of lipoprotein associated cholesterol in the concentrates ranged from 9.3 g/litre to 11.1 g/litre with the percentage of the cholesterol recovered from the bovine serum ranging from 52.2% to 61.4%.
  • the table also shows that the lipoprotein concentrates contained substantially reduced levels of ⁇ - globulin and ⁇ -globulin proteins compared to levels in the starting bovine serum. In particular, these proteins were reduced to levels ranging from 5% to 8.2% and 11.8% to 13% of that in the bovine serum, respectively.
  • KT-ABL-0301 1000.0 2.00 2000 1045 52.3% 81.40 81400 2062 2.5% 95 11.00 21.70 1.9
  • Example 3 Effect of varying the concentration of divalent metal cation
  • bovine serum samples from a number of different sources were obtained. Each sample was divided into 0.8 litre aliquots. For the purpose of precipitation of the lipoprotein, the aliquots were added to 1 litre centrifuge buckets and dextran sulfate at a concentration of 2.85g/ litre was added to each. The concentrations of the CaCl 2 added ranged from 25g/l to 56.3 g/1. The precipitate was collected by centrifuging the buckets and decanting off the supernatant.
  • the lipoprotein was precipitated and processed generally following the protocol described in Example 1 except that the dissolved lipoprotein was not concentrated due to the small volumes involved and the lipoprotein was subjected to dialysis to purify the lipoprotein rather than ultrafiltration. Diatomaceous earth was also not used except for one of the samples.
  • the percentage of total protein precipitated with a concentration of 30g/l or more ranged from 1.75% of the total protein in the bovine serum to 4.73% and typically, between about 2.09% and 3.89%.
  • the recovery rate of the lipoprotein associated cholesterol from the bovine serum ranged from 54.4% to 77.3% for these CaCl 2 concentrations.
  • the ratio of total protein to lipoprotein associated cholesterol recovered from the bovine serum was dependent on the initial concentration of the cholesterol in the bovine serum samples assayed. That is, higher ratios of protein to cholesterol were found for those samples that had a lower initial concentration of cholesterol.
  • Iable 2 Effect of varying calciumion concentration relative to dextran sulphate concentration on precipitation of other protein with lipoprofc ein from bovine serum
  • Trial 1 25.0 0.80 2.00 1.60 1.14 71.1% 80.00 64.00 3.36 5.3% 0.30 3.79 11.20 2.96
  • TYB O/N 44.0 0.80 1.46 1.17 0.80 68.6% 75.60 60.48 2.07 3.4% 0.24 3.34 8.63 2.58
  • TYB (6) 48.0 0.80 1.46 1.17 0.76 65.1% 75.60 60.48 1.90 3.1% 0.24 3.17 7.91 2.50
  • Fig.l and Fig. 2 show the effect of increasing CaCl 2 concentration on the total protein precipitated and the ratio of total protein to lipoprotein associated cholesterol, respectively.
  • Graphs illustrating the effect of increasing the CaCl 2 concentration relative to dextran sulfate concentration on the percentage of lipoprotein associated cholesterol recovered from the starting bovine serum are shown in Fig. 3.
  • Example 4 Effect of different divalent metal cations on precipitation of lipoprotein from bovine serum
  • Example 3 Similar results to those observed in Example 3 were obtained using CaCl ⁇ MgCl 2 and MgSO 4 for all parameters measured. Moreover, a decreasing ratio of total protein to lipoprotein associated cholesterol in the precipitates were also obtained with increasing concentrations of the salts. In contrast, substantially all the protein was precipitated from the bovine serum using CaSO , CaPO 4 and CaCO 3 and only low levels of lipoprotein were recovered from the precipitate.

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Abstract

L'invention concerne une méthode de précipitation d'une lipoprotéine à partir d'une source de sang, telle que du sérum ou du plasma, au moyen d'un cation métallique divalent en association avec un agent précipitant polyanionique, tel que le dextran-sulfate. Ledit cation métallique divalent sera généralement un cation métallique divalent du groupe IIA, tel qu'un ion calcium ou magnésium.
PCT/AU2003/001013 2002-08-09 2003-08-11 Methode de preparation d'une lipoproteine a partir d'une source de sang WO2004014942A1 (fr)

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AU2002950726 2002-08-09
AU2002950726A AU2002950726A0 (en) 2002-08-09 2002-08-09 A method of preparing lipoprotein from a blood source
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AU2002950730A AU2002950730A0 (en) 2002-08-12 2002-08-12 A method of preparing lipoprotein from a blood source

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Cited By (9)

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US7947813B2 (en) 2007-01-22 2011-05-24 Genentech, Inc. Polyelectrolyte precipitation and purification of proteins
EP2645102A3 (fr) * 2007-06-08 2014-01-22 Quest Diagnostics Investments Incorporated Analyse de lipoprotéines par mobilité différentielle de particules chargées
US8709818B2 (en) * 2007-06-08 2014-04-29 Quest Diagnostics Investments Incorporated Lipoprotein analysis by differential charged-particle mobility
US9250211B2 (en) 2010-12-30 2016-02-02 Quest Diagnostics Investments Incorporated Magnetic separation of lipoproteins using dextran sulfate
US9354200B1 (en) 2008-08-07 2016-05-31 Quest Diagnostics Investments Incorporated Detection apparatus for differential-charged particle mobility analyzer
CN110551207A (zh) * 2019-08-21 2019-12-10 广州蕊特生物科技有限公司 一种脂蛋白纯化方法
CN111955675A (zh) * 2020-09-01 2020-11-20 合肥工业大学 一种风味增强型血豆腐的加工方法
CN113176133A (zh) * 2021-03-15 2021-07-27 广州邦德盛生物科技有限公司 一种分离血浆或血清中蛋白质和脂类的方法及基质血清
CN114279778A (zh) * 2021-12-04 2022-04-05 南京岚煜生物科技有限公司 一种复合高值参考品的制备方法

Citations (3)

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