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US20040197891A1 - Removal of plasmin(ogen) from protein solutions - Google Patents

Removal of plasmin(ogen) from protein solutions Download PDF

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US20040197891A1
US20040197891A1 US10/477,519 US47751904A US2004197891A1 US 20040197891 A1 US20040197891 A1 US 20040197891A1 US 47751904 A US47751904 A US 47751904A US 2004197891 A1 US2004197891 A1 US 2004197891A1
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plasmin
ogen
support
mixture
amino acid
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Israel Nur
Liliana Bar
Malkit Azachi
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Omrix Biopharmaceuticals SA
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Omrix Biopharmaceuticals SA
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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
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • 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)
    • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin

Definitions

  • the present invention relates to a resin and method for the specific removal of plasmin(ogen) and its derivatives from protein solutions, where the resulting protein solution can be used for intravenous administration and for local applications, i.e. matrix support for sustained release and healing of wounds, either as a sole active component or combined with other pharmaceutical, acceptable drugs.
  • the removal of plasmin(ogen) would preserve the integrity and the function of the protein solution for longer incubation periods.
  • This invention is also related to the production of highly purified plasmin(ogen) for therapeutic use.
  • Plasmin(ogen) or its active molecule plasmin in the following plasmin(ogen), very frequently contaminates protein solutions, especially those extracted from animal fluids or animal organs.
  • the presence of plasmin(ogen) in a protein solution presents a multiple threat to its acceptance as a stable pharmaceutical product, due to the molecule's known proteolytic activity on various protein and peptides at arginyl and lysyl peptide bonds (Weinstein M. J., Doolittle R F. Differential specificities of the thrombin, plasmin and trypsin with regard to synthetic and natural substrates and inhibitors RF Biochim Biopliys Acta.
  • the first group is based on several consecutive purification steps that utilize the differential solubility, isoelectric point, or molecular size distribution Alkjaerisig N. (The purification and properties of human plasmin(ogen). Biochem. J. 1963, 93:171-182). Since their prime target was to purify plasmin(ogen), these methods totally distorted the composition of the protein solution.
  • the second group of methods is based on one step affinity purification. The purification is based on binding plasmin(ogen) to various synthetic ⁇ -amino carboxylic acid ligands that can bind onto the lysine binding sites on the plasmin(ogen) heavy chain.
  • plasmin(ogen) kringles consist of 5 triple loop disulfide bridges with internal sequence homology known as the plasmin(ogen) kringles, located on the NH 2 plasmin(ogen) heavy chain and far from the catalytic site located on the COOH light chain, bind fibrin(ogen).
  • affinity chromatography Another possibility for affinity chromatography is to bind plasmin(ogen) via the catalytic site, a potentially less specific binding since it may bind many proteins such as serine proteases having similar or lower affinity to arginyl and lysyl peptide bonds and basic amino acids.
  • plasmin(ogen) affinity chromatography is performed by a given ligand that chemically and ionically resembles co-amino-carboxylic acid or the substrate of the plasmin catalytic site.
  • the ligand is bound to the resin through an adequate spacer or linker.
  • an ideal affinity resin for the removal of plasmin(ogen) is not essentially the same resin found ideal for the purification of plasmin(ogen).
  • Such resins should contain a ligand that binds plasmin(ogen) at high affinity and has very low affinity to other proteins such as other serine proteases and especially very low affinity for fibrinogen which is the main protein in Plasma Cohn's fraction I or in cryoprecipitate.
  • the antifibrinolytic potency (ability to inhibit the binding of plasmin(ogen) to fibrinogen at high affinity) of the ⁇ -amino-carboxylic acids depends on the presence of free amino and carboxylic group and on the distance between the COOH-group and the carbon atoms to which the NH 2 -group is attached (Markwardt 1978) such as ⁇ -amino caproic acid (EACA), and p-amino benzamidine (PAMBA). Comparison between the antifibrinolytic activities of EACA and PAMBA showed that the latter is about three times more active.
  • Shimura et al (1984) designed a resin in which p-amino benzamidine was bound to microparticles of hydrophilic vinyl polymer via a spacer (linker) moiety.
  • Shimura et al were able to separate plasmin and plasmin(ogen) by high performance affinity chromatography.
  • lysine-resin Another resin, the lysine-resin, is manufactured and used for the affinity purification of plasmin(ogen).
  • the antifibrinolytic potency of lysine is very low and thus, also its binding affinity. It also binds to other proteins and its specificityis buffer dependent.
  • tranexamic acid is identified as a powerful ligand of plasmin, it is indicated that the anti-fibrinolytic effect of tranexamic acid is a result of not only the binding to plasmin(ogen), but also of the enhancement of cooperation of the natural antiplasmins. Therefore one would conclude that binding of tranexamic acid to a solid support will not only remove plasmin(ogen) from plasma but also the natural antiplasmins.
  • tranexamic acids may cause of formation of aggregates (conglomerates) with plasmin inhibitors. This understanding is based on the discrepancy, which can be found when comparing the anti-fibrinolytic activity of ⁇ -amino-caproic acid and tranexamic acid resulting in 98 and 91% inhibition in urokinase-stimulated plasma versus plasma that has been heparinized oral blood (65 and 39% for ⁇ -amino-caproic acid and tranexamic acid respectively—cf. tables 2 and 7 in the Moroz et al. paper).
  • tranexamic acid and ⁇ -amino-caproic acid are good candidates for high affinity ligands.
  • this ligands would block an affinity column as a result binding of plasmin and plasmin inhibitor complexes.
  • FIG. 1 shows a gradient gel SDS-PAGE (5-12% poly-acrylamide) of 7 ⁇ g of proteins eluted by the two methods (described in the material and method), with three different resins -TEA-Sepharose, Lys-Ceramic Hyper DF and Lys-Sepharose 4B.
  • the present invention is based upon the result that a rigid amino acid was surprisingly found as to be able for specifically binding plasmin(ogen).
  • “Specifically binding” within the context of the description of the invention means that out of a mixture containing proteins, such as plasmin(ogen) and fibrinogen essentially the plasmin(ogen) is removed from the mixture whereas fibrinogen is maintained almost unaffected in the mixture. Preferably at least 85 to 99% of the plasmin(ogen) is removed and at least 85% of fibrinogen remains in the mixture. More preferably plasmin(ogen) is removed to at least 98,0% to 99,9% or fibrinogen remains 95% by 99%.
  • the amino group of the amino acid and the carboxylic group of the amino acid are about 6-8 Angstroms, preferably about 7 Angstroms apart and the rigid amino acid is covalently bound to a support via the amino group of the amino acid.
  • tranexamic acid in its transconfiguration and 4-aminomethylbicyclo-[2.2.2.]-octane-1-carboxylic acid (EMBOCA).
  • ⁇ -amino-caproic acid did not work as well as tranexamic acid and that once the tranexamic acid is bound to a solid surface it looses all its extra plasmin(ogen) binding capacity (so-called cooperation) and the resin removes only plasmin(ogen) from plasma or plasma products. If ⁇ -amino-caproic acid is attached to the column its extra plasmin(ogen) activity still remains and still binds fibrinogen and other proteins from plasma, whereas the extra plasmin(ogen) activity of tranexamic acid according to the invention is totally abolished. However, the affinity of the tranexamic acid resin to plasmin(ogen) is not affected.
  • the rigid amino acid is attached to an appropriate spacer, in particular longer than 3 carbon atoms, and the support and the affinity material may deplete plasmin(ogen) from a mixture containing proteins without further altering the protein solution composition.
  • the removal can be done in presence of various buffers.
  • the method of the invention is also suitable for making pure fractions of plasmin(ogen) after elution from the affinity support.
  • the invention pertains to a method for specifically removing or isolating plasmin(ogen) in presence of fibrinogen from a mixture containing plasmin(ogen) by contacting the mixture with a rigid amino acid wherein the amino group of the amino acid and the carboxylic group of the amino acid are about 6-8 Angstroms, preferably about 7 Angstroms apart and the rigid amino acid is covalently bound to the support via the amino group of the amino acid.
  • the mixture is selected from the group consisting of body fluids such as blood; blood fractions, cryoprecipitate, cell cultures, animal tissue extracts, such as bovine lungs, bovine intestines or animal bone extracts gelatin, bovine serum albumin as well as animal derived water immiscible fats, such as lanoline (PC-phosphatidyl choline).
  • body fluids such as blood; blood fractions, cryoprecipitate, cell cultures, animal tissue extracts, such as bovine lungs, bovine intestines or animal bone extracts gelatin, bovine serum albumin as well as animal derived water immiscible fats, such as lanoline (PC-phosphatidyl choline).
  • the method of the invention can be used to obtain highly purified plasmin(ogen) from the respective mixtures.
  • the plasmin(ogen) can be eluted from the solid affinity material.
  • The, as such known, principles of solid phase extraction can be applied here as well.
  • the plasmin(ogen) can be eluted by a solution containing a ligand, which competes with binding sites of the rigid amino acid, e.g. tranexamic acid, at the plasmih(ogen) protein.
  • ligands are typically ⁇ -aminoacids, preferably lysine.
  • lysine may be employed in concentrations of 0.85% by weight to 0.99% by weight. Also other concentrations are possible, especially when the ionic strength of the elution medium is balanced by other ingredients e.g. electrolytes.
  • the plasmin(ogen) eluting from the solid phase can be made free of the elution buffer by a method, which extracts the buffer components for example dialysis.
  • the plasmin(ogen) obtainable according to the method of the invention is characterized by very high purity. The unique property becomes evident from the data: SUMMARY TABLE 1 Comparison of specific activity, purification factor and recovery of plasmin(ogen) from cryo-depleted FFP plasma using the preferred loading and elution conditions for each of the resins.
  • the purity of the eluates was assessed by SDS-gel electrophoresis.
  • the eluates of the three different resins (TEA-Sepharose, Lys-Ceramic Hyper DF and Lys-Sepharose 4B) underwent SDS-PAG by loading a 5-12% gradient of acrylamide and loading 7 ⁇ g protein per lane.
  • the resulting Coomassie Blue stained gel is showed in FIG. 1.
  • FIG. 1 shows a gradient gel SDS-PAGE (5-12% poly-acrylamide) of 7 ⁇ g of proteins eluted by the two methods (described in the material and method), with three different resins -TEA-Sepharose, Lys-Ceramic Hyper DF and Lys-Sepharose 4B. Lanes 1-Glu plasminogen; 2-fibrinogen; 3-Albumin; 4-Immunoglobulin G; 5-molecular wt.
  • Subject of the present invention is also a support having covalently bound a rigid amino acid wherein the amino group of the amino acid and the carboxylic group of the amino acid are apart about 6-8 Angstroms, preferably about 7 Angstroms.
  • the support for performing the method of the invention is preferably a chromatographic material which is able to bind a rigid amino acid wherein the amino group of the amino acid and the carboxylic group of the amino acid are apart about 6-8 Angstroms, preferably about 7 Angstroms.
  • the distance between the amino group and the carboxylic group is kept substantially constant by the rigid constitution of the amino acid.
  • the rigidity of the amino acid can be generated by alicyclic rings, preferably by a cyclohexan ring, wherein the amino and carboxyl group are arranged in 1,4 position of the alicyclic ring.
  • aromatic systems e.g. substituted benzoic acids or aniline substituted acetic acid are within the scope of the invention.
  • the support preferably has bound amino acids selected from the group consisting of tranexamic acid and EMBOCA.
  • the chromatographic material to be employed according to the method of the invention is e.g. a hydrophilic material such as agarose, cellulose, controlled pore glass, silica gels, dextranes or an organic artificial polymer such as based on polyacrylamides polystyrens.
  • a hydrophilic material such as agarose, cellulose, controlled pore glass, silica gels, dextranes or an organic artificial polymer such as based on polyacrylamides polystyrens.
  • Typical materials are commercially available under the trade names Sephacryl® (Pharmacia, Sweden), Ultragel® (Biosepara, France) TSK-Gel Toyopearl® (Toso Corp., Japan), HEMA (Alltech Ass. (Deerfield, Ill., USA), Eupergit® (Rohm Pharma, Darmstadt, Germany).
  • materials based on azlactones (3M, St. Paul, Minn, USA) can be used. Particularly preferred is Ag
  • the method according to the invention is performed by employing a particulate chromatographic material or a monolithic block-material.
  • the particulate material can be suspended in an appropriate medium and the resulting slurry can be used e.g. in a chromatographic column.
  • the method of the invention can also be performed in a batch.
  • the polymers may be used as particulate material or also in form of membranes.
  • the tranexamic acid is bound to the support preferably via a linker, in particular a bifunctional linker, between the support and tranexamic acid.
  • a linker in particular a bifunctional linker, it can be selected from the group consisting of N-hydroxy succinimide, BAPA, CNBr, epoxy, diaminodipropylamine (DADPA), 1,6 diaminohexane, succinic acid, 1,3 diamino-2-propanol, ethylendiamine (EDA), TNB, pyridyldisulfide, iodoacetamide, maleimide activated support or combinations thereof.
  • DADPA diaminodipropylamine
  • EDA ethylendiamine
  • TNB pyridyldisulfide
  • iodoacetamide maleimide activated support or combinations thereof.
  • the support for performing the method of the invention is preferably modified by a moiety which reacts with primary or secondary amino groups.
  • the mixture is incubated with the support for a sufficient time period, and eluted with a neutral aqueous solution containing sodium salts, calcium salts, buffer salts after contacting the mixture with the support having bound tranexamic acid.
  • the plasmin or plasmin(ogen) may be eluted with an aqueous solution containing a sufficient amount of lysine or an equivalent which competes with the covalently bound tranexamic acid.
  • Subject of the invention is a mixture derived from natural sources being substantially free of plasmin(ogen) and plasmin.
  • the mixture of the invention is blood, a blood derivative or blood fraction, cryoprecipitate.
  • a blood derivative of the invention is in particular a plasma derived blood clotting factor or mixture of blood clotting factors, such as FVIII, FIX, fibrinogen, fibronectin, ⁇ 1 -antitrypsin, anti-thrombin III, von Willebrand factor, albumin, immunoglobulin.
  • a plasma derived blood clotting factor or mixture of blood clotting factors such as FVIII, FIX, fibrinogen, fibronectin, ⁇ 1 -antitrypsin, anti-thrombin III, von Willebrand factor, albumin, immunoglobulin.
  • a support having tranexamic acid covalently bound is also subject of the present invention.
  • the support of the invention is preferably a chromatographic material, more preferred a hydrophilic chromatographic material such as dextranes or an organic artificial polymer such as mentioned above.
  • a very preferred support is Agarose® or Sepharose® to which tranexamic acid is bound.
  • the chromatographic material which forms the support may be a particulate material or a monolithic block-material. The latter is described in Hermanson et al, incorporated by reference (Hermanson G T, Mallia A K and Smith P K 1992 “Immobilization Affinity Ligand Techniques” pp. 454 Academic Press, Inc. San Diego, USA).
  • the tranexamic acid is bound to the support via a linker between the support and tranexamic acid.
  • a linker which is able to bind tranexamic acid.
  • Spacer arms or leashes are low molecular weight molecules that are used as intermediary linkers between a support or matrix and affinity ligand which is according to the invention the amino acid having a rigid structure and the amino group about 6-7 Angstrom apart from the carboxyl group.
  • the spacers comprise two functional groups on both ends for easy coupling to ligand and support.
  • the spacer is typically a hydrocarbon compound having two functional groups at its ends. One of the two ends is attached covalently to the matrix using conventional or per se known reactions. The second end is covalently linked to the ligand using another coupling procedure.
  • the linker is a bifunctional linker such as N-hydroxy succinimide, DAPA, CNBr, epoxy, diaminodipropylamine (DADPA), 1,6 diaminohexane, succinic acid, 1,3 diamino-2-propanol, ethylendiamine (EDA), TNB, pyridyldisulfide, iodoacetamide, maleimide activated support or combinations thereof.
  • DADPA diaminodipropylamine
  • EDA ethylendiamine
  • TNB pyridyldisulfide
  • iodoacetamide maleimide activated support or combinations thereof.
  • cryoprecipitate is subjected to affinity chromatography with an immobilized ligand to give an adsorbed fraction and an unadsorbed fraction.
  • the substance, which can be eluted from the adsorbed fraction, is plasmin(ogen).
  • the immobilized ligand may be any analogue, which can interact with the plasmin(ogen) lysine binding sites.
  • the process for preparation of immobilized ligands that are used according to the invention is disclosed underneath. The following examples are illustrative but not limiting.
  • the slurry mixture was stirred slowly for 1 h at room temperature, while adding to it 2.5 g succinic anhydride.
  • the succinylated gel was washed #sequentially with purified water. 1 M NaCl and again with purified water to remove the excess of unreacted succinic acid.
  • the immobilized succinilated DADPA was washed with 250 ml of purified #water, then excess water was suctionated dry to a moist cake and transferred to a 500-ml beaker.
  • the gel was resuspended in 25 ml 0.1 M MES Buffer, pH 4.7 and stirred slowly while 0.25 g of p-aminobenzamidine (Sigma) and 0.75 g EDC (Pierce) were added.
  • the pH of the reaction mixture was maintained at 4.7 for 1 h by adding continuously 0.5 M NaOH. #The reaction mixture was then left overnight at room temperature under continuous slow mixing.
  • the gel was washed successively with 0.5 L of each: purified water, 0.1 M sodium acetate, pH 4.7, 0.5 M sodium bicarbonate and purified water. Immobilized p-aminobenzamidine was stored until use in 0.02% sodium azide at 4° C.
  • the protein solution was washed with 3 cycles of 50 ml buffer at alternating pH's #(0.1 M acetate buffer pH 4 containing 0.5 M NaCl followed by washing with 0.1 M Tris-HCl buffer pH 8 containing 0.5 M NaCl).
  • the immobilized Arginine-Sepharose 4B was stored until use in 0.02% sodium azide at 4° C. #followed by washing for 1 h with 500 ml of purified water, added in aliquots, through a sintered glass filter.
  • 20 ml of coupling buffer (0.1 M NaHCO 3 pH 9.3 and 0.5 M NaCl) and the swelled gel were poured into two tubes containing Arginine. The mixtures were mixed in a plastic tube overnight at RT.
  • a 5 ml syringe cylinder with a diameter of 8.36 mm was packed with 1.5 ml (wet volume) of the following affinity resins described in example 1: immobilized ⁇ -aminohexanoic acid (Sepharose 4B using CNBr as a spacer), immobilized p-aminobenzamidine/Arginine/TEA (Agarose 4% using DADPA as a spacer), immobilized Arginine/TEA (Sepharose 4B using CNBr as a spacer), immobilized Arginine/TEA (Sepharose 6B using epoxy as a spacer) and immobilized L-Lysine (Ceramic HyperDF Hydrogel using epoxy as a spacer).
  • affinity resins described in example 1 immobilized ⁇ -aminohexanoic acid (Sepharose 4B using CNBr as a spacer), immobilized p-aminobenzamidine/Arginine/TEA (Aga
  • the optimized gel packing procedure was as follows: the packed gel was washed with 4 volumes of i) purified water, ii) 1 M NaCl, iii) purified water, iv) TLN 0.1 buffer (Tris 0.05 M Lysine 0.02 M, 0.1 M NaCl, pH 9.0), v) TLN 1 buffer (Tris 0.05 M, Lysine 0.02 M and 1 M NaCl, pH 9.0) and vi) purified water. All samples, loading and washing buffers were applied to the syringe cylinder following a centrifugation at 1000 rpm for 1 min at 25° C.
  • Table 3 illustrates both the efficiency of various gel types in the plasmin(ogen) removal and their capacity to retain fibrinogen in a concentrated cryoprecipitate.
  • the fibrinogen content was measured by a clotting time test while the plasmin(ogen) content was measured by chromogenic assay.
  • Cryoprecipitate was treated with aluminium hydroxide to adsorb the Vitamin K dependent clotting factors, and then incubated with a solvent detergent mixture (SD-1% Tri (n-butyl)phosphate, 1% Triton X-100) for 4 hours at 30° C. to inactivate lipid enveloped viruses.
  • SD-1% Tri (n-butyl)phosphate, 1% Triton X-100 a solvent detergent mixture
  • the SD reagents were removed by castor oil extraction and hydrophobic interaction chromatography, and the preparation was subsequently pasteurized (10 h at 60° C.) in the presence of sucrose and glycine as stabilizers.
  • sucrose and glycine were removed by diafiltration.
  • a column of 10 mm in diameter (Biorad, USA) was packed with either 6 ml (wet volume) of immobilized TEA (TEA Sepharose 6B) or immobilized Lysine (Ceramic HyperDF/Sepharose 4B) and washed with 4 volumes of each of the following solutions in sequence: i) purified water, ii) 1M NaCl, iii) purified water, iv) TLN 0.1 buffer (0.1 M NaCl, Lysine 0.02 M, Tris 0.05 M pH 9.0), v) TLN 1 buffer (1 M NaCl, Lysine 0.02 M, Tris 0.05 M pH 9) and vi) purified water. Equilibration was carried out with 4 volumes of BN1 or alternatively with phosphate buffer. The filtered cryoprecipitate was loaded into the column at a flow rate of 100 ⁇ l/min.
  • Table 4 illustrates the effectiveness of plasmin(ogen) removal by different kinds of resins and the recovery of fibrinogen.
  • the fibrinogen content was measured by the clotting time assay (Clauss assay) while the plasmin(ogen) content was measured by a chromogenic assay.
  • a column of 26 mm in diameter (Pharmacia, Sweden) was packed with 50 ml (wet volume) of immobilized TEA (TEA Sepharose 6B) and washed with 4 volumes of purified water and the same volume of TLN 0.1 buffer (0.1 M NaCl, Lysine 0.02 M, Tris 0.05 M pH 9.0), TLN 1 buffer (1 M NaCl, Lysine 0.02 M, Tris 0,05 M pH 9) and purified water.
  • Equilibration was carried out with 4 volumes of BN1 buffer (NaCl, Tri Na-citrate, CaCl 2 , pH 7.0) and the filtered BAC was passed through the column at a flow rate of 700 ⁇ l/ml.
  • the resulting product was sterile filtered through 0.2 ⁇ m filter.
  • glu-plasmin(ogen) can be used as an indicator of the total plasmin(ogen) in the plasma.
  • a column of 10 mm in diameter (Pharmacia, Sweden) was packed with 2 ml (wet volume) of immobilized TEA and washed with 4 volumes of purified water and the same volume of TLN-0.1 buffer (0.1M NaCl, Lysine 0.02M, Tris 0.05M pH 9.0), TLN-1 buffer (1M NaCl, Lysine 0.02M, Tris 0,05M pH 9.0) and purified water.
  • TLN-0.1 buffer 0.1M NaCl, Lysine 0.02M, Tris 0.05M pH 9.0
  • TLN-1 buffer (1M NaCl, Lysine 0.02M, Tris 0,05M pH 9.0
  • the Imunclone® Glu-plasmin(ogen) ELISA kit (American Diagnostica, Greenwich, Conn., USA) used in these experiments is an enzyme-linked sandwich immunoassay specific for the determination of native human glu-plasmin(ogen) levels.
  • the quantitation limit of the assay (according to the lowest calibration curve standard) in plasma or in plasma derivatives is 0.063 ⁇ g/ml.
  • Plasmin Activity a fibrinolytic assay was performed to semi-quantify plasmin activity in the eluates. Briefly, plasmin(ogen) free-fibrinogen (Enzyme Research) was incubated with various concentrations of normal pooled plasma (Unicalibrator, Stago) or purified plasmin(ogen) eluted from the affinity columns, in the presence of excess streptokinase. The time at which de degradation of the clot was complete was recorded and compared with the complete clot degradation of the sample.
  • Table 8b shows that an average of 99,5% of plasmin(ogen) was removed from the unbound fraction, mostly accounted for by the amount recovered in the eluate (table 8a).
  • TABLE 8a Effect of additional washing of TEA resin with 3 M sodium chloride (Method 1 vs Method 2) on the specific activity, purification and recovery of plasmin(ogen) from plasma.
  • Method 1 Method 1 Method 2 Method 2 Fraction Starting Eluate Starting Eluate Material Material Volume (ml) 40 13.2 40 20 Protein (mg/ml) 46.96 0.291 55.41 0.180 Glu-plasmin(ogen) 65.4 18.2 78.0 142.9 ( ⁇ g/ml) Plasmin(ogen) (IU/ml) not done ⁇ 0.5 ⁇ 1 Specific Activity 0.0014 0.625 0.0014 0.794 (mg plasmin(ogen)/mg protein Purification factor 446 567 Recovery of 91.8 91.6 Plasmin(ogen) %
  • Tables 9a and 9b compare the removal and purification of plasmin(ogen) by two different commercially available immobilized-lysine resins using two different purification methods with each. Although there is relatively little difference in the recovery of plasmin(ogen) in the eluates (9a), it can be seen that by using method 2 and the resin Ceramic HyperDF, a much higher purification of plasmin(ogen) is achieved.
  • Method 1 Eluate Eluate Ceramic Ceramic Hyper Sepharose Hyper Sepahrose Fraction Load DF 4B Load DF 4B Volume 40 8.1 7.2 40 8.2 9.8 (ml) Protein 55.94 0.347 0.345 60.05 0.0719 0.672 (mg/ml) Glu- 65.4 40.3 41.9 60.8 32.2 19.9 plas- min- ogen ( ⁇ g/ml) Specific 0.0012 0.116 0.121 0.001 0.444 0.030 Activity (mg plas- min- ogen/ mg protein) Puri- 97 101 444 30 fication factor Recov- 12.5 11.6 10.9 8.2 ery (%)

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US11/581,753 Expired - Lifetime US7641918B2 (en) 2001-05-21 2006-10-17 Plasma cryoprecipitate substantially free of plasminogen
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