AU2002214468A1 - Process for the preparation of latent antithrombin III - Google Patents

Process for the preparation of latent antithrombin III

Info

Publication number
AU2002214468A1
AU2002214468A1 AU2002214468A AU1446802A AU2002214468A1 AU 2002214468 A1 AU2002214468 A1 AU 2002214468A1 AU 2002214468 A AU2002214468 A AU 2002214468A AU 1446802 A AU1446802 A AU 1446802A AU 2002214468 A1 AU2002214468 A1 AU 2002214468A1
Authority
AU
Australia
Prior art keywords
process according
ammonium sulfate
range
sample
hepes
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
AU2002214468A
Inventor
Goran Karlsson
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Octapharma AG
Original Assignee
Biovitrum AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from SE0004086A external-priority patent/SE0004086D0/en
Application filed by Biovitrum AB filed Critical Biovitrum AB
Publication of AU2002214468A1 publication Critical patent/AU2002214468A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8128—Antithrombin III
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Description

Process for the preparation of latent antithrombin L±X
Field of the invention
The present invention relates to a process for the preparation of latent antithrombin III.
Background of the invention
Antithrombin III (AT) is a plasma glycoprotein with a total molecular weight of 58.1 kDa (Lebing et al, Vox Sang. 67, 117-124, 1994), that inhibits serine proteases in the coagulation cascade and thus plays a major role in the regulation of blood clotting. Antithrombin III is an inhibitor of Factors IXa, Xa, XI and Xlla, as well as of thrombin. Thus, AT regulates clot formation in different stages of the coagulation cascade. A small decrease of AT content in the blood is associated with an increased risk of thromboembolism. Concentrates of AT are used in the prophylaxis and treatment of thromboembolic disorders in patients with acquired or hereditary antithrombin deficiency. In addition, it has been reported that AT has a function in many other processes of the human body, for example in angiogenesis and inflammatory responses. The function of AT in these physiological processes is not fully understood. A particular form of antithrombin III, which was first characterized by Wardell et al (Biochemistry 36, 13133- 13142, 1997), is known as the latent form (L-AT) . L-AT and a selectively elastase cleaved variant have been shown to possess a strong antiangiogenic activity, and also to suppress tumor growth in mice that have been injected subcutaneously with a human neuroblastoma cell line (O'Reilly et al, Science 285, 1926-1928, 1999, and WO 00/20026) . Hence, L-AT must be considered a potential human anti-cancer drug. However, clinical evaluation of this potential drug remains to be performed. Purification of AT with affinity chromatography is done using purified heparin as solid phase bound ligand, as is known in the art. iller-Andersson et al (Thrombosis Research 5, 439-452, 1974) discloses the use of heparin- Sepharose to purify human AT. This chromatographic system has also been useful for the separation between AT and L- AT, where the decreased affinity of heparin for L-AT relative to AT makes it possible to resolve the two components, as described by Chang and Harper (Thrombosis and Haemostasis 77, 323-328, 1997) . Hydrophobic interaction chromatography has been used for the separation of native and latent forms of AT (Karlsson, G & Winge, S. (2001) Protein Expr. Purif. 21:149-155)
Induction of the latent form of AT has previously been performed as described by Wardell et al ( supra) , who obtained 50-60% L-AT by incubating AT in 0.25 M citrate, 10 mM Tris/HCl, pH 7.4, for 15 h in 60 °C.
Upon incubation of native antithrombin III at 60°C in medium or buffer only, aggregates of polymerized protein are often formed. The presence of these aggregates is detrimental to a high yield of latent antithrombin III, and should be avoided as far as possible .
An object of the present invention is then to provide a process for the preparation of latent antithrombin III, L-AT, from a solution of native antithrombin III, AT, which process gives a higher yield of the desired product than the prior art process.
A further object of the invention is to provide a process for the preparation of L-AT from AT, wherein the production of aggregates of AT polymers is kept to a minimum .
Another object of the invention is to provide such a process for the conversion of AT to L-AT, in which commonly available reagents and buffer solutions are used, and which is performed in vitro.
Still another object of the invention is to provide a process for the preparation of L-AT from AT, which is readily scaled up for industrial production of L-AT. Summary of the invention
The aforementioned and other objects of the invention are met by a process as defined in the claims. Thus, a process is provided, which comprises incubation of a solution of native antithrombin III in the presence of sulfate ions and a buffer selected from Good's zwitterionic buffers. It has surprisingly been found that these incubation conditions makes possible the recovery of L-AT from the process in yields that are substantially higher than those obtained by methods of the prior art (notably the citrate conditions of Wardell et al) , while avoiding possible aggregation problems.
Figure legends
Figure 1: Heparin affinity chromatography of antithrombin using a sodium chloride gradient, 0-2 (5-60 min) . The injected amount of protein was 100 μg for sample A-B, and 150 μg for sample C-D. All samples were incubated at 60 °C for 16 h, except for the reference AT sample A, which was not heat-treated (sample 7 in the example) . Sample B (sample 6 in the example) was incubated according to Wardell, ie in 0.5 M citrate. Samples C (sample 2 in the example) and D (sample 1 in the example) were incubated in 5 mM HEPES, pH 7.4, with 0.9 and 0.8 M ammonium sulfate respectively. Integration of the low affinity heparin- binding peak, eluting at 22 min, gave 44%, 71% and 89% of the total integrated area for samples B, C, and D, respectively. Native AT eluted at 39 min.
Figure 2: Native electrophoresis of antithrombin samples, using 12.5% polyacryl mide in a homogeneous gel. The amount of sample was 0.5 rø protein/lane, and the gels were silver-stained after running. All samples, except for lane 7, were incubated in 60 °C for 16 h.
Lane 1) 5 mM HEPES, 0.8 M ammonium sulfate, pH 7.4 Lane 2) 5 mM HEPES, 0.9 M ammonium sulfate, pH 7.4 Lane 3) 5 mM HEPES, 1.1 M ammonium sulfate, pH 7.4 Lane 4) 5 mM HEPES, 1.4 M ammonium sulfate, pH 7.4 Lane 5) 5 mM HEPES, 2.0 M ammonium sulfate, pH 7.4 Lane 6) 10 mol Tris/HCl, 0.5 M trisodium citrate, pH 7.4 (according to Wardell et al . 1997) Lane 7) Reference AT sample, not heat-treated
Lane 8) 25 mM sodium phosphate, 100 mM sodium chloride, pH 7.4 Lane 9) 25 mM HEPES, 0.8 M ammonium sulfate, pH 7.4 Lane 10) 5 mM HEPES, 0.5 M ammonium sulfate, pH 7.4 Lane 11) 5 mM HEPES, 2.0 M ammonium sulfate, pH 7.4 Lane 12) 5 mM HEPES, 0.8 M ammonium sulfate, pH 7.0 All lane numbers correspond to the sample numbers listed in the example below.
Detailed description of the invention
The invention provides a process for the preparation of latent antithrombin III (referred to as L-AT) , starting from a solution of antithrombin III in its native form (referred to as AT) . AT can be isolated from blood plasma by heparin-Sepharose chromatography as has been described.
According to the invention, the AT is then incubated in the presence of sulfate ions and a buffer. The incubation temperature and duration can be readily determined by the skilled person, but normal pasteurization conditions, such as a temperature of about 60 °C during about 16 hours, have been found to work well.
The sulfate ions are preferably provided in the form of a sulfate salt. Here, the use of an alkali metal sulfate, an alkaline earth sulfate or ammonium sulfate is preferred. Especially preferred is the use of ammonium sulfate. A suitable concentration of sulfate ions in the process according to the invention lies in the range from 0.5 to 2.0 M, preferably from 0.7 to 1 M, a concentration between 0.8 and 0.9 M being most preferred. Another component of the incubation mixture is a buffer selected from Good's zwitterionic buffers (Good et al, Biochemistry 5, 467-477, 1966) . Which of the indicated buffers to use in the process of the invention can be determined without undue experimentation, keeping in mind that the buffer should fulfil most or all of the following requirements: it should exhibit a pKa value of between about 6 and about 9, a maximum solubility in water and a minimum solubility in other solvents, produce a minimum of salt effects, be stable at the experimental conditions used, and not absorb light in the visible or ultraviolet spectral regions (so as not to interfere with spectrophotometric measurements). Good's zwitterionic buffers, including buffers such as HEPES, MES and PIPES, typically present the desired characteristics. The use of HEPES is particularly preferred in the process according to the invention. The widely used Tris buffer is unsuitable for the purposes of the invention. Preferred buffer concentrations are somewhat dependent on the buffer chosen, but typically lie in the range from 1 to 25 mM, more preferably from 2.5 to 10 mM, most preferably from 4 to 6 mM.
As indicated above, the pH of the incubation reaction should lie between pH 6 and pH 9, preferably between pH 7 and pH 8, most preferably between pH 7.4 and pH 7.6.
Following the incubation of AT under the conditions outlined above, separation of the L-AT thus obtained from remaining AT is preferably performed using heparin affinity chromatography. The L-AT exhibits a lower binding affinity to heparin than AT, eluting substantially faster and enabling easy separation of the two forms of antithrombin III.
The preparation of L-AT thus obtained is advantageously subjected to treatment for the inactivation or removal of pathogens, particularly in the form of viruses and prions . This can be done in any stage of the process using one of several methods for inactivation or removal known in the art, or combinations of such methods. Examples of such methods include chemical inactivation, heat inactivation, light inactivation, microwave inactivation and nano-filtration removal. A dead-end filtration procedure with a high salt content, like that described in WO96/00237, is particularly preferred, alone or in combination with other procedures. The removal and inactivation of pathogens can also be performed when the antithrombin III molecules are in the native state, before conversion to L-AT.
The invention is further illustrated by the following, non-limiting example.
EXAMPLE A laboratory sample of AT, > 95% pure, was obtained from Plasma Products, Pharmacia, Stockholm, Sweden. This sample was prepared according to known methods (Miller- Andersson et al, supra) and used for induction of the latent form of antithrombin.
Preparation of L-AT
The laboratory sample of AT was transferred to the following solutions:
Sample 1) 5 mM HEPES, 0.8 M ammonium sulfate, pH 7.4
Sample 2) 5 mM HEPES, 0.9 M ammonium sulfate, pH 7.4
Sample 3) 5 mM HEPES, 1.1 M ammonium sulfate, pH 7.4
Sample 4) 5 mM HEPES, 1.4 M ammonium sulfate, pH 7.4
Sample 5) 5 mM HEPES, 2.0 M ammonium sulfate, pH 7.4 Sample 6) 10 mmol Tris/HCl, 0.5 M trisodium citrate, pH 7.4 (according to Wardell et al . 1997)
Sample 7-8) 25 mM sodium phosphate, 100 mM sodium chloride, pH 7.4
Sample 9) 25 mM HEPES, 0.8 M ammonium sulfate, pH 7.4 Sample 10) 5 mM HEPES, 0.5 M ammonium sulfate, pH 7.4
Sample 11) 5 mM HEPES, 2.0 M ammonium sulfate, pH 7.4
Sample 12) 5 mM HEPES, 0.8 M ammonium sulfate, pH 7.0
Sample 13) 5 mM HEPES, 0.8 M ammonium sulfate, pH 7.8
All buffers listed above were adjusted to the desired pH at room temperature; 1 M HCl was used for adjustment of sample 6, while 1 M sodium hydroxide was used for pH adjustment of all other samples. AT at a final concentration of 6 mg/ l was incubated in the solutions (samples 1-13) in glass tubes for 16 h at 60°C (except for sample 7, which was kept in a fridge at about 8°C) and transferred to a solution containing 50 mM Tris/HCl, 50 mM sodium chloride, pH 7.4, using small gelfiltration columns (NAP-5 Amersham Pharmacia Biotech, Uppsala, Sweden) .
The formation of L-AT in the samples was analyzed by heparin affinity chromatography, and the presence of aggregates was analyzed by native electrophoresis.
Heparin affinity chromatography
This method was performed based on Chang and Harper ( supra) . A HPLC equipped with an TSK Heparin® column (Tosohaas, Stuttgart, Germany, 7.5 i.d. x 75 mm, 10 μm, 1000 A) was used. Eluting buffers were 20 mM Tris/HCl buffer, pH 7.4 (buffer A) and 2 M sodium chloride in 20 mM Tris/HCl buffer, pH 7.4 (buffer B) . A linear gradient was run (0-5 min of 0% B, 5-60 min 0-100% B, 60-90 min 0% B) . The flow rate was 0.4 ml/min and detection was carried out by measuring the absorbance at 280 nm.
Native polyacrylamide gel electrophoresis
Electrophoresis was performed using a 12.5% polyacrylamide homogeneous Phast® gel (Amersham Pharmacia
Biotech, Uppsala, Sweden) employing the recommended running parameters. 0.5 μg protein in 1 μl was loaded in each lane. A diamino silver staining was performed according to the booklet from Pharmacia & Upjohn (Phast System™, Technical Note No 2, Two-dimensional electrophoresis with PhastGel™ separation media, Pharmacia LKB Biotechnology AB, Uppsala, Sweden) , except that use was made of a slightly stronger fixation solution, containing 50% ethanol, 10% acetic acid and 40% water.
Antithrombin activity
Sample 2 (incubation in 0.9 M ammonium sulfate) was analyzed regarding biological AT activity with the thrombin chro ogenic peptide substrate (S-2238) (Chromogenix, Molndal, Sweden), according to Handeland et al . (Scand J. Haematol . 31, 427-436, 1983). The assay solution consisted of thrombin, heparin, chromogenic substrate and sample, and the response after incubation was recorded as a change in absorbance at 405 nm.
Results
Heparin affinity chromatography gave elution of native AT at 39 min (about 0.9 M sodium chloride) and the main latent peak eluted at 22 min (about 0.3 M sodium chloride) (figure 1A-1B) . Integration of the low heparin-binding peak indicated a yield of 44% (figure IB) for the sample prepared according to Wardell' s method (sample 6), while incubation in 0.9 and 0.8 M ammonium sulfate (samples 2 and 1, respectively) yielded 71% and 89% respectively of the total integrated area (figure 1C-1D) . Table 1 shows that the percentage of formed L-AT decreases at increased concentration of ammonium sulfate/HEPES or at a higher pH value.
Native electrophoresis of AT incubated at 60 °C in phosphate/NaCl (sample 8) gave a strong formation of aggregates, and only a minor part of the protein remained in the monomeric form (figure 2, lane 8) . AT incubated according to Wardell (figure 2, lane 6), as well as the not incubated AT (figure 2, lane 7), gave no aggregates. Incubation in 0.5 M ammonium sulfate (sample 10) induced a strong aggregation (figure 2, lane 10), while 0.8 M (sample 1) only gave a minor part of aggregates (figure 2, lane 1) . Ammonium sulfate at a concentration of 0.9 - 2.0 M (samples 2-5) resulted in no visible aggregates (figure 2, lanes 2- 5) . At pH 7.0, a lot of aggregates were observed (figure 2, lane 12) , while a pH of 7.8 gave a smaller amount of aggregates (data not shown) . Antithrombin activity assay on sample 2 (with 0.9 M ammonium sulfate) showed that 34% of the original specific activity remained; this should be compared with the 29% yield of high affinity heparin-binding AT upon analysis of the same sample by affinity chromatography (Table 1) .
Table 1: Heparin affinity chromatography. Formation of 5 L-AT in various sample buffers after 16 h incubation in 60 °C.
% AT with
Sample low heparin
Incubation solution no1 affinity mmol Tris/HCl, 0.5 M citrate, pH 7.4 (Wardell -)) 66 44* mM Hepes, 0.5 M ammonium sulfate, pH 7.4 10 99 mM Hepes, 0.8 M ammonium sulfate, pH 7.4 1 89 mM Hepes, 0.9 M ammonium sulfate, pH 7.4 2 71* mM Hepes, 1.1 M ammonium sulfate, pH 7.4 3 56* mM Hepes, 1.4 M ammonium sulfate, pH 7.4 4 49* mM Hepes, .2.0 M ammonium sulfate, pH 7.4 5 48* 5 mM Hepes, 0.8 M ammonium sulfate, pH 7.4 9 70 mM Hepes, 0.8 M ammonium sulfate, pH 7.0 12 99 mM Hepes, 0.8 M ammonium sulfate, pH 7.8 13 65
1 According to the example
* No visible aggregates when analyzed by native electrophoresis (see Fig. 2)
10
Experimental conclusions
By incubation of AT in 5 mM HEPES, pH 7.4, containing 0.8 or 0.9 M ammonium sulfate in 60 °C for 16 h, about 85- 90% and 70-75% respectively of AT was transformed to the
15 latent form. Native electrophoresis showed a small part of aggregates at 0.8 M ammonium sulfate and no visible aggregates at 0.9 M. In a purification procedure, such small amounts of aggregate can be easily removed by gel filtration or similar techniques. 0 The optimal concentration for the conversion of AT to L-AT using ammonium sulfate is 0.8-0.9 M. The conversion will also yield good results between 0.7 and 1 M, and some results between 0.5 and 2.0 M. For formation of L-AT, a process using 0.5-2.0 M ammonium sulfate, preferably 0.8- 0.9 M, and up to 25 mM HEPES, preferably not more than 10 mM, at a pH near 7.4 has been found to give the most pleasing results. The percentage of L-AT formed will decrease at a higher concentration of ammonium sulfate/HEPES or at a higher pH value. In addition, for the prevention of formation of aggregates, it is necessary not to use too low an ammonium sulfate concentration or too low a pH value .

Claims (16)

1. A process for the preparation of latent antithrombin III, comprising incubation of a solution of native antithrombin III in the presence of sulfate ions and a buffer selected from Good's zwitterionic buffers.
2. A process according to claim 1, wherein said sulfate ions are provided as a salt selected from ammonium sulfate, alkali metal sulfates and alkaline earth sulfates.
3. A process according to claim 2, wherein said sulfate salt is ammonium sulfate.
4. A process according to any one of claims 1-3, wherein the concentration of said sulfate ions is within the range from 0.5 to 2.0 M.
5. A process according to claim 4, wherein the sulfate ion concentration is within the range from 0.7 to 1 M.
6. A process according to claim 5, wherein the sulfate ion concentration is within the range from 0.8 to 0.9 M.
7. A process according to any one of claims 1-6, wherein said buffer comprises a HEPES buffer.
8. A process according to any one of claims 1-7, wherein the concentration of said buffer is within the range from 1 to 25 mM.
9. A process according to claim 8, wherein the buffer concentration is within the range from 2.5 to 10 mM.
10. A process according to claim 9, wherein the buffer concentration is within the range from 4 to 6 mM.
11. A process according to any one of claims 1-10, wherein the pH value is within the range from 6 to 9.
12. A process according to claim 11, wherein the pH value is within the range from 7 to 8.
13. A process according to claim 12, wherein the pH value is within the range from 7.4 to 7.6.
14. A process according to any one of claims 1-13, further including a treatment for inactivation or removal of pathogens, especially viruses and prions .
15. A process according to claim 1, wherein the native antithrombin III has been treated for inactivation or removal of pathogens, especially viruses and prions.
16. A process according to claim 14 or 15, wherein said treatment comprises one of, or a combination of, methods selected from chemical inactivation, heat inactivation, light inactivation, microwave inactivation and nano-filtration removal.
AU2002214468A 2000-11-08 2001-11-08 Process for the preparation of latent antithrombin III Abandoned AU2002214468A1 (en)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
SE0004086A SE0004086D0 (en) 2000-11-08 2000-11-08 Preparation process
SE0004086 2000-11-08
US25214800P 2000-11-20 2000-11-20
US60252148 2000-11-20
PCT/SE2001/002473 WO2002038610A1 (en) 2000-11-08 2001-11-08 Process for the preparation of latent antithrombin iii

Publications (1)

Publication Number Publication Date
AU2002214468A1 true AU2002214468A1 (en) 2002-05-21

Family

ID=26655296

Family Applications (1)

Application Number Title Priority Date Filing Date
AU2002214468A Abandoned AU2002214468A1 (en) 2000-11-08 2001-11-08 Process for the preparation of latent antithrombin III

Country Status (15)

Country Link
EP (1) EP1332159A1 (en)
JP (1) JP2004522707A (en)
KR (1) KR20030057545A (en)
AU (1) AU2002214468A1 (en)
BR (1) BR0115188A (en)
CA (1) CA2428055A1 (en)
CZ (1) CZ20031277A3 (en)
EE (1) EE200300218A (en)
HU (1) HUP0301766A3 (en)
IL (1) IL155539A0 (en)
MX (1) MXPA03004030A (en)
NO (1) NO20032048L (en)
PL (1) PL360410A1 (en)
RU (1) RU2003117016A (en)
WO (1) WO2002038610A1 (en)

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030205538A1 (en) 2002-05-03 2003-11-06 Randel Dorian Methods and apparatus for isolating platelets from blood
US7832566B2 (en) 2002-05-24 2010-11-16 Biomet Biologics, Llc Method and apparatus for separating and concentrating a component from a multi-component material including macroparticles
US20060278588A1 (en) 2002-05-24 2006-12-14 Woodell-May Jennifer E Apparatus and method for separating and concentrating fluids containing multiple components
US7845499B2 (en) 2002-05-24 2010-12-07 Biomet Biologics, Llc Apparatus and method for separating and concentrating fluids containing multiple components
SE0203770D0 (en) * 2002-12-19 2002-12-19 Biovitrum Ab Method of separation
DK1848473T3 (en) 2005-02-07 2013-09-02 Hanuman Llc Plasma concentrations device
US7694828B2 (en) 2005-04-27 2010-04-13 Biomet Manufacturing Corp. Method and apparatus for producing autologous clotting components
US8567609B2 (en) 2006-05-25 2013-10-29 Biomet Biologics, Llc Apparatus and method for separating and concentrating fluids containing multiple components
EP2146794B1 (en) 2007-04-12 2016-10-19 Biomet Biologics, LLC Buoy suspension fractionation system
US8328024B2 (en) 2007-04-12 2012-12-11 Hanuman, Llc Buoy suspension fractionation system
WO2009108890A1 (en) 2008-02-27 2009-09-03 Biomet Biologics, Llc Methods and compositions for delivering interleukin-1 receptor antagonist
WO2009111338A1 (en) 2008-02-29 2009-09-11 Biomet Manufacturing Corp. A system and process for separating a material
US8313954B2 (en) 2009-04-03 2012-11-20 Biomet Biologics, Llc All-in-one means of separating blood components
US9011800B2 (en) 2009-07-16 2015-04-21 Biomet Biologics, Llc Method and apparatus for separating biological materials
US8591391B2 (en) 2010-04-12 2013-11-26 Biomet Biologics, Llc Method and apparatus for separating a material
US9642956B2 (en) 2012-08-27 2017-05-09 Biomet Biologics, Llc Apparatus and method for separating and concentrating fluids containing multiple components
US9950035B2 (en) 2013-03-15 2018-04-24 Biomet Biologics, Llc Methods and non-immunogenic compositions for treating inflammatory disorders
US9895418B2 (en) 2013-03-15 2018-02-20 Biomet Biologics, Llc Treatment of peripheral vascular disease using protein solutions
US10143725B2 (en) 2013-03-15 2018-12-04 Biomet Biologics, Llc Treatment of pain using protein solutions
US10208095B2 (en) 2013-03-15 2019-02-19 Biomet Manufacturing, Llc Methods for making cytokine compositions from tissues using non-centrifugal methods
US20140271589A1 (en) 2013-03-15 2014-09-18 Biomet Biologics, Llc Treatment of collagen defects using protein solutions
US9550028B2 (en) 2014-05-06 2017-01-24 Biomet Biologics, LLC. Single step desiccating bead-in-syringe concentrating device

Also Published As

Publication number Publication date
MXPA03004030A (en) 2004-02-12
WO2002038610A1 (en) 2002-05-16
HUP0301766A2 (en) 2003-08-28
KR20030057545A (en) 2003-07-04
CA2428055A1 (en) 2002-05-16
RU2003117016A (en) 2004-12-10
NO20032048D0 (en) 2003-05-07
IL155539A0 (en) 2003-11-23
PL360410A1 (en) 2004-09-06
EP1332159A1 (en) 2003-08-06
CZ20031277A3 (en) 2003-08-13
NO20032048L (en) 2003-05-07
HUP0301766A3 (en) 2005-12-28
BR0115188A (en) 2004-02-03
JP2004522707A (en) 2004-07-29
EE200300218A (en) 2003-08-15

Similar Documents

Publication Publication Date Title
US20020090711A1 (en) Process for preparing latent antithrombin III
AU2002214468A1 (en) Process for the preparation of latent antithrombin III
KR100451266B1 (en) Purification Method of α-1 Protein Inhibitor Using Novel Chromatography Separation Conditions
EP0317376B2 (en) Preparation of a concentrate of high-purity human factor IX and of other plasma proteins
Evans et al. Heparin binding site, conformational change, and activation of antithrombin
JP2009524622A (en) Purification and use of wound healing aids
JPH0764751B2 (en) Process for producing alpha-1-proteinase inhibitor
US4379087A (en) Method of preparing alpha-1-proteinase inhibitor
US5777081A (en) Process for producing an inter-alpha-trypsin inhibitor concentrate for therapeutic use and concentrate thus obtained
JP4950053B2 (en) Isolation method of haptoglobin
US5583111A (en) Thrombin inhibitors
EP0282363B1 (en) Method for preparation of a concentrate of alpha-1-antitrypsin from a human plasma fraction, and its use as a pharmaceutical
ZA200303492B (en) Process for the preparation of latent antithrombin III.
Karlsson et al. Separation of latent, prelatent, and native forms of human antithrombin by heparin affinity high-performance liquid chromatography
JPH03218399A (en) Urine-derived anticoagulant substance, its production and medical composition containing the same substance
Karlsson et al. Separation between the α and β forms of human antithrombin by hydroxyapatite high-performance liquid chromatography
Karlsson Pasteurization of antithrombin without generation of the prelatent form of antithrombin
AU777294B2 (en) Process for the purification of antithrombin-III
Karlsson et al. Preparative conversion of native human antithrombin to the latent form
US6777541B2 (en) Protein purification II
US20030180925A1 (en) Method for making purified plasminogen activator-inhibitor type 1 (PAI-1) and purified PAI-1 made therefrom
WO2004056870A2 (en) Method for separation of antithrombin
CA2328605A1 (en) Protein z-dependent protease inhibitor
WO2000034453A1 (en) PURIFICATION OF α1-PROTEINASE INHIBITOR
WO2000043412A1 (en) Compositions containing highly purified heparin cofactor ii and method for separating the same