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  Factor viii separationUSPTO Application #: 20070215475Title: Factor viii separation Abstract: The claims describe methods for isolating functionally active Factor VIII using a membrane-based separation system containing a separation membrane to create a first and second interstitial volume between at least two restriction membranes. One or more stabilizing agents are added to the sample and/or an interstitial volume. A solvent in the first interstitial volume maintains FVIII in a desired charge state. Applying a potential between the first and second interstitial volumes separates FVIII on one side of the separation membrane from unwanted molecules on the other side of the separation membrane. These methods may also be used as a substitute for one or more steps in a conventional purification scheme for the separation of native or recombinant FVIII. (end of abstract) Agent: Cooley Godward Kronish LLP Attn: Patent Group - Washington, DC, US Inventors: Elizabeth Jean Seabrook, Thomas Norman Turton, Brendon Conlan USPTO Applicaton #: 20070215475 - Class: 204540000 (USPTO) Related Patent Categories: Chemistry: Electrical And Wave Energy, Non-distilling Bottoms Treatment, Electrophoresis Or Electro-osmosis Processes And Electrolyte Compositions Therefor When Not Provided For Elsewhere, Barrier Separation (e.g., Using Membrane, Filter Paper, Etc.), Ion Selective, Biological Material Prepared, Recovered, Or Treated (e.g., Urine, Etc.) The Patent Description & Claims data below is from USPTO Patent Application 20070215475. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims benefit of the Australian Provisional Application No. PR 6388 filed Jul. 13, 2001. FIELD [0002] The present application relates to methods and apparatus for the separation of clotting factors from blood or recombinant sources, particularly separation of Factor VIII from plasma. BACKGROUND [0003] Human Factor VIII (FVIII) is a 265 kDa glycoprotein which circulates in plasma (0.1 .mu.g/mL) bound to von Willebrand factor (vWf). The glycoprotein is highly sensitive to proteolytic processing which brings about both its activation and destruction, therefore regulating its role as a co-factor in the coagulation cascade (blood clotting). Activated FVIII (FVIIIa) is a co-factor in the activation of Factor X to Factor Xa. A deficiency in FVIII may lead to the bleeding disorder Haemophilia A. [0004] The average industrial yield of FVIII from plasma is 140 to 270 international units (IU) per litre of plasma (1 IU is the average amount of FVIII activity found in 1 mL of pooled plasma=0.2 .mu.g). The first step of FVIII conventional purification is typically cryoprecipitation (traditional Cohn fractionation) which yields 40-50% of FVIII. The cryoprecipitate containing the FVIII is then treated using chromatography, typically immunoaffinity and ion exchange. Minimizing the loss of FVIII at each of the processing steps is desired to improve yield since current supply of suitable FVIII is inadequate. Hemasure Denmark A/S has developed technology in an attempt to combat current problems with traditional Cohn fractionation of plasma. Hemasure uses a high capacity gel filtration step to replace the initial cryoprecipitation step reported to provide a step yield of 60-70% FVIII with a total process yield of 200 IU of FVIII/litre plasma. J. Dam, Downstream, vol. 31, p. 65 (December 1999). Although this method results in an improved yield, there are still significant losses of FVIII in the process. Recombinant production is another source of FVIII, but this source has not replaced FVIII obtained from natural sources. Current purification schemes are time consuming, result in a significant loss of FVIII, and do not adequately remove pathogens, particularly viruses, without adversely affecting FVIII activity or yield. [0005] It has been reported that several thousand different proteins coexist in plasma. Obtaining a given protein from such a complex mixture can be difficult, especially if the given protein must retain its biological activity in its isolated state. Currently, it is very difficult to purify or separate FVIII in reasonable quantities with good yields from plasma. As an example, for plasma having an average protein concentration of 70 mg/mL, FVIII (.about.0.1 .mu.g/mL) constitutes approximately only 0.00014% of total plasma protein. The presence of FVIII in such low concentrations in plasma or recombinant sources usually requires large amounts of plasma or other sources to obtain reasonable commercial yields. Hence, production costs are increased and typically require process step reduction. [0006] FVIII is a relatively unstable protein in plasma. As a result, standard purification technology applied to FVIII separation has difficulty obtaining a method for obtaining large amounts of biologically active FVIII. Current processes involve stabilizing the FVIII preparation at the end of the purification scheme with stabilizing agents, the most common being human albumin. However, addition at the end of the purification process may be too late to protect the activity of the separated FVIII. "Kogenate-F", a recombinant FVIII (rFVIII) therapeutic product formulated with sucrose, may eliminate the need to add human albumin to the preparation. [0007] Viral contamination of FVIII preparations is also a potential problem. Typically, solvent detergent (SD), pasteurization, Methylene Blue (MB) and UV treatment or a combination thereof are used to inactivate viruses in FVIII preparations. However, traditional viral removal steps often result in loss or inactivation of FVIII. SUMMARY [0008] The present application relates to various methods and apparatus for isolating functionally active Factor VIII using a membrane-based electrophoresis separation system. [0009] In one aspect, these methods use an electrophoresis apparatus containing a separation membrane to create a first and second interstitial volume between at least two restriction membranes. One or more stabilizing agents are added to the sample and/or an interstitial volume. A solvent in the first interstitial volume maintains FVIII in a desired charge state. Applying a potential between the first and second interstitial volumes separates FVIII on one side of the separation membrane from unwanted molecules on the other side of the separation membrane. [0010] These and other features of the claims will be appreciated from review of the following detailed description of the application along with the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS [0011] FIG. 1 is a block diagram of a method for isolating functionally active Factor VIII using a membrane-based electrophoresis separation system; [0012] FIG. 2 illustrates the movement of FVIII antigen (FVIII:Ag) in S1 of the electrophoresis system at various pH values over time; [0013] FIG. 3 illustrates the movement of FVIII:Ag in S2 of the electrophoresis system at various pH values over time; [0014] FIG. 4 shows an isoelectric focusing Western blot of FVIII from a highly purified product probed with mouse anti-human FVIII. The results indicate a true pI of 6.2 for FVIII from this source which demonstrates the movement results at various pH values; [0015] FIG. 5 illustrates the movement of FVIII:Ag in S1 and S2 of an electrophoresis apparatus using albumin as a stabilizing agent in the running buffer (final concentration of albumin at 10 mg/mL); [0016] FIG. 6 illustrates the movement of FVIII:Ag in S1 and S2 of an electrophoresis apparatus using Synthamin 17 as a stabilizing agent in the running buffer (final concentration of Synthamin 17 at 0.05 g/mL); [0017] FIG. 7 illustrates the movement of FVIII:Ag in the S1 and S2 of the electrophoresis apparatus using Synthamin 17 as a stabilizing agent in the running buffer (final concentration of Synthamin 17 at 0.025 g/mL); [0018] FIG. 8 illustrates the movement of FVIII:Ag in S1 of an electrophoresis apparatus at various pH values using neat plasma as starting material; [0019] FIG. 9 is an isoelectric focussing (IEF) blot of porcine parvo virus (PPV) where Lane 1 is a pI marker stained with ponceaus S stain, lane 2 is PPV preparation stained with ponceaus S stain, lane 3 is PPV preparation probed with mouse anti-porcine parvo virus; Continue reading... Full patent description for Factor viii separation Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Factor viii separation patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Factor viii separation or other areas of interest. ### Previous Patent Application: Electrodialysis system and process Next Patent Application: Electrophoresis chip and electrophoresis unit having the same Industry Class: Chemistry: electrical and wave energy ### FreshPatents.com Support Thank you for viewing the Factor viii separation patent info. IP-related news and info Results in 0.98918 seconds Other interesting Feshpatents.com categories: Tyco , Unilever , Warner-lambert , 3m |
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