Chemistry: electrical and wave energy – Processes and products – Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
2000-04-11
2002-06-11
Snay, Jeffrey (Department: 1743)
Chemistry: electrical and wave energy
Processes and products
Electrophoresis or electro-osmosis processes and electrolyte...
Reexamination Certificate
active
06402913
ABSTRACT:
TECHNICAL FIELD
The present invention relates to the separation of biomolecules from plasma, particularly human plasma.
BACKGROUND ART
Human plasma contains approximately 3000 proteins with a variety of functions and potential therapeutic uses. Tight control of plasma available for blood fractionation means that the supply of important therapeutic agents like IgG is severly curtailed. This together with methodology which ends in very low yields and takes three to five days contributes to the international shortfall of major plasma fractions.
The present inventors have found that rapid isolation times, high recoveries and high-resolution make Gradiflow™ technology a viable alternative purification technology to conventional Cohn precipitation and column chromatography [1, 2].
Albumin and IgG both have enormous importance in medicine and therefore are of considerable commercial value. Albumin alone has an estimated annual global market value of $US1.5 billion [3]. Conventional purification protocols are cumbersome and expensive with low yields and long processing times [4].
Albumin is the most abundant protein component (50 mg/mL) in human plasma and functions to maintain whole blood volume and oncotic pressure. Albumin also regulates the transport of protein, fatty acids, hormones and drugs [4]. Clinical uses include blood volume replacement during surgery, treatment of shock, serious bums and other medical emergencies and the stabilisation of other pharmaceutical products.
Albumin has a molecular mass of 67 kDa and an isoelectric point (pI) of approximately 4.9. The protein consists of a single subunit and is globular in shape [5]. Conventional purification schemes use the Cohn ethanol precipitation method and result in only 50% recovery.
Immunoglobulin G (IgG) is the most abundant of the immunoglobulins, representing almost 70% of the total immunoglobulin component in human serum. The concentration of IgG in normal plasma is approximately 10 mg/mL [6]. The IgG plays an essential role in the immune response and have clinical uses including treatment of snake and spider bites, neurological disorders and IgG is commonly used in analytical or diagnostic kits.
The gamma-globulins have a molecular mass of approximately 150 kDa and consist of four chains, two of which are light and two of which are heavy [6]. Immunoglobulins are traditionally isolated using Cohn ethanol precipitation or alternatively affinity chromatography [7].
Alpha-1-antitrypsin is an acid glycoprotein of 54 kDa with an isoelectric point of 4.8 and is used in the treatment of hereditary emphysema [8]. Conventional purification schemes utilise a combination of Cohn fractionation and column chromatography with the major difficulty being the removal of albumin from &agr;-1-antitrypsin preparations [9]. Current production schemes provide a yield of approximately 30% and much of this is contaminated with albumin. The present inventors have adapted Gradiflow™ to provide an alternative technique for producing highly pure &agr;-1-antitrypsin with a yield of above 70%. This strategy also exemplifies Gradiflow™ technology's use in isolating protease inhibitors.
Gradiflow™ Technology
Gradiflow™ technology utilises molecular characteristics of size and charge to isolate protein [1] with the resolution of two-dimensional electrophoresis and the throughput of preparative chromatography. Proteins exist as charged molecules above or below their isoelectric point (pI). In the Gradiflow™ the net charge on a macromolecule is controlled by the choice of buffer pH. The proteins are separated in an electric field by charge and/or size differences [2].
The present inventors have found that the Gradiflow™ technology can be adapted to purify a number of different biomolecular components from plasma. The present inventors have devised methodology for the rapid isolation of albumin, IgG and &agr;-1-antitrypsin from a single volume of plasma in a four-phase process with high yield and low cost.
Disclosure of Invention
In a general aspect, the present invention relates to the sequential separation of a number of biomolecules present in a plasma sample using four major separation phases or processes.
In a first aspect, the present invention consists in a method of separating components from plasma, the method comprising the steps:
Phase I—Removal of albumin, &agr;-1-antitrypsin and small contaminants
(a) placing the plasma in a first solvent stream, the first solvent stream being separated from a second solvent stream by a first electrophoretic separation membrane having a molecular mass cut-off less than the molecular mass of albumin and a restriction membrane having a molecular mass cut-off less than the first electrophoretic separation membrane;
(b) selecting a buffer for the first solvent stream having a pH greater than the pI of albumin;
(c) applying an electric potential between the two solvent streams causing movement of albumin and &agr;-1-antitrypsin through the first electrophoretic membrane into the second solvent stream while biomolecules having a molecular mass greater than albumin and &agr;-1-antitrypsin are substantially retained in the first solvent stream, or if entering the first electrophoresis membrane, being substantially prevented from passing through the first electrophoresis membrane, wherein biomolecules in the plasma having a molecular mass less than albumin and &agr;-1-antitrypsin are caused to move through the first separation membrane and the restriction membranes to a waste collection;
(d) optionally, periodically stopping and reversing the electric potential to cause movement of biomolecules having a molecular mass greater than albumin and &agr;-1-antitrypsin having entered the first electrophoresis membrane to move back into the first solvent stream, wherein substantially not causing any albumin or &agr;-1-antitrypsin that have entered the second solvent stream to re-enter first solvent stream;
(e) maintaining steps (c) and optionally (d) until the desired amount of albumin and &agr;-1-antitrypsin have been collected as an albumin/&agr;-1-antitrypsin pool and biomolecules having a molecular mass less than albumin and &agr;-1-antitrypsin have been removed from the first solvent stream to form a treated plasma;
Phase II—Removal of large contaminants
(f) placing the treated plasma in a third solvent stream, the third solvent stream being separated from a fourth solvent stream by a second electrophoretic separation membrane having a molecular mass cut-off less than the molecular mass of immunoglobulins;
(g) selecting a buffer for the third solvent stream having a pH above neutral;
(h) applying an electric potential between the third and fourth solvent streams causing movement of biomolecules having a molecular mass less that that of immunoglobulins in the treated plasma through the second electrophoretic separation membrane into the fourth solvent stream while immunoglobulins and other biomolecules having a molecular mass greater than immunoglobulins are substantially retained in the third solvent stream, or if entering the second electrophoresis separation membrane, being substantially prevented from passing through the second electrophoresis separation membrane;
(i) optionally, periodically stopping and reversing the electric potential to cause movement of immunoglobulins and other biomolecules having a molecular mass greater than immunoglobulins having entered the second electrophoresis separation membrane to move back into the third solvent stream, wherein substantially not causing any biomolecules having a molecular mass less than immunoglobulins that have entered the fourth solvent stream to re-enter third solvent stream;
(j) maintaining steps (h) and optional (i) until the desired amount of biomolecules having a molecular mass less than immunoglobulins have been removed from the third upstream to form an immunoglobulins concentrate;
(k) removing the biomolecules from the fourth solven
Conlan Brendon
Gilbert Andrew Mark
Nair Chenicheri Hariharan
Baker & McKenzie
Gradipore Limited
Siefke Samuel P.
Snay Jeffrey
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