Liquid purification or separation – Processes – Chromatography
Reexamination Certificate
2001-06-04
2004-06-08
Kim, John (Department: 1723)
Liquid purification or separation
Processes
Chromatography
C210S198200, C210S252000, C210S321600, C210S635000, C210S646000, C210S650000, C210S651000, C436S073000, C436S161000, C436S177000, C436S178000, C436S528000, C436S529000, C530S413000, C530S414000, C530S417000
Reexamination Certificate
active
06746607
ABSTRACT:
The present invention relates to the use of an adsorbent gel combining the properties of size exclusion and affinity chromatographies (AdSEC, for “Adsorptive Size Exclusion Chromatography”).
The principle of an AdSEC gel results from the fusion of two chromatographic techniques: size exclusion and affinity, so as to obtain supports combining the most advantageous properties thereof.
Size exclusion chromatography (gel filtration) allows the separation of molecules according to their steric bulk alone during their passive diffusion in a molecular sieve (gel). The largest molecules cannot penetrate the crosslinked matrix and are consequently excluded more rapidly from the column. This technique possesses the characteristic feature of not exhibiting interactions between the support and the molecules, and therefore of being relatively only slightly sensitive to the biochemical conditions (pH, ionic strength) of the solution. On the other hand, because of its principle of diffusion, the limiting factors for its use are generally a long operation time (because low flow rates are used), as well as a relatively limited deposition of samples (1 to 5% of the column volume).
Affinity chromatography is based on molecular interactions between the support (matrix onto which affinity ligands are grafted) and the molecules to be separated. Among these affinity ligands, immobilized metal ions, introduced in 1975 by Porath et al. (Nature, 1975, 258, 598-599), represent a method of separation based on the interactions (coordination bonds) between biomolecules in solution and metal ions immobilized on a support; Zn(II), Cu(II), Ni(II) and Co(II) ions are the most commonly used. This is described as immobilized metal ion affinity chromatography (IMAC).
The combined use of the principles of size exclusion and affinity chromatographies (AdSEC) has been discussed by Porath et al. (Int. J. of Bio-Chromatogr., 1997, 3, 9-17). These authors have shown that iminodiacetic derivatives of dextran bearing metal ions as affinity ligand allow size exclusion and are capable of effectively concentrating solutions by their properties of adsorption and affinity. These authors have shown that an AdSEC gel column having a volume of 5 ml could bind a high percentage of compounds having a molecular weight of between 5 kDa and 50 kDa and concentrate them about 1000 fold in a single operation.
Such supports make it possible to adsorb the smallest molecules (having affinity for the grafted ligand) at high rates and volumes (not permitted in gel filtration). Moreover, during the synthesis of the adsorbent gel, the threshold of accessibility to the affinity ligand may be modulated during the synthesis of the gel according to the size of the biomolecule to be removed or to be purified.
Terminal renal insufficiency currently affects 22,000 people in France of which 20,000 are treated by iterative hemodialysis. Only 1800 can hope to undergo transplants each year, knowing that a quarter of them will return within 5 years to hemodialysis because of a rejection while waiting for a new transplant.
The survival of the uremic individual, all methods considered, can exceed 25 years if they do not suffer from a severe cardiovascular condition. In this case, the quality of survival is profoundly impaired over the years by the osteoarticular complications of terminal uremia, at the forefront of which there are described erosive arthropathies subsequent to depositions of &bgr;2-microglobulin (&bgr;2-M).
The mechanism of onset of these arthropathies begins as soon as the renal insufficiency responsible for accumulation of &bgr;2-microglobulin appears. This protein, having a molecular weight of 11,800 Da, will accumulate in the body over the years and become selectively deposited at the level of the cervical disks, of the shoulders, of the hips and of the wrists. Cardiac and digestive depositions have been reported. These depositions will make fragile the joint and the adjacent bone up to total destruction of the joint. Thus, a breakdown of the vertebral bodies is observed which can cause medullary compression with loss of control of the four members, irreversible articular luxations, loss of prehension in the hands and pseudofractures of the hip. Ductal nerve compressions are observed such as the carpal tunnel syndrome.
These complications irremediably lead the uremic individual toward invalidity and the bedridden state which conventional methods of dialysis cannot prevent. A transplant allows these lesions to be stabilized.
To effectively prevent these complications, it is important to be able to effectively purify the polluting components of blood, in particular &bgr;2-microglobulin, which are synthesized daily by the body and which are not, or not sufficiently, removed by the defective kidneys in dialyzed patients.
The purification of these various biomolecules can only be done on artificial membranes during dialysis, which are currently not sufficiently effective in spite of purification by filtration and nonspecific membrane adsorption.
The existing techniques for removing biomolecules, including &bgr;2-microglobulin, are currently of 3 types:
1. Removal of Biomolecules by Hemodialysis
Hemodialysis is a technique intended for subjects suffering from partial or complete renal insufficiency (FIG.
1
). It consists in extracorporeal treatment of blood, providing the same functions as the kidney using a membrane process. The essential part of the hemodialyzer (
1
) is an exchange membrane, on either side of which circulate countercurrentwise the patient's blood and the dialyzate obtained from the hemodialysis generator (
2
). This technique allows the purification of the small molecular weight compounds polluting the blood, such as urea, amino acids, inorganic salts, which are normally removed by the kidney. In the case of serum &bgr;2-microglobulin, the various dialysis membranes commonly used possess two antagonistic properties:
capture of &bgr;2-microglobulin by nonspecific adsorption on the membrane,
generation of &bgr;2-microglobulin by detachment of this molecule which is noncovalently associated with the surface of nucleated blood cells in the major histocompatibility complex type I.
The degree of generation of &bgr;2-microglobulin is one of the criteria which define the biocompatibility of the membranes. Thus, endowed with these two antagonist properties, some membranes lead overall, during a hemodialysis session, to an increase in the concentration of &bgr;2-microglobulin, whereas others reduce it.
However, regardless of the membranes used, these results level out over periods of over one year. Thus, it has been observed that the plasma level of &bgr;2-microglobulin in uremic patients after fifteen months of dialysis was invariably increased to be between 40 and 50 mg/l (against 1 to 2 mg/l in healthy patients). Such problems of biocompatibility also exist for the other biomolecules.
2. Removal of the Biomolocules by Hemofiltration
Once per month, the dialyzed individual is subjected to an ultrafiltration session. The module used (
1
) possesses a higher cut-off than in hemodialysis (average cut-off of 40 kDa) and allows the removal, by filtration, of the small molecules from plasma, including the smallest proteins, such as &bgr;2-microglobulin (FIG.
2
). During an ultrafiltration session, the loss of plasma water is compensated by an equivalent supply of physiological saline (
3
).
The qualitative results, with respect to the removal of &bgr;2-microglobulin (purification and generation of this molecule by ultrafiltration membranes), are similar to those obtained in hemodialysis. There is thus a great influence of the nature of the membrane and of the duration of the hemofiltration. While some membranes appear to remove more &bgr;2-microglobulin over 5 hours (one session), a leveling out of the results is also observed over time. At the quantitative level, it appears that about 50% of the serum &bgr;2-microglobulin is removed per hemofiltration session. However, even if this technique is more effective for the purificatio
Legallais Cécile
Moriniere Philippe
Pitiot Olivier
Vijayalakshmi Mookambeswaran
Centre National de la Recherche Scientifique
Kim John
LandOfFree
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