The invention relates to a method for producing an aqueous albumin solution from a starting albumin solution, which contains stabilizer molecules, which are capable of occupying binding sites of the albumin, wherein in a method for increasing the albumin binding capacity (ABiC) for other molecules, for example those with physiological effects, at least a portion of the stabilizer molecules is removed from the albumin of the starting albumin solution and separated from the starting albumin solution.
In many severe diseases with resultant organ failure, the mismatch between the capacity of the blood vessels and the volume contained therein plays a central role. If the capacity is too high or the volume is too low, blood pressure drops result and the organs are not sufficiently flushed with blood. If the capacity is too small or the volume too large, blood pressure rises with consequent heart insufficiency and/or lung oedema.
Both sudden and slow volume loss in the blood vessels may be responsible for blood pressure drops with minimum perfusion. Sudden loss in volume may occur when bleeding occurs. Slow volume losses result, for example, from fluid loss by transudation from the vascular bed into the intercellular space due to reducing concentrations of albumin as an oncotic protein (for example when synthesis is affected in liver disease). However, both sudden and slow increases in the capacity of the vessels may also be responsible for blood pressure drops. Sudden vessel expansion results, for example, from an acute cascade of vasodilatory textile hormones, such as histamine, bradykinin, kallikrein, leukotrienes or prostaglandins, which occur during anaphylactic shock. Slow expansions result from a chronic pressure rise in the portal artery linked to an increased presence of vasodilatories in the arterioles of the splanchnic network and result in hepatorenal syndrome with ascites formation via an enlargement of capacity.
In each case there is a mismatch between the capacity and volume, which can be influenced by two treatment strategies.
Firstly, an attempt can be made to increase the intravasal volume by administering crystalloid or colloidal volume replacement solutions. If the mean arterial pressure is not thereby brought into the range which allows sufficient blood flow to all organs, then in the second step “pressors” (vasocontrictors, for example catecholamine) are used which narrow the vessels. Such a vasoconstriction is used particularly when vessel tonus is lost, as occurs in liver disease and sepsis, to try and further reduce the capacity of the vessel system.
In diseases which cause acute or chronic vasodilation through a higher level of vasodilators, maintaining a sufficient blood pressure by infusion for long term volume increase is not possible without vasoconstrictors. Examples of such intensive care medical problems are liver failure with a pressure drop and hepatorenal problems (secondary kidney failure in liver failure due to blood flow problems) or sepsis. Both cases are linked to a high mortality and are expensive medically.
Existing solutions recommend the administration of volume replacement fluids in combination with vasoconstrictors. The time which existing volume replacement fluids spend in the vessels is limited, however. Crystalloid (salt-containing) infusion solutions diffuse quickly into the intercellular space. Volume replacement solutions with polymers, for example starch solutions (hydroxyethyl starch, HAES) or gelatin solutions (Gelafundin) are effective in the vessels for longer, as they have water-binding properties which keep the plasma liquid in the vessel and thus can increase the intravasal volume for a longer period. However, problems arise with artificial polymers due to incompatibility.
A particularly suitable volume replacement means is a solution of the natural colloid serum albumin. Serum albumin has been used in the medical field for decades as a plasma expander and is considered to be the best tolerated biologically and thus the most preferred volume expansion medium, albeit the most expensive.
Solutions of human serum albumin for infusion are commercially available. However, those solutions must be supplemented with stabilizers to allow pasteurization and storage, to avoid the spontaneous polymerization of the albumin. Usually, N-acetyl tryptophan and octanoic acid or their sodium salts are used, alone or in combination. These stabilizers have a very high binding affinity for the albumin molecule and occupy and block important binding sites for the biological function of the albumin.
Meta-analysis has shown that the use of serum albumin solutions in intensive care when compared to other plasma volume replacement solutions was linked to increased mortality (Cochrane meta-analysis in BMJ 1998; 317, p 235-240). With the exception of a few particular indications, then, existing albumin infusion solutions appear to have no clinical advantage. How the production method (fractionation with subsequent pasteurization and stabilization) of existing serum albumin solutions adversely affects the theoretical ideal properties of the serum albumin as a plasma expander, is not currently known. The literature makes inconsistent mention that the stabilizers N-acetyl tryptophan and octanoic acid could under certain circumstances have damaging side effects. Thus, it would be desirable if these stabilizers could be removed before administering the albumin solution to a patient, as these occupy and block binding sites which are required for important functions of the albumin with a high affinity. However, stabilizer-free albumin solutions suffer from the problem mentioned above of spontaneous polymerization of the albumin and thus the poor storage properties of such solutions.
The biologically important ability of human serum albumin to bind ligands is treated in many publications. A comprehensive overview can be found, inter alia, in J. Peters jr., All about Albumin, Academic Press, San Diego, New York, Boston, London, Sydney, Tokyo Toronto, 1996, and in Pacifici G M, Viani A, Methods of Determining Plasma and Textile Binding of Drugs, Clin Pharmacokinet, 1992, 23 (6): 449-468. Because of the enormous variety of methods for determining the albumin binding capacity, the results are difficult to compare and an interpretation as regards its medical relevance is practically impossible.
A novel method for documenting the binding behaviour of albumin is constituted by a measurement of the albumin binding capacity (ABiC) for dansylsarcosin (Klammt S, Brinkmann B, Mitzner S, Munzert E, Loock J, Stange J, Emmrich J, Liebe S, Albumin Binding Capacity (ABiC) is reduced in commercially available Human Serum Albumin preparations with stabilizers, Zeitschrift für Gastroenterologie, Supplement 2001, 39: 24-27). These methods are based on measuring the ultrafiltered part of the test marker dansylsarcosin under predetermined experimental conditions and the relationship of this binding capacity to a reference albumin.
In a comparison between healthy blood donors and patients with serious liver diseases, a significant reduction in the binding capacity of serum albumin was observed which was explained by the greater occupation of the serum albumin binding sites by endogenous ligands as a result of liver detoxification malfunctions in the patients under investigation. It is known that the binding behaviour of commercially available preparations of human serum albumin towards particular model markers (for example Ibuprofen) is also dramatically limited.
It is also known that in a ligand-free albumin, the binding capacity for dansylsarcosin can be reduced from 100% to about 60% if N-acetyl tryptophan is added stepwise in amounts of up to a molar ratio of 1:1 (measured using the ABiC in accordance with Klammt et al, 2001).
The technical and medical literature contains many publications regarding the purification of albumin from donor plasma or from biotechnologically produced (recombinant) albumin. These publications are, however, are primarily concerned with the purest possible preparation of the albumin fraction and the removal of other protein components or potentially toxic components from the blood plasma or, in the case of recombinant production, from the vector system.
The removal of low molecular weight ligands such as stabilizers in commercial serum albumin solutions was carried out up to 1967 using Goodman's methods (Goodman D S, Science, 125, 1996, 1957) based on extraction with a mixture of iso-octane and acetic acid, or William's methods (Williams E J and Foster J F, J Am Chem Soc, 81, 965, 1959), based on spontaneous lipid layer formation in highly acidic media. Both methods are extremely time-consuming and not suitable for the production of therapeutic preparations because of potential toxicity. Albumin solutions produced using those methods have very poor stability on storage.
Since 1967, free fatty acids have been added to albumin solutions as stabilizers, such as octanoic acid, and removed from the albumin solution by rendering it highly acidic and then treatment with activated charcoal. The method was initially published by Chen et al, Journal of Biological Chemistry, volume 212, no 2, 25, January, p 173-181, 1967. In that method, the albumin solution is acidified in distilled water using an acid (HCl) to a pH of 3 or less to unfold the albumin molecule by breaking hydrogen bonds and also to protonate the corresponding fatty acids. This loosens the bond between albumin and the fatty acid to such an extent that the fatty acid can diffuse to the activated charcoal as a small molecule. Next, the albumin solution is mixed with activated charcoal and stirred for 1 hour in an ice bath using a magnetic stirrer. Next, the activated charcoal is separated by centrifuging the mixture at 20200 g. In this method, various fatty acids can be removed. This standard procedure (until now) for the removal of fatty acids is based on detailed investigations of the various conditions such as the pH and the mass ratios of activated charcoal to albumin, wherein the standard procedure described above is by far the most successful. The removal of stabilizers from albumin molecules was thus only achieved by breaking the structure of the albumin molecule and an associated reduction in the binding affinity in a highly acidic medium. Substantial reduction of free fatty acids from human serum albumin at higher pHs of more than 3 was not successful.
An important disadvantage of the method is the structural alteration of the albumin molecule by the considerable acidification in aqueous medium. Herein, not only the loop-forming bonds between amino acids which are separated from each other are cut, but also the hydrophobic binding pockets are opened up, which leads to increased adsorption of the albumin on the activated charcoal which is used. Chen et al note albumin losses of 20% in the charcoal pellet in their method. The method is unsuitable for the primary production of commercial therapeutic albumin solutions as the structural alteration in the albumin molecule triggers a spontaneous polymerization of the human serum albumin on storage.
In a quarter of current medical applications for human serum albumin (HSA) as a volume replacement medium (in total about 200 tonnes per year), in addition to colloid-osmotic properties, intact binding properties for toxins (for example benzodiazepine) play a major role, namely for indications associated with liver disease. This property is, however, limited in commercial preparations by stabilizers (N-acetyl tryptophan and octanoic acid) which occupy binding sites, which is reflected in a reduced albumin binding capacity (ABiC).
USPTO Backgrounds
USPTO Backgrounds