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MONACO

”Monolithic Adsorbent Columns for EXTRA Corporeal Medical Devices and Bioseparations – MONACO-EXTRA”

Call: FP7-PEOPLE-2007-3-1-IAPP

The main purpose of the project is to manufacture novel materials for extracorporeal blood purification, to characterize their physical and physico-chemical properties and evaluate their performance in the processes of removal of toxins like cytokines or cancer cells from human blood. In this project natural or synthetic polymer hydrogels produced by cryopolymerization will be used to create new macroporous adsorbents permeable for blood cells and proteins. By means of incorporation of small carbon or polymeric particles with high adsorption capacity into the cryogel matrices, the composite blood-compatible materials targeted against the above toxins or cells will be developed and tested for blood purification.

Partners:

University of Brighton, United Kingdom (coordinator)
MAST Carbon Technology Ltd, United Kingdom
Protista Biotechnlogy AB, Sweden
Polymerics GmbH, Germany
Lund University, Sweden
Universität fur Weiterbidung Krems, Austria
Brighton and Sussex University Hospitals, United Kingdom

Background information to the project

It is estimated by the World Health Organization (WHO) that there are 1.5 million patients worldwide in need of liver support therapy. Until now there was no system available to solve the complications of liver failure according to the clinical practice requirements. Hemodialysis, the artificial kidney with about 60 million treatments of renal insufficiency per year cannot remove protein-bound toxins which must be metabolized by the liver. The market for liver support is estimated to be substantial: 700 million USD in the USA and 1.4 billion USD worldwide. The existing methods for plasma perfusion intended to remove the toxins suffer from complications such as fibrinogen adsorption and activation of complement on the adsorbent materials. There is, therefore, a need for development of new haemoperfusion adsorbents that would fulfill a number of requirements such as a biocompatibility, very low flow resistance, high mechanical and chemical stability, a large surface area-to-volume ratio, ease of functionalisation and large interconnected pores. Good candidates for such adsorbents are cryogels widely studied at Protista Biotechnology AB as chromatographic materials for protein and cell separation.

The other separation technique highly required in medicine is cytapheresis, or removal of certain types of blood cells from the whole blood. Extracorporeal filtering devices were applied to treat autoimmune diseases such as systemic lupus erythematosus, ulcerative colitis and Crohn’s disease by means of extraction of white blood cells from the circulation and modulating immune responce. Obviously, the filter materials should allow for unrestricted passage of the rest of blood cells and very low thrombogenicity. The macroporous structure of cryogels permeable for cells offers good prospects for development of new adsorbents both for plasma perfusion and for cytapheresis. These matarials are under development in the course of the project.

Detailed description of the project

Extracorporeal blood purification systems are based on the use of physicochemical processes such as convection and diffusion – in filtration and/or dialysis or adsorption – in haemoperfusion. Dialysis and filtration are widely used for treatment of chronic renal failure, hepatic failure, multi-organ failure, sepsis and acute poisoning. Although haemoperfusion in principle is more advantageous than the two other techniques as it, at least in theory, removes only target species from the blood stream and does not require large volumes of expensive replacement fluids, in reality it has been mostly limited to treatment of acute poisoning with low molecular substances. This is mainly due to poor haemocompatibility of adsorbents – activated carbon and porous polymers, which requires their coating with a layer of a more biocompatible material. This coating improves biocompatibility but severely affects efficiency and rate of adsorption, particularly of larger molecules[1]. To solve this problem, blood plasma can be separated from blood cells and treated by adsorption. Some elegant systems have been designed for plasma purification, in which suspensions of adsorbents rather than stationary columns are used, one such system being developed and patented by Partner 6[2].

Recent technological advances in activated carbon manufacturing by pyrolysis of porous synthetic polymer precursors achieved by Partner 2 led to development of medical adsorbents (jointly with Partner 1) which can be used for direct contact with blood without additional coating in granual and monolithic form[3]. Partners 1, 2 and 7 have also shown that selectivity of adsorption towards large target molecules such as inflammatory cytokines or lipopolysaccharide (endotoxin) can be improved by fine tuning of pore size and structure of activated carbon. Even higher selectivity and specificity of extracorporeal adsorption has been achieved by surface functionalisation of porous cellulose adsorbens produced by Partner 4 and Partner 6 with antibodies against target substances such as inflammatory cytokines.

Pheresis, or apheresis, from Greek ‘taking away’, means separating some components of blood from others. Whilst plasmapheresis – a complete separation of plasma from blood cells - like adsorption targets molecular solutes, cytapheresis is aimed at separation of certain types of cells from blood by either centrifugation or filtration. It is also used to remove leukocytes from stored blood. It has long been recognised that the specific removal of pathologically significant cells or formed elements from the blood, to improve a wide variety of clinical conditions, is an attractive notion. Cytapheresis has been shown recently to have clinical efficacy in a number of immune and cardiovascular diseases, chronic inflammatory disorders, organ transplantation, viral hepatitis and cancer[4]. Importantly, cytapheresis is often chosen when other therapies failed. So far, the Japanese S&T has been leading in this field.

Extracorporeal continuous-flow centrifugation, using devices such as the Celltrifuge, has been used for several decades to reduce a patient’s leukocyte load in Acute Myeloid Leukaemia (AML), but this is very non-specific, removing the entire buffy coat mechanically, and thus a wide range of cell types, many of which are not pathological. More recently, a device consisting of a non-woven polyester fibre surrounding a porous core (Cellsorba) has been used to treat, inter alia, rheumatoid arthritis by removal of leukocytes and thus a large number of immunoreactive lymphocytes. Again, this was a non-specific treatment which removed most white cells in order to eliminate the small number of T-helper and T-cytotoxic cells actually involved in the disease. The filter also removed most of the platelets that passed through it, although subsequent blood parameters were reported as normal[5]. Nevertheless, marked clinical improvement was reported. Subsequently, this device was applied to other autoimmune diseases such as systemic lupus erythematosus, ulcerative colitis and Crohn’s disease[6]. It has recently been approved for clinical use in Japan specifically for the treatment of active ulcerative colitis. Again, in all these cases the improvement was presumably due to the removal of specific reactive CD4 and CD8 T-cells amongst the total white cell population removed. Examples of other experimental applications of cytapheresis include removal of leukocytes after coronary artery bypass in order to reduce post-operative organ dysfunction, which, although successful in terms of leukocyte removal, seemed to provide little clinical improvement treatment of neurological diseases such as multiple sclerosis, as evidenced by use in the model syndrome - experimental encephalomyelitis, and a variety of autoimmune diseases4. The problem of the non-selective nature of this technique has been addressed by the functionalisation of the filter support and attachment of a biological ligand, such as antibody against target antigen or Protein A (an avid ligand for IgG antibody). An obvious application has been the use of anti-CD4 antibody to remove CD4+ T-lymphocytes4c or the more widely applicable functionalisation with Protein A to capture antibody-coated cells suggested by Partners 3 and 5[7]. This allows a more targeted means of cell depletion, and presents a much wider range of potential diseases which might be treated. Apheresis, or plasma perfusion over Protein A adsorption column has been approved by FDA for treatment of rheumatoid arthritis. Alternative means of specific cell separation, such as flow cytometry or immunomagnetic beads, are not applicable to clinical situations as the cells themselves need to be modified. A wide variety of other potential applications of cytapheresis could be, and have been envisioned, such as the removal of bacterial load from blood in septicaemia, removal of metastatic cells bearing tumour specific antigens from blood, or the recovery of a range of pluripotent stem cells for clinical use.

Despite its high potential, apheresis has not yet achieved wide clinical applications for a number of reasons. In order to be usable as a routine extracorporeal cell-specific filter device, its ideal characteristics should include a very low flow resistance, high mechanical and chemical stability, a large surface area-to-volume ratio, ease of functionalisation, large interconnected pores which can allow unrestricted cell passage, control of pore size during manufacture, very low leukocyte activation and very low thrombogenicity. Large surface area will allow use of another physicochemical separation mechanism, that is, of adsorption, which enhances the separation efficiency of cytapheresis. Current filter devices and other apheresis techniques do not meet all, or many of these criteria. Creating a large surface area usually leads to a high flow resistance or vice versa, and column packings such as Sepharose present both inter- and intra- bead pores of very different diameters, ranging from nanometers to hundreds of microns. Furthermore, such filters tend to foul easily when used with cellular fluids, and bind platelets readily, increasing the risks of thrombosis. It is also desirable to have a monolithic column for therapeutic cytapheresis which allows direct contact with blood with continuous on-line cell separation.

A majority of chromatographic matrices, which may be of use in cytapheresis, are hydrogels. Here a polymer matrix is cross-linked either by covalent or secondary bonding, and expanded within an aqueous phase, which prevents gel instability and collapse. Examples range from agarose gels used to separate DNA fragments, through beaded matrices such as Sepharose, to natural products such as alginate. Such gels are inappropriate for cytapheresis for a number of reasons. Their pore sizes are too small or too variable for the passage of whole cells, and in some gels are not well interconnected, yielding high tortuosity and large flow resistance. They cannot conveniently be manufactured in monolithic form, and while beading is useful in, for instance, protein chromatography, it is not appropriate for cytapheresis because of the very variable paths presented by inclusion and exclusion. Thirdly, many conventional hydrogels are not mechanically stable, with their structure completely collapsing after drying and re-hydrating. Use of monolithic columns for chromatographic separations has been already suggested[8] but so far they have not been used for cell separations due to the failure of achieving an appropriate pore structure. These problems will be overcome by employing (i) supermacroporous polymeric cryogels rather than conventional hydrogels and (ii) making them in a continuous (monolithic) form using a cryopolymerisation technology developed by Partners 3 and 5[9]. Cryogels have been shown to have some unique properties, most importantly mechanical, chemical and osmotic stability, and interconnected macro- or super-macropores capable of allowing passage and separation of whole in a chromatographic regime[10]. They have also shown shape memory as they can be repeatedly dried and re-swollen in the solvent acquiring the same shape in which they were synthesised. This suggests that cryogels might provide an ideal structure to act as supports for cell-specific extracorporeal cytapheresis.



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[1] Mikhalovsky SV. in: Microspheres, Microcapsules and Liposomes, Vol. 2, R. Arshady, Ed., Citus Books, London, 1999, p. 133.

[2] Falkenhagen D, et al. Ther Apher Dial (2006) 10: 154.

[3] Howell CA, et al. Biomaterials (2006) 27: 5286; Sandeman SR, et al. Biomaterials (2005) 26: 7124.

[4] a. Yamaji K, Tsuda H, Hashimoto H. Therap Apher (2001) 5: 287; b. Nakane S, et al. Multiple Sclerosis (2003) 9: 579; c. Onodera H, et al. Therap Apher (2003) 7: 329; d. Shibata H, et al. Therap Apher (2003) 7: 44.

[5] Kondoh T, et al. Artif Organs (1991) 15: 180

[6] Takenaka Y. Artif Organs (1996) 20: 914.

[7] Kumar A, et al. J Immunol Methods (2003) 283: 185.

[8] Svec F, Frechet JM. Science (1996) 273: 205.



[9] Plieva FM, et al. J Separation Sci (2004) 27: 828.

[10] Dainiak MB, et al. J Chrom A (2006) 1123: 145.







For any questions please contact Professor Sergey Mikhalovsky (University of Brighton, UK): S.Mikhalovsky@bton.ac.uk







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