This invention relates to magnetically responsive superpar ©magnetic particles, way of producing said particles and their use.

More spe ifically the invention describes a method for the production of superparamagnetie particles which are made of a metal oxide surrounded by a surface isyer of a biologically acceptable polymer. In this form the particles msy be used as a contrast agent medical diagnostics . m particular m Magnetic Resonan e Imaging (MRI). They may also be used as a matrix for biological molecules, thus functioning as a separation device. Another area for their use is by combining said particles with a drug which after injection into the blood stream is stopped m an predetermined ares using an external magnet,

The invention is based on magnetically responsive particles which are superparamagnetie, indicating that they are not permanently magnetized when subjected to a magnetic field. This means that the particles, after withdrawing of the magnetic field, are easil resυspended, Using magneti¬ cally responsive particles within the areas mentioned above, it is of utmost importance that the particles are superparamagnetie m order to avoid permanent aggregation,

Magnetic particles has for long times been discussed as a extremely effective separation device (Hirschbem et al. Chemtech . March 1982 , 172- 179). However, prior art regarding magnetic particles lack one or several of the demands needed m order for the technology to obtain general accept¬ ance,

The above mentioned superparamagnetie property is thereby one of the most important.

I hakissios (US Patent 4.115,634. Sep. 19, 1978) describes magnetic polymer particles m the size range 10-100 μxn where the polymer material is covalently crosslmked,

Avramea3 & Gυesdon (US atent 4,241,176, Dec , 23, 19801 describes magnetic polymer particles where magnetite has been entrapped in a poly- acryla ide-agarose polymer in an emulsion process, The sue of the spheres is 50-500 urn,

idder et al (US Patent 4,230,686. Oct. 28, 1980) describes a ay of producing magnetic particles where the magnetite is entrapped into albumin/protei -A with subsequent covalent stabilization of the polymer mixture.

Ugelstad et al (PCT HO83 00Q14) describes a ay of producing particles where the magnetic material is precipitated within prefabricated polymer particles of a defined size.

Czerlinski (US Patent 4,454,234. June 12, 1984) describes magnetite particles which has been surface coated with a crosslinked polymer of acrylamide,

Moldsy (US Patent 4,462,773. Jun.6, 1984) described sway of pro¬ ducing magnetite particles with a size of 10-70 nm, which are surface coated with a dextran polymer. Dextran has a high molecular weight and is not degraded in the body, thus particles produced in this way are less suitable for medical applications. After production these particles are further processed by centrifiigation at 2S.000 rpm, alternatively the particles are allowed to pass a gel chromatography column in connection with the couphng of affinity ligands. Both of these methods maybe used in laboratory scale production, however, the technologies are not suitable in the production of particles to be used in large scale separation of fermentation suspensions ⁵ or the production of superparamagnetie particles for the use as a contrast agent, as discussed in this invention.

As is discussed in the Molday patent above, the particles msy not be separated with conventional magnets. This is also seen in the examples pre¬ sented, where only examples referring to the separation of cells or cell or anells are described. In the separation of cells or cell organells, a large, number of magnetic particles are associated to the surface of one cell, whereby the total amount of magnetic material attached to a. cell makes it responsive to the magnetic field obtained by conventional cobalt -samarium magnets.

Schroder &τ Borrebaeck (EPC 83901116.0) describes the entrappment of magnetite particles within a carbohydrate matrix by an emulsion process with subsequent stabilization try crystallization of the carbohydrate polymer.

Chagnon et al (EP 0 126 995) describes a process for the production of magnetic particles in a two step procedure.: in the first step the magnetic particles are produced, and in step two, these particles are, after extensive washing, subjected to a surface coating with one or several silicons polymers.

Two steps in the process of Chagnon thus differs significantly from the present invention:

1, On Page 20 is described the ratio between FeCl2 and FeCl ₃ to be used in order to obtain an optimal recoveiy. In the text and in the examples, the best particles are achieved using a ratio Fe ²⁺ /Fe ³⁺ of 2/1 or 4/1. If Chagnon is using a ratio of Fe ²⁺ Fe ⁵⁺ of 1/2 or 1/4, heterogeneous particles which "bleeds", finally being totally dissolved in the washing steps (page 20, line 15-25) is obtained. This is in contrast to our own results where optimal particles are obtained with ratios of Fe ²⁺ /Fe ^s+ contradictory to Chagnon,

2, On page 20 is described how the recently prepared particles are washed in an faCl solution in order to ensure that the final product is resuspe dable to particles in the size of interest. However, this is not necessary according to the present invention.

3, On page 35 is described h w Chagnon prior to the surface coating of silicone has to suspend the particles by powerful homogenization in order to obtain a final product consisting of particles which upon suspension forms a monodisperse suspension,

This is also an experience from the inventors of the present invention: if you don't perform the precipitation of metal salts to metal oxide in the presence of a protective colloids, such as starch, the ferromagnetic particles obtained have a size of 5-50 μn , making them unusable for the purposes as described in this invention.

Using superparamagnetie particles in the areas, as described in this invention, the size of the particles and the choice of polymer is of great importance.

Within medical diagnostics there is primarily a wish to obtain an contrast agent for the liver and the spleen. This can be accomplished using injection of particles into the blood stream having a size of 0.4-1.0 μm, since particles having this size are eliminated from the blood stream by these organs.

Using superparamagnetie particles within biotechnology separation it is also a wish to have particles in the same size range due to the large area per volume obtained. As an example, reducing the size of a. particle from 10 to 0.5 μm, the area accessible for attachment of affinity ligands is enhanced 20 times, as calculated on a defined volume packed spherical particles.

The size should, however , not be below 0.3 μm , since the magnetically

responsive particle thus contains too small amounts of magnetic material to be attracted be a magnetic field. Furthermore, particles having a size below 0.3 μrn possess particle/liquid interactions resulting in a suspension behaving more like a magnetic fluid and not as a suspension where the particles easily can be retrieved.

Despite what is known about magnetic separation, as described in the above mentioned patents/patentapplications, the magnetic separation technology has not yet been used in large scale separation, such as in fermentation, since the particles has to be to produced in a simple way in large quantities with the subsequent attachment of affinity ligands. In large scale separation, separation of e.g. human proteins produced by genetically engineered bacteria in fermentation volumes of several thousands of liters is discussed.

In the separation process, under the influence of the magnetic field, the superparamagnetϊc particles will aggregate, however, due to the superpara¬ magnetie property, the particles will be resuspended as soon as the magnetic field is switched off, thereby allowing for the affinity attached protein to be recovered.

Another item using the present invention is that the structure of the particle (a core of metal oxide surface coated with a polymer) renders a system where the affinity ligand is associated only to the surfe.ee of the particle. Thus, the final product is economically advantageous as compared to macroporous particles since the amount of affinity ligand (e.g. a monoclonal antibody) used can be reduced. Furthermore, the surface association of the monoclonal antibody renders rapid adsorption of the substance of interest due to the lack of the diffusion barriers.

Thus, magnetic separation using the particles according to the present invention will give the following advantages:

- the number of separation steps can be reduced

- higher yield

- faster processing

This combination thus leads to reduced costs for the separation using the particles according to the present invention.

By the use of the process for the fabrication of superparamagnetie particles according to the present invention, it is now possible to achieve this.

Thus, the present invention relates to superparamagnetie particles, made of a core of magnetite, surrounded by a pharmacologically acceptable carbohydrate polymer, the superparamagnetie particle having a size of 0.1- 2.0 μm produced by precipitation of iron salts dissolved in a starch solution,

The process for fabrication of the superparamagnetie particles according to the present invention is based on optimization of parameters resulting in particles which all fulfills the demands mentioned above. Thus, the process renders a high yield where further secondary purification steps in order to obtain optimal superparamagnetie particles having the adequate size and magnetic- properties are not needed.

Thus, the process according the this invention, results in particles in a. high yield in a simple one step procedure.

The process is based on the well known technology of precipitating iron salts in alkali, whereby the metal oxide is formed, However , this precipitation is performed in a way that both the starch and the iron salts simultaneously are added to the alkali solution (e.g, MaOH) while sonicating, The obtained solution is neutralized to pH 7. The suspension obtained, containing a. monodisperse suspension of superparamagnetie particles, having a size of 0.2-2.0 μm, is thereby ready for use.

For the person skilled in the art it is now easy the use the suspension for vaπoijs purposes, As an example, the suspension, containing a monodispers suspension of superparamagnetie particles in a solution of carbohydrates in an alkali or neutral environment, directly or after a concentration step, can be emulsified in an organic solvent, a crosslinker may added to the emulsion or the water solution of superparβmagnetic particles, whereafter these ere stabilized into a water -insoluble three dimensional crosslinked superpara¬ magnetie microsphere.

EXAMPLE 1.

2.7 g FeCl ₃ x 7 H ₂ Q, 4.5 g FeCl ₂ x 4 H ₂ 0 and 3.0 g of starch is dissolved in 10 ml of water by gentle heating, This solution is added dropwise to 100 ml of 1,0 M T'JaOH while sonicating. After adding all of the iron -chloride /starch solution the suspension is sonicated for another 5 minutes where¬ after the suspension is neutralized with HC1 to pH 7.0,

The size of the obtained monodisperse suspension of superparamagnetie

particles is measured in an Coulter Counter Multisizer with the following result:

Average size: 0.775 μm.

99% of the particles is found in the size range 0.35 - 1.22 μm.

Dry weight: 25 mg/ml, where 85%. is magnetite.

Number of particles /ml: 7 x 10 ^δ