Extraction and purification of (APO)ferritin from blood plasma and plasma fractions

Abstract

The invention relates to a method for extracting (apo)ferritin, comprising the steps: (i) providing blood plasma or a plasma fraction as an (apo)ferritin containing fluid, in particular a plasma fraction obtained from side fractions of plasma fractionation according to Cohn or to Kistler-Nitschmann, (ii) performing a heat treatment of the fluid under conditions allowing precipitation of proteins other than apoferritin or ferritin, (ii-b) removing precipitated proteins from the fluid, preferably by performing at least one centrifugation, (iii) performing ion-exchange chromatography, in particular anion-exchange chromatography, under conditions allowing elution of ferritin and / or apoferritin, (iii-b) performing at least one filtration with the elution of step iii. allowing concentration of (apo)ferritin, and (iv) performing positive affinity chromatography under conditions allowing elution of ferritin and / or apoferritin. Further, the invention relates to (apo)ferritin obtained by said method and said (apo)ferritin for use in a method of treating or preventing an acute or chronic inflammation, its use in preparation of magnetoferritin, or its use for treating iron overload caused by genetic disorders. Finally, the invention refers to the use of the method for extraction and / or purification of (apo)ferritin from blood, blood plasma and / or a fraction thereof.

Description

[0001] Extraction and purification of (apo)ferritin from blood plasma and plasma fractions

[0002] The present invention relates to a method for extracting (apo)ferritin. The method comprises the steps: (i) providing blood plasma or a plasma fraction as an (apo)ferritin containing fluid, in particular a plasma fraction obtained from side fractions of plasma fractionation according to Cohn or to Kistler-Nitschmann, (ii) performing a heat treatment of the fluid under conditions allowing precipitation of proteins other than apoferritin or ferritin, (ii-b) removing precipitated proteins from the fluid, preferably by performing at least one centrifugation, (iii) performing ionexchange chromatography, in particular anion-exchange chromatography, under conditions allowing elution of ferritin and / or apoferritin, (iii-b) performing at least one filtration with the elution of step (iii), allowing concentration of (apo)ferritin, and (iv) performing positive affinity chromatography under conditions allowing elution of ferritin and / or apoferritin.

[0003] Further, the invention relates to (apo)ferritin obtained by said method and said (apo)ferritin for use in a method of treating or preventing an acute or chronic inflammation, its use in the preparation of magnetoferritin, or its use in method for treating iron overload caused by genetic disorders. Finally, the invention refers to the use of the inventive method for extraction and / or purification of (apo)ferritin from blood, blood plasma and / or a fraction thereof.

[0004] Ferritin is a well-known protein which is involved in the iron metabolism. Ferritin is a universal intracellular protein that stores iron - up to 4,500 iron atoms - and releases it in a controlled fashion. The protein is produced by almost all living organisms, including algae, bacteria, higher plants, and animals. In humans, it acts as a buffer against iron deficiency and iron overload. Ferritin is found in most tissues as a cytosolic protein, but small amounts are secreted into the serum where it functions as an iron carrier. Plasma ferritin is also an indirect marker of the total amount of iron stored in the body, hence serum ferritin is used as a diagnostic test for iron- deficiency anemia (Wang et al., "Serum ferritin: Past, present and future", Biochim Biophys Acta 2010; 1800 (8): 760-769). Iron-containing ferritin can e.g. be used to treat anemia.

[0005] Ferritin is a globular protein complex consisting of 24 protein subunits forming a hollow nanocage structure with multiple metal-protein interactions (Theil, "Ferritin protein nanocages - the story", Nanotechnology Perceptions, 2012, 8(1 ):7- 16). It is the primary intracellular iron-storage protein in both prokaryotes and eukaryotes, keeping iron in a soluble and non-toxic form. Ferritin that is essentially free of iron ions and preferably essentially free of other inorganic ions, is called apoferritin and may operate as iron catcher of non-transferrin-bound or other free iron or of other free inorganic ions.

[0006] The quaternary structure of apoferritin is formed by 20 similar protomers that occupy the comers of a pentagon dodecahedron (relative molecular mass 480,000). Iron, especially Fe(OH)3, can be incorporated into the hollow spherical quaternary structure (cage structure) of apoferritin. Further, apoferritin as an “organic shell” may also be applied for diverse nanotechnological applications, e.g. as nanoreactor.

[0007] Le Xue et al. (Magnetoferritin: Process, Prospects, and Their Biomedical Applications, Int. J. Mol. Sci. 2019, 20(10):2426), for example, describe the synthesis of magnetoferritin from ferritin and its biomedical applications in imaging technologies, diagnosis and for drug delivery. Comparably, Tan and Cheng D. (Using Magnetoferritin Nanoprobes for Both Nuclear and Magnetic-Resonance Imaging. Nanomedicine, 2016, 12(1 ), 9-11.) report an application of magnetoferritin in MRI / nuclear tumor targeted imaging.

[0008] WO 2012 / 012786 relates to the deposition of high concentrations of anions within the ferritin cage (apoferritin) and to its therapeutic use. The description focuses on the preparation of anion supplemented apoferritin, its origin (or the origin of other protein cages) is not reported. As example for therapeutic use, an application of ferritin incorporating radioisotopes for targeting cancer cells is outlined.

[0009] Ferritin is composed of two types of sub-units, H and L. Ferritin H has a potent ferroxidase activity that catalyses the oxidation of iron. Ferritin L plays a role in iron nucleation and protein stability. Ferritin transports iron the bone marrow for the synthesis of haemoglobin and its incorporation into the erythrocytes. Therefore, ferritin is of some interest in the detection and may be even treatment of anaemias, caused by different reasons. In the human body, in an adult, iron is typically maintained within a range of 4 to 5 g by a strict control of its absorption, mobilization, storage, and recycling. Iron excretion is not actively controlled, and skin desquamation is the major mechanism described so far, often accounting for about 1 to 2 mg per day. 90% of iron is often recycled in the human body (e.g. at the degradation of red blood cells), often only 10% is supplemented from the nutrition. Often, 70% of total body iron is bound in heme compounds, about 29% is stored as ferritin and hemosiderin, often <1 % is incorporated in heme-containing-enzymes, and often less than 0.2% of iron circulate in the plasma, while being bound to transferrin. 25-30 mg of iron are used daily for the generation of haemoglobin.

[0010] Because of its ability to participate directly as a donor or acceptor in electron transfer reactions, iron may become toxic by the generation of highly reactive free radicals that cause lipid peroxidation, DNA strand breaks, and protein modifications that may result in cell death (Harrison PM, Arosio P. The ferritins: molecular properties, iron storage function and cellular regulation. Biochim. Biophys. Acta 1996; 1275: 161- 203. [PubMed: 8695634]). Oxidative stress is the cause of many diseases of civilization known today, such as arteriosclerosis, heart attacks, cancer, rheumatism and Alzheimer's disease. Already in catalytic amounts only, free iron can convert naturally formed hydrogen superoxide into hydroxyl radicals, causing oxidative stress (F. Kieffer; Eisenuberladung und oxidativer Stress. Forschende Komplementarmedizin 1 May 1995; 2 (5): 259-267.).

[0011] Ferritin provides a source of metabolic active iron and serves as a radical cytoprotective protein, storing iron that is not consumed for immediate metabolic use. It was shown that the induction of ferritin protects cells from oxidative stress (Regan RF et al.: Ferritin induction protects cortical astrocytes from heme-mediated oxidative injury. Neuroscience. 2002;113(4):985, PMID: 12182902, Shackelford RE et al.: Pharmacological manipulation of ataxia-telangiectasia kinase activity as a treatment for Parkinson’s disease. Medical Hypotheses (2005) 64, 736-741 ). For this reason, ferritin, and especially apoferritin, is of some interest in neurodegenerative diseases, like Parkinson’s disease. Iron is often associated with oxidative stress that may be associated with neurodegeneration.

[0012] This mechanism of neuronal death is proposed as a cause of Parkinson’s disease (PD). Although most of researchers agree with this, controversies remain regarding the amounts of iron involved in this process. According to non-destructive methods of assessment of the concentration of the total iron in substantia nigra (SN), there is no difference between Parkinson’s disease and control.

[0013] However, there is no need for an increase of the total iron in parkinsonian SN to trigger the oxidative stress but only of the non-ferritin-bound labile iron.

[0014] Recent studies suggest an increase of this iron in Parkinson’s disease (Friedman A et al.: Iron as a cause of Parkinson’s disease - a myth or a well-established hypothesis? Parkinsonism Relat Disord. 2009 Dec; 15 Suppl 3:S.212-4. doi: 10.1016 / S1353-8020(09)70817-X; Mostile G. Iron and Parkinson's disease: A systematic review and meta-analysis. Mol Med Rep. 2017 May; 15(5):3383-3389.). This might play a role in other inflammation sites as well. The successful removal of iron at those sites of a chronic inflammation might reduce the level of inflammation, therefore the oxidative stress in the tissue and therefor the tissue damage. This can be of interest due to the fact, that the current therapy for iron overload or oxidative stress is using iron chelators (Garringer et al., “Effect of Systemic Iron Overload and a Chelation Therapy in a Mouse Model of the Neurodegenerative Disease Hereditary Ferritinopathy”, PlosOne, 2016, 11 (8):e0161341.). For this reason, ferritin, and especially apoferritin, is of high interest for use in and improvement of such therapies.

[0015] AU 2012 362244 refers to methods for isolation, use and analysis of ferritin from plant and animal material. Among other strategies, an isolation of ferritin comprising a step of heat-denaturation of the soluble fraction at temperatures below 80 °C is described. Further specifications are not disclosed nor any reference to human and / or (blood) plasma sources. The isolated ferritin is suggested for use in administration to humans or animals and treating iron deficiency. Apoferritin or any reduction of iron is not discussed.

[0016] DD 129216 discloses a method for preparation of RNase-free ferritin for molecular biological applications, where ferritin may be used as marker for proteins or nucleic acids due to its microscopically visible iron core. Therefore, commercial ferritin is converted into apoferritin by reduction in a known manner via reducing agents. The apoferritin is purified by chromatography with a G 100 or G 200 molecular sieve column. Then, apoferritin in a 1 % solution is converted back into ferritin by incorporation of iron(lll), which is subsequently further purified by ultracentrifugation. Purity and characteristics of the intermediately obtained apoferritin is not further assessed.

[0017] WO 2003 / 094849 relates to ferritin fusion proteins, i.e. ferritin fused with a protein, peptide, antibody or the like, for use in vaccines and therapeutic applications. Such applications my include the delivery of drugs or imaging technologies. The fusion of ferritin and the supplemented protein does not make use of hollow structure or its capability of incorporating ions but is performed at the C terminus or at the N terminus of ferritin.

[0018] A low-cost protocol for standardized production of iron-free apoferritin with high protein recovery and suitable conformation for nanotechnology applications is reported by Moglia et al. in Journal of Inorganic Biochemistry, 2018, 188: 184-190. Focus of this publication is a comparison of methods to eliminate iron from ferritin in order to obtain apoferritin and its characterization.

[0019] US 5,358,722 discloses ferritin analogs comprising an apoferritin protein shell and a core substantially devoid of ferrihydrite, e.g. of inorganic composition such as aluminum hydroxide or organic composition such as acetaminophen.

[0020] JP-A 2004-217984 relates to a separating agent, which continuously and selectively or non-selectively separates metal ions from an aqueous solution containing various metal ions, and a method for treating an aqueous solution containing metal ions.

[0021] Reduction of iron ions (also designatable as ferric ions) such as in a body fluid, such as blood or blood plasma or cerebrospinal fluid, is often achieved by means of addition of chelating agents. Using higher concentrations of chelators for such purpose, however, bears several drawbacks. It is laborious and often challenging to remove chelators, which are typically soluble agents, from a fluid.

[0022] Further, chelators are often toxic and thus undesired in body fluids. Despite of the low efficacy, they have some unwanted side effects (Parvu et al., “Ferritin level changes and erythroid improvement in a group of adult, polytransfused patients treated with Deferasirox”. Clujul Med., 2018, Jul;91 (3):288-292).

[0023] Therefore, there is an unmet need for providing improved and inclusive means for obtaining purified ferritin and / or apoferritin, especially in research grade quality and amounts from scalable methods as well as from available resources, such as blood plasma and plasma fractions.

[0024] Surprisingly, it has been found that the isolation from blood plasma and plasma fractions, especially from a Cohn-Fraction, provides a basis for the production of research grade (apo)ferritin and allows an immediate obtainment of purified (apo)ferritin, when using a combination of heat treatment, ion-exchange chromatography and positive affinity chromatography. Even though ferritin and apoferritin usually are present in extremely low concentrations in body liquids and Cohn-Fractions compared to other proteins, and thus, the purification requires a particularly suitable and effective strategy, the suggested method surprisingly results both in high yields and high quality of ferritin and / or apoferritin. Furthermore, it was found that such obtained purified (apo)ferritin is suitable for different uses and treatments which could not previously be served with apoferritin from other sources.

[0025] Disclosed is a method for extracting (apo)ferritin, wherein said method comprises at least the following steps: i. providing an (apo)ferritin containing fluid, ii. performing a heat treatment of the fluid under conditions allowing precipitation of proteins other than apoferritin or ferritin, iii. performing ion-exchange chromatography, in particular anion-exchange chromatography, under conditions allowing elution of ferritin and / or apoferritin, and iv. performing positive affinity chromatography under conditions allowing elution of ferritin and / or apoferritin.

[0026] Accordingsly, a first aspect of the present invention relates to a method for extracting (apo)ferritin, wherein said method comprises the following steps: i. providing blood plasma or a plasma fraction as an (apo)ferritin containing fluid, in particular a plasma fraction obtained from side fractions of plasma fractionation according to Cohn or to Kistler-Nitschmann, ii. performing a heat treatment of the fluid under conditions allowing precipitation of proteins other than apoferritin or ferritin, ii-b. removing precipitated proteins from the fluid, preferably by performing at least one centrifugation, iii. performing ion-exchange chromatography, in particular anion-exchange chromatography, under conditions allowing elution of ferritin and / or apoferritin, iii-b. performing at least one filtration with the elution of step iii. allowing concentration of (apo)ferritin, and iv. performing positive affinity chromatography under conditions allowing elution of ferritin and / or apoferritin.

[0027] As used herein, in the context of the present invention, the term “(apo)ferritin” may be understood in a broad way as ferritin and / or apoferritin alone or in combination in any given ratio. Thus, (apo)femtin may be mainly ferritin, or it may be mainly apoferrtin, or it may be a combination in a ratio of e.g. approximately 10:90, or 25:75, or 50:50, or 75:25, or 90:10.

[0028] In a preferred embodiment, the concentration step iii-b. of the inventive method is: performing ultrafiltration and diafiltration (UF / DF) with the elution of step iii. allowing concentration of (apo)ferritin.

[0029] Examples for performing a heat treatment, ion-exchange chromatography, ultrafiltration and diafiltration (UF / DF) and positive affinity chromatography within the inventive method are provided in the Example section below.

[0030] Surprisingly, it was found that the combinations of the disclosed steps i.-iv., particularly combined with steps ii-b. and / or iii-b., immediately result in a high yield of purified (apo)femtin in research grade quality. The preparation of the (apo)femtin containing fluid, the performance of a heat treatment, the performance of an ionexchange chromatography, preferably followed by performance of ultrafiltration and diafiltration, and the performance of a positive affinity chromatography, positively complement each other and improve the overall result and product.

[0031] A (apo)ferritin containing fluid may be any fluid that contains ferritin and / or apoferritin. It may be of natural origin or may be obtained by heterologous expression. Further, the (apo)ferritin containing fluid may further contain any reducing agent that enables reduction Fe3+ions to Fe2+ions.

[0032] Ferritin may be any ferritin. Ferritin may be human, animal, fungal, plant or bacterial ferritin or a combination of two or more thereof. In a preferred embodiment, ferritin is mammalian ferritin. In a preferred embodiment, ferritin is human ferritin. Ferritin comprises at least two bound iron ions (i.e., is loaded with two or more iron atoms). Typically, ferritin comprises at least two, at least five, at least ten, at least 20 bound iron ions, at least 50 bound iron ions or at least 100 bound iron ions. Apoferritin, however, is essentially free of iron atoms.

[0033] Human ferritin may comprise a light chain (e.g., such designated as UniProtKB - P02792 (FRIL_HUMAN) or a sequence homologue having a sequence homology of at least 90%, in particular at least 99%, thereof) and a heavy chain (e.g., such designated as UniProtKB - P02794 (FRIH_HUMAN) or a sequence homologue having a sequence homology of at least 90%, in particular at least 99%, thereof) loaded with two or more, typically three or more, five or more, ten or more iron ions, 20 or more iron ions, 50 or more iron ions or 100 or more iron ions.

[0034] Human ferritin may also comprise mitochondrial ferritin e.g., such designated as UniProtKB - Q8N4E7 (FTMT_HUMAN) or a sequence homologue having a sequence homology of at least 90%, in particular at least 99%, thereof), loaded with two or more, typically three or more, five or more, ten or more iron ions, 20 or more iron ions, 50 or more iron ions or 100 or more iron ions. Ferritin may also be a globular protein complex comprising a number of (in human ferritin typically 24) protein subunits forming a nanocage with multiple metal-protein interactions.

[0035] According to one aspect of the present disclosure, the (apo)ferritin-containing fluid is a body fluid. The (apo)ferritin-containing fluid may be any body fluid. The body fluid may be a fluid from any animal species including humans. It may be obtained from a vertebrate (e.g., mammals including humans, birds, fishes, amphibia, reptiles, etc.) or from an invertebrate (e.g., arthropod (e.g., insects, spiders, Crustacea, crustaceans, etc.), Mollusca, etc.).

[0036] According to one aspect of the present disclosure, the ferritin-containing fluid is obtained from a vertebrate or an invertebrate (e.g, an insect), a plant, a bacterium or an archaea. Preferably, the (apo)ferritin-containing fluid is a vertebrate, in particular a mammal body fluid. In one aspect, the body fluid is a human body fluid.

[0037] In some aspects of the present disclosure, the (apo)ferritin-containing fluid is selected from the group consisting of blood, blood plasma or a fraction thereof, and hemolymph or a fraction thereof. Preferably, the body fluid is selected from the group consisting of mammal blood, mammal blood plasma or a fraction thereof. Preferably, the body fluid is selected from the group consisting of human blood, human blood plasma or a fraction thereof. In a preferred aspect of the present disclosure, the body fluid is human blood. In an embodiment of the invention, the body fluid is human blood plasma or a fraction thereof.

[0038] In one aspect of the present disclosure, the (apo)ferritin-containing fluid is a unit of stored blood. Preferably, the body fluid is a unit of mammal stored blood. Preferably, the body fluid is a unit of human stored blood. In one embodiment of the invention, the (apo)ferritin-containing fluid is a unit of stored plasma. Preferably, the body fluid is a unit of mammal stored plasma. Preferably, the body fluid is a unit of human stored plasma.

[0039] In one aspect of the present disclosure, the (apo)ferritin-containing fluid is a body fluid selected from the group consisting of blood, blood plasma or a fraction thereof, ascites, liquor cerebrospinalis, or the ferritin-containing fluid is selected from the group consisting of intracellular or extracellular liquids, cell culture fluids (e.g. supernatants of cell cultures), homogenates or organs or tissues, hemolymph or a fraction thereof, and plant extracts. As used herein, cell cultures may be any cell cultures known in the art such as, e.g, vertebrate cell cultures (e.g., human cell cultures, mammalian cell cultures), invertebrate cell cultures (e.g., insect cell cultures), plant cell cultures, fungal cell cultures (e.g., yeast cell cultures) or bacterial cell cultures. Preferably, the (apo)ferritin-containing fluid is a unit of stored blood or plasma.

[0040] In a preferred aspect of the present disclosure, the (apo)ferritin containing fluid in step i. of the inventive method is a body fluid, preferably a body fluid selected from the group consisting of blood, blood plasma or a fraction thereof, ascites, liquor cerebrospinalis, or the (apo)ferritin containing fluid is selected from the group consisting of intracellular or extracellular liquids, cell culture fluids, homogenates or organs or tissues, hemolymph or a fraction thereof, and plant extracts, in particular wherein the (apo)ferritin containing fluid is a unit of stored blood or plasma. In one embodiment of the invention the (apo)ferritin containing fluid is blood plasma or a plasma fraction.

[0041] In a preferred embodiment of the invention, the (apo)ferritin containing fluid is a plasma fraction, in particular a plasma fraction obtained from side fractions of plasma fractionation according to Cohn or to Kistler-Nitschmann. The methods of plasma fractionation according to Cohn or to Kistler-Nitschmann rely on sequential precipitation steps at distinct temperatures, different ethanol concentrations and various pH to segregate bulk plasma proteins, resulting in various fractions, mainly aiming in preparation of human serum albumin and (intravenous) immunoglobulins.

[0042] (Apo)Ferritin is, compared to other proteins in such fraction, only present in very low concentrations. This results preferably in a protein preparation strategy to concentrate the protein ahead of the chromatographic purification. By reducing the total volume using UF / DF (ultrafiltration / diafiltration) or protein precipitation (e.g. ammonium sulphate precipitation), the concentration of (apo)ferritin can be successfully increased. Another positive effect of applying a large-pore membrane during UF / DF is the reduction of non-specific proteins.

[0043] An (apo)ferritin containing fluid may be provided by any means. It may be a stored from the plasma fractionation process obtained from a human or animal body (donor) or produced by gene technologies. Storage may be any kind of storage such as, e.g., storage at room temperature (RT), storage in an cool environment (e.g. in a fridge at a temperature in the range of 1 to 10 °C), storage in the frozen state (e.g. in a freezer at a temperature in the range of -25 to -1 °C or -90 °C to -60 °C or in liquid nitrogen at around -196 °C), or storage of a freeze dried state at any temperature, preferably below 30 °C. Accordingly, storage may be at a temperature range of 15 °C to 30 °C, 1 °C to 10 °C, -25 °C to 25 °C, -70 °C to -15 °C, -90 °C to -60 °C, -200 °C to -80 °C, at around -196 °C or below -196 °C.

[0044] Storage may be storage for at least one or more minutes (min), for at least an hour (h), for at least six hours, for at least twelve hours, for at least a day (d), for at least a week, for at least a month, for at least two months, for at least six months, or for at least a year.

[0045] Provision of an (apo)ferritin-containing fluid freshly obtained from a human or animal body (donor) may be performed by any means. Preferably such provision is conducted by means which do not involve a (severe) health risk for the human or animal. For example, such provision may be blood sampling optionally followed by blood fractioning. Blood sampling may be, for instance venous blood sampling or arterial blood sampling. In another preferred embodiment of the inventive method, the (apo)ferritin containing fluid further comprises one or more chaotropic agents decreasing interactions between apoferritin or ferritin sub-units and / or releasing iron ions from ferritin.

[0046] Preferably, such chaotropic agent is selected from the groups consisting of urea, n-butanol, ethanol, guanidinium chloride, barium salts, thiocyanates, perchlorates, lithium acetate, magnesium chloride, magnesium sulfate, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and combinations of two or more thereof. Particularly preferred, such chaotropic agent is urea.

[0047] A chaotropic agent may be used in any concentration suitable for support reducing activity. For instance, a chaotropic agent may be used in a concentration of from 0.01 to 10 M (molar), of from 0.1 to 9 M, of from 0.5 to 8 M, or of from 1 to 5 M. This concentration may be the concentration of one or each chaotropic agent or may be the concentration of the sum of chaotropic agents.

[0048] Preferably, the chaotropic agent may be removed from the (apo)ferritin-containing fluid after its iron content is decreased. This may optionally be performed in a further step. During investigation, the conditions for heat treatment of (apo)ferritin containing fluid, in particular suspended Fraction I V-4, to remove the main impurities were analyzed.

[0049] In a preferred embodiment of the inventive method the heat treatment is conducted at a pH in the range from 4.5 to 6.0, in particular at a pH in the range around 4.8.

[0050] In a preferred embodiment of the inventive method the heat treatment is conducted at 50 to 110 C, preferably at 60 to 90 C, more preferably at 65 to 85 °C, even more preferably at 70 to 80 °C, in particular at around 75 °C.

[0051] In a preferred embodiment of the inventive method the heat treatment is conducted for 1 min to 4 h, preferably for 2 min to 2 h, more preferably for 5 min to 60 min, even more preferably for 7 min to 30 min, most preferably for 10 to 20 min, in particular for around15 min.

[0052] In one embodiment, the heat treatment is conducted at 70 to 80 °C for 10 to 20 min, in particular for around 15 min. Particularly, said heat treatment is conducted at a pH in the range from 4.5 to 6.0, preferably at a pH in the range around 4.8. In another embodiment, the heat treatment is conducted at around 75 °C for 10 to 20 min, in particular for around 15 min. Particularly, said heat treatment is conducted at a pH in the range from 4.5 to 6.0, preferably at a pH in the range around 4.8.

[0053] Examples for performing the heat treatment within the inventive method are provided in the Example section below.

[0054] In another preferred embodiment of the invention, said inventive method is performed until an (apo)ferritin purity of >60% (w / v), in particular >70% (w / v), preferably >80% (w / v) is obtained.

[0055] Such purity may be determined by any suitable means known in the art, e.g. it may be determined by ELISA (Enzyme-linked Immunosorbent Assay) in comparison with Bradford Assays or BCA (bicinchoninic acid) Assays.

[0056] Further, also methods have been developed in order to analyze the total iron content of the obtained product, as well as the iron binding capacity.

[0057] Overall, the inventive method for the extraction of (apo)ferritin positively combines the steps of preparing the (apo)ferritin containing fluid, performing a heat treatment, performing an ion-exchange chromatography, preferably followed by performance of ultrafiltration and diafiltration, and performing a positive affinity chromatography. The combinations of such steps show a high degree of positive complementation and an interlock among the steps, immediately resulting in a high yield of purified (apo)femtin in research grade quality. Thus, the overall method as well as the result and product are significantly improved. After using the method invented here, a high yield of purified (apo)femtin in research grade quality will immediately be obtained, even from sources of comparatively low total (apo)ferritin content, such as blood plasma and plasma fractions.

[0058] A further aspect of the invention, is (apo)femtin obtainable from, preferably (apo)ferritin obtained from, the inventive method as disclosed. Such (apo)ferritin comprises ferritin and / or apoferritin, which is essentially free of iron, and it is of high quality and purity and it is obtained from its source in high yields.

[0059] Yet another aspect of the invention is (apo)ferritin, preferably obtained from the inventive method, for use in a method of treating or preventing an acute or chronic inflammation, in particular chronic neurologic inflammation, by acting as iron scavenger and thereby reducing oxidative stress caused by free iron, in particular free iron released from tissue or blood. Inflammatory diseases related to local iron overload include e.g. Still’s disease, Hemophagocytic syndrome (macrophage activation syndrome or lympho-histiocytic syndrome), systemic juvenile idiopathic arthritis (SJIA), Kawasaki’s disease, Cataract syndrome in ferritin L hyperferritinemia.

[0060] Further, the invention refers to (apo)ferritin, preferably obtained from the inventive method, for use in the preparation of magnetoferritin, wherein said (apo)ferritin is supplemented with Ferrit.

[0061] In a preferred embodiment, such prepared magnetoferritin is used as drug carrier and inductively excitable at the site of action, in particular at a tumor site.

[0062] In a particularly preferred embodiment, such drug carrier is combined with monoclonal antibodies and / or cancer targeting molecules.

[0063] While biological or biomedical applications of magnetoferritin concerning imaging and diagnosis are known in the art, herein an inventive use of magnetoferritin and its particular properties further for use in therapeutic applications and medical treatment, especially with regard to tumor therapy, is suggested. Due to the magnetic properties of magnetoferritin an inductive excitation of the molecule is possible. Such excitation results in immediate heat creation of the magnetoferritin itself and its surrounding.

[0064] Thus, inductive excitation may be used for local heating and subsequent destruction of tissue by heat, in particular destruction of tumor tissue by local heating. Any temperature above physiological values ("boiling") is a reliable means of killing or eliminating (malignant) cells and tissue. Such suggested application is both effective and targeted and is associated with few side effects.

[0065] Tissue may be any body tissue that interacts with the magnetoferritin or absorbs the magnetoferritin, in particular malignant tissue, especially tumor tissue. Such tumor tissue may be any kind of tumor tissue in any region or organ of a body, including e.g. astrocytomas, blastomas, carcinomas, lymphomas, melanomas, sarcomas.

[0066] Monoclonal antibodies may be any monoclonal antibodies know in the art and may include e.g. rituximab (Mabthera), trastuzumab (Herceptin), cetuximab (Erbitux), pertuzumab (Perjeta), bevacizumab (Avastin), adalimumab (Humira). Cancer targeting molecules may be any cancer targeting molecules known in the art and may include peptides, enzymes, small molecules, liposomes, nucleic acids, carbohydrates, antibodies, nanostructures.

[0067] Another aspect of the invention is (apo)ferritin, preferably obtained from the inventive method, for use in a method of treating iron overload caused by genetic disorders, in particular caused by Hemochromatosis. Genetic disorders causing iron overload include, e.g. Hemochromatosis (all types), African iron overload, Sickle cell disease, Major [3-thalassemia, Sideroblastic anemia, enzyme deficiencies (e.g. pyruvate kinase, G6PD) or disorders of transporting proteins such as Atransfemnemia or Aceruloplasminemia.

[0068] In addition, the invention refers to a use of the inventive method for extraction and / or purification of (apo)ferritin from a body fluid, in particular from blood, blood plasma and / or a fraction thereof, preferably a plasma fraction obtained from side fractions of plasma fractionation according to Cohn or to Kistler-Nitschmann.

[0069] It will be understood that the definitions and preferred embodiments as laid out in the context of the method herein also mutatis mutandis apply to the obtainable or obtained (apo)ferritin. Further, it will be understood that the definitions and preferred embodiments as laid out in the context of the method herein also mutatis mutandis apply to the medical and non-medical uses and methods.

[0070] The invention is not limited to the embodiments described herein and the aspects emphasized therein. Rather, within the range indicated by the claims, a large number of modifications are possible which are within the scope of skill in the art.

[0071] The invention is further illustrated by the figures, examples and claims.

[0072] Brief Description of the Figures

[0073] Figure 1 shows an exemplary outline of the manufacturing procedure of the inventive method, extracting and purifying (apo)femtin, structured by process steps. Starting material is a Fraction IV-4 from human plasma (Paste IV-4), obtained during plasma fractionation (step i. of the inventive method). This material is suspended and filtered (second process step), afterwards heat treated according to step ii. of the inventive method and filtered (third process step) and then treated via ionexchange chromatography according to step iii. of the inventive method (here: anion exchange chromatography AEX, fourth process step). Further a fifth process step of ultrafiltration and diafiltration (UF / DF) follows according to step iii-b. of the inventive method and the performance of a positive affinity chromatography according to step iv. of the inventive method, before finally obtaining (apo)ferritin as product.

[0074] Figure 2 shows the reduction of ROS in a cell culture by addition of apoferritin. ROS (reactive oxygen species) is introduced into a cell culture via hydrogen peroxide H2O2 - first column. Afterwards, different increasing concentrations of apoferritin are added to the cell culture, resulting in reduction of fluorescence signal and thus, in reduction of ROS, as show in the subsequent columns (H2O2 + apoferritin). As a control, apoferritin alone, applied in the same concentrations does not change the fluorescent signal of the cell culture (last set of columns, apoferritin).

[0075] Figure 3 shows the influence of different pH values, created by suspension in different buffers, on the heat treatment of a plasma fraction with regard to the solubility of (apo)femtin and its concentration in the solution. Analysis was performed before and after a heat treatment, (see Example 1.1 )

[0076] Figure 4 shows changes of total protein (TP) (first columns), ferritin (second columns) and albumin (third columns) after heat treatment of the plasma fraction at 60, 65, 70, 75 and 80 °C for 10, 15, 20, 25 and 30 min (see Example 1.2). Black arrow: Recommendation for further heat treatments.

[0077] Examples

[0078] The Examples show a manufacturing procedure for the extraction and purification of (apo)ferritin. Starting materials were received in a plastic container (up to 12.5 kg) under frozen conditions on dry ice from a Plasma fractionator: Fraction IV-4 from pooled human plasma (Cohn-Fraction), which contains -136 ng / g paste ferritin (concentration of ferritin in suspended Fraction IV-4 -38 pg / L) was selected as starting material and was stored below -35 °C until use.

[0079] Example 1 : Conditions for heat treatment

[0080] Example 1.1: Testing of Buffer Conditions for Heat Treatment in Cohn Fraction IV-1-4

[0081] In order to find the optimal conditions for heat treatment, the suspended plasma fraction (Cohn-Fraction IV-1-4) was rebuffered using a desalting column. 1 mL paste suspensions of Fraction IV-1-4 were heated at 68 °C for 10 min. The concentration of ferritin was analyzed before and after heating. Results are shown in Figure 3. The concentration was largely constant in a pH range of 4.5 to 6.0. Outside this pH range, a matrix effect could not be excluded. Below a pH of 3.5, a disassembling of the ferritin cage occurs, and the iron ions are released in the form of free iron. Used buffers and according pH values were as follows:

[0082] Table 1. Overview of tested pH range, established by application of different buffers

[0083] Example 1.2: Thermostability of Ferritin in Cohn Fraction IV-4

[0084] The heating time together with the temperature of the suspension must be optimized in order to minimize negative effects caused by aggregates in the further processing steps (filtration, concentration). In order to evaluate the thermostability of ferritin and apoferritin ((apo)femtin) in suspension heat treatment was conducted at different temperatures and for different durations. 1 mL paste suspensions of Fraction IV-4 were heated at 60, 65, 70, 75 and 80 °C for 10, 15, 20, 25 and 30 min. The treated suspensions were analyzed for their total protein (TP), albumin and ferritin contents, outlined in Figure 4. Albumin is the main impurity in this fraction (compare Table 2) and showed the strongest thermostability among the impurities. The concentrations without heat treatment were normalized as 100%. The quantities of TP, albumin and ferritin post treatment were calculated in % and are shown in Figure 4.

[0085] The concentration of ferritin is stable during heat treatment, independent of temperature and incubation time. All other proteins were significantly denatured and precipitated. The optimal conditions for the best yield of ferritin and highest impurity removal were at 75 °C for 15 min. The amount of TP was reduced from 100% (14.23 mg / mL, w / o heat treatment) to 2.91 % (0.41 mg / mL), the amount of Albumin from 100% (9 mg / mL, w / o heat treatment) to 0.05% (0.005 mg / mL, bdl), and ferritin was at a constant level of 98.55% (37.5 ng / mL), with 100% having a concentration of 38.05 ng / mL (without heat treatment).

[0086] For the suspended Fraction I V-4, the optimal conditions for heat treatment are at a temperature of 75 °C for 15 min and a buffer with pH between 4.5-6.0. The paste suspension was performed in a sodium acetate buffer pH 4.8, therefore the dissolved paste can be directly exposed to heat treatment without prior re-buffering. This effectively precipitates the maximal amount of unwanted proteins, leaving (apo)ferritin unaffected. However, also other conditions are possible, also in view of the treated fraction or fluid.

[0087] Example 2: Chromatographic Purification of Ferritin

[0088] Tested materials were Cation Exchange Chromatography (CEX) with UnoSphere S or Nuvia HR-S columns, Anion Exchange Chromatography (AEX) with MacroPrep DEAE or Nuvia HP-Q columns, and other Affinity Material. Analyzed parameters influencing the chromatography and the running behavior of samples were pH, buffer, load volume, run conditions and performance of heat treatment.

[0089] In case of AEX and CEX, the preliminary heat treatment step leads to an increased ferritin purity (factor of ~40) in eluate fractions. With the column using resin Nuvia HR-S, only 40-60% of the loaded ferritin could be detected. ~20% was identified in the flow through, and ~40% in eluate fraction. In comparison, AEX resins (MacroPrep DEAE and Nuvia HP-Q) detected 85%-100% of loaded ferritin. 83-95% was measured in the flow through, and 5-17% in the eluate fraction. The resins Nuvia HP-Q and Nuvia HR-S were tested with suspended Fraction IV-4 with and without prior heat treatment. The experiments with affinity material were carried out without heat treatment.

[0090] Experiments were also carried out with the ferritin affinity column (CV 4.5 mL). Samples showed that the impurities are > 90% in the flow through fraction and the purity of ferritin is much higher compared to the AEX and the CEX resin results.

[0091] Example 3: Extraction and purification of (apo)ferritin

[0092] Example 3.1 : Paste Suspension of Fraction IV-4

[0093] The whole paste dissolution process was performed in a PE container (VWR) with a diameter of 30 cm at 2 to 8 °C. The paste suspension buffer (0.15 M sodium acetate buffer pH 4.8) was used in a ratio of 1 :5 (1 part paste to 4 parts buffer) and pre-cooled to 2 to 8 °C in the refrigerator before use. The buffer was weighed with a balance (12 kg). Afterwards, the buffer was added to the tank. The frozen paste was removed from the -35 °C freezer and weighed (3 kg). Weights were noted in a paste suspension protocol. The weighed paste was added stepwise to the suspension buffer. The suspension in the container was continuously stirred at 345 min-1 for 240 min using the mixer (Heidolph, RZR 2041 ) and the temperature was maintained between 2 to 8 °C using the Lauda thermostat (Serial No: A02013). Before starting a 240 min stirring time, the pH was adjusted to pH 4.8 with concentrated acetic acid 99.5% (v / v) and the conductivity was checked. The final volume at the end of the suspension step (approximately 15.0 L) was measured and aliquoted (5 x 0.5 mL) with a paste suspension lot number. The suspended paste was ready for the following deep filtration steps.

[0094] Table 2. Overview of absolute (Concentration [mg / mL]) and relative (Percentage of total protein TP [%]) protein amounts in 88 g of suspended Fraction IV-4 (V=236.3 mL).

[0095] Example 3.2: Filtration of the paste using Filtrox System

[0096] The filtration set up was prepared for filtering the suspended paste. The depth filtration step was carried out at room temperature using a CH33P layer capsule from Filtrox (5" Capsule, area: 0.0127 m2). The CH33P was first conditioned with 500 mL deionized water and 150 mL of 0.15 M sodium acetate pH 4.8. Later, all residual liquid was removed from the capsule and tubes by running with reverse flow, until no liquid was left in the system.

[0097] The paste suspension was dispensed into a clean beaker, the volume of the suspension was measured before starting the filtration. The suspension beaker was placed on a magnetic stirrer and stirred at 150 min-1during the filtration process through the CH33P filter with a peristaltic pump pressure of < 2 bar. The filtrate was collected in a separate clean tank (filtered ~1.9 L) and each filtration cycle took approximately 15-18 min. Since the total volume of the 3 kg suspended paste was around 15 L and the maximum volume per capsule was 2.6 L, the filter was changed six times to finish one lot of paste suspension. The tube and capsule were washed with water and 0.15 M sodium acetate buffer before use. The whole process for 3 kg suspended paste filtration was completed in 120-150 min and required in total up to 6 capsules.

[0098] The final filtrate (FT) was sampled (~12.5 L) in clean glass bottles and stored at 2 to 8 °C for heat treatment the next day or stored below -35 °C in a 1 L sterile bottle for later use. Once the process ended, the tubes were cleaned with 500 mL deionized H2O and 150 mL of 20% (v / v) EtOH.

[0099] Example 3.3: Heat Treatment of suspended Fraction IV

[0100] The filtrate was stored at 2 to 8 °C overnight in refrigerator. The water bath was prewarmed for starting the heat treatment process at 75 °C. The pre-warm heating element was set at 56 °C. The filtrate was taken out from the fridge and weighed (3.500 g in the steel container for one batch of heat treatment process).

[0101] Once the water bath reached 75 °C, the paste suspension in the steel container was placed in the water bath. The heating element was clamped on the steel sidewall, set to 56 °C, and the suspension stirring started. The water bath was covered with aluminum film as a thermo-protective layer. 30-45 min later, the suspension temperature reached 75 °C and it was held at 75 °C for 15 min to finish the heat treatment step. The temperature was monitored and documented using a temperature logger.

[0102] Afterwards, the Lauda thermostat (serial no: A02013) was pre-set at 4 °C for a cooling step. The steel container with the hot suspension was placed in an ice bath bucket for cooling down by using Lauda thermostat and stirred at 150 min-1for approximately 60 min until the suspension temperature reached 4 °C.

[0103] The centrifuge was pre-set at 4 °C before starting the centrifugation step. The cooled heat-treated suspension was dispensed into 4 x 1 centrifugation vessels (0.9 L) and centrifuged at 4000 min-1for 20 min at 4 °C. The clear supernatants were combined in one fresh beaker. If the supernatant was cloudy and not transparent on visual inspection, then the centrifugation cycle was repeated one more time. The supernatant volume was measured. The supernatant was filtered through a CH33P capsule (Filtrox). The system was conditioned with 500 mL deionized H2O and 150 mL of 0.15 M sodium acetate buffer pH 4.8. All residual liquid was removed from the system by running with reverse flow, until no liquid was left. Afterwards, the supernatant of heat-treated suspension (in beaker) was placed on a magnetic stirrer and stirred at 150 min-1at room temperature. The filtration was performed. The volume before and after the filtration procedure was measured.

[0104] The filtrate was additionally filtrated through a 0.45 pm membrane and the volume noted. The filtrate was named and labeled. In total, 4 sub-pools were performed and pooled. Finally, aliquots from the pooled heat-treated filtrates were taken (5x 500 pL) and stored below -35 °C. The remaining filtrate was stored at 2 to 8°C for chromatographic purification step next day. Table 3. Overview of heat treatment method

[0105] Example 3.4: Preparation of Chromatography

[0106] To prepare the feed, pH and conductivity are adjusted before starting the chromatography run. The feed preparation process was held in a PE container (VWR) with a diameter of 30 cm, empty PE container weighed (Balance: ID: PP- 2022-0289) before starting the feed preparation process. The heat-treated filtrate used was stored overnight at 2 to 8 °C and removed from the fridge and filtered through a 0.22 pm membrane (Lot: MP213814G2 [Merck]). For one AEX run, 6.2 L heat-treated filtrate was used. In total, three AEX runs were performed for one lot. pH and conductivity were adjusted to 8.0 and 3.5 mS / cm (electrode: Hanna Instruments) under continuously stirring with a four-wing stirrer at 210 min’1. 3.5 M Tris (range 170 - 200 mL) was used to adjust to the target pH in the heat-treated filtrate. The pH value before (4.5-4.8) and after adjustment (8.0) was measured.

[0107] 10 mM Tris / HCI pH 8 (= dilution buffer) was used to set the target conductivity. Normally the conductivity range of the heat-treated filtrate was quite high (around 6.00-6.80 mS / cm). The starting conductivity value was noted in the protocol. To reach the 3.5 mS / cm conductivity, the heat-treated filtrate was diluted with around 16 to 18 L of 10 mM Tris / HCI pH 8.0. The buffer used was weighed and the printout added to the protocol. The volume of dilution buffer and the conductivity were noted in the protocol. Finally, the pH was checked again and re-adjusted if required by adding 3.5 M Tris. The final conductivity and pH values were documented in the protocol and the data were logged in the PC system.

[0108] The PE container with the prepared feed was weighted and the pre-determined tare weight of the tank was subtracted from the total weight. For the calculation of injection volume, it was assumed that 1 ,000 g is equal in volume to 1 ,000 mL. 1 ,350 mL of feed must remain in the tank to make sure that no air is drawn onto the column during the run. Therefore, to determine the loading volume, the total weight (= volume) minus 1 ,350 mL was calculated and 3.02 mL was calculated for sample injection to clear the buffer from the line. The load was named, labeled, aliquoted (5 x 500 pL, storage below -35 °C).

[0109] Example 3.5: Anion Exchange Chromatography Packing of Vantage L Laboratory Column (250x22 mm), used for Nuvia HP-Q (CV

[0110] 69.6 mL), was carried out as follows:

[0111] The suspension was poured directly into the prepared Vantage L Laboratory Column (Merck: 96220250) (prepared according to the manufacturer’s instruction manual with the bottom adjuster). Then, the column was closed with the upper adjuster and packed in normal mode with a 0.15 M NaCI solution (several times for 5 min with 26 mL / min flowrate until bed height was constant). The upper adjuster was lowered to the upper level of the packed resin. For Quality Control of the column, asymmetry determination was carried out with a 0.15 M NaCI solution, with a flowrate of 3.35 mL / min (100 cm / h). The conductivity peak was determined by injection of 1 mL of 1 M NaCI in a 1 mL loop. The packing process was successful, if the as value was within the range of 0.8 to 1 .8.

[0112] The method was loaded into ChromLab software (Bio-Rad) for each chromatography run. A main program was loaded, and for individual runs only the variables (load volume, collection window flow-through) were adjusted and saved for each run under the respective run name. All the buffers and solutions used were filtered through a 0.22 pm membrane filter and degassed. The column was washed with 2 CV H2O and equilibrated with 2 CV equilibration buffer (10 mM Tris / HCI pH 8) before starting the run.

[0113] Overview program setting:

[0114] • Equilibration step (EQ): 10 mM Tris / HCI pH 8, Valve A2, flow rate 13 mL / min, 1 CV

[0115] • Sample application: Feed Fraction IV-4, Valve: Sample pump, flow rate 13 mL / min, 1 CV

[0116] • Wash step: 10 mM Tris / HCI pH 8, Valve: A2, flow rate 13 mL / min, 2 CV

[0117] • Elution step: 10 mM Tris / HCI + 500 mM NaCI pH 8, Valve: Pump B (100%), flow rate 2 mL / min, 3 CV

[0118] • Regeneration step: 2 M NaCI, Valve: A3, flow rate 8.50 mL / min, 2 CV

[0119] • Wash step: H2O, Valve: A4, flow rate 8.5 mL / min, 2 CV

[0120] • CIP step:1 M NaOH, Valve: A6, flow rate 8.5 mL / min, 2 CV

[0121] • Wash step: H2O, Valve: A4, flow rate 8.5 mL / min, 3 CV

[0122] • Storage step: 20% (v / v) EtOH, Valve A7, flow rate 5 mL / min, 3 CV Sample application:

[0123] Heat-treated Fraction IV-4 was used for the run. The procedures for feed preparation and calculation of loading volume for the chromatographic run are described above in detail. In the ChromLab program the volume was added with the flow rate 13 mL / min for sample application.

[0124] Sample collection:

[0125] The sample collection window was set with respect to the total volume of load / feed volume of run. The collection window was with a flow through (FT 1 , FT2 and FT3) interval of 6x 8 mL, from the beginning of the run. Where a wash peak was eluted, the samples were collected from an intensity higher than 75 mAU with a fraction volume of 6 mL. The eluate fraction was collected in 6 mL fraction sizes starting at an intensity of 40 mAU. Further program steps were not collected. The FT1 , FT2, FT3, wash and elution fractions were pooled and mixed carefully. Aliquots were prepared for analysis. The remaining main sample (eluate fraction) and aliquots from flow-through, wash and eluate fraction were frozen and stored below -35 °C.

[0126] The left overload (load left) in the PE container was for two days at room temperature. The volume was measured, aliquots prepared and stored at -35 °C.

[0127] Table 4: Chromatographic program with Nuvia HP-Q column.

[0128] Example 3.6: Ultrafiltration / Diafiltration UF / DF (Amicon® Stirrer Cell)

[0129] An Amicon® Stirrer Cell (Merck) was used for rapid sample concentration and buffer exchange steps through a 100 kDa membrane under an external compressed gas source (air at 10 PSI and ~0.9 bar), while stirring with a magnetic stirrer at 200 min’1. The equipment was assembled, and the filtration was performed at room temperature according to the Merck user guide. Three eluate fractions from (pooled) Nuvia HP-Q runs were removed from -35 °C storage and thawed at 37 °C for approximately 15 min in a water bath. Afterwards, the samples were pooled in the Amicon® stirrer cell. At this point, the protein solution looked clear, no precipitation visible after thawing at 37 °C for 15 min. The membrane was washed in H2O for 1 h, the shiny side of the membrane facing down. The water was changed 3 times. In addition, the Amicon® Stirred Cell was washed with water before use. The membrane (shiny side up) was placed on the bottom of the Amicon® Stirred Cell and the Amicon® Stirred Cell was washed with H2O (75 mL) and 10 mM Tris / HCI pH 7.2 (200 mL) again.

[0130] Amicon Procedure

[0131] The thawed elution fractions from three Nuvia HP-Q runs were placed in the Amicon® Stirred Cell (max. 200 mL) and the cell was closed. For each elution fraction the volume and the total volume together were noted in the protocol. The samples were mixed for a few minutes using a magnetic stirrer at 200 min-1, the cell opened and aliquots for analysis were taken (Load: 3 x 70 pL, 3 x 100 pL, 1 x 400 pL).

[0132] Again, the Amicon® cell was closed, placed back on the magnetic stirrer, and attached with air pressure (target: 0.5-1 bar, ~10 PSI). The pressure which was set on the system was noted in the protocol as well as the stirring speed (200 min-1). The sample was concentrated until a volume of < 30 mL was left. The Amicon® cell was opened and the actual volume was checked with a disposable pipette. If the total sample volume exceeded 200 mL, the remaining sample could be added in the cell and the steps as just described were repeated until a volume of approximately 20 mL remained. Next, the buffer was exchanged by adding 100 mL of 10 mM Tris / HCI pH 7.2. Settings and volumes of Tris buffer were documented. The cell was closed and stirring at 200 min-1was started. The air pressure was attached, and the values of both stirring speed and air pressure were documented.

[0133] The air pressure was stopped at a volume of ~20 mL and the retentate was transferred to a 50 mL falcon. The exact time and volume were measured. The stirrer, cell and membrane were intensively washed with 4x 5 mL of 10 mM Tris / HCI pH 7.2 and combined with the sample. Total volume of retentate [mL] was measured and noted in the protocol (target: ~40 mL). The pH and conductivity of the retentate were checked. The pH was adjusted with 1 M HCI to pH 7.20 (± 0.05) and the conductivity with 10 mM Tris / HCI pH 7.2 to < 5 mS / cm (target: 3-5 mS / cm). The final pH and conductivity were documented, and the value was logged in the system. Afterwards, the retentate was centrifuged with the centrifuge Megafuge ST4R Plus at 4000 min-1and 4 °C for 10 min. If a pellet was visible, the supernatant was transferred into a new 50 mL tube. Next, an additional filtration was done through a 0.2 pm membrane (32 mm syringe filter with 0.8 / 0.2 pm Supor® membrane, ref: 4658). Beforehand, the membrane was saturated for 5 min with Albumin, final concentration 2 g / L in H2O (Kedrion, infusion solution, 200 g / L, PC: 04150093766171 SN: 84064763506105) and afterwards washed with 10 mL H2O and 20 mL Tris / HCI pH 7.2 buffer.

[0134] The final volume was measured; the sample was named, labelled and aliquoted for analysis (retentate / load CH: 3 x 70 pL, 3 x 100 pL, 1 x 400 pL). The retentate / load was ready for affinity chromatography run. For this purpose, the loading volume was calculated by deducting 4.5 mL from the final volume. In addition, the volume of the flow-through fraction was measured and aliquots for analysis (FT: 8 x 500 pL) were taken. The aliquots were stored at below -35 °C.

[0135] Example 3.7: Affinity Chromatography

[0136] The final purification and polishing step were performed using a ferritin affinity column. Packing of ferritin-affinity resin (Gdtec column, CV 530 pL) was carried out as follows:

[0137] The suspension was poured directly into the prepared Gdtec column (prepared according to the manufacturer’s instruction manual, with the bottom adjuster). Then, the column was closed with the upper adjuster and packed in normal mode with 1x PBS (firstly for 5 min at 0.33 mL / min (100 cm / h), then several times for 2 minutes with 0.5 to 1.55 mL / min flowrate until the bed height was constant). The upper adjuster was lowered to the upper level of the packed resin. For Quality Control of the column, asymmetry determination was carried out with a 0.15 M NaCI solution, with a flowrate of 0.33 mL / min (100 cm / h). The conductivity peak was determined by injecting 53 pL of 1 M NaCI in a 100 pL loop. The packing process was successful if the As value was within the range of 0.8-1 .8.

[0138] Chromatographic Set-Up

[0139] The method was loaded in ChromLab software (Bio-Rad) for each chromatography run. A main program was loaded, and for individual runs, only the variable (load volume) was adjusted and saved for each run under its respective name. All buffers and solutions used were filtered and degassed in advance. The column was manually washed with 40 CV equilibration buffer (PBS pH 7.3) before starting the run.

[0140] Overview program setting:

[0141] • Equilibration step (EQ): PBS pH 7.3, Valve 2 (A2), flow rate 0.3 mL / min, 5 CV

[0142] • Sample application: Retentate UF / DF, Valve 1 (A1 ), flow rate 0.09 mL / min

[0143] • Wash step 1 : PBS pH 7.3, Valve: 2 (A2), flow rate 0.19 mL / min, 20 CV

[0144] • Wash step 2: 0.1 M acetate buffer pH 5.0, Valve: 3 (A3), flow rate

[0145] 0.19 mL / min, 15 CV

[0146] • Wash step 3: 0.1 M acetate buffer pH 4.5, Valve: 4 (A4), flow rate

[0147] 0.19 mL / min, 15 CV

[0148] • Elution step 1 : 2 mM citrate + 150 mM NaCI pH 2.5, Valve: 5 (A5), flow rate 0.19 mL / min, 25 CV

[0149] • Elution step 2: 2 mM citrate pH 2.0, Valve: 6 (A6), flow rate 0.19 mL / min, 25 CV

[0150] • CIP step 2: 0.1 M NaOH, Valve: 7 (A7), flow rate 0.19 mL / min, 5 CV

[0151] • Wash step: H2O, Valve: 5 (A5), flow rate 0.19 mL / min, 10 CV

[0152] • Storage step: PBS + 0.05% (w / v) sodium azide, Valve B (100% B), flow rate 0.3 mL / min, 5 CV

[0153] Sample application:

[0154] Retentate from the UD / DF step was used for the run. The procedure for feed preparation and calculation of loading volume for chromatographic run were described above in detail. In the ChromLab program the volume was added with the flow rate 0.09 mL / min for sample application. A minimum of 1.0 mL feed must remain in the tube to make sure that no air is drawn onto the column during the run. The feed was stored at 2 to 8 °C during the run.

[0155] Sample collection:

[0156] The whole sample was collected during the run with a volume of 4 mL for flow- through, 3 mL for the wash steps and 1 mL for elution, CIP and wash (H2O). The storage step was not collected. For immediate neutralization of the pH in the collected eluate fractions, 8 pL (eluate 1 condition pH 2.5) and 10 pL (eluate 2 condition pH 2.0) of a 1 M sodium carbonate solution was placed in each of the tubes. The next day, pH paper was used to check the pH target value of 7 to 7.5 and if necessary, adjusted with 1 M HCI or 1 M sodium carbonate solution. The individual fractions collected after the run were analyzed by SDS PAGE under reducing conditions. For this purpose, the eluate fractions were applied undiluted onto the gel. In addition, a molecular size standard (8 pL), a ferritin standard (1.5 pg), the load of the run (1 :8 dilution), the combined flow-through and wash fraction (1 :8 dilution) were loaded. The SDS PAGE was performed. After analysis of the gel, the (apo)ferritin fractions were pooled, named, labeled, and aliquots taken for analysis, and the remaining main sample and aliquots were frozen and stored below -35 °C. The same was done for flow-through / wash fraction and for analytical purpose, single fractions before and after the (apo)ferritin fraction. The volume of the remaining load (load remaining) in the tube was measured and stored at -35 °C. The affinity chromatography run was programmed as follows:

[0157] Table 5. Chromatographic program with ferritin affinity column

[0158] Example 3.8: Analytics and Data Analysis

[0159] Characterization of a biotechnological / biological products include the determination of physicochemical properties, identity, purity & impurities, potency, and quantity. Table 6 shows the analytical methods used for process sample analysis.

[0160] Table 6. Overview on the main Analytical Methods

[0161] Calculation of Total Protein (TP), Ferritin and Iron content in process samples To calculate the results for the process samples (Bradford, MicroBCA, iron assay, iron binding assay, ferritin ELISA), the median from the relevant duplicate measurements were taken and the results summarized. Fehler! Verweisquelle konnte nicht gef unden werden. Fehler! Verweisquelle konnte nicht get unden werden.Tne purity of the product was calculated by taking account in proportion of the total protein value and ferritin content.

[0162] Step Yield and Process Yield calculation

[0163] The step yield was calculated by taking account the ratio from load (100%) and fraction of each process step. Process yield was calculated from the beginning of the process until the final product, the median from the single measurements were taken and the results were summarized.

[0164] Protein Detection by Bradford Method

[0165] The Bradford protein assay was used to determine the concentration of total protein in a sample. The Quick Start™ Bradford Assay Kit was used according to the manufacturer’s protocol. Briefly, samples were mixed with Bradford reagent, incubated for 5 min at 37 °C, and absorbance was measured at 595 nm. As a reference, a certified (CE) “Normal & Abnormal Correctness Control" (LABOR + TECHNIK Eberhard Lehmann GmbH) was used, with verified ranges for total protein. For the range 125 - 1 ,000 pg / mL protein (Bradford high), 5 pL of diluted sample were mixed with 250 pL reagent and then processed as described above. For the range of 1.25 - 20 pg / mL protein (Bradford low), 150 pL of diluted sample were mixed with 150 pL reagent and then processed as described above. SDS-PAGE, Coomassie Staining and Western Blotting

[0166] SDS-PAGE and Western Blot analysis were performed using the Mini-PROTEAN® tetra cell system and 4-20% precast polyacrylamide gels (Bio-Rad, Mini-Protean TGX Stain-free gels 4-20%). For a non-reducing SDS-PAGE, the samples were diluted 1 :4 with Laemmli buffer (non-reducing) and then loaded onto the gel. For a reducing SDS-PAGE the Protein samples were diluted 1 :4 in Laemmli buffer with 10% DTT, and incubated for 5 min at 95 °C. For an SDS-PAGE, the lanes were loaded with 0.59 to 4 pg of protein per well. 0.3 to 1.5 pg of protein per well were used for Western Blots.

[0167] Each gel was loaded with standard lanes of ferritin and the Precision Plus Protein™ Dual Color Pre-Stained Protein standard. Electrophoresis was carried out at 80 - 200 V with 1x running buffer for 1 h in a refrigerator at 4 °C.

[0168] For Coomassie staining, SDS-PAGE gels were incubated in Bio Safe™ Coomassie G-250 stain solution at room temperature for 1 h on a rocking table. Gels were washed and de-stained with distilled water. Transfer onto nitrocellulose membranes was performed by wet blotting in 1x Transfer buffer (10x buffer diluted 1 :10 with 20% v / v methanol) using the Mini Trans-Blot® Electrophoretic Transfer System at 350 mA for 1 h. Since the transfer system is set-up outside the refrigerator, a sealed ice unit was added to the buffer tank to cool the system and prevent overheating and temperature fluctuations. Then transfer membranes were blocked in 5% (w / v) dried skimmed milk in TBST buffer for 1 h and incubated overnight with the primary antibody (diluted in 5% (w / v) dried skimmed milk in TBST buffer) at 4 °C. After washing steps using TBST buffer (3 x 5 minutes), membranes were incubated with a secondary antibody conjugated to horseradish peroxidase or alkaline phosphatase (in 5% [w / v] dried skimmed milk in TBST buffer) for 1 h at room temperature. Protein bands were detected using SeramunBlau prec® membrane substrate (for Horseradish peroxidase conjugated antibodies) or SeramunPurple prec® membrane substrate (for Alkaline Phosphatase conjugated antibodies) according to the manufacturers’ instructions.

[0169] Native PAGE and Iron Staining

[0170] Native-PAGE was performed using the Mini-PROTEAN® tetra cell system and 4- 20% precast polyacrylamide gels. The samples were diluted 1 :2 with 2 x Native buffer and then loaded onto the gel. For a Native-PAGE, the lanes were loaded with 2 to 3.5 pg of protein per well. Each gel was loaded with standard lanes of ferritin, Light-chain ferritin, Heavy-chain ferritin and the Precision Plus Protein™ Dual Color Pre-Stained Protein standard. Electrophoresis was carried out at 50 V for at least 30 min and at 80 - 200 V with 1x Running buffer for Native PAGE (without SDS) for ~1.5 h in a refrigerator at 4 °C. For iron staining, Native PAGE gels were incubated in 20 mL of Solution 1 (0.1 M potassium hexa-cyano-ferrate II) at room temperature in the dark on a rocking table for ~10 min. Gels were washed with distilled water and incubated in 20 mL of Solution 2 (10% methanol v / v, 10% trichloroacetic acid w / v) for 30-90 min at room temperature on a rocking table. Gels were washed and de-stained with distilled water and a picture taken for documentation. These gels could be further used for Coomassie staining. For this purpose, Native-PAGE gels were incubated in Bio Safe™ Coomassie G-250 stain solution at room temperature for 30 min on a rocking table. Gels were washed and de-stained with distilled water overnight in the refrigerator at 4 °C. Transfer onto nitrocellulose membranes was performed by wet blotting as described above.

[0171] ELISA (Total Ferritin)

[0172] The following enzyme immunoassay test uses a typical one-step capture or ‘two- site-binding’ type assay. Ferritin from the sample and standards are allowed to bind simultaneously to the plate and to the horseradish peroxidase (HRP) conjugate. The washing and decanting steps remove any unbound HRP conjugate. After the washing step, the enzyme substrate is added. The enzymatic reaction is terminated by addition of the stopping solution. All reagents must be at room temperature before use.

[0173] 1 . Pipette 20 pL of each calibrator, control and sample into corresponding wells in duplicate.

[0174] 2. Pipette 200 pL of the conjugate working solution into each well

[0175] 3. Incubate on an orbital shaker (approx. 600 min-1) for 30 minutes at room temperature

[0176] 4. Wash the wells 5 times with 300 pL of diluted wash buffer per well and tap the plate firmly against absorbent paper.

[0177] 5. Pipette 150 pL of TMB substrate into each well at timed intervals.

[0178] 6. Incubate on an orbital shaker (approx. 600 min-1) for 10-15 minutes at room temperature.

[0179] 7. Pipette 50 pL of stopping solution into each well. 8. Read the plate on a microwell plate reader at 450 nm within 20 minutes after addition of the stopping solution.

[0180] Iron Assays for Determination of Fe2+and Fe3+

[0181] Fe2++ 2,2-Bipyridin [Fe-(bipy)s]2+

[0182] Scheme of Fe-(bipyridine)3 complex (formula 1 ):

[0183] 10 pl samples were pipetted into wells of the microplate and 20 pl of 1 M HCI were added to each well. For Fe3+KSCN or NH4SCN and for Fe2+2,2-Bipyridin were used as dye reagent and were added to each well. The absorptions at 493 nm I 520 nm in the Multiscan FC Reader were measured and used as the collated building of FeSCN and Fe(bipyridine). The standard calibrators were set for Fe3+: 54.75, 109.5, 219, 545, 1090 pM and Fe2+: 5, 10, 20, 50, 100, 200, 500 pM. The linearity was shown to be R2> 0,995.

[0184] Iron Binding capacity

[0185] To assess the quality and potency of final product (apo)ferritin, the iron binding capacity of (apo)ferritin was determined by titration with ferric nitrilotriacetic acid. Stock solution of 100 mM Fe(lll)NTA: 16.4 mg NaHCOs

[0186] 4.1 mg trisodium nitrilotriacetic acid (NTA) 18.4 mg ferric (III) chloride in 1 mL water (Keep anoxi!)

[0187] Solution A: 1 :200 Dilution in water: 500 pmol / L

[0188] Solution B: 0.3 mol / L Tris-HCI, pH 8

[0189] 50 pL aliquots of solution A and 450 pL samples of (apo)ferritin subunits (1 .5 ng) in solution B were added in tube. The samples were incubated for 1 h at 20-25 °C in Thermomixer 300 min’1. 260 pL of samples were transferred to a microtiter plate and the absorbance measured at 450 nm. The generated (apo)ferritin product (#1113) with 116.88 pg / mL “Ferritin” was able to bind 507.8 pmol / L of Fe2+ions. That corresponds to 2,085 bound molecules of iron per ferritin molecule with the following formula:

[0190] X = Ratio of Iron molecule per one ferritin moleculecFerritin = Concentration ferritin [ng / mL] from ELISAcbound iron = Molar concentration of iron [pmol / L] from Iron Binding Assay M = Molecular mass ferritin, 480 kDa

[0191] In summary, it was experimentally evidenced that a method based on performing a heat treatment, an ion-exchange chromatography and a positive affinity chromatography with a (apo)ferritin containing fluid as claimed enables the immediate extraction and purification of (apo)ferritin. As exemplified, a fraction from plasma fractionation as (apo)femtin containing fluid is a particularly suitable starting material.

Claims

Patent Claims1. A method for extracting (apo)ferritin, wherein said method comprises the following steps: i. providing blood plasma or a plasma fraction as an (apo)femtin containing fluid, in particular a plasma fraction obtained from side fractions of plasma fractionation according to Cohn or to Kistler- Nitschmann, ii. performing a heat treatment of the fluid under conditions allowing precipitation of proteins other than apoferritin or ferritin, ii-b. removing precipitated proteins from the fluid, preferably by performing at least one centrifugation, iii. performing ion-exchange chromatography, in particular anion- exchange chromatography, under conditions allowing elution of ferritin and / or apoferritin, iii-b. performing at least one filtration with the elution of step iii. allowing concentration of (apo)femtin, and iv. performing positive affinity chromatography under conditions allowing elution of ferritin and / or apoferritin.

2. The method of claim 1 wherein the concentration step iii-b is performing ultrafiltration and diafiltration (UF / DF) with the elution of step iii. allowing concentration of (apo)ferritin.

3. The method of any of claims 1 or 2, wherein the (apo)ferritin containing fluid further comprises one or more chaotropic agents decreasing interactions between apoferritin or ferritin sub-units and / or releasing iron ions from ferritin.

4. The method of any of claims 1 to 3, wherein the heat treatment is conducted at a pH in the range from 4.5 to 6.0, preferably at a pH in the range around 4.8.

5. The method of any of claims 1 to 4, wherein the heat treatment is conducted at 50 to 110 °C, preferably at 60 to 90 °C.

6. The method of any of claims 1 to 5, wherein the heat treatment is conducted for 1 min to 4 h, preferably for 5 min to 60 min.

7. The method of any of claims 1 to 6, wherein said method is performed until an (apo)ferritin purity of >60% (w / v), in particular >70% (w / v), preferably >80% (w / v) is obtained.

8. (Apo)Ferritin obtained from a method of any of claims 1 to 7.

9. (Apo)Ferritin obtained from a method of any of claims 1 to 7 for use in a method of treating or preventing acute or chronic inflammation, in particular chronic neurologic inflammation, by acting as iron scavenger and thereby reducing oxidative stress caused by free iron, in particular free iron released from tissue or blood.

10. (Apo)Ferritin obtained from a method of any of claims 1 to 7 for use in the preparation of Magneto-Ferritin, wherein said (apo)femtin is supplemented with ferrit.

11. (Apo)Ferritin of claim 10, wherein the prepared Magneto-Ferritin is used as drug carrier and inductively excitable at the site of action, in particular at a tumor site.

12. (Apo)Ferritin of claim 11 , wherein the drug carrier is combined with monoclonal antibodies and / or cancer targeting molecules.

13. (Apo)Ferritin obtained from a method of any of claims 1 to 7 for use in a method of treating iron overload caused by genetic disorders.

14. Use of the method of any of claims 1 to 7 for extraction and / or purification of (apo)ferritin from blood, blood plasma and / or a fraction thereof, preferably a plasma fraction obtained from side fractions of plasma fractionation according to Cohn or to Kistler-Nitschmann.

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