Methods for the isolation of human haptoglobin, individual human hp isoforms, and human albumin and haptoglobin from human cohn v fraction, and, product

Abstract

METHODS FOR THE INSULATION OF HUMAN HAPTOGLOBINE, INDIVIDUAL HP HUMANA ISOFORMS, AND HUMAN ALBUMIN AND HAPTOGLOBINE FROM FRACTION V OF COHN HUMANA, AND, PRODUCT. The invention provides a method for the isolation of haptoglobin from a sample comprising fraction Cohn V, wherein said method comprises anion exchange chromatography of said sample. The product can be further purified and / or subjected to one or more steps of inactivation or viral reduction. Haptoglobin alone can then be formulated for pharmaceutical use.

Description

[0001] Haptoglobin (Hp) is an acute-phase response protein synthesized primarily in the liver. It is also synthesized in other tissues such as arterial walls, the endometrium, and the peritoneum.

[0002] The function of the Hp core is as a hemoglobin (Hb) binding protein, necessary for the terminal processing and removal of free Hb, mainly in the reticuloendothelial system (RES) of the liver. This system allows the iron present in the Hb component to be conserved.

[0003] The heme molecule (ferroprotoporphyrin IX) found in Hb is an indispensable element in a wide variety of other protein systems (e.g., enzymes such as cyclooxygenase [COX] and nitric oxide synthase [NOS]) that act according to the properties of the polypeptide(s) to which it is bound. However, excess free heme can cause cell damage and tissue injury (e.g., in the kidneys, lungs, and central nervous system - CNS), since it catalyzes the formation of reactive oxygen species, which in turn causes oxidative stress.

[0004] Intravascular hemolysis occurs physiologically, but it also accelerates as a serious complication in several autoimmune, infectious (e.g., malaria), and inherited (e.g., sickle cell disease) diseases. During hemolysis, free vascular Hb is captured by Hp and transported to the RES of the liver. Some processing may also occur in monocyte macrophages. However, free vascular Hb can be rapidly transformed into met-Hb, which readily releases the potentially toxic heme component. Free vascular heme is captured by albumin or hemopexin (Hx) and transported to the liver for degradation in the RES. When large amounts of heme accumulate (e.g., in a blood clot or vascular deposit), clearance mechanisms are completely overwhelmed or unable to access the free heme, resulting in tissue damage.If the amount of red blood cell lysis saturates the heme / Hb removal system, Hb will begin to appear in the urine (glomerular filtration by the kidneys).

[0005] Hp has a tetrameric structure comprising two α and two β chains, linked by disulfide bonds. The β chain (245 amino acids) has a mass of approximately 40 kDa (of which approximately 30% w / w is carbohydrate) and is shared by all phenotypes. The α chain exists in two forms: α1 (83 amino acids, 9 kDa) and α2 (142 amino acids, 17.3 kDa), and therefore Hp occurs as three phenotypes, referred to as Hp1-1, Hp2-1, and Hp2-2. Hp1-1 contains two α1 chains, Hp2-2 contains two α2 chains, and Hp2-1 contains one α1 and one α2 chain. Hp1-1 has a molecular mass of 100 kDa, or 165 kDa when complexed with Hb. Hp1-1 exists as a single isoform and is also referred to as the Hp dimer. Hp2-1 has an average molecular mass of 220 kDa and forms coating polymers. Hp2-2 has an average molecular mass of 400 kDa and forms cyclic polymers. Each different polymeric form is a different isoform. A PCR methodology was outlined (Koch et al. 2002, Clin. Chem.48: 1377-1382) to study the polymorphism of Hp.

[0006] Hp has been identified as a potential treatment for renal disorders caused by hemolysis. It is potentially useful therapeutically as a means of removing free hemoglobin; the complexes thus formed have potential additional benefits as anti-inflammatory, antioxidant, or angiogenic agents. However, Hp is considered difficult to isolate in large quantities while retaining its biological activity. A variety of protocols for the isolation of Hp have been described. A common theme is affinity chromatography. Affinity ligands used include monoclonal antibodies (Katnik and Jadach, 1993, Arch. Immunol. Ther. Exp. (Warz) Vol. 41: 303-308 and Tseng et al. 2004, Protein Expr. Purif. 33: 265-273), hemoglobin (Liau et al. 2003, J. Chromatogr. Analyt, Technol. Biomed. Life Sci. 790: 209-216 and Concanavalin A (Katnik et al. 1995, Eur. J. Clin. Chem. Clin. Biochem.33: 727-732).

[0007] Although monoclonal antibodies based on affinity ligand protocols result in a reasonably pure product, only low yields have been reported. Such isolation methods are also not well suited to large-scale production processes, as a result of the need for large quantities of affinity ligand. In particular, the required quantities of monoclonal antibodies are likely to be too expensive and difficult to obtain. Monoclonal antibodies also suffer from the potential drawback of being selective for the various Hp isoforms. The use of Hb as the affinity ligand has been more successful. Good yields of reasonably homogeneous Hp have been obtained; however, as is the case with all affinity ligand-based protocols, process scaling is difficult and often not cost-effective.Another drawback of affinity ligand-based protocols is that, if the Hp product is intended for use as a pharmaceutical product, the source of the affinity ligand must be controlled. In this respect, Hb may not be acceptable to regulators, since it originates from red blood cells. It is also necessary to monitor and control ligand leaching, as the presence of the affinity ligand in the product may raise concerns about its safe use in patients.

[0008] Selective precipitation of Hp by a plant root extract has been described (Nayak et al. 2002, Pro. Pep. Lett. 9: 503-510). However, like affinity chromatography, this protocol is unsuitable for large-scale commercial production.

[0009] HPLC (High Performance Liquid Chromatography) based techniques have also been used on a porcine serum ammonium sulfate precipitate (Yang and Mao, 1999, J. Chromatogr. B. Biomed. Sci. Appl., 731: 395-402). Again, this protocol is only considered suitable for small-scale Hp isolation.

[0010] An alternative approach to Hp isolation has been to use ion-exchange chromatography. US 4,061,735 and US 4,137,307 described the isolation of Hp via anion-exchange chromatography of ammonium sulfate precipitates from human plasma, or from derivatives of Cohn fractions IV, IV-1 or IV-4 derived from human plasma. The precipitation of ammonium sulfate is established to remove transferrin, albumin and other undesirable proteins. It is essential for this method that the ion-exchange substrate be a strong ion-exchange substrate, such as QAE-Sephadex® or GE cellulose. This method is unsuitable for large-scale commercial isolation of Hp because of the immense quantities of contaminated salt (ammonium sulfate) that would have to be discarded. Batch purification methods using an anion exchanger are not equally ideal for incremental use.

[0011] The isolation of Hp from rabbit plasma precipitated in sodium acetate-acetic acid using DE-52 microgranular cellulose as an anion exchange substrate (DEAE cellulose) has been reported (Basler and Burrel, 1983 Inflammation 7(4): 387-400). Preparative Isoelectric Focusing was required to obtain a reasonable degree of product purity. However, although a maximum yield between 50 and 70% was mentioned, data analysis indicates that the yield was actually 44% and the purification was only three times. This protocol is not suitable for large-scale commercial use because of its complexity.

[0012] The object of the present invention is to improve what is currently available for the isolation of Hp in one or more of the following aspects: yield and / or purity of Hp and its reproducibility, simplicity of the process and suitability for use on a large and / or commercial scale in terms of economic viability.

[0013] It has now been observed that Hp can be isolated from a previously unknown fraction of human plasma as a source of Hp using an anion exchange substrate in a simple method that results in high reproducible yields and is capable of economical use on a large scale. Importantly, the isolation method must be usable in the purification of Hp for therapeutic uses (i.e., the final product must be sufficiently pure and free of contaminants for administration to humans).

[0014] The isolation of human plasma components is well documented. Methods for isolating proteins such as albumin and immunoglobulins have been known for many years. A common first step is plasma fractionation. The most commonly used fractionation method is the Cohn method (Cohn, EJ et al. 1946 J. Am. Chem. Soc. 68: 459) and its modifications (e.g., Kistler and Nitschmann, 1962, Vox Sang, 7, pp. 414-424). This process begins with cryoprecipitation to remove some of the coagulation factors, followed by celite treatment. The resulting plasma lake is treated with increasing concentrations of ethanol and the precipitate fraction A+1 (a combination of fractions I, II, and III) and fraction IV. The reduction in pH of the supernatant of fraction IV and the drop in temperature from -5 °C (± 1 °C) to -10 °C (± 3 °C) cause the precipitation of Cohn's fraction V.

[0015] Traditionally, Cohn's V fraction has been considered to be primarily a source of albumin. However, it has now been understood that Cohn's V fraction is also a useful source of Hp and, moreover, is a source from which Hp can be successfully and relatively easily isolated.

[0016] In a first aspect, the present invention therefore provides a method for isolating haptoglobin from a sample comprising Cohn's V fraction, wherein said method comprises anion exchange chromatography of said sample.

[0017] By “haptoglobin (Hp)” is meant all phenotypes (including all isoforms) of Hp. Hp isolated using the method of the invention will potentially contain all Hp isoforms present in the starting sample. The exact composition of the isolate will ultimately be prescribed by the source phenotypes. Consequently, if pooled plasma samples are used, all isoforms will be isolated. A methodology has been devised for studying Hp polymorphism (haptoglobin typing using the Phastsystem, Hansson et al., Application Note 373 for the Phastsystem, Amersham Biosciences), and thus the skilled person should be able to determine which isoforms are present in the isolate. The method can be used to determine which of the various sizes of Hp isoforms are present.The use of hemoglobin to bind haptoglobin and thus provide a pseudoperoxidase activity that can be detected in a gel can be replaced by Western blotting with antibody detection (immunostain).

[0018] It has been verified, from the use of these techniques and others, such as high-performance size exclusion chromatography (HPLC-SEC assay), Hp ELISA, Hb binding assay and turbidimetric readings, that the different forms of Hp give different signals in the different assays. For example, lower molecular weight Hp forms give a lower signal in ELISA, and a higher signal by turbidimetric analysis, than higher molecular weight forms. The assays of choice for comparing the quantities of the different forms are, therefore, Western blots, HPLC-SEC and the Hb binding assay.

[0019] Different Hp isoforms are thought to have different biological effects, as demonstrated by in vitro observations and population studies in certain disease states. From a study of these data, it is likely that the lower molecular weight forms isolated from fraction V have a greater anti-inflammatory effect than the higher molecular weight forms. It is equally clear from the analysis of a different Hp species that binding to human Hb may well be species-specific. Knowledge of these differences has allowed for the optimization of the fractionation method and access to different Hp isoforms for in vitro research and in vivo efficacy studies.

[0020] By “isolation” it is meant that preferably at least 50% of the Hp present in the starting sample of fraction V is present in the product of the method of the invention. Preferably at least 65%, and most preferably at least 80% of the Hp present in the starting sample is present in the product of the method of the invention. The Hp obtained using the method of the invention will preferably be at least 70% pure, more preferably at least 80% pure, and most preferably 90% pure. It should be noted that, as with all isolation procedures, increases in purity are frequently associated with decreases in yield. The added stages to ensure viral safety may also reduce overall recovery.

[0021] The qualified person shall be familiar with the techniques by which the purity / yield of an Hp isolate of the invention can be determined. These should include HPLC-SEC chromatography analysis (for samples having a purity level greater than 40%), an Hp-Hb binding assay (based on the method of Katnik et al., 1995, ibid.) or in combination with the HPLC-SEC assay when the sample is sufficiently pure, or with spectrophotometry at 280 nm with an average extinction coefficient for plasma proteins (Hansson et al., Application Note 373 for the Phastsystem, Amersham Biosciences). The specific activity of the product can also be determined using the Hb binding assay in combination with HPLC-SBC and the extinction coefficient when determined from the amino acid sequence.

[0022] By “Cohn V fraction” is meant the plasma fraction designated Cohn V fraction in the Cohn fractionation method (Cohn, 1946, ibid.) and any modification of said method, including the modified method of Kistler and Nitschmann (1962, ibid.). A skilled person should readily understand that the fraction is Cohn V fraction and should be able to prepare this fraction without undue burden. Briefly, this process may involve plasma cryoprecipitation, celite treatment, then staged exposure to 19% ethanol at pH 5.85 and -5 °C, 40% ethanol at pH 5.85 and -5 °C, and finally 40% ethanol at pH 4.8 and -8 °C, with the precipitated material being removed at each stage. The final precipitate is Cohn V fraction. Any fractions equivalent in composition to Cohn's fraction V, obtained in an alternative manner or known by alternative terminology, are considered to be covered by the invention.The main constituent of fraction V is albumin. Hp and transferrin are also present in appreciable quantities, and alpha-1-acid glycoprotein can be detected at low levels. Trace amounts of other proteins are also present. Plasma can be obtained from any suitable biological source, although mammalian blood plasma is preferred. Human blood plasma is the most preferred.

[0023] By “sample comprising Cohn’s V fraction” it is denoted that the majority of the proteinaceous component of the sample is Cohn’s V fraction.

[0024] Anion exchange chromatography denotes a chromatographic method whereby separation is achieved based on charge, specifically negative charge. Ion exchange chromatography uses a charged solid support that binds the molecules of a sample applied to the solid support. The unbound molecules can be the target molecules, and thus the ion exchange step can be considered similar to a filtration step that removes unwanted molecules. Alternatively, the target molecules can be those that remain bound, the unbound molecules being unwanted. The latter is the more usual approach, since the elution of the bound molecules from the solid support can then be controlled / selective and thus a better quality product can be obtained. Anion exchange chromatography techniques commonly use substrates such as, but not limited to, dextran, cellulose and their modifications that are positively charged.These substrates may comprise part of the solid support (e.g., a coating) or may form the entirety of the solid support. The solid support may be in particulate form (e.g., a resin), but non-particulate supports (e.g., filter papers or gels) may also be used. Particulate substrates are typically, though not always, thickened in columns.

[0025] When the term “substrate” or the expression “anion exchange substrate” is used, it should be interpreted as referring to substrates in a form suitable for use in anion exchange techniques.

[0026] A sample to be subjected to anion exchange chromatography is applied to the substrate. Based on charge interactions, the (negatively charged) molecules within the sample bind to the substrate. Washing the substrate therefore removes unbound or weakly bound molecules. Controlled / selective elution of bound molecules can be achieved by passing solutions of increasing salt concentration over the substrate, as this breaks the charge interactions between the substrate and the bound molecules. The pH of the elution solution can also be altered to induce elution, since this will change the charge present on the bound molecule and the substrate. The weaker the charge interaction between the molecule and the substrate, the lower the salt concentration required to break the interaction and thus induce the elution of that molecule from the substrate.By carefully controlling the salt concentration, selective elution of bound molecules can be achieved.

[0027] The intensity of the charge interaction can be modified by the choice of material for the solid support. For example, QAE-Sephadex® or GE cellulose are strong anion exchange substrates, while DEAE-cellulose and DEAE-Sephadex® are weak anion exchange substrates.

[0028] A person skilled in the art will be well acquainted with anion exchange techniques and tools and should be able to design and implement protocols specific to their needs. Of particular use in the method of the invention are weak anion exchange substrates, and of exceptional use is DEAE Sepharose® (Amersham). Sepharose® is the name commonly used for agarose beads. Other suitable anion exchange resins include cellulose, dextran, and polymer-based beads.

[0029] By “weak” is meant anion exchange substrates with a weak buffering capacity. The buffering capacity of any particular anion exchanger is easily determined by acid-base titration of the substrate, the resulting titration curve indicating the intensity and amplitude of the buffering capacity.

[0030] Thus, in a preferred embodiment, the invention provides a method for isolating haptoglobin from a sample comprising Cohn's V fraction, wherein said method comprises anion exchange chromatography of said sample using a weak anion exchange substrate.

[0031] In a more preferred embodiment, the invention provides a method for isolating haptoglobin from a sample comprising Cohn fraction V, wherein said method comprises anion exchange chromatography using DEAE agarose.

[0032] Typically, the sample will be loaded onto the anion exchange substrate in a suitable loading solution. The anion exchange substrate and loading conditions should be such that Hp is bound to the substrate while albumin, the main constituent of Cohn fraction V, remains unbound or only weakly bound. Albumin can then be removed from the substrate using a suitable washing buffer before selective elution of Hp. For ease of processing, the anion exchange substrate will preferably be in the form of a column. However, the method of the invention is not limited to column chromatography.

[0033] Similarly, for ease of processing, the same components are preferably used in all different buffers, with only the quantities or concentrations of the individual components varying between the different buffers.

[0034] Thus, in a preferred embodiment, the invention provides a method for isolating haptoglobin from a sample comprising Cohn's V fraction, wherein said method comprises: a) loading said sample onto an anion exchange substrate, and b) selectively eluting haptoglobin therefrom.

[0035] One or more washing steps may also be included to reduce undesirable molecules in the Hp eluate. The purpose of a washing step is to pass a suitable buffer through the substrate, which will elute unbound or very weakly bound molecules from the sample (e.g., albumin) without inducing elution of the target molecule (Hp). Most commonly, one or more washing steps will be included between the loading step of the sample containing the Cohn V fraction into the anion exchange substrate and the elution step of the Hp contained therein. However, washing steps may be included in the middle of the different elution steps, especially if other potentially useful molecules have to be eluted before the elution of Hp.

[0036] Ideally, the anion exchange substrate is washed to remove unbound material before Hp is eluted from it.

[0037] Thus, in a preferred embodiment, the invention provides a method for isolating haptoglobin from a sample comprising Cohn's V fraction, wherein said method comprises: a) loading the sample onto an anion exchange substrate, b) washing the substrate to remove unbound or weakly bound contaminants, and c) selectively eluting Hp from the anion exchange substrate.

[0038] In another aspect, the invention provides a method for isolating albumin and haptoglobin from a sample comprising Cohn's V fraction, the method comprising: a) loading the sample onto an anion exchange substrate, followed by b) washing the substrate to selectively remove albumin, and followed by c) selectively eluting the haptoglobin from the substrate.

[0039] A skilled person will be aware of appropriate loading, washing, and loading buffers and will be able to formulate suitable buffers (in terms of constituents and their concentrations and pH) to achieve either loading, washing of bound Hp, or selective elution of Hp from the particular anion loading substrate in use. The skilled person will be able to optimize these parameters without undue overload. Loading, washing, and elution conditions should be selected in such a way that no unnecessary damage occurs to the Hp. Typical loading, washing, and elution buffers comprise a pair of buffer components and a salt. Suitable pairs of buffer components include, but are not limited to, sodium acetate and acetic acid, sodium citrate and citric acid, citric acid and sodium phosphate, and succinic acid and sodium hydroxide. A preferred pair of buffer components is sodium acetate and acetic acid.Suitable salts include sodium chloride, potassium chloride, and sodium sulfate. Sodium chloride is a preferred salt.

[0040] Since a wide variety of salts and buffer components can be used, loading, washing, and eluting buffers can alternatively be defined in terms of their conductivity. A skilled person will be able to formulate buffers with any suitable salts and buffer components provided the correct buffer conductivity is obtained. A skilled person should also note that the conductivity required for each type of buffer may depend on the anion exchange substrate used.

[0041] Loading and washing plugs are often the same. However, a qualified person will be able to plan separate loading and washing plugs from their common general knowledge, should this become necessary.

[0042] Regarding the washing of a weak anion exchange substrate to which Hp is bound without causing significant elution of Hp, a conductivity between 0.1 and 3.0 mS / cm is preferred, more preferably the conductivity will be between 0.5 and 2.5 mS / cm and, most preferably, between 1.0 and 2.0 mS / cm.

[0043] For washing with Hp-bound Sepharose® DEAE without significant Hp elution, a conductivity between 0.7 and 2.7 mS / cm is preferred, more preferably between 1.1 and 2.3 mS / cm, and most preferably between 1.2 and 2.2 mS / cm.

[0044] As an example, a washing buffer with this more preferred conductivity comprises 5 mM sodium acetate and 15 mM sodium chloride adjusted to pH 4.6 with acetic acid.

[0045] For the elution of Hp from a weak anion exchange substrate, a conductivity between 8.0 and 15.0 mS / cm is preferred, more preferably between 9.5 and 13.5 mS / cm, and most preferably between 10.5 and 12.5 mS / cm.

[0046] For the elution of Hp from DEAE Sepharose®, a conductivity between 9.0 and 14.0 mS / cm is preferred, more preferably between 10.0 and 13.0 mS / cm, and most preferably between 10.5 and 12.5 mS / cm. As an example, an elution buffer with this most preferred conductivity comprises 5 mM sodium acetate and 113.5 mM sodium chloride adjusted to pH 4.6 with acetic acid.

[0047] The pH of loading, washing, and eluting buffers is also important. pH can alter the buffer conductivity, depending on the buffer constituents used, and can also induce elution (desired or not) of the target substrate molecule. In the case of washing buffers for washing Hp bound to a weak anion exchanger, a pH range between 3 and 7 is preferred. More preferred is a pH between 4 and 6, and most preferable is a pH between 4.2 and 5.0. For a buffer comprising 5 mM sodium acetate and 15 mM sodium chloride at a conductivity of 1.7 ± 0.5 mS / cm, the pH should be 4.6 ± 0.1 (adjusted with acetic acid). In the case of eluting Hp from a weak anion exchanger, a pH range between 3 and 7 is preferred. The most preferred pH is between 4 and 6, and the most preferable is between 4.2 and 5.0. For a buffer comprising 5 mM sodium acetate and 113.5 mM sodium chloride at a conductivity of 11.5 ± 1.0 mS / cm, the pH should be 4.6 ± 0.1 (adjusted with acetic acid).A qualified technician will be aware of the relationship between pH and conductivity, and between pH and the degree of elution, and will be able to predict the precise pH ranges that are appropriate for the buffers and substrates being used, and the function they are performing. Furthermore, common general knowledge will allow for the optimization of buffer parameters without undue overload.

[0048] In a more preferred embodiment, the invention provides a method for isolating haptoglobin from a sample containing Cohn's V fraction, wherein said method comprises: a) applying said sample to a DEAE agarose anion exchange substrate, b) washing said substrate with a sodium acetate / acetic acid / sodium chloride washing buffer of a conductivity between 1.2 and 2.3 mS / cm and a pH between 4.5 and 4.7, and c) eluting the haptoglobin from said substrate with a sodium acetate / acetic acid / sodium chloride elution buffer of a conductivity between 10.5 and 12.5 mS / cm and a pH between 4.5 and 4.7.

[0049] Depending on the other constituents present in the sample, it may be necessary to perform the elution in stages to obtain Hp with a high degree of purity. Contaminants that bind to the substrate less strongly than Hp can be eluted first by choosing the appropriate initial elution conditions. Similarly, the elution buffer used to elute Hp should be chosen so that it does not remove contaminants that bind to the substrate more strongly than Hp does. For example, albumin and transferrin bind to DEAE Sepharose more weakly than Hp does, and are eluted before Hp. On the other hand, α1-acid glycoprotein (AAG) has been observed to bind more strongly to DEAE Sepharose than Hp does, and may remain bound to the column after Hp has been eluted.In general, the more negatively charged a particular protein is, the more strongly it will bind to the anion exchange resin, and the higher the salt concentration required to elute it. Any eluted proteins other than Hp can be discarded or, if they are potentially useful, retained for further processing.

[0050] The qualified technician will be familiar with techniques for monitoring the eluate to enable the progress of the elution to be followed and to ascertain what is being eluted in the various fractions. For example, UV spectroscopy can follow the progress of the elution in real time. Techniques such as HPLC SEC chromatography or SDS-PAGE can be used to detect the presence and identity of impurities. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-ToF) of HPLC fractions or SDS-PAGE bands can also be used to identify the proteins present. Known proteins can be monitored with antibody-based detection methods [e.g., enzyme-linked immunosorbent assay (ELISA), radial immunodiffusion (RID), and turbidimetric determinations].

[0051] The method of the invention can also be used to obtain the separation and isolation of particular Hp isoforms. Separation can be achieved based on charge. These differences in charge are likely to be a result of the varying glycosylation patterns in the different isoforms. The lower the negative charge of an isoform, the lower the salt concentration required to elute that isoform. Typically, salt concentrations increase during the elution step, and thus the less negatively charged isoforms elute first. By monitoring the eluate via normal routes, a skilled technician should be able to identify discrete elution peaks corresponding to the discrete isoforms and consequently isolate these discrete isoforms.

[0052] Thus, in another aspect, the present invention provides a method for isolating the individual haptoglobin isoforms from a sample comprising Cohn's V fraction, wherein said method comprises anion exchange chromatography of said sample.

[0053] The use of Cohn fraction V as the starting material may also have an effect on the Hp isoforms that are ultimately isolated. It has been observed that the proportions of Hp phenotypes present in fraction IV and fraction V of the Cohn fractionation process differ. Therefore, in the isolation of Hp from fraction V, a different Hp product may be produced than the Hp product produced using fraction IV. Specifically, the proportion of isoform 1-1 was observed to be greater than the proportion of isoforms 2-2 in the product derived from fraction V compared to a product derived from fraction IV. This is assumed to be a result of the precipitation of heavier isoforms when the pH is reduced at high ethanol concentration during the fractionation process.

[0054] The previously preferred embodiments of the invention apply, mutatis mutandis, to this aspect of the invention. When considering the term “isolation” in this aspect, it should be borne in mind that any production must be calculated with respect to the particular isoform.

[0055] A serious problem with prior art methods for isolating Hp from plasma has been their inadequacy for scaling up isolation to a large / commercial scale. As can be seen from the Examples, it has now been observed that anion exchange chromatography can be used to obtain a high-yield, high-purity preparation of Hp from Cohn fraction V, without the need for other processing steps such as Preparative Isoelectric Focusing. Thus, the method of the invention is capable of being used on a large / commercial scale and is economically viable at that scale.

[0056] By “wide scale” it is denoted that isolation is obtainable from starting sample volumes on the order of thousands of liters. Alternatively, wide scale refers to starting plasma batch sizes of at least 1000 liters, more preferably at least 3000 liters, and most preferably at least 6000 liters.

[0057] The previously preferred embodiments of the invention apply, mutatis mutandis, to this aspect of the invention. A qualified person should be able to apply the previously examined embodiments to large-scale production without undue overhead.

[0058] Anion exchange chromatography can be used to isolate a significant proportion of Hp in Cohn fraction V. The direct product of the anion exchange step of the method of the invention can be subjected to procedures to further purify it and / or to concentrate the preparation. A skilled person should know and be able to apply suitable procedures or devise alternatives. Examples of suitable procedures include, but are not limited to, diafiltration, ultrafiltration, through-flow chromatography, metal chelate chromatography, hydroxyapatite chromatography, and dedicated viral inactivation / reduction procedures.

[0059] If the Hp is intended for pharmaceutical use, the isolate and / or purified Ap may need to undergo further processing to remove any biological or chemical contaminants that may have remained in the sample. Such procedures are well known in the art and a skilled person should be able to apply their common general knowledge and perform routine tests to enable them to formulate isolated / purified Hp that is suitable for pharmaceutical use.

[0060] For example, an effective method of further purifying Hp has been observed to be through the use of hydrophobic interaction chromatography (HIC) sorbents. Several HIC sorbents, such as phenyl sepharose (high-substituted, low-substituted, and high-performance), butyl sepharose, and octyl sepharose, have been investigated. In all cases, Hp was observed to bind less strongly than the contaminants. A skilled individual should be able to choose a suitable HIC ligand for their needs, and should know that, by modifying the ionic strength of the feed and equilibrium buffers, molecules with different affinities for an HIC ligand should be collected in different fractions. For example, it may be preferable to choose conditions where Hp passes through while contaminants remain bound, while in other situations the opposite may be true.The qualified person must also be informed of the factors that affect HIC purification, such as pH and residence time. Residence time is especially critical in cases where the product is in the throughflow. This is because shorter residence times can allow contaminants to pass through, thus contaminating the product.

[0061] Suitable HIC buffers have an ammonium sulfate concentration between 0.5 M and 1.5 M, preferably between 0.8 M and 1.2 M, and most preferably between 0.9 M and 1.1 M, and a pH between 5 and 9, preferably between 6 and 8, and most preferably between 6.5 and 7.5. Suitable flow rates for HIC should be no more than 10 cm / minute, preferably between 0.5 and 5 cm / minute, and most preferably between 1 and 3 cm / minute.

[0062] Diafiltration can be used to adjust the salt concentration or pH to be suitable for pharmaceutical use. Biological contaminants such as viruses and prions can be removed by known virus filtration techniques, known chemical disinfection techniques (viral inactivation), and / or known pasteurization or heat treatment techniques. For example, we observed that large-scale viral removal and / or inactivation of a solution containing Hp is possible using solvent detergent treatment as outlined in EP-A 0131740, provided that the Hp is treated at a pH in the range of pH 5 to 9. It is also possible to filter the Hp produced by the purification methods described herein through one or more suitable viral filters, for example filters with pore sizes of about 20 nm, and thus theoretically ensure the removal of potentially pathogenic viruses.If solvent detergent treatment is used to inactivate the viruses, an additional step may be included in the method to remove the solvent detergent reagents. The qualified person should be versed in such methods. As an example, anion exchange chromatography may be used. A suitable anion exchange chromatography step should be identical to those examined here regarding the initial purification of Hp. More specifically, a solvent detergent reagent removal step should comprise diluting the solvent detergent-treated sample appropriately to maintain conductivity below 3 mS / cm, loading the treated sample onto an anion exchange substrate (e.g., a DEAE agarose substrate), washing the substrate to selectively remove unbound or weakly bound contaminants, and selectively eluting the Hp from the anion exchange substrate.The use of anion exchange chromatography for the removal of solvent detergent reagents can also lead to improved purity of the final product.

[0063] If pasteurization or heat treatment is used, the use of stabilizers is considered. Stabilizers should include, but are not limited to, sugars, sugar alcohols, ascorbic acid, and amino acids. Methods for removing stabilizers, if necessary, are well known in the art. If a chemical disinfectant (e.g., solvent / detergent) is used, removal will also likely be necessary. Methods for removing chemical disinfectants are well known in the art. The particular order of the above-mentioned procedures is not considered important, but specific orders may be more advantageous than others in terms of convenience and cost. For example, it may be preferable to perform pasteurization with stabilizers or to perform a chemical disinfection step before a filtration or dialysis step, which could be scheduled to remove the stabilizers or disinfecting agents.Blood products intended for use as pharmaceuticals will preferably undergo at least two viral inactivation steps. Hp can then be subsequently formulated for clinical use.

[0064] In a formulation of Hp suitable for pharmaceutical use, the Hp must be substantially free of chemical and biological contaminants, to the extent that the levels in the formulation are not considered harmful to a patient. Ideally, the levels of any contaminants will be substantially lower than the minimum levels required by Regulatory Bodies for pharmaceutical products.

[0065] “Biological contaminants” refers to biological entities capable of inducing pathologies in a patient. These entities include, but are not limited to, viruses, prions, bacteria, fungi, spores, and cells.

[0066] By “chemical contaminants” are meant molecules that are likely to induce adverse reactions if administered to patients.

[0067] In yet another aspect, the invention provides products obtained by any and all of the methods of the invention described herein above.

[0068] Any and all combinations of the preferred aspects examined in this report are covered by the invention, even if not explicitly stated. The invention will be further described with reference to the following Figures, in which:

[0069] Figure 1 shows the results of an example of a laboratory-scale Hp isolation experiment. The three lines correspond to conductivity (Cond), pH (pH), and UV absorbance (UV). The protein elution peaks are labeled with R / T albumin, Hapto peak 1, Hapto peak 2, and AAG (al-acid glycoprotein).

[0070] Figure 2 shows the HPLC analysis of the Hp peaks (peak 1 and peak 2) for Hapto011. Figure 2a, peak 1. Figure 2b, peak 2.

[0071] Figure 3 shows the results of an example of a commercial-scale Hp isolation experiment (Production Operation). The three lines correspond to conductivity (Cond), pH (pH), and UV absorbance (UV).

[0072] Figure 4 shows the HPLC analysis of the Hp peaks (peak 1 and peak 2) for the Production Operation. Figure 4a, peak 1. Figure 4b, peak 2. EXAMPLE 1 Preparation of Cohn's V Fraction

[0073] The plasma was subjected to controlled thawing at -0.5 °C to 2 °C during which some proteins precipitated. The supernatant was collected, treated with Celite, and then filtered to remove other undesirable proteins. The resulting supernatant was adjusted to a pH of 5.85 with acetate buffer, and 17 to 21% v / v ethanol was added. The temperature was controlled during the resulting precipitation between -4 °C and -6 °C. These conditions are similar to those used in the second stage of the Kistler-Nitschmann process, and thus the precipitate included fraction 1 and precipitate A of that process. The precipitate was referred to as A+1. The supernatant was further fractionated by adjusting the ethanol concentration between 38 and 42%. The precipitated proteins are collectively known as fraction IV in the Kistler-Nitschmann process. Adjusting the pH to 4.85 and the temperature between -7 °C and -13 °C caused fraction V to precipitate. EXAMPLE 2 Anion exchange columns and preparation of buffers and solutions. Columns:

[0074] Examples 1 and 2, DEAE Sepharose® (Amersham) 12.5 cm bed height on an Amersham 16 / 20 XK column.

[0075] Examples 3 and 4, commercial scale DEAE Sepharose® (Amersham) column.

[0076] Equilibrium / washing buffer: 5 mM sodium acetate, analar (0.68 g / L sodium acetate trihydrate), 15 mM sodium chloride, analar (0.88 g / L) prepared in Pyrogen-Free Water (PFW) pH: 4.6 ± 0.1 (pH adjusted with glacial acetic acid, analar) Conductivity: 1.7 ± 0.5 mS / cm

[0077] For 400 liters of wash / equilibration buffer, 272 g of sodium acetate trihydrate, 351 kg of sodium chloride, and 110 g of glacial acetic acid were added to a suitable vessel (at least 500 liters capacity). The ingredients were dissolved and compounded to 400 liters with suitable water. pH and conductivity readings were taken. Acceptable range: pH 4.5 to 4.7. Conductivity: 1-2 mS / cm. HP elution buffer: sodium acetate 5 mM, analyte (0.68 g / liter of sodium acetate trihydrate); sodium chloride 113.5 mM, analyte (6.63 g / L). pH: 4.6 ± 0.1. Conductivity: 11.5 ± 1 mS / cm

[0078] For 500 liters of Elution Buffer, 340 g of sodium acetate trihydrate, 3.32 kg of sodium chloride, and 110 g of glacial acetic acid were added to a suitable vessel (at least 500 liters capacity). The ingredients were dissolved and compounded to 500 liters with suitable water. pH and conductivity readings were taken. Acceptance range: pH 4.5 to 4.7. Conductivity: 10.5 to 12.5 mS / cm. Elution buffer: AAG sodium acetate 5 mM, analyte (0.68 g / liter of sodium acetate trihydrate). sodium chloride 212 mM (12.40 g / liter) pH: 4.6 ± 0.1 Conductivity: 19.5 ± 2.5 mS / cm

[0079] For 400 liters of AAG Elution Buffer, 272 g of sodium acetate trihydrate, 4.96 kg of sodium chloride, and 110 g of glacial acetic acid were added to a suitable vessel (at least 500 liters capacity). The ingredients were dissolved and compounded to 400 liters with suitable water. pH and conductivity readings were checked. Acceptable range: pH 4.5 to 4.7. Conductivity: 17 to 22 mS / cm. EXAMPLE 3 Laboratory-scale isolation of Hp

[0080] The isolation of Hp from Cohn fraction V was performed on a laboratory scale. The anion exchange substrate DEAE-Sepharose® Fast Flow was thickened to a bed height of 12.5 cm in a 25 ml column housed in an Amersham BioSciences 16 / 20 XK column. This is a scaled-down version (1 / 4000) of a commercial-scale column.

[0081] The Cohn fraction V solution was loaded onto the column. The loading of Cohn fraction V was reduced from 250 ml to 150 ml to save processing time. After loading was complete, the column was washed with equilibrium buffer. Albumin was eluted in the flow through the load. Hp elution was then induced by developing elution buffer through the column. The buffer flow rate was maintained at 4.0 ml / minute throughout the process. Figure 1 shows the presence of the protein in the eluate when monitored by UV absorbance as a function of the volume of buffer developed through the column. The conductivity and pH of the eluate are also shown. As can be observed, Hp eluted as two peaks after the albumin was eluted.

[0082] The samples corresponding to the two Hp peaks were further analyzed by HPLC. Figure 2 shows that Peak 1 (Figure 2a) had more Hp 1-1 than the other forms and that Peak 2 (Figure 2b) was richer in the higher Hp forms (Hp 2-2 and 2-1) than Peak 1. In this example, for peak 1, the purification factor was 40 times, the purity was 72%, and the yield was 80%. Peak 2 had a purity of 40%. EXAMPLE 4 LARGE-SCALE Hp ISOLATION

[0083] The isolation of Hp from Cohn fraction V was performed on a commercial scale using a full-scale DEAE-Sepharose® column (200-liter column). The Cohn fraction V solution (1100 liters) was loaded onto the column. Once loading was complete, the column was washed with washing buffer. This caused the albumin to be eluted. Hp was then eluted with elution buffer. The buffer flow rate was maintained at 14 liters / minute, which is equivalent to 4.0 ml / minute on a laboratory scale. Figure 3 shows the presence of protein in the eluate (monitored by UV absorbance) as a function of the volume of buffer development through the column. The chromatogram begins at the end of the albumin elution peak. As can be observed, Hp eluted as two peaks after the albumin was eluted.The similarity of this line compared to laboratory-scale experience highlighted the suitability of the invention's method for proportional elevation for commercial Hp isolation.

[0084] The samples corresponding to the two Hp peaks were further analyzed by HPLC. Figure 4 shows that Peak 1 (Figure 4a) had more Hp 1-1 than the other forms and that Peak 2 (Figure 4b) was richer in the higher Hp forms (Hp 2-2 and 2-1) than Peak 1. The purity of peak 1 was 67%, and that of peak 2 was lower. The yield of peak 1 was approximately 74%. As only one sample of peak two was taken, the yield of this peak could not be evaluated. Furthermore, by comparing the analytical results of Production with the analytical results, it can be observed that the elevation does not affect the quality of the product. EXAMPLE 5 PURIFICATION OF Hp IN BUTYL SEPHORESIS

[0085] The Hp fraction obtained from Example 4 (peak 1) was further purified in butyl sepharose. A pre-condensed MiPrep® Butyl ff at 20 ml was equilibrated with 180 ml of 1.0 M ammonium sulfate in 50 mM Na2HPO4 buffer, pH 7.0, at a flow rate of 4.5 ml / minute (135 cm / hour). 40 ml of peak 1 from Example 4 were then loaded at 3.5 ml / minute. The column was then washed with 20 ml of the equilibrium buffer. The throughflow and wash (haptoglobin product) were collected in a vessel. Bound molecules (contaminants) were separated by washing the column with deionized water. The throughflow (Hp) was analyzed by HPLC-SEC. Of the spike areas, 98% of the total protein was Hp, and 67% of that protein was Hp with a molecular weight of approximately 100 kDa.

[0086] Analogous experiments were performed using high-substitution, low-substitution, and high-performance phenyl sepharose and octyl sepharose columns. High-substitution phenyl sepharose gave a purity of 99% and a yield of 32%. The remaining sorbents gave purities between 96% and 97% and yields between 32% and 61%.

[0087] Optimization experiments using HiPrep® Butyl ff showed that a) the pH should be between 6 and 8, b) the ammonium sulfate concentration should be between 0.8 M and 1.2 M, c) the feed rate should be between 1 and 3 cm / minute. EXAMPLE 6 HP STABILITY

[0088] The Hp produced by the methods of the invention have been shown to be resilient to a wide range of pH conditions, making feasible other purification schemes requiring acidic or alkaline conditions. In one example, 3 ml of Hp from peak 1 in Example 4 were incubated with 3 ml of buffer (200 mM glycine for pH 2, 3 and 4, 200 mM NH4HCO3 for pH 11). Samples were taken at 0, 4, 7, 24, 48 and 72 hours. The samples were immediately neutralized with 1 M Tris-HCl, pH 7.0. The samples were then analyzed by silver-stained SDS-PAGE and the analysis results showed that Hp can withstand a pH range of 4 to 11 for 72 hours. However, at pH 2, complete aggregation occurred within 4 hours, and at pH 3, partial degradation was observed (as evidenced by the extra bands on the SDS-PAGE).

[0089] It has also been shown that the Hp produced by the methods of the invention is stable at 4 °C for periods exceeding 18 months, without the addition of any protein, monosaccharide or disaccharide excipient. Extreme temperatures, specifically those above 40 °C, produce aggregation of the Hp after a short or longer period, depending on the temperature. For example, incubation of the Hp at 60 °C for 24 hours resulted in complete aggregation, as shown by HPLC-SEC. EXAMPLE 7 HP Solvent Detergent Treatment

[0090] Peak 1 of Example 4 was treated with detergent solvent following the method of patent number EP-A 0131740. Briefly, 4.32 g of polysorbate 20, 1.16 g of tri-n-butyl phosphate (TnBP) and 14.52 g of WFI (water for injection) were vigorously mixed for 15 minutes. 8.7 g of this mixture were added to 150 ml of peak 1 of Example 4, and incubated at 25 °C for 30 minutes. The sample was then diluted to 475 ml with WFI. The diluted sample was then loaded onto a 30 ml DEAE column with buffers as per Example 3. Unbound molecules and detergent solvent (SD) reagents were separated by choline washing. Hp was eluted as in Example 3. The Hp obtained was analyzed by a) SDS-PAGE, which showed that most of the contaminants were removed, and by the hemoglobin binding assay, which showed that 63% of the active Hp was recovered in the product.The remaining 37% were lost during the washing stage, thus showing that SD treatment does not affect Hp activity.

[0091] It is also possible to filter the Hp produced by the purification methods described herein, using one or more 20 nm virus filters, thus theoretically guaranteeing the removal of potentially pathogenic viruses, including uncoated viruses, which may not have been inactivated by SD treatment.

Claims

1. Method for isolating human haptoglobin (Hp) from human Cohn fraction V, the method characterized in that it comprises: a) loading said human Cohn fraction V onto an anion exchange substrate, and b) selectively eluting human Hp from the same.

2. Method according to claim 1, characterized in that all isoforms of human Hp present in the human Cohn fraction V are isolated from the human Cohn fraction V.

3. Method according to claim 1 or claim 2, characterized in that it comprises an anion exchange chromatography of said human Cohn fraction V using a weak anion exchange substrate.

4. Method according to claim 3, characterized in that the said weak anion exchange substrate is DEAE agarose.

5. A method according to any one of claims 1 to 4, characterized in that it comprises: a) loading said human Cohn fraction V onto an anion exchange substrate, b) washing the substrate to remove unbound or weakly bound contaminants, and c) selectively eluting human Hp from the anion exchange substrate.

6. A method according to any one of claims 1 to 5, characterized in that the loading and washing steps use buffers with conductivity between 0.1 and 3.0 mS / cm.

7. Method according to claim 6, characterized in that the loading and washing steps use buffers with conductivity between 1.0 and 2.0 mS / cm.

8. A method according to any one of claims 1 to 7, characterized in that the loading and washing steps use pH buffers between 3 and 7.

9. Method according to claim 8, characterized in that the loading and washing steps use pH buffers between 4.2 and 5.

0.

10. Method according to any one of claims 1 to 9, characterized in that the elution step uses buffers with conductivity between 8.0 and 15.0 mS / cm.

11. Method according to claim 10, characterized in that the elution step uses conductivity buffers between 10.5 and 12.5 mS / cm.

12. Method according to any one of claims 1 to 11, characterized in that the elution step uses pH buffers between 4 and 6.

13. Method according to claim 12, characterized in that the elution step uses pH buffers between 4.2 and 5.

0.

14. Method according to any one of claims 1 to 13, characterized in that the loading and / or washing and / or eluting buffers comprise sodium acetate, acetic acid and sodium chloride.

15. Method according to any one of claims 1 to 14, characterized in that it comprises: a) applying said human Cohn fraction V to a DEAE agarose anion exchange substrate, b) washing said substrate with a sodium acetate / acetic acid / sodium chloride washing buffer of a conductivity between 1.2 and 2.3 mS / cm and a pH between 4.5 and 4.7, and c) eluting the human Hp from said substrate with a sodium acetate / acetic acid / sodium chloride elution buffer of a conductivity between 10.5 and 12.5 mS / cm and a pH between 4.5 and 4.

7.

16. Method for isolating individual human Hp isoforms from human Cohn fraction V, the method characterized in that it comprises anion exchange chromatography of said human Cohn fraction V.

17. Method according to any one of claims 1 to 16, characterized in that the eluate is monitored to identify the elution of discrete human Hp isoforms.

18. Method according to claim 17, characterized in that the human Hp1-1 isoform is preserved in the final product.

19. Method for isolating human albumin and haptoglobin from human Cohn fraction V, the method characterized in that it comprises: a) loading human Cohn fraction V onto an anion exchange substrate, followed by b) washing the substrate to selectively remove albumin, and followed by c) selectively eluting human haptoglobin from the substrate.

20. A method according to any one of claims 1 to 19, characterized in that it further comprises at least one concentration and / or purification step.

21. Method according to claim 20, characterized in that at least one concentration and / or purification step is selected from diafiltration, ultrafiltration, through-flow chromatography, metal chelate chromatography and hydroxyapatite chromatography.

22. Method according to claim 20, characterized in that at least one purification step is hydrophobic interaction chromatography.

23. A method according to any one of claims 1 to 22, characterized in that it further comprises at least one contaminant removal step.

24. Method according to claim 23, characterized in that the contaminant removal step is a virus inactivation or removal step.

25. Method according to claim 24, characterized in that the said virus inactivation or removal step comprises solvent detergent treatment and / or virus filtration.

26. Method according to any one of claims 1 to 25, characterized in that it comprises: (a) anion exchange chromatography of human Cohn fraction V; (b) viral inactivation by detergent-solvent treatment and chromatographic removal of detergent-solvent reagents; (c) virus filtration; and (d) formulation in a physiological buffer; wherein step (b) and step (c) may be carried out in any order.

27. Method according to claim 23, characterized in that said contaminant removal step is a step for removing a biological contaminant by pasteurization or heat treatment, and wherein said pasteurization or heat treatment is carried out in the presence of stabilizers selected from the group consisting of sugars, sugar alcohols, ascorbic acid and amino acids.