| Purification of immunoglobulins -> Monitor Keywords |
|
|
 
Purification of immunoglobulinsRelated Patent Categories: Liquid Purification Or Separation, Processes, ChromatographyPurification of immunoglobulins description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070151928, Purification of immunoglobulins. Brief Patent Description - Full Patent Description - Patent Application Claims
TECHNICAL FIELD [0001] The present invention relates to the field of antibody preparation, and more specifically to a separation matrix for isolation of antibodies. The invention also encompasses a chromatography column that comprises the novel matrix and a method of isolating antibodies. BACKGROUND [0002] The immune system is composed of many interdependent cell types that collectively protect the body from bacterial, parasitic, fungal, viral infections and from the growth of tumour cells. The guards of the immune system are macrophages that continually roam the bloodstream of their host. When challenged by infection or immunisation, macrophages respond by engulfing invaders marked with foreign molecules known as antigens. This event, mediated by helper T cells, sets forth a complicated chain of responses that result in the stimulation of B-cells. These B-cells, in turn, produce proteins called antibodies, which bind to the foreign invader. The binding event between antibody and antigen marks the foreign invader for destruction via phagocytosis or activation of the complement system. Five different classes of antibodies, or immunoglobulins, exist: IgA, IgD, IgE, IgG, and IgM. They differ not only in their physiological roles but also in their structures. From a structural point of view, IgG antibodies are a particular class of immunoglobulins that have been extensively studied, perhaps because of the dominant role they play in a mature immune response. [0003] The biological activity, which the immunoglobulins possess, is today exploited in a range of different applications in the human and veterinary diagnostic, health care and therapeutic sector. In fact, in the last few years, monoclonal antibodies and recombinant antibody constructs have become the largest class of proteins currently investigated in clinical trials and receiving FDA approval as therapeutics and diagnostics. Complementary to expression systems and production strategies, purification protocols are designed to obtain highly pure antibodies in a simple and cost-efficient manner. [0004] Traditional methods for isolation of immunoglobulins are based on selective reversible precipitation of the protein fraction comprising the immunoglobulins while leaving other groups of proteins in solution. Typical precipitation agents being ethanol, polyethylene glycol, lyotropic i.e. anti-chaotropic salts such as ammonium sulphate and potassium phosphate, and caprylic acid. Typically, these precipitation methods are giving very impure products while at the same time being time consuming and laborious. Furthermore, the addition of the precipitating agent to the raw material makes it difficult to use the supernatant for other purposes and creates a disposal problem, which is particularly relevant when speaking of large-scale purification of immunoglobulins. [0005] Ion exchange chromatography is another well-known method of protein fractionation frequently used for isolation of immunoglobulins. However, since the charged ion exchange ligands will react with all oppositely charged compounds, the selectivity of ion exchange chromatography may be somewhat lower than other chromatographic separations. [0006] Protein A and Protein G affinity chromatography are popular and widespread methods for isolation and purification of immunoglobulins, particularly for isolation of monoclonal antibodies, mainly due to the ease of use and the high purity obtained. Used in combination with ion exchange, hydrophobic interaction, hydrxyapatite and/or gel filtration steps, especially protein A-based methods have become the antibody purification method of choice for many biopharmaceutical companies. However, despite their common usage, there is a growing need and demand for effective alternatives addressing familiar problems associated with protein A-based media, such as cost, leakage and instability at increased pH values. [0007] Hydrophobic interaction chromatography (HIC) is also a method widely described for isolation of immunoglobulins. However, hydrophobic matrices require an addition of lyotropic salts to the raw material to make the immunoglobulin bind efficiently. The bound antibody is released from the matrix by lowering the concentration of lyotropic salt in a continuous or stepwise gradient. If a highly pure product is the object, it is recommended to combine the hydrophobic chromatography with a further step. Thus, a disadvantage of this procedure is the necessity to add lyotropic salt to the raw material as this gives a d problem and thereby increased cost to the large-scale user. For other raw materials than cell culture supernatants such as whey, plasma, and egg yolk the addition of lyotropic salts to the raw materials would in many instances be prohibitive in large-scale applications as the salt could prevent any economically feasible use of the immunoglobulin depleted raw material. An additional problem in large-scale applications would be the disposal of several thousand litres of waste. [0008] Thiophilic adsorption chromatography was introduced by J. Porath in 1985 (J. Porath et al; FEBS Letters, vol. 185, p. 306, 1985) as a new chromatographic adsorption principle for isolation of immunoglobulins. In this paper, it is described how divinyl sulphone activated agarose coupled with various ligands comprising a free mercapto-group show specific binding of immunoglobulins in the presence of 0.5 M potassium sulphate, i.e. a lyotropic salt. It was postulated that the sulphone group, from the vinyl sulphone spacer, and the resulting thioether in the ligand was a structural necessity to obtain the described specificity and capacity for binding of antibodies. It was however later shown that the thioether could be replaced by nitrogen or oxygen if the ligand further comprised an aromatic radical (K. L. Knudsen et al, Analytical Biochemistry, vol. 201, p. 170, 1992). Although the matrices described for thiophilic chromatography generally show good performance, they also have a major disadvantage in that it is needed to add lyotropic salts to the raw material to ensure efficient binding of the immunoglobulin, which is a problem for the reasons discussed above. [0009] Other thiophilic ligands coupled to epoxy activated agarose have been disclosed in (J. Porath et. al. Makromol. Chem., Makromol. Symp., vol. 17, p. 359, 1988) and (A. Schwarz et. al., Journal of Chromatography B, vol. 664, pp. 83-88, 1995), e.g. 2-mercaptopyridine, 2-mercaptopyrimidine, and 2-mercaptothiazoline. However, all these affinity matrices still have inadequate affinity constants to ensure an efficient binding of the antibody without added lyotropic salts. [0010] U.S. Pat. No. 6,498,236 (Upfront Chromatography) relates to isolation of immunoglobulins. The method disclosed involves the steps of contacting a solution that comprises a negatively charged detergent and contains immunoglobulin(s) with a solid phase matrix, whereby at least a part of the immunoglobulins becomes bound to the solid phase matrix; and contacting the solid phase matrix with an eluent in order to liberate the immunoglobulin(s) from the solid phase matrix. The immunoglobulin-containing solution is further characterised by having a pH in the range of 2.0 to 10.0, a total salt content corresponding to an ionic strength of at the most 2.0, and lyotropic salts in a concentration of at the most 0.4 M. The detergent present in the solution is believed to suppress the adherence of other biomolecules to the matrix, and may be exemplified by octyl sulphate, bromphenol blue, octane sulphonate, sodium laurylsarcosinate, and hexane sulphonate. The solid phase matrix is defined by the formula M-SP1-L, wherein M designates the matrix backbone, SP1 designates a ligand comprising a mono- or bicyclic aromatic or heteroaromatic moiety. [0011] Liu et al (Yang Liu, Rui Zhao, Dihua Shangguan, Hongwu Zhang, Guoquan Liu: Novel sulfinethazine ligand used for one-step purification of immunoglobulin G from human plasma, Journal of Chromatography B, 792 (2003) 177-185) investigated the affinity of sulfinethazin (SMZ) to human IgG. Thus, a ligand is disclosed, which comprises a sulphonyl group wherein the R group is a heterocyclic ring. According to this article, SMZ was immobilised on monodisperse, non-porous, cross-linked poly(glycidyl methacrylate) beads. The beads were then used in high-performance affinity chromatography for isolation of IgG from human plasma. Maximal adsorption was achieved at pH 5.5. The beads presented minimal non-specific interaction with other proteins. Thus, the ligands were capable of adsorbing antibodies, while their interaction with other proteins was just sufficient to provide retardation thereof in the adsorption buffer used. However, as is well known, ester compounds such as methacrylate are easily hydrolysed at increased pH values. Consequently, similar to Protein A and Protein G matrices, the therein disclosed separation matrix would be expected to unstable at the commonly used cleaning in place (cip) procedures. [0012] U.S. Pat. No. 4,725,355 relates to a body fluid purification medium comprising a support and an adsorbent, which includes at least one sulfa drug, for adsorbing and removing a pathogenic substance in a body fluid. The sulfa drug is a chemotherapeutic agent, and more specifically a sulfonamide characterised by aromatic R group(s). The medium can be provided in a body fluid flow path provided in a container between body fluid inlet and outlet ports. [0013] EP 0 197 521 relates to an immunoglobulin adsorbent and an adsorption apparatus. More specifically, an adsorbent for immunoglobulin is disclosed, which adsorbent comprises a hydroxyl-containing water-insoluble carrier to which a diamine compound has been attached. The diamine compound is represented by the general formula: NH.sub.2(CH.sub.2).sub.nNH.sub.2 wherein n is an integer having a value of 3 to 9. The compound has been attached through a silane coupling agent or a derivative thereof, with a heterocyclic compound being attached to the diamine through a difunctional reagent. Thus, the R groups are aromatic structures. [0014] However, there is still a need of alternative methods for purification of antibodies or antibody constructs, which observe the demands of purity, safety, potency and cost effectiveness. BRIEF DESCRIPTION OF THE PRESENT INVENTION [0015] Accordingly, one aspect of the present invention is a separation matrix, which enables adsorption of antibodies at low ionic strengths at pH values around neutral. This can be achieved by the separation matrix as defined in claim 1. [0016] Another aspect of the present invention is a separation matrix, which enables highly selective adsorption of antibodies. [0017] A specific aspect of the present invention is a separation matrix to which antibodies are adsorbed, while other proteins are allowed to pass without any essential interaction. [0018] A further aspect of the present invention is to a process of preparing a matrix for separation of antibodies, which comprises functional groups that enable adsorption of antibodies by thiophilic, hydrophobic and/or hydrogen bond interactions, which method makes it easy to vary the ligand structure. This can be achieved by immobilisation of amines and/or polyamines to a porous support and a subsequent step of sulphonylating said immobilised amines. [0019] Yet another aspect of the invention is to a method of isolating antibodies from a liquid by adsorption thereof to a separation matrix, which method does not require any addition of detergent to achieve adsorption. [0020] Further aspects and advantages of the invention will appear from the detailed description that follows. BRIEF DESCRIPTION OF THE DRAWINGS Continue reading about Purification of immunoglobulins... Full patent description for Purification of immunoglobulins Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Purification of immunoglobulins patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Purification of immunoglobulins or other areas of interest. ### Previous Patent Application: Metal oxides dispersant composition Next Patent Application: Water purification device and method Industry Class: Liquid purification or separation ### FreshPatents.com Support Thank you for viewing the Purification of immunoglobulins patent info. IP-related news and info Results in 0.16523 seconds Other interesting Feshpatents.com categories: Canon USA , Celera Genomics , Cephalon, Inc. , Cingular Wireless , Clorox , Colgate-Palmolive , Corning , Cymer , 174 |
 * Protect your Inventions * US Patent Office filing 
PATENT INFO |
|
