IL268878B2 - Scaled-up methods for purifying antibodies - Google Patents

Description

SCALED-UP METHODS FOR PURIFYING ANTIBODIES FIELD AND BACKGROUND OF THE INVENTIONThe present invention, in some embodiments thereof, relates to methods and, kits for purifying antibodies.Monoclonal antibodies (mAb's) are currently the recombinant proteins most commonly used as therapeutics; they were the largest selling class of biologics in the USA in 2012. The dramatic increase in their expression levels from low milligram to multi-gram concentration per liter, together with the multi-hundred kilogram to ton quantities in which some of them will be required, pose an on-going challenge for industrial purification methods capable of efficiently capturing mAb's from complex mixtures. This is generally achieved via ProA chromatography as the initial capturing step, commonly resulting in high recovery yields (~95%), purity (>95%), while removing the majority of host DNA, viral contaminants and leached ProA.These remarkable features have made ProA chromatography the gold standard for antibody manufacturing. However, there is motivation for the development of more economic alternatives since ProA resins suffer from high costs relative to non­affinity polymeric supports (e.g. ion exchangers). This motivation is further justified when considering the current and future global biotech demands (i.e. many tons of purified mAb's per year) representing hundreds of different therapeutic mAb's under development, all aimed at targeting various cancers, autoimmune and inflammatory disorders.It has been argued that, the use of ProA, and of chromatographic strategies in general, represent an inherent "productivity bottleneck" for industrial purification of mAb's, which can account for up to 80% of the total manufacturing cost thus making any antibody capturing method not entailing: (a) ProA as a ligand and/or (b) chromatography as the primary capturing step, an attractive alternative for future pharmaceutical needs.Background art includes Patchornick et al., Bioconjugate Chemistry, 2013, Volume 24, pages 1270-1275; Guse et al., J. Chromatogr A. (1994) 661, 13-23; Manske et al., J. Immunol Methods (1997) 2008, 65-73; Follman and Fahrner J.

Chromatogr A. (2004) 1024, 79-85 and Ghosh and Wang, J. Chromatogr A. (2006) 1107, 104-109.Additional background art includes WO2018/207184.

SUMMARY OF THE INVENTIONAccording to an aspect of the present invention there is provided a method of isolating an antibody, the method comprising:(a) contacting a hydrophobic chelator, a non-ionic detergent and metal ions so as to generate an aggregate comprising the hydrophobic chelator, the detergent and the metal ions;(b) contacting the aggregate with a medium comprising the antibody under conditions that allow partitioning of the antibody into the aggregate; and subsequently(c) filtering the medium comprising the aggregate, thereby isolating the antibody.According to an aspect of the present invention there is provided a method of preparing an aggregate, the method comprising:(a) contacting a hydrophobic chelator, a non-ionic detergent and metal ions so as to generate a first aggregate comprising the hydrophobic chelator, the detergent and the metal ions;(b) contacting the aggregate with a medium comprising an antibody under conditions that allow a first fraction of the antibody to participate into the aggregate;(c) isolating the antibody from the aggregate;(d) disassociating the aggregate;(e) isolating the hydrophobic chelator; and subsequently(f) contacting the hydrophobic chelator, a non-ionic detergent and metal ions so as to generate a second aggregate comprising the hydrophobic chelator, the detergent and the metal ions, thereby preparing the aggregate.

According to embodiments of the invention, the disassociating the aggregate comprises contacting the aggregate with a water soluble chelator under conditions that allows disassociation of the aggregate.

According to embodiments of the invention, the water soluble chelator comprises EDTA or EGTA.According to embodiments of the invention, the isolating the antibody from the aggregate comprises filtering the medium comprising the aggregate.According to embodiments of the invention, the aggregate has a diameter of greater than 500 nM.According to embodiments of the invention, the aggregate has a diameter of between 500-3000 nM.According to embodiments of the invention, the medium comprises a cell lysate.According to embodiments of the invention, the cell lysate is a whole cell lysate.According to embodiments of the invention, the medium comprises a hybridoma medium.According to embodiments of the invention, the medium comprises serum albumin.According to embodiments of the invention, the cell lysate is devoid of organelles greater than about 2 microns.According to embodiments of the invention, the conditions of step (b) comprise having a level of salt below 100 mM.According to embodiments of the invention, the method further comprises solubilizing the antibody following step (b).According to embodiments of the invention, the isolating the antibody comprises solubilizing the antibody.According to embodiments of the invention, the solubilizing is effected with a buffer having a pH between 3-6.According to embodiments of the invention, the solubilizing is effected with a buffer having a pH between 3.8 and 4.According to embodiments of the invention, the buffer further comprises a salt.

According to embodiments of the invention, the buffer is a carboxylic buffer.

According to embodiments of the invention, the buffer comprises an amino acid.According to embodiments of the invention, the carboxylic buffer is selected from the group consisting of isoleucine, valine, glycine and sodium acetate.According to embodiments of the invention, the non-ionic detergent is a polysorbate surfactant.According to embodiments of the invention, the polysorbate surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate and polysorbate 80.According to embodiments of the invention, the hydrophobic chelator comprises 8-Hydroxyquinoline.According to embodiments of the invention, the hydrophobic chelator comprises a phenanthroline.According to embodiments of the invention, the phenanthroline is selected from the group consisting of N-(1,10-Phenanthrolin-5-yl)methanamide) (Phen-C1), N-(1,10-Phenanthrolin-5-yl)ethanamide) (Phen-C2), N-(1,10-Phenanthrolin-5- yl)propanamide) (Phen-C3), N-(1,10-Phenanthrolin-5-yl)butanamide) (Phen-C4), N- (1,10-Phenanthrolin-5-yl)pentanamide) (Phen-C5), N-(1,10-Phenanthrolin-5- yl)hexanamide) (Phen-C6), N-(1,10-Phenanthrolin-5-yl)heptanamide) (Phen-C7), N- (1,10-Phenanthrolin-5-yl)octanamide) (Phen-C8), N-(1,10-Phenanthrolin-5- yl)nonanamide) (Phen-C9) and N-(1,10-Phenanthrolin-5-yl)decanamide) (Phen-C10).According to embodiments of the invention, the phenanthroline is selected from the group consisting of bathophenanthroline, N-(1,10-Phenanthrolin-5- yl)hexanamide) (Phen-6), N-(1,10-Phenanthrolin-5-yl)decanamide) (Phen-C10) and N-(1,10-Phenanthrolin-5-yl)octanamide) (Phen-C8).According to embodiments of the invention, the phenanthroline is bathophenanthroline.According to embodiments of the invention, the metal ions are divalent metal ions.

According to embodiments of the invention, the divalent metal ions are selected from the group consisting of Zn2+, Fe2+, Mn2+, Ni2+ and Co2+.

According to embodiments of the invention, the divalent metal ions are selected from the group consisting of Zn2+ and Fe2+.According to embodiments of the invention, the hydrophobic chelator is present in the aqueous solution at a concentration in the range of about 0.1% to about 10% (v/v).According to embodiments of the invention, the metal ions are present in the aqueous at a concentration in the range of about 0.1 % about 10% (v/v).According to embodiments of the invention, the cell lysate is derived from a bacterial cell.According to embodiments of the invention, the cell lysate is derived from a mammalian cell.According to embodiments of the invention, the mammalian cell is a Chinese Hamster Ovary cell (CHO).According to embodiments of the invention, the antibody is a humanized antibody.According to embodiments of the invention, the antibody is a recombinant antibody.According to embodiments of the invention, the antibody is selected from the group consisting of IgA, IgD, IgE, IgM and IgG.According to embodiments of the invention, the IgG is IgG1, IgG2, IgG3 or IgG4.Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGSSome embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.In the drawings:FIGs.1A-B: Yield of the engineered-micelle platform for Ab purification as compared to ProA or Protein G (ProG) resins. Centrifugation: Lanes 1-2: hIgG (control); lanes 3-4 and 5-6: supernatant composition obtained after purification of hIgG with Protein A or Protein G spin columns; Lanes 7-8: supernatant composition after purification of hIgG with Tween-20 detergent aggregates. Filtration: As in Centrifugation, but by applying filtration, i.e.: the BSA+IgG mixture is added to preformed Tween-20:bathophenanthroline:Fe2+] aggregates, incubated for 5 minutes and filtration is applied (0.22 micron filters). The resulting precipitate is washed with mM NaCl and an extraction buffer is added. The system is further incubated for 2­minutes at 20-42 °C and filtration is applied again (0.22 micron filters). Lanes 5-show the composition of the filtrate obtained via this protocol. BSA, H & L are bovine serum albumin and the reduced heavy and light chains of the target antibody, respectively. Gels are Coomassie blue stained.FIGs. 2A-D - Supernatant composition (after IgG capture & extraction) with indicated detergents, the: [Bathophenanthroline):Fe2+] complex and human IgG (hIgG) as the target. BSA, H & L are bovine serum albumin and the reduced heavy and light chains of the target antibody, respectively. Gels are Coomassie blue stained.FIG. 3: Purification of human IgG (hIgG) in the presence of indicated chelators. Lanes 1-2: Control: Supernatant composition containing the recovered hIgG after its capture with: [Tween-20:bathophenanathroline:Fe2+:PEG-6000] aggregates and further extraction with 50 mM Ile at pH 3.8 (15 minutes at 32 °C); lanes 3-4, 5-6, 7-8, and 9-10 as in lanes 1-2, but with the presence of indicated chelators concentrations during the IgG capturing step. H, L denote the reduced heavy and light chains of the target antibody, respectively. Gels are Coomassie stained.

FIG. 4. Steps associated with chelator recycling. Step I: Removal of the water­soluble detergent (Tween-20) and antibodies. Step II: Dissociation of the red [(batho)3:Fe2+] hydrophobic complex with excess of the water-soluble chelator: EDTA. Step III: Crystals observed at low temperature.FIGs. 5A-B. A. Red crystals observed after washing Tween-20 aggregates with NaCl. B. Comparison in absorbance between freshly prepared: red [(batho)3:Fe2+] complex and dissolved crystals shown in A.FIGs. 6A-B. A. Mass spectrometry analysis of pure (control) batho crystals and B. regenerated colorless crystals shown in Figure 4.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTIONThe present invention, in some embodiments thereof, relates to methods and, kits for purifying antibodies. In particular, the methods relate to an alternative route for antibody capturing without the use of the common ligand, Protein A (ProA).Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.Purification of antibodies typically uses Protein A (proA) chromatography as the initial capturing step. However, proA chromatography is very expensive creating a "productivity bottleneck".The present inventors uncovered a new method of purifying antibodies based on the use of hydrophobic chelators, non-ionic detergents and metal ions (see WO2018/207184).In order to adapt those methods to ones that could be used for large-scale antibody purification, the present inventors have now surprisingly shown that a filtration step can be used to replace the small-scale laboratory centrifugation step. Furthermore, the present inventors showed that the hydrophobic chelator could be recycled and used in a second step of purification thereby enhancing the yield of purified antibody per unit weight of hydrophobic chelator.Thus, according to a first aspect of the present invention there is provided a method of isolating an antibody, the method comprising: (a) contacting a hydrophobic chelator, a non-ionic detergent and metal ions so as to generate an aggregate comprising the hydrophobic chelator, the detergent and the metal ions;(b) contacting the aggregate with a medium comprising the antibody under conditions that allow partitioning of the antibody into the aggregate; and subsequently(c) filtering the medium comprising the aggregate, thereby isolating the antibody.The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof(such as Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH and VL) that are capable of binding to an epitope of an antigen.Suitable antibody fragments contemplated by the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as "light chain"), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as "heavy chain"), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv (scFv), a disulfide- stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’)2.As used herein, the terms "complementarity-determining region" or "CDR" are used interchangeably to refer to the antigen binding regions found within the variable region of the heavy and light chain polypeptides. Generally, antibodies comprise three CDRs in each of the VH (CDR HI or HI; CDR H2 or H2; and CDR H3 or H3) and three in each of the VL (CDR LI or LI; CDR L2 or L2; and CDR L3 or L3).The identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by Kabat et al. (See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989.), a compromise between Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now AccelrysTM, see, Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268; and world wide web site www(dot)bioinf-org(dot)uk/abs), available complex crystal structures as defined by the contact definition (see MacCallum et al., J. Mol. Biol. 262:732-745, 1996) and the "conformational definition" (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008).As used herein, the "variable regions" and "CDRs" may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches.Functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows:(i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;(ii) single chain Fv ("scFv"), a genetically engineered single chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.(iii) disulfide-stabilized Fv ("dsFv"), a genetically engineered antibody including the variable region of the light chain and the variable region of the heavy chain, linked by a genetically engineered disulfide bond.(iv) Fab, a fragment of an antibody molecule containing a monovalent antigen­binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CH1 domains thereof;(v) Fab’, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);(vi) F(ab’)2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments held together by two disulfide bonds); and(vii) Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.

In one embodiment, the antibody is a polyclonal antibody.In another embodiment, the antibody is a monoclonal antibody.In still a further embodiment, the antibody is a recombinant antibody.In still a further embodiment, the antibody is a humanized antibody.In still further embodiments, the antibody is IgA, IgD, IgE and IgG (e.g. IgG1, IgG2, IgG3 or IgG4).In still further embodiments, the antibody is IgM.Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly.These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.Fv fragments comprise an association of VH and VL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659­(19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single­chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-5(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:5(1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)].Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 8(1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed cells, can be removed, e.g., by centrifugation or ultrafiltration. Where the antibody is secreted into the medium, supernatants from such expression systems can be first concentrated using a commercially available protein concentration filter, e.g., an Amicon™ or Millipore Pellicon™ ultrafiltration unit.Lysis of the cells may be performed by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration. Where the antibody is secreted, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, e.g., an Amicon™ or Millipore Pellicon™ ultrafiltration unit. Where the antibody is secreted into the medium, the recombinant host cells can also be separated from the cell culture medium, e.g., by tangential flow filtration.In one embodiment, the medium in which the antibody is comprised is a cell lysate.As used herein, the term "cell lysate" refers to an aqueous solution of cellular biological material which comprises the antibody, wherein a substantial portion of the cells of the cellular material have become disrupted and released their internal components.In one embodiment, the cell lysate is prepared from whole cells.In the case of a whole cell lysate, it will be appreciated that following cell membrane disruption, the cell lysate may be treated so as to remove organelles greater than about 2 microns (e.g. cell nucleii). Thus, for example the whole cell lysate may be centrifuged so as to precipitate cell nucleii from the cell lysate. Exemplary centrifugation conditions include 1-5 minutes at 500-1000 x g (e.g. 2 min. at 985 x g).The cell lysate may be prepared from any cell that expresses an antibody. The cells may be eukaryotic (e.g. mammalian, plant, fungus) or prokaryotic (bacteria).In another embodiment, the cells secrete antibody into the cell medium.The cell may be genetically modified so as to express the antibody. In another embodiment, the cell is not genetically modified.Exemplary cells that are contemplated include, but are not limited to gram negative bacterial cells, such as E. Coli; gram positive bacterial cells such as Bacillus brevis, Bacillus subtilis, Bacillus megaterium and Lactobacilli (e.g. Lactobacillus zeae/casei or Lactobacillus paracasei); yeast cells such as Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Schizosaccharomyces pombe, Schwanniomyces occidentalis, Kluyveromyces lactis, and Yarrowia lipolytica; filamentous fungii such as Trichoderma and Aspergillus; insect cells; mammalian cells including Chinese hamster ovary (CHO) cells and plant cells.In one embodiment, the cells have been immortalized and are part of a cell line - e.g. hybridoma. As mentioned, the isolation method of this aspect of the present invention is carried out by contacting the medium comprising the antibody with aggregates of non-ionic detergent, hydrophobic chelator and metal ions.Examples of cell media for culturing antibody producing cells include hybridoma media - e.g. serum-free hybridoma media. Such media are readily available from Companies such as Gibco, Thermo Fisher Scientific and Sigma- Aldrich.In one embodiment, the media comprises a serum albumin such as horse serum albumin (HAS) or bovine serum albumin (BSA).Preferably the serum albumin is present at a concentration of less than 0.mg/ml - for example between 0.1-0.5 mg/ml.Prior to the isolation step, the medium comprising the antibody may optionally be clarified.As used herein, the term "clarified" refers to a sample (i.e. a cell suspension) having undergone a solid-liquid separation step involving one or more of centrifugation, microfiltration and depth filtration to remove host cells and/or cellular debris. A clarified fermentation broth may be a cell culture supernatant. Clarification is sometimes referred to as a primary or initial recovery step and typically occurs prior to any chromatography or a similar step.As mentioned the first step of the isolation comprises generation of an aggregate comprising a hydrophobic chelator, a non-ionic detergent and metal ions.The term "non-ionic detergent" refers to detergents that comprise uncharged, hydrophilic headgroups. Some non-ionic detergents are based on polyoxyethylene or a glycoside. Common examples of the former include Tween, Triton, and the Brij series. These materials are also known as ethoxylates or PEGlyates and their metabolites, nonylphenol. Glycosides have a sugar as their uncharged hydrophilic headgroup. Examples include octyl thioglucoside and maltosides. HEGA and MEGA series detergents are similar, possessing a sugar alcohol as headgroup.According to a particular embodiment, the non-ionic detergent is a polysorbate sufactant. Examples of such include, but are not limited to of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.In one embodiment, the non-ionic detergent is polysorbate 20.Other exemplary non-ionic detergents contemplated by the present invention include those that belong to the pluronic family e. g. F-68 and F-127.As used herein, the term "chelator" refers to a compound which binds metal ions from solution, by the formation or presence of two or more separate co-ordinate bonds between a polydentate ligand and a single central atom. The chelator of this aspect of the present invention is capable of chelating the metal ion which is used for the isolation. Preferably, the chelator binds electrostatically (non-covalently) to the metal ion. According to a particular embodiment, the chelator is capable of chelating metal ions with a ratio of chelator to metal of 2:1 or greater.The hydrophobicity of the chelator is such that it is capable of partitioning into the aggregates of the non-ionic detergent. In one embodiment, the chelator is capable of embedding into the aggregates of the non-ionic detergent.In one embodiment, the hydrophobic chelator comprises at least 8 carbons (for example in a chain, or in a ring) and does not comprise charged groups.In some embodiments, the hydrophobic chelator is 8-Hydroxyquinoline or a derivative thereof. Exemplary derivatives of 8-Hydroxyquinoline include, but are not limited to 2-methyl-8-hydroxyquinoline (CH3-HQ), 5,7-dichloro-2-methyl-8- hydroxyquinoline (Cl2-CH3-HQ), 5,7-dibromo-8-hydroxyquinoline (Br2-HQ), 5- sulfo-7-iodo-8-hydroxyquinoline (ferron) and 5-sulfo-8-hydroxyquinoline (SO3H- HQ).In some embodiments, the hydrophobic chelator comprises a phenanthroline, for example a 1,10-Phenanthroline. Other phenanothrolines are also contemplated which have not been substituted with hydrophilic substituents.Exemplary hydrophobic phenanthrolines include, but are not limited to bathophenanthroline, and N-(1,10-Phenanthrolin-5-yl)alkylamide), with the alkyl being from 1-10 carbon atoms in length. Exemplary N-(1,10-Phenanthrolin-5- yl)alkylamide) compounds include N-(1,10-Phenanthrolin-5-yl)methanamide) (Phen- C1), N-(1,10-Phenanthrolin-5-yl)ethanamide) (Phen-C2), N-(1,10-Phenanthrolin-5- yl)propanamide) (Phen-C3), N-(1,10-Phenanthrolin-5-yl)butanamide) (Phen-C4), N- (1,10-Phenanthrolin-5-yl)pentanamide) (Phen-C5), N-(1,10-Phenanthrolin-5- yl)hexanamide) (Phen-C6), N-(1,10-Phenanthrolin-5-yl)heptanamide) (Phen-C7), N- (1,10-Phenanthrolin-5-yl)octanamide) (Phen-C8), N-(1,10-Phenanthrolin-5- yl)nonanamide) (Phen-C9), N-(1,10-Phenanthrolin-5-yl)decanamide) (Phen-C10).In some such embodiments, the phenanthroline is selected from the group consisting of bathophenanthroline, N-(1,10-Phenanthrolin-5-yl)hexanamide) (Phen- 6), N-(1,10-Phenanthrolin-5-yl)decanamide) (Phen-C10) and N-(1,10-Phenanthrolin- 5-yl)octanamide) (Phen-C8).Herein throughout, an "alkylamide" describes a –NH-C(=O)-R, wherein R is alkyl.The term "alkyl" describes a saturated aliphatic hydrocarbon including straight chain and branched chain groups. Preferably, the alkyl group has 1 to 20 carbon atoms in length. Whenever a numerical range; e.g., "1-20", is stated herein, it implies that the group, in this case the alkyl group, may contain 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms. More preferably, the alkyl is a medium size alkyl having 1 to 10 carbon atoms. The alkyl group may be substituted or unsubstituted. Substituted alkyl may have one or more substituents, whereby each substituent group can independently be, for example, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, and heteroaryl. Additional substitutents may include, for example, hydroxyalkyl, trihaloalkyl, heteroalicyclic, amine, halide, sulfonate, sulfoxide, phosphonate, hydroxy, alkoxy, aryloxy, thiohydroxy, thioalkoxy, thioaryloxy, cyano, nitro, azo, sulfonamide, C-carboxylate, O-carboxylate, N-thiocarbamate, O-thiocarbamate, urea, thiourea, N-carbamate, O-carbamate, C- amide, N-amide, guanyl, guanidine and hydrazine, as long as the functionalities of the chelator are maintained.In some embodiments, the phenanthroline is Phen-C10 or Phen-C8.Additional examples of hydrophobic chelators include acidic organophosphorus chelators, for example DEHPA, EHEHPA and DTMPPA; neutral organophosphorus chelators, for example TBP and tri-n-octylphosphine oxide (TOPO), bifunctional organophosphorus chelators, for example CMPO and N,N,N',N'-tetraoctyl-3-oxamentanediamide (TOGDA); basic chelators, for example tri-n-octylamine (TOA) and tricaprylmethylammonium chloride. Other chelators known to those of skill in the art may also be used, including hydroxyoximes, for example 5,8-diethyl-7-hydroxy-6-dodecane oxime and 2-hydroxy-5-nonylacetophenon oxime, crown ethers, for example di-t-butyl-dicyclohexano-18- crown-6, and dithiosemicarbazone.According to some embodiments, the hydrophobic chelator is present in the aqueous solution at a concentration in the range of about 0.1% to about 10% (v/v), such as, for example, about 0.5% to about 10% (v/v), about 1% to about 10% (v/v) such as for example about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of 20mM solution of chelator.In some embodiments, the metal ion is a divalent metal ion.In some embodiments, the divalent metal ion is selected from the group consisting of Zn2+, Fe2+, Mn2+, Ni2+ and Co2+. Preferably, the divalent metal ion Zn2+ or Fe2+.In some embodiments, the metal ion is present in the aqueous solution at a concentration in the range of about 0.1% to about 10% (v/v), such as, for example, about 0.5% to about 10% (v/v), about 1 % to about 10% (v/v), about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% of 50mM solution of metal ion.The conditions of the incubation are such that aggregates are formed comprising the metal ion, the hydrophobic chelator and the non-ionic detergent.

Thus, for example, generation of aggregates is typically carried out at a temperature of about 0 ºC to about 25 ºC and more preferably from about 4 ºC to about 25 ºC. The aggregates of this aspect of the present invention are typically between 10-500 nM, 10-200 nM, 1-100 mM or 10-100 mM.The concentration of salt (e.g. NaCl) in the aggregates is typically, below 1mM and more preferably below 50 mM. In one embodiment, the concentration of salt is below 40 mM, below 30 mM, below 20 mM, below 10 mM or even below 5 mM. Exemplary ranges include 20-100 mM, 20-50mM, 0-50mM, 0-40 mM, 0-30 mM, 0­mM, 0-20 mM. In one particular embodiment, the concentration of salt is about mM.In some embodiments, contacting the non-ionic detergent with a hydrophobic chelator is performed prior to contacting with a metal ion.In other embodiments, contacting the non-ionic detergent with a hydrophobic chelator is performed concomitantly to contacting with a metal ion.In still further embodiments, the hydrophobic chelator is contacted initially with the metal ion and then subsequently with the non-ionic detergent.Preferably, the aggregates that are formed are greater than 500 nM in diameter.Preferably, the aggregates that are formed are greater than 1000 nM in diameter.Preferably, the aggregates that are formed are greater than 2000 nM in diameter.Typical contemplated ranges are between 500-3000 nM in diameter, 1000­3000 nM in diameter or 500-2000 nM in diameter.

Once aggregates are formed, they are contacted with the cell lysate under conditions that allow partitioning of the antibody (present in the cell lysate) into the aggregate.Once this happens (seconds to hours – for example 5 minutes to 1 hour), precipitation of the complex is facilitated by filtration.The filters are selected according to the size of the aggregates.

In one embodiment, the filters are 0.2 micron filters, 0.22 micron filters or even 0.45 micron filters.According to a particular embodiment, the filters are 0.1 micron filters.Following the filtration, the antibody may be released from the pelleted complex i.e. solubilized.Initially, the pellet may be washed – for example in a low salt solution (e.g. below 50 mM e.g. 20 mM NaCl solution).Extraction may be effected with a buffer having a pH between 3-6, and more preferably between 3.8-5. In one embodiment, the buffer is a carboxylic buffer, examples of which include, but are not limited to sodium acetate and sodium citrate. An exemplary pH of sodium acetate is about pH 4.6.In another embodiment, the buffer comprises an amino acid. In one embodiment, the buffer comprises a single amino acid. In another embodiment, the buffer comprises at least two amino acids.In one embodiment, the amino acid is one which can competes for (i) hydrophobic interactions between the antibody side chains and the detergent aggregate (e.g. valine or isoleucine); (ii) ionic and/or H-bond interactions between the antibody side chains and the detergent aggregate (e.g. aspartic acid, glutamic acid or arginine); or (iii) metal chelation interactions between the antibody side chains and the detergent aggregate (e.g. histidine).In a particular embodiment, the amino acid buffer is glycine, valine or isoleucine. In another embodiment, the amino acid buffer is isoleucine.An exemplary pH of amino acid buffers is about pH 3.8 or pH 4.The sample may be heated for a length of time that enhances extraction - for example (1-60 minutes), 1 minute, 5 minutes, 10 minutes. The temperature is selected such that it does not have an impact on the activity of the extracted antibody and does not cause the detergent aggregate to undergo dissolution. An exemplary temperature is between 25-35 °C. According to a particular embodiment, the sample is heated for 5 minutes at 32 °C.To enhance the purity of the released antibody, salt may be added to the buffer (e.g. between 5-50 mM NaCl or 10-20 mM NaCl). To enhance the amount of antibody released from the complexed pellet, the present inventors contemplate using buffers which do not contain salt. It will be appreciated however, that the purity of the released antibody may then be compromised.As mentioned, the present inventors contemplate reusing the metal chelator to purify additional antibodies as further described herein below.Thus, according to another aspect of the present invention there is provided a method of preparing an aggregate, the method comprising:(a) contacting a hydrophobic chelator, a non-ionic detergent and metal ions so as to generate a first aggregate comprising the hydrophobic chelator, the detergent and the metal ions;(b) contacting the aggregate with a medium comprising an antibody under conditions that allow a first fraction of the antibody to participate into the aggregate;(c) isolating the antibody from the aggregate;(d) disassociating the aggregate;(e) isolating the hydrophobic chelator; and subsequently(f) contacting the hydrophobic chelator, a non-ionic detergent and metal ions so as to generate a second aggregate comprising the hydrophobic chelator, the detergent and the metal ions, thereby preparing the aggregate.Steps (a), (b) and (c) have been described herein above, although it will beappreciated that step (b) can be facilitated by centrifugation (e.g. ultra-centrifugation), instead of (or together with) the filtration.In order to dissociate the aggregate, the detergent is solubilized by adding salt to the medium (which comprises residual antibodies, that were not extracted duringthe purification process). Exemplary salts include NaCl (e.g. at concentrations between 0.25-1M) andAmmonium sulfate, AS. The complex may then be dissociated using a water-soluble chelator (e.g. EDTA or EGTA) that could compete with the hydrophobic chelator on binding to the metal ions. The chelator can optionally be added together with an alcohol (e.g. methanol). The solution is then heated to atemperature between 80 degrees and 100 degrees, for example 95 degrees for between 2-5 minutes.Once disassociated, the hydrophobic chelator (e.g. bathophenanthroline) can be recrystallized so as to exclude any residual antibody which has not been removed during the first round of purification.

Recrystallization of bathophenanthroline is accomplished due to its extensive planar aromatic system. This inherent planarity and lipophilic nature of the chelator are ideal for promoting pi-pi (Π- Π) stacking between bathophenanthrolines and thus represent the driving force for its rapid crystal growth in aqueous media. The presence of EDTA does not interfere with the above, since EDTA is charged and as such, is repelled from the highly lipophilic faces of the growing crystals.The recrystallized, purified hydrophobic chelator can then be reused to generate additional aggregates, which in turn can be used to aid in the purification of additional antibodies.Depending on the intended use of the antibody that is isolated and optionally solubilized, the protein (either membrane or cytosolic) or agent that is bound thereto, may be subjected to further purification steps. This may be effected by using a number of biochemical methods which are well known in the art. Examples include, but are not limited to, fractionation on a hydrophobic interaction chromatography (e.g. on phenyl sepharose), ethanol precipitation, isoelectric focusing, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, anion exchange chromatography, cation exchange chromatography, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, hydroxylapatite chromatography, gel electrophoresis, dialysis, viral inactivation (e.g. viral filtration) and ultrafiltration.Examples of additional purification steps (and the order they may be carried out) are summarized in Figure 5B.Anion-exchange chromatography is a process that separates substances based on their charges using an ion-exchange resin containing positively charged groups, such as diethyl-aminoethyl groups (DEAE). In solution, the resin is coated with positively charged counter-ions (cations). Anion exchange resins will bind to negatively charged molecules, displacing the counter-ion.Cation-exchange chromatography is a process that separates substances based on their charges using an ion-exchange resin containing negatively charged groups, such as carboxymethyl (CM), sulfoethyl(SE), sulfopropyl(SP), phosphate(P) and sulfonate(S). In solution, the resin is coated with negatively charged counter-ions (anions). Cation exchange resins will bind to positively charged molecules, displacing the counter-ion.

The phrase "viral inactivation", as used herein, refers to a decrease in the activity of adventitious enveloped viruses in a particular sample ("inactivation"). Such decreases in the activity of enveloped viruses can be on the order of about 3 log reduction factor (LRF) preferably of about 4 LRF, more preferably of about 5 LRF, even more preferably of about 6 LRF.Any one or more of a variety of methods of viral inactivation can be used including heat inactivation (pasteurization), pH inactivation, solvent/detergent treatment, UV and γ-ray irradiation and the addition of certain chemical inactivating agents such as β-propiolactone or e.g., copper phenanthroline as in U.S. Pat. No. 4,534,972, the entire teaching of which is incorporated herein by reference.Methods of pH viral inactivation include, but are not limited to, incubating the mixture for a period of time at low pH, and subsequently neutralizing the pH. In certain embodiments the mixture will be incubated at a pH of between about 2 and 5, preferably at a pH of between about 3 and 4, and more preferably at a pH of about 3.6.The pH of the sample mixture may be lowered by any suitable acid including, but not limited to, citric acid, acetic acid, caprylic acid, or other suitable acids. The choice of pH level largely depends on the stability profile of the antibody product and buffer components. It is known that the quality of the target antibody during low pH virus inactivation is affected by pH and the duration of the low pH incubation. In certain embodiments the duration of the low pH incubation will be from 0.5hr to 2hr, preferably 0.5hr to 1.5hr, and more preferably the duration will be about 1hr. Virus inactivation is dependent on these same parameters in addition to protein concentration, which may limit inactivation at high concentrations.Thus, the proper parameters of protein concentration, pH, and duration of inactivation can be selected to achieve the desired level of viral inactivation.In certain embodiments viral filtration is performed. This can be achieved via the use of suitable filters. A non-limiting example of a suitable filter is the Ultipor DV50™ filter from Pall Corporation. In certain embodiments, alternative filters are employed for viral inactivation, such as, but not limited to, Sartorius filters, Viresolve™ filters (Millipore, Billerica, Mass.); Zeta Plus VR™ filters (CUNO; Meriden, Conn.); and Planova™ filters (Asahi Kasei Pharma, Planova Division, Buffalo Grove, 111.).

Ultrafiltration is described in detail in: Microfiltration and Ultrafiltration: Principles and Applications, L. Zeman and A. Zydney (Marcel Dekker, Inc., New York, N.Y., 1996); and in: Ultrafiltration Handbook, Munir Cheryan (Technomic Publishing, 1986; ISBN No. 87762-456-9). A preferred filtration process is Tangential Flow Filtration as described in the Millipore catalogue entitled "Pharmaceutical Process Filtration Catalogue" pp. 177-202 (Bedford, Mass., 1995/96). Ultrafiltration is generally considered to mean filtration using filters with a pore size that allow transfer of protein with average size of 50kDa (for example) or smaller. By employing filters having such small pore size, the volume of the sample can be reduced through permeation of the sample buffer through the filter while antibodies are retained behind the filter.Diafiltration is a method of using ultrafilters to remove and exchange salts, sugars, and non-aqueous solvents, to separate free from bound species, to remove low molecular-weight material, and/or to cause the rapid change of ionic and/or pH environments. Microsolutes are removed most efficiently by adding solvent to the solution being ultrafiltered at a rate approximately equal to the ultratfiltration rate. This washes microspecies from the solution at a constant volume, effectively purifying the retained antibody. In certain embodiments of the present invention, a diafiltration step is employed to exchange the various buffers used in connection with the instant invention, optionally prior to further chromatography or other purification steps, as well as to remove impurities from the antibody.In one embodiment, the antibody which is isolated is crystallized.As used herein the term "crystallizing" refers to the solidification of the molecule of interest so as to form a regularly repeating internal arrangement of its atoms and often external plane faces.Several crystalization approaches which are known in the art can be applied to the sample in order to facilitate crystalization of the molecule of interest. Examples of crystallization approaches include, but are not limited to, the free interface diffusion method [Salemme, F. R. (1972) Arch. Biochem. Biophys. 151:533-539], vapor diffusion in the hanging or sitting drop method (McPherson, A. (1982) Preparation and Analysis of Protein Crystals, John Wiley and Son, New York, pp 82-127), and liquid dialysis (Bailey, K. (1940) Nature 145:934-935).

Presently, the hanging drop method is the most commonly used method for growing macromolecular crystals from solution; this approach is especially suitable for generating protein crystals. Typically, a droplet containing a protein solution is spotted on a cover slip and suspended in a sealed chamber that contains a reservoir with a higher concentration of precipitating agent. Over time, the solution in the droplet equilibrates with the reservoir by diffusing water vapor from the droplet, thereby slowly increasing the concentration of the protein and precipitating agent within the droplet, which in turn results in precipitation or crystallization of the protein.The agents used for purifying the antibody may be provided as a kit.It is expected that during the life of a patent maturing from this application many relevant hydrophobic chelators will be developed and the scope of the term hydrophobic chelator is intended to include all such new technologies a priori.As used herein the term "about" refers to ± 10 %.The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".The term "consisting of" means "including and limited to".The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.Various embodiments and aspects of the present invention as delineated herein above and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262;3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. EXAMPLE 1 Purification of antibody by filtration MATERIALS Bovine serum albumin, 4,4'-dinonyl-2,2'dipyridyl, Protein A HP Spin-Trap, Protein AB Spin-Trap, isoleucine, FeSO4:heptahydrate, NaCl, NiBr2, ZnCl2, MnCl2, CuSO4, MgCl2, CaCl2, Tween-20 (Polysorbate 20), Ex-CELL 610-HSF medium, ethylenediaminetetraacetic acid (EDTA), 1,2-dihydroxybenzene (catechol), histidine (His), and imidazole were all purchased from Sigma-Aldrich; glycine (Bio-lab), PM2700 ExelBand 3-color Broad range protein marker (Smobio, Taiwan), bathophenanthroline (GFS chemicals) (batho), 4'-diphenyl-2,2'dipyridyl (Santa Cruz Biotechnology, CA, USA), human IgG (hIgG) (Lee-Bio-solutions, 340-21, MO, USA), mouse IgG (Equitech, SLM66, TX, USA). METHODS 2+ Preparation of [Tween-20:chelator:M2+] aggregates Tween-20 aggregates were obtained by mixing equal volumes of medium #and medium #2 as follows. For medium #1, 30 μL of 20mM amphiphilic chelator in methanol was added to 270 μL of 0.25 mM Tween-20 in DDW with vigorous vortexing to achieve a final volume of 300 μL. An equal volume of medium #2, containing FeSO4, CuSO4, NiBr2, ZnCl2, MnCl2, MgCl2 or CaCl2 (all 1 mM) in mM NaCl, was then added with vigorous vortexing. After 5 min incubation at room temperature, the mixture was centrifuged (13K, 2 min.), the supernatant discarded and the resulting pellet washed with 20mM NaCl in DDW and analyzed. In order to control pH, Tween-20 aggregates were prepared as described above but with the addition of pH buffers: 50 mM acetic acid (pH 3, 4, and 5) or 50 mM Tris(hydroxymethyl)aminomethane (Tris) (pH 6, 7, 8, or 9). Chelators phen-C10 and 4,4'diphenyl-2,2'-dippyridyl (dipyridyl phenyl) were tested with Ni2+, Fe2+, Zn2+ and Mn2+ (all under identical conditions). Antibody capture using conjugated Tween-20 micelles Freshly prepared, conjugated Tween-20 micellar aggregates were resuspended in 100 μL serum-free medium (Ex-CELL 610-HSF, NaHCO3 buffer: pH~6.72) containing 5% PEG-6000, the target IgG (1 mg/mL) and BSA (0.5 mg/mL). After 5­min incubation at room temperature, centrifugation (13K, 2 min.) was applied, the supernatant discarded and pellets were briefly washed with 100 μL of cold 20 mM NaCl. An additional centrifugation step followed (13K, 2 min.), the supernatant was removed and pellets were analyzed by SDS-PAGE. General extraction protocol of IgG's from conjugated Tween-20 micellar aggregates Pellets containing target IgG were incubated with 100 μL of 50 mM glycine (pH 3.8), 30 mM NaCl for 5 minutes at 32 °C. Centrifugation followed (13K, minutes) and the supernatant was carefully removed for analysis on SDS-PAGE gels. 2+ Preparation of [Tween-20:batho:Fe2+] aggregates at different pH Tween-20 aggregates were prepared as described above but in the presence of pH buffers: 50 mM acetic acid (for pH 3, 4, and 5) or 50 mM Tris(hydroxymethyl)aminomethane (Tris) (for pH 6, 7, 8, or 9). Dynamic light scattering (DLS) The intensity-weighted size distributions of extracted human and mouse IgG samples were determined using the autocorrelation protocol of the Nanophox instrument (Sympatec GmbH, Germany). Densitometry Bands on Coomassie stained gels were quantified using the ImageJ (NIH) program. RESULTS Current findings show that, purification of IgG's with: [Tween- 20:bathophenanthroline:Fe2+] detergent aggregates can rely on filtration rather than on centrifugation (Figures 1A-B). Under these circumstances, IgG’s are relatively pure and lead to overall greater recovery yields (Figure 1).Implementation of the purification protocol to other members of the Tween family and to other surfactant families (e.g. Brij, Triton, Pluronic) show that IgG purification is accomplished (Figures 2A-D).It has been found that purification can be performed in the presence of different chelators (e.g. EDTA, Catechol, Histidine, imidazole) and lead to relatively pure antibody and good recovery yields (Figure 3).

EXAMPLE 2 Chelator recycling The general strategy for recycling the chelator: bathophenanthroline (batho) via recrystallization is illustrated in Figure 4In brief, pellets comprised of Tween-20 (polysorbate-20), the red hydrophobic complex: [(batho)3:Fe2+] and residual antibodies (that were not extracted during the purification process), were resuspended in 0.25-1M NaCl to solubilize the detergent (Tween-20) and IgG's while keeping the majority of the highly hydrophobic [(batho)3:Fe2+] red complex, insoluble. (Figure 4 - Step I) Following a short spin, the supernatant was discarded and the resulting pellets were found to be comprised of red crystals (Figure 5A) that exhibited identical absorption (λmax=533nm) to the freshly prepared [(batho)3:Fe2+] red complex. (Figure 5B). Addition of MeOH and molar excess of EDTA (pH 7) allowed total dissolution of the red crystals and loss of the solution red color (Figure 4 - Step II), presumably due to the presence of EDTA. The latter, is a strong water-soluble chelator that could compete with batho on binding to Fe2+ ions thereby leading to the dissociation of the: [(batho)3:Fe2+] complex and generation of the colorless [EDTA-Fe2+] complex. Slow cooling of the solution promoted the growth of colorless crystals (Figure 3 - Step III) comprised of the regenerated chelator. (Figures 6A-B). Recycling protocol: 1. Add 400 µL of 0.5 M NaCl to the resulting pellet after the IgG extraction step.2. Vortex for 1 minute and apply centrifugation (13K, 5 minutes, room temp).3. Remove the supernatant.4. Add MeOH (50 µL) and 0.2 M EDTA (200 µL, pH 7) in DDW.5. Incubate for 2-5 minutes at 95 °C until the solution becomes colorless and clear.6. Add 800 µL of DDW.7. Allow the system to cool to room temperature (e.g. 15 minutes) and then, to -ºC or -18 ºC (if needed).8. Apply centrifugation and exclude the supernatant (13K, 5 min., room temp.)9. Wash crystals with cold DDW (500 µL), centrifuge and discard the water.

Quantification of recycling efficiency: 1. Dissolve crystals with MeOH to a final known volume.2. Add excess Fe2+ (e.g. 100 mM).3. Measure absorbance at 530-533 nm (representing the: λmax of the [(batho)3:Fe2+] red complex).

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as anadmission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims (29)

1.268878/

2.Claims: 1. A method of preparing an aggregate, the method comprising: (a) contacting a bathophenanthroline chelator, a non-ionic detergent and iron ions so as to generate a first aggregate comprising said bathophenanthroline chelator, said detergent and said iron ions; (b) contacting said aggregate with a medium comprising an antibody under conditions that allow a first fraction of said antibody to participate into said aggregate; (c) isolating said antibody from said aggregate; (d) disassociating said aggregate; (e) isolating said bathophenanthroline chelator; and subsequently (f) contacting said bathophenanthroline chelator, a non-ionic detergent and iron ions so as to generate a second aggregate comprising said bathophenanthroline chelator, said detergent and said iron ions, thereby preparing the aggregate. 2. The method of claim 1, wherein said disassociating said aggregate comprises contacting said aggregate with a water soluble chelator under conditions that allows disassociation of said aggregate.

3. The method of claim 2, wherein said water soluble chelator comprises EDTA or EGTA.

4. The method of claim 1, wherein said isolating said antibody from said aggregate comprises filtering said medium comprising said aggregate.

5. The method of claim 1, wherein said aggregate has a diameter of greater than 5nM.

6. The method of claim 5, wherein said aggregate has a diameter of between 500-30nM. 32 268878/

7. The method of claim 1, wherein said medium comprises a cell lysate.

8. The method of claim 7, wherein said cell lysate is a whole cell lysate.

9. The method of claim 1, wherein said medium comprises a hybridoma medium.

10. The method of claim 1, wherein said medium comprises serum albumin.

11. The method of claim 7, wherein said cell lysate is devoid of organelles greater than about 2 microns.

12. The method of claim 1, wherein said conditions of step (b) comprise having a level of salt below 100 mM.

13. The method of claim 1, wherein said isolating said antibody comprises solubilizing said antibody.

14. The method of claim 13, wherein said solubilizing is effected with a buffer having a pH between 3-6.

15. The method of claim 14, wherein said buffer further comprises a salt.

16. The method of claim 14, wherein said buffer is a carboxylic buffer.

17. The method of claim 14, wherein said buffer comprises an amino acid.

18. The method of claim 16, wherein said carboxylic buffer is selected from the group consisting of isoleucine, valine, glycine and sodium acetate.

19. The method claim 1, wherein said non-ionic detergent is a polysorbate surfactant. 33 268878/

20. The method of claim 19, wherein said polysorbate surfactant is selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

21. The method of claim 1, wherein said bathophenanthroline chelator is present in said aqueous solution at a concentration in the range of about 0.1% to about 10% (v/v).

22. The method of claim 1, wherein said iorn ions are present in said aqueous at a concentration in the range of about 0.1 % about 10% (v/v).

23. The method of claim 7, wherein said cell lysate is derived from a bacterial cell.

24. The method of claim 7, wherein said cell lysate is derived from a mammalian cell.

25. The method of claim 24, wherein said mammalian cell is a Chinese Hamster Ovary cell (CHO).

26. The method of any one of claims 1-25, wherein said antibody is a humanized antibody.

27. The method of any of claims 1-25, wherein said antibody is a recombinant antibody.

28. The method of any one of claims 1-25, wherein said antibody is selected from the group consisting of IgA, IgD, IgE, IgM and IgG.

29. The method of claim 28, wherein said IgG is IgG1, IgG2, IgG3 or IgG4. Dr. Hadassa Waterman Patent Attorney G.E. Ehrlich (1995) Ltd. 35 HaMasger Street, 13th Floor, Sky Tower 6721407 Tel Aviv