WO2015017723A2 - Protein a fragment crystals and cross-linked crystals and methods of use thereof - Google Patents

Abstract

ZDPA crystals and ZDPA cross-linked protein crystals (CLPCs) are described. Methods of preparing and using are also disclosed.

Description

PROTEIN A FRAGMENT CRYSTALS AND CROSS-LINKED

CRYSTALS AND METHODS OF USE THEREOF

CROSS REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application 61/860,451, filed on July 31, 2013, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Protein A is a 40-60 kDa surface protein originally found in the cell wall of the bacteria Staphylococcus aureus. It has found use in biochemical research because of its ability to bind immunoglobulins. Protein A bind proteins from many mammalian species, most notably immunoglobulin Gs (IgGs). It binds with the fragment crystallizable (Fc) region of

immunoglobulins through interaction with the heavy chain. Recombinant Staphylococcal Protein A is often produced in E. coli for use in immunology and other biological research. Protein A is often coupled to other molecules such as a fluorescent dye, enzymes, biotin, colloidal gold or radioactive iodine without affecting the antibody binding site. It is also widely utilized coupled to magnetic, latex and agarose beads. Protein A is often immobilized onto a solid support and used as a reliable method for purifying total IgG from crude protein mixtures such as serum or ascites fluid, or coupled with one of the above markers to detect the presence of antibodies. Immunoprecipitation studies with Protein A conjugated to beads are also commonly used to purify proteins or protein complexes indirectly through antibodies against the protein or protein complex of interest. In addition, it is widely used in purification of monoclonal antibodies from wide variety of sources.

[0003] The gene for Protein A was sequenced in 1984, and was found to consist of three regions with different functions. The N-terminal part is a signal peptide sequence (Ss), the central portion contains five homologous IgG-binding domains (E, D, A, B and C) and the C-terminal region anchors the protein to the bacterial cell wall (regions X and M). Sequence similarity between the five IgG-binding domains varies between 65-90%. Each IgG-binding domain of Protein A can be expressed separately; each folds independently into a three helix bundle that can bind the Fc portion of IgG. The B-domain is the most thoroughly studied of the five domains. Two amino acid substitutions were designed in the B-domain to improve its stability. This modified variant has been given the name Z-domain.

[0004] The Z-domain, like the B-domain, consists of 58 amino acids. Both proteins are nearly identical in structure and consist of a three-helix bundle. The Z-domain has found several uses in the field of protein purification. Most commonly, the nucleotide sequence of the Z-domain is added to an expression vector to create a fusion protein with the protein of interest. This fusion protein, once expressed, can be purified by commercially available chromatography media that has covalently linked IgG or Fc protein. Typically, the nucleotide of the Z-domain is repeated twice in the expression vector to produce a dimeric Z-domain fusion protein. It has been determined that dimeric Z-domain (often referred to as ZZ) binds IgG more strongly than its monomer due to a lower off-rate achieved through the avidity effect.

[0005] In addition to use in purification, Z-domain fusion proteins have been used as detection tags in immunoblotting, as a solubilizing fusion partner, and as an aid in the correct folding of the protein to which it is fused.

SUMMARY OF THE INVENTION

[0006] The invention provides new and improved compositions of, and methods for making and using, Protein A Z-Domain cross-linked protein crystals. The invention relates, in part, to the preparation of the Z-Domain of Protein A (ZDPA) cross -linked protein crystals (CLPCs), e.g., produced so as to develop an innovative system (e.g., chromatography system) for the purification of antibodies. ZDPA CLPCs offer the advantages of highly concentrated Z-Domain of Protein A activity combined with high stability and chemical resistance. The condensed Z- Domain concentration reduces column size (if used), buffer volume and process time.

Moreover, the cross-linking of Z-domain crystals can prevent or decrease the leaching of Z- Domain during purification (e.g., using chromatography), e.g., of immunoglobulins (e.g., antibodies). Altogether, this can reduce antibody production time and cost.

[0007] The invention relates to crystals of Z-Domain and cross-linked forms ("ZDPA-CLPC or CLPC") or derivatives thereof and their uses to purify immunoglobulins/antibodies or corresponding Fab or Fc fragments, e.g., polyclonal antibodies, monoclonal antibodies from cell culture, therapeutic antibodies, antibodies from bacterial culture, serum, plasma, the use of such crystals of Z-Domain for immunoprecipitation, and extracorporeal devices.

[0008] Disclosed herein are compositions containing a crystalline form of ZDPA. An embodiment of this is a composition containing a cross-linked crystalline (CLPC) form of ZDPA. In a further embodiment, ZDPA crystals are cross-linked with glutaraldehyde. In some embodiments, ZDPA crystal is cross-linked with about 0.02% to about 4% (w/v) glutaraldehyde. In yet other embodiments, ZDPA crystal is cross-linked with about 1.00% (w/v) glutaraldehyde.

[0009] The compositions disclosed herein are more active than non-crystalline forms of ZDPA because they have a higher binding capacity. In an embodiment of this, the binding capacity of the crystalline forms of ZDPA is at least about 100% higher than the binding capacity of the soluble immobilized form at pH 7. In yet another embodiment, the cross-linked ZDPA crystal has at least about 150% of the binding capacity of the non-crystalline immobilized form of ZDPA.

[00010] In an embodiment of the invention, the ZDPA crystals of the invention are stable

(retain their binding capacity) at about pH 2 to about pH 12.

[00011] In another embodiment the ZDPA crystals disclosed herein have 0.0% protein leaching when compared to immobilized non-crystalline ZDPA.

[00012] Also disclosed herein are the use of the crystalline ZDPA compositions in a prepacked column as column material, or in a membrane (impregnated), or in an extracorporeal device.

[00013] In still another embodiment of the invention, disclosed herein are kits containing the crystalline ZDPA compositions disclosed herein. Such kits may contain other reagents, purification apparatus, and instructions for using the crystalline ZDPA compositions described herein.

[00014] In one embodiment, crystalline ZDPA can be pre-packed in a column to be used as a purification material of antibodies and antibody fragments, e.g., purification of monoclonal antibodies from mammalian cell culture, purification of monoclonal antibodies expressed in transgenic milk or polyclonal antibodies generated in serum.

[00015] Additionally disclosed are methods of producing protein crystals from

recombinant soluble ZDPA. Compositions, e.g., pharmaceutical compositions, including the crystals of ZDPA and cross-linked forms thereof ("CLPC") are also disclosed.

[00016] In one aspect, the invention provides cross-linked ZDPA crystals. The cross- linking agent can be multifunctional, and in certain embodiments, the agent is a bifunctional agent, such as glutaraldehyde. In certain embodiments, the ZDPA crystals are cross-linked with glutaraldehyde at a concentration that does not substantially alter binding capacity, e.g., at a concentration of at least about 0.02% (w/v). In some embodiments, the level of cross-linking of the ZDPA crystal is equivalent to that produced by treatment with 0.02% (w/v) glutaraldehyde. The level of cross-linking can be determined by methods known in the art or disclosed herein, e.g., determining the level of protein leaching.

[00017] The invention further provides ZDPA crystals that have a higher binding capacity, e.g., at least about 100%, 200%, 300%, 400%, 500%, or more compared to soluble ZDPA.

[00018] The invention further provides a stabilized, e.g., cross-linked ZDPA crystal, wherein said stabilized crystal retains a binding capacity and/or stability, in acidic conditions at least 2-, 3-fold higher than the binding capacity and/or stability retained by a soluble ZDPA in similar acidic conditions (e.g., an acidic pH of about 2 to 3). In some embodiments, the stabilized ZDPA crystal has at least about 200%, 300%, 400% more binding capacity and/or stability than a soluble ZDPA in acidic conditions.

[00019] The invention further provides a stabilized, e.g., cross-linked, ZDPA crystal, wherein said stabilized crystal retains a binding capacity and/or stability, in the presence of a protease, at least 2-, 3-fold higher than the binding capacity and/or stability retained by a soluble ZDPA in similar conditions. In some embodiments, the stabilized ZDPA crystal has at least about 200%, 300%, 400% more binding capacity and/or stability than a soluble ZDPA in the presence of a protease. The protease can be chosen from one or more of, e.g., pepsin, chymotrypsin or pancreatin.

[00020] In other embodiments, the binding capacity of the stabilized or soluble ZDPA is measured after exposing the stabilized crystal or soluble ZDPA to acidic conditions and/or a protease for a predetermined length of time, e.g., at least one, two, three, four or five hours.

[00021] In a related aspect, the invention features a cross-linked, ZDPA crystal which is substantially stable in variable pH conditions (e.g., about pH 2.0 or 3 to about pH 7.5 or about pH 8.5 to about pH 10-14), and/or in the presence of a protease, e.g., a protease can be chosen from one or more of the following: pepsin, chymotrypsin or pancreatin. In still other embodiments, the cross-linked crystal retains its binding capacity at least about 2-, 3-fold higher than the binding capacity retained by a soluble ZDPA in acidic conditions (e.g., an acidic pH of about 2 to 3) and in the presence of a protease, as described herein. In other embodiments, the stabilized ZDPA crystal is at least 200%, 300%), 400%) more stable than a soluble ZDPA in acidic conditions (e.g., an acidic pH of about 2 to 3) and in the presence of a protease, as described herein.

[00022] Also disclosed herein are Compositions, e.g., pharmaceutical compositions that include the crystals and/or the cross-linked ZDPA crystals.

[00023] In some embodiments, the crystals include ZDPA having a sequence identical or substantially identical to ZDPA (SEQ. ID 1).

[00024] In another aspect, the invention provides membranes impregnated with cross- linked protein crystals, devices, systems and methods of producing and using the same for a variety of suitable applications including, for example, the purification of antibodies and antibody fragments and removal of immunoglobulins during dialysis therapy. In this regard, the ZDPA impregnated membranes of the present invention can bind to the antibodies and antibody fragments. This can effectively minimize the amount of buffer needed for purification, thus minimizing costs. [00025] In another embodiment, the present invention provides a material including a membrane impregnated with about 3.25 mg/cm2 or less of a cross-linked protein crystal.

Preferably, the membranes are impregnated with cross-linked ZDPA crystals ("ZDPA-CLPC"). The membranes impregnated with ZDPA-CLPCs can be used to isolate and purify

immunoglobulins and can be reused similar to immobilized ZDPA.

[00026] In still another embodiment, the present invention provides a method of producing a membrane impregnated with cross-linked protein crystals, preferably ZDPA-CLPC. The method includes preparing a membrane casting solution. The casting solution includes a polymeric base material, such as polyurethane, in a solvent, such as l-methyl-2-pyrrolidinone ("NMP"), dimethylformamide ("DMF"), the like or combinations thereof. The membrane casting solution can also include a bulking agent, such as zirconium oxide, and an agent, such as polyvinylpyrrolidone ("PVP") to render the membrane more hydrophilic.

[00027] The casting solution is then mixed with a suitable amount of ZDPA-CLPC, such that the membrane is impregnated with about 3.25 mg/cm2 ZDPA-CLPC. The solution can then be spread on a support material, such as a synthetic mesh material, and immersed into an aqueous media under suitable conditions, thus forming a membrane precipitate. The membrane precipitate is subsequently dried in a glycerol solution, preferably a mixture of glycerol and water at a ratio of 40: 60.

[00028] An advantage of the present invention is to provide improved protein

impregnated membranes suitable for use in a variety of different applications such as antibody purification and immunoprecipitation.

[00029] A further advantage of the present invention is to provide improved materials capable of binding antibodies without the need for any inert support unlike immobilized ZDPA.

[00030] In still another aspect, the invention provides a method of reducing

immunoglobulin concentration (disorder associated with elevated immunoglobulin

concentration) in a subject by administering a composition, e.g., a pharmaceutical composition that includes ZDPA crystals, e.g., cross-linked ZDPA crystals, as disclosed herein. In one embodiment of this aspect, the ZDPA crystals are stabilized by a cross-linking agent, such as glutaraldehyde. Administration of the composition can cause a reduction of immunoglobulin concentration by at least about 10%, at least about 20%, at least about 30%, or at least about 40% or more. In some embodiments, the composition is administered orally or via an extracorporeal device. In a further embodiment, the extracorporeal device is a catheter, e.g., a catheter coated with ZDPA crystals. In still another embodiment, the method of reducing immunoglobulin concentration in a mammal includes a step of assaying the immunoglobulin concentration in a biological sample of the mammal, such as a blood, plasma, or a serum sample.

[00031] In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, that includes ZDPA crystals, e.g., cross-linked ZDPA (e.g., the crystals and/or cross-linked crystals, as disclosed herein).

[00032] In yet another aspect, the invention provides a method of treating a mammal by administering/as a sorbent in dialysis equipment (extracorporeal device) an effective amount of a pharmaceutical composition that includes ZDPA crystals, e.g., cross-linked ZDPA crystals (e.g., the crystals and/or cross-linked crystals, as disclosed herein).

[00033] In yet another embodiment, the crystallizing step includes concentrating the purified protein, followed by adding precipitation reagents thereby forming crystallized protein. The crystallization step can additionally include contacting the crystallized protein with a cross- linking agent, e.g., a cross-linking agent disclosed herein (e.g., glutaraldehyde). The

concentration of crosslinking agent used can be in the range of about 0.01% to 20% w/v;

typically, about 0.02%> to 10%> w/v; more typically about 0.5%>, 1% or 2% w/v.

[00034] In other embodiments, the yield of the crystallized protein in the preparation is at least about 50%>, 60%>, 70%>, 80%> of the specific protein found in the soluble ZDPA suspension. In other embodiments, the yield of the crystallized protein is at least about 90%, 95% or higher of that found in the soluble preparation. In yet other embodiments, the yield of the crystallized protein is at least about 50%>, 60%>, 70%>, 80%> of that found in the soluble preparation.

[00035] The invention further provides protein crystals, (e.g., ZDPA crystals) produced by the methods disclosed herein.

[00036] In some aspects, the disclosure features ZDPA crystals.

[00037] In some aspects, the disclosure features ZDPA cross-linked protein crystals

(CLPCs),e.g., cross-linked as described herein.

[00038] In some aspects, the disclosure features a composition containing ZDPA crystals and another ingredient (e.g., buffer, e.g., Tris buffer).

[00039] In some aspects, the disclosure features a composition containing ZDPA cross - linked protein crystals (CLPCs) and another ingredient (e.g., buffer, e.g., Tris buffer).

[00040] In some aspects, the disclosure features a method of making ZDPA crystals, e.g., as described herein.

[00041] In some aspects, the disclosure features a method of making ZDPA cross -linked protein crystals (CLPCs), e.g., as described herein. [00042] In some aspects, the disclosure features a kit containing ZDPA crystals and another component (e.g., instructions for use).

[00043] In some aspects, the disclosure features a kit containing ZDPA cross -linked protein crystals (CLPCs) and another component (e.g., instructions for use).

[00044] In some aspects, the disclosure features a method of using ZDPA crystals, e.g., as described herein, e.g., to purify an immunoglobulin, e.g., an antibody, e.g., a therapeutic antibody. In some embodiments, leaching of ZDPA is prevented or decreased (e.g., decreased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%), about 100% or about 2 fold, about 5 fold, or about 10 fold as compared to the amount of leaching when ZDPA in non-crystal form is used under the same conditions.

[00045] In some aspects, the disclosure features a method of using ZDPA cross -linked protein crystals (CLPCs), e.g., as described herein, e.g., to purify an immunoglobulin, e.g., an antibody, e.g., a therapeutic antibody. In some embodiments, leaching of ZDPA is prevented or decreased (e.g., decreased by about 10%>, about 20%>, about 30%>, about 40%>, about 50%>, about 60%, about 70%, about 80%, about 90%, about 100% or about 2 fold, about 5 fold, or about 10 fold as compared to the amount of leaching when ZDPA in non-crystal form or when ZDPA crystals in non-cross-linked form are used under the same conditions.

[00046] In some aspects, the disclosure features a method of using ZDPA crystals, e.g., as described herein, e.g., to purify an immunoglobulin, e.g., an antibody, e.g., a therapeutic antibody. In some embodiments, binding capacity of ZDPA per mL is increased (e.g., increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%), about 90%), about 100% or more or about 2 fold, about 5 fold, or about 10 fold or more as compared to the amount of binding when ZDPA in non-crystal form is used under the immobilized conditions (e.g., using a support).

[00047] In some aspects, the disclosure features a method of using ZDPA cross -linked protein crystals (CLPCs), e.g., as described herein e.g., to purify an immunoglobulin, e.g., an antibody, e.g., a therapeutic antibody. In some embodiments, binding capacity of ZDPA is increased (e.g., increased by about 10%>, about 20%>, about 30%>, about 40%>, about 50%>, about 60%), about 70%), about 80%>, about 90%>, about 100%), or more or about 2 fold, about 5 fold, or about 10 fold or more as compared to the amount of binding when ZDPA in non-crystal form or when ZDPA crystals in non-cross-linked form are used under the immobilized conditions (using a support).

[00048] In some aspects, the disclosure features a column (e.g., chromatography column) containing ZDPA crystals. [00049] In some aspects, the disclosure features a column (e.g., chromatography column) containing ZDPA cross -linked protein crystals (CLPCs).

[00050] In some aspects, the disclosure features a membrane (e.g., holofiber system) containing ZDPA crystals.

[00051] In some aspects, the disclosure features a membrane (e.g., holofiber system) containing ZDPA cross -linked protein crystals (CLPCs).

[00052] Disclosed herein are methods of crystallizing ZDPA in which the ZDPA is solubilized or concentrated, and to the solubilized or concentrated protein is added precipitating reagents to form crystals.

[00053] Disclosed herein are processes for the purification of immunoglobulins which include the steps of (a) mixing a medium containing immunoglobulins with a buffer solution having a pH in the range of about pH 7.0 to pH 10 and containing a combination of cations and anions to provide a buffered immunoglobulin medium; (b) contacting said buffered

immunoglobulin medium with an immobilized ZDPA (ZDPA-CLPC: ZDPA is immobilized by crystallizing and cross-linking ZDPA molecules) adsorbent to adsorb the immunoglobulins present in said buffered immunoglobulin medium upon said immobilized ZDPA adsorbent; (c) washing the ZDPA-CLPC adsorbent having immunoglobulins adsorbed thereon with said buffer solution; (d) contacting said ZDPA-CLPC adsorbent having immunoglobulins adsorbed thereon with a buffer solution having a pH in the range of about pH 2 to pH 6 to remove the adsorbed immunoglobulins from the ZDPA-CLPC adsorbent; and (e) recovering the removed

immunoglobulins in substantially pure form. In a further embodiment of this process, the process is accomplished in a column containing the crystalline compositions of ZDPA described herein. In yet another embodiment of the process, the contacting of the buffered

immunoglobulin medium with the ZDPA-CLPC adsorbent is accomplished in a column containing ZDPA-CLPC adsorbent. In another embodiment of this process, the medium containing immunoglobulins is a normal mammalian serum, or immune mammalian serum, such as plasma or ascites fluid. In other embodiments the immunoglobulin medium is obtained from a hybridoma, tissue culture fluid, cell culture fluid, a mammalian cell culture fluid, bacterial cell culture fluid, transgenic source fluid, a plant extract, or a yeast culture fluid.

[00054] In some embodiments of the process for purification, the buffer solution will have a pH in the range of about pH 7.0 to about pH 10 and the buffer is a glycine buffer, borate buffer, tris (hydroxymethyl) aminomethane buffer, or phosphate buffer. In yet other embodiments of the process of purification, the buffer solution has a concentration range of about 0.01M to about 0.25M, or about 0.05M to about 0.5M. [00055] In still other embodiments of the process for purification, the buffer solution has a pH in the range of about pH 2 to about pH 6 and has a concentration in the range of about 0.01M to 0.25M. In some embodiments of the process of purification, the buffer solution is an acetic acid-acetate buffer and has a pH in the range of about pH 2 to about pH 6.

[00056] In another embodiment of the process for purification, the buffer solution contains potassium ions and phosphate ions in a concentration of about 1.0M to about 1.5M.

[00057] In yet another embodiment of the process for purification, the buffer solution contains ammonium ions and phosphate ions in a concentration of about 1.0M to about 1.5M. In still another embodiment of the process for purification, the buffer solution contains ammonium ions and sulfate ions in a concentration of about 1.0M to about 1.5M. In some embodiments of the process for purification the buffer solution contains sodium ions and sulfate ions in a concentration of about 1.0M to about 1.25M. In still other embodiments the buffer solution contains sodium ions, phosphate ions and chloride ions in a concentration of about 1.0M to about 1.25M.

[00058] In further embodiments of this process for purification, the immobilized ZD PA adsorbent is crystalline and cross-linked ZDPA without any support. In yet other embodiments the adsorbent is crystalline and cross-linked ZDPA without any support attached to magnetic particles.

[00059] Disclosed herein is a method for purifying a protein from a contaminated solution using ZDPA chromatography including the steps of : (a) adsorbing the protein to purified to an immobilized solid phase crystallized and cross-linked ZDPA; (b) removing contaminants bound to the solid phase by washing the solid phase with an electrolyte solvent having a pH in the range from about 5 to about 7 and containing an electrolyte selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC),

tetrapropylammonium chloride and tetrabutylammonium chloride; and (c) recovering the protein from the solid. In some embodiments of this method the protein to be purified is an

immunoglobulin, antibody, or fragment thereof. In still further embodiments of this method, the protein to be purified is a polycolonal antibody, monoclonal antibody, or chimeric antibody or fragment thereof. In other embodiments of this method the solid phase is a crystalline ZDPA in a column or is a crystalline cross-linked ZDPA in a column.

[00060] In some embodiments of the method for purifying a protein from a contaminated solution, the electrolyte solvent contains tetramethylammonium chloride (TMAC) or

tetraethylammonium chloride (TEAC). In still further embodiments of this method, the concentration of the electrolyte in the electrolyte solvent is in the range from about 0.1 to about 1.0 M or in the range from about 0.25 to about 0.5 M.

[00061] In other embodiments of the method for purifying a protein from a contaminated solution, the contaminants are Chinese Hamster Ovary Proteins (CHOP). In some embodiments of this method the contaminated solution comprises Harvested Cell Culture Fluid (HCCF) comprising a recombinant antibody.

[00062] In embodiments of the method for purifying a protein from a contaminated solution, the elution buffer of step (c) involves eluting the protein using an elution buffer having a pH in the range from about 2.0 to about 5.0 or from about 2.5 to about 3.5.

[00063] Disclosed herein is a method for purifying a protein which has been produced a produced by a Chinese Hamster Ovary (CHO) cell using ZDPA chromatography including the steps of: (a) adsorbing the protein to ZDPA immobilized which is done by crystallizing and crosslinking of ZDPA to create a solid phase; (b) removing Chinese Hamster Ovary Protein (CHOP) contaminants bound to the solid phase by washing the solid phase with an electrolyte solvent comprising an electrolyte selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride and tetrabutylammonium chloride; and (c) recovering the protein from the solid phase.

[00064] Also disclosed herein is a method for purifying a protein that has been produced by a Chinese Hamster Ovary (CHO) cell using ZDPA chromatography including the steps of: (a) adsorbing the protein to ZDPA immobilized which was done by crystallizing and cross-linking to create a solid phase; (b) removing Chinese Hamster Ovary Protein (CHOP) contaminants bound to the solid phase by washing the solid phase with an electrolyte solvent having a pH in the range from about 5 to about 7 and containing an electrolyte solvent selected from the group consisting of tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride and tetrabutylammonium chloride, wherein the concentration of the electrolyte in the electrolyte solvent is in the range from about 0.25 to about 0.5 M; and (c) recovering the protein from the solid phase.

[00065] Disclosed herein is a method of treating a disorder associated with elevated immune globulins concentration in a mammal, the method comprising administering ZDPA crystals to the mammal in an amount sufficient to reduce one or more symptoms associated with the disorder. In some embodiments of this method, the disorder is related to immune function.

[00066] Disclosed herein is a method of producing a membrane, the method includes the steps of : (a) preparing a casting solution composed of a polymeric base material in a solvent; (b) adding a sufficient amount of a cross-linked protein crystal to the casting solution; (c) applying the membrane casting solution to a support material; (d) immersing the membrane casting solution and the support material in an aqueous media; (e) forming a membrane composite material; (f) and drying the membrane composite material with a fluid medium. In some embodiments of this method the polymeric base material is composed of a suitable polymer including polyurethane. In yet other embodiments of this method, the cross-linked protein crystal includes a protein selected from the group consisting of ZDPA, Protein G, Protein L and combinations thereof. In still other embodiments of this method, the solvent is selected from the group consisting of 1 -methyl -2 -pyrrolidinone, dimethylformamide and combinations thereof. In some embodiments of this method the fluid medium includes glycerol.

[00067] Disclosed is a method of producing a ZDPA impregnated membrane, including the steps of: (a) forming a membrane casting solution including polyurethane and a bulking agent in a solvent; (b) adding the ZDPA-CLPC to the membrane casting solution; (c) processing the membrane casting solution in an aqueous media; (d) and forming a membrane precipitate impregnated with the ZDPA-CLPC. In some embodiments of this method the bulking agent is selected from the group consisting of zirconium oxide, zirconium phosphate, carbon and combinations thereof. In another embodiment the method of producing an impregnated membrane includes the additional step of drying the membrane precipitate with a glycerol solution.

[00068] Disclosed herein is a material capable of removing immunoglobulins from dialysate or body fluids during dialysis therapy, the material including a membrane impregnated with cross-linked protein crystal wherein the membrane has been dried with a fluid medium. In an embedment of this method the cross-linked protein crystal includes a protein selected from the group consisting of ZDPA, Protein G, Protein L and combinations thereof. In still further embodiments the membrane includes a bulking agent selected from the group consisting of zirconium oxide, zirconium phosphate, carbon and combinations thereof. In still other embodiments of this method, the fluid medium includes glycerol.

[00069] Disclosed herein is a device for removing immunoglobulins from a dialysate or body fluid used during dialysis therapy, wherein the device includes: a body defining an interior with an inlet and an outlet, the interior containing a layer of a membrane impregnated with a cross-linked protein crystal wherein the membrane has been dried with a fluid medium. In some embodiments of this device, the cross-linked protein crystal includes a protein selected from the group consisting of ZDPA, Protein G, Protein L and combinations thereof. In other

embodiments of this device the fluid medium includes glycerol. [00070] Disclosed herein is a system for providing dialysis therapy, the system

comprising a device capable of removing immunoglobulins during dialysis, wherein the device includes a body defining an interior with an inlet and an outlet, and the interior contains a layer of a membrane impregnated with cross-linked protein crystal wherein the membrane has been dried with a fluid medium. In embodiments of this system the cross-linked protein crystal includes a protein selected from the group consisting of ZDPA, Protein G, Protein L and combinations thereof. In other embodiments the fluid medium includes glycerol.

[00071] Disclosed herein is a method of providing dialysis therapy, the method including the steps of: (a) passing a dialysis fluid or body fluid through a device including a layer of a membrane impregnated with cross-linked protein crystal wherein the membrane has been dried with a glycerol solution; and (b) removing a therapeutically effective amount of

immunoglobulins from the dialysis fluid or body fluids. In an embodiment of this method, the cross-linked protein crystal includes a protein selected from the group consisting of ZDPA, Protein G, Protein L and combinations thereof.

[00072] Disclosed herein are compositions including crystalline form of ZDPA. In some embodiments the ZDPA is produced by recombinant means or is chemically synthesized. In some embodiments the ZDPA has a full length native protein sequence. In still other embodiments, the ZDPA contains at least one antibody binding domain. In yet other

embodiments, the ZDPA is chemically modified. In other embodiments the ZDPA has a mutation or is a functional derivative of a native ZDPA.

[00073] Disclosed herein are compositions including cross-linked crystalline form of

ZDPA. In some embodiments the composition is cross-linked with glutaraldehyde.

[00074] Disclosed herein is a method of purifying an immunoglobulin including the step of binding said immunoglobulin to a composition including crystalline form of ZDPA and/or cross-linked crystalline form of ZDPA. In some embodiments the immunoglobulin is an antibody, a monoclonal antibody, a Fab fragment, a Fc fragment, a single chain antibody, a chimeric antibody, a fully human antibody, or a humanized antibody. In other embodiments the immunoglobulin belongs to class IgG. In still other embodiments of this method the

immunoglobulin is chemically modified.

[00075] Disclosed herein is a method of making crystalline form of ZDPA using the batch crystallization method including the steps of: (a) placing ZDPA in deionized water at a 1 : 1 protein reagent ratio, wherein said reagent is selected from the group consisting of a composition containing 4 M ammonium sulfate, 0.1 M citrate buffer pH 5.0. In some embodiments of this method the ZDPA in step (a) is at concentration of about 5 mg/ml or about 300 mg/ml ZDPA in deionized water. In still other embodiments of this method, the ZDPA in step (a) is at concentration of about 50 mg/ml or about 120 mg/ml ZDPA in deionized water.

[00076] Disclosed herein is a method of making cross-linked crystalline form of ZDPA comprising mixing crystalline form of ZDPA with glutaraldehyde. In some embodiments of this method, the glutaraldehyde is at a final concentration of about 0.2 to about 4%. In other embodiments of this method the glutaraldehyde is at a final concentration of about 2%.

[00077] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the case of conflict, the present specification, including definitions, controls. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[00078] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below.

DESCRIPTION OF THE DRAWINGS

[00079] Figure 1. ZDPA crystals in batch. Recombinant ZDPA (10 mg/ml in H20) was crystallized in 1 ml batch with Wizard II crystallization screen.

[00080] Figure 2. Cross-linked ZDPA crystals. ZDPA crystals were cross-linked with glutaraldehyde 2% for 3 hours at room temperature.

DETAILED DESCRIPTION

[00081] The present invention relates to compositions comprising crystalline ZDPA and cross-linked ZDPA, and methods of making and using such crystalline compositions for separating an immunoglobulin/ antibodies or an Fc-fragment, Fab fragment, single-chain antibodies and monoclonal antibodies from microorganisms, serum, plasma, mammalian cell culture. The crystalline compositions described herein have the ability of binding at least one immunoglobulin through the Fc-portion of the latter, from a liquid containing the

immunoglobulins in a mixture of other substances.

[00082] Without wishing to be bound by any particular theory, the invention is characterized in that the liquid containing said immunoglobulin/antibodies is contacted with a solid phase consisting of crystalline cross-linked ZDPA substance which is insoluble in said liquid and which has at least one or more immunoglobulin or its Fc-fragment binding regions thereto. The Fc-part of the immunoglobulin or the Fc-fragment is able to bind the ZDPA-CLPC so that said ZDPA-CLPC is bound to the Fc-part of said immunoglobulin/antibody or to the Fc- fragment but not the contaminating substances in the liquid, whereupon the liquid with the remaining contaminants is separated from the solid phase and the bound antibodies is optionally also separated from said solid phase.

[00083] The method of the present invention distinguishes from known methods used for the same purpose in that it avoids complicated multistage operations using, inter alia, ion exchange chromatography and also additional solid support such as agarose etc. By means of the new method, the immunoglobulin/antibody/fragment in question is obtained in surprisingly pure form in a very simple manner under convenient conditions and with a high yield. The ZDPA- CLPC in question is extremely valuable since it is able to reversibly bind immunoglobulins specifically with a very high binding capacity when compared to conventional immobilized ZDPA because the bond is effected at the Fc-part of the immunoglobulin.

[00084] The immunoglobulin in question can derive from different animal species, primarily vertebrates, preferably mammals. As is known, immunoglobulins may belong to different immunoglobulin classes, such as class A (IgA), D (IgD), E (IgE), G (IgG) and M (IgM). One valuable aspect of the invention is that it can be applied in connection with ZDPA- CLPC capable of binding themselves to immunoglobulins belonging to the IgG class, since this class, inter alia, quantitatively dominates among the immunoglobulins. The Fc-part of immunoglobulins can be split off therefrom by known enzymatic methods, whereby free Fc- fragments are obtained. The Fc-part of a specific immunoglobulin is often structurally similar in different animal species. Since, for example, ZDPA from S. aureus reacts with the Fc-part of IgG, the method of the present invention often enables IgG from different animal species to replace one another.

[00085] In accordance with the invention, there should be used when practicing the method a ZDPA-CLPC (polymeric substance) insoluble in the liquid, whereby the polymeric substance has at least one immunoglobulin or its Fc-fragment binding region thereto. The ZDPA-CLPC can thus not be dissolved out of the solid phase or removed there from during washing operations. Preferably, the ZDPA-CLPC is formed by cross-linking of ZDPA crystals by means of bonds of a covalent nature.

[00086] The ZDPA-CLPC can be used in a column consisting of cross-linked ZDPA in crystalline particulate form which is pre-packed in columns that provide for easy affinity purification of polyclonal antibodies, monoclonal antibodies, Fab fragments from various sources. The crystalline cross-linked ZDPA is prepared using a cross-linking method that results in excellent protein stability and binding characteristics. The columns are intended for traditional gravity- flow procedures. [00087] Alternatively, the ZDPA-CLPC can be bound to the polymer (solid surface) or magnetic particles by means of methods conventionally used when binding polypeptides, e.g., proteins, to polymeric substances, e.g. by means of cyanogen halide, isocyanates etc. The insoluble polymeric substances used may be such as are generally available for similar purposes, i.e., polymers with functional groups which can be used when binding proteins to polymers. Examples of such functional groups are hydroxyl groups, mercapto groups, primary and secondary amino groups, carbonyl groups, hydrazide groups, diazo groups and carboxyl groups. These groups can be used when forming bridges by conventional methods from the polymer to a protein, which in this case is the ZDPA-CLPC. The polymer, which is insoluble in the liquid used, may, however, swell in said liquid. For example, it may swell in water when an aqueous liquid is used. The polymer may consist of a three-dimensional network obtained, for example, by cross-linking a polymer such as a polysaccharide. Thus, very different polymers can be used, for example, cellulose, agarose, polyaminostyrene, cross-linked polymers (for example cross- linked polysaccharides such as dextran cross-linked with epichlorohydrin (Sephadex® ) or with diepoxides (for example with 1,4-butanediol diglycide ether)) or starch or cellulose derivatives or polyvinyl alcohol cross-linked with epichlorohydrin or diepoxides. Other examples are insoluble polymers obtained by reacting tetraethylenepentamine or hexamethylenediamine with epichlorohydrin or diepoxides. Another example is cross-linked polyacrylamide polymer substituted by p-aminophenyl groups (Enzacryl®).

[00088] The solid phase may exist in crystalline cross-linked ZDPA or embedded in polymeric substance or membrane. In many instances it may be suitable to use the ZDPA in particulate form. Other examples include a polymeric test tube wall to which is bound a ZDPA- CLPC to purify immunoglobulin or its Fc-fragment, e.g., IgG or its Fc-fragment.

[00089] The substances from which the relevant immunoglobulin/antibody/fragments is separated by the method described herein may be of widely differing character. Thus, such immunoglobulins may be purified away from polypeptides (e.g., proteins), polysaccharides, nucleic acids or low molecular weight substances from the microorganisms from which the relevant antibody was produced.

[00090] The method of the present invention is carried out in the presence of a liquid. The liquid used is primarily an aqueous liquid, e.g., an aqueous buffered NaCl solution having a suitable pH, e.g., in the proximity of the neutral point.

[00091] The relevant antibodies/fragments thereof bound to the ZDPA-CLPC in accordance with the invention may readily be released from the CLPC under mild conditions, e.g., by changing the pH or ion strength. [00092] Crystals of ZDPA (ZDPA -CLPC) as described herein can bind antibodies or its

Fab fragments and can be used in purification or in immunology or immunoprecipitation studies from various sources without the need for any solid support. Crystals of ZDPA (ZDPA -CLPC) can also reduce immune circulating complexes or IgG from plasma by immunoadsorption in an extracorporeal device in a mammal. Methods of using ZDPA/CLPC crystals to purify antibodies are described herein. Additionally, ZDPA crystals and cross-linked crystals (CLPCs) are provided, as are compositions comprising and using the same. Additionally disclosed are methods of producing large quantities of protein crystals/CLPCs from soluble form of ZDPA.

[00093] Definitions. In order that the present invention may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[00094] As used herein "Whole Antibody or Antibody Fragment" means a whole antibody or antibody fragment, e.g., a single-chain Fv fragment or Fab antibody fragment, according to this invention, is a functional antibody or antibody fragment, i.e., that is able to recognize and bind to its specific antigen in vitro or in vivo, and may initiate any subsequent actions associated with antibody-binding, e.g., Direct Cytotoxicity, Complement-Dependent Cytotoxicity (CDC), Antibody-Dependent Cytotoxicity (ADCC).

[00095] The term "Antibody" means a glycoprotein of approximate MW 150 kD, that is produced by the humoral arm of the immune system of vertebrates in response to the presence of foreign molecules in the body. Antibodies are essential for the prevention and resolution of infection by microorganisms, e.g. parasites, bacteria and viruses. Antibodies perform this function by recognizing and binding, in a highly specific manner, protein (or, sometimes, other organic molecules including polysaccharides, glycoproteins, lipids, or nucleic acids)

configurations called antigens (or epitopes), including those on invading microorganisms and their products. Antibodies bind their target antigens through highly specific interactions between hypervariable domains, called antigen-binding sites, on the antibody, and the epitope itself. Upon binding to the antigen, antibodies activate one or more of the many effector systems of the immune system that contribute to the neutralization, destruction and elimination of the infecting microorganism, or other antigen-containing entity, e.g. cancer cell.

[00096] Antibodies are also used for the treatment of cancer, inflammation,

cardiovascular disease, and transplant rejection, by virtue of their specific binding and subsequent neutralization of the cellular targets, which are involved in disease states. For example, monoclonal antibody Infliximab binds to tumor necrosis factor and neutralizes its role in inflammation by blocking its interaction with cell surface receptor; while Rituximab targets malignant B lymphocytes by binding to their cell surface CD20 antigen.

[00097] A single antibody molecule has a structure composed of two identical heavy chains (each of approximate MW 50 kD) covalently bound to each other, and two identical light chains (each of approximate MW 25 kD), each covalently bound to one of the heavy chains. The four chains are arranged in a classic "Y" motif. The bottom "leg" of the "Y" is called the Fc region ("c" stands for "crystallizable" or, alternatively, "complement-binding") and is used to anchor the antibody within cell membranes, and also to bind macrophage cells and activate complement. The two "arms" at the top of the "Y" are called Fab regions (the "ab" stands for "antigen-binding"). Each Fab region contains a constant region (at the juncture of the Fab and the Fc regions) and a variable region (which extends to the tip of the "Y"). Each variable region contains identical antigen-binding sites (at regions within the variable regions called

"hypervariable" regions) at each tip of the "Y". Thus, each Fab region has one antigen-binding site, and the complete antibody molecule therefore has two antigen-binding sites (i.e., is "bivalent"). The two antigen-binding sites on a naturally occurring antibody are identical to each other, and therefore the antibody is specific for one antigen (i.e., is "monovalent").

[00098] A number of molecular fragments of antibody molecules have been isolated to date. These do not occur naturally, but are engineered from one or more complete antibody molecules. These fragments include Fab fragments (a single Fab that is isolated from a complete antibody by digestion with the enzyme papain), and F(ab')2 fragments (two Fabs covalently-bound to each other, produced by digesting the antibody with the enzyme pepsin). Fab fragments are monospecific, while F(ab')2 fragments are bispecific. Recently, a number of engineered antibody fragments have been introduced. These include double-stranded Fv (dsFv) fragments and single-chain Fv (scFv) fragments (the "v" stands for "variable" in both cases). A dsFv fragment consists of an Fab fragment minus the constant regions, i.e., consisting only of the variable regions of a heavy and light chain covalently bound to each other. A scFv fragment is a single polypeptide chain, consisting of the variable region of a heavy chain linked via a peptide linker to the variable region of a light chain. Classically, both dsFv and scFv fragments are monovalent (and thus mono-specific). However, two dsFv fragments or two scFv fragments can themselves be linked to form a bispecific fragment (which would be analogous to an F(ab')2 fragment without the constant regions). Furthermore, it is possible to link two dsFv fragments or scFv fragments with different antigen-binding sites (i.e., different specificities), to form a bispecific fragment. Such fragments may be used as either research tools or therapeutic or diagnostic reagents. [00099] There are five classes of antibodies (also called immunoglobulins) in humans:

IgG, IgM, IgA, IgD, and IgE, each with its own unique characteristics and function. IgG, IgD, and IgE are all made up of one antibody molecule, while IgA can be made up of one, two or three such molecules and IgM consists of five. Furthermore, in humans, there are four subclasses of IgG (IgGl, IgG2, IgG3, or IgG4), and two subclasses each of IgM and IgA (1 and 2, respectively). For example, the monoclonal antibody Rituximab (Rituxan™) is an IgGl antibody.

[000100] Though naturally occurring antibodies are derived from a single species, engineered antibodies and antibody fragments may be derived from more than one species of animal, i.e., may be chimeric. To date, mouse (murine)/human chimeric antibodies have been generated, though other species' combinations are possible. Chimeric antibodies have been further broken down into two subtypes: chimeric and humanized. Chimeric murine/human antibodies contain approximately 75% human and 25% mouse amino acid sequences, respectively. The human sequences represent the constant regions of the antibody while the mouse sequences represent the variable regions (and thus contain the antigen-binding sites) of the antibody. The rationale for using such chimeras is to retain the antigen specificity of the mouse antibody but reduce the immunogenicity of the mouse antibody (a murine antibody would cause an immune response against it in species other than the mouse) and thus be able to employ the chimera in human therapies. Chimeric antibodies also include those which comprise CDR regions from different human antibodies. CDR regions, also called hypervariable regions, are sequences within the variable regions of antibody molecules that generate the antigen- binding sites. CDR regions are so-named because the binding site is complementary in shape and charge distribution to the epitope recognized on the antigen.

[000101] Alternatively, chimeric antibodies comprise framework regions from one antibody and CDR regions from another antibody. Chimeric antibodies also include those which comprise CDR regions from at least two different human antibodies. Humanized antibodies contain approximately 90% (or more) human amino acid sequences. The only murine sequences present are those for the hypervariable region (that are the actual antigen- binding sites contained within the variable region). Humanized antibodies have minimal mouse immunogenicity as compared with chimeric antibodies.

[000102] There are generally two types of antibodies that can be distinguished by their specificities: polyclonal antibodies and monoclonal antibodies. Polyclonal antibodies are those that are found as the immunoglobulin fraction of blood, and are essentially a polyclonal mixture of many different types of antibodies specific for the different antigens the individual has been exposed to (i.e., they originate from many different clones of B lymphocytes (or B cells), the cell that produces antibodies).

[000103] Monoclonal antibodies are antibodies of a single specificity, i.e., that are derived from a single clone of B lymphocytes (B cells). These antibodies have exquisite specificity for their target antigens and also can be produced in high amounts (i.e., high titres). They are useful as markers for specific antigens (e.g., cancer antigens), as diagnostic agents (e.g., in assays to detect viruses like HIV-1), and as therapeutic agents. Whole monoclonal antibodies are those that have a classic molecular structure that includes two complete heavy chains and two complete light chains. This is distinguished from antibody fragments, such as Fab, F(ab')2, Fc fragments, dsFv fragments, and scFv fragments.

[000104] Traditionally, monoclonal antibodies have been produced by fusing the antibody- producing B cell with an immortal hybridoma cell to generate B cell hybridomas, which continually produce monoclonal antibodies in cell culture. Another method that is traditionally used to generate monoclonal antibodies involves the expression of the monoclonal antibodies in bacterial cell culture using phage-display technology. Currently, however, monoclonal antibodies may be produced in vivo in large quantities in genetically-modified animals, such as cows and goats (Genzyme Transgenics), pigs and rabbits (Medarex, PPL Therapeutics), and chickens (Tranxenogen), and in plants, such as tobacco and corn (Epicyte, Integrated Protein Technologies, Meristem Croptech, and others). For example, large amounts of monoclonal antibodies can be found in the milk of genetically-modified goats (Genzyme Transgenics). ZDPA-CLPC can be used to purify antibodies from all such sources according to this invention. Furthermore, as a result of transgenics, mice have been modified to contain and express the entire human B cell genome (which encodes human antibodies). Therefore, such transgenic mice (Abgenix) are a source of human antibodies can be purified using ZDPA-CLPC according to this invention. It should be noted that glycosylation is specific to the animal that is producing the antibodies. For example, human antibodies from sources other than humans will have subtly different glycosylation profiles. Therefore, the whole antibodies or single-chain Fv antibody fragments or Fab antibody fragments of this invention may display modified glycosylation or be deglycosylated. Antibodies which can be purified using ZDPA-CLPC according to this invention also include derivatized antibodies. Such antibodies include those derivatized with polyethylene glycol or at least one carbohydrate moiety or least one methyl or ethyl group.

[000105] Clinically relevant antibodies may also be classified according to the therapeutic area in which they are to be employed. Such antibodies include, for example, those for treating cancers (e.g., pancreatic cancer), inflammatory diseases (e.g., autoimmune diseases, arthritis), cardiovascular diseases (e.g., strokes), infectious disease (e.g., HIV/AIDS), respiratory diseases (e.g., asthma), tissue transplantation rejection and organ transplantation rejection. Such antibodies also include antibodies for radioimmunotherapy. Antibodies which may be purified according to the present invention include, for example, Abciximab, Palivizumab, Murumonab- CD3, Gemtuzumab, Trastuzumab, Basiliximab, Daclizumab, Etanercept and Ibritumomab tiuxetan.

[000106] As used herein "Aqueous-organic solvent mixture" - means a mixture comprising n% organic solvent, where n is between 1 and 99 and m% aqueous, where m is 100- n.

[000107] As used herein "Biocompatible polymers" means polymers that are non- antigenic (when not used as an adjuvant), non-carcinogenic, non-toxic and which are not otherwise inherently incompatible with living organisms. Examples include: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters) such as poly (lactic acid) or PLA, poly (lactic-co-glycolic acid) or PLGA, poly (β- hydroxybutryate), poly (caprolactone) and poly (dioxanone); poly (ethylene glycol), poly ((hydroxypropyl)methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof.

[000108] As used herein "Biodegradable polymers" means polymers that degrade by hydrolysis or solubilization. Degradation can be heterogenous— occurring primarily at the particle surface, or homogenous— degrading evenly throughout the polymer matrix.

[000109] As used herein, a "biological sample" is biological material collected from cells, tissues, organs, or organisms, for example, to detect an analyte. Exemplary biological samples include a fluid, cell, or tissue sample. Biological fluids include, for example, serum, blood, plasma, saliva, urine, or sweat. Cell or tissue samples include biopsy, tissue, cell suspension, or other specimens and samples, such as clinical samples.

[000110] As used herein, a Co-polymer means apolymer made with more than one monomer species.

[000111] As used herein, an Emulsifier is a surface active agent which reduces interfacial tension between polymer coated crystals and a solution.

[000112] A "crystal" is one form of the solid state of matter, comprising atoms arranged in a pattern that repeats periodically in three dimensions (see, e.g., Barret, Structure of Metals, 2nd ed., McGraw-Hill, New York (1952)). A crystal form of a polypeptide, for example, is distinct from a second form— the amorphous solid state. Crystals display characteristic features including shape, lattice structure, percent solvent, and optical properties, such as, e.g., refractive index.

[000113] As used herein a "Cross-linked crystal form of protein have the property that the cross-linked protein crystals remain insoluble and in the solid state when added to solution.

[000114] An "extracorporeal device" is a structure that is not within the body for bringing a body fluid in contact with ZDPA crystals in the treatment of an individual. Preferably, an extracorporeal device is a device used for dialysis, including kidney dialysis, a device for continuous arteriovenous hemofiltration, an extracorporeal membrane oxygenator, or other device used to filter waste products from the bloodstream. Similarly, components of devices to filter waste products are encompassed by the term, including a tube, a porous material, or a membrane, for example. In particular, an extracorporeal device may be a dialysis device. It may also be a membrane of a dialysis device.

[000115] A "functional fragment" of ZDPA is a portion of a ZDPA polypeptide that retains one or more binding activities towards antibodies or its Fab fragments of ZDPA, such as the ability to be used in purification of antibodies. As used herein, a functional fragment may comprise terminal truncations from one or both termini, unless otherwise specified. For example, a functional fragment may have 1, 2, 4, 5, 6, 8, 10, 12, 15, or 20 or more residues omitted from the amino and/or carboxyl terminus of a ZDPA polypeptide. Preferably, the truncations are not more than 20 amino acids from one or both termini. A functional fragment may optionally be linked to one or more heterologous sequences or mutations to any amino acids in the original native sequence of ZDPA.

[000116] The term "individual" or "subject" refers to any mammal, including but not limited to, any animal classified as such, including humans, non human primates, primates, baboons, chimpanzees, monkeys, rodents (e.g., mice, rats), rabbits, cats, dogs, horses, cows, sheep, goats, pigs, etc.

[000117] Insoluble and stable form of a protein. A form of a protein which is insoluble in aqueous solvents, organic solvents or aqueous-organic solvent mixtures and which displays greater stability than the soluble form of the protein. According to an alternate embodiment of this invention, the phrase "insoluble and stable form of a protein" may be a protein which is insoluble in dry and wet formulations. In any embodiment, the cross-linked protein crystals may be active in insoluble form. [000118] The term "isolated" refers to a molecule that is substantially free of its natural environment. For instance, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term refers to preparations where the isolated protein is sufficiently pure to be administered as a therapeutic composition, or at least 70% to 80% (w/w) pure, more preferably, at least 80%> 90%> (w/w) pure, even more preferably, 90 to 95% pure; and, most preferably, at least 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% (w/w) pure.

[000119] As used herein, the term "about" refers to up to + 10%> of the value qualified by this term. For example, about 50 mM refers to 50 mM + 5mM; about 4% refers to 4% + 0.4%.

[000120] As used herein, "macromolecular substrate" means a large biomolecule, such as a protein or a carbohydrate having a molecular weight of at least 600-700 Daltons, which is also a substrate for a reaction catalyzed by the protein constituent of a cross-linked protein crystal.

[000121] The term "Organic solvents" means any solvent of non-aqueous origin.

[000122] The protein constituents of the cross-linked protein crystal formulations of this invention may be naturally or synthetically modified. They may be glycoproteins, unmodified proteins or contain other modifications.

[000123] As used herein, "Protein activity", means an activity selected from the group consisting of binding, catalysis, or activities which generate a functional response within the environment in which the protein is used, such as the binding of immunoglobulins,

immunoprecipitation, or combinations thereof.

[000124] The term "soluble form of protein" means individual protein molecules in solution and dissociated from a crystal lattice.

[000125] The terms "therapeutically effective dose," or "therapeutically effective amount," refer to that amount of a compound that results in prevention, delay of onset of symptoms, or amelioration of symptoms of an immune-related condition, such as adult thrombocytopenic purpura. A therapeutically effective amount will, for example, be sufficient to treat, prevent, reduce the severity, delay the onset, and/or reduce the risk of occurrence of one or more symptoms of a disorder associated with elevated immunoglobulins/IgGs or its subclasses concentrations. The effective amount can be determined by methods well known in the art.

[000126] The terms "treatment," "therapeutic method," and their cognates refer to treatment of an existing disorder and/or prophylactic/preventative measures. Those in need of treatment may include individuals already having a particular medical disorder, as well as those at risk or having, or who may ultimately acquire the disorder. The need for treatment is assessed, for example, by the presence of one or more risk factors associated with the development of a disorder, the presence or progression of a disorder, or likely receptiveness to treatment of a subject having the disorder. Treatment may include slowing or reversing the progression of a disorder.

[000127] The term "Polymer" means a large molecule built up by the repetition of small, simple chemical units. The repeating units may be linear or branched to form interconnected networks. The repeat unit is usually equivalent or nearly equivalent to the monomer.

[000128] Polymeric carriers as used herein mean polymers used for encapsulation of ZDPA-CLPC for purification or for delivery of such ZDPA-CLPC, including biological delivery. Such polymers include biocompatible and biodegradable polymers. The polymeric carrier may be a single polymer type or it may be composed of a mixture of polymer types. Polymers useful as the polymeric carrier, include for example, poly (acrylic acid), poly

(cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters) such as poly (lactic acid) or PLA, poly (lactic-co-glycolic acid) or PLGA, poly (β-hydroxybutryate), poly (caprolactone) and poly (dioxanone); poly (ethylene glycol), poly

((hydroxypropyl)methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, natural and synthetic polypeptides, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, modified starches such as amylose starch, amylopectin starch, hydroxyethyl starch, methacrylate starch, and other starches, and any conventional material that will encapsulate protein crystals.

[000129] As used herein, ZDPA refers to a polypeptide from Staphylococcus aureus strains. ZDPA are a group of polypeptides known in the art capable of binding immunoglobulin or its Fc-fragments. ZDPA is a cell wall component produced by several strains of

Staphylococcus aureus that consists of a single polypeptide chain of molecular weight 42,000 and contains little or no carbohydrate. It consists of six regions, five of which bind IgG. ZDPA binds specifically to the Fc region of immunoglobulin molecules, especially IgG. The ZDPA molecule contains four high affinity (Ka = 108/M) binding sites capable of interacting with the Fc region of several species. The molecule is heat-stable and retains its native conformation when exposed to denaturing reagents such as 4 M urea, 4 M thiocyanate and 6 M guanidine hydrochloride.

[000130] ZDPA has been used extensively for the isolation of IgG from several species of mammals. However, the interaction between ZDPA and IgG is not equivalent for all species. Even within a species, ZDPA interacts with some subgroups of IgG and not others. For instance, human IgGi, IgG₂ and IgG₄ bind strongly, whereas IgG₃ does not bind and mouse IgGi binds poorly to ZDPA. There are also many instances in which monoclonal antibodies do not bind to ZDPA such as the majority of rat immunoglobulins. Despite its variable binding characteristics, ZDPA possesses IgG binding properties that make it ideal for affinity purification of IgG. One molecule of immobilized protein A can bind at least two molecules of IgG.

[000131] Another protein, Protein G, a cell surface protein from Group G Streptococci, is a type III Fc receptor and binds IgG with a non-immune mechanism similar to that of protein A. A recombinant form of the protein produced in E. coli, from which the albumin-binding region of the native protein has been genetically deleted, can also be used for purification of IgGs. Recombinant protein G contains two Fc-binding regions.

[000132] Another antibody binding protein, Protein L, is a cell surface protein of bacterial species Peptostreptoccus magnus and is found to bind Ig through L chain interaction. It consists of 719 amino acid residues. The molecular weight of Protein L purified from the cell walls of Peptostreptoccus magnus was first estimated as 95kD by SDS-PAGE in the presence of reducing agent 2-mercaptoethanol, while the molecular weight was determined to be 76kD by gel chromotography in the presence of 6 M guanidine HCl. Unlike ZDPA and Protein G, which bind to the Fc region of immunoglobulins (antibodies), Protein L binds antibodies through light chain interactions. Since no part of the heavy chain is involved in the binding interaction, Protein L binds a wider range of antibody classes than ZDPA or G. Protein L binds to representatives of all antibody classes, including IgG, IgM, IgA, IgE and IgD. Single chain variable fragments (ScFv) and Fab fragments also bind to Protein L. Protein L binding is restricted to those antibodies that contain kappa light chains. Given the specific requirements for effective binding, the main application for immobilized Protein L is purification of monoclonal antibodies from ascites or cell culture supernatant that are known to have the kappa light chain.

[000133] The present invention has applicability to isoforms of ZDPA, and glycoforms of those isoforms.

[000134] ZDPA is used to prepare the crystals which are used in methods described herein. The crystals provided herein may be formed from polypeptides comprising amino acid sequences of ZDPA, or a functional fragment of ZDPA that retains antibody binding region(s). Preferably, the ZDPA retains at least one functional binding characteristic of a naturally occurring ZDPA, e.g., retains one or more of the ability to bind at least one antibody.

[000135] Alternatively, recombinant ZDPA may be used to form the crystals and methods provided herein. In some instances, recombinant ZDPA encompass or are encoded by sequences from a naturally occurring ZDPA sequence. Further, ZDPA comprising an amino acid sequence that is homologous or substantially identical to a naturally occurring sequence are herein described. Also, ZDPA encoded by a nucleic acid that is homologous or substantially identical to a naturally occurring ZDPA-encoding nucleic acid are provided and may be crystallized and/or administered as described herein.

[000136] Polypeptides referred to herein as "recombinant" are polypeptides which have been produced by recombinant DNA methodology, including those that are generated by procedures which rely upon a method of artificial recombination, such as the polymerase chain reaction (PCR) and/or cloning into a vector using restriction enzymes. "Recombinant" polypeptides are also polypeptides having altered expression, such as a naturally occurring polypeptide with recombinantly modified expression in a cell, such as a host cell.

[000137] ZDPA polypeptides useful for forming ZDPA crystals may be expressed in a host cell, such as a host cell comprising a nucleic acid construct that includes a coding sequence for a ZDPA polypeptide or a functional fragment thereof. A suitable host cell for expression of ZDPA may be yeast, bacteria, fungus, insect, plant, or mammalian cell, for example, or transgenic plants, transgenic animals or a cell-free system. Preferably, a host cell is capable of glycosylating the ZDPA polypeptide if necessary, capable of disulfide linkages, capable of secreting the ZDPA, and/or capable of supporting multimerization of ZDPA polypeptides.

Preferred host cells include, but are not limited to E. coli (including E. coli Origami B and E. coli BL21), Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Bacillus subtilis, Aspergillus, Sf9 cells, Chinese hamster ovary (CHO), 293 cells (human embryonic kidney), and other human cells. Also transgenic plants, transgenic animals including pig, cow, goat, horse, chicken, and rabbit are suitable hosts for production of ZDPA.

[000138] For recombinant production of ZDPA, a host or host cell should comprise a construct in the form of a plasmid, vector, phagemid, or transcription or expression cassette that comprises at least one nucleic acid encoding an ZDPA or a functional fragment thereof. A variety of constructs are available, including constructs which are maintained in single copy or multiple copy, or which become integrated into the host cell chromosome. Many recombinant expression systems, components, and reagents for recombinant expression are commercially available, for example from Invitrogen Corporation (Carlsbad, CA); U.S. Biological

(Swampscott, MA); BD Biosciences Pharmingen (San Diego, CA); Novagen (Madison, WI); Stratagene (La Jolla, CA); and Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), (Braunschweig, Germany).

[000139] Recombinant expression of ZDPA is optionally controlled by a heterologous promoter, including a constitutive and/or inducible promoter. Promoters such as, e.g., T7, the alcohol oxidase (AOX) promoter, the dihydroxy-acetone synthase (DAS) promoters, the Gal 1,10 promoter, the phosphoglycerate kinase promoter, the glyceraldehyde-3 -phosphate dehydrogenase promoter, alcohol dehydrogenase promoter, copper metallothionein (CUP1) promoter, acid phosphatase promoter, CMV and promoters polyhedrin are also appropriate. The particular promoter is selected based on the host or host cell. In addition, promoters that are inducible by methanol, copper sulfate, galactose, by low phosphate, by alcohol, e.g., ethanol, for example, may also be used and are well known in the art.

[000140] A nucleic acid that encodes ZDPA may optionally comprise heterologous sequences. For example, a secretion sequence is included at the N-terminus of an ZDPA polypeptide in some embodiments. Signal sequences such as those from a Mating Factor, BGL2, yeast acid phosphatase (PHO), xylanase, alpha amylase, from other yeast secreted proteins, and secretion signal peptides derived from other species that are capable of directing secretion from the host cell may be useful. Similarly other heterologous sequences such as linkers (e.g., comprising a cleavage or restriction endonuclease site) and one or more expression control elements, an enhancer, a terminator, a leader sequence, and one or more translation signals are within the scope of this description. These sequences may optionally be included in a construct and/or linked to the nucleic acid that encodes ZDPA. Unless otherwise specified, "linked" sequences can be directly or indirectly associated with one another.

[000141] Similarly, an epitope or affinity tag such as Histidine, HA (hemagglutinin peptide), maltose binding protein, AviTag®, FLAG, or glutathione-S-transferase may be optionally linked to the ZDPA polypeptide. A tag may be optionally cleavable from the ZDPA after it is produced or purified. A skilled artisan can readily select appropriate heterologous sequences, for example, match host cell, construct, promoter, and/or secretion signal sequence.

[000142] ZDPA homologs or variants differ from a ZDPA reference sequence by one or more residues. Structurally similar amino acids can be substituted for some of the specified amino acids, for example. Structurally similar amino acids include: (I, L and V); (F and Y); (K and R); (Q and N); (D and E); and (G and A). Deletion, addition, or substitution of amino acids is also encompassed by the ZDPA homologs described herein. Such homologs and variants include (i) polymorphic variants and natural or artificial mutants, (ii) modified polypeptides in which one or more residues is modified, and (iii) mutants comprising one or more modified residues.

[000143] A ZDPA polypeptide or nucleic acid is "homologous" (or is a "homolog") if it is at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a reference sequence. If the homolog is not identical to the reference sequence, it is a "variant." A homolog is "substantially identical" to a reference ZDPA sequence if the nucleotide or amino acid sequence of the homolog differs from the reference sequence (e.g., by truncation, deletion, substitution, or addition) by no more than about 1, 2, 3, 4, 5, 8, 10, 20, 50 or more residues, and retains (or encodes a polypeptide that retains) the ability to bind to immunoglobulins/antibodies or its Fab fragments. Fragments of a ZDPA may be homologs, including variants and/or substantially identical sequences. By way of example, homologs may be derived from various sources of ZDPA, or they may be derived from or related to a reference sequence by truncation, deletion, substitution, or addition mutation. Percent identity between two nucleotide or amino acid sequences may be determined by standard alignment algorithms such as, for example, Basic Local Alignment Tool (BLAST) described in Altschul et al., J. Mol. Biol, 215:403 410 (1990), the algorithm of Needleman et al, J. Mol. Biol, 48:444 453 (1970), or the algorithm of Meyers et al., Comput. Appl. Biosci. 4: 11 17 (1988). Such algorithms are incorporated into the BLASTN, BLASTP, and "BLAST 2 Sequences" programs (reviewed in McGinnis and Madden, Nucleic Acids Res. 32:W20-W25, 2004). When utilizing such programs, the default parameters can be used. For example, for nucleotide sequences the following settings can be used for "BLAST 2 Sequences": program BLASTN, reward for match 2, penalty for mismatch 2, open gap and extension gap penalties 5 and 2 respectively, gap x dropoff 50, expect 10, word size 11, filter ON. For amino acid sequences the following settings can be used for "BLAST 2 Sequences": program BLASTP, matrix BLOSUM62, open gap and extension gap penalties 11 and 1 respectively, gap x dropoff 50, expect 10, word size 3, filter ON. The amino acid and nucleic acid sequences for ZDPA that are appropriate to form the crystals described herein, may include homologous, variant, or substantially identical sequences.

Preparation of Cross-linked Protein Crystals - Protein Crystallization

[000144] Protein crystals are grown by controlled crystallization of protein out of aqueous solution or aqueous solution-containing organic solvents. Conditions to be controlled include, for example, the rate of evaporation of solvent, the presence of appropriate co-solutes and buffers, pH and temperature. A comprehensive review of the various factors affecting the crystallization of proteins has been published by McPherson, Methods EnzymoL, 114, pp. 112- 20 (1985). McPherson and Gilliland, J. Crystal Growth, 90, pp. 51-59 (1988) have compiled comprehensive lists of proteins and nucleic acids that have been crystallized, as well as the conditions under which they were crystallized. A compendium of crystals and crystallization recipes, as well as a repository of coordinates of solved protein and nucleic acid structures, is maintained by the Protein Data Bank at the Brookhaven National Laboratory [http//www. pdb.bnl.gov; Bernstein et al, J. Mol. Biol, 112, pp. 535-42 (1977)]. These references can be used to determine the conditions necessary for crystallization of a protein, as a prelude to the formation of an appropriate cross-linked protein crystal, and can guide the crystallization strategy for other proteins. Alternatively, an intelligent trial and error search strategy can, in most instances, produce suitable crystallization conditions for many proteins, provided that an acceptable level of purity can be achieved for them [see, e.g., C.W. Carter, Jr. and C.W. Carter, J. Biol. Chem., 254, pp. 12219-23 (1979)].

[000145] The large single crystals which are needed for X-ray diffraction analysis are not required for use in the methods described herein. Microcrystalline showers are suitable.

[000146] For example, the cross-linked protein crystals may have a longest dimension between about 0.01 μιη and about 500 μιη, alternatively, between 0.1 μιη and about 50 μιη. They may also have a shape selected from the group consisting of: spheres, needles, rods, plates, such as hexagons and squares, rhomboids, cubes, bipryamids and prisms.

[000147] In general, crystals are produced by combining the protein to be crystallized with an appropriate aqueous solvent or aqueous solvent containing appropriate crystallization agents, such as salts or organic solvents. The solvent is combined with the protein and may be subjected to agitation at a temperature determined experimentally to be appropriate for the induction of crystallization and acceptable for the maintenance of protein activity and stability. The solvent can optionally include co-solutes, such as divalent cations, co-factors or chaotropes, as well as buffer species to control pH. The need for co-solutes and their concentrations are determined experimentally to facilitate crystallization.

[000148] In an industrial-scale process, the controlled precipitation leading to

crystallization can best be carried out by the simple combination of protein, precipitant, co- solutes and, optionally, buffers in a batch process. As another option, proteins may be crystallized by using protein precipitates as the starting material. In this case, protein

precipitates are added to a crystallization solution and incubated until crystals form. Alternative laboratory crystallization methods, such as dialysis or vapor diffusion, can also be adopted. McPherson, supra and Gilliland, supra, include a comprehensive list of suitable conditions in their reviews of the crystallization literature.

[000149] Occasionally, incompatibility between the crosslinking agent and the

crystallization medium might require exchanging the crystals into a more suitable solvent system.

[000150] Many of the proteins for which crystallization conditions have already been described, may be used to prepare cross-linked protein crystals according to this invention. It should be noted, however, that the conditions reported in most of the above-cited references have been optimized to yield, in most instances, a few large, diffraction quality crystals.

Accordingly, it will be appreciated by those of skill in the art that some degree of adjustment of these conditions to provide a high yielding process for the large scale production of the smaller crystals used in making cross-linked protein crystals may be necessary.

Preparation of Cross-linked Protein Crystals - Crosslinking of Protein Crystals

[000151] Stabilized crystals. Once ZDPA crystals have been grown in a suitable medium they can be optionally stabilized, such as by cross-linking. Cross-linking results in stabilization of the crystal lattice by introducing covalent links between the constituent protein molecules of the crystal. This makes possible transfer of the protein into an alternate environment that might otherwise be incompatible with the existence of the crystal lattice or even with the existence of intact protein. ZDPA crystals may be cross-linked through, e.g., lysine amine groups, thiol (sulfhydryl) groups, and carbohydrate moieties. Cross-linked crystals are also referred to herein as "ZDPA-CLPC," "CLPC-ZDPA," or "CLPC."

[000152] A cross-linked crystal may alter the stability (e.g., pH, temperature, mechanical and/or chemical stability), the pH profile of antibody binding, the solubility, the uniformity of crystal size or volume, the rate of release of bound antibody from the crystal, and/or the pore size and shape between individual molecules in the underlying crystal lattice.

[000153] Advantageously, cross-linking or stabilizing according to the present invention is carried out in such a way that the crystals comprise a ZDPA that shows at least about 60%, 80%, 100%, 150%, 200%, 250%, 300% or more of the binding capacity/per mL of crystals (binding of antibodies) as compared to immobilized ZDPA per mL of the gel. Stability may be increased by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300% or more as compared to soluble or immobilized ZDPA. Stability can be measured under conditions of storage, such as pH stability, temperature stability, stability against proteases, dissolution stability, and as in vitro column stability, for example.

[000154] In some embodiments, cross-linking slows the dissolution of the ZDPA polypeptides in the crystal into solution, effectively immobilizing the protein molecules into microcrystalline particles. Upon exposure to a trigger in the environment surrounding the cross- linked protein crystals, such as under conditions of use rather than storage, the protein molecules slowly dissolve, releasing active ZDPA polypeptide and/or increasing ZDPA activity. The rate of dissolution is controlled, for example, by one or more of the following factors: the degree of cross-linking, the length of time of exposure of protein crystals to the cross-linking agent, the rate of addition of cross-linking agent to the protein crystals, the nature of the cross-linker, the chain length of the cross-linker, pH, temperature, presence of sulfahydryl reagents like cysteine, glutathione, the surface area of the cross-linked protein crystals, the size of the cross-linked protein crystals, and the shape of the cross-linked protein crystals.

[000155] Cross-linking can be achieved using one or a combination of a wide variety of cross-linking agents, including a multifunctional agent, at the same time (in parallel) or in sequence. Upon exposure to a trigger in the surrounding environment, or over a given period of time, the cross-links between protein crystals cross-linked with such multifunctional cross- linking agents lessen or weaken, leading to protein dissolution or release of activity.

Alternatively, the cross-links may break at the point of attachment, leading to protein dissolution or release of activity. See U.S. Patent Nos. 5,976,529 and 6,140,475.

[000156] In some embodiments, the cross-linking agent is a multifunctional cross-linking agent having at least 2, 3, 4, 5, or more active moieties. In various embodiments, the agent may be chosen from glutaraldehyde, succinaldehyde, octanedialdehyde, glyoxal,

dithiobis(succinimidylpropionate), 3,3' dithiobis(sulfosuccinimidylpropionate), dimethyl 3,3 '- dithiobispropionimidate · HC1, N-succinimidyl-3 -(2-pyridyldithio)propionate,

hexamethylenediamine, diaminooctane, ethylenediamine, succinic anhydride, phenylglutaric anhydride, salicylaldehyde, acetimidate, formalin, acrolein, succinic semialdehyde,

butyraldehyde, dodecylaldehyde, glyceraldehyde, and trans-oct-2-enal.

[000157] Additional multifunctional cross-linking agents include halo triazines, e.g., cyanuric chloride; halo-pyrimidines, e.g., 2,4,6-trichloro/bromo pyrimidine; anhydrides or halides of aliphatic or aromatic mono- or di-carboxylic acids, e.g., maleic anhydride,

(meth)acryloyl chloride, chloroacetyl chloride; N-methylol compounds, e.g., N-methylol chloro- acetamide; di-isocyanates or di-isothiocyanates, e.g., phenylene-l,4-di-isocyanate and aziridines. Other cross-linking agents include epoxides, such as, for example, di-epoxides, tri-epoxides and tetra-epoxides. In one embodiment, the cross-linking agent is glutaraldehyde, a bifunctional agent, and glutaraldehyde is used alone or in sequence with an epoxide. Other cross-linking reagents (see, for example, the 1996 catalog of the Pierce Chemical Company) may also be used, at the same time (in parallel) or in sequence with reversible cross-linking agents, such as those described below.

[000158] According to an alternate embodiment of this invention, cross-linking may be carried out using reversible cross-linking agents, in parallel or in sequence. The resulting cross- linked protein crystals are characterized by a reactive multi-functional linker, into which a trigger is incorporated as a separate group. The reactive functionality is involved in linking together reactive amino acid side chains in a protein and the trigger consists of a bond that can be broken by altering one or more conditions in the surrounding environment (e.g., pH, presence of reducing agent, temperature, or thermodynamic water activity).

[000159] The cross-linking agent may be homofunctional or heterofunctional. The reactive functionality (or moiety) may, e.g., be chosen from one of the following functional groups (where R, R', R", and R'" may be alkyl, aryl or hydrogen groups):

I. Reactive acyl donors, such as, e.g.: carboxylate esters RCOOR', amides RCONHR', Acyl azides RCON₃, carbodiimides R-N=C=N-R\ N hydroxyimide esters, RCC-C-NR', imidoesters R-C=NH2⁺ (OR'), anhydrides RCC-C-COR', carbonates RO-CO-O-R', urethanes RNHCONHR', acid halides RCOHal (where Hal=a halogen), acyl hydrazides RCO NR'R", and O acylisoureas

RCO-0-C=NR'(-NR"R'")

II. Reactive carbonyl groups, such as, e.g.: aldehydes RCHO and ketones RCOR', acetals RCO(H₂)R', and ketals RR'C02 R'R" (Reactive carbonyl containing functional groups known to those well skilled in the art of protein immobilization and cross-linking are described in the literature (Pierce Catalog and Handbook, Pierce Chemical Company, Rockford, 111. (1994); S. S. Wong, Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla. (1991));

III. Alkyl or aryl donors, such as, e.g.: alkyl or aryl halides R-Hal, azides R-N3, sulfate esters RSO3 R', phosphate esters RPO(OR'3), alkyloxonium salts R₃ 0+, sulfonium R₃ S+, nitrate esters RON0₂, Michael acceptors RCR'=CR"'COR", aryl fluorides ArF, isonitriles RN+=C- haloamines R₂ N-Hal, alkenes, and alkynes;

IV. Sulfur containing groups, such as, e.g.: disulfides RSSR', sulfhydryls RSH, and epoxides R₂ CACR' ₂; and

V. Salts, such as, e.g.: alkyl or aryl ammonium salts R₄ N+, carboxylate RCOO-, sulfate ROSO3-, phosphate ROPO3", and amines R₃ N.

[000160] Reversible cross-linking agents, for example, comprise a trigger. A trigger includes an alkyl, aryl, or other chain with an activating group that can react with the protein to be cross-linked. Those reactive groups can be any variety of groups such as those susceptible to nucleophilic, free radical or electrophilic displacement including halides, aldehydes, carbonates, urethanes, xanthanes, and epoxides among others. For example, reactive groups may be labile to acid, base, fluoride, enzyme, reduction, oxidation, thiol, metal, photolysis, radical, or heat.

[000161] Additional examples of reversible cross-linking agents are described in T. W. Green, Protective Groups in Organic Synthesis, John Wiley & Sons (Eds.) (1981). Any variety of strategies used for reversible protecting groups can be incorporated into a cross-linker suitable for producing cross-linked protein crystals capable of reversible, controlled solubilization.

Various approaches are listed, in Waldmann's review of this subject, in Angewante Chemie Inl. Ed. Engl, 35:2056 (1996).

[000162] Other types of reversible cross-linking agents are disulfide bond-containing cross-linkers. The trigger breaking cross-links formed by such cross-linking agents is the addition of reducing agent, such as cysteine, to the environment of the cross-linked protein crystals. Exemplary disulfide cross-linking agents are described in the Pierce Catalog and Handbook (1994-1995). Examples of such cross-linkers and methods are disclosed in U.S. Patent No. 6,541,606, relevant portions of which are incorporated herein by reference.

[000163] In addition, cross-linking agents which cross-link between carbohydrate moieties or between a carbohydrate moiety and an amino acid may also be used.

[000164] The concentration of the cross-linking agent may be from about 0.01% to 20%, about 0.02%) to 10%>, or about 0.05%> to 5% w/v in solution. Typically, the crosslinking agent is about 0.5% or about 1% w/v. For example, the concentration of the cross-linking agent may be, e.g., about 0.01%, 0.02%, 0.05%, 0.075%, 0.1%, 0.2%, 0.3%, 0.5%, 1%, 2%, 3%, 3.5%, 4%, 5%), 6%), 7%), 8%), 9%), 10%), 15%), or 20%> w/v in solution. It may be necessary to exchange buffers prior to cross-linking. Crystals, including CLPCs, may be optionally lyophilized or otherwise formulated.

[000165] The crystals, including the cross-linked crystals described herein are useful in the methods of treatment and methods to reduce IgG levels of circulating immune complexes such as in immune thrombocytopenic purpura. The ZDPA crystals are also useful in methods relating to purification processes (e.g., monoclonal antibody purification, polyclonal antibody purification, purification of immunoglobulins, IgGs and its subtypes, Fab fragments, single chain antibodies etc. from various sources either in column format or impregnated in a membrane or in polymers or coating or immobilized on a support. The crystals described herein can be applied to these uses, based on one or more properties of the stabilized ZDPA crystals described above,

[000166] Drying of Crystals/CLPCs of ZDPA. Crystals of ZDPA are dried by removal of water, organic solvent or liquid polymer by drying means including drying with N2, air or inert gases, vacuum oven drying, lyophilization, washing with a volatile organic solvent followed by evaporation of the solvent, evaporation in a fume hood, tray drying, fluid-bed drying, spray drying, vacuum drying, or roller drying. Typically, drying is achieved when the crystals become a free-flowing powder. Drying may be carried out by passing a stream of gas over wet crystals. The gas may be selected from the group consisting of: nitrogen, argon, helium, carbon dioxide, air or combinations thereof.

[000167] In principle, dried crystals can be prepared by lyophilization. However, this technique involves rapid cooling of the material and can be applied only to freeze stable products. In one embodiment, the aqueous solution containing a crystalline/CLPC ZD PA is first frozen to between -40 and -50°C, followed by removal of the under vacuum.

[000168] Production of crystals of ZDPA/CLPC, or formulations or compositions comprising such crystals. In one aspect, crystals of ZDPA/CLPC, or formulations or compositions comprising such crystals are disclosed. Such compositions can be prepared according to the following process:

[000169] First, the ZDPA is crystallized. Next, excipients or ingredients selected from sugars, sugar alcohols, viscosity increasing agents, wetting or solubilizing agents, buffer salts, emulsifying agents, antimicrobial agents, antioxidants, and coating agents are added directly to the mother liquor. Alternatively, the mother liquor is removed, after which the crystals are suspended in an excipient solution for a minimum of 1 hour to a maximum of 24 hours. The excipient concentration is typically between about 0.01 and about 10% (w/w). The ingredient concentration is between about 0.01 and about 90% (w/w). The crystal concentration is between about 0.01 and about 99% (w/w).

[000170] The mother liquor is then removed from the crystal slurry either by filtration or by centrifugation. Subsequently, the crystals are washed optionally with solutions of about 50 to 100%) (w/w) of one or more organic solvents such as, for example, ethanol, methanol, isopropanol or ethyl acetate, either at room temperature or at temperatures between about -20°C to about 25°C.

[000171] The crystals are then dried either by passing a stream of nitrogen, air, or inert gas over them. Alternatively, the crystals are dried by air drying, spray drying, lyophilization or vacuum drying. The drying is carried out for a minimum of about 1 hour to a maximum of about 72 hours after washing, until the moisture content of the final product is below about 10% by weight, most preferably below about 5% by weight. Finally, micronizing (reducing the size) of the crystals can be performed if necessary. [000172] According to one embodiment of this invention, when preparing crystals of ZDPA/CLPC, or formulations or compositions comprising such crystals, enhancers, such as surfactants, are not added during crystallization. Excipients or ingredients are added to the mother liquor after crystallization, at a concentration of between about 1 and about 10% (w/w), alternatively at a concentration of between about 0.1 and about 25% (w/w), alternatively at a concentration of between about 0.1 and about 50% (w/w). The excipient or ingredient is incubated with the crystals in the mother liquor for about 0.1 to about 3 hrs, alternatively the incubation is carried out for about 0.1 to about 12 hrs, alternatively the incubation is carried out for about 0.1 to about 24 hrs.

[000173] In another embodiment of this invention, the ingredient or excipient is dissolved in a solution other than the mother liquor, and the crystals are removed from the mother liquor and suspended in the excipient or ingredient solution. The ingredient or excipient

concentrations and the incubation times are the same as those described above.

[000174] Another advantage of the present invention is that crystals of ZDPA/CLPC, or formulations thereof that are encapsulated within polymeric carriers to form compositions comprising microspheres can be dried by lyophilization. Lyophilization, or freeze-drying allows water to be separated from the composition. The ZDPA/CLPC crystal composition is first frozen and then placed in a high vacuum. In a vacuum, the crystalline water sublimes, leaving the ZDPA/CLPC crystal composition behind, containing only the tightly bound water. Such processing further stabilizes the composition and allows for easier storage and transportation at typically encountered ambient temperatures.

[000175] Spray drying allows water to be separated from the crystal preparation. It is highly suited for the continuous production of dry solids in either powder, granulate or agglomerate form from liquid feedstocks as solutions, emulsions, and pumpable suspensions. Spray drying involves the atomization of a liquid feedstock into a spray of droplets and contacting the droplets with hot air in a drying chamber. The sprays are produced by either rotary (wheel) or nozzle atomizers. Evaporation of moisture from the droplets and formation of dry particles proceed under controlled temperature and airflow conditions. Relatively high temperatures are needed for spray drying operations. However, heat damage to products is generally only slight, because of an evaporative cooling effect during the critical drying period and because the subsequent time of exposure to high temperatures of the dry material may be very short. Powder is discharged continuously from the drying chamber. Operating conditions and dryer design are selected according to the drying characteristics of the product and the powder specification. Spray drying is an ideal process where the end product must comply with precise quality standards regarding particle size distribution, residual moisture content, bulk density and particle shape.

[000176] ZDPA-CLPC useful in the methods of this invention may be combined with an excipient. According to this invention, an "excipient" acts as a filler or a combination of fillers used in pharmaceutical compositions. Examples of excipients are described in Handbook of Pharmaceutical Excipients, published jointly by the American Pharmaceutical Association and the Pharmaceutical Society of Great Britain, and further examples are set forth below. Preferred excipients included in this category are: Salts of either 1) amino acids such as glycine, arginine, aspartic acid, glutamic acid, lysine, asparagine, glutamine, proline; 2) carbohydrates, e.g., monosaccharides such as glucose, fructose, galactose, mannose, arabinose, xylose, ribose; 3) disaccharides, such as lactose, trehalose, maltose, sucrose; 4) polysaccharides, such as maltodextrins, dextrans, starch, glycogen; 5) alditols, such as mannitol, xylitol, lactitol, sorbitol; 6) glucuronic acid, galacturonic acid; 7) cyclodextrins, such as methyl cyclodextrin,

hydroxypropyl-P-cyclodextrin and alike; 8) inorganic molecules, such as sodium chloride, potassium chloride, magnesium chloride, phosphates of sodium and potassium, boric acid, ammonium carbonate and ammonium phosphate; 9) organic molecules, such as acetates, citrate, ascorbate, lactate; 10) emulsifying or solubilizing/ stabilizing agents like acacia, diethanolamine, glyceryl monostearate, lecithin, monoethanolamine, oleic acid, oleyl alcohol, poloxamer, polysorbates, sodium lauryl sulfate, stearic acid, sorbitan monolaurate, sorbitan monostearate, and other sorbitan derivatives, polyoxyl derivatives, wax, polyoxyethylene derivatives, sorbitan derivatives; and 11) viscosity increasing reagents like, agar, alginic acid and its salts, guar gum, pectin, polyvinyl alcohol, polyethylene oxide, cellulose and its derivatives propylene carbonate, polyethylene glycol, hexylene glycol, tyloxapol. A further preferred group of excipients includes sucrose, trehalose, lactose, sorbitol, lactitol, inositol, salts of sodium and potassium such as acetate, phosphates, citrates, borate, glycine, arginine, polyethylene oxide, polyvinyl alcohol, polyethylene glycol, hexylene glycol, methoxy polyethylene glycol, gelatin,

hydroxypropyl-P-cyclodextrin, polylysine, polyarginine.

[000177] In one embodiment of this invention, the excipient is selected from the group consisting of: salts, alcohols, carbohydrates, proteins, lipids, surfactants, polymers and polyamino acids. In another embodiment, the excipient is selected from the group consisting of: Protamine, polyvinylalcohol, cyclodextrins, dextrans, polyamino acids, such as polyarginine, polylysine and poly glutamate, polyethylene glycol and dendrimers, polymers such

polycarbophil, alginate. [000178] Compositions. ZDPA crystals, including cross-linked crystals are provided as a composition, such as a pharmaceutical composition (see, e.g., U.S. Patent No. 6,541,606, describing formulations and compositions of protein crystals). Pharmaceutical compositions comprising ZDPA crystals include the ZDPA crystal with one or more ingredients or excipients, including, but not limited to sugars and biocompatible polymers.

[000179] The ZDPA-CLPC may be administered as a crystal in a composition as any of a variety of physiologically acceptable salt forms, and/or with an acceptable pharmaceutical carrier and/or additive as part of a pharmaceutical composition. Physiologically acceptable salt forms and standard pharmaceutical formulation techniques and excipients are well known to persons skilled in the art (see, e.g., Physician's Desk Reference (PDR) 2003, 57th ed., Medical Economics Company, 2002; and Remington: The Science and Practice of Pharmacy, eds.

Gennado et al, 20th ed, Lippincott, Williams & Wilkins, 2000). For the purposes of this application, "formulations" include "crystal formulations." Other useful ingredients and excipients for the ZDPA crystal compositions include the following:

[000180] Biocompatible polymers. Biocompatible polymers are polymers that are non- antigenic (when not used as an adjuvant), non-carcinogenic, non-toxic and which are not otherwise inherently incompatible with living organisms may be used in the ZDPA crystal compositions described herein. Examples include: poly (acrylic acid), poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly (depsipeptide), poly (esters) such as poly (lactic acid) or PLA, poly (lactic-co-glycolic acid) or PLGA, poly (β-hydroxybutryate), poly

(caprolactone) and poly (dioxanone); poly (ethylene glycol), poly

((hydroxypropyl)methacrylamide, poly [(organo)phosphazene], poly (ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin, alginate, cellulose and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfated polysaccharides, blends and copolymers thereof.

[000181] Biodegradable polymers, i.e., polymers that degrade by hydrolysis or

solubilization may be included in ZDPA crystal compositions. Degradation can be heterogenous (occurring primarily at the particle surface), or homogenous (degrading evenly throughout the polymer matrix).

[000182] Ingredients such as one or more excipients or pharmaceutical ingredients or excipients may be included in ZDPA crystal compositions. An ingredient may be an inert or active ingredient. [000183] The present invention is applicable to IgG's in general, regardless of source. Preferred IgG's for the purposes of this invention are human and mouse IgG classes. The proteins from which the species of interest may be separated are other immunoglobulins, such as, for example, IgM and IgE, and other proteins such as, for example, albumins. The binding affinity of these proteins for protein A is known to be much less than that of IgG's.

[000184] Any inorganic salt which is soluble in an aqueous medium may be used.

Examples include alkali and alkaline earth metal halides and sulfates. Positively charged ions such as ammonium may be substituted for the metallic ions. The salt must also be nonreactive toward the immunoglobulins, the protein A or any support to which the protein A is bound. The salt concentration may range from about 0.5 M up to the solubility limit, preferably from about 1.0 M to about 4.0 M. The exact pH of the solution is not critical and can vary widely within the range from approximately neutral to mildly alkaline. Thus, the pH may be greater than or equal to approximately 7.0, preferably from about 8.5 to about 9.5.

[000185] The salt is preferably used as part of a buffer solution, the buffering effect created by either the salt itself or by a separate component in the mixture. Conventional buffers can be used, appropriately selected to achieve the desired pH.

[000186] For immobilization purposes, the ZDPA is crystallized and cross-linked without any solid support, such as the packing material in an affinity chromatography column.

[000187] When the present invention is used to enhance separation in affinity

chromatography, it is preferable to equilibrate the column packing by repeated washings with a buffer solution containing a high salt concentration, and also to dilute the sample mixture in the same buffer solution before adding it to the column. The dilution may also vary widely, although dilutions ranging from about 1 : 1 to about 1 :20 are preferred. As the buffer solution passes through the column, non-binding proteins will be carried with the buffer solution which thereby separates them from the bound immunoglobulins. The recovery of the immunoglobulins is then achieved by elution with an acidic buffer, preferably having a pH ranging from about 2.0 to about 5.0, more preferably from about 2.5 to about 4.0.

[000188] The nature of the column is not critical and can vary widely, ranging from an open column to a pressurized column. The strong binding inherent in the invention permits an effective separation to be achieved by the use of an open column.

[000189] A "sample containing a target protein" as used in this specification refers to any sample containing the target protein, whether naturally derived or artificially prepared. The sample can include mixtures of different liquids, a liquid and a solid, or different solids. Examples of a sample containing a target protein can include blood, serum, ascites fluid, milk, a tissue sample, a cell culture, a cell lysate, or the supernatant of a cell culture.

[000190] "Support" as used in this specification refers to anything to which a ZDPA can be immobilized to obtain an affinity chromatography medium, regardless of its form or the material from which it is made. Examples include agarose, which is often used in affinity

chromatography; cellulose; and polyacrylamide.

[000191] "Chromatography medium" as used in this specification refers to a stationary phase used for chromatographic purification regardless of its configuration (column or planar), state (liquid or solid), or the material from which it is made. Common examples of

chromatography media used for protein purification include an ion exchange resin, an affinity stationary phase, and a gel permeation stationary phase.

[000192] "Coupled" as used in this specification refers to the direct contact as well as the disposition of a linker gene or protein between two or more types of genes or proteins. It also includes the coupling through covalent or non-covalent bonds.

[000193] "Target protein" as used in this specification refers to any protein to be purified. An example of a target protein can be an antibody.

[000194] A "fusion protein" refers herein to any combination of two or more separate polypeptides. The fusion protein includes a combination of two or more separate polypeptides wherein the two or more polypeptides are covalently linked. The fusion protein includes a combination of two or more separate polypeptides wherein the two or more separate

polypeptides are non-covalently linked. The two or more separate polypeptides may directly contact with each other without any mediator. The two or more separate polypeptides may be mediated by a mediator such as a linker peptide.

[000195] The method disclosed herein uses the high binding specificity between a ZDPA and a target protein such as antibody.

[000196] In an embodiment, the method includes contacting a sample containing a target protein to a support having immobilized thereon a crystalline cross-linked ZDPA, wherein the target protein is a antibody and the ZDPA-CLPC has specific binding affinity for the antibody, and wherein the contact is under conditions such that the ZDPA binds to the antibody; removing components of the sample that are not bound; and then removing the target protein/antibody from the support. In some embodiments, the sample is a cell lysate, a cell culture, the supernatant of a cell culture, or a biological fluid containing the target protein. In some embodiments, the support having immobilized thereon a cross-linked ZDPA is configured as an affinity chromatography column. [000197] In the method and column for the purification of proteins disclosed herein, a disposable column may be used. The disposable column may have a diameter of about 0.1 to about 1.0 mm, about 0.3 to about 0.7 mm, or about 0.5 mm. The column may be placed in a glass test tube of about 16x 125 mm. In an embodiment, a disposable column having the diameter of 0.5 mm is placed in a 16^x 125 mm glass test tube. The chromatography medium, e.g., ZDPA-CLPC, may be packed into the column according to any method known in the art. In an embodiment using a disposable column, a sufficient volume of degassed buffer/water is added to the column to fill it up to the reservoir (wide -mouth) portion, then any air bubbles are eliminated from the column. After that, the gel (ZDPA-CLPC) can be packed into the column with degassed 50% gel slurry, and degassed buffer solution (or water) at room temperature. Sufficient volume of degassed gel slurry can be added to obtain the desired settled gel volume. The gel can be permitted to settle down in the column for at least 30 minutes. The packed column can be stored and used at 4° C.

[000198] Removing the target protein from the support can comprise eluting the target protein from the support. Eluting the target protein can be conducted by elution at a pH that lowers the binding affinity between the ZDPA-CLPC and the antibody such that the target protein/antibody is removed from the support (ZDPA -CLPC).

[000199] The first step in the process of the present invention requires a buffer having a pH in the range of about pH 7.0 to pH 10 and a combination of monovalent cations and polybasic anions in a concentration of about 0.01M to 2M. Any buffer may be used to provide the desired pH. For example phosphate buffer, glycine buffer, borate buffer or tris buffer can be used. The concentration of buffer should be in the range of about 0.01M to 0.25M. In addition, salts of NaCl, KC1, tetramethylammonium chloride (TMAC), tetraethylammonium chloride (TEAC), tetrapropylammonium chloride and tetrabutylammonium chloride etc can be added to the buffer in the concentration range of about 0.05M to 2.0M.

[000200] Where the monovalent cations are potassium ions or sodium ions and the polybasic anions are phosphate ions the potassium ions and phosphate ions can be provided by the use of potassium phosphate either in the form of tripotassium phosphate, K 3 PO 4 , dipotassium hydrogen phosphate, K 2 HPO 4 or monopotassium dihydrogen phosphate, KH 2 PO 4 , since the pH of the medium controls the proportion of the various phosphate ions which are present. The potassium ions and phosphate ions should be present in a concentration in the range of about 0.6M to 1.75M. A concentration of about 1.0M to 1.5M has been found especially satisfactory. [000201] Other combinations of monovalent cations and polybasic anions which have adequate solubilities at the high concentrations used in the present invention, include ammonium phosphates in concentrations of about 1.0M to 1.5M, ammonium sulfates in concentrations of about 1.0M to 1.5M and sodium sulfates in concentrations of about 1.0M to 1.25M. Other combinations may be used as well so long as the salts do not precipitate at the concentrations used.

[000202] As pointed out above, the adsorbent (ZDPA-CLEC) is preferably used in a column to facilitate contact with the immunoglbulins to be purified. Prior to application of the medium containing the impure immunoglobulins to the column, the column containing ZDPA- CLEC is equilibrated with several bed volumes of buffer containing the combination of to the column, the column is equilibrated with several bed volumes of buffer containing the combination of monovalent cations and polybasic anions at concentrations in the range of about 0.01M to 4M. This ensures that the environment is optimum for binding the immunoglobulins to the column. The medium containing the immunoglobulins to be purified, such as an immune serum or other source of immunoglobulins is mixed with the buffer containing the combination of monovalent cations and polybasic anions. The resulting mixture is then applied to the column, resulting in adsorption of the immunoglobulins to the column. The column is then washed with additional buffer containing the combination of monovalent cations and polybasic anions in order to elute from the column impurities which are not strongly adsorbed to the column. The immunoglobulins on the other hand are strongly adsorbed to the column because of the enhanced affinity of the adsorbent for the immunoglobulins as a result of the presence of the buffer containing the combination of monovalent cations and polybasic anions. Following removal of the undesired impurities by washing with the same buffer solution, the purified immunoglobulins are eluted from the column by means of a buffer having an acidic pH, namely a pH in the range of about pH 2.0 to pH 6.0. At pH 6.0 a part of the immunoglobulins, principally the IgG 1 fraction, is eluted. As the pH is lowered the remainder of the

immunoglobulins, including the IgG 2a and IgG 2b fractions, is eluted. The immunoglobulins can be eluted using a pH 2.0 buffer, which is effective to elute all of the immunoglobulins. However, if desired, a fraction of the immunoglobulins can be eluted at pH 6.0. Various other fractions can be eluted, if so desired, by lowering the pH between pH 6.0 and pH 2.0. By lowering the pH in steps, it is possible to isolate purified fractions of immunoglobulins which contain specific immunoglobulins as desired. Any buffer can be used for elution. For example an acetic acid-acetate buffer or glycine.HCl buffer can be used for this purpose. A buffer concentration in the range of about 0.01M to 0.25M can be used. A buffer concentration of about 0.1M to 0.2M is especially preferred.

[000203] The immunoglobulins or fractions thereof can be bound to a short column of ZDPA-CLEC when compared to immobilized ZDPA bound to support such as agarose for example. The isolated immunoglobulins or fractions thereof can be recovered in yields which are as much as ninety percent (90%). Even binding obtained using the most sophisticated techniques previously available can be improved by as much as 2 to 10 %.

[000204] In certain embodiments, the primary recovery sample is subjected to affinity chromatography to further purify the antibody of interest away from HCPs. In certain embodiments the chromatographic material is capable of selectively or specifically binding to the antibody of interest. Non-limiting examples of such chromatographic material include:

ZDPA, Protein G, Protein L. In specific embodiments, the affinity chromatography step involves subjecting the primary recovery sample to a column comprising a suitable ZDPA resin. ZDPA resin is useful for affinity purification and isolation of a variety antibody isotypes, particularly IgGl, IgG2, and IgG4.

[000205] A non-limiting example of a suitable column packed with ZDPA-CLPC is about 1.0 cm diameterx about 21.6 cm long column (~17 mL bed volume). This size column can be used for small scale purifications and can be compared with other columns used for scale ups. For example, a 20 cm><21 cm column whose bed volume is about 6.6 L can be used for larger purifications. Regardless of the column, the column can be packed using a ZDPA-CLPC.

[000206] In certain embodiments it will be advantageous to identify the dynamic binding capacity (DBC) of the ZDPA resin in order to tailor the purification to the particular antibody of interest. For example, but not by way of limitation, the DBC of a ZDPA-CLPC column can be determined either by a single flow rate load or dual-flow load strategy. The single flow rate load can be evaluated at a velocity of about 300 cm/hr throughout the entire loading period. The dual-flow rate load strategy can be determined by loading the column up to about 35 mg protein/mL resin at a linear velocity of about 300 cm/hr, then reducing the linear velocity by half to allow longer residence time for the last portion of the load.

[000207] In certain embodiments, the ZDPA column can be equilibrated with a suitable buffer prior to sample loading. A non-limiting example of a suitable buffer is a PBS or

Tris/NaCl buffer, pH of about 7.2-7.4. A non-limiting example of suitable equilibration conditions is PBS buffer pH 7.4 or 25 mM Tris, 100 mM NaCl, pH of about 7.2. Following this equilibration, the sample can be loaded onto the column. Following the loading of the column, the column can be washed one or multiple times using, e.g., the equilibrating buffer. Other washes, including washes employing different buffers, can be employed prior to eluting the column. For example, the column can be washed using one or more column volumes of 20 mM citric acid/sodium citrate, 0.5 M NaCl at pH of about 6.0. This wash can optionally be followed by one or more washes using the equilibrating buffer. The ZDPA column can then be eluted using an appropriate elution buffer. A non-limiting example of a suitable elution buffer is an acetic acid/NaCl buffer, pH of about 3.5. Suitable conditions are, e.g., 0.1 M acetic acid, pH of about 3.5 or 0.2M glycine.HCl buffer, pH 2.0. The eluate can be monitored using techniques well known to those skilled in the art. For example, the absorbance at OD280 can be followed. Column eluate can be collected starting with an initial deflection of about 0.5 AU to a reading of about 0.5 AU at the trailing edge of the elution peak. The elution fraction(s) of interest can then be prepared for further processing. For example, the collected sample can be titrated to a pH of about 5.0 using Tris (e.g., 1.0 M) at a pH of about 10. Optionally, this titrated sample can be filtered and further processed.

[000208] The present invention provides protein impregnated materials suitable for use in a variety of applications including, for example, the removal of immune globulins from blood, plasma, serum, cell culture during purification or immunoprecipitation and methods of producing and using same. Such protein impregnated membranes can also be used in therapeutic, diagnostic and other industrial applications.

[000209] In an embodiment, the present invention includes membranes impregnated with a cross-linked protein crystal. The cross-linked proteins can include any suitable cross-linked protein made from a variety of suitable proteins. In an embodiment, the cross-linked proteins can include proteins capable of removing antibodies or the like from serum, plasma, blood, cell culture, such as, ZDPA, Protein G, Protein L, like proteins or combinations thereof. Preferably, the cross-linked protein crystal is a ZDPA-CLPC as described herein.

[000210] In an embodiment, the present invention includes a polymeric membrane, impregnated with the cross-linked protein crystals, preferably ZDPA-CLPC, alone or in combination with other cross-linked protein crystals such Protein G or Protein L. It is believed that the use of ZDPA-CLPC impregnated membranes will provide a number of benefits over currently available immobilization technologies including, for example: 1) Better ZDPA containment without any leaching; 2) Reduced cartridge/column size resulting in enhanced ease of use; 3) Ease of use during cartridge/column manufacture; and 4) Increased safety over the existing system (due to better containment of ZDPA in the cartridge).

[000211] The cross-linked protein impregnated membranes of the present invention can be made in a variety of suitable ways. In general, a polymer-based membrane casting solution is first prepared and then mixed with the desired amount and types of cross- linked protein crystals. It should be appreciated that the membrane casting solution can be made from any suitable polymer-based materials. Once formed and mixed with the cross-linked protein, the membrane casting solution is applied to a support material by, for example, spreading on the support material, and subjected to one or more precipitation and washing sequences to form a composite membrane which can be subsequently dried prior to use.

[000212] In an embodiment, the casting solution is composed of a polymeric base material, such as polyurethane or the like, in any suitable solvent including, for example, l-methyl-2- pyrrolidinone ("NMP"), dimethylformamide ("DMF"), the like or combinations thereof. The casting solution can also include additional other components, such as a bulking agent, a hydrophilic agent (e. g., an agent that can render the membrane more hydrophilic), the like or combinations thereof. In an embodiment, the bulking agent can include zirconium oxide, zirconium phosphate, carbon, the like or combinations thereof. The bulking agent can be added in a sufficient amount to control the porosity of the membrane. The bulking agent can be added in an amount of up to about 80% of the total dry weight of the membrane, preferably about 50% of the total dry weight of the membrane. In an embodiment, the bulking agent and the cross- linked protein crystal are added in equal amounts or at least approximately equal amounts.

[000213] In an embodiment, the hydrophilic agent is polyvinylpyrrolidone ("PVP"), the like or combinations thereof. The hydrophilic agent can be added in any suitable amount to enhance the hydrophilic nature of the membrane.

[000214] The casting solution is then mixed with a suitable amount of a cross-linked ZDPA. In an embodiment, the cross-linked ZDPA-CLPC is added to the membrane in an amount effective to provide a desired level of binding activity. In an embodiment, the membrane is impregnated with about 3.25 mg/cm2 or less of the cross-linked protein. In this regard, the cross-linked protein crystal can amount to about 80% or less of the membrane weight.

[000215] The resultant membrane solution is then applied to a support, such as a synthetic mesh material, and immersed into water or other suitable media, such as a mixture of isopropyl alcohol and water, preferably a 50: 50 ratio of isopropyl alcohol ("IP A") to water. A polymer membrane composite material can then be precipitated under suitable conditions. For example, a suitable amount of NMP can be added during water precipitation to control the rate of precipitation. In this regard, the rate of precipitation can be decreased, thus resulting in a more porous polymeric matrix of the membrane. [000216] The ZDPA-CLPC of the present invention may be administered through an extracorporeal device or catheter, such as for delivery of ZDPA-CLPC to a patient. Catheters, for example, urinary catheters, may be coated with compositions containing ZDPA-CLPC crystals.

[000217] The following examples provide illustrative embodiments of the invention. One of ordinary skill in the art will recognize the numerous modifications and variations that may be performed without altering the spirit or scope of the present invention. Such modifications and variations are encompassed within the scope of the invention. The Examples do not in any way limit the invention.

EXAMPLES

Example 1. INTRODUCTION

[000218] The ZDPA cross-linked crystals (CLPCs) were produced so as to develop an innovative ZDPA chromatography system for the purification of antibodies. ZDPA CLPCs will offer the advantages of highly concentrated ZDPA activity combined with high stability and chemical resistance. The condensed ZDPA concentration will reduce column size, buffer volume and process time. Moreover, the cross-linking of ZDPA crystals will prevent the leaching of ZDPA during chromatography. Altogether, this will reduce antibody production time and cost.

[000219] The Examples herein describe the crystallization in a batch and the cross-linking of recombinant ZDPA.

PURPOSE

[000220] To develop cross-linked ZDPA crystals for antibody purification.

EQUIPMENT AND MATERIALS

• ZDPA, Custom Synthesized, Standard Chemical Peptide Synthesis Service, GenScript USA, Inc., Sequence :

VDNKFNKEQQNAFYEILHLPNLNEEQR AFIQSLKDDPSQSANLLAEAK LNDAQAPK. Purity as determined by reverse phase HPLC : >98%

• Ammonium Sulfate, Omni Pur, Catalog Number 2150, Lot Number

UE20GZEMS. • Citric Acid, anhydrous, ACS 99.5+%, Stock Number 36664, Lot Number A20X027.

• Sodium Citrate, Dihydrate, BDH, Catalog Number BDH0288-500G, Lot Number 111830

• pH conductivity meter, Denver Instrument, Model 220.

• 8453 UV-Visible Spectrophotometer, Agilent Technologies.

• Hydrochloric Acid Solution, Fisher Scientifics, Cat. No. A481-212.

• Sodium cacodylate trihydrate, Sigma-Aldrich, Cat. No. C0250.

• Sodium chloride, Fisher Scientific, Cat. No. S271-3.

• Tris(hydroxymethyl)aminomethane (Trizma Base), Sigma-Aldrich, Cat. No. T- 4661.

• DI (Reverse Osmosis & De-Ionization) H₂0. PROCEDURE

[000221] Crystallization of recombinant ZDPA was carried out with a batch crystallization method. The ZDPA crystals were made in approximately 0.5 ml batch size.

Solution preparation.

3 M Ammonium sulfate, 0.2 M Citric Acid/Sodium Citrate

[000222] 39.6 g of ammonium sulfate was dissolved in DI H20 to a final volume of 95ml. Upon complete dissolution, 0.7 grams of Citric Acid, anhydrous and 1.86 grams of Sodium Citrate, Dihydrate were dissolved into the same solution. The pH of the solution is made equal to 5.0 using the pH meter, and DI H20 added to a final volume of 100ml.

ZDPA crystallization in batches.

[000223] ZDPA crystallization was set up in batches with recombinant ZDPA from GenScript USA. The ZDPA sample was used at a concentration of approximately 10 mg/ml. Crystallization was performed in 1.5M ammonium sulfate, 0.05M citric acid, pH 5.0 in a 500 μΐ microbatch. The batch was incubated at room temperature without tumbling during 21 days.

[000224] Crystal ZDPA was spun down in a microcentrifuge for 5 minutes at 13,000 rpm and the mother liquor discarded. The crystals were washed once with 1.5M ammonium sulfate, 0.05 Citric Acid, pH 5.0, respun, the wash buffer discarded, and the crystals stored at 5C. RESULTS

Batch crystals of ZDPA.

[000225] As shown in Figure 1, recombinant ZDPA was crystallized in 0.5 ml batches with the final concentration of ammonium sulfate at 1.5M and citric acid at 0.05 M, pH 5.0.

Crystallization in batches produced cubic-shaped crystals. The cubic-shaped crystal size was about 10 microns.

Example 2.

Cross-linking of ZDPA crystals.

[000226] The crystals from Example 1 were resuspended in 1ml of 1.5M ammonium sulfate, 0.05M citric acid, pH 5.0, and was cross-linked with glutaraldehyde (the final glutaraldehyde concentration was 2%). The sample was immediately vortexed for 90 minutes, followed by 90 minutes of end-over end mixing. After cross-linking, the ZDPA sample was quenched by incubation in 1ml of 1M Tris-HCl, pH 9 for one hour while mixed end-over-end at room temperature. The crystals were then washed 3 times in 1 ml of 10 mM Tris-HCl buffer pH 7 (centrifugation was carried out at 4,500 rpm for 5 minutes), and the ZDPA cross-linked crystals (CLPCs) were resuspended in 1 ml of 10 mM Tris-HCl buffer pH 7.

RESULTS

[000227] As shown in Figure 2, cross-linking ZDPA produced amalgams of cubic-shaped crystals. The size of these amalgams was between 80-120 microns.

Example 3.

PURPOSE

[000228] The purpose of the experiment was to determine the binding capacity of the cross- linked ZDPA crystals using Human IgG.

EQUIPMENT AND MATERIALS

Equipments: Table Top Centrifuge: Eppendorf Centrifuge 5415D

Eppendorf Tubes 1 ml

Balance: Mettler Toledo AG285

Material

Cross-linked ZDPA crystals: From Example 2.

Human IgG: ICN Biomedical, Inc. Cat. # 64145

Phosphate Buffered Saline (PBS) tablets: Sigma Cat. # P- 4417

Glycine: Fluka # 50046

O. lN NaOH: Acros Cat # 12419-0010

PROCEDURE

Buffer preparation

Phosphate Buffered Saline (PBS):

[000229] 1 tablet was dissolved in 200ml of distilled water to obtain phosphate buffered saline.

0.2 M glycine pH 2.0:

[000230] 7.5gm of glycine was dissolved in 90ml of water. pH was adjusted to 2.0 with IN HCl. Volume was made up to 100ml using DI water. Check pH and adjusted to 2.0 again if necessary.

Experimental procedure:

[000231] 1.67 milligrams of cross-linked ZDPA crystals (CLPCs) were used in this experiment. Five hundred microliters of monoclonal antibody (10 mgs/ml) in PBS was added to the cross-linked ZDPA. The resulting slurry was mixed overnight, along with a negative control, i.e., 500 microliters of monoclonal antibody alone. Both tubes were spun down in a microcentrifuge for 10 minutes (13K rpm), and the supernatant from the mixture was removed for further analysis. The supernatant and the negative control were analyzed by absorbance at 280 nanometers. Subtraction of A280 of the standard by the supernatant gives the amount of monoclonal antibody bound to the cross-linked ZDPA.

RESULTS

[000232] Binding capacity of cross-linked ZDPA crystals was calculated as amount of monoclonal antibody bound and eluted per microgram of cross-linked ZDPA. A total of 1.13 milligrams of monoclonal antibody bound to 1.67 micrograms of cross-linked ZDPA, giving a binding capacity of 677 milligrams per gram of crosslinked ZDPA.

CONCLUSIONS

[000233] From these experiments, the binding capacity was calculated to be 677mg of monoclonal antibody per gram of Cross-linked ZDPA crystals.

Example 4.

Leaching: pH controlled solubility of cross-linked Protein A crystals.

[000234] Solubility of various cross-linked ZDPA crystals is examined following a decrease in pH from 7.5 to 2.0. The cross-linked crystals are incubated at 1 mg/ml in 50 mM glycine-HCl (pH 2.0). Aliquots are removed after 5 hour incubation at 37°C with stirring. Soluble protein concentration is measured at OD280 nm after separation of the undissolved cross-linked crystals by centrifugation at 2000 rpm and filtration of the supernatant through 0.22 μιη filter.

Example 5.

[000235] This Example describes the determination of human immunoglobulin G (IgG) binding capacity of ZDPA CLPCs in columns.

PROCEDURE

Solution preparation

Phosphate buffered saline solution

[000236] One phosphate buffered saline (PBS) tablet from Sigma-Aldrich is dissolved in 200 ml DI H20, so as to prepare the following PBS solution: 0.01 M phosphate buffer, 2.7 mM KC1, 137 mM NaCl at pH 7.4.

1 M Tris-HCl buffer pH 8.5

[000237] 12.11 g of Trizma Base is dissolved in 80 ml DI H20. The pH is adjusted to 8.5 with 1 N HC1 solution, and the solution is adjusted to 100 ml with DI H20. 0.2 M glycine buffer pH 2

[000238] 1.5 g of glycine is dissolved in 80 ml DI H20. The pH is adjusted to 2 with 1 N HC1 solution, and the solution is adjusted to 100 ml with DI H20.

M citric acid buffer pH 3

[000239] 1.9 g of citric acid anhydrous is dissolved in 80 ml DI H20. The pH is adjusted to 3 with 1 N NaOH solution, and the solution is adjusted to 100 ml with DI H20.

N NaOH solution

[000240] 100 ml of 0.5 N NaOH solution is diluted with 400 ml DI H20.

Human IgG binding capacity ofZDPA CLPCs

Determination of IgG binding capacity in columns

[000241] An empty Waters Sep-Pak® Vac column is packed with ZDPA CLPCs (the bed volume is approximately 500 μΐ with a ZDPA CLPC).

[000242] The column is equilibrated with 5 ml PBS, and loaded with 2000 mg of human IgG from ICN Biomedicals at a concentration of 20 mg/ml in PBS.

[000243] The flow-through is collected in 10 ml aliquots, and the protein concentration is determined by spectrophotometry at 280 nm.

[000244] The column is then washed with 5 ml PBS, and 1 ml fractions collected.

[000245] Human IgG is eluted with 5 ml of 0.2 M glycine buffer pH 2, and the glycine eluate is collected in 1 ml aliquots and assayed for protein concentration by spectrophotometry at 280 nm. Each eluted fraction is adjusted to physiologic pH with 100 μΐ of 1 M Tris-HCl buffer pH 8.5.

[000246] After the glycine elution, the column is regenerated with 5 ml of 0.1 M citric acid/NaOH buffer pH 3, which is collected after its elution from the column.

[000247] Finally, the column is cleaned with 3 ml of 0.1 M NaOH solution. Fractions of 1 ml are collected and the column is washed with 5 ml PBS.

[000248] After the A280 spectrophotometric assays, the flow-through fractions, as well as the fractions of the PBS washes and of the NAOH washes are pooled separately.

Example 6.

[000249] In this experiment, the effects of post treatment on binding capacity of the

ZDPA-CLPC are evaluated. The ZDPA-CLPC impregnated membranes are made by forming a membrane casting solution with polyurethane in a specific type of solvent; adding ZDPA-CLPC to the casting solution; precipitating an impregnated membrane composite material in a suitable media; and optionally processing the composite material by drying prior to use. Specific processing conditions, such as the type of membrane casting solvent e.g. DMF, amount of ZDPA-CLPC, precipitation bath media (50/50 IP A/water) and post treatment conditions (wet never dried), for each of the test impregnated membranes are identified and are tested. The ZDPA-CLPC binding capacity is tested for each of the test impregnated membranes,

a. Example 7.

[000250] In this experiment, two groups of test impregnated membranes are made, namely Groups A and B. Group A membranes (e. g., A1-A2) are made from polyurethane in a NMP solvent. Group B membranes (e. g., B1-B2) are made from polyurethane in a DMF solvent. The impregnated membranes are about 1 inch in diameter. Specific processing conditions, such as the type of membrane casting solvent e.g. DMF or NMP, amount of ZDPA-CLPC, precipitation bath media (50/50 IP A/water or water alone) and post treatment conditions (wet never dried or 40% glycerol dried), exposure to gamma radiation ( 15 to 40 kGy) for each of the test impregnated membranes are identified and are tested. Once formed, a membrane from each group is exposed to gamma radiation at certain dosages. The other membrane in each group is used as a control with no exposure to gamma-radiation. The antibody binding capacity of each of the membranes is then tested with a antibody test solution. The results will provide the retention of the binding capacity and stability to organic solvents and gamma-radiation exposure of ZDPA-CLPC.

Sequences:

The 58-amino acid sequence of ZDPA is shown below (SEQ ID NO: 1).

VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAK LNDAQAPK

Claims

WHAT IS CLAIMED IS:

1. A composition comprising crystalline form of ZDPA.

2. A composition comprising cross-linked crystalline form of ZDPA.

3. A method of purifying an immunoglobulin comprising contacting said immunoglobulin using the composition of claim 1 or claim 2.

4. The method of claim 3 wherein said immunoglobulin is an antibody.

5. The method of claim 4 wherein said antibody is a therapeutic antibody.

6. The method of claim 4 wherein said antibody is a monoclonal antibody.

7. The method of claim 3 wherein said immunoglobulin is a Fab fragment.

8. The method of claim 3 wherein said immunoglobulin is an Fc fragment.

9. The method of claim 3 wherein said immunoglobulin is a single chain antibody.

10. The method of claim 3 wherein said immunoglobulin is a chimeric antibody.

11. The method of claim 3 wherein said immunoglobulin is a fully human antibody.

12. The method of claim 3 wherein said immunoglobulin is a humanized antibody.

13. The method of claim 3 wherein said immunoglobulin belongs to class IgG..

14. The composition of claim 2 wherein the composition is cross-linked with glutaraldehyde.

15. A kit comprising the composition of claims 1 or 2.

16. The kit of claim 15 wherein the kit contains written instructions describing the use of crystalline and/or cross-linked crystalline form of ZDPA.

17. A column containing the composition of claims 1 or 2.

18. The composition of claim 2, wherein said crystal is more active (binding capacity) than an immobilized form of ZDPA.

19. The composition of claim 2, wherein said crystals are active (binding capacity) and stable at about pH 2 to about pH 12.

20. The composition of claim 2, wherein said crystals have 0.0% protein leaching when compared to immobilized ZDPA.

21. The composition of claim 1 or 2, wherein the composition is used in a pre-packed column column material.

22. The composition of claim 1 or 2, wherein the composition is used in a membrane

(impregnated).

23. The composition of claim 1 or 2, wherein the composition is used in an extracorporeal device.

24. A process for the purification of immunoglobulins which comprises:

(a) mixing a medium containing immunoglobulins with a buffer solution having a pH in the range of about pH 7.0 to pH 10 and containing a combination of cations and anions to provide buffered immunoglobulin medium;

(b) contacting said buffered immunoglobulin medium with an immobilized ZDPA (ZDPA- CLPC: ZDPA is immobilized by crystallizing and crosslinking ZDPA molecules) adsorbent to adsorb the immunoglobulins present in said buffered immunoglobulin medium upon said immobilized ZDPA adsorbent;

(c) washing the ZDPA-CLPC adsorbent having immunoglobulins adsorbed thereon with said buffer solution;

(d) contacting said ZDPA-CLPC adsorbent having immunoglobulins adsorbed thereon with a buffer solution having a pH in the range of about pH 2 to pH 6 to remove the adsorbed immunoglobulins from the ZDPA-CLPC adsorbent; and

(e) recovering the removed immunoglobulins in substantially pure form.

25. A process according to claim 24 wherein the contacting of said buffered immunoglobulin medium with said ZDPA-CLPC adsorbent is accomplished in a column of said ZDPA-CLPC adsorbent.

26. A process according to claim 24 wherein said medium containing immunoglobulins is a normal mammalian serum.

27. A process according to claim 24 wherein said medium containing immunoglobulins is an immune mammalian serum.

28. A process according to claim 24 wherein said medium is a mammalian plasma.

29. A process according to claim 24 wherein said medium is mammalian ascites fluid.

30. A process according to claim 24 wherein said medium is obtained from a hybridoma.

31. A process according to claim 24 wherein said medium is a tissue culture fluid.

32. A process according to claim 24 wherein said medium is a cell culture fluid.

33. A process according to claim 24 wherein said medium is a mammalian cell culture fluid.

34. A process according to claim 24 wherein said medium is a bacterial cell culture fluid.

35. A process according to claim 24 wherein said medium is a transgenic source fluid.

36. A process according to claim 24 wherein said medium is a plant extract containing immunoglobulins .

37. A process according to claim 13 wherein said medium is a yeast culture fluid.

38. A method of making crystalline form of ZDPA using the batch crystallization method comprising the steps of:

(a) placing ZDPA in deionized water at a 1 : 1 protein reagent ratio, wherein said reagent is selected from the group consisting of a composition containing 2 M ammonium sulfate in citrate buffer pH 5; and

(b) incubating until crystal formation occurs.

39. A method according to claim 38 wherein the ZDPA in step (a) is at concentration of about 7 mg/ml ZDPA in deionized water.

40. A method of making cross-linked crystalline form of ZDPA comprising mixing crystalline form of ZDPA with glutaraldehyde.

41. A method according to claim 40 wherein the glutaraldehyde is at a final concentration of about 2%.

42. A method of purification comprising contacting the substance to be purified with the composition of claims 1 or 2.