WO2005090403A2

WO2005090403A2 - Method and apparatus for antibody purification

Info

Publication number: WO2005090403A2
Application number: PCT/US2005/008032
Authority: WO
Inventors: Michael Gramer; Darrell Page; Robert Wojciechowski; Grant Adams; Chad Sinkler
Original assignee: Biovest International, Inc.
Priority date: 2004-03-12
Filing date: 2005-03-14
Publication date: 2005-09-29
Also published as: WO2005090403A3

Abstract

An automated method and apparatus for purifying antibodies which utilize pre-sanitized or pre-sterilized, disposable cultureware is disclosed.

Description

METHOD AND APPARATUS FOR PROTEIN PURIFICATION

RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 60/552,895 (filed March 12, 2004) entitled "Method and Apparatus for Protein Purification". The entire content of the above-referenced application is incorporated herein by reference.

BACKGROUND OF THE INVENTION With the increased use of proteins, such as antibodies, in clinical diagnostics and therapy, the need has arisen for more efficient, rapid and sterile purification methods. Conventional purification techniques typically involve manually passing mixtures containing proteins through a suitable column to selectively adsorb the proteins from the mixture. The adsorbed proteins are then eluted from the column in purified form. Manual methods for purifying proteins have their drawbacks. For example, these methods can be labor intensive, time consuming and typically use multiple columns which are manually packed with resin and sterilized prior to each purification run. The manual steps involved in these methods also include a high risk of contamination. Accordingly, it is an object of the present invention to provide a fully automated process and apparatus for the purification of proteins which is less labor intensive and involves a pre-sterilized, disposable cultureware set to remove the risk of contamination.

SUMMARY OF THE INVENTION The present invention provides an automated protein purification method and apparatus which utilize pre-sanitized or pre-sterilized disposable cultureware, such as pre-sanitized or pre-sterilized disposable selection means, diafiltration module, liquid reservoirs, valves, tubing, and collection vessels, which can be packaged together for single use and then disposed of. Accordingly, the present purification method and purification apparatus (also referred to as an Autovaxid Purification Module™) are capable of purifying proteins, such as antibodies, in a highly efficient and contaminant- free manner. Specifically, the automated protein purification method and apparatus of the present invention minimize the need for operator intervention and provide a completely disposable flowpath to eliminate the need for cleaning and to eliminate the potential for cross-over contamination. Therefore, the present invention provides an automated method and apparatus for purifying proteins in a less labor intensive manner compared to manual purification methods, thus, reducing purification time and increasing efficiency. In one embodiment, the purification method of the invention begins with the automated step of loading a protein-containing aqueous medium, e.g., an antibody- containing aqueous medium, onto a pre-sanitized, preferably a pre-sterilized, disposable selection means to absorb the protein onto the selection means. The selection means for use in the present method can include, for example, a column packed with an affinity resin, such as an anti-IgM resin, a Protein A, a Protein G, or an anti-IgG resin. In another embodiment, the protein-containing aqueous medium and the selection means can be pre-treated prior to loading. For example, the protein-containing aqueous medium can be automatically heated and degassed before loading. The selection means can be washed, pre-eluted, and/or pre-neutralized prior to loading. After loading, the selection means is typically washed to remove residual contaminants contained within the aqueous medium, such as residual proteins from host cells used to produce the protein to be purified, e.g., host cell proteins, nucleic acids and endotoxins. After washing, the bound protein is then eluted into an aqueous medium. The step of eluting can be accomplished, for example, by either changing the pH or the salt concentration of the solution which is loaded onto the selection means. For example, an acidic solution can be added to the selection means to produce a protein- containing acidic eluate. An example of an appropriate acidic solution for eluting includes a solution of approximately 0.05 to 0.5 M of an acid (such as, glycine or citrate) at a pH of about 2 to 5. Alternatively, the step of eluting can include adding a solution to the selection means which alters the salt concentration of the aqueous medium loaded onto the selection means. In one embodiment, the step of eluting the protein is facilitated by the use of a photometer. Upon eluting the protein into an aqueous medium, the eluted purified protein can be automatically deposited into a pre-sterilized, disposable collection vessel and removed from the automated purification apparatus. Alternatively, the eluted purified protein can undergo further automated processing. In one embodiment, the eluted purified protein, e.g., an antibody, is transferred to an acidic solution. This transfer to an acidic solution can be accomplished by using, for example, a diafiltration module contained within the automated apparatus. The diafiltration module is a membrane-based ultrafiltration module installed within the automated purification module which utilizes the tangential flow filtration principle. Accordingly, the eluted protein is diafiltered against an acidic solution (e.g., a solution of approximately 0.1 M glycine at a pH of about 2.0 to 5). In another embodiment, the method includes the step of holding the eluted protein in the acidic solution for approximately less than 16 hours to inactivate any susceptible virus that may be contained within the solution. For example, the protein is held in the acidic solution for approximately 15.5 hours, 15 hours, 14.5 hours, 14 hours, 13.5 hours, 13 hours, 12.5 hours, 12 hours, 11.5 hours, 11 hours, 10.5 hours, 10 hours, 9.5 hours, 9 hours, 8.5 hours, 8 hours, 7.5 hours, 7 hours, 6.5 hours, 6 hours, 5.5 hours, 5 hours, 4.5 hours, 4, hours, 3.5 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour or less, hi another embodiment, the protein is held in the acidic solution for less than 1 hour, for example for approximately 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 29 minutes, 28 minutes, 27 minutes, 26 minutes, 25 minutes, 24 minutes, 23 minutes, 22 minutes, 21 minutes, 20 minutes or less, e.g., 19, 18, 17, 16, 15, 14, 13, 12 ,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute. In another embodiment, the protein-containing acidic solution is then neutralized by transferring it to a basic solution, for example, by diafiltering against a basic solution (e.g., a solution of approximately 10 mM citrate at a pH of about 5 to 7). In another embodiment, the protein-containing basic solution is transferred to a final buffer solution, for example, by diafiltering against a final buffer solution (e.g., a. solution of approximately 0.2 - 0.9% saline (NaCl)). In another embodiment, the protein-containing final buffer solution can be further purified, for example, by filtering the solution, for example, filtering it through a filter having a pore size of approximately 0.22 μm. In another embodiment, the purification method further includes the step of conjugating the purified protein (e.g., antibody) to an adjuvant, such as Keyhole limpet hemocyanin (KLH). Accordingly, the present invention can be used to produce a variety of vaccines. In a particular embodiment, the invention provides a method for producing an antibody vaccine, particularly an antibody against a tumor cell, such as a B cell for the treatment of lymphoma. In still another embodiment, the present invention includes an automated apparatus for purifying a protein from a protein-containing aqueous medium, comprising pre-sanitized or pre-sterilized, disposable cultureware attached to an automated means for controlling liquid flow through the cultureware. For example, the pre-sanitized or pre-sterilized, disposable cultureware includes a selection means; multiple, liquid reservoirs; a means for flowing liquid from the reservoirs and into the selection means; a means for diverting the effluent from the selection means; and a means for collecting effluent from the selection means. The pre-sanitized or pre-sterilized, disposable cultureware can further include a diafiltration module; a means for flowing liquid from the reservoirs and into the diafiltration module; a means for flowing liquid between the selection means and the diafiltration module; a means for diverting the effluent from the diafiltration module; and a means for collecting effluent from the diafiltration module, e.g., at least two disposable reservoirs. In a particular embodiment, the means for flowing liquid (such as, wash buffer, elution buffer, or neutralization solution) into the selection means or diafiltration module includes a series of pre-sanitized or pre-sterilized, disposable valves and tubing which connect the reservoirs to the selection means or diafiltration module and which allow liquid from only one reservoir at a time to pass through the selection means or diafiltration module. Alternatively, the valves and tubing which connect the reservoirs to the selection means and diafiltration module allow liquid from more than one reservoir at a time to pass through the selection means. In one embodiment, the valve includes a disposable outer body through which flexible tubing is threaded. A cammed shaft is mated with the body and a motor drives the shaft to open and close the tubing. Multiple tubing lines can be controlled by one motor/shaft. The pre-sanitized or pre-sterilized tubing lines contain the fluid and maintain sterility. The tubing and outer body housing are disposed of at the end of use. In another embodiment, the purification apparatus of the present invention includes a means for monitoring the effluent from the selection means or diafiltration module, such as a probe for measuring the pH, absorbance at a particular wavelength, or conductivity of the effluent.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows a schematic representation of one embodiment of the automated purification apparatus, wherein the selection means, e.g., the affinity column, and the diafiltration module of the apparatus are connected to multiple liquid reservoirs. The reservoirs each contain liquid, such as a wash buffers and an elution buffer for delivery to the selection means and acidic, basic and final buffer solutions for delivery to the diafiltration module. Elution of the purified protein from the selection means can be aided by a photometer. The apparatus further includes a means for flowing liquid from the reservoirs into the selection means or into the diafiltration module, for example, valves and tubing which connect the reservoirs to the selection means or to the diafiltration module. The flow of the liquid is diverted by valves which transfers the eluted protein-containing solution from the selection means to the diafiltration module and then to the pre-sterilized removable collection vessel. DETAILED DESCRIPTION OF THE INVENTION The present invention provides an automated method and apparatus for purifying proteins, particularly antibodies. As used herein, the terms "automation" and "automated" are used interchangeably and refer to the controlled operation of an apparatus, process, or system by mechanical or electronic means. Automated methods of the invention include sequential, pre-determined steps, which are internally controlled by software driven servo-actuators. Thus, the methods are standardized, efficient and free of human error. In addition to automation, the method and apparatus of the present invention utilize pre-sanitized or pre-sterilized, disposable cultureware components. As used herein, "cultureware" refers to components which come in contact with the protein- containing aqueous medium, the purified protein, or any liquid involved in the purification process. The cultureware includes a pre-packed, disposable, pre-sanitized or pre-sterilized selection means, e.g., a column packed with resin, which separates the protein from the contaminants contained in the protein-containing aqueous medium. The cultureware can further include a pre-sanitized or pre-sterilized diafiltration module which further serves to purify the protein, as well as pre-sanitized or pre-sterilized, disposable liquid reservoirs, valves, tubing, and collection vessels. As used herein, the terms "diafiltration module" and "tangential flow filtration cassette" are used interchangeably and generally refer to membrane-based ultrafiltration devices. The diafiltration module works on the tangential flow filtration principle whereby molecules over 50,000 daltons, such as proteins (e.g., antibodies such as IgG and IgM), cannot pass through the membrane but small molecules, such as buffers, can pass through. The diafiltration module is used to exchange one buffer for another and is a more efficient substitute for dialysis. In one embodiment, the diafiltration module contains a membrane having about 50cm² area and a normal molecular weight limit or cutoff of 50,000 daltons. As used herein, the terms "pre-sanitized" and "sanitized" are used interchangeably and generally refer to components which have been cleaned to reduce the presence of contaminating substances and typically packaged (to remain sanitized), before use. Components which have been pre-sanitized may also be pre-sterilized or sterile. As used herein, the term "pre-sterilized" or "sterile" are used interchangeably and generally refer to components which are free from viable contaminating organisms and typically packaged (to remain sterile), before use. Accordingly, the pre-sanitized cultureware utilized in the present invention is free of contaminants which can contaminate the purification process, such contaminants can include viable microorganisms. Moreover, the method of the invention does not require the steps of sanitizing or sterilizing the cultureware to be used, since the cultureware is already or has previously been sanitized/sterilized and is ready for use. Still further, because the cultureware is disposable, the method does not require re-sanitizing or re-sterilizing components after use. As used herein, the -term "disposable" refers to components which are designed to be used and then thrown away. For example, the pre-sanitized cultureware of the present invention can be designed to be used for a single purification run and then thrown away. Accordingly, the present invention provides the advantage of eliminating the time-consuming and labor intensive steps of pre-sanitizing or pre-sterilizing and pre-assembling the cultureware used to purify the protein. The pre-sanitized or pre-sterilized, disposable selection means is chosen according to the particular type of purification method used, such as immuno-afrfinity chromatography, affinity chromatography, ionic exchange chromatography, hydrophobic interaction chromatography, or size exclusion chromatography. Suitable selection means for these types of purification processes are well known in the art including, for example, an affinity column packed with an anti-IgM resin, a Protein A, a Protein G, or an anti-IgG resin, an ion exchange column containing a charged particle (matrix) which binds reversibly to particular proteins (e.g., a Nydac VHP-Series Protein Ion-Exchange column with a polystyrene-divinylbenzene copolymer bead and a. chemically attached hydrophilic surface), a column packed with a hydrophobic absorbent, such as cellulose, cross-linked dextrose (Sephadex), or a column containing cross-linked polystyrene with pores of varying sizes. In a particular embodiment, the selection means comprises a pre- sterilized affinity purification column, e.g., a column approximately 1.5 to 2.5 x 10 cm in length which is pre-packed with approximately 4 to 10 ml of resin, such as ar affinity ligand (binding substance), such as Protein A, Protein A analogs, Protein G, anti-IgG or anti-IgM resin. Affinity chromatography (AC) is a technique enabling purification of a biomolecule with respect to biological function or individual chemical structure- The substance to be purified is specifically and reversibly adsorbed to a ligand which., is immobilized by a covalent bond to a chromatographic bed material (matrix). Samples are applied under favorable conditions for their specific binding to the ligand. Substances of interest are consequently bound to the ligand while unbound substances are washed away. Recovery of molecules of interest can be achieved by changing experimental conditions to favor desorption. The ligand, Protein A, is a group specific ligand which binds to ttie Fc region of most IgG. It is synthesized by some strains of staphylococc s aureus and can be isolated from culture supernatants then insolubilised by coupling to agarose t> eads or silica. An alternative method is to use whole bacteria of a strain which carries large amounts of Protein A on the bacterial cell surface. Both types of gel preparation are available commercially (Pharmacia; Calbiochem). Alternatively, a recombinant form of Protein-A can be used (ProSep-rA, Millipore). An alternative to Protein A is Protein G (Anal. Chem. (1989) 61(13):1317). Protein G is a cell surface-associated protein from streptococcus that binds to IgG with high affinity. It has three highly homologous IgG-binding domains. Anti-IgM antibody can also be used as part of the selection means of the present invention to purify antibodies. In a particular embodiment, the anti-IgM antibody includes a mouse anti-human IgM monoclonal antibody attached to sepharose by cyanogen bromide (CNBr). In one embodiment, the ligand, e.g., the affinity resin, is immobilized on a solid phase. By "solid phase" is meant a non-aqueous matrix to which the ligand can adhere, such as a solid phase comprising a glass or silica surface. The solid phase may be a purification column or a discontinuous phase of discrete particles. In a particular embodiment, the solid phase is a controlled pore glass column or a silicic acid column. Optionally, the solid phase is coated with a reagent (such as glycerol) which prevents nonspecific adherence of contaminants to the solid phase. Affinity ligands and methods of binding them to solid support materials are well known in the purification art. See, e.g., Affinity Separations: A Practical Approach (Practical Approach Series), Paul Matejtschuk (Editor), Irl Pr: 1997; and Affinity Chromatography, Herbert Schott, Marcel Dekker, New York: 1997. Proteins which can be purified by the present invention include various forms of proteins, such as tumor antigens and antibodies. As further used herein, "tumor antigen" describes apolypeptide expressed on the cell surface of specific tumor cells, e.g., an idiotypic tumor antigen expressed on the surface of B cells, and which can serve to identify the type of tumor. An epitope of the tumor antigen can be any site on the antigen that is reactive with an antibody or T cell receptor. Other examples of tumor antigens include, but are not limited to human epithelial cell mucin (Muc-1 ; a 20 amino acid core repeat for Muc-1 glycoprotein, present on breast cancer cells and pancreatic cancer cells), the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA), the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-A/Mart-1, gplOO, HER2/neu, EBN-LMP 1 & 2, HPN-F4, 6, 7, prostatic serum antigen (PSA), alpha-fetoprotein (AFP), CO17-1 A, GA733, gp72, p53, the ras oncogene product, HPN E7 and melanoma gangliosides, as well as any other tumor antigens now known or identified in the future. The term "antibody," as referred to herein, includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chain thereof. Various antibody isotypes are also encompassed by the invention, including IgG, IgM, IgA, IgD, and IgE. The antibodies of this invention can be isolated from a number of sources, including without limitation, serum of immunized animals, ascites fluid, hybridoma or myeloma supernatants, conditioned media derived from culturing a recombinant cell line that expresses the immunoglobulin molecule and from all cell extracts of immunoglobulin producing cells. A purified protein, e.g., antibody, of the present invention is substantially free from host cell contaminants such as host cell proteins, nucleic acids and endotoxins. In a particular embodiment of the invention, the automated method involves purifying a protein using the steps outlined in Example 1 and the apparatus shown in Figure 1. Specifically, prior to loading the protein-containing aqueous medium, the selection means, e.g., an affinity column of approximately 1.5 to 2.5. x 10 cm in length packed with approximately 4 to 10 ml of resin, is washed. The selection means can be washed with liquid stored in the liquid reservoirs which are connected to the selection means with pre-sanitized valves and tubing. For example, the selection means can be washed with phosphate buffer saline (PBS) or a neutral buffer at a pH of about 7.2 to remove impurities, such as preservatives found in the pre-packed, pre- sterilized, disposable column. The size of the column may vary based on the type of protein being purified. For example, methods for purifying IgM antibodies use a column about 2.5 cm in diameter, while methods for purifying IgG antibodies use a column about 1.5 cm in diameter. In addition, before loading, the column can be pre-eluted with an elution buffer stored in the liquid reservoirs, such as a buffer at a low pH of about 2.4 to 3.0. The column can then be equilibrated to neutralize or increase the pH using, e.g., PBS, and is ready for loading. The protein-containing aqueous medium or supernatant is then loaded onto the pre-sterilized, disposable selection means. This can be done after adjusting the supernatant or, more preferably, is done without adjusting the supernatant. The appropriate rate for loading can be determined as is known in the art and generally involves loading at a rate of at least 0.5 to 2.5 ml/min, preferably about 5.0 ml/min. In a particular embodiment, the medium is heated and/or degassed prior to loading to reduce or eliminate the amount of dissolved gas which can accumulate in the separation means and hinder its ability to bind protein. For example, the protein- containing aqueous medium is heated to about room temperature and degassed. Once loaded, the selection means can be washed with a wash solution that is stored in a liquid reservoir to remove any residual contaminants contained in the aqueous medium, such as residual proteins from the host cells which were used to produce the protein to be purified, e.g., contaminants such as host cell proteins, nucleic acids and endotoxins. The appropriate volume and solution for removing contaminants can be determined as is known in the art. In a particular embodiment, the column is washed using a buffer, such as PBS, until the ultraviolet (UN) absorbance of the effluent is about zero as measured using standard photometric procedures. The protein is then eluted, for example, by using an acidic solution, thereby producing a protein-containing acidic eluate. In another embodiment, the salt concentration of the loaded column is changed. To elute by changing the pH of the loaded column, an acidic elution buffer can be added, such as an elution buffer containing approximately 0.05 to 0.5 M of an acid and at a pH of about 2.0 to 5.0. The appropriate volume and rate of the elution buffer can be determined by one of ordinary skill in the art. In a particular embodiment, the elution buffer is added to the column at approximately 1.0 to 2.0 ml/min for a total of about four (4) column volumes. Further, the type of elution buffer depends on the type of protein to be purified and can also be determined based on the techniques known in the art. For example, for purification of an IgM antibody, the elution buffer may comprise approximately 0.1 M glycine at about pH 2.4. For purification of an IgG antibody, the elution buffer may comprise approximately 0.1 M citrate at approximately pH 3.0. Those of ordinary skill in the art can determine the optimum molarity and pH based on the ranges and teachings provided herein. In a particular embodiment, the elution of the purified protein from the selection means can be aided by a monitoring device. For example, the absorbance at a particular wavelength of the eluate can be monitored using a photometer to determine the appropriate concentration of the eluate. Methods for eluting proteins by using a photometer are well known in the art. Generally, collection of the peaks containing the purified protein begins when the ultraviolet (UV) absorbance of the eluate begins to increase from baseline (zero). Collection continues until the UN absorbance returns to its baseline. In a particular embodiment, the volume of the peak fractions collected is about 10 to 25 ml and the peaks are collected in a pre-sanitized or pre-sterilized, reservoir contained within the purification apparatus. Following elution, the purified protein (e.g., the antibody) can be collected in a pre-sterilized, disposable collection vessel and removed from the purification apparatus. In another embodiment, the eluted protein can undergo further processing by the automated purification apparatus. For example, the eluted protein can be transferred to an acidic solution, e.g., an acidic solution containing approximately 0.1 M glycine at a pH of approximately 2.4, to ensure that the previous buffer that the protein was solubilized in has been replaced by the acidic solution. Enough volume is used so that the buffer exchange efficiency is theoretically greater than or equal to about 99.5%. Once in the acidic solution (e.g., 0.1 M glycine at pH 2.4), the protein- containing solution is treated (held) for less than approximately 16 hours at these conditions in order to inactivate any susceptible virus that may be present. However, holding the protein-containing solution for longer than 16 hours may result in degradation of the protein. Protein degradation is caused directly by the low pH which unfolds the protein irreversibly or by proteases which are more active at low pH. Degradation of proteins can be measured by using assays which characterize the structure of the proteins, such as gel electrophoresis and high performance liquid chromatography (HPLC). Degradation of proteins can also be measured by protein activity, such as potency, toxicity, or content, in a biological assay, such as in vitro cell receptor binding assays or in vitro antigen content assays. Accordingly, in one embodiment, the protein is held in the acidic solution for approximately 15.5 hours, 15 hours, 14.5 hours, 14 hours, 13.5 hours, 13 hours, 12.5 hours, 12 hours, 11.5 hours, 11 hours, 10.5 hours, 10 hours, 9.5 hours, 9 hours, 8.5 hours, 8 hours, 7.5 hours, 7 hours, 6.5 hours, 6 hours, 5.5 hours, 5 hours, 4.5 hours, 4, hours, 3.5 hours, 3 hours, 2.5 hours, 2 hours, 1.5 hours, 1 hour or less. In another embodiment, the protein is held in the acidic solution for less than 1 hour, for example for approximately 55 minutes, 50 minutes, 45 minutes, 40 minutes, 35 minutes, 30 minutes, 29 minutes, 28 minutes, 27 minutes, 26 minutes, 25 minutes, 24 minutes, 23 minutes, 22 minutes, 21 minutes, 20 minutes or less, e.g., 19, 18, 17, 16, 15, 14, 13, 12 ,11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 minute. The protein-containing acidic solution can further be neutralized by transferring it to a neutral or basic solution to neutralize the effects of the previous low pH treatment, for example, a solution containing approximately 10 mM citrate at a pH of about 5.3. During this step, a continual and gradual rise in pH occurs over the course of less than approximately 16 hours, e.g., approximately 30 minutes. In a particular embodiment, neutralization is completed within about 29-30 minutes, preferably within about 28-29 minutes, and more preferably within about 25-28 minutes of transferring the protein from the acidic solution. In another embodiment, the purified protein (e.g., antibody) is transferred into an appropriate buffer, such as a saline buffer having approximately 0.2 - 0.9% saline (NaCl). In a particular embodiment, enough volume is used so that the buffer exchange efficiency is theoretically greater than or equal to about 99.5%. In an additional embodiment, the protein (e.g., antibody) contained in the final buffer solution is filtered through a filter, for example, a filter having a pore size of approximately 0.22 μm. The purified protein is automatically deposited into a pre- sterilized collection vessel and removed from the automated purification apparatus. In a particular embodiment, the purified protein (e.g., antibody) is stored in a solution containing approximately 0.2 - 0.9% saline or further processed. In another embodiment the step of transferring the eluted antibody to different solutions occurs automatically using a pre-sterilized diafiltration module. Diafiltration is the fractionation process that washes smaller molecules through a membrane and keeps molecules of interest in the retentate. Diafiltration can be used to remove salts or exchange buffers. In discontinuous diafiltration, the solution is concentrated, and the lost volume is replaced by new buffer. Concentrating a sample to half its volume and adding new buffer four times can remove over 96% of the salt, h continuous diafiltration, the sample volume is maintained by the inflow of new buffer while the salt and old buffer are removed. Greater than 99% of the salt can be removed by adding up to seven volumes of new buffer during continuous diafiltration. In a particular embodiment, the diafiltration module contains a filtration membrane of approximately 50cm² areas having a normal molecular weight limit or cutoff of 50,000 daltons. Specifically, the diafiltration module is used to further purify the protein (e.g., the antibody) and uses the tangential flow filtration principle whereby molecules over 50,000 daltons (e.g., the antibodies, such as IgG and IgM) cannot pass through the membrane but small molecules, such as buffers, can pass through. Accordingly, the diafiltration module is used to exchange one buffer for another and is a more efficient substitute for dialysis. In a particular embodiment, the diafiltration module is sterilized using a solution containing approximately 0.1 N Sodium Hydroxide at a crossflow or feed rate of approximately 20-40 mL/min. This crossflow rate is maintained throughout the process. The 0.1 N Sodium Hydroxide is flushed out of the system using a solution containing approximately 0.1 M Glycine at a pH of about 2.4. After sanitization is complete, the protein-containing solution which was eluted from the selection means is introduced into the diafiltration module. In a particular embodiment, the present invention provides an automated method of producing a vaccine by purifying a protein and conjugating the protein to an adjuvant. More particularly, the invention provides a method for producing an autologous vaccine, i.e., a vaccine, such as an antibody vaccine, against a self-protein or idiotype (ID) antigen, such as a tumor antigen. In a particular embodiment, the antigen is a B cell antigen, such as an antibody expressed on B cell tumors (e.g., lymphomas). Accordingly, the vaccine is used to target one specific molecule which is expressed by B-cell lymphoma cells. Moreover, since each vaccine produced is patient-specific, the one time, disposable use of the cultureware used in the invention is particularly advantageous. As used herein, the term "adjuvant" refers to any substance which enhances the immune-stimulating properties of an antigen. Examples of adjuvants include, for example, keyhole limpet hemocyanin (KLH), bovine serum albumin, (BSA), and β₂ -glycoprotein I. Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as adjuvants, as well as bovine gamma globulin or diphtheria toxoid. KLH is a respiratory protein found in mollusks. Its large size (M.W. 8-9 x 10⁶ Da) makes it very immunogenic and the large number of lysine residues available for conjugation make it very useful as a carrier for haptens. The phylogenic separation between mammals and mollusks increases the immunogenicity and reduces the risk of cross-reactivity between antibodies against the KLH carrier and naturally occurring proteins in mammalian samples. KLH is obtainable both in its native form, for conjugation via amines, and succinylated, for conjugation via carboxyl groups.

Succinylated KLH may be conjugated to a hapten containing amine groups (such as a peptide) via cross-linking with carbodiimide between the newly introduced carboxyl groups of KLH and the amine groups of the hapten. Protocols for conjugating haptens to carrier proteins may be found in Antibodies: A Laboratory Manual, E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold Spring Harbor, NY, 1988) pp. 78-87. Accordingly, in one embodiment, the invention provides an automated method of producing a vaccine by purifying an antibody and conjugating the antibody to KLH. Methods for conjugating proteins (e.g., antibodies) to adjuvants (e.g., KLH) are known in the art. In general, conjugation is achieved by mixing the purified protein and the adjuvant with an appropriate catalyst under the appropriate conditions. In a particular embodiment, it may be necessary to take a sample of the purified protein (e.g., antibody) for off-line determination of the antibody concentration before the adjuvant (e.g., KLH) is added. In another particular embodiment, gluteraldehyde is added to begin the conjugation. Since gluteraldehyde is unstable, it may be added manually and can be added in concentrated form or a more dilute form. Adding gluteraldehyde in dilute form may require a diafiltration or dialysis step. After several hours, the conjugation is quenched. In a particular embodiment, the conjugation is quenched by adding glycine. The conjugated protein (e.g., antibody) can be purified further using techniques known in the art, such as dialysis. In a particular embodiment, the conjugated protein is dialyzed using saline for injection (SFI) and the product is recovered. In yet another aspect, the present invention provides an automated apparatus for obtaining a purified protein, e.g., a purified antibody, from a protein- containing aqueous medium. In one embodiment, the apparatus has the configuration shown in Figure 1. The purification apparatus comprises a pre-sanitized or pre- sterilized, disposable cultureware set, as discussed above. With reference to Figure 1, the cultureware set includes, for example, a selection means (e.g., a purification column), a diafiltration module, multiple liquid reservoirs, means for flowing liquid from the reservoirs and into the selection means and the diafiltration module, a means for diverting the effluent from the selection means and the diafiltration module, e.g., at least two reservoirs. The cultureware is capable of being installed into the purification apparatus via a single motion or "snap-on" technique and comprises mechanical and electrical interfaces for communicating with the purification apparatus. In a particular embodiment, the selection means, e.g., the affinity column, and the diafiltration module of the apparatus are connected to multiple liquid reservoirs. The reservoirs each contain liquid, such as a wash buffer, an elution buffer, or a neutralization solution, for delivery to the selection means or the diafiltration module. Accordingly, the apparatus further includes pre-sanitized or pre-sterilized means for flowing liquid from the reservoirs into the selection means, for example, pre-sterilized valves and tubing which connect the reservoirs to the selection means. The valves and tubing may allow liquid from only one reservoir at a time to pass through the selection means. Alternatively, the valves and tubing allow for liquid from more than one reservoir to pass through the selection means. In a particular embodiment, the apparatus includes a pre-sanitized or pre- sterilized means for diverting the effluent from the selection means into the diafiltration module or into a waste container. Similarly, the apparatus includes a pre-sanitized or pre-sterilized means for diverting the effluent from the diafiltration module into the pre- sterilized collection vessel or into a waste container. The purification method and apparatus of the present invention can be used in conjunction to provide a fully automated method for purifying proteins.

The present invention is further illustrated by the following examples which should not be construed as further limiting.

EXAMPLES

Example 1 : Automated Purification Technique for IgM and IgG

In a particular embodiment of the invention, the automated method involves purifying an IgM or IgG antibody using the steps outlined below and using the apparatus shown in Figure 1. Supernatant Source: Filtered supernatant containing the unpurified antibody (either IgG or IgM) is produced in an automated cell culture device (e.g., the AUTONAXID™ cell culture module). Once the desired quantity of antibody has been produced (e.g., approximately 100 mg of antibody) the automated purification module is connected to the cell culture device. The filtered supernatant is transferred to the automated Purification Module by means of a pump.

Column Set-Up: For IgM, a pre-sterilized, disposable, glass 2.5 x 10 cm column is packed with anti-IgM chromatography resin and snapped into the automated apparatus which also includes a UN monitor and electronic data recorder. Using the automated apparatus as shown in Figure 1, PBS is flowed through the column at a pump rate of 5 m /min. This same pump rate is used throughout the process. The column is pre-eluted with 0.1 M glycine at pH 2.4. The column is then equlibrated with PBS.

For IgG, a pre-sterilized, disposable 1.5 x 10 cm column is packed with Protein A chromatography resin and snapped into the automated apparatus which also includes a UN monitor and chart recorder. Using the automated apparatus as shown in Figure 1, the system is primed with PBS at a pump rate of 5mL/min. The column is pre- eluted with 0.1 M citrate at a pH of 3.0. The column is then equlibrated with PBS.

Purification: For IgM, the filtered supernatant is passed through a heater to warm it to ambient temperature prior to entering the column. Once all of the supernatant has entered the column, the column is washed with PBS to remove unbound impurities. The column is then further washed with PBS at a pH of 5. The purified IgM is eluted from the column using 0.1 M glycine at a pH of 2.4. The UN absorbing peak containing the purified IgM is collected and sent to a mixing chamber for further processing . For IgG, the filtered supernatant is passed through a heater to warm it to ambient temperature prior to entering the column. Once all of the supernatant has entered the column, the column is washed with PBS to remove unbound impurities. The purified IgM is eluted from the column using 0.1 M citrate at a pH of 3.0. The UN absorbing peak containing the purified IgG is collected and sent to a mixing chamber for further processing.

Sanitization of Diafiltration Apparatus: A membrane based ultrafiltration device is installed in the automated purification module. This cassette or device contains a membrane of 50cm area having a normal molecular weight limit or cutoff of 50,000 daltons. This device works on the tangential flow filtration principle whereby molecules over 50,000 daltons, such as the IgG and IgM, cannot pass through the membrane but small molecules, such as buffers can pass through. The tangential flow filtration cassette is used to exchange one buffer for another and is a more efficient substitute for dialysis. After installation, the tangential flow filter (TFF) cassette and system is sanitized using 0.1N Sodium Hydroxide at a crossflow or feed rate of 20- 40mL/min. This crossflow rate is maintained throughout the process. After sanitization is complete the 0.1N Sodium Hydroxide is flushed out of the system using 0.1M Glycine at a pH of 2.4. The antibody is then introduced into the system.

Low pH Treatment: The remaining steps are the same for either IgM or IgG. The antibody is diafiltered against enough volume of 0.1M Glycine pH 2.4 to ensure that the previous buffer that the antibody was solubilized in has been replaced by the 0.1M Glycine pH 2.4. Enough volume is used so that the buffer exchange efficiency is theoretically greater than or equal to 99.5%. Once in 0.1M Glycine pH 2.4, the antibody is treated (held) for 30 minutes at these conditions in order to inactivate any susceptible virus that may be present. Neutralization: After the 3 O minute treatment the antibody is diafiltered against lOmM Citrate at a pH of 5.3. The antibody is diafiltered against sufficient lOmM Citrate at a pH of 5.3 to neutralize the effects of the previous low pH treatment.

Final Buffer Exchanfie: After neutralization, the antibody is diafiltered into 0.2- 0.9%) Saline (NaCl). Again, enough volume is used so that the buffer exchange efficiency is theoretically greater than or equal to 99.5%. Once in the final buffer the antibody is filtered through a 0.22//.m filter and removed from the automated purification module. This filtered purified antibody can be stored in 0.2 - 0.9% Saline or further processed.

The foregoing particular embodiment is summarized below.

Purification Module

1) Prosep A column used for IgG purification

4mL Prosep-rA Column 1.5cm diam 5mL/min Sanitization: 0.3% HC1 pH 1.5

Pre-Equilibration: PBS pH 7.2

Pre-Elution: 0.1M Citrate pH 3.0

Equilibration: PBS pH 7.2

Load: Undiluted Culture Harvest Post Load Wash: PBS pH 7.2

Elution: 0.1M Citrate pH 3.0

Discard after one use 2) 1D12-CL4B (anti-IgM antibody) column used for IgM purification

9mL lD12-Sepharose 4B Column 2.5cm diam 5mL/min Pre-Equilibrate: PBS pH 7.2 Pre-Elution: 0.1M Glycine pH 2.4 Equilibration: PBS pH 7.2 Load: Undiluted Culture Harvest Post Load Wash 1 : PBS pH 7.2 Wash 2: PBS pH 5 Elution: 0.1M Glycine pH 2.4 Discard after one use

3) Material coming from 1 and 2 enters diafiltration module of the cultureware. Diafiltration using Pellicon XL Biomax 50 screen channel A. This would also include the 30 minute low pH hold to inactivate susceptible virus.

Pellicon XL Biomax 50 membrane area = 50cm² 20-40mL/min cross flowrate

Sanitize: 0. IN NaOH

Hold: > 30 minutes at pH 2.4 Flush: 0.1M Glycine pH 2.4

Fill Mix Chamber with Glycine pH 2.4

Precondition: 0.1M Glycine pH 2.4

Fill Mix Chamber with Ab

Diafilter against: 0.1M Glycine pH 2.4 Hold:

Diafilter Antibody against lOmM Citrate pH 5.3

Diafilter Antibody against 0.2% to 0.9% Saline

Remove Antibody from system through 0.22μm Filter 1 OmL/min

Discard Pellicon after one use

Equivalents Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Any combination of the embodiments disclosed in the dependent claims are also contemplated to be within the scope of the invention.

Incorporation by Reference All patents, pending patent applications and other publications cited herein are hereby incorporated by reference in their entirety.

Claims

We claim:

1. An automated method for purifying an antibody from an antibody-containing aqueous medium, comprising: (a) loading the medium onto a pre-sterilized, disposable selection means to absorb the antibody onto the selection means; (b) eluting the antibody into an aqueous medium; and (c) collecting the antibody in a pre-sterilized, disposable collection vessel to form a purified antibody.

2. The method of claim 1, further comprising the step of transferring the antibody into an acidic solution.

3. The method of claim 2, wherein the transfer is achieved by diafiltration.

4. The method of claim 2, wherein the antibody is held in the acidic solution.

5. The method of any one of claims 2-4, further comprising the step of transferring the antibody to a basic solution.

6. The method of claim 5, wherein the wherein the transfer is achieved by diafiltration.

7. The method of claim 5, further comprising the step of transferring the antibody to a final buffer solution.

8. The method of claim 8, wherein the wherein the transfer is achieved by diafiltration.

9. The method of any of the preceding claims, further comprising the step of washing the selection means with at least one solution prior to loading the medium.

10. The method of any of the preceding claims, further comprising the step of washing the selection means with at least one solution prior to eluting the antibody.

11. The method of claim 10, wherein the at least one solution comprises the use of PBS, glycine, and/or citrate.

12. The method of any of the preceding claims, further comprising the step of warming and or degassing the medium prior to loading the medium onto the selection means.

13. The method of any of the preceding claims, wherein the step of eluting the antibody comprises the use of a solution containing glycine or citrate.

14. The method of claim 13, wherein the solution comprises approximately 0.1 M glycine at a pH of about 2.4 or approximately 0.1 M citrate at a pH of about 3.0.

15. The method of any of the preceding claims, wherein the step of eluting the antibody comprises the use of a photometer.

16. The method of claim 2, wherein the step of transferring the antibody into an acidic solution comprises the use of a solution containing glycine.

17. The method of claim 16, wherein the solution comprises approximately 0.1 M glycine at a pH of about 2.4.

18. The method of claim 5, wherein the step of transferring the antibody to a basic solution comprises the use of a solution containing citrate.

19. The method of claim 18, wherein the basic solution comprises approximately 10 mM citrate at a pH of about 5.3.

20. The method of claim 7, wherein the step of transferring the antibody to a final buffer solution comprises the use of solution containing saline.

21. The method of claim 20, wherein the final buffer solution comprises approximately 0.2 - 0.9% saline.

22. The method of claim 7, further comprising the step of filtering the antibody contained in the final buffer solution prior to collecting the antibody in a pre- sterilized, disposable collection vessel.

23. The method of any one of the preceding claims, wherein the selection means comprises a column packed with an affinity resin.

24. The method of claim 23, wherein the affinity resin is anti-IgM resin.

25. The method of claim 23, wherein the affinity resin is selected from the group consisting of a Protein A, Protein G, and anti-IgG resin.

26. The method of any one of the preceding claims, further comprising the step of conjugating the purified antibody to an adjuvant.

27. An automated method for producing a vaccine, comprising: (a) purifying an antibody from an antibody-containing aqueous medium, comprising: (i) loading the medium onto a pre-sterilized, disposable selection means to absorb the antibody onto the selection means; (ii) eluting the antibody into an aqueous medium; (iii) collecting the antibody in a pre-sterilized, disposable collection vessel to form a purified antibody; and (b) conjugating the purified antibody to an adjuvant.

28. The method of claim 27, further comprising the step of transferring the antibody into an acidic solution prior to conjugating the antibody.

29. The method of claim 28, wherein the transfer is achieved by diafiltration.

30. The method of claim 28, wherein the antibody is held in the acidic solution.

31. The method of any one of claims 27-30, further comprising the step of transferring the antibody to a basic solution prior to conjugating the antibody.

32. The method of claim 31, further comprising the step of transferring the antibody to a final buffer solution.

33. The method of claim 27, wherein the vaccine is an autologous vaccine.

34. The method of claim 33, wherein the antibody is expressed on B cell tumor cells.

35. An automated apparatus for purifying an antibody from an antibody-containing aqueous medium, comprising pre-sterilized, disposable cultureware attached to an automated means for controlling liquid flow through the cultureware.

36. The apparatus of claim 35, wherein the cultureware comprises (a) a pre-sterilized, disposable selection means, (b) multiple, pre-sterilized, disposable liquid reservoirs, (c) pre-sterilized, disposable means for flowing liquid from the reservoirs and into the selection means, (c) a pre-sterilized, disposable means for diverting the effluent from the selection means, and (d) a pre-sterilized, disposable means for collecting effluent from the selection means.

37. The apparatus of either claims 35 or 36, wherein the selection means is an affinity resin.

38. The apparatus of claim 37, wherein the affinity resin is selected from the group consisting of anti-IgM resin, Protein A, Protein G, and anti-IgG resin.

39. The apparatus of any one of claims 35-38, wherein the means for flowing liquid into the selection means comprises a series of pre-sterilized, disposable valves and tubing which connect the reservoirs to the selection means and which allow liquid from only one reservoir at a time to pass through the selection means.

40. The apparatus of any one of claims 35-39, wherein the means for flowing liquid into the selection means comprises a series of pre-sterilized, disposable valves and tubing which connect the reservoirs to the selection means and which allow liquid from more than one reservoir at a time to pass through the selection means.

41. The apparatus of any one of claims 35-40, further comprising a means for diafiltering the purified antibody.

42. The apparatus of any one of claims 35-41, further comprising a means for monitoring the effluent from the selection means.

43. The apparatus of claim 42, wherein the means for monitoring involves measuring the pH, absorbance at a particular wavelength, or conductivity of the effluent.

44. The apparatus of any one of claims 35-43, wherein the multiple liquid reservoirs each contain a wash buffer, an elution buffer, or a neutralization solution.

45. The apparatus of any one of claims 35-44, wherein the pre- sterilized, disposable means for collecting effluent from the selection means includes at least two disposable reservoirs.