WO2015038205A1

WO2015038205A1 - Surface plasmon resonance spectroscopy method to detect residual polymer flocculants in cell culture feed streams

Info

Publication number: WO2015038205A1
Application number: PCT/US2014/041673
Authority: WO
Inventors: Nripen Singh; David Beattie
Original assignee: Emd Millipore Corporation
Priority date: 2013-09-16
Filing date: 2014-06-10
Publication date: 2015-03-19

Abstract

Methods for detecting residual polymer flocculant or stimulus responsive polymer in cell culture feed streams produced from the purification of biomolecules such as proteins, polypeptides, antibodies, vaccines and the like, by a stimuli responsive polymer, such as a solubilized or soluble polymer to capture the desired biomolecules from a solution/suspension by a precipitation mechanism. Surface Plasmon resonance spectroscopy is used to capture and detect residual polymer to levels below 1 ppm.

Description

Surface Plasmon Resonance Spectroscopy Method to Detect Residual Polymer Flocculants in Cell Culture Feed streams

This application claims priority of U.S. Provisional Application Serial No. 61/878,240 filed September 16, 2013, the disclosure of which is incorporated herein by reference.

Embodiments disclosed herein relate to the purification of biomolecules . More particularly, embodiments disclosed herein relate to methods for detecting residual polymer (e.g., polymer flocculant or stimulus responsive polymer) in cell culture feed streams produced from the purification of biomolecules such as proteins, polypeptides, antibodies, vaccines and the like, by a stimuli responsive polymer, such as a solubilized or soluble polymer to capture the desired biomolecules from a solution/suspension by a precipitation mechanism.

BACKGROUND

The general process for the manufacture of biomolecules, such as proteins, particularly recombinant proteins, typically involves two main steps: (1) the expression of the protein in a host cell, followed by (2) the purification of the protein. The first step involves growing the desired host cell in a bioreactor to effect the expression of the protein. Some examples of cell lines used for this purpose include Chinese hamster ovary (CHO) cells, myeloma (NSO) bacterial cells such as e-coli and insect cells. Once the protein is expressed at the desired levels, the protein is removed from the host cell and harvested. Suspended particulates, such as cells, cell fragments, lipids and other insoluble matter can be removed from the protein-containing fluid by filtration or centrifugation, resulting in a clarified fluid containing the protein of interest in solution as well as other soluble impurities .

The second step involves the purification of the harvested protein to remove impurities which are inherent to the process. Examples of impurities include host cell proteins (HCP, proteins other than the desired or targeted protein) , nucleic acids, endotoxins, viruses, protein variants and protein aggregates.

Conventional techniques for this purification include several chromatography steps, which can include affinity, ion exchange, hydrophobic interaction, etc. on solid matrices such as porous agarose, polymeric or glass or by membrane based adsorbers .

Efficient and ergonomic large-scale purification of biomolecules such as, e.g., therapeutic proteins including antibodies, is an increasingly important consideration for the biotechnology and pharmaceutical industries. Generally, the purification processes are elaborate and expensive and include many steps. For example, typically, in the case of proteins, proteins are produced using cell culture methods, e.g., using either mammalian or bacterial cell lines engineered to produce the proteins of interest by insertion of a recombinant plasmid containing the gene encoding that protein. In general, following the expression of the target protein, its separation from one or more undesired components including, for example, host cell proteins, media by-products and DNA, poses a formidable challenge. Such separation is especially important when the therapeutic proteins are meant for use in humans and have to be approved by the Food and Drug Administration (FDA) .

It has been demonstrated that certain polymers are especially useful in the purification of biomolecules from one or more impurities in a sample. For example, the use of polyelectrolyte polymers in flocculation to purify proteins is well established. This can be accomplished with a wide range of polymers, with the only general characteristic being the polymer must have some level of interaction with a species of interest (e.g., a target molecule or an impurity) . Further, U.S. Publ. Nos. 2008/0255027, 2009/0036651, 2009/0232737 and 2011/0020327 (the disclosures of which are incorporated herein by reference) , discuss the use of certain polymers, referred to as "smart polymers" which are soluble in an aqueous based solvent under a certain set of conditions such as pH, temperature and/or salt concentration and are rendered insoluble upon a change in one or more of such conditions and subsequently precipitate out.

Flocculation of cell culture harvest has been widely used to enhance clarification throughput and downstream filtration operations. This can be accomplished in a variety of ways including polymer treatment, chemical treatment (changes in pH) or the addition of a surfactant. Precipitation using flocculants can be used to selectively recover target proteins or to remove impurities while leaving the protein product in solution. Flocculants are effective in aggregating and precipitating cells, cell debris and proteins because of the interaction between the charges on the biomolecules and on the polyelectrolytes , creating insoluble complexes, and subsequent bridging of insoluble complexes either by residual charge interaction or through hydrophobic patches on the complexes to form target clusters.

Although the polymers can be precipitated out of solution, the precipitation step does not always remove the entire polymer present in the sample or the biological material- containing stream, thereby resulting in the presence of residual amounts of the polymer in a sample containing the biomolecule of interest. Detection of residual amounts of polymer is especially crucial when the biomolecule of interest is a therapeutic protein, e.g., when the protein is meant for use in humans and requires government approval (FDA) , since toxicity can result if the level of residual polymer is too high. However, the existing methods are unable to provide detection less than 1 ppm with a reliable accuracy in the presence of protein of interest such as antibody, process related impurities such as host cell proteins (HCP) , DNA, and colloidal particulates.

To overcome these present challenges associated with sample preparation, embodiments disclosed herein utilize a Surface Plasmon Resonance (SPR) spectroscopy method to detect the presence of polymer residuals in the presence of a biomolecule of interest (e.g., antibody) in addition to host cell proteins, DNA and colloidal particles. In accordance with certain embodiments, polymer residual analyte is selectively bound to a substrate used in SPR under optical conditions in the presence of antibody and process related impurities, if present. The captured residual polymer is detected, and the response units generated by the spectrometer are indicative of the amount of residual polymer present. SUMMARY

Embodiments disclosed herein relate to a method of detecting residual amounts of polymer used for enriching a biomolecule of interest in a sample. In addition, embodiments disclosed herein enable direct, label-free, real time detection, do not require sample processing, and can be carried out using equipment commonly found in laboratories.

In accordance with certain embodiments, a method of detecting residual amounts of polymer in a sample comprising a biomolecule of interest is provided, where the polymer is used for separating the biomolecule of interest from one or more impurities. The polymer may bind one or more impurities or it may bind the biomolecule of interest, thereby enabling separation of the biomolecule of interest from the one or more impurities. In certain embodiments, the polymer may bind both the biomolecule of interest and the one or more impurities, where the biomolecule of interest is subsequently selectively eluted while the one or more impurities remain bound to the polymer, again thereby enabling separation of the biomolecule of interest from the one or more impurities. The solution comprising the separated biomolecule of interest can be analyzed for residual polymer by surface Plasmon resonance spectrometry .

In certain embodiments, the method comprises contacting the sample with the polymer, causing the polymer to bind with the biomolecule of interest and/or impurities in the sample, and detecting the unbound residual polymer remaining in the sample with surface Plasmon resonance spectroscopy, wherein the response unit (RU) generated by the spectrometer is indicative of the amount of residual polymer in the solution comprising the biomolecule of interest.

In some embodiments, a polymer is a traditional polymer flocculant. In some embodiments, the polymer is used for clarification (i.e., binding to one or more impurities in a sample containing a biomolecule of interest and one or more impurities) . In other embodiments, the polymer is used for capture (i.e., binding to the biomolecule of interest) .

In some embodiments, a polymer is a stimulus responsive polymer. In some embodiments, the polymer is used for clarification (i.e., binding to one or more impurities in a sample containing a biomolecule of interest and one or more impurities) . In other embodiments, the polymer is used for capture (i.e., binding to the biomolecule of interest) . In still other embodiments, a polymer binds to both the biomolecule of interest and one or more impurities, where the biomolecule of interest is subsequently selectively eluted from the polymer, whereas the one or more impurities remain bound to the polymer.

DETAILED DESCRIPTION

In certain embodiments, a polymer is a stimulus responsive polymer. In certain embodiments, the polymer is used for clarification (i.e., binding to one or more impurities in a sample containing a biomolecule of interest and one or more impurities) . In certain embodiments, the polymer is used for capture (i.e., binding to the biomolecule of interest) . In still other embodiments, a polymer binds to both the biomolecule of interest and one or more impurities, where the biomolecule of interest is subsequently selectively eluted from the polymer, whereas the one or more impurities remain bound to the polymer.

Accordingly, in certain embodiments, a method for detecting residual amounts of traditional polymer flocculant or stimulus responsive polymer in a sample is provided, where the method comprises the steps of: (1) contacting a solution containing a biomolecule of interest and one more impurities with a stimulus responsive polymer; (2) applying a stimulus to the solution, thereby to precipitate the polymer and bound biomolecule; (3) removing the precipitate from the solution; (4) eluting the biomolecule; and (4) capturing and detecting residual polymer in the eluted sample using surface Plasmon resonance spectroscopy, by selectively capturing any residual polymer on an SPR sensor substrate, wherein the response units generated by the SPR is indicative of the amount of residual polymer present.

In certain embodiments, a method for detecting residual amounts of a stimulus responsive polymer comprises the steps of: (1) contacting a solution containing a biomolecule of interest and one or more impurities with a stimulus responsive polymer, where the polymer forms complexes with the one or more impurities under a set of conditions; (2) adding a stimulus to the solution, thereby to precipitate the complexes; (3) removing the precipitate from the solution; and (4) detecting residual polymer in the remaining solution containing the biomolecule of interest using surface Plasmon resonance spectroscopy,, by selectively capturing any residual polymer on an SPR sensor substrate, wherein the response units generated by the SPR is indicative of the amount of residual polymer present.

In certain embodiments, a method for detecting residual amounts of a traditional polymer flocculant or stimulus responsive polymer comprises the steps of: (1) contacting a solution containing a biomolecule of interest and one or more impurities with a stimulus responsive polymer, where the polymer forms complexes with both the biomolecule of interest as well as the one or more impurities under a first set of conditions; (2) adding a stimulus to the solution, thereby to precipitate the complexes; (3) subjecting the precipitate to a second set of conditions, thereby to selectively elute the biomolecule of interest from the complex; and (4) detecting residual polymer in the solution containing the biomolecule of interest using surface Plasmon resonance spectroscopy, by selectively capturing any residual polymer on an SPR sensor substrate, wherein the response units generated by the SPR is indicative of the amount of residual polymer present.

In certain embodiments, there is a wash step between steps (2) and (3) .

Residual polymer remaining with the recovered biomolecule can be detected by capturing residual polymer on a sensor substrate and subjecting the sensor substrate to surface Plasmon resonance spectrometry. The results of the detection can be used to assess whether the residual polymer is below a certain predetermined level, such as 1 ppm. Surface Plasmon resonance spectroscopy measures adsorption of materials onto planar substrates, such as silver or gold substrate surfaces (the sensor chip) having molecules immobilized thereon. The surface plasmon resonance (SPR) phenomenon occurs when polarized light, under conditions of total internal reflection, strikes an electrically conducting (e.g., gold) layer at the interface between media of different refractive index: the glass of a sensor surface (high refractive index) and a buffer (low refractive index) . A wedge of polarized light, covering a range of incident angles, is directed toward the glass face of the sensor surface. Reflected light is detected. Electric field intensity, known as an evanescent wave, is generated when the light strikes the glass. This evanescent wave interacts with, and is absorbed by, free electron clouds in the substrates, generating electron charge density waves called plasmons and causing a reduction in the intensity of the reflected light. The resonance angle at which this intensity minimum occurs is a function of the refractive index of the solution close to the gold layer on the opposing face of the sensor surface.

Residual polymer in the samples of the embodiments disclosed herein bind to the immobilized molecules on the sensor surface, and the refractive index at the interface between the sensor surface and the solution is altered to a degree proportional to the change in mass at the surface. This alteration can be detected in real time, and the generated signal is indicative of the amount or concentration of bound polymer.

Suitable sensor substrates include glass substrates coated with a metal such as gold or silver. The metal layer is covered with a suitable matrix such as a dextran or carboxylated dextran matrix, which acts as an immobilized substrate to which molecules can be attached. The matrix can be modified with suitable ligands depending upon the polymer desired to be bound, such as carboxymethylated dextran covalently attached to a gold surface via carboxyl groups. Suitable sensor chips include CM5 and CM7 sensor chips associated with a Biacore SPR spectrometer. In certain embodiments, the spread in capture levels for residual polymer is broader for the CM7 chip, and requires additional regeneration, and thus the CM5 chip is preferred. Other substrates include surface modified with various charged polymers including poly (acrylic acid), poly (methacrylic acid), poly (vinyl ) sulfonic acid), poly (styrene) sulfonic acid, DNA modified including amine-modified ssDNA, hydrophobic groups such as butyl methacrylate, phenyl methacrylate, hexyl methacrylate, etc.

Suitable agents for regenerating sensor surfaces include a mix of NaOH, NaCl and Tween-20. In order to circumvent the problem of gel formation within hours of preparation of NaOH/NaCl/Tween-20 solution, two stock regeneration solutions of 3M NaCl/100 mM NaoH and 3M NaCl/1% Tween-20 can be prepared. Equal volume aliquots of these two solutions can be mixed and immediately injected across the sensor surface to regenerate the surface.

In certain embodiments, the SPR spectrometer includes a spectrophotometer for receiving a first signal and a second signal from the sensor surface, the second signal being received at a time after residual polymer in the sample to be tested is captured on the sensor surface. A processing unit calculates and compares the properties of the first received signal and the second received signal to determine the presence of the polymer.

In certain embodiments, the stimulus responsi e polymer is responsive to a salt stimulus or a pH stimulus. In a particular embodiment, the polymer is responsive to a multivalent anion stimulus.

Exemplary stimulus responsive polymers include, but are not limited to, poly (diallyldiamine ammonium chloride), Polyethylenimine, polyvinylpyridine, polyvinylamine, polyallylamine .

Exemplary stimulus responsive polymers also include, but are not limited to, polyvinylamine, polyallylamine, polyvinylpyridine, copolymers of vinylpyridine and polymers modified with a hydrophobic group.

In certain embodiments, the polymer is poly (4-vinyl pyridine) .

In other embodiments, the polymer is polyvinylamine. In still other embodiments, the polymer is polyallylamine. In still further embodiments, the polymer is a polyallylamine or polyvinylamine polymer modified with a hydrophobic group. In a particular embodiment, the polyallylamine has a molecular weight of 150 kDa, where 30% of its amine groups are covalently modified through a reaction with benzylchloride . In another particular embodiment, the polymer is poly (diallyldiamine ammonium chloride) .

In certain embodiments, the stimulus responsive polymer comprises a polyelectrolyte backbone modified with one or more hydrophobic groups. In certain embodiments, the stimulus responsive polymer is a polymer disclosed in US Publ . No. 2011/0313066, the disclosure of which is hereby i corporated by reference. In certain embodiments, the stimulus responsive polymer comprises the following structure:

wherein the polymer comprises a carbon containing poly- electrolyte backbone; R i and R2 are charged groups which form a part of the backbone; R3 is a hydrophobic group attached to a charged group in the backbone; and n is the number of monomeric units in the polymer, wherein n is equal to or greater than 2. In certain embodiments, the stimulus responsive polymer comprises the following

structure :

4 R5 wherein the polymer comprises a carbon containing poly- electrolyte backbone; Ru R2 and R3 are charged groups which form a part of the backbone; R4 is a hydrophobic group attached to a charged group in the backbone; R5 is a group possessing a charge opposite of the charge found in Ru R2 or R3 ; and n is the number of monomeric units in the polymer, wherein n is equal to or greater than 2. In certain embodiments, the stimulus responsive polymer comprises the following structure:

wherein the polymer comprises a carbon containing poly- electrolyte backbone; R 1, R2 and R3 are amine groups which form a part of the backbone; R4 is a phenyl group which is attached to 5% to 75% of the Ru R2 or R3 groups; R5 is a cation exchange group modifying 5% to 50% of the RD R2 or R3 groups; and n is the number of monomeric units in the polymer, wherein n is egual to or greater than 2.

In certain embodiments, the polymer is responsive to addition of multivalent anions, for example, phosphate or citrate ions.

In certain embodiments, the polymer is soluble under a certain set of process conditions such as one or more of pH, salt concentration, temperature, light, or electrical field, and is able to interact and complex with insoluble impurities (cells, debris, etc.) and a fraction of the soluble impurities, and is rendered insoluble and precipitates out of solution upon a change in conditions (temperature, salt concentration, light, electrical field, or pH) , e.g. a stimuli responsive polymer. Only when precipitated out of solution, the polymer is capable of reversibly binding to one or more desired biomolecules within the stream (protein, polypeptide, etc) in an unclarified cell broth. The precipitate can then be removed from the stream, such as by being filtered out from the remainder of the stream and the desired biomolecule is recovered such as by selective elution from the precipitate. The stream is then discarded removing with it the great majority of the impurities of the mixture such as cell culture media, anti foam materials, additives, and soluble components.

The precipitate that contains the polymer, impurities such as cells and cell debris, host cell proteins, DNA and the like and the desired biomolecule, can be washed one or more times to ensure that any impurities in the liquid remaining in the precipitate or entrapped in or on the polymer have been removed. The biomolecule of interest can then be recovered, such as by selective elution of the target biomolecule from the precipitate by altering the ionic strength and/or pH conditions of the solution while the impurities, including soluble and insoluble material, remain complexed with the precipitated polymer. The purified target biomolecule is recovered in the elution pool and the precipitated polymer- impurity complex is discarded.

Suitable conditions for selective polymer capture include high pH (e.g, . pH greater than 7) and high salt concentration (e.g., salt concentration greater than 150 mM, preferably greater than 200 mM, most preferably 300 mM or greater) , where polymer capture can be as much as thirty times greater than protein capture. PURIFICATION

In certain embodiments, one or more polymers soluble in a liquid phase are used to selectively bind to one or more desired biomolecules in a solution/suspension by a precipitation mechanism and which polymer can also be removed, if present, in any excess, by the same mechanism. By way of example, this can best be described in the context of protein purification although it can be used to purify any solute from complex mixtures as long as the mechanism of removal applies to the specific solute of interest.

The one or more polymers can be used in excess unlike flocculants and can be recovered essentially completely from the mixture by the precipitation action. This allows one to operate the purification step with greater windows of use and without having to calculate the precise amount of material that needs to be used.

Certain polymers undergo changes in properties as a result of changes in the environment (stimuli) in which they are in, i.e. stimuli responsive polymers. The most common polymer property to change as a result of a stimulus is solubility and the most common stimuli relating to solubility are temperature, salt concentration and pH. As an example, a polymer may remain in solution as long as the pH, salt level or temperature is maintained within a certain range but it will precipitate out of solution as soon as the condition is changed outside of said range. Certain polymers, such as poly(N-vinyl caprolactam) , poly (N-acryloylpiperidine) , poly(N- vinylisobutyramide) , poly (N-substituted acrylamide) including [poly (N-isopropylacrylamide) , poly (N, ' -diethylacrylamide) , and poly (N-acryloyl- ' -alkylpiperazine) ] and hydroxyalkylcellulose are examples of polymers that exhibit solubility changes as a result of changes in temperature. Other polymers, such as copolymers of acrylic acid and methacrylic acid, polymers and copolymers of 2 or 4- vinylpyridine and chitosan exhibit changes in solubility as a result of changes in pH or salt. For example, the mixture comprising the biomolecule of interest and one or more impurities may be provided at a set of pH conditions. One or more polymers soluble in the mixture under the set of pH conditions and capable of reversibly and selectively binding to the biomolecule may be added to the mixture and mixed throughout the mixture. The set of pH conditions in the mixture is then changed to a pH effective to precipitate the one or more polymers and to neutralize the one or more polymers to an extent sufficient to cause impurities bound to the one or more precipitated polymers by charge interaction to solubilize. The set of pH conditions is further changed in the mixture to approach the isoelectric point of the biomolecule, thereby causing the biomolecule to bind to the one or more polymers by hydrophobic interaction. The precipitated polymer and bound biomolecule can be separated from the mixture and residual polymer can be captured and detected by SPR.

For example, monoclonal antibodies can be captured by a polymer. Such monoclonal antibodies are by their very nature hydrophobic. However, at low pH, (e.g., a pH of about 2), the monoclonal antibodies carry a positively charged counter ion that renders the Mab soluble in aqueous solutions. This positively charged counter ion is not permanent; if it is shed by the Mab at higher pH (i.e., the Mab is no longer a salt), the Mab becomes hydrophobic and thus insoluble in aqueous solutions. As the pH is raised, each repeat unit of the Mab having a positively charged counter ion is neutralized, one-by- one, until ultimately, enough of the positively charged counter ions have been shed to render the Mab predominantly hydrophobic. The hydrophobic Mab then precipitates and binds to the polymer by hydrophobic interaction. As some of these polymers may not have an ability to selectively bind or elute the desired molecules of interest they need to be modified with ligands or chemical groups that will complex with the desired molecule and hold it in complex and then release the desired molecule under the appropriate elution conditions. Suitable chemical groups can include but are not limited to carboxylated groups and pyridine groups formed as part of the polymer or attached to the polymer. Ligands such as chemical mimics of affinity ligands may be used. Such ligands include but are not limited to natural ligands or synthetic ligands such as mercaptoethylpyridine (MEP) , mercaptoethylpyrazine, MEB, 2-aminobenzimidazole (ABI), AMBI, 2-mercapto-benzoic acid (MBA), 4-amino-benzoic acid (ABA), 2-mercapto-benzimidazole (MBI) and the like.

Depending upon the polymer used, the process used can vary .

In certain embodiments, most of the insoluble impurities, such as cells and cell debris, can be removed from the liquid before the capture polymer is used. This may be done by classic methods such as centrifugation of the cell batch and/or clarification through depth filters and the like. Optionally, and preferably, the impurities or at least a portion of the impurities are removed via an impurity removing soluble polymer as described below. Such polymers are dissolved and added to the liquid and precipitated to remove the impurities upon a change in stimuli such as temperature, pH, salt concentration and the like.

Suitable temperature sensitive soluble polymers include but are not limited to functional copolymers of N- isopropylacrylamide, functionalized agarose and functionalized polyethylene oxide.

Suitable pH sensitive soluble polymers include but are not limited to cationic polyelectrolytes and anionic polyelectrolytes. Suitable cationic polyelectrolytes are selected from the group consisting of chitosan, polyvinylpyridines , primary amine containing polymers, secondary amine containing polymers and tertiary amine containing polymers. Suitable anionic polyelectrolytes selected from the group consisting of copolymers of acrylic acid, methacrylic acid and methyl methacrylate.

In certain embodiments, at least some of the cell mass and larger impurities are removed by filtration, centrifugation or the like and then one or more of the soluble polymers that are capable of binding to the molecule of interest are added to the remaining fluid. The fluid may either be preconditioned or the fluid can be conditioned upon addition of the polymer (s) or the polymer (s) can be added to a carrier liquid that is properly conditioned to the temperature or pH or other stimulus. The polymer (s) is allowed to circulate thoroughly with the fluid and then the stimulus is changed (change in pH, temperature, salt concentration, etc.) and the desired biomolecule and polymer (s) precipitate out of solution. The desired biomolecule is then recovered from the polymer (s) such as by elution and the like.

In certain embodiments, an impurity removing polymer system can be used as described below to remove the impurities without any pretreatment such as clarification, filtration, centrifugation or the like, by adding one or more of the impurity removing soluble polymer (s) such as poly(N-vinyl caprolactam) , poly (N-acryloylpiperidine) , poly(N- vinylisobutyramide) , poly (N-substituted acrylamide) including [poly (N-isopropylacrylamide) , poly (N, ' -diethylacrylamide) , and poly (N-acryloyl- ' -alkylpiperazine) ] , hydroxyalkylcellulose, copolymers of acrylic acid and methacrylic acid or methacrylic acid and methyl methacrylate, polymers and copolymers of 2 or 4-vinylpyridine and chitosan to the starting fluid. The fluid may either be preconditioned or the fluid can be conditioned upon addition of the impurity removing polymer (s) or the polymer (s) can be added to a carrier liquid that is properly conditioned to the temperature or pH or other stimulus. The impurity removing polymer (s) is allowed to circulate thoroughly with the fluid and then the stimulus is changed (change in pH, temperature, salt concentration, etc) and the impurities and polymer (s) precipitate out of solution. The remaining liquid that contains the desired molecule and perhaps some amount of impurities (other proteins, viruses, etc) that are not removed by the selected polymer (s) are then recovered such as by filtering it through a suitably sized membrane (microfiltration or ultrafiltration) , centrifugation where the desired molecule is in the supernatant that is recovered and the like. This can be conducted in the bioreactor, especially if it is a disposable plastic bioreactor or it can be done in a separate container such as a stainless steel vat or tank or plastic bag, tank or the like.

Then one or more of the soluble polymers that are capable of binding to the molecule of interest is added to the remaining fluid. The soluble binding or capture polymer (s) such as poly(N-vinyl caprolactam), poly (N-acryloylpiperidine) , poly (N-vinylisobutyramide) , poly (N-substituted acrylamide) including [poly (N-isopropylacrylamide) , poly(N,N^r- diethylacrylamide) , and poly (N-acryloyl- ' -alkylpiperazine) ] , hydroxyalkylcellulose, copolymers of acrylic acid and methacrylic acid or methacrylic acid and methyl methacrylate, polymers and copolymers of 2 or 4-vinylpyridine and chitosan contain a functional group and/or ligand that binds to the biomolecule of interest. The fluid may either be preconditioned or the fluid can be conditioned upon addition of the polymer (s) or the polymer (s) can be added to a carrier liquid that is properly conditioned to the temperature or pH or other stimulus. The polymer (s) is allowed to circulate thoroughly with the fluid and then the stimulus is changed (change in pH, temperature, salt concentration, etc) and the desired biomolecule and polymer (s) precipitate out of solution. The desired biomolecule is then recovered from the polymer (s) such as by elution.

The processes will generally involve having one or more conditions of the liquid of the mixture, at the correct pH, temperature or salt concentration or other condition used to cause the polymer (s) to become soluble and then adding the polymer (s) either directly or already solubilized in a carrier liquid, such as water, to the mixture. In many instances, the mixture will be at the proper condition to allow the polymer (s) to be simply added to the mixture.

In other instances, the mixture may need to be conditioned or modified to be at the desired condition. This modification or conditioning can be by modifying the mixture first and then adding the polymer (s), by adding the polymer (s) to a carrier liquid that is conditioned to the desired state and simply adding it to the mixture such that the carrier liquid is sufficient to cause the mixture to thus reach that condition or to do both. The conditions of the liquid in the mixture are then changed (pH, temperature, salt content, combinations thereof, etc) that causes the polymer (s) to become insoluble and precipitate out of the mixture as a dispersed solid suspension. The mixture and the suspended insoluble polymer (s) are then mixed to ensure that the entities of the mixture and the insolubilized polymer (s) have sufficient and intimate contact with each other. The insoluble polymer (s) bind the one or more desired biomolecules it contacted while in the mixture and continue to bind to it thereafter until elution conditions are met to remove the biomolecule from the polymer. The precipitate is separated such as by centrifugation or filtration or gravity and time with the liquid portion being decanted. The recovered polymer/desired biomolecule ( s ) is washed one or more times to remove any residual impurities or contaminants and then the biomolecule ( s ) is eluted from the polymer under conditions that cause the biomolecule entity to release from the polymer so it can be recovered and subjected to further processing. Residual polymer then can be detected by surface plasmon resonance spectroscopy.

One polymer or a blend of polymers may be used and it is meant to cover both embodiments whenever the term polymer, polymer (s) or one or more polymers is used hereafter.

As discussed above, the polymer may be added directly to the mixture either as is or in a conditioned state that enhances the solubility of the polymer as it is added. Alternatively, it can be added to a carrier liquid in which it is soluble and which carrier preferably is also compatible with the mixture. One such carrier liquid is water, water adjusted to a specific pH using acid or base, another is an aqueous based solution such as saline, physiological buffers or blends of water with an organic solvent such as water/alcohol blends. The selection of carrier liquid is dependent on the mixture to which it is added as to what is preferred and tolerated. The polymer is added to the carrier liquid that either has already been conditioned (such as pH adjusted or heated to a desired temperature or heated to a desired temperature with the addition of one or more salts or cooled to the desired temperature with or without one or more salts) or it can be added and then the carrier is conditioned to cause the solubilizing of the polymer in the carrier . The carrier/soluble polymer blend is then added to the mixture.

The mixture may be contained in a mixing vessel such as a tapered bottom metal (preferably stainless steel more preferably 304 or 316L stainless steel) or glass or plastic bag, vat or tank. Alternatively, especially when a cell culture or microbial or yeast culture, it may be the bioreactor or fermentor in which the cells have been grown. It may also be a disposable bioreactor or fermentor or a disposable mixing bag such as a plastic bag as is available from EMD Millipore Corporation of Billerica, Mass. The mixture and polymer are brought into intimate contact through a mixing action that may be done by a magnetic stirred bar, a magnetic driven mixer such as a NovAseptic® mixer available from EMD Millipore Corporation of Billerica, Mass., a Lightning-type mixer, a recirculation pump, or a rocking motion closed mixing bag or bioreactor or fermentor, such as is shown in US 2005/0063259A1 or an airlift type of mixer or reactor in which rising bubbles in the liquid cause a circulatory pattern to be formed .

Alternatively, the mixture and polymer (either by itself or in a carrier) can be in separate containers and mixed in line in a static blender. The blend can either then go to a container or to a centrifuge or a filter where the precipitated polymer and its bound one or more entities is separated from the remainder of the mixture and then is further processed.

In another embodiment, the mixture and polymer (either by itself or in a carrier) are blended together in the container holding the mixture and further mixed in line in a static blender. The blend can either then go to a container or to a centrifuge or to a filter where the precipitated polymer and its bound one or more entities is separated from the remainder of the mixture. Then the precipitated polymer (whichever contains the target or desired biomolecule) is further processed .

Using centrifugation, one can easily and quickly separate the precipitated polymer from the remainder of the liquid mixture. After centrifugation, the supernatant, generally the remainder of the mixture is drawn off. The precipitated polymer is further processed. If desired the supernatant may be subjected to one or more additional polymer precipitation steps to recover even more of the desired biomolecule.

Simple decantation may also be used if desired.

Filtration can be accomplished in a variety of manners. Depending upon the size of the polymer as it is precipitated; one may use one or more filters of varying sizes or asymmetries. The selection of type and size of filter will depend on the volume of precipitate to be captured.

Membrane based filters, preferably microporous membranes can be used in the embodiments disclosed herein. Such filters are generally polymeric in nature and can be made from polymers such as but not limited to olefins such as polyethylene including ultrahigh molecular weight polyethylene, polypropylene, EVA copolymers and alpha olefins, metallocene olefinic polymers, PFA, MFA, PTFE, polycarbonates, vinyl copolymers such as PVC, polyamides such as nylon, polyesters, cellulose, cellulose acetate, regenerated cellulose, cellulose composites, polysulfone, polyethersulfone, polyarylsulfone, polyphenylsulfone, polyacrylonitrile, polyvinylidene fluoride (PVDF) , and blends thereof. The membrane selected depends upon the application, desired filtration characteristics, particle type and size to be filtered and the flow desired. Suitable membrane based filters include DURAPORE® PVDF membranes available from Millipore Corporation of Billerica Mass., MILLIPORE EXPRESS® and MILLIPORE EXPRESS® PLUS or SH PES membranes available from EMD Millipore Corporation of Billerica Mass.

Depending on the mixture, polymer and the nature of biomolecule may be hydrophilic or hydrophobic. Suitable membranes are hydrophilic and are low in protein binding.

The membrane may be symmetric in pore size throughout its depth such as DURAPORE® PVDF membranes available from EMD Millipore Corporation of Billerica Mass., or it may be asymmetric in pore size through its thickness as with MILLIPORE EXPRESS^® and MILLIPORE EXPRESS^® PLUS or SH PES membranes available from EMD Millipore Corporation of Billerica Mass. It may contain a prefilter layer if desired, either as a separate upstream layer or as an integral upstream portion of the membrane itself.

The pore size of the membrane can vary depending upon the polymer and mixture selected. Generally, it has an average pore size of from about 0.05 micron to 5 microns, preferably from about 0.05 micron to about 1 micron, more preferably from about 0.05 to about 0.65 micron.

The membrane filter may run in a dead-end or normal flow (NF) format or a tangential flow (TFF) format. The choice is dependent on a number of factors, primarily the user's preference or installed filtration equipment as either works. A TFF process and equipment is preferred when large amounts of polymer and molecule are to be recovered as TFF is less subject to clogging or fouling than NF methods.

In a first step, the clarified mixture may be either conditioned to the correct parameter (s) to maintain the capture polymer of choice in solution or if the conditions of the mixture are already such that the polymer (s) become soluble in the mixture, no further conditioning may be required. Alternatively, the polymer (s) may be added as a solid to an unconditioned mixture and then the mixture (containing the solid polymer (s) ) may be conditioned to the correct parameters to dissolve the capture polymer (s) in the mixture. In a second step, the polymer (s) may be mixed with the mixture in the stream for desirable amount of time to create suitable distribution to make intimate contact with all the constituents of the mixture. In a third step, the conditions of the liquid in the mixture are then changed (pH, temperature, salt content, combinations thereof, etc) to cause the polymer (s) to become insoluble and precipitate out of the mixture as a dispersed solid suspension while retaining the biomolecule. The mixture and the precipitated polymer (s) are then separated from each other in a fourth step. As discussed above the precipitate and remaining mixture may be separated by centrifugation or filtration.

The precipitate can then optionally be washed one or more times with water, a buffer or an intermediate wash solution as are known in the art to remove any impurities from the precipitate or any non-specifically bound impurities from the precipitate .

The desired biomolecule is then recovered. Preferably it eluted from the polymer such as by the addition of a buffer at a pH (acidic or basic depending on the molecule and the polymer used) and the salt concentration or temperature of the solution is changed to allow for the recovery of the desired molecule free of the polymer.

Residual polymer in the resulting eluted solution containing the desired molecule can then be detected by capturing any residual polymer on a suitable sensor substrate, and detecting the captured polymer using surface Plasmon resonance spectroscopy.

An exemplary process uses a pH dependent polymer such as poly (4-vinylpyridine-co-styrene) , (10% mol styrene) which has an affinity for the desired biomolecule in the insoluble state. In a first step, the mixture is clarified from a harvested broth so as to remove most of the insoluble impurities such as cells, cell debris and the like. The mixture is then either conditioned to the correct pH (in this case to a pH below about 5.0) to maintain the polymer of choice in solution before, during or after the introduction of the polymer or it is already at the desired condition in a second step. In a third step which may occur separately before, simultaneously or after the second step, the polymer is added to a carrier liquid under conditions that allow it to go into solution and then mixed to make intimate contact with all the constituents of the mixture so that the polymer can complex with the desired molecule (for example a IgG molecule) .

In a fourth step, the mixture conditions are changed to cause the polymer to precipitate out of solution in the form of a dispersed solid suspension.

If desired, one may conduct one or more additional steps to ensure that all polymer has been removed from the mixture by subjecting the mixture to a step containing a material that will remove any residual polymer from the mixture such as ion exchange resin, activated carbon, alumina, diatomaceous earth and the like. Typically however the polymer is removed on the first precipitation and such additional steps are not necessary.

As discussed above, the precipitate and remaining mixture may be separated by centrifugation or filtration in a fifth step. Optionally, the polymer and complex are washed one or more times while being kept under conditions such that the polymer/complex precipitate remains undissociated .

To recover the molecule, the conditions are changed, such as by lowering the pH of the solution, so as to solubilize the polymer and to break the complex between the ligand and the molecule in a sixth step. In a seventh step, the solution conditions are changed, such as by increasing the ionic strength to selectively cause the polymer to precipitate out of the solution, thereby leaving substantially all of the biomolecule of interest in solution. The supernatant (liquid) is removed from the precipitated polymer by filtration or centrifugation or the like and is recovered in an eighth step.

The biomolecule of interest after having been recovered, may undergo one or more known additional process steps such as chromatography steps including but not limited to ion exchange, hydrophobic interaction or affinity chromatography, various filtration steps such as microfiltration ultrafiltration, high performance tangential flow filtration (HPTFF) with or without charged UF membranes, viral removal/inactivation steps, final filtration steps and the like. Alternatively, the eluted biomolecule of interest may be used as is without the need for further purification steps. Also the biomolecule of interest may undergo further purification without the need for chromatography steps.

Residual polymer can be measured by SPR in accordance with the embodiments disclosed herein at any point or at multiple points during each of these additional steps. The results of the detection can be used to assess whether the residual polymer is below a certain predetermined level, such as 1 ppm.

In a further embodiment, all process steps after cell harvest/clarification through affinity chromatography are eliminated. A biological process under this embodiment would consist of capture of the biomolecule directly from the clarified mixture via the polymer-based purification step, separation of the biomolecule from the polymer and the remainder of the mixture, two or more steps of viral removal or inactivation such as removal through viral filters or inactivation through treatment with heat, chemicals or light, a compounding step into the correct formulation and a final filtering before filling the compounded biomolecule into its final container for use (vial, syringe, etc) .

In any of the embodiments, the biomolecule such as a protein thus recovered may be formulated in a pharmaceutically acceptable carrier and is used for various diagnostic, therapeutic or other uses known for such molecules.

CLARIFICATION

In certain embodiments, a liquid phase or solubilized polymer that has a capability, such as affinity or charge or hydrophobicity and the like, to remove undesirable soluble and suspended impurities from a fluid containing a desirable biomolecule of interest is used. Suitable polymers have an affinity or electrostatic ability. The biomolecule of interest is then recovered and further processed as desired or required .

More specifically, certain embodiments relate to the process of using one or more polymers soluble in a liquid phase to remove impurities from a solution/suspension by a precipitation mechanism and which polymer can also be removed, if present, in any excess, by the same mechanism. By way of example, this idea can best be described in the context of protein purification although it can be used to purify any solute from complex mixtures as long as the mechanism of removal applies to the specific solute of interest.

Certain polymers, such as poly (N-isopropylacrylamide) , agarose, polyethylene oxide, etc. are examples of polymers that exhibit solubility changes as a result of changes in temperature. Other polymers, such as certain catonic and anionic polyelectrolytes, especially poly (4-vinylpyridine) , poly (2-vinylpyridine) , copolymers of 4-vinyl pyridine or 2- vinyl pyridine with other monomers such as styrene, butyl methacrylate, etc., chitosan and copolymers of acrylic acid or methacrylic acid with other monomers such as methyl methacrylate are examples of polymers that exhibit changes in solubility as a result of changes in pH and/or salt concentration.

The precise mechanism is not currently known. It may be that the polymer (s) interact with the entity or entities while in a soluble state and continue to bind to them upon precipitation. It may also be that the polymer and/or entity (s) bind to one another as the polymer is in the process of precipitating. It may be another mechanism as yet unknown to the inventor at this time. The inventors do not wish to be bound to any particular theory of what mechanism is being used; any all such mechanisms and phenomena are encompassed herein .

Although not bound by any particular theory, it is believed that at low pH, negatively charged impurities bind to the positively charged polymer in solution. As the pH is raised, the polymer-impurity bound complex becomes insoluble and precipitates. The polymer does not bind the Mabs in this case, because the pH is not raised to a level high enough that the Mabs become hydrophobic and precipitate; instead the pH is low enough at all times such that the Mabs remain sufficiently positively charged and remain in solution.

For example, a method for purifying a biomolecule of interest such as a monoclonal antibody from a mixture containing a member selected from the group consisting of host cell protein, cell, cell fragment, nucleic acid, virus, pyrogen and endotoxin impurities may comprise providing the mixture at a set pH, adding to the mixture one or more polymers, such as polyvinylpyridine and copolymers of vinylpyridine, solubilizable in the mixture under the set pH, mixing the one or more solubilized polymers throughout the mixture, thereby causing the impurities to bind to the one or more polymers by charge interaction; changing the pH in the mixture to a pH effective to precipitate the one or more polymers and bound impurities, while maintaining the pH below the isoelectric point of the biomolecule of interest (e.g., the monoclonal antibody); and recovering the biomolecule of interest. Residual polymer in the recovered solution containing the biomolecule of interest can be captured and detected by SPR.

Depending upon the polymer used, the process used can vary. However, the processes will generally involve having one or more conditions of the liquid or the mixture, such as a cell broth, at the correct pH, temperature, temperature and salt concentration or other condition used to cause the polymer (s) to become soluble and then adding the polymer (s) either directly or already solubilized in a carrier liquid, such as water, to the mixture. In many instances the mixture will be at the proper condition to allow the polymer (s) to be simply added to the mixture. In other instances, the mixture may need to be conditioned or modified to be at the desired condition. This modification or conditioning can be by modifying the mixture first and then adding the polymer (s), by adding the polymer (s) to a carrier liquid that is conditioned to the desired state and simply adding it to the mixture such that the carrier liquid is sufficient to cause the mixture to thus reach that condition or to do both. The mixture and the solubilized or soluble polymer (s) are then mixed to ensure the polymer (s) is solubilized, and that the entities of the mixture and the solubilized polymer (s) have sufficient and intimate contact with each other. The conditions of the liquid in the mixture are then changed (pH, temperature, salt content, combinations thereof, etc.) that causes the polymer (s) to become insoluble and precipitate out of the mixture as a solid while either still remaining bound to the one or more entities it contacted while soluble in the mixture or to bind to the entities as it precipitates and continue to bind to it thereafter. The precipitate and remaining mixture are then separated such as by centrifugation or filtration or gravity and time with the liquid portion being decanted. Depending on what was bound to the precipitate, it is either disposed of (if it bound to impurities) or treated (such as by elution and or washing) one or more times to remove any residual impurities or contaminants and then sanitized for reuse .

One polymer or a blend of polymers may be used and it is meant to cover both embodiments whenever the term polymer, polymer (s) or one or more polymers is used hereafter. As discussed above the polymer may be added directly to the conditioned mixture. Alternatively, it can be added to a carrier liquid in which it is soluble and which carrier preferably is also compatible with the mixture, as discussed previously.

The mixture may be contained in a mixing vessel such as a tapered bottom metal (preferably stainless steel more preferably 304 or 316L stainless steel) or glass vat or tank. Alternatively, especially when a cell culture or microbial or yeast culture is used, it may be the bioreactor or fermentor in which it has been grown. It may also be a disposable bioreactor or fermentor or a disposable mixing bag such as a plastic bag as is available from EMD Millipore Corporation of Billerica, Mass. The mixture and polymer are brought into intimate contact through a mixing action that may be done by a magnetic stirred bar, a magnetic driven mixer such as a NovAseptic® mixer available from EMD Millipore Corporation of Billerica Mass., a Lightning-type mixer, a recirculation pump, or a rocking motion closed mixing bag or bioreactor or fermentor, such as is shown in US 2005/0063259A1 or an airlift type of mixer or reactor in which rising bubbles in the liquid cause a circulatory pattern to be formed.

Alternatively, the mixture and polymer (either by itself or in a carrier) can be in separate containers and mixed in line in a static blender. The blend can either then go to a container or to a centrifuge or a filter where the polymer is caused to precipitate and the precipitated polymer and its bound one or more entities is separated from the remainder of the mixture. Then at least the remainder of the mixture is further processed. In another embodiment, the mixture and polymer (either by itself or in a carrier) are blended together in the container holding the mixture and further mixed in line in a static blender. The blend is then treated to cause precipitation of the polymer and its bound entity (s) . It can either then go to a container or to a centrifuge or to a filter where the precipitated polymer and its bound one or more entities is separated from the remainder of the mixture. Then at least the filtrate is further processed.

Using centrifugation, one can easily and quickly separate the precipitated polymer from the remainder of the liquid mixture. After centrifugation, the supernatant, generally the remainder of the mixture is drawn off. Either the drawn off mixture or the precipitated polymer or both if desired is further processed.

Simple settling of the precipitated solids and decantation of the supernatant fluid may also be used if desired .

Filtration can be accomplished in a variety of manners. Depending upon the size of the polymer as it is precipitated; one may use one or more filters of varying sizes or asymmetries.

The selection of type and size of filter will depend on the volume of precipitate to be captured and whether one wishes to further process the precipitated polymer or just the remainder of the mixture, as discussed previously. The membrane filter may run in a deadend or normal flow (NF) format or a tangential flow (TFF) format. The choice is dependent on a number of factors, primarily the user's preference or installed filtration equipment as either works. Depth filters such as the MILLISTAK+® depth filters, in either lenticular or POD format, or POLYGARD® wound filters available from EMD Millipore Corporation of Billerica Mass. allows one to trap a large volume of precipitated polymer due to its asymmetric structure and large holding capacity. This can be useful when the polymer is designed to remove impurities and to leave the target or desired biomolecule in the liquid of the remaining mixture.

The change in stimuli may be gradual or it may be done substantially instantaneous. For example, a change in pH can be done by slowly adding a pH changing material to the liquid to change the pH slowly over a span of several minutes or even hours. Alternatively, for example, a suitable amount of pH changing material can be added to the liquid at one time to cause the change in pH to occur more rapidly. More control has been found in general with incremental changes rather than immediate changes for most processes.

Examples of cationinc polyelectrolytes that exhibit this selective solubility behavior include chitosan, polyvinylpyridines (PVPs) and copolymers of PVPs such as poly (2 vinylpyridine) (P2VP) or poly (4 vinylpyridine) (P4VP) , polyvinylpyridine-co-styrene (PVP-S) , polyvinylpyridine-co- butyl methacrylate (PVP-BMA) as well as other primary, secondary and tertiary amine-containing polymers. These polymers are soluble at a pH lower than about 6-7 and are insoluble at a pH greater than about 5-7. When in solution, these polymers will precipitate if the pH is raised above this critical range (pH=5^~7) . In the context of protein purification, a solution of said cationic polyelectrolyte can be added to a fluid containing a biomolecule of interest, such as a protein in the presence of other impurities. This fluid can be for example a cell culture fluid. The polymer is added to the fluid either as a solution in a carrier liquid at a pH of about 4.5 or as a solid particulate in which the fluid is either modified to a pH of about 4.5 either before, during or after the introduction of the polymer to it (as further described below) so that the polymer binds all the negatively charged impurities, such as cells, cell fragments, nucleic acids, viruses, host cell proteins, pyrogens and endotoxins. The biomolecule of interest does not interact with the polymer given its positive net charge due to its basic pi. The pH is then raised to 5-7 or more if desired and the polymer precipitates out of solution, carrying with it all the impurities as well as any excess polymer. The precipitate can then be easily removed by centrifugation or filtration, resulting in a "purified" biomolecule containing solution.

An example of anionic polyelectrolytes that exhibit this solubility behavior is a class of copolymers of acrylic acid and methyl methacrylate or methacrylic acid and methyl methacrylate. These polymers are soluble at a pH greater than about 4-7 and insoluble at a pH lower than about 4-7. These polymers can also be used to purify proteins from complex mixtures in a bind and elute mode. For instance, a solution of these polymers can be added to a fluid containing a protein of interest in the presence of other impurities wherein the pH of the fluid is at or above about 4-7. Under these conditions, the negatively charged polymer binds the positively charged protein of interest (basic pi) while it repels the negatively charged impurities. The pH of the fluid is then lowered below about 4-7 to effect precipitation of the polymer-protein complex and any excess polymer. The precipitate can then be washed to remove any soluble or loosely bound impurities while the pH is kept below about 4-7. The protein can be subsequently eluted from the polymer with an elution buffer at high salt concentrations and a pH below about 4-7 to recover the purified protein.

Examples of a temperature sensitive polymer is agarose, which is often used in chromatography, hydroxyalkylcelluloses such as hydroxypropylcellulose ; polymers and copolymers containing N-isopropylacrylamide monomer, polyethylene oxide, etc. The temperature can then be reduced or raised to cause the polymer to gel and/or precipitate out of solution.

In some cases, such as with agarose, these polymers are generally insoluble at room temperature and are soluble in water or other solvents at temperatures generally between about 80 to 120°C. They can be simply heated to cause them to dissolve, added to the mixture and then cooled to cause them to precipitate. In other cases, such as with polymers and copolymers containing N-isopropylacrylamide monomer, the polymer is soluble at a temperature below about 30 to 35°C. and will precipitate out of solution when the temperature is raised above this range.

In the case of agarose the use of gel-inhibiting agents such as various salts can depress the solubility temperature to lower temperatures, often to room temperature if desired.

Salts that can be used include lithium chloride and zinc chloride. Bases, such as sodium hydroxide or lithium hydroxide can also be used to depress the gelling temperature or to eliminate it altogether. Although the melting point for agarose is about 92 degrees, the gelling temperature is about 43 degrees. This gelling temperature can be manipulated by the modification of the agarose molecule as described above or by the addition of the above salts or by a combination thereof. For example, a cationic ligand can be attached to agarose in an amount such that the gelling temperature of the modified polymer is about 20° C. degrees with or without the addition of the above salts. The modified agarose is added, in solution at a temperature about 25°C, to the mixture (also at a temperature of about 25°C) to bind the constituents and then the temperature of the mixture is lowered to below 20 °C thereby gelling the modified agarose with the constituents.

With some polymers, such as polyvinylpyridine, polyvinylpyridene-co-styrene and the like, there may be residual monomer left in the polymer as supplied. It is desirable to remove any free monomer before using the polymer. One such method is to place the polymer as purchased in an oven, preferably with an inert or low oxygen gas atmosphere such as by purging the oven several times during the process with argon or nitrogen, and maintain it at an elevated temperature (generally between 100 and 200°C, preferably about 120°C.) and under a vacuum so as to drive off all monomer present (generally about 24 hours) .

Additionally, with some polymers, such as polyvinylpyridine, polyvinylpyridene-co-sytrene and the like, it is desirable to select higher molecular weight polymers (200, 000 molecular weight or higher) as they have been found to more freely precipitate out of solution than lower molecular weight polymers. This means that one can be sure that no residual polymer is left in the solution after precipitation. In some instances, precipitation by itself may be slow or incomplete. In those instances, one can repeat the process of changing the stimuli conditions two or more times, add precipitant enhancers such as glass beads, salts and the like, vary the temperature of the process and the like to enhance the precipitation.

Typical polymer concentrations in the carrier solvent are between 1-20% by weight depending on the viscosity of the solution. It is preferred that the concentration be as high as possible to minimize dilution of the feedstock. Practically, polymer solutions in the 10-20% are preferred to achieve a good balance between viscosity and dilution of the feedstock. The final concentration of the polymer in the feedstock may depend on the amount of impurities in the feedstock but it is typically between 0.01% to 2% by weight and more specifically between 0.05% and 0.1%.

In some processes one may use two or more polymers either simultaneously or sequentially to enhance the impurity removal. For example, one may use chitosan as the first polymer and conduct a first purification step. This fluid is separated from the precipitated chitosan/impurities and then treated with a second polymer such as a polyvinylpyridine to further remove impurities.

The recovered biomolecule may then undergo one or more additional processing steps depending on whether it is contained within the liquid of the mixture or is bound to the precipitated polymer.

A method of sequential precipitation may be used to recover the biomolecule of interest. In such a method, a first precipitation as described above would be used to remove impurities and the precipitated polymer/impurities mass would be separated from the target biomolecule containing solution. The solution would then be mixed with a stimuli responsive polymer containing a ligand capable of binding to the biomolecule of interest at a solution condition at which the polymer is soluble. Following methods described previously, the solution conditions would be changed so as to precipitate the polymer and bound biomolecule. The polymer/biomolecule would then be separated as previously described, the biomolecule eluted or otherwise separated from the polymer, and the recovered biomolecule further processed as needed.

As the biomolecule of interest is in the liquid, it may, if needed or desired undergo one or more known process steps including but not limited to chromatography steps such as ion exchange, hydrophobic interaction or affinity chromatography, various filtration steps such as high performance tangential flow filtration (HPTFF) , viral removal/inactivation steps, final filtration and the like. Alternatively, the biomolecule of interest present in the liquid may be used as is without the need for further purification steps.

The chromatography may be column based using solid bead media or monoliths or through a membrane absorber or chromatography device. The step if desired can be a classic bind/elute or a flow through mode of chromatography.

Also the biomolecule of interest may undergo further purification without the need for chromatography steps such as through the use of high performance tangential flow filtration using one or more charged membranes.

Additionally, in several embodiments, no further purification is required. One may if desired add additional steps to ensure that viruses have been removed or inactivated or to be sure no residual precipitate remains.

A further variation uses an affinity step to bind and then elute the desired biomolecule. Affinity ligands such as Protein A either on a solid matrix such as a bead or membrane may be used.

In one embodiment, the current process simply replaces a clarification step and prefilter step in a normal biological product process train.

In another embodiment, it replaces clarification, prefiltration and at least one chromatography step by directly purifying the biomolecule of interest from the starting materials .

In an additional embodiment, it replaces a cell harvest or biomolecule collection step by being added directly to the bioreactor or fermentor. This also eliminates the need for clarification, prefiltration and potentially at least one chromatography step by directly purifying the biomolecule of interest from the starting materials.

Residual polymer remaining in the solution containing the biomolecule of interest then can be detected by surface plasmon resonance spectroscopy in accordance with the embodiments disclosed herein. The results of the detection can be used to assess whether the residual polymer is below a certain predetermined level, such as 1 ppm.

Clarification + Purification

In certain embodiments, the polymer is a soluble polymer capable of irreversibly binding to insoluble particulates and a subset of soluble impurities and also capable of reversibly binding to one or more desired biomolecules in an unclarified biological material containing stream, and can be used to purify one or more desired biomolecules from such a stream without the need for prior clarification.

In certain embodiments, a stimuli responsive polymer such as a selectively soluble polymer capable of selectively and reversibly binding to one or more desired biomolecules in an unclarified biological material containing stream can be used to purify one or more desired biomolecules from such a complex mixture of materials including the biomolecule ( s ) of interest and various impurities such as other proteins (host cell proteins) , DNA, virus, whole cells, cellular debris and the like without the need for prior clarification of the stream.

The liquid phase or solubilized polymer has a capability even when precipitated, such as affinity or charge or hydrophobicity and the like, to selectively and reversibly bind to at least one or more biomolecules of interest and optionally one or more impurities in an unclarified liquid. The biomolecule of interest is then selectively eluted from the polymer preferably while the polymer is preferably retained in its solid or precipitated form with any impurities still attached to it. The biomolecule is then recovered for further processing, and residual polymer remaining in the solution containing the biomolecule of interest can be detected by surface Plasmon resonance spectroscopy in accordance with the embodiments disclosed herein. The results of the detection can be used to assess whether the residual polymer is below a certain predetermined level, such as 1 ppm.

More specifically, in certain embodiments, one or more polymers soluble in a liquid phase selectively bind to one or more desired biomolecules in a solution/suspension by a precipitation mechanism and which polymer can also be removed, if present, in any excess, by the same mechanism. By way of example, this idea can best be described in the context of protein purification although it can be used to purify any solute molecule from complex mixtures as long as the mechanism of removal applies to the specific solute of interest.

Certain polymers, such as poly(N-vinyl caprolactam) , poly (N-acryloylpiperidine) , poly (N-vinylisobutyramide) , poly (N-substituted acrylamide) including [poly(N- isopropylacrylamide) , poly (N, ' -diethylacrylamide) , and poly(N- acryloyl-N' -alkylpiperazine) ] and hydroxyalkylcellulose are examples of polymers that exhibit solubility changes as a result of changes in temperature. Other polymers, such as copolymers of acrylic acid and methacrylic acid, polymers and copolymers of 2 or 4-vinylpyridine and chitosan exhibit changes in solubility as a result of changes in pH or salt.

As some of these polymers may not have an ability to selectively bind or elute the desired molecules of interest and/or impurities, they need to be modified with ligands or chemical groups that will complex with the desired molecule and hold it in complex and then release the desired molecule under the appropriate elution conditions. Suitable chemical groups can include but are not limited to carboxyl groups and pyridine groups formed as part of the polymer or attached to the polymer. Ligands such as chemical mimics of affinity ligands may be used. Such ligands include but are not limited to natural ligands or synthetic ligands such as mercaptoethylpyridine (MEP) , mercaptoethylpyrazine, MEB, 2- aminobenzimidazole (ABI), AMBI, 2-mercapto-benzoic acid (MBA), 4-amino-benzoic acid (ABA), 2-mercapto-benzimidazole (MBI) and the like.

Depending upon the polymer used, the process used can vary .

Suitable pH sensitive soluble polymers include but are not limited to cationic polyelectrolytes and anionic polyelectrolytes . Suitable cationic polyelectrolytes are selected from the group consisting of chitosan, polyvinylpyridines , primary amine containing polymers, secondary amine containing polymers and tertiary amine containing polymers. Suitable anionic polyelectrolytes selected from the group consisting of copolymers of acrylic acid, methacrylic acid and methyl methacrylate.

One can use unclarified cell culture fluid containing the biomolecule of interest along with cell culture media components as well as cell culture additives, such as anti- foam compounds and other nutrients and supplements, cells, cellular debris, host cell proteins, DNA, viruses and the like. Moreover, the process can be conducted, if desired, in the bioreactor itself.

The fluid may either be preconditioned to a desired stimulus such as pH, temperature or other stimulus characteristic or the fluid can be conditioned upon addition of the polymer (s) or the polymer (s) can be added to a carrier liquid that is properly conditioned to the required parameter for the stimulus condition required for that polymer to be solubilized in the fluid. The polymer (s) is allowed to circulate thoroughly with the fluid and then the stimulus is applied (change in pH, temperature, salt concentration, etc) and the desired biomolecule and polymer (s) precipitate out of solution. The polymer and desired biomolecule ( s ) is separated from the rest of the fluid and optionally washed one or more times to remove any trapped or loosely bound contaminants. The desired biomolecule is then recovered from the polymer (s) such as by elution and the like. Preferably, the elution is done under a set of conditions such that the polymer remains in its solid (precipitated) form and retains any impurities to it during the selective elution of the desired biomolecule. Alternatively, the polymer and biomolecule as well as any impurities can be solubilized in a new fluid such as water or a buffered solution and the biomolecule be recovered by a means such as affinity, ion exchange, hydrophobic, or some other type of chromatography that has a preference and selectivity for the biomolecule over that of the polymer or impurities. The eluted biomolecule is then recovered and if desired subjected to additional processing, either traditional batch like steps or continuous flow through steps if appropriate. Residual polymer can be captured and detected by SPR either before or after the additional processing.

The soluble polymer (s) such as poly(N-vinyl caprolactam) , poly (N-acryloylpiperidine) , poly (N-vinylisobutyramide) , poly (N-substituted acrylamide) including [poly(N- isopropylacrylamide) , poly (N, ' -diethylacrylamide) , and poly(N- acryloyl-N' -alkylpiperazine) ] , hydroxyalkylcellulose, copolymers of acrylic acid and methacrylic acid or methacrylic acid and methyl methacrylate, polymers and copolymers of 2 or 4-vinylpyridine and chitosan may contain a functional group and/or ligand that binds to the biomolecule of interest or it may act by hydrophobic action or other such well known chromatographic type actions with the biomolecule.,

The processes will generally involve having one or more conditions of the liquid of the mixture, at the correct pH, temperature, light, electrical field or salt concentration or other condition used to cause the polymer (s) to become soluble and then adding the polymer (s) either directly or already solubilized in a carrier liquid, such as water or buffered solution, to the mixture. In some instances, the mixture will be at the proper condition to allow the polymer (s) to be simply added to the mixture.

In other instances, the mixture may need to be conditioned or modified to be at the desired condition. This modification or conditioning can be by modifying the mixture first and then adding the polymer (s), by adding the polymer (s) to a carrier liquid that is conditioned to the desired state and simply adding it to the mixture such that the carrier liquid is sufficient to cause the mixture to thus reach that condition or to do both.

The conditions of the liquid in the mixture are then changed (pH, temperature, salt content, combinations thereof, etc) that causes the polymer (s) to become insoluble and precipitate out of the mixture as a dispersed solid suspension. The mixture and the suspended insoluble polymer (s) are then mixed to ensure that the biomolecules of interest in the mixture and the insolubilized polymer (s) have sufficient and intimate contact with each other and some of the impurities of the mixture. In most instances the impurities are the insoluble materials such as whole cells or cellular debris. Most if not all soluble impurities are removed with the liquid of the mixture when it is separated from the precipitant. In some instances, the insoluble polymer (s) bind the one or more desired biomolecules it contacts while in the mixture and continues to bind to it thereafter until elution conditions are met to remove the biomolecule from the polymer. In others the polymer (s) bind to one or more impurities such as cells or cellular debris and entrain the biomolecule along with the impurities during its shift to a precipitate. Lastly, in some embodiments both the biomolecule and impurities are simply entrained out of the mixture by the precipitation of the polymer ( s ) .

The precipitate is separated such as by centrifugation or filtration or gravity and time with the liquid portion being decanted. The recovered polymer is optionally washed one or more times to remove any loosely bound residual impurities or contaminants and then the biomolecule ( s ) is eluted from the polymer under conditions that cause the biomolecule entity to release from the polymer so it can be recovered and subjected to further processing or use. Preferably, the elution conditions are such that the polymer remains in its solid or precipitated form and the eluted biomolecule is separated from the polymer by simple filtration, using a filter that allows the biomolecule through but retains the solid polymer upstream.

One polymer or a blend of polymers may be used in and it is meant to cover both embodiments whenever the term polymer, polymer (s) or one or more polymers is used hereafter.

As discussed above with respect to purification and clarification, the polymer may be added directly to the mixture either as is or in a conditioned state that allows the polymer to be solubilized as it is added. Alternatively, it can be added to a carrier liquid in which it is soluble or dispersable and which carrier preferably is also compatible with the mixture. Suitable carriers are as discussed above with respect to the other embodiments.

The mixture may be contained in a mixing vessel such as a tapered bottom metal (preferably stainless steel more preferably 304 or 316L stainless steel) or glass or plastic bag, vat or tank. Alternatively, especially when a cell culture or microbial or yeast culture, it may be the bioreactor or fermentor in which the cells have been grown. It may also be a disposable bioreactor or fermentor or a disposable mixing bag such as a plastic bag as is available from EMD Millipore Corporation of Billerica, Massachusetts. The mixture and polymer are brought into intimate contact through a mixing action that may be done by a magnetic stirred bar, a magnetic driven mixer such as a NovAseptic® or a Mobius® mixer available from EMD Millipore Corporation of Billerica, Massachusetts, a Lightning-type mixer, a recirculation pump, or a rocking motion closed mixing bag or bioreactor or fermentor, such as is shown in US 2005/0063259A1 or US 7, 377, 686 or an airlift type of mixer or reactor in which rising bubbles in the liquid cause a circulatory pattern to be formed.

Alternatively, the mixture and polymer (either by itself or in a carrier) can be in separate containers and mixed in line in a static blender. The blend can either then go to a container or to a centrifuge or a filter where the precipitated polymer and its bound one or more biomolecule entities is separated from the remainder of the mixture and then is further processed.

In another embodiment, the mixture and polymer (either by itself or in a carrier) are blended together in the container holding the mixture and further mixed in line in a static blender. The blend can either then go to a container or to a centrifuge or to a filter where the precipitated polymer and its bound one or more biomolecule entities and cells or other impurities are separated from the remainder of the mixture. Then the precipitated polymer is further processed to recover the biomolecule of interest.

Using centrifugation, one can easily and quickly separate the precipitated polymer from the remainder of the liquid mixture. After centrifugation, the supernatant, generally the remainder of the mixture is drawn off. The precipitated polymer is further processed.

If desired, the supernatant may be subjected to one or more additional polymer precipitation steps to recover even more of the desired biomolecule.

Simple decantation may also be used if desired.

The use of settling due to density differences may also be used and the separated materials decanted or otherwise separated from each other after that.

Filtration can be accomplished in a variety of manners.

Depending upon the size of the polymer as it is precipitated; one may use one or more filters of varying sizes or asymmetries. The selection of type and size of filter will depend on the volume of precipitate to be captured.

Membrane based filters, preferably microporous membranes can be used. Such filters are generally polymeric in nature and can be made from polymers such as but not limited to olefins such as polyethylene including ultrahigh molecular weight polyethylene, polypropylene, EVA copolymers and alpha olefins, metallocene olefinic polymers, PFA, MFA, PTFE, polycarbonates, vinyl copolymers such as PVC, polyamides such as nylon, polyesters, cellulose, cellulose acetate, regenerated cellulose, cellulose composites, polysulfone, polyethersulfone, polyarylsulfone, polyphenylsulfone, polyacrylonitrile, polyvinylidene fluoride (PVDF) , and blends thereof. The membrane selected depends upon the application, desired filtration characteristics, particle type and size to be filtered and the flow desired. Suitable membrane based filters include DURAPORE® PVDF membranes available from EMD Millipore Corporation of Billerica Massachusetts, MILLIPORE EXPRESS® and MILLI PORE EXPRESS® PLUS or SH PES membranes available from EMD Millipore Corporation of Billerica Massachusetts. Prefilters, depth filters and the like can also be used in these embodiments such as Polygard® prefilters (Polygard CE prefilters) and depth filters (Polygard CR depth filters) available from EMD Millipore Corporation of Billerica Massachusetts .

Depending on the mixture, polymer and the nature of biomolecule, the filter, such a membrane, may be hydrophilic or hydrophobic. Suitable membranes are hydrophilic and are low in protein binding.

The membrane may be symmetric in pore size throughout its depth such as DURAPORE® PVDF membranes available from EMD Millipore Corporation of Billerica Massachusetts, or it may be asymmetric in pore size through its thickness as with MILLIPORE EXPRESS® and MILLIPORE EXPRESS® PLUS or SH PES membranes available from EMD Millipore Corporation of Billerica Massachusetts. It may contain a prefilter layer if desired, either as a separate upstream layer or as an integral upstream portion of the membrane itself.

The filter or prefilter or depth filter may be formed of non-membrane materials such as continuous wound fiber, fibrous mats (Millistak+® pads) and/or non woven materials such as Tyvek® plastic paper.

The pore size of the membrane can vary depending upon the polymer and mixture selected. Generally, it has an average pore size of from about 0.05 micron to 5 microns, preferably from about 0.05 micron to about 1 micron, more preferably from about 0.05 to about 0.65 micron. Prefilters and depth filters often are not rated by pore size but to the extent that they are they may have a pore size of from about 0.22 micron to about 10 micron.

The filter be it membrane, non woven, pad or other form may run in a deadend or normal flow (NF) format or a tangential flow (TFF) format. The choice is dependent on a number of factors, primarily the user's preference or installed filtration equipment as either works. A TFF process and equipment is preferred when large amounts of polymer and molecule are to be recovered as TFF is less subject to clogging or fouling than NF methods.

In certain embodiments, in a first step the unclarified mixture is either conditioned to the correct parameter (s) so as to maintain the capture polymer of choice in solution when added or if the conditions of the mixture are already such that the polymer (s) become soluble in the mixture, no further conditioning may be required. Alternatively, the polymer (s) may be added as a solid to an unconditioned mixture and then the mixture (containing the solid polymer (s) ) may be conditioned to the correct parameters to dissolve the polymer (s) in the mixture. Likewise, the polymer can be added to a carrier liquid and added at the correct conditions to the mixture. The mixture itself may also be preconditioned or it may rely on the carrier to condition it upon its introduction. In a second step, the polymer (s) is mixed with the mixture in the stream for desirable amount of time to create suitable distribution to make intimate contact with all the constituents of the mixture. In a third step, the conditions of the liquid in the mixture are then changed (pH, temperature, salt content, combinations thereof, etc) to cause the polymer (s) to become insoluble and precipitate out of the mixture as a dispersed solid suspension while retaining the biomolecule and cells or other impurities. The rest of the mixture and the precipitated polymer (s) are then separated from each other in a fourth step. As discussed above the precipitate and remaining mixture may be separated by centrifugation, decantation or filtration.

The desired biomolecule is then recovered. Preferably it is eluted from the polymer such as by the addition of a buffer at a pH (acidic or basic depending on the molecule and the polymer used) and/or the salt concentration or temperature of the solution is changed to allow for the recovery of the desired molecule free of the polymer. Preferably the elution conditions are such that the polymer remains in its solid (precipitated) form although it can if desired or needed be rendered soluble again.

In certain embodiments, the process may be applied to a pH dependent polymer such as poly (4-vinylpyridine-co-styrene) , (10% mol styrene) which has an affinity, be it chemical, electrical, phobic/philic, etc. for the desired biomolecule in the insoluble state. In a first step, the mixture is either conditioned to the correct pH (in this case to a pH below about 5.0) to maintain the polymer of choice in solution before, during or after the introduction of the polymer or it is already at the desired condition in a second step. In a third step which may occur separately before, simultaneously or after the second step, the polymer is added to a carrier liquid under conditions that allow it to go into solution and then mixed to make intimate contact with all the constituents of the mixture so that the polymer can complex with the desired molecule (for example a IgG molecule, cells, etc.) .

If desired one may conduct one or more additional steps to ensure that all polymer has been removed from the mixture by subjecting the mixture to a step containing a material that will remove any residual polymer from the mixture such as ion exchange resin, activated carbon, alumina, diatomaceous earth and the like. Typically however the polymer is removed on the first precipitation and such additional steps are not necessary .

As discussed above, the precipitate and remaining mixture may be separated by centrifugation, decantation or filtration in a fifth step. Optionally, the polymer and complex are washed one or more times while being kept under conditions such that the polymer/biomolecule ( s ) /cells precipitate remains undissociated .

To recover the molecule, the conditions are changed, such as by lowering the pH or changing the ionic strength of the solution, so as to break the complex between the polymer and the biomolecule in a sixth step. The elution liquid is removed from the precipitated polymer by filtration, decantation or centrifugation or the like and the eluted biomolecule is recovered in a seventh step. Optionally, one can solubilize the polymer and then change the conditions of the liquid such that the bond between the biomolecule and polymer is broken (such as pH or ionic change) , then change the conditions again such that the polymer and impurities are reprecipitated and the biomolecule remains in solution. The biomolecule is then recovered by filtration, decantation, centrifugation and the like. The biomolecule of interest after having been recovered, may undergo one or more known additional process steps such as chromatography steps including but not limited to ion exchange, hydrophobic interaction or affinity chromatography, various filtration steps such as microfiltration, ultrafiltration, high performance tangential flow filtration (HPTFF) with or without charged UF membranes, viral removal/inactivation steps, final filtration steps and the like. Alternatively, the eluted biomolecule of interest may be used as is without the need for further purification steps. Also the biomolecule of interest may undergo further purification without the need for chromatography steps.

In a further embodiment, at least the step of clarification and preferably the steps of clarification and affinity chromatography are eliminated. A biological process under this embodiment would consist of capture of the biomolecule directly from the unclarified mixture via the polymer-based purification step, separation of the biomolecule from the polymer and the remainder of the mixture, two or more steps of viral removal or inactivation such as removal through viral filters or inactivation through treatment with heat, chemicals or light, a compounding step into the correct formulation and a final filtering before filling the compounded biomolecule into its final container for use (vial, syringe, etc) .

In any of the embodiments the biomolecule such as a protein thus recovered may be formulated in a pharmaceutically acceptable carrier and is used for various diagnostic, therapeutic or other uses known for such molecules.

The mixture that is the starting material of the process will vary depending upon the cell line in which it was grown as well as the conditions under which it is grown and harvested. For example, in most CHO cell processes the cells express the molecule outside of the cell wall into the media. One tries not to rupture the cells during harvest in order to reduce the amount of impurities in the mixture. However, some cells during growth and harvesting may rupture due to shear or other handling conditions or die and lyse, spilling their contents into the mixture. In bacteria cell systems, the biomolecule is often kept with the cellular wall or it may actually be part of the cellular wall (Protein A) . In these systems, the cell walls need to be disrupted or lysed in order to recover the biomolecule of interest.

In any of the embodiments the protein thus recovered may be formulated in a pharmaceutically acceptable carrier and is used for various diagnostic, therapeutic or other uses known for such molecules.

ANTIBODIES

The mixture that is the starting material of the process will vary depending upon the cell line in which it was grown as well as the conditions under which it is grown and harvested. For example, in most CHO cell processes the cells express the molecule outside of the cell wall into the media. One tries not to rupture the cells during harvest in order to reduce the amount impurities in the mixture. However, some cells during grow and harvesting may rupture due to shear or other handling conditions or die and lyse, spilling their contents into the mixture. In bacteria cell systems, the biomolecule is often kept with the cellular wall or it may actually be part of the cellular wall (Protein A) . In these systems the cell walls need to be disrupted or lysed in order to recover the biomolecule of interest.

The target molecule to be purified can be any biomolecule, preferably a protein, in particular, recombinant protein produced in any host cell, including but not limited to, Chinese hamster ovary (CHO) cells, Per.C6® cell lines available from Crucell of the Netherlands, myeloma cells such as NSO cells, other animal cells such as mouse cells, insect cells, or microbial cells such as E. coli or yeast. Additionally, the mixture may be a fluid derived from an animal modified to produce a transgenic fluid such as milk or blood that contains the biomolecule of interest. Optimal target proteins are antibodies, immunoadhesins and other antibody-like molecules, such as fusion proteins including a C_H2/C_H3 region. In particular, this product and process can be used for purification of recombinant humanized monoclonal antibodies such as (RhuMAb) from a conditioned harvested cell culture fluid (HCCF) grown in Chinese hamster ovary (CHO) cells expressing RhuMAb.

Antibodies within the scope of the embodiments disclosed herein include, but are not limited to: anti-HER2 antibodies including Trastuzumab (HERCEPTIN®) (Carter et al . , Proc. Natl. Acad. Sci. USA, 89:4285-4289 (1992), U.S. Pat. No. 5,725,856); anti-CD20 antibodies such as chimeric anti-CD20 "C2B8" as in U.S. Pat. No. 5,736,137 (RITUXAN®) , a chimeric or humanized variant of the 2H7 antibody as in U.S. Pat. No. 5,721,108, Bl, or Tositumomab (BEXXAR®) ; anti-IL-8 (St John et al . , Chest, 103:932 (1993), and International Publication No. WO 95/23865); anti-VEGF antibodies including humanized and/or affinity matured anti-VEGF antibodies such as the humanized anti-VEGF antibody huA4.6.1 AVASTIN®. (Kim et al . , Growth Factors, 7:53-64 (1992), International Publication No. WO 96/30046, and WO 98/45331, published Oct. 15, 1998); anti-PSCA antibodies (WO01/40309) ; anti-CD40 antibodies, including S2C6 and humanized variants thereof (WO00/75348) ; anti-CDlla (U.S. Pat. No. 5, 622, 700, WO 98/23761, Steppe et al . , Transplant Intl. 4:3-7 (1991), and Hourmant et al . , Transplantation 58:377-380 (1994)); anti-IgE (Presta et al . , J Immunol. 151:2623-2632 (1993), and International Publication No. WO 95/19181); anti-CD18 (U.S. Pat. No. 5,622,700, issued Apr. 22, 1997, or as in WO 97/26912, published Jul. 31, 1997); anti-IgE (including E25, E26 and E27; U.S. Pat. No. 5, 714, 338, issued Feb. 3, 1998 or U.S. Pat. No. 5,091,313, issued Feb. 25, 1992, WO 93/04173 published Mar. 4, 1993, or International Application No. PCT/US98/13410 filed Jun . 30, 1998, U.S. Pat. No. 5,714,338); anti-Apo-2 receptor antibody (WO 98/51793 published Nov. 19, 1998); anti-TNF- antibodies including cA2 (REMICADE®) , CDP571 and MAK-195 (See, U.S. Pat. No. 5,672,347 issued Sep. 30, 1997, Lorenz et al . J. Immunol. 156(4) :1646- 1653 (1996), and Dhainaut et al . Crit. Care Med. 23(9):1461- 1469 (1995)); anti-Tissue Factor (TF) (European Patent No. 0 420 937 Bl granted Nov. 9, 1994); anti-human ₄β₇ integrin (WO 98/06248 published Feb. 19, 1998); anti-EGFR (chimerized or humanized 225 antibody as in WO 96/40210 published Dec. 19, 1996); anti-CD3 antibodies such as OKT3 (U.S. Pat. No. 4,515,893 issued May 7, 1985); anti-CD25 or anti-tac antibodies such as CHI-621 (SIMULECT®) and (ZENAPAX®) (See U.S. Pat. No. 5,693,762 issued Dec. 2, 1997); anti-CD4 antibodies such as the cM-7412 antibody (Choy et al . Arthritis Rheum 39(l):52-56 (1996)); anti-CD52 antibodies such as CAMPATH-1H (Riechmann et al . Nature 332:323-337 (1988)); anti- Fc receptor antibodies such as the M22 antibody directed against FcyRI as in Graziano et al . J. Immunol . 155(10) :4996- 5002 (1995); anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14 (Sharkey et al . Cancer Res. 55 (23Suppl) : 5935s- 5945s (1995); antibodies directed against breast epithelial cells including huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al . Cancer Res. 55(23): 5852s-5856s (1995); and Richman et al . Cancer Res. 55(23 Supp) : 5916s-5920s (1995)); antibodies that bind to colon carcinoma cells such as C242 (Litton et al . Eur J. Immunol. 26(1): 1-9 (1996)); anti-CD38 antibodies, e.g. AT 13/5 (Ellis et al . J Immunol. 155 (2) : 925-937 (1995)); anti- CD33 antibodies such as Hu M195 (Jurcic et al . Cancer Res 55(23 Suppl) : 5908s-5910s (1995) and CMA-676 or CDP771; anti- CD22 antibodies such as LL2 or LymphoCide (Juweid et al . Cancer Res 55(23 Suppl) : 5899s-5907s (1995)); anti-EpCAM antibodies such as 17-1A (PANOREX®) ; anti-GpIIb/IIIa antibodies such as abciximab or c7E3 Fab (REOPRO®) ; anti-RSV antibodies such as MEDI-493 (SYNAGIS®) ; anti-CMV antibodies such as PROTOVIR®; anti-HIV antibodies such as PR0542; anti- hepatitis antibodies such as the anti-Hep B antibody OSTAVIR®; anti-CA 125 antibody OvaRex; anti-idiotypic GD3 epitope antibody BEC2; anti-orv^3 antibody VITAXIN®; anti-human renal cell carcinoma antibody such as ch-G250; ING-1; anti-human 17- 1A antibody (3622W94); anti-human colorectal tumor antibody (A33) ; anti-human melanoma antibody R24 directed against GD3 ganglioside ; anti-human squamous-cell carcinoma (SF-25) ; and anti-human leukocyte antigen (HLA) antibodies such as Smart ID10 and the anti-HLA DR antibody Oncolym (Lym-1) . The preferred target antigens for the antibody herein are: HER2 receptor, VEGF, IgE, CD20, CDlla, and CD40.

Aside from the antibodies specifically identified above, the skilled practitioner could generate antibodies directed against an antigen of interest, e.g., using the techniques described below.

The antibody herein is directed against an antigen of interest. Preferably, the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disease or disorder can result in a therapeutic benefit in that mammal. However, antibodies directed against non-polypeptide antigens (such as tumor- associated glycolipid antigens; see U.S. Pat. No. 5,091,178) are also contemplated. Where the antigen is a polypeptide, it may be a transmembrane molecule (e.g. receptor) or ligand such as a growth factor. Exemplary antigens include those proteins described in section (3) below. Exemplary molecular targets for antibodies encompassed by the embodiments disclosed herein include CD proteins such as CD3, CD4, CD8, CD 19, CD20, CD22, CD34, CD40; members of the ErbB receptor family such as the EGF receptor, HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Macl, pl50,95, VLA-4, ICAM-1, VCAM and ν/β3 integrin including either a or β subunits thereof (e.g. anti-CDlla, anti-CD18 or anti-CD lib antibodies); growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, or any of the other antigens mentioned herein. Antigens to which the antibodies listed above bind are specifically included within the scope herein.

Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for generating antibodies. For transmembrane molecules, such as receptors, fragments of these (e.g. the extracellular domain of a receptor) can be used as the immunogen. Alternatively, cells expressing the transmembrane molecule can be used as the immunogen. Such cells can be derived from a natural source (e.g. cancer cell lines) or may be cells which have been transformed by recombinant techniques to express the transmembrane molecule.

Other antigens and forms thereof useful for preparing antibodies will be apparent to those in the art.

Polyclonal antibodies can also be purified. Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, for example, maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues) , N-hydroxysuccinimide (through lysine residues) , glutaraldehyde, succinic anhydride, SOCI₂, or R^!N=C NR, where R and R¹ are different alkyl groups. Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later the animals are boosted with Ys to 1/10 the original amount of antigen or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different cross-linking reagent. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

Monoclonal antibodies are of interest and may be made using the hybridoma method first described by Kohler et al . , Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

In the hybridoma method, a mouse or other appropriate host animal, such as a hamster or macaque monkey, is immunized as hereinabove described to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. Lymphocytes then are fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies : Principles and Practice, pp. 59-103 (Academic Press, 1986)). The hybridoma cells thus prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) , the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium) , which substances prevent the growth of HGPRT-deficient cells.

Suitable myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Among these, suitable myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al . , Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) . After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies : Principles and Practice, pp. 59-103 (Academic Press, 1986)) . Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal .

The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, Pro-Sep® Protein A media available from Millipore Corporation of Billerica, Mass., hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Preferably the Protein A chromatography procedure described herein is used.

DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the monoclonal antibodies) . The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells . The DNA also may be modified, for example, by substituting the coding sequence for human heavy- and light- chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4, 816, 567; Morrison, et al . , Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.

Typically such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen- combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

In a further embodiment, monoclonal antibodies can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al . , Nature, 348:552-554 (1990). Clackson et al . , Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al . , Bio/Technology, 10:779-783

(1992) ), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al . , Nuc . Acids. Res., 21:2265-2266

(1993) ). Thus, these techniques are viable alternatives to traditional hybridoma techniques for isolation of monoclonal antibodies . A humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al . , Nature, 321:522-525 (1986); Riechmann et al . , Nature, 332:323- 327 (1988); Verhoeyen et al . , Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable-domain sequences. The human sequence which is closest to that of the rodent is then accepted as the human FR for the humanized antibody (Sims et al . , J. Immunol., 151:2296 (1993)). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may be used for several different humanized antibodies (Carter et al . , Proc. Natl. Acad. Sci . USA, 89:4285 (1992); Presta et al . , J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., the analysis of residues that influence the ability of the candidate immunoglobulin to bind its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen (s), is achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

Alternatively, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been described that the homozygous deletion of the antibody heavy-chain joining region (J_H) gene in chimeric and germ-line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al . , Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al . , Nature, 362:255- 258 (1993); Bruggermann et al . , Year in Immuno . , 7:33 (1993); and Duchosal et al . Nature 355:258 (1992) . Human antibodies can also be derived from phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381 (1991); Marks et al . , J. Mol. Biol., 222:581-597 (1991); Vaughan et al . Nature Biotech 14:309 (1996) ) .

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al . , Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al . , Science, 229:81 (1985)) . However, these fragments can now be produced directly by recombinant host cells. For example, the antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab'>2 fragments (Carter et al . , Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) . See WO 93/16185.

Multispecific antibodies have binding specificities for at least two different antigens. While such molecules normally will only bind two antigens (i.e. bispecific antibodies, BsAbs) , antibodies with additional specificities such as trispecific antibodies are encompassed by this expression when used herein.

Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al . , Nature, 305:537-539 (1983)) . Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al . , EMBO J., 10:3655-3659 (1991) .

According to another approach described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. The preferred interface comprises at least a part of the C_H3 domain of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan) . Compensatory "cavities" of identical or similar size to the large side chain (s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine) . This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers .

Bispecific antibodies include cross-linked or "heteroconj ugate" antibodies. For example, one of the antibodies in the heteroconj ugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4, 676, 980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089) . Heteroconj ugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al . , Science, 229: 81 (1985) describe a procedure wherein intact antibodies are proteolytically cleaved to generate F(ab'>2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The Fab' fragments generated are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to the Fab '-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Recent progress has facilitated the direct recovery of Fab'-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al . , J Exp. Med., 175: 217-225 (1992) describe the production of a fully humanized bispecific antibody F(ab'>2 molecule. Each Fab' fragment was separately secreted from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was able to bind to cells over expressing the ErbB2 receptor and normal human T cells, as well as trigger the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al . , J. Immunol., 148 (5) : 1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers . This method can also be utilized for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism for making bispecific antibody fragments. The fragments comprise a heavy-chain variable domain (V_H) connected to a light-chain variable domain (V_L) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, the V_H and V_L domains of one fragment are forced to pair with the complementary V_L and V_H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al . , J. Immunol., 152:5368 (1994). Alternatively, the antibodies can be "linear antibodies" as described in Zapata et al . Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (V_H-C_H1-V_H-C_H1 ) which form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Antibodies with more than two valencies are contemplated.

For example, tri specific antibodies can be prepared. Tutt et al. J. Immunol. 147: 60 (1991).

The simplest and most straightforward immunoadhesin design combines the binding domain (s) of the adhesin (e.g. the extracellular domain (ECD) of a receptor) with the hinge and Fc regions of an immunoglobulin heavy chain. Ordinarily, when preparing the immunoadhesins , nucleic acid encoding the binding domain of the adhesin will be fused C-terminally to nucleic acid encoding the N-terminus of an immunoglobulin constant domain sequence, however N-terminal fusions are also possible .

Typically, in such fusions the encoded chimeric polypeptide will retain at least functionally active hinge, C_H2 and C_H3 domains of the constant region of an immunoglobulin heavy chain. Fusions are also made to the C-terminus of the Fc portion of a constant domain, or immediately N-terminal to the C_H1 of the heavy chain or the corresponding region of the light chain. The precise site at which the fusion is made is not critical; particular sites are well known and may be selected in order to optimize the biological activity, secretion, or binding characteristics of the immunoadhesin .

In certain embodiments, the adhesin sequence is fused to the N-terminus of the Fc domain of immunoglobulin d (IgGi) . It is possible to fuse the entire heavy chain constant region to the adhesin sequence. However, more preferably, a sequence beginning in the hinge region just upstream of the papain cleavage site which defines IgG Fc chemically (i.e. residue 216, taking the first residue of heavy chain constant region to be 114), or analogous sites of other immunoglobulins is used in the fusion. In a particularly preferred embodiment, the adhesin amino acid sequence is fused to (a) the hinge region and C_H2 and C_H3 or (b) the C_H1, hinge, C_H2 and C_H3 domains, of an IgG heavy chain.

For bispecific immunoadhesins , the immunoadhesins are assembled as multimers, and particularly as heterodimers or heterotetramers. Generally, these assembled immunoglobulins will have known unit structures. A basic four chain structural unit is the form in which IgG, IgD, and IgE exist. A four chain unit is repeated in the higher molecular weight immunoglobulins; IgM generally exists as a pentamer of four basic units held together by disulfide bonds. IgA globulin, and occasionally IgG globulin, may also exist in multimeric form in serum. In the case of multimer, each of the four units may be the same or different.

Various exemplary assembled immunoadhesins within the scope herein are schematically diagrammed below: AC_L-AC_L; (a) AC_H- (AC_H, AC_L-AC_H, AC_L-V_HC_H, or V_LC_L-AC_H) ; (b) AC_L-ACH- (AC_L-ACH, AC_L-VHCH, V_LC_L-AC_h, or V_LC_L-V_HC_H) (c) AC_L-VHCH- (AC_H, or AC_L-V_HC_H, or V_LC_L-AC_H) ; (d) V_LC_L-ACH- (AC_L-VHCH, or V_LC_L-ACH) ; and (e)

(A-Y)_n- (V_LC_L-V_HC_H)₂, (f) wherein each A represents identical or different adhesin amino acid sequences;

V_L is an immunoglobulin light chain variable domain; V_H is an immunoglobulin heavy chain variable domain;

C_L is an immunoglobulin light chain constant domain;

C_H is an immunoglobulin heavy chain constant domain; n is an integer greater than 1;

Y designates the residue of a covalent cross-linking agent. In the interests of brevity, the foregoing structures only show key features; they do not indicate joining (J) or other domains of the immunoglobulins, nor are disulfide bonds shown. However, where such domains are required for binding activity, they shall be constructed to be present in the ordinary locations which they occupy in the immunoglobulin molecules .

Alternatively, the adhesin sequences can be inserted between immunoglobulin heavy chain and light chain sequences, such that an immunoglobulin comprising a chimeric heavy chain is obtained. In this embodiment, the adhesin sequences are fused to the 3 ' end of an immunoglobulin heavy chain in each arm of an immunoglobulin, either between the hinge and the C_H2 domain, or between the C_H2 and C_H3 domains. Similar constructs have been reported by Hoogenboom, et al . , Mol . Immunol.

28:1027-1037 (1991) . Although the presence of an immunoglobulin light chain is not required in the immunoadhesins , an immunoglobulin light chain might be present either covalently associated to an adhesin-immunoglobulin heavy chain fusion polypeptide, or directly fused to the adhesin. In the former case, DNA encoding an immunoglobulin light chain is typically coexpressed with the DNA encoding the adhesin-immunoglobulin heavy chain fusion protein. Upon secretion, the hybrid heavy chain and the light chain will be covalently associated to provide an immunoglobulin-like structure comprising two disulfide-linked immunoglobulin heavy chain-light chain pairs. Methods suitable for the preparation of such structures are, for example, disclosed in U.S. Pat. No. 4, 816, 567, issued 28 Mar. 1989.

Immunoadhesins are most conveniently constructed by fusing the cDNA sequence encoding the adhesin portion in-frame to an immunoglobulin cDNA sequence. However, fusion to genomic immunoglobulin fragments can also be used (see, e.g. Aruffo et al., Cell 61:1303-1313 (1990); and Stamenkovic et al . , Cell 66:1133-1144 (1991)). The latter type of fusion requires the presence of Ig regulatory sequences for expression. cDNAs encoding IgG heavy-chain constant regions can be isolated based on published sequences from cDNA libraries derived from spleen or peripheral blood lymphocytes, by hybridization or by polymerase chain reaction (PCR) techniques. The cDNAs encoding the "adhesin" and the immunoglobulin parts of the immunoadhesin are inserted in tandem into a plasmid vector that directs efficient expression in the chosen host cells.

In other embodiments, the protein to be purified is one which is fused to, or conjugated with, a C_H2/C_H3 region. Such fusion proteins may be produced so as to increase the serum half-life of the protein. Examples of biologically important proteins which can be conjugated this way include renin; a growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1- antitrypsin; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrands factor; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA) ; bombesin; thrombin; hemopoietic growth factor; tumor necrosis factor-alpha and -beta; enkephalinase ; RANTES (regulated on activation normally T-cell expressed and secreted) ; human macrophage inflammatory protein (MIP-1-alpha) ; a serum albumin such as human serum albumin; Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbial protein, such as beta-lactamase ; DNase; IgE; a cytotoxic T-lymphocyte associated antigen (CTLA) , such as CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF) ; receptors for hormones or growth factors; Protein A or D; rheumatoid factors; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF) , neurotrophin-3 , -4, -5, or -6 (NT- 3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF- β; platelet-derived growth factor (PDGF) ; fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF) ; transforming growth factor (TGF) such as TGF-alpha and TGF- beta, including TGF-βΙ, TGF-p2, TGF-p3, TGF-p4, or TGF-βδ; insulin-like growth factor-I and -II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD3, CD4, CD8, CD19, CD20, CD34, and CD40; erythropoietin; osteoinductive factors; immunotoxins ; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs) , e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay accelerating factor; viral antigen such as, for example, a portion of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins such as CDlla, CDllb, CDllc, CD18, an ICAM, VLA-4 and VCAM; a tumor associated antigen such as HER2, HER3 or HER4 receptor; and fragments of any of the above-listed polypeptides.

The following examples are offered by way of illustration and not by way of limitation. The disclosures of all citations in the specification are expressly incorporated herein by reference.

Examples

Example A. Generation of Standard Curve for Quantitation of pDADMAC (polydiallyldimethyl ammonium chloride) hereby

referred as Polymer B Samples and Demonstration of Biosensor Ability to Detect Residual Polymer in IgG Samples.

A CM5 sensor chip and PBS adjusted to pH 8.5 and containing 350 mM NaCl was used. At the end of each binding cycle, the surface of the chip was regenerated with one injection each of 50mM NaOH, 750mM H₃P0₄, and 750 mM H₃P0₄/3M NaCl. pDADMAC (polydiallyldimethyl ammonium chloride) -HCCF (harvest cell culture fluid) was prepared at six concentrations (0.1 to 100 ppm) in running buffer (PBS with 300mM NaCl, pH 8.5) and each sample was injected across the sensor surface for three minutes. Under these buffer conditions, this span of polymer concentrations produced a wide range of capture levels and initial capture rates:

0 50 100 150 200

Time (s)

The entire set of polymer concentrations was tested times to determine the reproducibility in the responses:

To generate a standard curve, the initial capture rates (highlighted by the dashed lines in the figure below) were plotted against polymer concentration:

Example B. Detection of Residual Polymer on Sensor Surface for Varying Dose in Feed Sample and Identity of Residuals Across Various Steps of Downstream Processing. Cell culture harvest containing MAb05 was pretreated by using cationic Polymer A flocculant. Polymer A was added to cell culture broth at a final concentration between 0.025 and 0.075%, % (w/v) at an addition rate 0.05 L/min. The final pH of the solution was in the range of 7.0-7.2. The pretreated cell culture suspensions were mixed for approximately 30 minutes and filtered with Clarisolve^® filters. The clarified CHO fermentation culture supernatants served as base loading materials for all of the chromatographic steps. For ProA experiments, neutralized ProA eluate contained 100 mM sodium acetate buffer together with neutralizing Tris buffer at pH 6 and conductivity of ~4 mS/cm. Cation exchange eluate contains mainly 50 mM sodium acetate, 135 mM sodium chloride buffer at pH 5.0 with conductivity of about 17 mS/cm. Anion exchange flow through contains mainly 25 mM Tris buffer at pH 7 with conductivity of about 3 mS/cm. The test resins include ProSep® Ultra Plus Affinity Chromatography Media, Fractogel® EMD SO₃ (S) and Fractogel® EMD TMAE Hicap (M) Chromatography Media purchased from Merck KGaA, Darmstadt, Germany. Finally, the residual polymer in the filtrates and chromatography pools were determined using SPR.

The polymer-containing samples (feed, after protein A elution, after cation exchange elution and after anion exchange elution) were similarly tested for binding to the CM5 sensor surface. Each sample was diluted 1/10 in running buffer (PBS with 300mM NaCl, pH 8.5) and then in a four-fold dilution series. Below is an example data set showing the responses for the 1/10 dilution of the polymer samples:

0,03 6% poiyA ¾¾d AEX oiot 0.020% poiyA eed OOAA siui.

Time (s)

0.075% poiyA feed GEX eiui

aResidual polymer concentration for pDADMAC treated feeds

bResidual polymer concentration for pDADMAC treated feeds after Protein A Residual polymer concentration for pDADMAC treated feeds after CEX

^Residual polymer concentration for pDADMAC treated feeds after AEX

Example C . Capture of Smart polymer hereby referred to as Polymer B on Sensor Surface and Identity of Conditions that Subsequently Removes Residual Polymer for the Surface.

Shown below is the response for lOppm polymer B (prepared from 1000 ppm polymer B + 13.8 mg/ml IgG stock solution into running buffer) injected across the sensor

Polymer B was readily captured on the surface and remained bound as the surface was washed with running buffer. For efficient sample analysis, all polymer B from the surface must be removed prior to testing another sample. However, acidic conditions could be deleterious for polymer B, so regeneration with low-pH conditions was not feasible, and the commonly used alternatives such as individual base, salt or detergent solutions were ineffective. Accordingly, a combination of NaCl/NaOH/Tween-20 was used. A three-minute injection of this mixture of 50 mM NaOH, 3M NaCl and 0.5% Tween-20 effectively regenerated the surface after capture of 10 ppm polymer B again :

0 100 200 300 400 500 600 700

Time (s)

Example D . Generation of Standard Curve for Quantitation of Polymer B Samples and Demonstration of Biosensor Ability to Detect Polymer B in IgG Samples.

Cell culture harvest was pretreated by using cationic Polymer B stimulus responsive polymer. Unconditioned cell culture containing Mab05 was treated with a Polymer at concentrations of 0-0.2% (w/v) at an addition rate 0.05 L/min in the presence of 50 mM stimulus reagent, sodium phosphate, which binds the free flocculant molecules, makes them precipitate and becomes easier to be removed by the subsequent clarification step. The final pH of the solution was in the range of 7.0-7.2. The pre-treated cell culture suspensions were mixed for approximately 30 minutes and filtered with Clarisolve^® filters. The clarified CHO fermentation culture supernatants served as base loading materials for all of the chromatographic steps. For ProA experiments, neutralized ProA eluate contained 100 mM sodium acetate buffer together with neutralizing Tris buffer at pH 6 and conductivity of ~4 mS/cm. Cation exchange eluate contains mainly 50 mM sodium acetate, 135 mM sodium chloride buffer at pH 5.0 with conductivity of about 17 mS/cm. Anion exchange flow through contains mainly 25 mM Tris buffer at pH 7 with conductivity of about 3 mS/cm. The test resins include ProSep® Ultra Plus Affinity Chromatography Media, Fractogel® EMD S0₃ (S) and Fractogel® EMD TMAE Hicap (M) Chromatography Media purchased from Merck KGaA, Darmstadt, Germany. Finally, the residual polymer in the filtrates and chromatography pools were determined using SPR.

Analysis conditions and approach: Analyses were performed at 25°C using a Biacore 2000 optical biosensor equipped with a CM5 sensor chip and equilibrated with 20 mM tris, 300 mM NaCl, pH 8.5. After each binding cycle, the sensor surface was regenerated with two 3-minute injections of 3 M NaCl, 50 mM NaOH, 0.5% Tween-20 (consisting of two solutions mixed immediately before the first regeneration inj ection) .

Pre-coating of glass sampling vials and plastic pipet tips with Polymer B.

1. An aliquot of the (1000 ppm polymer B + 13.8 mg/ml IgG in sodium acetate pH 5.0) stock solution was diluted 10 fold in the tris buffer and the sample re-adjusted to pH 8.5 (some precipitate was apparent at this concentration in this buffer) .

2. A set of glass vials and plastic pipet tips were filled with this polymer B solution and allowed to sit for four hours, after which the vials and tips were twice each rinsed quickly with the tris-based running buffer. 3. Using the pre-coated pipet tips, samples (dilutions for standard curve and four unknowns) were prepared immediately in these vials after rinsing.

Generation of Standard Curve.

The (1000 ppm polymer B + 13.8 mg/ml IgG) stock solution was diluted into the tris-based running buffer to prepare six polymer B concentrations. Preparation of each concentration involved only one sample transfer, for example, the 10 ppm solution was 10 yL of 1000 ppm stock solution diluted into 990 yL buffer and the 0.5 ppm solution was 0.5 yL stock solution diluted into 999.5 yL buffer. (This means that potentially significant error can occur at the low concentrations since it is challenging to pipet volumes of ^~1 yL accurately.)

Each of the six concentrations was tested immediately after preparation and then four more times (replicates sampled about 2.7 hours apart) . The following figure shows the replicate responses obtained for each concentration. The dashed line indicates the region of responses used to calculate the slope for the standard curve:

polymer B (ppm)

Evaluating the Lower Limit of Polymer B Detection.

Ten 4.0 mL vials compatible with the sample racks in the Biacore 2000 instrument (and corresponding pipet tips) were pre-coatd with polymer B (as described above) and rinsed quickly with running buffer immediately before sample preparation and testing. Each sample was tested twice (with the duplicate measurements about 3.5 hours apart).

S m les est d this e eriment were. fi al cone ip rn)

4 ml ning buffer * 0 pi iO O pm polymer B * 0

Z.M ml f¾!Wi¾ buffe ^■5· 0.4 μΐ lOOOp m polymer 8 _* n.Bmgiml. igG 0,1

3,999 ml unni g buffer ··*· S μί. lOOuppm polymer S 0.2

3.§988 ml. runnine: buffe * 1.2 pt 10OO m pob/O B 0.3

3,S 8 ml r nning buffer + 1.6 pi lOOO m ol mer 8 ^■s- 13,Smgmi. ¾G 0.4

ml rurmii*§ & ffm 2,0 pt i OQpp polymer B * .8m§mi I9G 0.6

3.99TIS ml. run ing buffer 2.4 pi. IbOO pm ot m^f B · I3,8mgml ¾G 0.8

3,9972 mi merging buffer * 2,8 p lOOO pm polymer B ^■s- a.emsmi. IgG 0,7

3. *968 mL nnng butler ·*· 3.2 pi lOObpprn polymer 8 * 13.8?n§ml I9G 0,8

3.9964 ml run ing buffer *3.6 μί. IQOOppm lym r B * 13.8iP§?^'mi loG 0.9

The responses are shown below. For the most part, the responses increased in intensity with concentration and the duplicate responses at each concentration were fairly reproducible :

0 20 40 60 BO 100 120 140

Time (s) Below is the standard curve generated from these low concentrations:

lower-concentration polymer B standard curve

1.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 O.i 0.9

polymer B (ppm)

These results indicate that concentrations of <1 ppm polymer B are observable using this approach and the responses can be used to quantitate the level of polymer B at these low concentrations .

Example E . Detection of Residual Polymer A (poly (diallyldimethyl ammonium chloride) ) on the Sensor Surface of Varying Dose in the Feed Sample and Identity of Residuals Across Various Steps of downstream Processing.

Using pre-coated vials and pipet tips, the four polymer B saples (0.2% polymer B feed, 0.2 % Polymer B after protein A elution, 0.2 % Polymer B after cation exchange elution and 0.2 Polymer B after anion exchange elution) were was diluted 1/5 in tris-based running buffer. Each sample was tested four times (once immediately after preparation and then about 2.7 hours apart) .

Below are the responses for the four samples:

Time (s)

The dashed lines indicate the regions used to calculate the slope of capture on the sensor surface. The rectangular box highlights how much material remained captured on the surface after the end of the sample injection. Under these analysis conditions, only one sample (0.2 % polymer B after protein A elution) displayed an observable level of material captured on the surface. To determine the concentration of polymer B in the 0.2% polymer B after protein elution sample, this sample was diluted 1/5 and 1/12.5 into running buffer (using pre- coated glass vials and pipet tips) and tested for capture on the sensor surface. Both concentrations were tested four times :

0 20 40 60 80 100

Time (s)

The overlay of the quadruplicate tests of each concentration demonstrates that the pre-coating step helped minimize loss o polymer B from this elution sample. The slopes from these two sample concentrations can be used to calculate the polymer B concentration in the stock "0.2% polymer B after protein A elution" sample:

0.2% polymer B protein A elution = 6.3 ± 0.2 ppm polymer B (using standard curve generated from 0.1 to 0.9 ppm Poly B)

0.2% polymer B protein A elution = 8.1 ± 0.2 ppom polymer B (using standard curve generated from 0.5 to 10 ppm polyB) .

The similarity in the values obtained using the two standard curves provides additional evidence for the reliability of this approach. Accordingly, by pre-coating glass vials and plastic pipet tips with a high concentration of polymer B, reproducible responses were obtained for generating standard curves of polymer B concentrations, as well as for testing feed and elution samples.

Using pre-coated glass vials and plastic pipet tips, the four polymer B treated feed and elution samples were tested at five-fold dilution for capture on the sensor surface. Only the "0.2 polymer B protein A elution" appeared to contain observable levels of polymer B. The amount of polymer B in this sample was determined (by testing dilutions of 5x and 12.5X) to be approximately 7 ppm.

feistoi Pah

{p f

S rni Ρΐ%? » - HCCF t4i l ^ s i m PDA:J«A€ r¾»<b arte ΌΕΚ

Claims

What is claimed:

1. A method for detecting residual amounts of a stimulus responsive polymer used for separating a biomolecule of interest from a mixture containing one or more impurities, the method comprising:

a. providing the mixture at a set of conditions,

b. adding to said mixture one or more polymers soluble in said mixture under the set of conditions and capable of reversibly and selectively binding to the biomolecule,

c. mixing the one or more solubilized polymers throughout the mixture;

d. precipitating the one or more polymers by changing the set of conditions in the mixture, such that the precipitated polymers bind selectively and reversibly to the biomolecule;

e. separating the precipitated polymer and bound biomolecule from the mixture;

f . eluting the bound biomolecule from the polymer; and g. detecting, by surface Plasmon resonance spectroscopy, residual polymer in the resulting solution containing the biomolecule of interest, wherein the response unit of the spectroscopy is indicative of the amount of residual polymer in the solution comprising the biomolecule of interest.

2. The method of claim 1, wherein said set of conditions is a set of pH conditions.

3. The method of claim 1, wherein said one or more polymers selected from the group consisting of polyvinylpyridine and copolymers of vinylpyridine .

4. The method of claim 1, wherein said biomolecule of interest is a monoclonal antibody.

5. The method of claim 1, wherein said detection comprises selectively binding residual polymer to the surface of a sensor chip, and subjecting said chip to surface Plasmon resonance spectroscopy.

6. A method for detecting residual amounts of a stimulus responsive polymer used for separating a biomolecule of interest from a mixture containing one or more impurities, the method comprising:

a. providing the mixture at a set of conditions,

b. adding to said mixture one or more polymers solubilizable in said mixture under the set of conditions,

c. mixing the one or more solubilized polymers throughout the mixture;

d. precipitating the one or more polymers and one or more bound impurities out of solution by changing the set of conditions in the mixture;

e. recovering the biomolecule of interest; and

f. detecting, by surface Plasmon resonance spectroscopy, residual polymer in the solution containing the biomolecule of interest, wherein the response unit of the spectroscopy is indicative of the amount of residual polymer in the solution comprising the biomolecule of interest.

7. The method of claim 6, wherein said impurities are selected from the group consisting of host cell protein, cell, cell fragment, nucleic acid, virus, pyrogen and endotoxin impurities.

8. The method of claim 6, wherein said one or more polymers is selected from the group consisting of polyvinylpyridine and copolymers of vinyl pyridine.

9. The method of claim 6, wherein said set of conditions is a set of pH conditions.

10. The method of claim 6, wherein said one or more polymers selected from the group consisting of polyvinylpyridine and copolymers of vinylpyridine .

11. The method of claim 6, wherein said biomolecule of interest is a monoclonal antibody.

12. The method of claim 6, wherein said detection comprises selectively binding residual polymer to the surface of a sensor chip, and subjecting said chip to surface Plasmon resonance spectroscopy.

13. A method for detecting residual amounts of a stimulus responsive polymer used for separating a biomolecule of interest from a mixture containing soluble and insoluble impurities, the method comprising:

a. providing the mixture at a set of conditions;

b. adding one or more polymers or copolymers to said mixture, soluble in said mixture under the set of conditions and capable of reversibly and selectively binding to the biomolecule, and capable of irreversibly binding to said insoluble impurities;

c. mixing the one or more solubilized polymers throughout the mixture;

d. precipitating the one or more polymers, insoluble impurities and said biomolecule out of solution by changing the set of conditions in the mixture;

e. separating the precipitate from the mixture;

f. selectively eluting the biomolecule from the precipitate; and

g. detecting, by surface Plasmon resonance spectroscopy, residual polymer in the solution containing the eluted biomolecule of interest, wherein the response unit of the spectroscopy is indicative of the amount of residual polymer in the solution comprising the biomolecule of interest .

14. The method of claim 13, wherein said impurities are selected from the group consisting of host cell protein, cell, cell fragment, nucleic acid, virus, pyrogen and endotoxin impurities.

15. The method of claim 13, wherein said one or more polymers is selected from the group consisting of polyvinylpyridine and copolymers of vinyl pyridine.

16. The method of claim 13, wherein said set of conditions is a set of pH conditions.

17. The method of claim 13, wherein said one or more polymers selected from the group consisting of polyvinylpyridine and copolymers of vinylpyridine .

18. The method of claim 13, wherein said biomolecule of interest is a monoclonal antibody.

19. The method of claim 13, wherein said detection comprises selectively binding residual polymer to the surface of a sensor chip, and subjecting said chip to surface Plasmon resonance spectroscopy.