WO2003100379A2

WO2003100379A2 - Methods and reagents for removing contaminants from biomolecules

Info

Publication number: WO2003100379A2
Application number: PCT/US2003/016454
Authority: WO
Inventors: James G. Lazar; Peter A. Clausen
Original assignee: Marligen Biosciences, Inc.
Priority date: 2002-05-23
Filing date: 2003-05-23
Publication date: 2003-12-04
Also published as: AU2003241612A8; WO2003100379A3; AU2003241612A1

Abstract

Methods and reagents are provided for removing contaminants from preparations containing a target biomolecule. Binding molecules are prepared that selectively bind contaminating molecules that co-purify with the target biomolecule and are used as affinity reagents to remove the contaminating molecules from the target biomolecule. Affinity reagents also are provided that are useful for removing contaminants from a preparation containing a target biomolecule.

Description

METHODS AND REAGENTS FOR REMOVING CONTAMINANTS FROM BIOMOLECULES

FIELD OF THE INVENTION

The invention provides methods of removing contaminants from target biomolecules, particularly contaminant biomolecules that co-purify with the target biomolecule during affinity chromatography. The invention further provides methods of identifying the contaminants and designing, making, and using antibodies or specific binding entities to remove the contaminants from the target biomolecule. The invention also provides reagents that may be used to detect contaminants in a target biomolecule. In addition the invention provides a kit containing such antibodies or binding entities and optionally, other components.

BACKGROUND

Many processes in biological and pharmaceutical research require highly purified biomolecules such as proteins, enzymes, carbohydrates, lipids, nucleic acids, and the like. These biomolecules often are purified from crude preparations, such as cell lysates or cell culture media by means of affinity chromatography (Cuatrecasas et al. Proc Natl Acad Sci USA; 1968 ; 61(2):636- 43). Affinity chromatography uses a matrix that is bound to a binding agent, such as an antibody, that will in turn bind the target biomolecule. A crude mixture containing the target biomolecule is mixed with the affinity matrix either as a batch or by running the mixture over a column containing the affinity matrix. Under appropriate conditions, the target biomolecule binds to the matrix and is separated from the remaining material. After the remaining material has been removed, the target biomolecule is eluted from the matrix using appropriate conditions. Affinity matrices are commercially available from many suppliers and materials are available for researchers to create their own custom affinity matrices.

A major problem with affinity purification is that additional, unwanted, entities co-purify with the target biomolecule because the affinity matrices often have unwanted specificity for molecules other than the target biomolecule (Den et al. J Chromatogr 1975;l l l(l):217-22). Removing these contaminants from the target biomolecule then requires additional steps. These procedures are often expensive, tedious, and time-consuming; and also increase the potential for a significant loss in yield of the target biomolecule. Strategies for removing contaminants include various procedures to optimize binding and elution conditions and/or sequential rounds of purification over one or more different affinity matrices. Such measures often are unsuccessful.

Another strategy to reduce the possibility of cellular contaminants is the use of specific enzymes to cleave proteins of interest from the affinity matrix. One example of this strategy is the tev protease system (Kapust et al. Protein Expr Purif; 2001; 19(2):312-8). In this system the protein is engineered to incorporate a "tag" or additional sequence portion that binds to the affinity matrix. The process of incorporating a tag into a recombinant protein is a common procedure in the art of protein expression and purification; in the tev protease system a tev protease cleavage site is engineered in the expression construct to lie between the tag sequence and the protein of interest. After binding samples containing expressed proteins to an affinity matrix, the protein is cleaved from the matrix with tev protease and eluted. A drawback of this approach is that the use of enzymes is not cost- effective and such strategies often require the use of conventional chromatography to remove contaminating proteases, salts and buffer components. A modification of the enzyme-based strategy employs inteins (self cleaving peptide sequences) and circumvents the need for expensive enzymes. However, this approach suffers a limitation in that it requires the use of much larger tags (up to 50kDa) relative to the 6-his tag (<lkDa).

Today, there is great interest in producing large numbers of recombinant biomolecules, particularly proteins for proteomic studies, interaction studies, and for pharmaceutical development. In order to produce recombinant protein, it is common practice to clone proteins into DNA expression vectors to be used for producing useful quantities of proteins in bacteria, yeast, insect cells, as well as in mammalian cells or cell lines. Generally, protein sequences are cloned into DNA- based expression vectors that can be transformed, transfected or infected into the appropriate host cells for expression. A wide variety of such vectors generally are available from commercial sources. Many of the commercially available vectors contain special sequences that encode a protein tag that is cloned in-frame with the coding sequence of the gene of interest (Ford et al. Protein ExprPurif, 1991 2(2-3):95-107 ). The tag portion of expressed protein-tag fusions is then exploited for purposes of affinity purification. Common tags include polyhistidine, glutathione, Strep II, GST peptide, FLASH, FLAG, maltose binding protein, Myc, S-peptide, polyglutamine and others.

One of the most common tags is the 6X polyhisitidine tag (his-tag) (Janknecht et al. Proc Natl Acad Sci U S A; 1991; 15;88(20):8972-6) and one of the most common hosts is the bacterium E. coli. The his-tag is extremely popular due to the fact that his-tagged proteins can be easily purified under non-denaturing or denaturing conditions using widely available nickel-chelate affinity media (Ni- NTA HisSorb Strips and Plates, Qiagen). The nickel-bound affinity media is very cost-effective since it can be regenerated at low cost. Proteins that are expressed with other tags are purified on traditional antibody-based affinity columns (anti- FLAG for example) or on matrices that utilize the natural binding affinity between the tag and its binding partner (for example glutathione and glutathione-S- transferase (Smith et al. Gene; 1988; 15;67(l):31-40 ) or streptavidin-biotin). A major drawback of current affinity-based purification approaches is that, in any specific purification scheme, other cellular components will co-purify with the tagged proteins. These contaminants that co-purify with the target protein may interact non-specifically with the affinity matrix material or with the purification moiety that is attached to the matrix. For example, it is well known that purification of polyhistidine-tagged proteins on agarose-based nickel affinity matrix is characterized by co-purification of untagged proteins due to non-specific binding to the agarose portion of the matrix. Similarly, antibody-based affinity matrices are characterized by non-specific binding to the IgG moiety of the affinity matrix. These contaminants are difficult to remove since they may contain biochemical similarities to the target biomolecule and they may interfere with downstream processing, analysis, and use of the target biomolecule. For example, proteins that are targets for therapeutic development must be extremely pure to ensure that any pharmaceutical effect is due solely to the target protein and not to one of the contaminants. Crystallization of proteins to determine structure also requires extremely pure preparations. Many other applications including proteomic analysis and studies of protein-protein and protein-nucleic acid interactions require proteins of high purity.

When his-tagged proteins are grown in E. coli and purified on nickel- chelate affinity matrices, it has been observed that a number of E. coli host proteins often co-purify with the his-tagged protein of interest. Two well-defined examples are the E. coli GroΕl and SlyD proteins (Keresztessy et al., Biochem J., 1996 15; 314 ( Pt l):41-7. Mitterauer, et al, Biochem J. 1999 15; 342 ( Pt 1):33- 39.) Sample contamination by GroΕl and SlyD is widely problematic as these proteins interfere with analysis and use of the target biomolecule and with other downstream applications.

SUMMARY OF THE INVENTION

It is therefore an object of this invention to provide methods for removing contaminants from preparations containing a desired biomolecule.

It is a further object of this invention to provide methods for removing contaminants from preparations containing a desired biomolecule where those contaminants co-purify from an affinity matrix that binds the desired biomolecule.

It is a further object of the invention to provide a plurality of binding molecules that selectively bind contaminants that co-purify from an affinity matrix that binds a desired biomolecule, but where the plurality of binding molecules does not bind the desired biomolecule.

It is a further object of the invention to provide an affinity matrix that contains this plurality of binding molecules. In accomplishing these objects there is provided a method for purifying a desired biomolecule from a biological sample containing contaminants, comprising contacting the sample with an affinity matrix, where the affinity matrix comprises a plurality of binding molecules that selectively bind a plurality of the contaminants. In another embodiment, there is provided a method for purifying a desired biomolecule from a biological sample containing a plurality of contaminating molecules comprising collecting contaminating molecules that co-purify with a target biomolecule, preparing a plurality of binding molecules that selectively bind the contaminants, preparing an affinity matrix comprising the binding molecules; and contacting the sample with the affinity matrix.

The binding molecules may comprise one or more antibodies. The identity of one or more of the contaminants may be known or unknown. One or more of the contaminating molecules may be identified following collection. The antibodies may be obtained by immunizing one or more animals with a plurality of molecules derivable from contaminants contained in a biological sample. Antibodies also may be obtained from a phage display library of antibodies that are selected against a plurality of molecules derivable from contaminants contained in a biological sample. The binding molecules may be derived from contaminants that are present in a biological sample that does not contain significant amounts of the desired biomolecule. The biological sample may have been depleted of the desired biomolecule. In another embodiment, one or more of the binding molecules may be prepared by screening against fragments of one or more of the contaminating molecules.

In another aspect of the invention there is provided a composition comprising a plurality of contaminating molecules derivable from a biological sample that comprises a desired biomolecule, where the plurality of contaminating molecules co-purifies with the desired biomolecule, and where the composition is substantially free of the desired biomolecule.

In a further aspect of the invention there is provided a plurality of binding molecules that selectively bind a plurality of contaminating molecules derivable from a biological sample that comprises a desired biomolecule, where the plurality of contaminating molecules co-purifies with the desired biomolecule, and where the plurality of binding molecules does not bind the desired biomolecule. The binding molecules may be used to prepare an affinity matrix. The plurality of binding molecules may comprise monoclonal and/or polyclonal antibodies, which may be recombinant antibodies. The antibodies may be camelid antibodies. The binding molecules may be labeled with a detectable label.

In yet another aspect of the invention, there is provided a method for detecting the presence of contaminating molecules in a sample comprising a target biomolecule, comprising contacting the sample with a composition comprising labeled binding molecules of the type described above. In another aspect of the invention there is provided a method for purifying a desired biomolecule from a biological sample containing contaminants, comprising ontacting the sample with a first population of binding molecules that selectively bind a plurality of the contaminants and contacting the resulting composition with an affinity matrix, where the affinity matrix comprises a second population of binding molecules that selectively bind the first population of binding molecules.

In accordance with another object of the mvention, there is provided a kit for carrying out any of the methods described above. Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description..

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the procedure for collecting the contaminants of the present invention. A lysate that does not contain a target biomolecule is applied to an affinity matrix under conditions such that the target molecule would bind to the affinity matrix. The flow-through is discarded. The affinity matrix is washed under conditions that would be used if a target biomolecule were bound to the affinity matrix. An elution buffer is then applied to the affinity matrix that would elute the target biomolecule from the affinity matrix, if present. In this example, the elution buffer elutes the contaminants from the affinity matrix that have non- specifically bound to the affinity matrix.

Figure 2 shows identification of contaminants. Contaminants that are eluted from the affinity matrix are separated by 2-dimensional gel electrophoresis, the locations of the contaminants on the gel are visualized by staining, the stained spots cut from the gel and solubilized, digested with trypsin, and the fragments identified by mass spectrometry. The patterns generated by the mass spectrometry are compared to a comprehensive database and individual contaminants are identified. Figure 3 shows preparation of immunogen from a mixture of contaminants. To prepare immunogen from the mixture of contaminants, the contaminants are mixed with a carrier protein such as keyhole limpet hemocyanin (KLH) and a cross-linking agent such as EDC (l-Ethyl-3-[3- Dimethylaminopropyljcarbodiimide Hydrochloride). EDC catalyzes a conjugation reaction between carboxylic acid groups on the contaminants and amino groups on the KLH. After conjugation, the conjugate is mixed with an appropriate adjuvant such as Freund's complete or incomplete adjuvant.

Figure 4 shows preparation of polyclonal antibodies from contaminant- KLH immunogen. Antibodies to the contaminants are prepared by immunizing animals with immunogen, allowing the animals to produce antibodies to the contaminants, and then purifying anti-contaminant antibodies from the animal serum. A pre-immune bleed is first obtained, and the animals are then immunized and boosted by methods and protocols well known in the art. Several immune bleeds are obtained and tested for titer against the purified contaminants. The high titer bleeds are pooled and an irnmunoglobulin fraction is obtained by adding saturated ammonium sulfate to 50% saturation. Precipitated immunoglobulins are pelleted by centrifugation, washed with 50% ammonium sulfate and then resuspended in a small volume of phosphate buffered saline. Figure 5 shows preparation of a contaminant affinity matrix. A matrix with available primary amine groups is mixed with a solution of contaminants and the cross-linking agent EDC. The EDC catalyzes the formation of a bond between the amine groups on the matrix and the carboxylic acid groups on the contaminants and results in the formation of an affinity matrix comprising covalently bound contaminants.

Figure 6 shows purification of antibodies with high affinity for contaminants. The irnmunoglobulin fraction is applied to the contaminant affinity matrix and the antibodies are allowed to bind to the immobilized contaminants. The affinity matrix is washed with a neutral buffer such as phosphate buffered saline (PBS) to remove unbound and low affinity antibodies. An elution buffer is then applied to the affinity matrix to elute the high affinity anti-contaminant antibodies. The elution buffer could be a low pH glycine buffer or may be phosphate buffered saline containing a high concentration of a chaotropic agent such as 4M guanidine-HCl. Figure 7 shows preparation of an anti-contaminant affinity matrix. High affinity anti-contaminant antibodies are oxidized with sodium periodate to oxidize the carbohydrate on the Fc region of the antibodies resulting in the creation of reactive aldehyde groups on the Fc region of the antibodies. The oxidized antibodies are then reacted with a hydrazide-derivitized matrix. The aldehyde groups on the oxidized antibodies react with the hydrazide groups on the matrix forming a stable hydrazone linkage. Excess or unbound antibodies are washed away.

Figure 8 shows affinity purification of contaminants — removal of contaminants from a target biomolecule. A mixture of contaminants and a target biomolecule such as might be obtained from a first affinity purification of the target biomolecule is applied to the anti-contaminant affinity matrix. The contaminants in the mixture are captured onto the affinity matrix while the target biomolecule does not bind and is recovered in a purified form.

DETAILED DISCRETION OF THE INVENTION

Methods and reagents are provided for removing contaminants from preparations containing a target biomolecule, advantageously where it is desirable that the target biomolecule be as pure as possible. One aspect of the invention provides methods for removing contaminants from a biological sample by collecting contaminating molecules that co-purify with the target biomolecule, preparing an immunogen with the collected contaminating molecules, immunizing an animal with the immunogen, collecting serum from the animal, purifying the antibodies directed against the contaminating molecules by affinity chromatography, preparing an affinity matrix with the purified antibodies, and removing contaminants from a preparation containing the target biomolecule with the affinity matrix. Another aspect of the invention provides methods for removing contaminants from a biological sample by identifying the contaminating molecules that co-purify with a target biomolecule by a suitable method, preparing an immunogen with fragments of the identified contaminating molecules, immunizing an animal with the immunogen, collecting serum from the animal, purifying the antibodies directed against the contaminating molecules by affinity chromatography, preparing an affinity matrix with the purified antibodies, and removing contaminants from a preparation of the target biomolecule with the affinity matrix. The invention also provides reagents that are useful for removing contaminants from a preparation containing a target biomolecule, advantageously where it is desirable that the target biomolecule be as pure as possible. Such reagents include: a mixture of molecules comprising contaminating molecules that co-purify with a target biomolecule; an immunogen prepared with a mixture of contaminants that co-purify with a target biomolecule; an affinity matrix prepared with a mixture of contaminating molecules that co-purify with a target molecule; anti-serum containing antibodies directed against a mixture of contaminating molecules that co-purify with a target biomolecule; a solution comprising an irnmunoglobulin fraction of antiserum comprising a mixture of antibodies directed against a mixture of contaminatants that co-purify with a target biomolecule; a solution comprising a mixture of antibodies directed against a mixture of contaminants that co-purify with a target biomolecule obtained by affinity purification; and an affinity matrix comprising antibodies directed against a mixture of contaminants that co-purify with a target molecule. The invention also provides reagents that can be used to detect contaminants in a purified target biomolecule...

Definitions and abbreviations. In the description that follows, a number of terms used in the field of biochemical assays in general and proteases in particular are utilized.

Target biomolecule

As used herein, "target biomolecule" refers to a molecule to be purified. The molecule to be purified can be any peptide, protein, carbohydrate, nucleic acid or any fragment or complex or combination thereof whether recombinant or natural. The biomolecule can be for any therapeutic, diagnostic, or research purpose and can be naturally occurring or can be designed and produced by genetic engineering techniques. It is not necessary that the target biomolecule to be purified be now known or designed, since a similar procedure can be followed for all general classes of biomolecules.

Sample

As used herein, "sample" refers to any material that might contain a target biomolecule, and includes, but is not limited to human and animal tissues, cultured cells, cultured or naturally occurring microorganisms, bodily fluids, blood, serum, extracts from any of these materials, and the like. The sample need not contain only the biological material. The sample may also consist of material on or be in a physical matrix.

Lysate

As used herein, "lysate" refers to a form of the sample in which the sample has been treated in some fashion to increase the availability of the target biomolecule. Lysates may be prepared by heating, sonication, treatment with detergents, forcing the sample through a small orifice under high pressure or other techniques commonly used in the art.

Contaminants

As used herein "contaminants" refers to all molecules that co-purify with a target biomolecule. Contaminants are, in general, proteins or peptides, but may also be carbohydrates, lipids, nucleic acids, or fragments or complexes or combinations, thereof.

Tag

As used herein "tag" refers to a specific moiety that is attached to a target biomolecule to facilitate the purification of the target biomolecule. The tag may be covalently or non-covalently bound to the target biomolecule and may be a natural or non-natural. The tag may comprise protein, peptides, carbohydrates, lipids, nucleic acids, or fragments or complexes or combinations, thereof. Common tags of the present invention include polyhistidine, glutathione, streptavidin, biotin, Strep II, glutathione-S-transferase, FLASH, FLAG, maltose binding protein, Myc, S-peptide, polyglutamine and others. The tag may be a naturally occurring part of a biomolecule or the target biomolecule may be produced with a tag through recombinant methods. For example, proteins with a glutathione-S-transferase tag may be produced by expressing recombinant proteins from pGEX expression vectors available from Amersham Biosciences.

Matrix

As used herein, a "matrix" may be any porous or non-porous material or matrix suitable for attaching molecules such as proteins, peptides, nucleic acids, carbohydrates and the like. Commonly used matrices include 1-10% cross-linked agarose, polyacrylamide, cross-linked dextran, cellulose, polystyrene, polymethacrylic acid, and the like. Other matrices of the invention may comprise nylon, nitrocellulose, diazonitrocellulose, glass, silicon, polyvinyl chloride, polypropylene, polyethylene, agar, starch, or any other material that allows for the immobilization of molecules. The matrix can be formed in beads, filters, membranes, flat surfaces, tubes, channels, wells, sheets, microspheres, columns, fibers and the like. The matrix may also comprise a magnetic, paramagnetic or non-magnetic, metal to allow for easier handling and separation of the matrix.

Affinity Ligand

As used herein, "affinity ligand" refers to any molecule or mixture of molecules, of low or high molecular weight, that can be attached to a matrix and that specifically interacts with the proteins, peptides, nucleic acids, carbohydrates or the like of interest. Examples of classes of commonly used affinity ligands include nucleotides, oligo- and polynucleotides, amino acids, peptides, proteins, lectins, immunoglobulins, carbohydrates, dyes, chelated metal ions, vitamins, and other small molecules. Examples of specific affinity ligands include adenosine triphosphate (ATP), oligo dT, poly rA, NADP, D-tryptophan, concanavalin A, wheat germ lectin, Protein A, gelatin, ovalbumin, xylose, mannose, heparin, Cibachron Blue F3G-A, aminophenylboronic acid, Iminodiacetic acid - nickel (II) chelate, oxamic acid, and biotin. Other affinity ligands are known in the art and are encompassed by the invention, as are affinity ligands that are discovered in the future.

Affinity matrix As used herein, "affinity matrix" refers to an affinity ligand attached to a matrix. The affinity ligand is generally attached to the matrix through covalent interactions, but may also be bound to the matrix through non-covalent interactions. In order to improve the interaction between the affinity ligand and the target biomolecule, a spacer may be introduced between the matrix and the affinity ligand. The spacer is generally 4-20 atoms long and may be first attached to either the matrix or the affinity ligand. The affinity matrix may also comprise a combination of affinity ligands, bound covalently and/or non-covalently. For example, an affinity matrix may be prepared by first binding Protein A to a matrix through covalent interactions and then binding a specific irnmunoglobulin to the Protein A through non-covalent interactions. An affinity matrix may also be prepared by binding a mixture of affinity ligands to a matrix simultaneously or in series.

Binding Molecules

In the context of the present invention, a "binding molecule" is a type of molecule that can selectively capture or bind a contaminant from a solution containing a plurality of contaminants. Examples of binding molecules are polyclonal and monoclonal antibodies, antibody fragments, proteins, peptides, nucleic acids, oligonucleotides, lectins, carbohydrates, and the like. A binding molecule also may be a molecule that binds to or recognizes another molecule that binds the contaminants. For example, anti-rabbit IgG is a binding molecule that may be used to capture a rabbit antibody-contaminant complex. Similarly, Protein G may be used to capture a goat antibody-contaminant complex.

Detectable label

As used herein, a "detectable label" is a moiety that can generate a signal either directly or indirectly. In the present invention, a wide variety of signal moieties are possible including, for example, radioactive isotopes, fluorescent labels, chemiluminescent labels, bioluminescent labels, and enzyme labels. The signal moieties also can be haptens that are recognized by secondary reagents such as antibodies, peptides, direct chemical interactions, and by other methods that are well known in the art. The signal moiety may also be an oligonucleotide or nucleic acid that can be detected by hybridization, polymerization, ligation and/or amplification by methods well known in the art.

The signal moiety may also comprise two chromophores bound in close proximity to utilize a phenomemon called fluorescence resOnance energy transfer (FRET). When illuminated with light of the appropriate wavelength, one chromophore absorbs a photon and then exists in the excited state. The energy from the excited chromophore is transferred to an acceptor molecule when the two molecules are in close proximity to each other. This transfer prevents the excited chromophore from releasing the energy in the form of a photon of light thus quenching the fluorescence of the chromophore. When the acceptor molecule is not sufficiently close, the transfer does not occur and the excited chromophore may then fluoresce. Such pairs of interacting signal moieties are well known in the art. A similar phenomenon known as luminescence resonance energy transfer (LRET) occurs between sensitized lanthanide metals and acceptor dyes and may be used in the present invention.

The present invention provides methods and materials for purifying recombinant proteins and other biomolecules from co-purifying contaminants during affinity purification and also provides methods and reagents for the detection of contaminants in preparations containing the target biomolecule.

Methods for Removing Identified Contaminating-Proteins

Protein Identification

In one aspect, the invention provides methods for removing contaminating biomolecules that co-purify with a target biomolecule of interest during affinity purification. The target biomolecule may be present in a crude preparation such as a cellular lysate or culture medium and the contaminating biomolecules may be identified through conventional biochemistry techniques.

For example, recombinant proteins containing a glutathione-S-transferase tag produced in insect cells can be purified from insect cell lysate. Similar methods may be used with any cell system used to produce recombinant proteins, such as bacterial, viral, mammalian and transformed cell systems. The methods also can be used with other affinity column purification systems, such as are commonly known and used in the art.

For the purpose of defining the contaminating proteins and producing antibodies, a cell lysate or cell culture medium that does not contain any affinity tags or recombinant biomolecules advantageously may be used so that all of the binding sites on the affinity matrix are available to bind co-purifying contaminants. The column and conditions are chosen that will elute the target biomolecule. The cell lysate is brought into contact with the affinity matrix and contaminating entities are allowed to bind under conditions similar to those used for binding the target biomolecule. The matrix is washed under conditions similar to those used for washing the matrix when the target biomolecule is bound. After washing, those biomolecules that have bound to the column are eluted using standard conditions. The recovered biomolecules are then separated and identified using standard procedures. If the cell lysate did not contain the target biomolecule or a tag then the material eluted from the affinity matrix will consist entirely of contaminating biomolecules. If the cell lysate contained the target biomolecule or a specific tag that was bound by the affinity matrix, then the material eluted from the matrix will contain both the target biomolecule and the contaminating biomolecules. Contaminating proteins (and the target biomolecule if present) may be separated by, for example, elecrrophoresis on ID or 2D polyacrylamide gels and can be visualized by chemical or immunochemical staining. Proteins are recovered from gel bands or spots and fragmented, for example by enzymatic digestion (using trypsin, for instance) to generate peptide fragments. The fragment masses and fragment sequences are obtained by mass spectrometry or other methods as are well known in the art. Sequence data of the protein fragments can be compared to DNA and protein databases to identify the full protein sequence. If a match is not found, the peptide sequence itself can be used in the next step.

Antibodies and other binding moieties

Once the contaminants have been identified by peptide sequence and database analysis, peptides can be designed and made for the development of mono- or polyclonal antibodies. Alternatively, isolated intact proteins, protein fragments, or peptides can be used to generate antibodies using conventional techniques. A decision can be made as to which of the proteins are important contaminants based on the quantity and properties of the contaminants.

Antibodies can be made by any method known in the art. They can be made in any useful animal or in a cell system, such as hybridoma cells. Both monoclonal and polyclonal antibodies can be prepared and especially favored are polyclonal antibodies prepared in camelid species that lack light chains (Muyldermans, S., JBiotechnol. 2001 Jun;74(4):277-302.; Muyldermans, S., et al, Protein Eng. 1994, 7(9): 1129-35.) Antibodies can also be prepared using recombinant antibody techniques and phage display. (Maynard J, Georgiou G. Annu Rev Biomed Eng 2000 (2):339-76.)

The scope of the invention also includes fragments of antibodies, natural or synthetic peptides, aptamers and other materials that can be used to bind the contaminating biomolecules such as synthetic dyes. (Clark SL, Remcho, VT Electrophoresis 2002 23(9):1335-1340 and Lowe CR, Burton SJ, Pearson JC, Clonis YD, Stead V. J Chromatogr. 1986 Apr 11;376:121-30). Peptides that bind the contaminating materials can be identified using phage display methods thar are known in the art. For example, the contaminating materials can be bound to a solid surface, which is then contacted with a library of phage each displaying a peptide at the phage surface. The peptide advantageously is a cyclic peptide, but this is not essential for practicing the invention. Phage that bind to the contaminating material on the solid surface may be eluted and used to obtain supplies of peptides that specifically bind the contaminating materials. Kits for peptide phage display are commercially available from, for example, new England Biolabs (Beverley, MA). The binding peptides may be characterized if necessary, for example by sequencing the DNA encoding the peptide insert in the phage particle, or may be used without further identification by expressing the peptides from the insert in the phage DNA. In instances when the peptides are identified they may then be produced in large quantities by conventional methods such as, for example, solid phase peptide synthesis.

Once prepared, the antibodies or binding entities can be bound individually or as a mixture to affinity matrices. Samples containing recombinant protein(s) of interest can be passed through or over the matrix and the contaminants preferentially bind to the matrix, thereby separating the pure recombinant protein from the contaminants. The removal of contaminants may take place as a pre-clearing step prior to affinity tag purification, concomitant with purification using an affinity tag, or after affinity tag purification. Similarly antibody-contaminant purification can be performed on proteins or biomolecules that are to be purified and do not have a tag but are bound to an affinity matrix by another process. As a post affinity purification step, removal of contaminating proteins may be coupled with a desalting or buffer exchange step. In the above forms, the affinity matrices may be in any suitable format now known or that may be developed in the future, for example, any of the cuπently available supports (sepharose beads, magnetic beads, filters etc). The support advantageously is a bonded fiber matrix.

In an alternative 2-step procedure, the antibodies are added directly to the crude recombinant protein or cell lysate. The mixture may then be passed over a protein A or protein G column that removes the antibodies and any contaminants bound to the antibodies. It is intended that the recombinant protein of interest will not bind to immobilized protein A- or G. A similar procedure may be performed by first biotinylating the antibodies, adding the biotinylated antibodies to the protein mixture, and removing biotinylated antibody, and antibody-antigen complexes using immobilized avidin, streptavidin, neutravidin or any similar method.

. Methods for Removing Unidentified Contaminating-Proteins In another aspect the invention can be used to remove contaminating proteins and biomolecules that have not been identified. For such contaminants, a cell system advantageously is chosen which provides or is expected to provide optimum production of the desired protein. Using the desired expression system, a cell lysate, cell culture medium or other source material is passed through an affinity matrix such as agarose-nickel. Again, a lysate that does not contain a recombinant protein or a protein tag may be used so as to allow all binding sites on the affinity matrix to bind the co-purifying contaminants to the fullest extent. The lysate flow-through is recovered, and after washing under standard conditions, the bound contaminants are recovered by conventional methods (in this case pH or imidizole buffer). The contaminants are then used to directly immunize animals for the production of polyclonal antibodies without further identification. The contaminants may be desalted, concentrated, digested, modified, and/or mixed with adjuvant as necessary to achieve the appropriate conditions for immunization. The advantage of this mode of the invention over the previous mode is that it results in the production of antibodies to contaminants that may not be seen, resolved, or detected on ID of 2D gels or by other standard biochemical methods. Polyclonal antibodies can be made in any animal but are preferably made in camel species so that antibodies without light chains can be obtained. The scope of the invention also includes fragments of antibodies, natural or synthetic peptides, aptamers and other materials that can be used to bind the contaminating biomolecules such as synthetic dyes. (Clark et al, Electrophoresis 2002 23(9): 1335-1340 and Lowe et al, J Chromatogr. 1986 Apr 11;376:121-30)

III. Other Uses of Methods and Antibodies of the Invention

In another aspect, the antibodies obtained from these procedures are useful for detecting and measuring the contaminants in purified biomolecules such as recombinant proteins and for optimizing purification conditions to minimize the presence of contaminants. The scope of the present invention encompasses antibodies or binding entities obtained from this procedure and their use for identifying contaminants in purified biomolecule preparations. Labeled antibodies can be used to detect contaminants in a purified biomolecule and to optimize a process so as to decrease the relative amount of contaminating proteins.

In another aspect, the invention can be used in similar modes to purify other types of biomolecules whether natural or recombinant, and with or without tag sequences. Similar affinity purification schemes are used in the art to purify many kinds of proteins and biomolecules. Using normal procedures of protein chemistry and molecular biology, the methods and compounds of the present invention can readily be adapted to all such purification procedures. For example, the biomolecule to be purified may not be a recombinant protein expressed in a cell system but could be one that is in its natural medium such as blood, milk, saliva, urine, or other fluid, or from a whole animal or organ, including biopsy material. The biomolecule could also be found in a plant or in plant organs or cells, the biomolecule could be natural or recombinant. The biomolecule could be one that is normally produced by cells or one for which increased production can be induced by addition of a chemical or hormone.

The methods of the present invention could be used in the production and purification of proteins, biomolecules, or antibodies in transgenic animals or plants. In any such purification scheme, the material from which the biomolecule or protein is being purified is treated in an appropriate manner for purification such as grinding, sonicating, dialysing, centrifuging, concentrating, or filtering as well as lysing; and the contaminating proteins used to make antibodies as above. When the target biomolecule is a naturally occurring entity (as opposed to a recombinant biomolecule), the co-purifying contaminants need to be identified prior to the generation of antibodies so as not to generate antibodies to the target biomolecule. Alternatively, production of the target biomolecule may be suppressed or prevented by standard techniques (for example knock-out or anti- sense techniques), and the affinity reagents then can be made without identification of the individual components.

The mvention can be practiced with both highly specific affinity reagents such as antibodies and also with non-specific affinity reagents such as dyes (Cibacron Blue for example binds albumin and transferrin), Heparin, gelatin, lysine, concavalin A, sugars, and any other material that can be used to preferentially bind specific types or classes of biomolecules.

The invention described is superior to other methods currently available because it permits removal of contaminants from purified proteins that cannot be removed with current available technology. A major advantage of this technology is that contaminating entities need not be identified in order to prepare antibodies or an affinity matrix that can remove the contaminants. The invention can be provided commercially in the form of a kit containing one or more antibodies and optionally other components of the procedure and methods including additional antibodies, an expression system, an affinity purification matrix, detection methods, solutions, and so forth.

The following examples are provided for the purposes of illustration of the invention and do not in limit its scope. The references sited are hereby incorporated by reference as though included in their entirety.

EXAMPLES

Example 1: Identification of e.coli proteins that have affinity for nickel that may co-purify with recombinant proteins of interest.

Expression of recombinant proteins in a host cell such as E. coli taxes the host cell machinery and may lead to elevated production of any of a number of cellular proteins that are either directly involved in protein synthesis, or that directly or indirectly regulate normal cellular homeostasis. For this reason, lysates from E. coli that have been driven to express a recombinant protein were chosen. Competition of recombinant protein and host protein for binding sites on the nickel affinity matrix is avoided by not incorporating the HIS affinity tag in the coding region of the recombinant protein.

A: Transform pBAD expression vector coding for a thioredoxin gene

(pBAD/Thio-TOPO (Invitrogen cat #K370-01) into TOP 10 e. coli cells (Invitrogen C4040-10). B: Induce expression from pBAD vector with 0.2% arabinose for four hours. Take cell samples before and after induction and lyse by sonication in Tris (e.g. lOmM pH 8) or phosphate buffer (PBS pH 8) containing 200-300mM

NaCl and non-ionic detergent (Triton X-100, n-octosylglucosamine). C: Add imidizole to lysate to final concentration of 5mM. Bind the lysate to affinity column and wash the column extensively with phosphate buffer or Tris buffer pH 8.0 containing 25 mM imidazole. D: Elute bound proteins with 1 column volume of phosphate or Tris buffer containing 200mM imidazole or 2 mM EDTA. E: Separate proteins eluted from the affinity column using 2-D gel electrophoresis.

1. Samples are mixed with buffer to yield final concentrations as follows: 7 M urea, CHAPS (4% w/v), DTE (65 mM), Resolytes 4-

8 (2 % v/v) and a trace of bromophenol blue.

2. Run samples on IPG (isoelectric focusing gel) strips (Amersham).

3. After the first dimension run, the strips are equilibrated in order to resolubilize the proteins and to reduce -S-S- bonds. Strips are equilibrated with 100 ml of a solution containing Tris-HCl (50 mM) pH 8.4, urea (6 M), glycerol (30% v/v), SDS (2% w/v) and DTE (2% w/v) for 12 min. -SH groups are subsequently blocked with 100 ml of a solution containing Tris-HCl (50 mM) pH 6.8, urea (6 M), glycerol (30% v/v), SDS (2% w/v), iodoacetamide (2.5% w/v) and a trace of Bromophenol Blue for 5 min.

4. 2^nd dimension PAGE gel: Gel composition and dimension: Dimension: 160 x 200 x 1.5 mm Resolving gel: Acrylamide/PDA (9-16% T / 2.6% C) Leading buffer: Tris-HCl (0.375 M) pH 8.8 Trailing buffer: Tris-glycine-SDS (25 mM-198 mM-0.1% w/v) pH 8.3 Additives: Sodium thiosulfate (5 mM) Polymerization agents: TEMED (0.05%) APS (0.1%) Overlay gels with butanol for 2 hours. After replacing the overlay with Tris-glycine-SDS (25 mM- 198 mM-0.1% w/v) pH 8.3 the gels are left overnight.) 5. IPG gel strips transfer: After the equilibration, the IPG gel strips are cut to size. The second dimension gels are over layered with a solution containing agarose (0.5% w/v) and Tris-glycine-SDS (198 mM-25 mM-0.1% w/v) pH 8.3 heated at about 70° C and the IPG gel strips are immediately loaded through it. Running conditions

Current: 40 mA/gel (constant) Voltage: The voltage is non- limiting, but usually requires 100 to 400 V. Temperature: 8-12° C Time: 5 hours F: Prepare samples for mass spec analysis

1 Following the 2^nd dimension, gels are visualized using sypro ruby dye.

2. Cut out bands/spots of interest at the margin of detectable stain, Measure gel slice volumes. Put in pre- washed 500 μl microfuge tube.

3. De-stain two times with 200 μl of 200 mM ammonium bicarbonate (NH4C03) pH 8: 50% AcN 45 min @ 37° C, then dry gel slice completely in SpeedVac.

4. Reduce: Add 100 μL of 2 mM TCEP (Tris(2- carboxyethyl)phosphine, Sigma #C4706) in 25 mM ammonium bicarbonate (pH 8.0) to the dried gel and incubate 15 minutes at 37°C with agitation; remove supernatant.

5. Alkylate: add 100 μL of 20 mM iodoacetamide in 25 mM ammonium bicarbonate (pH 8.0) and incubate for 30 minutes at 37°C in the dark; discard the supernatant; Wash gel band three times with 200 μL of 25 mM ammonium bicarbonate for 15 minutes, each with agitation; then dry gel slice completely in SpeedVac. 6. Re-hydrate gel slice in 20 μl or 1.5 X original gel slice volume (whichever is greater) of 0.02 μg/μl of Promega Sequencing grade modified trypsin in 50% AcN, 40 mM NH4C03 pH 8; 0.1% w/v n- octylglucoside (1-O-n-Octyl-beta-D-glucopyranoside) for 1 h at room temperature to allow the concentrated trypsin to diffuse into the gel slice.

7. Add additional 50 μl 50% AcN, 40 mM NH4C03 pH 8; 0.1% w/v n-octylglucoside (1-O-n-Octyl-beta-D-glucopyranoside), and incubate with agitation for 16-18 h @ 37° C. a. Remove the supernatant and put in fresh pre-washed tube

(extract 1) b. SpeedVac volume down to 40%-50% of original volume to remove AcN for subsequent ZipTip clean-up c. Use ZipTip (Millipore) to concentrate and clean the digested sample

G. Mass spec analysis of trypsinized proteins is carried out using MALDI- TOF mass spectrometry.

This approach is a useful tool for identifying proteins that may co-purify with any affinity molecule in any of the expression hosts.

As an alternative to the 2-D gel approach, proteins eluted from the affinity matrix may be directly digested with trypsin and analyzed using LC-ESI-MS (Biemann, K, Methods Enzymol. 1990, 193, 351-360) and/or LC-FT-MS (Martin S. et al, Analytical Chemistry Chem. 2000 Sep 15; 72(18): 4266-74).

Antibody production

Once peptide masses are obtained, they can be searched against the available protein databases such as are available at the National Library of Medicine searchable databases (www.ncbi.nlm.nih.gov). From the fragment mass patterns, protein identities are determined. Antigenic peptides are designed and used to generate monoclonal and polyclonal antibodies. Antibodies are made using standard methods such as those described in Antibodies: A Laboratory Manual, Ed Habor and David Lane, Cold Spring Harbor Laboratory Press, 1988. The steps in this process are schematically shown in Figure 1. Removal of contaminating biomolecules.

Once antibodies to contaminating biomolecules have been prepared, they are bound to an agarose adipic acid hydrazide (Amersham Biosciences) by methods known in the art (Phillips, TM and Dickens BF. Affinity and

Immunoaffinity Purification Techniques, Eaton Publishing, 2000). The antibodies are diluted to 1-100 μg/mL in 0.1 M sodium acetate, pH 5.0. The mixture is cooled to 4°C and an equal volume of 10 mM sodium periodate is added. The solution is mixed well and incubated for 45 minutes at 4°C in the dark. The reaction is stopped by adding 25 μL of glycerol per ml final volume. The oxidized antibodies are desalted into 0.1 M sodium phosphate, pH 7.2 with Sephadex G-50 and then added directly to the agarose adipic acid hydrazide. The oxidized antibody and the support are mixed well and incubated with end-over- end mixing for 16 hours. The gel is then washed 5 times in 0.1 M sodium phosphate, pH 7.2.

Antibody Labeling

Antibodies are labeled by methods well known in the art ( Hermanson, GT. Bioconjugate Techniques, Academic Press, 1996.). Antibodies are oxidized with sodium periodate and desalted into phosphate buffer as described in the previous section. Biotin hydrazide or biotin-LC-hydrazide are added to a final concentration of 5 mM and allowed to react for 2 hours at room temperature, followed by cooling to 0°C. An equal volume of 30 mM sodium cyanoborohydride is added. The biotinylated antibody is desalted into phosphate buffered saline using Sephadex G-25.

Example 2: Identification of E.coli proteins that have affinity for glutathione that may co-purify with recombinant proteins of interest. Glutathione-S-transferast (GST) fusion proteins are constructed by inserting a gene or gene fragment into the multiple cloning site of a GST expression vector such as the series of pGEX vectors from Amersham Biosciences. Expression of GST-fusion proteins in E. coli yields proteins with the GST moiety at the amino terminus and the protein of interest at the carboxyl terminus. The protein accumulates in the cell's cytoplasm. GST occurs naturally as a 26kD protein that can be expressed in E. coli with full enzymatic activity. GST fusion proteins are purified from bacterial lysates by affinity chromatography using immobilized glutathione. GST fusion proteins are captured by an affinity matrix and most impurities are removed by washing. Fusion proteins are purified by applying reduced glutathione to the affinity matrix. Proteins purified by this method are typically 80% pure. The remaining uncharacterized materials in the eluate are contaminating molecules that co-purify with the target proteins.

In this example, E. coli lysate is applied to a glutathione affinity matrix. The matrix is washed to remove proteins that do not bind to the glutathione affinity matrix. Proteins remaining on the matrix are eluted with reduced glutathione under conditions identical to those which one would use to purify the target protein. The molecules in the eluate therefore are the contaminating molecules that co-purify with the target protein. These molecules are then used to prepare an immunogen, the immunogen is used to immunize an animal, antibodies are raised to the immunogen, and the purified antibodies are used to remove contaminants from a target protein.

A: Preparation of E.coli sonicate

Pick a single colony of E. coli strain BL21 from a plate and inoculate 100 ml of 2X YTA medium. Incubate for 12-15 hours at 37°C with vigorous shaking. Dilute the culture 1 :100 into 1000 ml fresh pre-warmed YTA medium and grow at 30-37°C with shaking until the A600 reaches 0.5-2.0.

Add 100 mM IPTG to a final concentration of 0.5 mM and continue incubation for an additional 2-6 hours. Transfer the culture to appropriate centrifuge containers and centrifuge at 7700 x g for 10 minutes at 4°C to sediment the cells. Discard the supernatant and drain the cell pellet. Place on ice. A pipet is used to completely suspend the cell pellet by adding 50 ul of ice-cold IX PBS per ml of culture. Disrupt the suspended cells using an appropriately equipped sonicator fo rthe suspended volumes. Sonicate on ice in short bursts. Add 20% Triton X-100 to a final concentration of 1%. Mix gently for 30 minutes. Centrifuge at 12,000 x g for 10 minutes at 4°C. Transfer the supernatant to a fresh container.

B: Preparation of glutathione affinity matrix Transfer 1 ml Glutathione Sepharose 4B (Amersham Biosciences) to an appropriate container. Sediment the medium by centrifugation at 500 x g for 5 minutes. Decant the supernatant Add 10 ml ice-cold IX PBS to the Glutathione Sepharose 4B and mix thoroughly. Pellet the medium by centrifugation at 500 x g for 5 minutes. Decant the supernatant. Add 0.75 ml ice cold IX PBS to the pelleted Glutathione Sepharose 4B. Mix thoroughly.

C: Purification of non-specifically bound molecules.

Add the resuspended Glutathione Sepharose 4B to the bacterial sonicate from A . Incubate for 30 minutes at room temperature with end-over-end rotation. Centrifuge the mixture at 500 x g for 5 minutes. Carefully decant the supernatant. Wash the matrix with 10 ml of ice-cold IX PBS. Invert to mix. Sediment the medium by centrifuging at 500 x g for 5 minutes. Decant the supernatant. Repeat twice for a total of three washes. Elute the bound materials from the sedimented matrix by adding 1 ml of elution buffer (10 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0). M ix gently to resuspend the matrix. Incubate at room temperature for 10 minutes to eluted the materials from the matrix. Use gentle agitation or end-over-end rotation. Sediment the medium by centrifuging at 500 x g for 5 minutes. Decant the supernatant into a fresh tube. Repeat two times for a total of three elutions. Pool the eluates. D: Concentrate and desalt the co-purifying molecules.

The purified molecules that co-purify with a GST-labeled target protein are desalted into 0.1 M MES and concentrated to approximately 1 mg/ml using standard methods.

E: Preparation of immunogen and raising antibodies.

A portion of the purified contaminants are covalently bound to a carrier protein such as keyhole limpet hemocyanin (KXH) using the cross-linking agent EDC (l-Ethyl-3-[3-Dimethylaminopropyl]carbodiimide Hydrochloride) according to the manufacturer's instructions. Briefly, 2 mg KLH is reconstituted with 200 μl deionized water. Sodium chloride is added to the purified contaminants to bring the concentration to 0.9 M NaCl. 500 μl of contaminant solution is added to 200 μl carrier protein solution. 50 ul of a lOmg/ml solution of EDC is added to the contaminant/carrier solution to begin the conjugation reaction. The reaction is allosed to proceed for 2 hours at room temperature. Any precipitate is removed by centrifugation prior to further purification. The conjugate is purified by gel filtration using Sephadex G-50. The purified conjugate is diluted into complete or incomplete Freund's adjuvant and used for immunization. Polyclonal and monoclonal antibodies to the purified contaminants can be raised in any animals using standard methods such as those described in Antibodies: A Laboratory Manual. Ed Habor and David Lane, Cold Spring Harbor Laboratory Press, 1988. Advantageously, animals of the species camelid may be used. Antiserum from the animals can be tested for reactivity to the purified contaminants by binding to purified contaminants to a high binding ELISA plate (Corning) and detecting the bound antibody with a labeled secondary antibody. For example, rabbit antibodies raised against purified contaminants can be detected with an alkaline phosphatase-labeled anti- rabbit antibody raised in goats. Purification of antibodies

Antibodies are purified from the serum of immunized animals using methods well known in the art. First, nearly all immunoglobulins can be separated from serum using ammonium sulfate precipitation. A saturated solution of ammonium sulfate is slowly mixed with serum until the concentration of ammonium sulfate in the serum reaches 50% saturation. At this point, the immunogloblulin in the serum will have precipitated out and can be purified by centrifugation and removal of the supernatant, the pellet containing the precipitated irnmunoglobulin is dissolved in a small amount of IX PBS.

Further purification can be carried out using an affinity matrix prepared with the purified contaminants. The purified contaminants are covalently bound to an amine-containing matrix through the cross-linking agent EDC. Alternatively, the purified contaminants can be coupled to cyanogen bromide activated Sepharose 4B. Once the contaminants have been immobilized on the matrix, the affinity matrix can be used to specifically purify antibodies with high specificity to the purified contaminants.

G: Preparation of contaminant-removal affinity column.

The purified mixture of antibodies directed against the contaminants are oxidized with 10 mM sodium periodate for 30 minutes at room temperature in order to generate reactive aldhehyde groups on the Fc portion of the antibodies. Excess sodium periodate is removed by gel filtration. The antibodies are mixed with a matrix containing a hydrazide moiety and the aldehyde groups on the antibodies reacts covalently with the hydrazide moiety forming a stable hydrazone linkage. Such hydrazide-modified matrices are available commercially from vendors such as Pierce Biotechnology, Rockford, Illinois. Unreacted antibody is washed from the matrix, and the affinity matrix is packed into a column.

H: Removal of contaminants from a solution containing the target biomolecule. The solution containing the target biomolecule in a standard buffer such as

PBS is allowed to flow slowly through the affinity matrix packed in the column. Contaminants in the solution bind to antibodies immobilized on the affinity matrix while the target biomolecule passes through the column. The purified target biomolecule is collected. The contaminants bound to the column can be removed by washing the column in a low pH buffer such as 0.1 M glycine pH 3.0 or a buffer containing a chaotropic agent such as 4M guanidine hydrochloride in phosphate buffered saline.

Example 3 : Generation of peptides that bind contaminants. A mixture of contaminants is bound to the surface of Immunotubes (Nunc) following the manufacturer's instructions, and then blocked overnight with BSA in PBS to prevent non-specific phage binding to the tubes. An aliquot of the C7C complete phage library from New England Biolabs (Beverley, MA) is incubated in the tubes at 4°C for 45 minutes in PBS with 0.5 % BSA (binding buffer). After incubation, the tubes are washed with PBS/0.5% BSA 0.05% Tween (wash buffer) for a total of 6 washes. Phage that bound are eluted with 0.2M glycine (pH 2.2) for 8 minutes then neutralized with 50 uL of IM Tris-HCl (pH 9.0). The number of phage bound is determined and the remaining eluate is amplifed. The amplified phage is reincubated with the Immunotubes and the process is repeated for a total of five rounds of maturation. After the five rounds of selection, phage from each round are titered, and plaques are picked. The phage DNA may be amplified by PCR to obtain DNA encoding the binding peptides which can be expressed in E. coli or can be sequenced to identify the sequences of the binding peptides. The peptides are then prepared by, for example, solid phase peptide synthesis.

REFERENCES

I . Biemann, K., Methods Enzymol. 1990, 193, 351-360 2. Clark et al, Electrophoresis 2002 23(9):1335-1340.

3. Cuatrecasas et al. Proc Natl Acad Sci USA 1968:61(2):636-43.

4. Den et al. J Chromatogr 1975: 11 l(l):217-22

5. Cytotoxic contaminants in commercial Clostridium perfringens neuraminidase preparations purified by affinity chromatography. 6. Ford et al. Protein Expr Purif ^'1991 2(2-3):95-107

7. Harlow et al Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988.

8. Hermanson , GT. Bioconjugate Techniques, Academic Press, 1996.

9. Janknecht et al. Proc Natl Acad Sci USA 1991 88(20):8972-6. 10. Kapust et al. Protein Expr Purif 2000 19(2):312-8.

II. Keresztessy et al. Biochem J. 1996 Feb 15; 314 (Pt 1): 41-7.

12. Lowe et al, J Chromatogr. 1986 Apr 11;376:121-30.

13. Martin et al. Analytical Chemistry 2000 Sep 15;72(18):4266-74.

14. Maynard et al, Aiinu Rev Biomed Eng 2000; (2):339-76. 15. Mitterauer et al, Biochem J. 1999 Aug 15; 342 (Pt 1): 33-9.

16. Muyldermans et al, Protein Eng. 1994 Sep; 7(9): 1129-35.

17. Muyldermans, J Biotechnol. 2001 Jun; 74(4): 277-302. 18. Phillips et al. Affinity and Immunoaffinity Purification Techniques, Eaton Publishing, 2000.

19. Smith et al. Gene 1988 67(l):31-40.

Claims

What is claimed is:

1. A method for purifying a desired biomolecule from a biological sample containing contaminants, comprising contacting the sample with an affinity matrix, wherein said affinity matrix comprises a plurality of binding molecules that selectively bind a plurality of said contaminants.

2. The method according to claim 1 wherein said binding molecules comprise one or more antibodies.

3. The method according to any preceding claim, wherein the identity of one or more of said contaminants is unknown.

4. The method according to any preceding claim, wherein the identity of one or more of said contaminants is known.

5. The method according to any of claims 2-4, wherein said antibodies are obtained by immunizing one or more animals with a plurality of molecules derivable from contaminants contained in a biological sample.

6. The method according to any of claims 2-4, wherein said antibodies are obtained from a phage display library of antibodies that are selected against a plurality of molecules derivable from contaminants contained in a biological sample.

7. The method according to claim 5 or claim 6, wherein said molecules are derived from contaminants that are present in a biological sample that does not contain significant amounts of said desired biomolecule.

8. The method according to claim 7, wherein said biological sample has been depleted of said desired biomolecule.

9. A method for purifying a desired biomolecule from a biological sample containing a plurality of contaminating molecules comprising collecting contaminating molecules that co-purify with a target biomolecule; preparing a plurality of binding molecules that selectively bind said contaminants; preparing an affinity matrix comprising said binding molecules; and contacting said sample with said affinity matrix.

10. The method according to claim 9, wherein said binding molecules comprise antibodies.

11. The method according to claim 9 or claim 10, wherein one or more of said contaminating molecules is identified following collection.

12. The method according to any of claims 9-11, wherein one or more of said binding molecules is prepared by screening against fragments of one or more of said contaminating molecules.

13. A composition comprising a plurality of contaminating molecules derivable from a biological sample that comprises a desired biomolecule, wherein said plurality of contaminating molecules co-purifies with said desired biomolecule, and wherein said composition is substantially free of said desired biomolecule.

14. A plurality of binding molecules that selectively bind a plurality of contaminating molecules derivable from a biological sample that comprises a desired biomolecule, wherein said plurality of contaminating molecules co- purifies with said desired biomolecule, and wherein said plurality of binding molecules does not bind said desired biomolecule.

15. An affinity matrix comprising a plurality of binding molecules according to claim 14.

16. A composition according to claim 15 or 16, wherein said plurality of binding molecules comprises monoclonal and/or polyclonal antibodies.

17. A composition according to claim 16 wherein said antibodies comprise recombinant antibodies.

18. A composition according to claim 16 or 17 wherein said antibodies comprise camelid antibodies.

19. The composition according to claim 14, wherein said binding molecules are labeled with a detectable label.

20. A method for detecting the presence of contaminating molecules in a sample comprising a target biomolecule, comprising contacting said sample with a composition according to claim 19.

21. A method for purifying a desired biomolecule from a biological sample containing contaminants, comprising: contacting the sample with a first population of binding molecules that selectively bind a plurality of said contaminants; and contacting the resulting composition with an affinity matrix, wherein said affinity matrix comprises a second population of binding molecules that selectively bind said first population of binding molecules.