EP0698037A1

EP0698037A1 - Separation of anti-metal chelate antibodies

Info

Publication number: EP0698037A1
Application number: EP94916647A
Authority: EP
Inventors: Daniel E. Beidler
Original assignee: Hybritech Inc
Current assignee: Hybritech Inc
Priority date: 1993-05-11
Filing date: 1994-05-03
Publication date: 1996-02-28
Also published as: WO1994026772A1; HUT73396A; ZA943142B; JPH08509983A; HU9503232D0; AU6824494A; KR960702474A; CA2162671A1

Abstract

The present invention provides a method for the separation of anti-metal chelate antibodies from non-specific proteins, including antibodies, by applying a preparation containing the anti-metal chelate antibodies to a carboxylic acid derivatized solid support and eluting first with an elution buffer containing sufficient salt concentration to elute non-specific proteins but not sufficient to elute the anti-metal chelate antibodies and then increasing the salt concentration of the elution solution so as to elute the anti-metal chelate antibodies. In one embodiment, the carboxylic acid derivatized solid support is a carboxymethyl resin. Appropriate salts include sodium phosphate, sodium chloride, sodium acetate and sodium sulfate. The method can be used to separate monoclonal or polyclonal anti-metal chelate antibodies from non-specific proteins as well as to separate bifunctional anti-metal chelate antibodies from monoclonal anti-metal chelate antibodies and other non-specific proteins. The method is also useful for separating anti-metal chelate antibody fragments bearing antigen reactive regions from non-specific proteins.

Description

SEPARATION OF ANTI-METAL CHELATE ANTIBODIES

Background of the Invention This invention relates to methods of separating antibodies and, more specifically, to a method of separating anti-metal chelate antibodies from other antibodies and proteins.

The last twenty-five years have witnessed a revolution in the field of immunology. During the 1970's methods were developed of producing in quantity single species of antibodies, the proteins which the immune system uses to recognize, bind and eventually eliminate substances which are recognized as foreign. In order to specifically bind to the enormous range of potential antigens, each of the some 10⁵ to 10⁸ lymphocyte lineages which an individual possesses produces different antibodies with specificity for a different foreign substance, or antigen. By fusing a single antibody-producing lymphocyte with an immortalized cell, such as a cancer cell, it is now possible to make a single lymphocyte clone, each cell producing the same monoclonal antibody. The development of such hybridomas to produce monoclonal antibodies has had an enormous impact on the ability to both diagnose and treat a vast array of diseases. Antibodies comprise two identical pairs of a heavy and light peptide chain arranged in the shape of a "Y" . Each of the arms contains a site which binds to the antigen through various non-covalent interactions, including ionic interactions, hydrogen bonding and van der Waals forces, which result in an affinity between the antibody and its cognate antigen. This antigen-binding site is encompassed within an antigen reactive region which corresponds to the so called variable region of the antibody. In native antibodies, both antigen reactive binding regions will be identical. "Bifunctional antibodies" have been produced which express two different variable regions. These bifunctional antibodies can be produced chemically or by engineered cells which simultaneously express two different sets of genes encoding the antibody proteins. These gene products then assemble into a variety of species of antibodies exhibiting different combinations of antibody gene products including two which exhibit identical antigen- binding sites ("monofunctional antibodies") and one exhibiting dissimilar antigen-binding sites.

Bifunctional antibodies have great utility in allowing simultaneous binding to more than one antigen. As one example of such use, a bifunctional antibody having one arm specific for an antigen expressed on the surface of a tumor cell and one arm specific for an imaging or therapeutic moiety may be effectively used to target such a moiety to the site of a tumor. Among the moieties so used for such therapeutic and diagnostic purposes are metals in metal chelates. Antibodies having binding affinity for metal chelates are termed "anti-metal chelate antibodies."

The recent developments of antibody technology have generated a need for methods to purify antibodies from proteins and other contaminants and to isolate specific species of antibodies from other antibodies. Conventionally, two methods have been used to separate antibodies: ion exchange chromatography and affinity chromatography. Ion exchange chromatography utilizes a solid support to which charged functional groups are covalently attached. The ionic interaction of these charged groups with charges available on the surface of various proteins provides a means of separating many types or families of protein. A protein whose surface is negatively charged will likely bind to an anion exchanger which has positively charged functional groups, while a protein whose surface exposes predominately positive charges will likely bind to a cation exchanger. The binding of these proteins is influenced by pH, salt composition and concentration and as such these parameters can be utilized to isolate antibodies as a family from other types of proteins. While ion exchange chromatography has proved quite useful in certain applications, it has the critical limitation that antibodies having similar physical characteristics but distinct functional characteristics, such as antigen binding, are usually not differentiated. More specific purification can be achieved using an affinity column containing a resin to which is bound the cognate antigen or hapten of the antibody to be isolated. A hapten is a small molecule which is antigenic when attached to a carrier. The antibody preparation is passed over the column, with antibodies specific to the antigen binding to and being retained on the column. Because of the strength of the antigen-antibody binding, a solution containing the cognate is required to elute the antibody. For instance, as disclosed in U.S. Patent Number 5,112,951, anti-metal chelate antibodies show extended retention times on a sulfopropyl column, as compared with non-specific antibodies, in the absence of hapten. Moreover, retention times were similar for murine antibodies and their chimeric derivatives despite the fact that the murine antibodies have a much lower pi than do the chimerics. However, the retention time of anti-metal chelate antibodies in the presence of the hapten analog, ImM Co/EDTA, was dramatically reduced as compared to only slight changes with the non¬ specific antibodies. This longer retention time of anti- metal chelate antibodies and its abrogation in the presence of metal chelate hapten indicate that the binding of these antibodies to oxo acid resin reflects interaction in the antigen reactive binding region.

Despite its potential for separating antibodies with similar physical characteristics, affinity purification has certain serious drawbacks. For example, it may be difficult or impossible to separate the antibodies from the antigen or analog hapten with which they elute. In addition, the cognate and cognate-resin may be unavailable or costly. Even more importantly, the antibody may_^ be denatured by the severe elution conditions, such as extreme pH or the presence of chaotropiσ agents. There thus exists a need for an inexpensive and effective method for specifically isolating anti-metal chelate antibodies from non-specific antibodies or proteins which does not result in their being denatured during the process. Preferably, such a method should be effective in isolating polyclonal fractions enriched in anti-metal chelate antibodies as well as monoclonal antibodies and their bifunctional derivatives. The present invention satisfies these needs and provides related advantages as well.

Summary of the Invention

The present invention provides a method for the separation of anti-metal chelate antibodies from non- specific proteins, including antibodies, by applying a preparation containing the anti-metal chelate antibodies to a carboxylic acid derivatized solid support and eluting first with an elution buffer containing sufficient salt concentration to elute non-specific proteins but not sufficient to elute the anti-metal chelate antibodies and then increasing the salt concentration of the elution solution so as to elute the anti-metal chelate antibodies, wherein the pH of the elution buffer is 7.5 or below. In one embodiment, the carboxylic acid derivatized solid support is a carboxymethyl resin. Appropriate salts include sodium phosphate, sodium chloride, sodium sulphate and sodium acetate. Alternatively, the anti-metal chelate antibody can be selectively eluted with a solution containing a second non-cognate carboxylic acid having less affinity for the anti-metal chelate antibody than does the cognate hapten.

The method can be used to separate monoclonal or polyclonal anti-metal chelate antibodies from non-specific proteins as well as to separate bifunctional anti-metal chelate antibodies from monoclonal anti-metal chelate antibodies and other non-specific proteins without precipitating out the eluted antibodies. The method is also useful for separating anti-metal chelate antibody fragments bearing antigen reactive regions from non-specific proteins.

Brief Description of the Figures Figure 1 is a graph illustrating reactivity with a series of non-cognate carboxylic acids of an antibody raised against a metal chelate, Indium-benzyl EDTA.

Figure 2 is a graph of ultraviolet absorbance at 280nm over time showing the elution scan of a bifunctional anti- metal chelate antibody (BxBFA) chromatographed on a sulfopropyl derivatized column as described in Example I using sodium phosphate as the elutant salt.

Figure 3 is a graph of ultraviolet absorbance at 280nm over time showing the elution scan of a bifunctional anti- metal chelate antibody (BxBFA) chromatographed on a carboxymethyl derivatized column as described in Example II using sodium phosphate as the elutant salt .

Figure 4 is a graph of ultraviolet absorbance at 280nm over time showing the elution scan of a bifunctional anti- metal chelate antibody (ECA 001) chromatographed on a carboxymethyl derivatized column as described in Example III using sodium phosphate as the elutant salt.

Figure 5 is a graph of ultraviolet absorbance at 280nm over time showing the elution scan of a bifunctional anti- metal chelate antibody (BxBFA) chromatographed on a iminodiacetic acid derivatized column as described in Example IV using sodium phosphate as the elutant salt .

Figures 6a and 6b are graphs of ultraviolet absorbance at 280nm over time showing the elution scan of a bifunctional anti-metal chelate antibody (BxBFA) chromatographed on an iminodiacetic acid derivatized column as described in Example V using glutamic acid and/or glycine as the elutant .

Figure 7 is a graph of ultraviolet absorbance at 280nm over time showing the elution scan of a bifunctional anti- metal chelate antibody (ECA 001) chromatographed on a iminodiacetic acid derivatized column as described in Example VI using sodium sulfate as the elutant salt.

Figure 8 is a graph of ultraviolet absorbance at 280nm over time showing the elution scan of a bifunctional anti- metal chelate antibody (BxBFA) chromatographed on a glutamic acid derivatized column as described in Example VII using sodium phosphate as the elutant salt.

Detailed Description of the Invention

The present invention provides an effective method for separating anti-metal chelate antibodies wherein the hapten chelate is an carboxylic acid or acid derivative from non¬ specific proteins, which may include other non-specific antibodies. The method exploits the unexpected ability of anti-metal chelate antibodies to bind to a non-cognate antigen, a carboxylic acid moiety with at least some structural similarity to the cognate hapten chelate, but with reduced affinity for their antigen reactive regions, thus differentiating them from other non-specific antibodies and proteins. While antigens and haptens, or their analogs, such as derivatives and fragments, have been used on the solid phase of affinity columns, the present method is based on a binding between anti-metal chelate antibodies and a non-cognate hapten or analog through their antigen-binding site. It is, moreover, an advantage of the present invention that an elevated salt concentration is sufficient to elute the anti-metal chelate antibodies. As an additional advantage of the present invention, the pH of the elevated salt concentration can be 6.5 or above. Alternatively, anti-metal chelate antibodies can be selectively eluted with mono-, di- or tri-carboxylic acids having less affinity for the anti-metal chelate antibody than does the cognate hapten (for example In-benyzl-EDTA) . Further, no harsh or denaturing elution conditions, common to conventional affinity purification systems, are required. The invention is premised on the unexpected ability of anti-metal chelate antibodies to bind to negatively charged multi-oxygen resonance structures in a manner which reflects their immunological specificity, indicating that the binding is with their antigen reactive region, at or near the antigen-binding site. In particular, anti-metal chelate antibodies raised against oxo acid hapten chelates, such as carboxylic acids and their derivatives, exhibit attraction for non-cognate hapten carboxylic acids, such as those having one or two carboxylic acid functionalities situated so as to cooperate in bonding with the antigen reactive region of the antibody. For example, non-cognate haptens, including EDTA with non-Indium metals, and non-cognate carboxylic acids, having lesser affinity for the binding site of an antibody raised against metal benzyl-EDTA than does the cognate, including acetic, iminodiacetic, benzyl- EDTA, as shown in Figure 1 of the drawings, as well as glycine or other acids such as citric aspartic or glutamic acid.

Such non-cognate carboxylic acids can be attached to a solid support, to form a carboxylic acid derivatized solid support. When such a non-cognate carboxylic acid solid support is used as the solid phase in a chromatographic method, monoclonal anti-metal chelate antibodies can be separated from non-specific proteins and similarly polyclonal anti-metal chelate antibody enriched fractions can be obtained from antiserum. In addition to distinguishing monoclonal and polyclonal anti-metal chelate antibodies from non-specific proteins, the invention permits the separation of bifunctional anti- metal chelate antibodies from monofunctional anti-metal chelate antibodies, such as would be found together in the culture fluid from a polydoma. The various species of antibodies in this culture fluid exhibit discrete retention times when eluted with a gradient having an increasing salt concentration; the active bifunctional anti-metal chelate antibodies elute from the non-cognate carboxylic acid derivatized solid support at a lower salt concentration than the active monofunctional anti-metal chelate antibodies. This separation reflects the difference in avidity between a bifunctional antibody having a single anti-metal chelate binding site ("monovalent") and a monofunctional anti-metal chelate antibody having two antigen-binding sites ("bivalent") . Thus, the bifunctional anti-metal chelate antibodies can be effectively separated from the other, non- desired, species, as disclosed in U.S. Patent Number 5,112,951.

As used herein, the term "anti-metal chelate antibodies" refers to antibodies and antibody constructs well known in the art, such as chimeric, CDR-grafted, and humanized antibodies which have a variable region with a high affinity for at least one metal chelate or metal chelate analog wherein the hapten chelate is a carboxylic acid such as benzyl-EDTA. Generally the affinity of the anti-metal chelate antibody is greater than about 10⁶ L/M, preferably greater than 10⁷ L/M, most preferably greater than 10⁸ L/M. However, a particular anti-metal chelate antibody will, of course, exhibit differing affinities for different metal chelates. Antibodies not exhibiting such an affinity for metal chelate haptens wherein the hapten is carboxylic acid or acid derivative are referred to as "non¬ specific antibodies." Non-specific antibodies together with non-antibody proteins are termed "non-specific proteins." For a description of anti-metal chelate antibodies see U.S. Patent Number 4,722,892 and Reardan, et al . , Nature,

316:265-268 (1985) . Metal chelates include any metal ion in the (II) or (III) oxidation state, including radioactive isotopes, complexed with a polycarboxylate chelating agent, including but not limited to EDTA, DTPA, and DOTA, termed herein the "cognate acid" chelator. For a list of such metals, see Reardan, supra. For a discussion of chelating agents see U.S. Patent Number 4,678,667.

The method of the invention is also suited to the separation of anti-metal chelate antibody fragments which contain the antigen reactive region from non-specific proteins, including non-specific antibodies and their fragments. As used herein, the term anti-metal chelate antibodies includes fragments thereof which bear antigen reactive regions (Fab fragments including Fab; F(ab')₂ and Fab' ) .

The method of the present invention has several advantages over more conventional affinity chromatography methods for the purification of anti-metal chelate antibodies. As indicated, not only does the method permit separation of anti-metal chelate antibodies from non¬ specific antibodies, but the method also permits the differentiation of antibodies having different avidity, by virtue of their valence, for the carboxylic acid derivatized solid phase support. In addition, it is more cost effective, as carboxylic acid derivatized solid phase supports, buffers and salts are less expensive and more readily available than metal chelate resins and metal chelate haptens. Also; the column can be easily sanitized, depyrogenated, cleaned of accumulated protein and regenerated, as by treatment with 0.2 N sodium hydroxide.

Anti-metal chelate monoclonal antibodies bind much more tightly to such a non-cognate carboxylic acid derivatized solid support than do other proteins commonly found in tissue culture supernatants and, unexpectedly, bind even tighter than do non-specific monoclonal antibodies with higher pi's. The pi or "isoelectric point" of an individual protein or antibody is determined primarily by amino acid composition and is defined as the pH at which the net charge of that protein is zero. Above its isoelectric point, the protein has a net negative charge and below its isoelectric point, the protein has a net positive charge. At any given pH, a protein with a higher pi would be more positively charged, or conversely be less negatively charged, than a protein with a lower pi . Particularly among proteins with similar structure and physical characteristics, such as antibodies, a higher pi would be expected to predict stronger interaction with the negatively charged functional groups on a carboxylic acid derivatized solid support, as ionic forces generally account for the interaction of proteins with negatively charged functional groups. Yet, even among antibodies genetically engineered to have identical constant regions, the anti-metal chelate antibody will be retained longer on the non-cognate carboxylic acid derivatized solid support than a non-metal chelate specific antibody having a higher pi. By the same token at a higher pH the same antibody has a less positive charge and will be held less securely by the negatively charged functional groups on a carboxylic acid derivatized solid support. Despite this, it has been discovered that metal chelate antibody separations can be performed using non-cognate carboxylic acid supports at a pH of 7.5 and below.

As an indication that this behavior of anti-metal chelate antibodies on non-cognate derivatized solid supports exhibits immunologically specific binding, U.S. Patent Number 5,112,951 discloses that anti-metal chelate antibodies bearing the same antigen reactive binding region sequences, but having different constant regions and pi's, as in the case of chimeric antibodies and the native murine antibodies from which they were derived, exhibit the same extended retention time on the non-cognate oxo acid derivatized solid support. These unexpected observations indicate that pi does not, in this case, explain the primary behavior of antibodies having a metal chelate specificity. Even more conclusively, the presence of a metal chelate hapten in solution-phase will effectively eliminate the anti-metal chelate antibodies' unexpectedly strong binding to the non-cognate derivatized solid phase by shortening their retention times so as to be comparable to those of non-specific antibodies. Considering the small size of these competing solution phase metal chelate haptens, the site of interaction which accounts for this unexpected binding behavior of anti-metal chelate antibodies must be near or identical with the antigen-binding site of these anti-metal chelate antibodies.

The unexpectedly strong non-cognate oxo acid binding reaction and its abrogation in the presence of metal chelate haptens indicates that anti-metal chelate antibodies bind to non-cognate carboxylic acid derivatized solid supports by a mechanism which is distinct from normal mechanisms of cation exchange chromatography and that this new mechanism of interaction is related to the immunological specificity of these antibodies. Moreover, this unexpected behavior extends to bifunctional antibodies derived from anti-metal chelate antibodies. Such bifunctional antibodies have only a single metal chelate binding site, and it is this monovalence which accounts for their being measurably less well retained on a non-cognate carboxylic acid derivatized column than is the monofunctional anti-metal chelate antibody from which they were derived, which has two metal chelate hapten-binding sites. The interaction between anti-metal chelate antibodies and the non-cognate carboxylic acid derivatized solid support exhibits a behavior characteristic of immunologically specific binding and reflects an interaction at or near the antigen-specific binding site. However, the fact that only increasing salt concentration is required for their elution indicates that any affinity that these antibodies have for the non-cognate carboxylic acid derivatized solid phase support is low in comparison to their affinity for the metal chelate. It is believed that non-cognate carboxylic acid groups on the solid support may mimic the three dimensional spatial configuration or charge density pattern of a portion of the metal chelate hapten, thereby accounting for the antibody's affinity for the matrix, albeit lower than for the metal chelate itself. Such conformational similarity is plausible as the non- cognate carboxylic acid derivatized solid support does possess oxygen atoms with negative charges like the metal chelate hapten. This characteristic indicates that other negatively charged multi-oxygen resonance structures would also be suitable derivatives for use in the method of the invention. Whatever the basis of this interaction, the behavior of anti-metal chelate antibodies on the non-cognate carboxylic acid derivatized column can best be described as an interaction reflecting the antigen reactivity of the anti-metal chelate antibody. As illustrated in Figure 1, reactivity with a series of non-cognate carboxylic acids of an antibody raised against a metal chelate, Indium-benzyl EDTA, increases as the structure of the non-cognate acid increases in similarity to that of the acid in the metal chelate, benzyl-EDTA.

It will be noted that the relative affinities of a subject antibody for various candidate non-cognate acids can be determined empirically using elution from solid support columns and methods well known in the art . These methods are further illustrated in the Examples herein. One skilled in the art will appreciate that candidate non-cognate acids can also be selected by inspection simply by comparing the degree of similarity in the chemical structure of the candidates to that of the cognate hapten. As a general rule, it will be expected that the non-cognate acid having the greatest degree of structural similarity to the cognate hapten will have an affinity for the anti-metal chelate antibody which is closer to that of the cognate metal chelate.

Various anti-metal chelate antibodies are known or available. Examples include CHA255 and CHB235, which are monoclonal antibodies of murine origin which have particular affinity for an Indium-EDTA complex. See U.S. Patent No. 4,722,892. Anti-metal chelate antibodies and non-specific antibodies referred to herein are listed with their characteristics in Table I.

TABLE I

Antibody pi Specificity Characteristics

monofunctional

Both monoclonal and polyclonal anti-metal chelate antibodies can be made by methods known to those skilled in the art. For example, an antigen can be prepared by conjugating a chelating agent to a carrier in solution. The resulting solution is then mixed with a metal salt, such as indium citrate, and dialyzed. Alternatively, one could use gel filtration in place of dialysis. The amount of attached chelate can be determined from the absorbance or by radioactive titration. Hybridoma cells producing anti-metal chelate antibodies can be prepared by methods well known in the art. See, for example, Antibodies, a Laboratory Manual. (Harlow and Lane, eds. ) Cold Spring Harbor, New York (1988) . In the preparation of CHA255 and CHB235, for example, an indium-EDTA antigen was prepared. Keyhole limpet haemocyanin (9.3mg) was allowed to react in 265μL aqueous solution, pH 6.0, with (L) -SCN-C₆H₄-CH₂-EDTA (isothiocyanate benzyl-EDTA; ITCBE) for eight hours at 36°C. The resulting solution was mixed with 90μL of 0.1M indium citrate and dialyzed against ImM EDTA, 0.15M NaCl. From the absorbance of the thiourea group at 310nm, it was determined that there was approximately O.lmg of attached chelate per mg of protein. Spleen cells from BALB/c mice, multiply immunized with the antigen described above, were fused with a variant of the P3.653 myeloma cell line using the technique of Gerhard, Monoclonal Antibodies, (Kennett, et al . , eds.) Plenum Press New York (1980) . The resulting hybridomas were screened, using a solid phase second antibody radioimmunoassay, for their ability to bind In(III) aminobenzyl-EDTA according to the method of Wang, et al. , . Immunol. Meth. , 18:157 (1977) . Those hybridomas exhibiting high titer and relatively high affinity antibodies as determined by inhibition of binding by unlabelled antigen, were selected and injected intraperitoneally into BALB/c mice for ascites production. In the case of murine antibodies, the monoclonal anti-metal chelate antibodies were purified from mouse ascites by ion-exchange chromatography on DEAE- cellulose as described by Parham, et al . , J. Immunol. Meth. , 53:133 (1982) .

The binding constants of the antibodies for the chelates were determined by the method of Eisen, Meth. Med. Res. 10:106 (1964) . Briefly, the antibody and metal chelates were dialyzed to near equilibrium for 24 hours at 37°C in 0.05M 2-hydroyethyl-piperazine-ethanesulfonate (HEPES) , 0.1M NaCl, 0.1% NaN₃ and 0.1% bovine serum albumin at pH 7. The concentration of antibody-binding sites inside the dialysis bag was 10^~7 M and the concentration of free In(III) - (L) -aminobenzyl EDTA complex was in the same range. CHA255 and CHB235 have affinities (binding constants) for In(III) EDTA complex on the order of 10⁹ L/M and 10⁸ L/M, respectively.

Chimeric anti-metal metal chelate antibodies can be produced expressing, for example, a variable region of murine origin and constant regions of human origin. CDR- grafted anti-metal chelate antibodies can be produced expressing, for example, CDR's of murine origin and framework and constant regions of human origin. To prepare such chimeric or CDR-grafted antibodies, DNA sequences of the variable and constant regions can be obtained from genomic DNA. Genomic DNA may be prepared and cloned by a variety of conventional techniques such as those described in Basic Methods in Molecular Biolocry, (L.G. Davis, M.D. Dibner and J.F. Battey, eds.) , Elsevier, New York (1986) ; Feder, J. , et al. , Am. J. Hum. Genetics, 37:635-649 (1985) ; and Steffer, D. and Weinberg, R.A. , Cell 15:1003-1010

(1978) ; Beidler, et al. , J. Immunol., 141:4053-4060 (1988) . For example, the DNA sequences encoding the desired variable light and heavy chain regions may be obtained from cellular DNA of a murine hybridoma expressing a desired anti-metal chelate antibody, while the DNA sequence encoding for the constant region may be derived from human lymphocytes, preferably human peripheral blood lymphocytes. Cellular DNA may be isolated by standard procedures, the genomic DNA fragmented into restriction fragments by restriction endonucleases, and the resulting fragments cloned into suitable recombinant DNA cloning vectors and screened with radiolabeled or enzymatically labeled probes for the presence of the desired DNA sequences. Methods for incorporating DNA constructs containing the desired sequences into cloning vectors and expression vectors are now well known in the art and described by numerous references such as Eukaryotic Viral Vectors, (Y. Gluzman, ed.) Cold Spring Harbor Laboratories publications, Cold Spring Harbor, New York (1982) ,- Eukaryotic Transcription, (Y. Gluzman, ed.) Cold Spring Harbor, New York (1985) ; Sequence Specificity in Transcription & Translation, (R. Calendar and L. Gold, eds.) Allan R. Liss, Inc., New York (1985) ; Maximizing Gene Expression, (W. Reznikoff and L. Gold, eds.) Butterworths, New York (1986) ; Mammalian Cell Technology, (W. G. Thilly, ed.) Butterworths, New York (1986); J. Sambrook and M.J. Gething, Focus, (Bethesda Research Laboratories/Life Technologies, Inc.) 10 #3, pp. 41-48 (1988) .

Appropriate host cells, preferably eukaryotic cells, may be transformed to incorporate the expression vectors by any one of several standard transfeetion procedures well known in the art, including, for example, electroporation techniques, protoplast fusion and calcium phosphate precipitation techniques. Such techniques are generally described by Toneguzzo, F., et al . , Mol . and Cell Biol.. 6:703-706 (1986); Chu, G., et al . , Nucleic Acid Res., 15:1311-1325 (1987) ; Rice, D., et al . , Proc. Natl . Acad. Sci. USA, 79:7862-7865 (1979) ; and Oi, V., et al . , Proc. Natl. Acad. Sci. USA. 80:825-829 (1983) . Preferably, the recombinant expression vectors comprising the chimeric constructs are transfected sequentially into host cells. For example, the expression vectors comprising the chimeric light chain DNA constructs are first transfected into the host cells. Transformed host cells expressing the chimeric light chain polypeptides are then selected by standard procedures known in the art as described, for example, in Engvall, E. and Perlmann, P., Immunochemistry, 8:871-874 (1971) . The expression vectors comprising the chimeric heavy chain DNA constructs are thereafter transfected into the selected host cells. Alternatively, both the chimeric light and heavy chain expression vectors can be introduced simultaneously into the host cells or both chimeric gene constructs can be combined on a single expression vector for transfection into cells. Following transfection and selection, standard assays are performed for the detection of chimeric antibodies directed against desired metal chelates.

The method of the present invention finds particular utility in separating bifunctional anti-metal chelate antibodies from the monofunctional anti-metal chelate antibodies. Bifunctional antibodies exhibiting one specificity against metal chelates and the other against a different antigen can be obtained. See, for example, U.S. Patent Number 4, 722,892, U.S. Patent Number 4,475,893 and Martinis, et al . , in Protides of the Biological Fluids, (H. Peters, ed.) pp. 311-316, Pergamon Press, Oxford (1983) . For example, polydomas able to express bifunctional antibodies can be formed by fusing a cell secreting antibodies of the one specificity with a cell secreting antibodies of a different specificity. The heavy and light chains of the two antibodies then assemble to form a variety of antibody species including two active monofunctional, bivalent antibodies (corresponding to those of the parental cells) , an active bifunctional antibody having one antigen- binding site comprising the light and heavy chain of one parent and the other antigen-binding site comprising the light and heavy chain of the other parent, and various other inactive species. The term "active" refers to constructs in which each antigen-binding site is composed of a light and a heavy chain from the same parent, thus giving it the parental specificity. "Inactive" refers to those constructs which lack the binding specificity of either parent because one or both antigen-binding sites comprise a heavy chain from one parent and a light chain from the other parent. The culture fluid from these polydomas contains various antibody species, including both the active monofunctional antibodies as well as active bifunctional antibodies.

Polyclonal anti-metal chelate antibodies can be obtained by means known to those skilled in the art. See, for example, Ghose, et al . , Methods in Enzymology, 93:326-327 (1983) . Because such antiserum will contain a plurality of both anti-metal chelate antibodies and non¬ specific antibodies, the method of the present invention is of particular utility as it permits separation of anti-metal chelate antibodies from non-specific antibodies and proteins and allows identification of fractions enriched in anti- metal chelate antibodies. Such antiserum can contain anti- metal chelate antibodies having only low affinity for metal chelates whose retention time non-cognate carboxylic acid derivatized solid supports can overlap the retention time of other proteins found in the supernatant. The invention, therefore, is particularly suited to separate those antibodies having affinities for metal chelates of greater than 10⁸ L/M although it can also be used to separate those antibodies having affinities of 10⁷ L/M or even as low as 10⁶ L/M, from non-specific antibodies, although the resulting preparations may be accordingly less pure. The method is particularly well suited to preparing fractions having a high concentration of anti-metal chelate antibodies.

In accordance with the separation method of the present invention, a solution containing the anti-metal chelate antibodies, such as a cell culture supernatant, is dialyzed into a buffer solution, such as 50mM sodium phosphate, pH 7.5 and below, preferably between 5.6 and 6.8, most preferably 6.8, and applied to a column of a non-cognate carboxylic acid derivatized resin which has been equilibrated in a starting solution, usually in the same buffer solution or one having the same conductivity as that in which the antibody is applied. Preferably, the resin is a carboxymethyl ion exchange resin. Examples of commercially available carboxymethyl (CM) resins include TSK CM 5/PW (TosoHaas, Philadelphia, Pennsylvania) and (Bio-Rad Laboratories, Richmond, California) and Pharmacia CM Sepharose Fast Flow (Pharmacia Biotech Inc., Piscataway, New Jersey) . Examples of commercially available iminodiacetic acid (IDA) resins include Chelate Column (Poros, Cambridge, Massachusetts and TosoHaas) . Other non-cognate carboxylic acid derivatized solid supports can be used. Appropriate support materials include polymeric resins, such as polystyrene and polyester, glass and glass matrices, dextran and cellulose, and polymer-coated supports although others will be known to those skilled in the art. The non-cognate carboxylic acid can be conjugated to the solid support through means known to those skilled in the art. These carboxylic acids can be attached to resin through aliphatic, aromatic, or branched alkyls of varying length, provided that a carboxylic acid group is available. In one embodiment of the invention, such as is appropriate for separating monoclonal anti-metal chelate antibodies from non-specific antibodies and other non¬ specific proteins, bound material is eluted from the column using an elution solution with a linear gradient of increasing salt concentration, by means well known in the art. Various buffer salts can be used for this purpose including, but not limited to, sodium phosphate, sodium chloride sodium acetate and sodium sulfate. Preferably, a linear gradient to 300 mM sodium phosphate, pH 7.5 or below, is used. Alternatively, sodium phosphate buffer, pH 7.5 or below in conjunction with a sodium sulfate gradient can be employed. Collected eluate fractions can be assayed for protein by means well known in the art, such as, for example, ultraviolet absorbance. Anti-metal chelate antibodies elute from the column at a later retention time than do non-specific antibodies, thereby permitting their separation.

An alternative embodiment as is appropriate for separating active bifunctional anti-metal chelate antibodies from other antibodies and proteins such as would be found in the culture of a polydoma. When the culture fluid is applied to the non-cognate carboxylic acid derivatized solid phase support as described above and the eluants assayed for protein concentration, various peaks are evident. The bifunctional antibody will normally elute between the two parental types. The identity of the peak which corresponds to the active bifunctional can be determined by, for example, a modified ELISA requiring both activities to be present in a single moiety. The elution conditions can then be selected so as to permit separation of the active bifunctional anti-metal chelate antibodies from the other antibodies species.

In a further embodiment, the invention provides a method for obtaining elution fractions enriched in anti- metal chelate antibodies from a polyclonal antiserum. When the antiserum is run on a non-cognate carboxylic acid derivatized solid phase support, as described above, protein concentration approaches a Gaussian distribution. Anti- metal chelate activity, as determined by, for example, a quantitative ELISA determination, increases in the later eluting fractions. Thus, by running the antiserum on a non- cognate carboxylic acid derivatized solid phase support and selecting later eluting material, anti-metal chelate antibody-enriched material will be obtained.

Although the invention is described in the following examples under particular conditions, including the identity of the solid support, the composition of the buffer, nature of the gradient, the affinity of the particular antibodies and so forth, it will be clear to one skilled in the art that the particular eluant conditions to be used to separate the desired antibody can be determined empirically. For example, using the teaching herein, an eluent condition may be found empirically in which non-specific antibodies will not be retained on non-cognate carboxylic acid derivatized solid supports, yet at which anti-metal chelate antibodies will be retained. The anti-metal chelate antibody can be removed from the non-cognate carboxylic acid derivatized solid support by this same eluent having a salt concentration selected to discriminate between the two types of antibodies. The exact eluent conditions will depend on the type of derivatized support, eluent composition, and physical characteristics of both the anti-metal chelate antibodies and non-metal chelate antibodies being separated. The determination of appropriate elution conditions can then be applied to batch-mode purification, where the starting solution is chosen so as to prevent binding of non-specific proteins and the elution buffer is chosen to elute the desired anti-metal chelate antibody. The following examples are intended to illustrate but not limit the invention.

EXAMPLE I HPLC Chromatography of Anti-metal Chelate Antibodies On a Sulfopropyl Column

A sulfopropyl (SP) column (75 x 7.5mm), packed with 10 micron TSK SP 5PW resin beads, was purchased from BioRad, prepared according to the manufacturer's instructions and equilibrated in 50mM sodium phosphate buffer, at the desired pH for each run. The anti-metal chelate antibody, BxBFA, a murine/human chimeric monoclonal bispecific antibody which has a pi of 8.6 and specificities for In-benzyl EDTA metal chelate and tumor CEA, was diluted 1:3 with the equilibration buffer. lOOug of antibody was loaded onto the column and allowed to bind to the matrix. In each trial the antibody was eluted from the column using three or, in some cases four pumps to mix mono and dibasic phosphate solutions with water to obtain the desired combinations of pH and phosphate concentration. A series of linear gradients of increasing salt from 50mM to 300mM sodium phosphate over a period of 100 minutes, were run at a pH of 8.0, 7.3, 6.7, 6.2 or 5.5 and a flow rate of 1ml/ minute. The presence of antibody was determined by absorbance at 280nm using a Waters 490E (Milford, Massachusetts) variable wavelength ultraviolet detector.

As can be seen from Figure 2, the retention time of the antibody on the column increased as the pH was decreased. This occurs due to the increasingly positive charge held by the antibody at these lower pH values and their attraction for the negative charges on the sulfopropyl column. The retention time of BxBFA at pH 5.5 was about 50 minutes.

EXAMPLE II Separation of Anti-metal Chelate Antibodies On A Carboxymethyl Column A carboxymethyl (CM) column (75 x 7.5mm) packed with 10 micron TSK CM 5/PW resin beads was purchased from Bio-Rad Laboratories, prepared according to the manufacturer's instructions, equilibrated, and antibody was prepared and loaded onto the column as in Example 1. Also as in Example I a series of elutions were conducted at pH 8.0, 7.3, 6.7, 6.2 and 5.5. The elution times of the antibody from the carboxymethyl column are shown in Figure 3. Relative to the sulfopropyl column, the BxBFA never eluted from the carboxymethyl column at a pH of 5.5. Instead of pH 5.5, the BxBFA can be eluted from the carboxymethyl column at pH 6.2 with a retention of about 61 minutes, a longer retention time than that seen with the sulfopropyl column at pH 5.5 under the same gradient conditions. This stroner retention of the BxBFA by the carboxylmethyl column in comparison with the sulfopropyl column occurs despite the fact that the antibody becomes less positively charged at the higher pH. This reduction in ionic attraction means that forces other than the attraction of opposite charge are responsible for the longer retention times on the carboxymethyl column. Generally longer retention times on the carboxymethyl column also mean that a purification can be done at a higher pH, above pH 6 or even closer to the physiological pH of the starting cell culture material. In the case of antibodies produced through in vitro cell culture procedures, adjusting the pH of the starting material to pH about 5.5 or below can cause protein precipitates that include the antibody therein. EXAMPLE III Effect of Hapten on Anti-metal

A carboxymethyl (CM) column (75 x 7.5mm) packed with 10 micron TSK CM 5/PW resin beads was purchased from Bio-Rad Laboratories, and prepared according to the manufacturer's instructions. The column was equilibrated, a purified sample of ECA 001, a murine polydoma produced bispecific antibody with a pi of 6.5 and with specificities against In- benzyl EDTA metal chelate and tumor CEA, was prepared and loaded onto the column as in Example I. Elution proceeded as in Example I except that the pH of the solutions were 9.4, 8.3, 7.4, 6.8, 6.2 and 5.5. The elution times were as shown in Figure 4. The retention time for ECA 001 was about 5 minutes less at pH 6.2 than for the BxBFA in Example 2 above. The ECA 001 antibody has an attraction for the carboxymethyl column similar in principle to that of the BxBFA antibody, but since this antibody has more negative charges relative to the BxBFA, as can be seen by comparing their respective pi's, it is to some degree repelled by the negatively charged carboxymethyl column. Although the two antibodies have greatly different pi's, their common specificity against In-benzyl EDTA metal chelate accounts for similar retention on the column. As in Example 2 above, at pH 5.5 the phosphate elution was unable to remove the ECA 001 antibody from the column.

EXAMPLE IV

A iminodiacetic acid (IDA) column (75 x 7.5 mm) packed with 10 micron resin beads was purchased as a Chelate Column from TosoHaus and prepared according to the manufacturer's instructions. The column was equilibrated without metal loading, and anti-metal chelate antibody BxBFA was prepared and loaded onto the column as in Example I . A series of phosphate buffer elutions were conducted as in Example I except that the pH values were 9.0, 8.0, 7.3, 6.7, 6.2 and 5.5 and were monitored over time as shown in Figure 5. It was impossible to elute the antibody from the column even with a buffer solution of 500mM sodium phosphate having a pH of 9.4. It is presumed that the structure of iminodiacetic acid is too similar to that of benzyl-EDTA, the cognate acid chelator, to elute the metal chelate with a simple phosphate salt solution.

EXAMPLE V

A iminodiacetic acid (IDA) column (75 x 7.5 mm) packed with 10 micron resin beads was purchased as a Chelate Column from TosoHaus, and prepared according to the manufacturer's instructions. The column was equilibrated without metal loading, and metal chelate antibody BxBFA was prepared and loaded onto the column as in Example I . Elutions were conducted following the procedure of Example I except that a linear gradient from zero to 240mM of glutamic acid was used at pH values of 8.2, 7.2, 6.7, 6.2 and 5.6. The same procedure was followed using a glycine elution at pH values of 8.1, 7.3, 6.7, 6.2 and 5.6 The elution times were as shown in Figures 6a and 6b. The pH was maintained throughout the gradient with sodium phosphate buffers adjusted to a final concentration of 60mM. It is believed that the structures of glutamic acid and glycine were similar enough to that of the cognate metal/benzyl-EDTA to free the antibody from the column whereas a phosphate salt solution could not.

EXAMPLE VI

A iminodiacetic acid (IDA) column was prepared, equilibrated and loaded as in Example V, except that the antibody was a purified sample of ECA 001. Elutions were conducted following the procedure of Example I, but using a linear gradient of sodium sulfate to elute the column. Again the pH of the elution solution was maintained using 60 mM phosphate at a value of 9.4, 8.1, 7.4, 6.8, 6.2 or 5.2. The elution times were as shown in Figure 7. This example shows that sulfate acts as a non-cognate hapten in a manner like that of glutamic acid. EXAMPLE VII

A glutamic acid (GLU) column was prepared with 10 micron resin beads according to the manufacturer's instructions using a Tresyl 5/PW column (TosoHaus) . The column was equilibrated and loaded as in Example V, except that the antibody was BxBFA. Elutions were conducted following the procedure of Example I, but using a linear gradient of 50 to 300 mM sodium phosphate to elute the column at pH values of 8.1, 7.4, 6.9, 6.4 and 5.7. The elution times were as shown in Figure 8.

Although the invention has been described in terms of the presently preferred embodiments, it will be apparent to one skilled in the art that modifications can be made without departing from the spirit of the invention. Thus, the invention is limited only by the following claims.

Claims

WE CLAIM :

1. A method for obtaining eluant fractions enriched in anti-metal chelate antibodies that have a pi of from about 6 to about 9, from a preparation containing said anti- metal chelate antibodies and non-specific proteins, comprising the steps of : a. applying said preparation in a starting solution to a non-cognate mono-carboxylic acid derivatized solid support equilibrated in said starting solution; b. removing said non-specific protein from said support; c. adding an eluant solution containing either 1) a first non-carboxylic acid salt in an increased concentration sufficient to elute said anti-metal chelate antibodies from said support, wherein the pH of the column, the solution containing the preparation to be separated, and said eluant solutions are from about 6.2 to about 6.8; or 2) an elution solution containing a non-cognate hapten with affinity for said anti-metal chelate antibodies; and d. collecting the fraction eluted in step c,

2. The method of claim 1 wherein said acid derivative is carboxymethyl . 3. The method of claim 1 wherein said solid support is selected from the group consisting of polymer-coated supports, polystyrene, polyester, glass, dextran and cellulose.

4. The method of claim 1 wherein said anti-metal chelate antibodies are anti-metal chelate monoclonal antibodies.

5. The method of claim 1 wherein said eluant solution contains said first noncarboxylic acid salt and said removing step additionally comprises applying a solution containing a second non-carboxylic acid salt in a concentration sufficient to elute non-specific proteins from said support . 6. The method of claim 5 wherein said eluant solution contains said first non-carboxylic acid salt and said salt is selected from the group consisting of sodium phosphate, sodium chloride, sodium acetate and sodium sulfate. 7. The method of claim 5 wherein said first and said second carboxylic acid salts have the same chemical formula.

8. The method of claim 5 wherein said eluant solution contains said first non-carboxylic acid salt and said applying step is performed in said starting solution having sufficient salt concentration to prevent binding of a portion of said non-specific proteins without preventing binding of said anti-metal chelate antibodies.

9. The method of claim 1 wherein said anti-metal chelate antibody is a fragment bearing the antigen reactive region.

10. The method of claim 1 wherein said anti-metal chelate antibodies were raised against metal chelating agents that chelate with more than one carboxy group.

11. Eluant fractions enriched in anti-metal chelate antibodies prepared by the method of claim 1.

12. The method of claim 1, wherein said method effectively separates anti-metal chelate antibodies that have a pi of from about 6 to about 9 from non-specific proteins in a preparation. 13. An anti-metal chelate antibody separated by the method of claim 12.

14. A method of separating bifunctional anti-metal chelate antibodies that have a pi of from about 6 to about 9, from a preparation containing said bifunctional anti- metal chelate antibodies, monofunctional anti-metal chelate antibodies and non-specific proteins, wherein the anti-metal chelate antibodies were raised against metal chelating agents that chelate with more than one carboxy group, comprising the steps of: a. applying said preparation in a starting solution to a mono-carboxylic acid derivatized solid support equilibrated in said starting solution; b. adding an eluant solution containing a first non-carboxylic acid salt in a concentration sufficient to elute non-specific antibodies from said support without detaching said bifunctional anti-metal chelate antibodies and said monofunctional anti-metal chelate antibodies; c. adding a further eluant solution containing a second non-carboxylic acid salt in an increased concentration sufficient to elute said bifunctional anti-metal chelate antibodies from said support but not elute said monofunctional anti-metal chelate antibodies from said support, and wherein the pH of the column, the solution containing the preparation to be separated, and said eluant solutions are from about 6.2 to about 6.8; and d. collecting the fraction containing said bifunctional anti-metal chelate antibodies eluted in step c.

15. The method of claim 14 wherein said carboxylic acid derivative is carboxymethyl.

16. The method of claim 14 wherein said solid support is selected from the group consisting of polymer-coated supports, polystyrene, polyester, glass, dextran and cellulose. 17. The method of claim 14 wherein said salt is selected from the group consisting of sodium phosphate, sodium chloride, sodium acetate, and sodium sulfate.

18. The method of claim 14 wherein said anti-metal chelate antibody is a fragment bearing the antigen reactive region.

19. The method of claim 14 wherein said applying step is performed in starting solution of sufficient salt concentration to prevent binding of a portion of said non¬ specific antibodies without preventing binding of said bifunctional anti-metal chelate antibodies.

20. The method of claim 14, wherein said first and said second noncarboxylic acid salts have the same chemical

chelate antibody is a fragment bearing the antigen reactive region.

27. The method of claim 22 wherein said applying step is performed in said starting solution having sufficient salt concentration to prevent binding of a portion of said non-specific proteins without preventing binding of said anti-metal chelate antibodies.

28. Anti-metal chelate antibodies separated by the method of claim 22.