Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
  • C07K16/065—Purification, fragmentation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins

Definitions

• 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.
• 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.
• 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.
• 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.
• 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.”
• 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.
• 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.
• 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.
• 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 chaotropi ⁇ 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.
• 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.
• 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.
• the carboxylic acid derivatized solid support is a carboxymethyl resin.
• Appropriate salts include sodium phosphate, sodium chloride, sodium sulphate and sodium acetate.
• 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.
• 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.
• BxBFA bifunctional anti- metal chelate antibody
• 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 .
• BxBFA bifunctional anti- metal chelate antibody
• 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.
• ECA 001 bifunctional anti- metal chelate antibody
• 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 .
• BxBFA bifunctional anti- metal chelate antibody
• 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 .
• BxBFA bifunctional anti-metal chelate antibody
• 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.
• ECA 001 bifunctional anti- metal chelate antibody
• 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.
• BxBFA bifunctional anti- metal chelate antibody
• 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.
• 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.
• the pH of the elevated salt concentration can be 6.5 or above.
• 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.
• 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.
• 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.
• 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.
• 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”) .
• 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.
• 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.
• the affinity of the anti-metal chelate antibody is greater than about 10 6 L/M, preferably greater than 10 7 L/M, most preferably greater than 10 8 L/M.
• a particular anti-metal chelate antibody will, of course, exhibit differing affinities for different metal chelates.
• Non-specific antibodies are referred to as "non ⁇ specific antibodies.”
• Non-specific antibodies together with non-antibody proteins are termed “non-specific proteins.”
• non-specific proteins For a description of anti-metal chelate antibodies see U.S. Patent Number 4,722,892 and Reardan, et al . , Nature,
• 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.
• a polycarboxylate chelating agent including but not limited to EDTA, DTPA, and DOTA
• chelating agents see U.S. Patent Number 4,678,667.
• 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 .
• 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.
• 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.
• 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.
• 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.
• 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.
• Both monoclonal and polyclonal anti-metal chelate antibodies can be made by methods known to those skilled in the art.
• 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) .
• an indium-EDTA antigen was prepared.
• Keyhole limpet haemocyanin (9.3mg) was allowed to react in 265 ⁇ L aqueous solution, pH 6.0, with (L) -SCN-C 6 H 4 -CH 2 -EDTA (isothiocyanate benzyl-EDTA; ITCBE) for eight hours at 36°C.
• the resulting solution was mixed with 90 ⁇ L 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) .
• 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.
• 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% NaN 3 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 10 9 L/M and 10 8 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.
• 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
• 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.
• Appropriate host 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.
• 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 .
• the recombinant expression vectors comprising the chimeric constructs are transfected sequentially into host cells.
• 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.
• 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) .
• 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.
• 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 10 8 L/M although it can also be used to separate those antibodies having affinities of 10 7 L/M or even as low as 10 6 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.
• a solution containing the anti-metal chelate antibodies such as a cell culture supernatant
• 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.
• the resin is a carboxymethyl ion exchange resin.
• CM resins examples 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) .
• iminodiacetic acid (IDA) resins examples 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.
• 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.
• a linear gradient to 300 mM sodium phosphate, pH 7.5 or below is used.
• 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.
• the invention provides a method for obtaining elution fractions enriched in anti- metal chelate antibodies from a polyclonal antiserum.
• 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.
• anti-metal chelate antibody-enriched material will be obtained.
• 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.
• 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 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.
• the presence of antibody was determined by absorbance at 280nm using a Waters 490E (Milford, Massachusetts) variable wavelength ultraviolet detector.
• 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.
• 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.
• adjusting the pH of the starting material to pH about 5.5 or below can cause protein precipitates that include the antibody therein.
• CM carboxymethyl
• TSK CM 5/PW resin beads 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.
• 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.
• 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.
• IDA iminodiacetic acid
• Example VII 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.
• IDA iminodiacetic acid
• 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.

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• 1994
  • 1994-05-03 AU AU68244/94A patent/AU6824494A/en not_active Abandoned
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