Classifications

• • 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
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
• • 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

Definitions

• the invention relates to protein isolation and purification techniques.
• proteins may be separated based upon their molecular charge using ion exchange chromatography, whereby protein mixtures are applied to an oppositely charged, chromatographic matrix, and the various proteins bind to the matrix by reversible, electrostatic interactions.
• the adsorbed proteins are eluted, in order of least to most strongly bound, by increasing the ionic strength or by varying the pH of the elution buffer.
• Another general approach makes use of a protein's physical characteristics as a means of separation. For example, a protein may be separated based upon its size, using gel filtration. By this method, protein mixtures are applied to a gel-filtration column containing a chromatographic matrix of defined pore size. Proteins are eluted, generally with an aqueous buffer, collected as individual chromatographic fractions and analyzed.
• a protein for example, may be purified using an antibody specific for that protein or conversely an antibody may be purified using its specific antigen.
• the antibody or antigen is bound to a column substrate and a solution which includes the particular antigen or antibody applied to the column, allowing immunocomplex formation.
• Bound immunocomplex partners are then eluted by destabilizing the antigen-antibody complex, e.g., by exposure to buffers of very high ionic strength or high or low pH.
• immunocomplex formation may be exploited to purify the antigen or antibody by immunoprecipitation.
• Antigen-antibody complexes may be precipitated following aggregation, or alternatively, one of the binding partners may be covalently linked to a solid particle (such as Sepharose or agarose) and immunoaffinity complexes isolated by centrifugation. In either method, the protein of interest is then released from the complex, again, e.g., by exposure to buffers of high ionic strength or high or low pH.
• a solid particle such as Sepharose or agarose
• an individual antibody molecule includes two identical heavy (H) chains and two identical light (L) chains; each light chain is disulfide bonded to a heavy chain, and the heavy chains are disulfide bonded to each other to form the basic dimeric structure of the molecule.
• H heavy
• L light
• units made up of about 110 amino acids fold up to form compact domains, themselves held together by a single internal disulfide bond.
• the L chain has two domains, and the H chains have four or five domains.
• the first two N-terminal domains of the H chains interact with the two L chain domains, producing the "Fab domain", a portion of the molecule which directs specific antigen recognition and binding.
• the extreme C-terminal domains of the H chains (termed the C H 2 and C H 3 domains) interact to produce the "Fc domain", a portion of the molecule which directs a number of immunoglobulin functions including binding to cells, fixing complement, and traversing the placenta.
• lying between the Fab and Fc domains is a small number of amino acids which make up the hinge region, a flexible domain which facilitates the free movement of the antigen binding portion of the molecule.
• affinity chromatography is a highly effective method for purifying proteins which exploits specific interactions between the proteins to be purified and a solid phase immobilized ligand.
• the solid phase ligand has some unique chemical character which results in the selective adsorption of the protein of interest. Contaminant proteins either do not bind the solid phase or can be removed by washing the solid phase with appropriate solutions.
• elution of an affinity column has been accomplished by either of two methods: (i) washing the matrix with a solution of specific ligand resembling the immobilized ligand, or (ii) washing the matrix with solutions of very high ionic strength, or very high (>11) or very low ( ⁇ 3) pH.
• method (i) is more attractive than method (ii) because solutions of very high ionic strength, while usually not deleterious to proteins, are also usually not effective at desorbing proteins which bind to the affinity matrix tightly, and many proteins are labile to buffers sufficiently acid or basic to elute the protein of interest.
• method (i) is not applicable if a specific eluting ligand can not be found or if the use of a specific eluting ligand is infeasible for practical reasons, e.g. if the specific ligand is unstable or expensive. Elution of proteins from immobilized ligands in which the ligand itself is a protein can rarely be achieved by the use of an eluting solution containing a specific ligand. This is because the specific ligand usually must be a protein or peptide fragment, and elution is then infeasible for the practical reasons mentioned above.
• the instant invention describes a new method for eluting affinity matrices based on the use of inexpensive low molecular weight compounds which mimic the side chains of specific amino acids.
• the eluting compounds are chosen to mimic the side chains of specific amino acids participating in recognition of the ligand.
• the compounds may mimic the side chains of either the protein to be purified or the immobilized ligand protein.
• Suitable side chain mimetic compounds are chosen for their water solubility and compatibility with the stability of the protein to be isolated. Because protein-protein interactions are frequently stabilized by hydrophobic interactions, and particularly by aromatic hydrophobic interactions, the most favored elution reagents will be chosen from the group comprising side chain mimetics of histidine, tyrosine, tryptophan, and phenylalanine. In particular examples, imidazole is an appropriate choice for destabilizing interactions promoted by binding to histidine residues. Hydroxylated aromatic compounds such as phenols and phenolic compounds whose solubility in water is promoted by other substituents, such as aminophenols or hydroxybenzenesulfonates, are appropriate mimetics for tyrosine side chains.
• Substituted indoles such as indole-3-sulfate or indole-3-acetic acid, or benzimidazole salts are appropriate mimetics for tryptophan.
• Benzoate salts, phenylsulfonates, and water soluble heterocycles, such as nicotinic acid salts or nicotinamide, are appropriate mimetics for phenylalanine.
• Useful amino acid mimetics may also be chosen from those molecules which disrupt charged residue interactions. Examples of compounds useful for interfering with such charged residue interactions include guanidine salts to mimic arginine, alkylamines to mimic lysine, and alkanoic acids to mimic glutamate or aspartate.
• aliphatic hydrophobic residue interactions for example, aliphatic alcohols, aliphatic ethers of water soluble polyalkylene glycols, sulfate esters of aliphatic alcohols and aliphatic amines.
• Compounds with dual hydrophobic action such as phenols substituted with aliphatic groups, may also prove useful in mimicking side chain residues.
• the invention features a method of isolating a protein from a sample, involving (i) providing a first molecule which is capable of forming an affinity complex with the protein; (ii) contacting the sample with the first molecule under conditions which allow affinity complex formation; (iii) isolating the complex; (iv) treating the complex with a second molecule, the second molecule mimicking an amino acid residue of either the protein or the first molecule which is critical to the complex formation, so that the second molecule disrupts the complex, causing the release of the protein from the complex; and (v) isolating the protein.
• the second molecule mimics an amino acid side chain.
• the first molecule is protein A and the protein to be isolated is an antibody or an antibody fusion protein which includes a protein A-binding domain.
• the second molecule preferably mimics a histidine residue and is, for example, imidazole.
• the first molecule is an antibody and the protein to be isolated is a recombinant protein; or the first molecule is an antigenic protein and the protein to be isolated is an antibody which specifically binds that antigenic protein.
• mimetic an amino acid residue is meant being of the same or similar chemical composition to either the amino acid residue itself or to a part of the amino acid residue which is critical to that residue's ability to interact with a proteinaceous or nonproteinaceous affinity ligand (i.e., the "first molecule") .
• the portion of the amino acid mimicked is that residue's side chain. Because such a mimetic molecule is used as an elution reagent, the molecule should preferably also be water soluble and compatible with the stability of the protein to be purified.
• amino acid mimetic any amino acid mimetic is useful in the invention, those which disrupt hydrophobic protein- protein interactions are preferred; these include interactions involving the amino acids histidine, tryptophan, tyrosine, and phenylalanine. Preferable mimetics for these amino acids are described above.
• antibody fusion protein is meant a protein which includes at least a portion of an immunoglobulin Fc domain directly or indirectly covalently bonded to a non- immunoglobulin polypeptide.
• amino acid side chain is meant that moiety bound to the molecule's central carbon atom which determines the amino acid's identity.
• Preferable side chains according to the invention include those hydrophobic side chains which characterize histidine, tryptophan, tyrosine, and phenylalanine.
• protein A-binding domain is meant that portion of the immunoglobulin molecule which interacts with the Staphylococcus aureus cell wall component termed protein A. By crystallographic studies, this domain is most likely positioned at the C H 2/C H 3 cleft.
• affinity purification techniques may be modified such that amino acid mimetics are used as elution reagents in the final step of purification to release the protein of interest from the affinity complex. Because this approach is considerably gentler than more conventional elution techniques (e.g., elution steps based on drastic changes in solution pH) , applicant's method facilitates the isolation and purification of those proteins which are destroyed (e.g., irreversibly denatured) or reduced in activity by standard elution procedures. Moreover, because the amino acid mimetics represent convenient and inexpensive elution reagents, they may be utilized as alternative elution reagents even for the purification of more stable proteins.
• FIGURE 1 shows a schematic of the immunoglobulin fusion protein CD62 Rg.
• FIGURE 2A shows flow cytometric results of CD62 Rg binding to the surface of neutrophils.
• FIGURE 2B shows flow cytometric results of CD62 Rg binding to the surface of H3630 cells.
• FIGURE 2C shows flow cytometric results of CD62 Rg binding to the surface HSB2 cells.
• FIGURE 2D shows flow cytometric results of CD62 Rg binding to the surface of K562 cells.
• Human IgGl was purified by affinity chromatography 5 followed by elution with the histidine mimetic imidazole as follows.
• Human IgGl was loaded on protein A trisacryl beads (Pierce, Rockford, IL) , and washed with phosphate buffered saline. The beads were divided among several 0 small columns, and the columns were eluted with solutions containing imidazole at 1, 2, 3, 4, or 5M concentration, adjusted to a final pH of 6, 7, 8, or 9. The results are given in Table 1 as the percent of maximum elution obtained for any one pH.
• immunoglobulin fusion proteins as well as IgGl alone, were purified from protein A columns without loss of biological activity of the protein moiety fused to the immunoglobulin constant domain (in particular, see the purification of CD62 Rg below) .
• acidic elution conditions destroyed the activity (i.e., the ligand binding activity) known to reside in the portion of the protein fused to the immunoglobulin domain.
• the purification of these immunoglobulin fusion proteins involved an initial isolation of a crude preparation of a fusion protein (including the hinge, C H 2 and C H 3 domains of human IgGl joined to the extracellular domain of some surface antigen) which had been prepared by transfection of COS cells with the appropriate cDNA constructs.
• Media supernatants were collected from transfected cells which had been grown for a further 5 to 10 days, clarified by centrifugation, and adsorbed to protein A trisacryl or protein A agarose beads.
• the beads were collected, washed thoroughly with phosphate buffered saline containing 1% nonionic detergent (Nonidet P40 or Triton X-100) followed by buffer alone, then eluted with 4 M imidazole adjusted to pH 8 with acetic or hydrochloric acids.
• the eluted fusion proteins were dialyzed against buffer, or the imidazole was removed by two cycles of centrifugal ultrafiltration (Centricon 30, Amicon Corp., Beverly, MA) .
• CD62 protein chimeras were prepared by genetic fusion of the first four N-terminal extracellular domains of CD62 to the hinge domain of human IgGl as follows.
• CD62 cDNA sequences encoding the lectin (L) , epidermal growth factor (EGF) , and first two complement regulatory protein repeat elements (CR) were amplified in polymerase chain reactions using synthetic oligonucleotides designed to allow fusion to the human IgGl artificial splice donor sequences described previously (Aruffo et al., Cell 61. 1303-1313, 1990) ( Figure 1).
• the forward primer bore the sequence GGC GCC GAA GCT TCC ATG GCC AAC TGC CAA ATA GCC ATC TTG (SEQ ID NO:l), while the reverse primer bore the sequence GGC CAG ATC TCC CTG CAC AGC TTT ACA CAC TGG GGC TGG (SEQ ID NO:2); the sequence allowed the CD62 fragment to be inserted as a Hindlll to Bglll fragment into
• Hindlll- and BamHI-digested vector To amplify the DNA, 20 PCR cycles were conducted, consisting of 30 s at 94°C, 2 min at 45°C, and 3 in at 72°C, using the reaction buffer recommended by the enzyme vendors (US Biochemical, Cleveland, OH) , and Mlul-digested DNA prepared from a previously described endothelial cell expression library (Bevilacqua et al., Science 243 F 1160-1165, 1989).
• a schematic of the resultant fusion protein, termed CD62 Rg is shown in Figure 1.
• the CD62 Rg expression plas id was transfected into COS cells using DEAE dextran as previously described (Seed and Aruffo, Proc. Natl. Acad.
• This imidazole purification procedure was carried out as follows. Twelve hours following transfection, a fraction of the COS cells transfected with each construct were seeded onto flasks. Thirty-six hours post- transfection, the cells were washed with phosphate- buffered saline (PBS) and overlayed with cysteine- methionine-free media for 30 min. [ 35 S]-methionine and [ 35 S]-cysteine (TransLabel, ICN, Costa Mesa, CA) were added to a final concentration of 150 ⁇ Ci/ml, and the cells were allowed to incorporate the label overnight.
• PBS phosphate- buffered saline
• cysteine- methionine-free media for 30 min.
• [ 35 S]-methionine and [ 35 S]-cysteine TransLabel, ICN, Costa Mesa, CA
• CD7 Rg, CD8 Rg (Aruffo et al., Cell 61, 1303-1313, 1990), ELAM-1 Rg (Walz et al., Science 150:1132-1135, 1990), intact IgGl, or a COS cell preparation of a fragment of the IgGl corresponding to the Fc fragment present in CD62 Rg reacted with sulfatides.
• affinity purification techniques may be modified such that amino acid mimetics are used in the final step of purification as elution reagents to release the protein of interest from the affinity complex.
• amino acid mimetic elution step may be employed in any standard affinity purification procedure, e.g., to release a protein from a column-bound complex or from a complex included in an immunoprecipitate.
• the general elution technique described herein may be utilized for the purification of any protein, including those proteins whose interaction contact points or amino acid sequences are unknown.
• most or all of the exemplary amino acid mimetics listed above may be combined (solubility allowing) into a general purpose elution reagent which would disrupt any of a number of affinity complex interactions based on hydrophobic, aliphatic hydrophobic, and/or charged interactions.
• a general purpose elution reagent obviates the need to determine the amino acid sequence of specific affinity contact points or even of the protein to be isolated.
• a generally useful affinity interaction point would be that region of any given protein which directs antibody complex formation; the identity of this region could be determined either from the art or experimentally (e.g., by successive deletion or point mutation analysis of the protein followed by an assay of the mutant protein's ability to bind antibody, e.g., by immunoprecipitation or antibody column binding) . Examination of this region's amino acid sequence directs the choice of mimetics which may be utilized as elution reagents. For any given region of interaction, more than one candidate amino acid contact point may exist. Accordingly, more than one mimetic may be tested, alone or in combination, to determine which mimetic best disrupts the complex and directs the most efficient protein elution.
• GENERAL INFORMATION (i) APPLICANT: Seed, Brian (ii) TITLE OF INVENTION: AFFINITY PURIFICATION METHODS INVOLVING AMINO ACID MIMETICS AS ELUTION REAGENTS

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• 1993
  • 1993-09-27 JP JP6509231A patent/JPH08503698A/ja active Pending
  • 1993-09-27 WO PCT/US1993/009174 patent/WO1994007912A1/en not_active Application Discontinuation
  • 1993-09-27 CA CA 2146162 patent/CA2146162A1/en not_active Abandoned
  • 1993-09-27 EP EP93923139A patent/EP0663925A4/en not_active Withdrawn
  • 1993-09-27 AU AU52926/93A patent/AU5292693A/en not_active Abandoned
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