WO1988002776A1 - A novel immunoaffinity purification system - Google Patents

A novel immunoaffinity purification system Download PDF

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Publication number
WO1988002776A1
WO1988002776A1 PCT/US1987/002657 US8702657W WO8802776A1 WO 1988002776 A1 WO1988002776 A1 WO 1988002776A1 US 8702657 W US8702657 W US 8702657W WO 8802776 A1 WO8802776 A1 WO 8802776A1
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Prior art keywords
activated
support
protein
activated support
aldehyde
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PCT/US1987/002657
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French (fr)
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Peter Grandics
Susan Szathmary
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Peter Grandics
Susan Szathmary
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Publication of WO1988002776A1 publication Critical patent/WO1988002776A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/16Extraction; Separation; Purification by chromatography
    • C07K1/22Affinity chromatography or related techniques based upon selective absorption processes
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K17/00Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02Peptides being immobilised on, or in, an organic carrier
    • C07K17/06Peptides being immobilised on, or in, an organic carrier attached to the carrier via a bridging agent
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding

Definitions

  • I ⁇ rinunoaffinity chromatography is an important area of affinity chromatography which employs biospecific antigen-antibody interactions permitting a high degree of purification in a single step.
  • the rapidly growing area of monoclonal antibody production and manufacturing of recombinant biotherapeutics needs powerful and economical downstream processes to rapidly recover the products from dilute solutions in a high yield and purity.
  • Immunoaffini y chromatography would allow economical, single-step purification of these high valued-biologicals.
  • the improperly selected pH alters the native conformation of antigen (antibody) resulting in immunoadsorbents with diminished binding activity.
  • Binding of protein to an activated support having no spacer arm, such as CNBr, CDI, Tresyl, or FMP-activation can als diminish the biological activity of immobilized protein due to the so-called "wall effect" (steric hindrance) .
  • glutaraldehyde-activated matrices have several shortcomings. Glutaraldehyde is unstable and tend to polymerize. It does crosslink proteins thereby diminishing their biological activity. Immobilized protein also leaches off glutaraldehyde-activated resins. Activated supports having a high density of single monoaldehyde functionalities at the termini of extended (>2 atoms) spacer arms are thus needed that is the topic of the subject invention.
  • a major problem associated with the currently used activation chemistries is the leakage of immobilized ligand from the resin. All the currently used chemistries are unstable to varying extent since they are sensitive to nucleophylic or electrophylic reagents present in biological fluids and buffers.
  • the CNBr-technique is well-known for its ability to leach while other activation methods, such as the N-hydroxysuccinimide, CDI, Tresyl, FMP, divinylsulfone or glutaraldehyde-activation shed ligand at a slower rate, but this can become accelerated by exposure to electrophyles or nucleophyles. This is unacceptable in processing(purification) of biopharmaceuticals since the product would be contaminated with components of the immunoadsorbent.
  • the high affinity of antibodies for antigens are generally precludes elution in the solvent of application.
  • Desorption of antigen from immunoadsorbents is usually achieved by reagents such as chaotropic salts, mineral acids, urea, guanidine, aliphatic acids, alcohols or a combination of these compounds.
  • This kind of reagents adversely affect the biological activity of both the eluted antigen and immunoadsorbent leading to the deterioration of immunoadsorbent capacity and denaturatio of eluted proteins to various degree.
  • These reagents despite of being strong denaturants , are still unable to quantitatively displace antigens from immunoadsorbents. Denaturation of high-valued proteins and the deterioration of expensive immunoadsorbents provided a disincentive for wide-spread utilization of immunoaffinity purification.
  • Immunoaffinity purification would enhance the overall economy of these manufacturing processes by permitting the recovery of protein products rapidly, usually in one-step, in a high yield and purity. Immunoaffinity chromatography would be particularly advantageous in such cases where the protein product should be recovered from very dilute solutions, such as fermentation broth of secreting recombinant organisms.
  • the currently used immunoaffinity techniques clearly need improvement in order to realize the full potentials of immunoaffinity purification with particular emphasis on large-scale downstream processes.
  • nonleaching active supports capable of immobilization under physiological conditions without crosslinking of proteins are needed.
  • Nondenaturing elution methods are also required to quantitatively recover proteins from immunoadsorbents while preserving their biological activity.
  • the subject invention overcomes the above shortcomings of current immunoaffinity purification methods.
  • the second component a nondenaturing eluent permits quantitative recovery of antibodies (antigens ) from immunoadsorbents while preserving their biological activity.
  • a system has been developed for immunoaffinity purification of proteins (antibodies).
  • the invention comprises the method and the system for carrying out immunoaffinity purification.
  • the concept of this invention has broader applications to the purification of any antigen to which an antibody can be raised.
  • the method of this invention exemplified by the immunoaffinity purification of mouse anti-IgG, comprises in its most preferred form:
  • An activated affinity support having single monomeric aldehyde functionalities capable of reversible binding of proteins or ligands containing primary a ⁇ tino groups.
  • NaCNBHa coupling reagent
  • the addition of NaCNBHa (coupling reagent)to the antigen/ activated support mixture irreversibly attach the antigen to the support through extremely stable, secondary a ine linkages.
  • a nondenaturing elution medium represents the second tier of the invention . Since the elution medium is a rich source of both electrophylic and nucleophylic reagents, it is only compatible with coupling chemistries insensitive to these agents. This kind of chemistry is incorporated into the subject activated affinity support. The elution medium permits quantitative recovery of antibodies from immunoadsorbents while retaining their immunoreactivity.
  • the invention lends itself for immunoaffinity purification of proteins other than antibodies in their biologically active state.
  • Recombinant proteins as well as monoclonal antibodies could be purified from very dilute solutions (culture medium) in a single step to homogeneity, free from pyrogens. This would significantly enhance the overall economy of the production of advanced biotherapeutics. ⁇ DESCRIPTION OF THE PREFERRED EMBODIMENT
  • a purification system and components are provided for quantitative immunoaffinity purification of biologicals while preserving the biological activity.
  • the immunoaffinity purification system has two essential elements: an activated solid support to which an antigen or antibody can be conjugated.
  • the activated support ma be a spherical, e.g. beaded, or laminar, e.g. membrane-type, material into which single aldehyde functionalities have been introduced through extended spacer arms. Proteins or ligands containing primary amino groups are reversibly bound to the support in a wide pH range (between pH 1 and 14). Since the binding reaction is reversible, no deactivation of unreacted groups is necessary following the coupling procedure.
  • Immobilization can be achieved by adding a reducing agent, preferably sodium cyanoborohydride, to the antigen(antibody) / activated support suspension.
  • a reducing agent preferably sodium cyanoborohydride
  • the addition of the reducing agen stabilizes the Schiff-base, formed between the ligand and the resin, by reducing it to a secondary amine.
  • Secondary amine linkage is extremely stable; i-t is insensitive to both electrophylic and nucleophylic reagents present in biological fluids or buffer materials. No leakage of immobilized ligand is observed from this resin over a period of one year.
  • the coupling reagent is functional between pH 3 and pH 10, thus making the concept of protein immobilization under physiological conditions feasible.
  • the activated support has also been prepare in a magnetizable form by either adsorbing or chemically attaching colloid magnetic (ferrite) particles to the support. Magnetization of the affinity support allows magnetic separation substitute any of the usual procedures involving columns, filter or centrifuges.
  • Antibody to an antigen(antibod ) is coupled to the magnetic affinity support and then mixed into the crude biological process fluid, such as fermentation broth, blood or milk.
  • Antigen from the biological fluid firmly binds to the magnetizable beads containing the respective anti-antibody which are then separated from the rest of the solution through an externally-applied magnetic field.
  • Isolated beads are suspended in a washing solution to remove nonspecifically-adsorbed material and bound antigen is recovered in a highly-purified, active form by using the subject elution medium.
  • Elution of antigen ( immunoglobulin) from the immunoadsorbent column is performed with 2-3 times of the bed volume of elution medium.
  • the pH for the buffers will be usually in the range of about 3-10 and preferably in the range of about 4-8.
  • the pH is chosen to maintain the native conformation of both the immobilized and the eluted protein.
  • Various buffer materials may be used to achieve the desired pH such as Tris, Mes, Hepes, barbital and the like.
  • Room temperature or refrigeration is usually used to carry out elution of bound antigen(immunoglobulin) .
  • the temperature for the elution will generally range from about 0-50 C, more usually from about 4-22 C.
  • the elution medium employs a combination of electrolytes and low moleculr weight hydroxylic compounds to achieve the release of protein antigen from the immunoadsorbent in its biologically active form.
  • the electrolyte concentration in the subject elution medium will usually be in the range of about 0.5-5 M with respect to MgCl ⁇ . preferably in the range of about 1-4 M.
  • the elution medium contains glycerol in the range of 2-30%(v/v), preferably in the range of about 7-20%(v/v) .
  • Ethyleneglycol concentration i the medium will vary in the range of 2-30%(v/v), preferably in the range of 5-17%.
  • the concentration of ethanolamine in the medium will be in the range of 0.02-0.5 M, preferably in the range of 0.08-0.3 M.
  • the elution medium will also contain a disaccharide , such as maltose, trehalose or lactose in the concentration range of 0.02-0.5 M, preferably in the range of 0.05-0.3 M.
  • a disaccharide such as maltose, trehalose or lactose
  • the above ingredients ensure quantitative elution of antigenf antibody) from immunoadsorbents.
  • the elution medium may also contain additives, such as metal ions, reducing agents, cofactqrs, substrates, inhibitors or activators etc. , whenever a particular application requires it.
  • EXAMPLE 1 Polysaccharide affinity chromatography support(e.g. agarose, cellulose, dextran) was derivatized as described in U.S. Patent 4,423,208. The amino termini of spacer arms are reacted with 1 M glyoxal, 0.1 M NaCNBH 3 , pH 7.0, for 2 hours at room temperature. The aldehyde substitution of the resin is estimated by incubating an aliquot in 2 M ethylenediamine, 0.1 M NaCNBH3, pH 7.0, for 2 hours. The sample was washed with 2 M HCl, followed by distilled water and amino group concentration determined by the TNBS method (Cuatrecasas , P. (1970) J.Biol.Chem. 245, 3059).
  • the activated support was equilibrated with 0.1 M acetate buffer pH 4.5 at 4 C, and Protein A dissolved in the same buffer was added. The coupling was performed as set forth in Example 5.
  • Protein A immobilized to the activated support at a concentratio of 1.5 mg per ml resin exhibits an IgG(human) binding capacity o 14-15 mg per ml of gel.
  • the IgG binding capacity of soluble Protein A has been determined by a radial immunodiffusion method (Becker, W. (1969) Immunochemi ' stry 6, 539) and found to be 8 mg IgG(human) per mg of Protein A corresponding to two IgG binding sites per Protein A molecule.
  • Protein A immobilized to the aldehyde activated support exhibits a higher IgG binding capacity than those in solution. This suggest that Protein A may not be fully active under those conditions (pH 8.0 generally used for its immobilization and binding assay (radial immunodiffusion) . Immobilization of Protein A to aldehyde-activated cellulose under its physiological conditions seems to lock the protein into a more active conformation not observable under alkaline conditions.
  • the activated support was equilibrated with 0.1 M phosphate buffer , pH 7.0, and avidin, dissolved in the same buffer was added.
  • the coupling was performed as set forth in Example 3.
  • Avidin immobilized to the aldehyde-activated agarose at a concentration of 1 mg per ml of gel exhibits a binding capacity of 60 nmoles of d(+)biotin per ml resin. This indicates that no loss of biotin binding activity occurs upon immobilization of avidin to the aldehyde-activated support.
  • Avidin is an extremely stable protein; it can withstand strong acids (1 M HCl), denaturants (6 M guanidine) or elevated temperature (90 C for 30 in) . Yet, immobilization to a CNBr-activated support diminishes its biological activity.
  • EXAMPLE 7 Immunoaffinity purification by using the subject elution medium on a nonmagnetic immunoaffinity resin
  • Mouse IgG was immobilized on aldehyde-agarose at a concentratio of 1.1 mg IgG per ml of gel and 2 ml columns were packed. Purified antibodies specific to mouse IgG were applied to each column at saturating concentration. Fractions of 0.3 ml were collected and analysed for protein (OD280) and immunoreactivity on an enzyme-linked immunosorbent assay (ELISA, OD 92) (Lew,A.M. (1984) J. Immunol. Methods 72, 171). Following sample application, columns were washed with 8 volumes of 0.15 M NaCl and then eluted with 6 ml of elution medium. The flow-rate of both the sample application and elution was 12 ml/h.
  • the eluted antibody was collected and immediately dialyzed against several changes of 0.15 M NaCl for 6-8 hours in the cold. Protein was determined from the dialyzate by measuring optical density at 280 nm. Immunoreactivity of eluted anti-IgG was determined by ELISA. Serial two-fold dilutions both of the eluted and dialyzed anti-IgG(starting dilution 25-fold) in PBS were added to the wells of microtiter plates. Parallel dilutions of the initial anti IgG were also plated and used as a reference After overnight incubation at 4 C, the plates were washed three times with phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • Immunoreactivity of eluted anti-IgG was found to be the highest with the subject elution medium (Table 1), 96-98% of the initial (reference sample).
  • Antibody eluted with 1 M acetic acid displayed 27-32% of the initial immunoreactivity. Incomplete neutralization of eluted antibody resulted in a further, substantial, 10-18% loss of immunoreactivity upon dialysis.
  • Elution with 3 M NH4SCN affected the antibody activity less severely with the retention of 41-46% of the initial immunoreactivity.
  • Acidic elution not only leads to significant losses of antibody activity but also subtle changes in the conformation of IgG detectable by an enhanced susceptibility to peptic digestion (Rousseaux, J.et al.(1983) J.Immunol.Methods 141, 141). This allows a complete digestion of isolated IgG by pepsin to F(ab' ) 2 in 2 hours as opposed to 16 hours for the control antibody.
  • the deleterious effect of extreme pH on the immunoreactivity of immunoglobulin has been noticed and leads to the deterioration of immunosorbent capacity upon continued recycling CEveleigh,J. . and Levy,D.E. (1977) J. Solid-Phase Biochem. 2, 45).
  • chaotropic eluents were given preference by many investigators since the pH can be maintaned between 6 and 8. In spite of the lower yields, chaotropic eluents still have the advantage in terms of the higher immunoreactivity of eluted antibodies.
  • the subject invention elution medium has been found to elute anti-IgG from the immunoadsorbent with the highest yield and immunoreactivity.
  • the results suggest that this novel elution medium has the potential of becoming the universal nondenaturing eluent that would significantly expand the application range of immunoaffinity purifications.
  • Magnetic immunoaffinity beads were prepared according to Example 2 and 5. The beads were mixed into rabbit plasma containing antibodies to mouse IgG. After stirring at room temperature for 30 min,the mixture was passed through a glass tube (i.d. 9 mm), filled with a steel mesh, which was placed between the poles of small electromagnet. Magnetic beads were retained on the steel mesh and washed thoroughly with 0.15 M NaCl to remove nonspecifically adsorbed proteins. While the beads were still held on the steel mesh, specifically-bound proteins were eluted with the subject invention elution medium. Components of the Elution Medium were removed by dialysis in order to recover eluted anti-IgG. DISCUSSION
  • the subject invention eliminates the above problems by permitting immobilization of proteins to solid-phase supports under physiological conditions with minimal or no loss of biological activity (see Examples 4-6). This is of great significance in immobilized enzyme processes in which the overal economy of the procedure is determined by the activity of the immobilized enzyme preparation. Enhancement of the biological activity of a protein following immobilization has also been observed (Example 4), which has not been reported before for thi protein. The reason for these improvements, most likely, is that coupling can be performed right at the optimal, physiological pH of the protein in question. Also, the single aldehyde groups on the activated resin are not self-coupling; their reactivity towards proteins is low which precludes any major perturbation i the tertiary structure of proteins to occur upon coupling.
  • a further major point to consider is the nonleaching characteristics of the invented activated affinity support.
  • the extremely stable secondary amine linkage, established between th resin and the ligand, is insensitive to nucleophylic or electrophylic reagents present in buffers or biological extracts None of the previously used activated supports are leach-proof; they shed ligand at different rates which can be accelerated by exposure to elecrophylic or nucleophylic reagents. This is a serious drawback since the product, purified on an immunoadsorbent, will be contaminated with ligands leaching off the specific adsorbent.
  • the invented activated support eliminates these shortcomings and offers a life-span for the immunoadsorbent which is largely the function of the activit of immobilized proteins.
  • an activated support In order to become applicable in industrial processes, an activated support must fulfil additional criteria such as stability in handling and storage. All the currently used activated supports are unstable and need cold storage, sometimes freezing, and maintaning nonaqueous conditions with dry solvents or lyophylization. The shelf-life of these supports seldom exceeds 3-6 months. Coupling must be performed in the cold or, when coupling is suggested to be carried out at room temperature, (e.g.CDI method) the process is quite slow (16 hours).
  • the invented activated support is stable at room temperature, supplied as an aqueous suspension (no organic solvents are involved), and couples rapidly (in 2 hours) with equal efficiency both at room temperature and in the cold.
  • the shelf-life of the activated resin exceeds one year and it is sterilizable both by steam and NaOH, a feature none of the activated supports offers.
  • Magnetization of the activated support further expands its potential application range. It will permit direct immunoaffinity purification of protein products from biological process fluids, e.g. fermentation broth or tissue culture media, without resorting to expensive clarification procedures. In the manufacture of recombinant DNA biotherapeutics, the product purification costs may well exceed 50 % of the total manufacturing costs. Any cost savings in this area would improve the overall economy of these processes. Magnetic immunoaffinity purification techniques can eliminate expensive clarification procedures as well as the need of conventional purification methods .
  • the chemistry can also be applied to the derivatization of nylon membranes by monoaldehyde groups.
  • the invented monoaldehyde- activated nylon membranes are superior to prior art glutaraldehyde- activated membranes in their stability, and retention of biological activity of immobilized protein.
  • the immunoreactivity of protein immobilized to glutaraldehyde-activated membranes is 2-3 times lower than on the subject monoaldehyde-activated membrane.
  • Derivatized membrane catridges as extracorporeal devices are about to be introduced to the medicine to remove toxic metabolic byproducts from body fluids.
  • the critical step remains is the elution and recovery of the product in a biologically active form.
  • the currently available methods include lowering or raising the pH, the use of classical denaturing agents, such as urea, guanidine, etc. or high concentration of chaotropic agents, such as thiocyanate, iodide, perchlorate, etc.. These treatments are rather harsh and despite claims of recovery of activity, the success rate can be widely variable.
  • High capacity immunoadsorbents need immobilized antibody of high affinity whic makes recovery of antigen from the immunoadsorbent very difficult.
  • elution media Unlike other elution methods (acids, base, chaotropic eluents, urea, etc.) in which the elution media must be exchanged rapidly to minimize damage to th eluted protein, antibody exhibited no loss of immunoreactivity when kept for 24 hours in the subject elution medium.
  • the unique feature of the elution medium is that it significantly stabilize eluted protein while accomplishing complete displacement of immunoadsorbent-bound antigen.
  • affinity columns can be regenerated by a passage of 0.15 M NaCl. The use of the subject elution medium does not appear to affect the performance of the immunoaffinity column upon recycling.
  • the subject invention differs from prior art methods in that, first, affinity support having single monoaldehyde functionalities at the termini of extended spacer arms capable of binding proteins or ligands under its physiological conditions are used. This allows maximization of biological activity of immobilized protein enzymes or ligand.
  • the invented elution medium permits elution of immunoadsorbent-bound antigen(antibody) quantitatively while preserving its biological activity which is also a very important step forward over the prior art. It will be understood that in giving the preferred embodiment and application of the invention, the concept and scope of the invention will not be limited to the specific reagents but certain changes and modifications may be practical within the scope of the appended claims.

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Abstract

A stable activated affinity support having single aldehyde functionalities at the termini of extended spacer arms. The support allows covalent attachment of proteins and ligands under physiological conditions with a maximal retention of biological activity. This is examplified by the covalent immobilization of pepsin, avidin, Protein A and immunoglobulin G. The activated adsorbent is also prepared in a magnetically-responsive form. An elution buffer for recovering antigen or antibody from immunoadsorbents is described and an immunoaffinity purification system is established for quantitative recovery of antigens from immunoadsorbents while retaining their biological acivity.

Description

A NOVEL IMMU OAFFINITY PURIFICATION SYSTEM
BACKGROUND OF THE INVENTION Description of the Prior Art
Iπrinunoaffinity chromatography is an important area of affinity chromatography which employs biospecific antigen-antibody interactions permitting a high degree of purification in a single step. The rapidly growing area of monoclonal antibody production and manufacturing of recombinant biotherapeutics needs powerful and economical downstream processes to rapidly recover the products from dilute solutions in a high yield and purity. Immunoaffini y chromatography would allow economical, single-step purification of these high valued-biologicals.
The reason why the potentials of immunoaffinity chromatography remain to be realized in large-scale downstream processes lies both in the antigen (antibody) immobilization chemistries and the desorption methods used to recover the product from immunosorbents . The chemistries used for immobilization of ligands profoundly affect the performance of the immunoadsorbent. Immobilization of the ligands is usually performed in a narrow alkaline pH range which may be incompatibl with a variety of proteins including monoclonal antibodies. The improperly selected pH alters the native conformation of antigen (antibody) resulting in immunoadsorbents with diminished binding activity. Binding of protein to an activated support having no spacer arm, such as CNBr, CDI, Tresyl, or FMP-activation can als diminish the biological activity of immobilized protein due to the so-called "wall effect" (steric hindrance) .
In spite of its obvious significance, the concept of "protein immobilization under physiological conditions" has not been established in the literature. Instead, proteins had to conform to the immobilization chemistries resulting in, occassionally, a far less than optimal retention of biological activity. Chemistrieslike the glutaraldehyde or carbodiimide methods permit protein coupling under mild conditions but exhibit undesirable side-effects like extensive crosslinking of protein molecules and, they do not operate in a sufficiently wide pH range. With the advent of sodium cyanoborohydride used as a coupling reagent, aldehyde-based coupling chemistry offers the opportunity of protein coupling to solid supports in a wide pH range (pH 3-10) under physiological conditions. However, the generally used glutaraldehyde-activated matrices have several shortcomings. Glutaraldehyde is unstable and tend to polymerize. It does crosslink proteins thereby diminishing their biological activity. Immobilized protein also leaches off glutaraldehyde-activated resins. Activated supports having a high density of single monoaldehyde functionalities at the termini of extended (>2 atoms) spacer arms are thus needed that is the topic of the subject invention.
A major problem associated with the currently used activation chemistries is the leakage of immobilized ligand from the resin. All the currently used chemistries are unstable to varying extent since they are sensitive to nucleophylic or electrophylic reagents present in biological fluids and buffers. The CNBr-technique is well-known for its ability to leach while other activation methods, such as the N-hydroxysuccinimide, CDI, Tresyl, FMP, divinylsulfone or glutaraldehyde-activation shed ligand at a slower rate, but this can become accelerated by exposure to electrophyles or nucleophyles. This is unacceptable in processing(purification) of biopharmaceuticals since the product would be contaminated with components of the immunoadsorbent.
The high affinity of antibodies for antigens are generally precludes elution in the solvent of application. Desorption of antigen from immunoadsorbents is usually achieved by reagents such as chaotropic salts, mineral acids, urea, guanidine, aliphatic acids, alcohols or a combination of these compounds. This kind of reagents, however, adversely affect the biological activity of both the eluted antigen and immunoadsorbent leading to the deterioration of immunoadsorbent capacity and denaturatio of eluted proteins to various degree. These reagents, despite of being strong denaturants , are still unable to quantitatively displace antigens from immunoadsorbents. Denaturation of high-valued proteins and the deterioration of expensive immunoadsorbents provided a disincentive for wide-spread utilization of immunoaffinity purification.
Numerous therapeutically useful proteins are currently manufactured by recombinant DNA techniques while monoclonal antibody production is also constantly growing. Immunoaffinity purification would enhance the overall economy of these manufacturing processes by permitting the recovery of protein products rapidly, usually in one-step, in a high yield and purity. Immunoaffinity chromatography would be particularly advantageous in such cases where the protein product should be recovered from very dilute solutions, such as fermentation broth of secreting recombinant organisms. The currently used immunoaffinity techniques clearly need improvement in order to realize the full potentials of immunoaffinity purification with particular emphasis on large-scale downstream processes.
The necessary improvements relate to both the solid-phase immobilization of proteins and the elution techniques. First, nonleaching active supports capable of immobilization under physiological conditions without crosslinking of proteins are needed. Nondenaturing elution methods are also required to quantitatively recover proteins from immunoadsorbents while preserving their biological activity. The subject invention overcomes the above shortcomings of current immunoaffinity purification methods. We have developed an immunoaffinity chromatography system utilizing a nonleaching affinity support capable of immobilizing proteins under physiological conditions, thereby allowing maximization of their biological activities upo immobilization. The second component, a nondenaturing eluent permits quantitative recovery of antibodies (antigens ) from immunoadsorbents while preserving their biological activity. SUMMARY OF THE INVENTION
A system has been developed for immunoaffinity purification of proteins (antibodies). The invention comprises the method and the system for carrying out immunoaffinity purification. The concept of this invention has broader applications to the purification of any antigen to which an antibody can be raised. The method of this invention, exemplified by the immunoaffinity purification of mouse anti-IgG, comprises in its most preferred form: An activated affinity support having single monomeric aldehyde functionalities capable of reversible binding of proteins or ligands containing primary aπtino groups. The addition of NaCNBHa (coupling reagent)to the antigen/ activated support mixture irreversibly attach the antigen to the support through extremely stable, secondary a ine linkages. The coupling chemistry allows the binding reaction to occur between pH 3 and pH 10, thus making the concept of protein immobilization under physiological conditions feasible resulting in the maximization of biological activity of immobilized proteins. This feature makes the activated support to be a very attractive choice for immobilization of enzymes or other analytically or diagnostically useful proteins. A nondenaturing elution medium represents the second tier of the invention . Since the elution medium is a rich source of both electrophylic and nucleophylic reagents, it is only compatible with coupling chemistries insensitive to these agents. This kind of chemistry is incorporated into the subject activated affinity support. The elution medium permits quantitative recovery of antibodies from immunoadsorbents while retaining their immunoreactivity.
The invention lends itself for immunoaffinity purification of proteins other than antibodies in their biologically active state. Recombinant proteins as well as monoclonal antibodies could be purified from very dilute solutions (culture medium) in a single step to homogeneity, free from pyrogens. This would significantly enhance the overall economy of the production of advanced biotherapeutics. δ DESCRIPTION OF THE PREFERRED EMBODIMENT
In accordance with the subject invention, a purification system and components are provided for quantitative immunoaffinity purification of biologicals while preserving the biological activity. The immunoaffinity purification system has two essential elements: an activated solid support to which an antigen or antibody can be conjugated. The activated support ma be a spherical, e.g. beaded, or laminar, e.g. membrane-type, material into which single aldehyde functionalities have been introduced through extended spacer arms. Proteins or ligands containing primary amino groups are reversibly bound to the support in a wide pH range (between pH 1 and 14). Since the binding reaction is reversible, no deactivation of unreacted groups is necessary following the coupling procedure. Immobilization can be achieved by adding a reducing agent, preferably sodium cyanoborohydride, to the antigen(antibody) / activated support suspension. The addition of the reducing agen stabilizes the Schiff-base, formed between the ligand and the resin, by reducing it to a secondary amine. Secondary amine linkage is extremely stable; i-t is insensitive to both electrophylic and nucleophylic reagents present in biological fluids or buffer materials. No leakage of immobilized ligand is observed from this resin over a period of one year. The coupling reagent is functional between pH 3 and pH 10, thus making the concept of protein immobilization under physiological conditions feasible. This kind of support not only prevents immobilized ligands from leaching off the support but also permits maximization of biological activity of immobilized ligand. It is demostrated that the activated support retains a substantially higher biological activity of immobilized protein than those currently used for immobilization. This feature will have a significant impact on the economy of large-scale application of immobilized enzymes. The activated support has also been prepare in a magnetizable form by either adsorbing or chemically attaching colloid magnetic (ferrite) particles to the support. Magnetization of the affinity support allows magnetic separation substitute any of the usual procedures involving columns, filter or centrifuges. This modification significantly expands the utility of the subject immunoaffinity purification system sincemagnetic separation offers significant advantages over conventional procedures for purification of biologicals from crude systems, such as fermentation broth (culture media) containing cells or cell debris, colloid solutions or tissue extracts. To magnetize the activated support, colloid magnetic particles have been developed. Stabilization of colloid magnetite has been achieved by using polyethyleneglycol which can also be easily derivatized to enable magnetic particles to bind covalently to the activated support. Beyond stabilizing colloid magnetite, polyethyleneglycol appears to facilitate incorporation of magnetic particles into the affinity matrices allowing a very economical utilization of the colloid magnetic material. In this regard, polyethyleneglycol coating of magnetic particles represent an advance over the prior art hydrocarbone coating procedure.
Antibody to an antigen(antibod ) is coupled to the magnetic affinity support and then mixed into the crude biological process fluid, such as fermentation broth, blood or milk. Antigen from the biological fluid firmly binds to the magnetizable beads containing the respective anti-antibody which are then separated from the rest of the solution through an externally-applied magnetic field. Isolated beads are suspended in a washing solution to remove nonspecifically-adsorbed material and bound antigen is recovered in a highly-purified, active form by using the subject elution medium. Elution of antigen ( immunoglobulin) from the immunoadsorbent column is performed with 2-3 times of the bed volume of elution medium. The pH for the buffers will be usually in the range of about 3-10 and preferably in the range of about 4-8. The pH is chosen to maintain the native conformation of both the immobilized and the eluted protein. Various buffer materials may be used to achieve the desired pH such as Tris, Mes, Hepes, barbital and the like.
Room temperature or refrigeration is usually used to carry out elution of bound antigen(immunoglobulin) . The temperature for the elution will generally range from about 0-50 C, more usually from about 4-22 C.
The elution medium employs a combination of electrolytes and low moleculr weight hydroxylic compounds to achieve the release of protein antigen from the immunoadsorbent in its biologically active form.
The electrolyte concentration in the subject elution medium will usually be in the range of about 0.5-5 M with respect to MgCl. preferably in the range of about 1-4 M. The elution medium contains glycerol in the range of 2-30%(v/v), preferably in the range of about 7-20%(v/v) . Ethyleneglycol concentration i the medium will vary in the range of 2-30%(v/v), preferably in the range of 5-17%. The concentration of ethanolamine in the medium will be in the range of 0.02-0.5 M, preferably in the range of 0.08-0.3 M. The elution medium will also contain a disaccharide , such as maltose, trehalose or lactose in the concentration range of 0.02-0.5 M, preferably in the range of 0.05-0.3 M. The above ingredients ensure quantitative elution of antigenf antibody) from immunoadsorbents. The elution medium may also contain additives, such as metal ions, reducing agents, cofactqrs, substrates, inhibitors or activators etc. , whenever a particular application requires it.
EXPERIMENTAL
The following examples are offered by way of illustration and not by way of limitation. All percents are by volume unless indicated otherwise and all temperatures are centigrade. The following abbreviations are used: IgG: immunoglobulin G; PEG: polyethyleneglycol.
EXAMPLE 1 Polysaccharide affinity chromatography support(e.g. agarose, cellulose, dextran) was derivatized as described in U.S. Patent 4,423,208. The amino termini of spacer arms are reacted with 1 M glyoxal, 0.1 M NaCNBH3 , pH 7.0, for 2 hours at room temperature. The aldehyde substitution of the resin is estimated by incubating an aliquot in 2 M ethylenediamine, 0.1 M NaCNBH3, pH 7.0, for 2 hours. The sample was washed with 2 M HCl, followed by distilled water and amino group concentration determined by the TNBS method (Cuatrecasas , P. (1970) J.Biol.Chem. 245, 3059).
EXAMPLE 2 Magnetic beads having single aldehyde functionalities
1. Preparation of derivatized PEG: 5 G of PEG (Mw 6,000) was dissolved in 0.5 M Na2C03, pH 11, and 2 ml of divinylsulfone was added and stirred for 4 hours at room temperature. Derivatized PEG was separated from reactants by dialysis. The dialysed PEG was lyophilized.
2. Preparation of magnetizable particles coated with derivatized PEG: A 30%(w/v) solution of derivatized PEG was prepared in water. FeCl2 (0.5 G) and FeCl3 (1.35 G) was dissolved in 30 ml of PEG solution and the pH was adjusted to 10. The precipitated material was immediately mixed with suction-dried, activated matrices and incubated with agitation for 2 hours. The resin was then thoroughly washed with distilled water until the effluent remained clear of unbound magnetizable particles.
3. Preparation of magnetizable particles with underivatized PEG: A 30% (w/v) solution of PEG was prepared in distilled water. FeCl2 (0.5 G) and FeCl3 (1.35 G) was dissolved in 30 ml of the above solution and pH was adjusted to 9. The precipitated magnetizable particles were mixed thoroughly with the suction-dried matrices. Unadsorbed particles were removed by profuse washing with distilled water.
EXAMPLE 3 Preparation of monoaldehyde-derivatized nylon membrane Nylon membrane was partially hydrolyzed by acid (Inman.D.J. and Hornby, .E. (1974) Biochem. J. 137, 25) and liberated primary amino groups were reacted with 1 M glyoxal, 0.1 M NaCNBH3, pH 7.0, for 2 hours. The aldehyde substitution was determined as se forth in Example 1.
EXAMPLE 4 Immobilization of Protein A to the activated support (magnetic or nonmagnetic)
The activated support was equilibrated with 0.1 M acetate buffer pH 4.5 at 4 C, and Protein A dissolved in the same buffer was added. The coupling was performed as set forth in Example 5. Protein A immobilized to the activated support at a concentratio of 1.5 mg per ml resin exhibits an IgG(human) binding capacity o 14-15 mg per ml of gel. The IgG binding capacity of soluble Protein A has been determined by a radial immunodiffusion method (Becker, W. (1969) Immunochemi'stry 6, 539) and found to be 8 mg IgG(human) per mg of Protein A corresponding to two IgG binding sites per Protein A molecule. Protein A immobilized to the aldehyde activated support, however, exhibits a higher IgG binding capacity than those in solution. This suggest that Protein A may not be fully active under those conditions (pH 8.0 generally used for its immobilization and binding assay (radial immunodiffusion) . Immobilization of Protein A to aldehyde-activated cellulose under its physiological conditions seems to lock the protein into a more active conformation not observable under alkaline conditions. A commercial CNBr-immobilized Protein A, substituted to 2.5 mg Protein A per ml gel, under the same conditions bound only 11-12 mg human IgG per ml of resin.
EXAMPLE 5 Immobilization of pepsin to the activated support (magnetic or nonmagnetic) The activated support was equilibrated with 0.1 M acetate buffer, pH 4.0 and then pepsin, dissolved in the same buffer, was added. The coupling was performed as set forth in Example 5. Pepsin has been immobilized at a concentration of 3-4 mg per ml of resin. Immobilized pepsin retained approximately 80 % of the activity of the soluble enzyme. The best results on pepsin immobilization reported a 30-40 % retained enzyme activity following glutaraldehyde coupling (Tomono,T.et al.(1981)
Biochim.Biophys .Acta 660,186). The resulting immobilized pepsin is extremely stable; no change in the activity of the immobilized enzyme was found after incubation at 37 C for over a month. Had leakage of immobilized enzyme occured, enzymic activity could be detected in the supernatant that would also lead to the deterioration of immobilized enzyme activity. However, none of these has been observed.
EXAMPLE 6 Immobilization of avidin to the activated support (magnetic or nonmagnetic)
The activated support was equilibrated with 0.1 M phosphate buffer , pH 7.0, and avidin, dissolved in the same buffer was added. The coupling was performed as set forth in Example 3. Avidin immobilized to the aldehyde-activated agarose at a concentration of 1 mg per ml of gel exhibits a binding capacity of 60 nmoles of d(+)biotin per ml resin. This indicates that no loss of biotin binding activity occurs upon immobilization of avidin to the aldehyde-activated support. A CNBr-i mobilized avidin at a substitution of 2 mg avidin per ml of gel exhibited a biotin binding capacity of 85 nmoles per ml of resin (Kohanski.R.A. and Lane,M.D.( 1985) J.Biol. Chem.260, 5014) which represents a 70% retained biological activity of avidin. Avidin is an extremely stable protein; it can withstand strong acids (1 M HCl), denaturants (6 M guanidine) or elevated temperature (90 C for 30 in) . Yet, immobilization to a CNBr-activated support diminishes its biological activity.
EXAMPLE 7 Immunoaffinity purification by using the subject elution medium on a nonmagnetic immunoaffinity resin Mouse IgG was immobilized on aldehyde-agarose at a concentratio of 1.1 mg IgG per ml of gel and 2 ml columns were packed. Purified antibodies specific to mouse IgG were applied to each column at saturating concentration. Fractions of 0.3 ml were collected and analysed for protein (OD280) and immunoreactivity on an enzyme-linked immunosorbent assay (ELISA, OD 92) (Lew,A.M. (1984) J. Immunol. Methods 72, 171). Following sample application, columns were washed with 8 volumes of 0.15 M NaCl and then eluted with 6 ml of elution medium. The flow-rate of both the sample application and elution was 12 ml/h.
The eluted antibody was collected and immediately dialyzed against several changes of 0.15 M NaCl for 6-8 hours in the cold. Protein was determined from the dialyzate by measuring optical density at 280 nm. Immunoreactivity of eluted anti-IgG was determined by ELISA. Serial two-fold dilutions both of the eluted and dialyzed anti-IgG(starting dilution 25-fold) in PBS were added to the wells of microtiter plates. Parallel dilutions of the initial anti IgG were also plated and used as a reference After overnight incubation at 4 C, the plates were washed three times with phosphate-buffered saline (PBS). Wells were then coated with 5 mg/ml bovine serum albumin(BSA) in PBS at room temperature for 1 hour. Mouse IgG (10 ug/ml) in PBS containing 1 mg/ml BSA were added to each well for 30 min at room temperature and plates were washed three times with PBS. Peroxidase conjugated goat anti-mouse IgG( 1:1000 diluted in PBS containing mg/ml BSA) was then added to the wells and incubated at room temperature for 1 hour. Plates were washed 6 times with PBS and 50 mM phosphate/citrate buffer, pH 5.0, containing 0.4 mg/ml o-phenylenediamine and 0.012 % H2O2 was added. After 20 min the reaction was stopped and optical density measured at 492 nm. Immunoreactivity of eluted antibody was expressed as a percentag of the reference sample both calculated in OD492 units per mg of anti-IgG.
In parallel experiments, the recovery of bound immunoglobuli was carried out by using the subject elution medium, acidic (0.1 M or 1 M acetic acid) or chaotropic (3 M NH4SCN, pH 7.0) elution media. All the experimental conditions were identical to those descibed above. The acetic acid eluate was neutralized prior to dialysis. Among all the elution media, the subject elution medium recovered the highest amounts, 96-98 % of the bound antibody (Table 1). Among the acidic elution media, 0.1 M acetic acid (pH 2.8) failed to displace antibody efficiently from the column and was replaced by 1 M acetic acid (pH 2.2), thus permitting the recovery of 82-86% of bound anti-IgG. Chaotropic elution medium (3 M NH4SCN, pH 7.0) was less efficient in this regard recovering only 73-78% of bound immunoglobulin.
Immunoreactivity of eluted anti-IgG was found to be the highest with the subject elution medium (Table 1), 96-98% of the initial (reference sample). Antibody eluted with 1 M acetic acid displayed 27-32% of the initial immunoreactivity. Incomplete neutralization of eluted antibody resulted in a further, substantial, 10-18% loss of immunoreactivity upon dialysis. Elution with 3 M NH4SCN affected the antibody activity less severely with the retention of 41-46% of the initial immunoreactivity.
Acidic elution not only leads to significant losses of antibody activity but also subtle changes in the conformation of IgG detectable by an enhanced susceptibility to peptic digestion (Rousseaux, J.et al.(1983) J.Immunol.Methods 141, 141). This allows a complete digestion of isolated IgG by pepsin to F(ab' ) 2 in 2 hours as opposed to 16 hours for the control antibody. The deleterious effect of extreme pH on the immunoreactivity of immunoglobulin has been noticed and leads to the deterioration of immunosorbent capacity upon continued recycling CEveleigh,J. . and Levy,D.E. (1977) J. Solid-Phase Biochem. 2, 45). For this reason, chaotropic eluents were given preference by many investigators since the pH can be maintaned between 6 and 8. In spite of the lower yields, chaotropic eluents still have the advantage in terms of the higher immunoreactivity of eluted antibodies.
The subject invention elution medium has been found to elute anti-IgG from the immunoadsorbent with the highest yield and immunoreactivity. The results suggest that this novel elution medium has the potential of becoming the universal nondenaturing eluent that would significantly expand the application range of immunoaffinity purifications. TABLE 1 Comparative study on the elution of anti-mouse IgG from a mouse IgG immunoadsorbent
Elution Medium 1 M acetic acid 3 M NH4SCN Invention
Elution Medi
Antibody bound(mg) 0.91 0.91 0.91
Antibody eluted(mg) 0.76 0.68 0.88
Yield (%) 84 75 97
Immunoreactivity of 29 43 97 eluted IgG(% of initial value )
The data presented represent an average of 5 separate experiment Immunoreactivity was defined as OD492 units per mg of anti-IgG
EXAMPLE 8 Immunoaffinity purification by using the subject elution medium on a magnetic immunoaffinity resin
Magnetic immunoaffinity beads were prepared according to Example 2 and 5. The beads were mixed into rabbit plasma containing antibodies to mouse IgG. After stirring at room temperature for 30 min,the mixture was passed through a glass tube (i.d. 9 mm), filled with a steel mesh, which was placed between the poles of small electromagnet. Magnetic beads were retained on the steel mesh and washed thoroughly with 0.15 M NaCl to remove nonspecifically adsorbed proteins. While the beads were still held on the steel mesh, specifically-bound proteins were eluted with the subject invention elution medium. Components of the Elution Medium were removed by dialysis in order to recover eluted anti-IgG. DISCUSSION
Efforts to provide a procedure and a system in which quantitative immunoaffinity purification of proteins, while preserving their biological activity, can be performed have not been successful. Several major problems have been encountered upon setting up immunoaffinity chromatography procedures. First, the currently used immobilization chemistries fail to support protein immobilization under physiological conditions without significantly reducing the biological activity of immobilized protein. Since immunoaffinity chromatography has inherent limitations with respect to the maximal binding capacity of immunoadsorbents , it is of paramount importance to maximize the immunoreactivity of immobilized protein(antibody) . This factor has a major impact on the overall economy of large-scale immunoaffinity processes, since the binding capacity of an immunoadsorbent may readily vary by a factor of 10 as a function of the immobilization chemistry. The commonly used activation chemistries with a few exceptions like glutataldehyde or carbodiimide, which extensively crosslink proteins resulting in significant decrease in their biological activity, need alkaline coupling conditions that is incompatible with a number of proteins. Several monoclonal antibodies have been found to lose immunoreactivity up to 70 % when kept at pH 10 for 30 min (Underwood,P.A. and Bean,P. . (1985) J.Immunol.Methods 80, 189). This suggests that a substantial loss of immunoreactivity does occur under alkaline coupling conditions. Therefore, immobilization of monoclonal antibodies under physiological conditions is essential in order to avoid or minimize the loss o immunoreactivity during this process. The currently used activated resins contain highly reactive groups which can react with amino acid residues hidden inside the folded protein structures. This may led to significant changes in the tertiary structure of proteins upon immobilization and to a loss of their biological functions to various degrees.
The subject invention eliminates the above problems by permitting immobilization of proteins to solid-phase supports under physiological conditions with minimal or no loss of biological activity (see Examples 4-6). This is of great significance in immobilized enzyme processes in which the overal economy of the procedure is determined by the activity of the immobilized enzyme preparation. Enhancement of the biological activity of a protein following immobilization has also been observed (Example 4), which has not been reported before for thi protein. The reason for these improvements, most likely, is that coupling can be performed right at the optimal, physiological pH of the protein in question. Also, the single aldehyde groups on the activated resin are not self-coupling; their reactivity towards proteins is low which precludes any major perturbation i the tertiary structure of proteins to occur upon coupling. In fact, no retention of protein on the resin is observed in the absence of sodium cyanoborohydride which is indicative of a reversible interaction. Another important factor influencing the activity of immobilized protein is the presence of an extended spacer arm in the subject activated support. This molecular arm separates immobilized protein from the perturbing effects of the matrix, i.e. , the so-called wall-effect.
A further major point to consider is the nonleaching characteristics of the invented activated affinity support. The extremely stable secondary amine linkage, established between th resin and the ligand, is insensitive to nucleophylic or electrophylic reagents present in buffers or biological extracts None of the previously used activated supports are leach-proof; they shed ligand at different rates which can be accelerated by exposure to elecrophylic or nucleophylic reagents. This is a serious drawback since the product, purified on an immunoadsorbent, will be contaminated with ligands leaching off the specific adsorbent. This is unacceptable for two reasons: (i) the contamination excludes human therapeutic applications unless further purification is undertaken; (ii) leaching results in the deterioration of immunoadsorbent capacity which depreciate the economy of immunoaffinity purifications. The invented activated support eliminates these shortcomings and offers a life-span for the immunoadsorbent which is largely the function of the activit of immobilized proteins.
In order to become applicable in industrial processes, an activated support must fulfil additional criteria such as stability in handling and storage. All the currently used activated supports are unstable and need cold storage, sometimes freezing, and maintaning nonaqueous conditions with dry solvents or lyophylization. The shelf-life of these supports seldom exceeds 3-6 months. Coupling must be performed in the cold or, when coupling is suggested to be carried out at room temperature, (e.g.CDI method) the process is quite slow (16 hours). The invented activated support is stable at room temperature, supplied as an aqueous suspension (no organic solvents are involved), and couples rapidly (in 2 hours) with equal efficiency both at room temperature and in the cold. The shelf-life of the activated resin exceeds one year and it is sterilizable both by steam and NaOH, a feature none of the activated supports offers.
Magnetization of the activated support further expands its potential application range. It will permit direct immunoaffinity purification of protein products from biological process fluids, e.g. fermentation broth or tissue culture media, without resorting to expensive clarification procedures. In the manufacture of recombinant DNA biotherapeutics, the product purification costs may well exceed 50 % of the total manufacturing costs. Any cost savings in this area would improve the overall economy of these processes. Magnetic immunoaffinity purification techniques can eliminate expensive clarification procedures as well as the need of conventional purification methods .
The chemistry can also be applied to the derivatization of nylon membranes by monoaldehyde groups. The invented monoaldehyde- activated nylon membranes are superior to prior art glutaraldehyde- activated membranes in their stability, and retention of biological activity of immobilized protein. The immunoreactivity of protein immobilized to glutaraldehyde-activated membranes is 2-3 times lower than on the subject monoaldehyde-activated membrane. Derivatized membrane catridges as extracorporeal devices are about to be introduced to the medicine to remove toxic metabolic byproducts from body fluids. An improvement in the binding capacity of the affinity membranes would increase the useful life-span of the devices or the size of the devices could be reduced which can also be a critical factor, and the overall economy of these processes could be increased. Glutaraldehyde-activated membranes also shed immobilized ligand at a slow, but constant rate which is undesirable in human therapeutic applications. The improvement represented by the subject invention eliminates the above shortcomings .
After adsorption of the antigen or antibody to the immunoaffinity matrix, the critical step remains is the elution and recovery of the product in a biologically active form. The currently available methods include lowering or raising the pH, the use of classical denaturing agents, such as urea, guanidine, etc. or high concentration of chaotropic agents, such as thiocyanate, iodide, perchlorate, etc.. These treatments are rather harsh and despite claims of recovery of activity, the success rate can be widely variable. High capacity immunoadsorbents need immobilized antibody of high affinity whic makes recovery of antigen from the immunoadsorbent very difficult. Even strong denaturants, such as urea or guanidine have been found to be unable to fully displace antigens from these immunoadsorbents. Denaturing reagents may also completely abolish biological activity. Therefore it is essential to develo efficient but mild conditions .to effect the dissociation of antigen-antibody complexes. ' ,
In search for an alternative to the above elution conditions we have investigated the dissociating effect of electrolytes on antigen-antibody complexes. Magnesium salts have been reported t dissociate antigen-antibody complexes and found to elute antigen or antibody from immunoadsorbents. The elution, however, has not been complete nor has the antibody been recovered without loss o immunoreactivity. We have developed a composition in which a magnesium salt and low molecular weight hydroxylic compounds cooperate in displacing the antigen from the immunoadsorbent while the hydroxylic compounds also cooperate in the stabilization of eluted protein. Unlike other elution methods (acids, base, chaotropic eluents, urea, etc.) in which the elution media must be exchanged rapidly to minimize damage to th eluted protein, antibody exhibited no loss of immunoreactivity when kept for 24 hours in the subject elution medium. The unique feature of the elution medium is that it significantly stabilize eluted protein while accomplishing complete displacement of immunoadsorbent-bound antigen. After elution, affinity columns can be regenerated by a passage of 0.15 M NaCl. The use of the subject elution medium does not appear to affect the performance of the immunoaffinity column upon recycling.
The subject invention differs from prior art methods in that, first, affinity support having single monoaldehyde functionalities at the termini of extended spacer arms capable of binding proteins or ligands under its physiological conditions are used. This allows maximization of biological activity of immobilized protein enzymes or ligand. The invented elution medium permits elution of immunoadsorbent-bound antigen(antibody) quantitatively while preserving its biological activity which is also a very important step forward over the prior art. It will be understood that in giving the preferred embodiment and application of the invention, the concept and scope of the invention will not be limited to the specific reagents but certain changes and modifications may be practical within the scope of the appended claims.

Claims

We claim :
1. A stable aldehyde-derivatized, activated affinity support having single aldehyde functionalities on extended spacer arms capable of immobilization the desired protein or ligand under physiological conditions.
2. The activated support of claim 1, wherein the matrix of said activated support contains no silicium compounds and is insoluble or soluble in aqueous solutions.
3. The activated support of claim 1, wherein said insoluble activated support is of spherical, e.g. beaded, or of laminar, e.g. membrane-type structure.
4. The activated support of claim 1 , wherein said activated support not having spherical or laminar structures is embedded in a carrier having spherical or laminar structures.
5. The activated support of claim 1, wherein said aldehyde groups, located at the termini of spacer arms, are single aldehyde groups having no polymerized residues.
6. The activated support of claim 1, wherein said aldehyde activated groups reside at the termini of spacer arms of longer than 2 carbon atom equivalent.
7. The activated support of claim 1, wherein the immobilized protein is pepsin.
8. The activated support of claim 1, wherein the immobilized protein is avidin, avidin derivatives or streptavidin.
9. The activated support of claim 1, wherein the immobilized protein is Protein A.
10. A magnetizable colloid having polyethyleneglycol or derivatized polyethyleneglycol coating.
11. The method of claim 10, wherein said polyethyleneglycol- coated, magnetizable colloid particles are attached covalently or noncovalently to said activated aldehyde affinity supports for rapid separation of said supports from complex mixtures through applying an external magnetic field.
12. An elution medium comprising a combination of magnesium salts and low molecular weight hydroxylic compounds for recovering antibodies or antigens from immunoadsorbents as well as storing proteins therein.
13. A system for use in immunoaffinity binding, elution , e.g. purification, of proteins or ligands comprising in combination or separately:
1, (a) a stable aldehyde-derivatized affinity support of claim having single aldehyde functionalities for immobilization of proteins or ligands under physiological conditions, (b) an elution medium of claim 12 permitting quantitative recovery of antibodies or antigens from immunoadsorbents while preserving their immunoreactivity.
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0428412A2 (en) * 1989-11-16 1991-05-22 Ortho Diagnostic Systems Inc. Method of producing a metal sol reagent containing colloidal metal particles of a preselected size
WO1991015766A1 (en) * 1990-04-09 1991-10-17 Dynal A.S Antigen/anti-antigen cleavage
WO1991015768A1 (en) * 1990-04-06 1991-10-17 Syngene, Inc. Process and composition for performing dna assays
EP0575973A2 (en) * 1992-06-23 1993-12-29 Ibiden Co., Ltd. Method for producing optical fiber having formyl groups on core surface thereof
US5328985A (en) * 1991-07-12 1994-07-12 The Regents Of The University Of California Recombinant streptavidin-protein chimeras useful for conjugation of molecules in the immune system
US5447859A (en) * 1993-07-16 1995-09-05 Viagene Method for the purification or removal of retroviruses using sulfated cellulose
US5665539A (en) * 1991-07-12 1997-09-09 The Regents Of The University Of California Immuno-polymerase chain reaction system for antigen detection
EP0818237A1 (en) * 1995-03-01 1998-01-14 Kanegafuchi Kagaku Kogyo Kabushiki Kaisha Method for immobilizing ligands or compounds having ligands bonded thereto
WO2001081367A2 (en) * 2000-04-20 2001-11-01 Universite De Geneve Purification of polypeptides
EP0640216B1 (en) * 1991-03-20 2002-10-02 Marconi Optical Components Limited Separation method
EP2383035A4 (en) * 2008-12-03 2017-11-22 Kaneka Corporation Formyl group-containing porous support, adsorbent using same, method for producing same, and method for producing the adsorbent

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4140662A (en) * 1977-03-25 1979-02-20 Ortho Diagnostics, Inc. Attachment of proteins to inert particles
US4143203A (en) * 1976-03-19 1979-03-06 Amicon Corporation Particulate support material
US4241176A (en) * 1975-12-02 1980-12-23 Etablissement declare d'Utilite Publique diti Institut Pasteur Magnetic gel suitable to immunoenzymatic determinations
US4271264A (en) * 1976-09-13 1981-06-02 Modrovich Ivan Endre Stabilized liquid enzyme and coenzyme compositions
US4423208A (en) * 1982-02-24 1983-12-27 Peter Grandics Affinity gel-adsorbent
JPS60177265A (en) * 1984-02-24 1985-09-11 Japan Synthetic Rubber Co Ltd Detection of immunoglobulin of antibody by class
US4563431A (en) * 1981-11-13 1986-01-07 Hoechst Aktiengesellschaft Biologically active latex conjugate
US4681782A (en) * 1982-03-31 1987-07-21 Biostar Medical Products, Inc. Article for determining the presence of immune complexes
US4693985A (en) * 1984-08-21 1987-09-15 Pall Corporation Methods of concentrating ligands and active membranes used therefor

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4241176A (en) * 1975-12-02 1980-12-23 Etablissement declare d'Utilite Publique diti Institut Pasteur Magnetic gel suitable to immunoenzymatic determinations
US4143203A (en) * 1976-03-19 1979-03-06 Amicon Corporation Particulate support material
US4271264A (en) * 1976-09-13 1981-06-02 Modrovich Ivan Endre Stabilized liquid enzyme and coenzyme compositions
US4140662A (en) * 1977-03-25 1979-02-20 Ortho Diagnostics, Inc. Attachment of proteins to inert particles
US4563431A (en) * 1981-11-13 1986-01-07 Hoechst Aktiengesellschaft Biologically active latex conjugate
US4423208A (en) * 1982-02-24 1983-12-27 Peter Grandics Affinity gel-adsorbent
US4681782A (en) * 1982-03-31 1987-07-21 Biostar Medical Products, Inc. Article for determining the presence of immune complexes
JPS60177265A (en) * 1984-02-24 1985-09-11 Japan Synthetic Rubber Co Ltd Detection of immunoglobulin of antibody by class
US4693985A (en) * 1984-08-21 1987-09-15 Pall Corporation Methods of concentrating ligands and active membranes used therefor

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
AVRAMEAS, et al., "The Cross-linking of Proteins With Glutaraldehyde and its Use for the Preparation of Immuno-adsorbents", Immunochemistry, Vol. 6, pages 53-66, issued 1969 (Pergamon Press, England), see pages 53 and 62-65. *
TERNYNCK et al., "Effect of Electrolytes and of Distilled Water on Antigen-Antibody Complexes", Biochem. J., Vol. 125, pages 297-302, issued 1971 (Biochemical Society, Essex, England), see pages 297, 301 and 302. *
TERNYNCK, et al., "Polyacrylamide-Protein Immunoadsorbents Prepared With Glutaraldehyde", FEBS Letters, Vol. 23, No. 1, pages 24-28, issued June 1972 (New Holland Publishing Company, Amsterdam), see pages 24, 25, 27 and 28. *
WESTON et al., "Proteins Coupled To Polyacrylamide Beads Using Gludaraldehyde", Biochemical and Biophysical Research Communications, Vol. 45, No. 6, pages 1514-1580, issued 1971, (Academic Press, Inc, San Diego, Californa), see pages 1574, 1579 and 1580. *

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