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Purification of antibodies by affinity chromatography - ScienceDirect

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This review focusses on affinity purification of immunoglobulins, a methodology which is a powerful tool to obtain pure and intact antibodies. Affinity techniques allow antibody purification both in a single step chromatographic procedure as well as in complex purification protocols depending on the intention to use the target antibody.

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Journal Article

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Chapter 26 Affinity Chromatography: General Methods

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Affinity chromatography is one of the most diverse and powerful chromatographic methods for purification of a specific molecule or a group of molecule...

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Part of the Methods in Molecular Biology™ book series (MIMB, volume 21)

Abstract

Affinity chromatography is one of the most convenient methods of purifying biological molecules, with many wide ranging applications (, ). The main prerequisite is to have a ligand that specifically binds to the target molecule. This ligand must also have two key properties other than exquisite specificity: It must be capable of being immobilized in an active form to a solid matrix, and the ligand/target complex must be readily dissociable under conditions that release the target molecule in an active form. These criteria are often best fulfilled for protein targets by antibody ligands. Monoclonal antibodies are the ligands of choice for most purposes, especially when a protein of unknown properties is being characterized. Monoclonal antibodies fulfill all the above criteria and also afford the advantage of being available in essentially unlimited supply. Moreover, they are themselves readily purified, often by affinity chromatography. The main disadvantage of monoclonal antibody ligands is that they must possess affinities that are sufficiently high to allow efficient purification and sufficiently low to allow efficient elution. This balance cannot really be tailored, but must be obtained by screening a number of specific monoclonals until empirically one is obtained with the desired properties. Once an appropriate monoclonal antibody has been identified, then in principle the purification of the corresponding antigen is straightforward and is limited in quantity only by the availability of the starting material.

References

1. Jakoby, W B and Wilchek, M., eds. (1974) Affinity Techniques, in Methods zn Enzymology-Enzyme Purification:Part B Vol 34, Academic, New York

2. Wilchek, M., Miron, T., and Kohn, J. (1984) Affinity chromatography Methods Enzymol. 104, 3-55.

3. Harlow, E. and Lane, D (1988) Immunoaffinity purification, in Antibodies A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 511-552.

4. Hall, R., Osland, A., Hyde, J. E, Simmons, D. L., Hope, I. A. and Scaife, J. G. (1984) Processing, polymorphism, and biological significance of p190, a major surface antigen of the erythrocytic forms of Plasmodium falcrparum. Mol Biochem. Parasitol. 11, 61-80.

5. Hall, R, Hyde, J. E., Goman, M., Simmons, D. L, Hope, I A., Mackay, M, Scaife, J. G., Merkh, B., Richle, R, and Stocker, J (1984) Major surface antigen gene of a human malaria parasite cloned and expressed in bacteria Nature 311, 379-382.

6. Hope, I. A., Hall, R, Simmons, D. L., Hyde, J. E., and Scaife, J. G. (1984) Evidence for immunological cross-reaction between sporozottes and blood stages of a human malaria parasite. Nature 308, 191-194

7. Hope, I A. (1984) Investigation of potentially protective antigens of the human malaria parasite Piusmodium fulciparum. PhD Thesis, University of Edinburgh, Scotland, UK.

8. Ridley, R. G, Takacs, B., Etlinger, H, and Scaife, J. G. (1990) A rhoptry anttgen of Plasmodium falciparum is protective in Saimiri monkeys Parasitology 101, 187-192.

9. Holder, A. and Freeman, R R. (1981) Immunization against blood-stage rodent malaria using purified parasite antigens. Nature 294, 361-364.

10. King, C. H., Lett, R. R, Nanduri, J., Ibiary, S. E, Peters, P A S., Olds, G R, and Mahmoud, A. A. F. (1987) Isolation and characterization of a protective antigen for adjuvant-free immunization against Schistosoma mansoni. J Immunol. 139, 4218-4224.

11. Trager, W. and Jensen, J. B. (1976) Human malaria parasites in continuous culture. Science 193, 673-675.

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Part of the Methods in Molecular Biology™ book series (MIMB, volume 21) Affinity chromatography is one of the most convenient methods of purifying biological molecules, with many wide ranging applications (, ). The main prerequisite is to have a ligand that specifically binds to the target molecule.

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The production and use of specific antibodies as detection probes and purification ligands-often called immunodetection or immunotechnology-has revolutionized bioresearch and diagnostic technologies. Animals immunized with prepared antigens will produce specific antibodies against the antigen. Once they are purified (and possibly after labeling them with an enzyme or fluorescent tag), these antibodies can be used directly to probe the specific antigen in Western blotting, ELISA and other applications.

Antiserum from an immunized animal can be used directly for certain applications, but more often some form of antibody purification is required to obtain an antibody probe that is effective for multiple types of detection methods. This article summarizes the main approaches and tools available for accomplishing antibody purification.

Introduction

Antibody purification involves selective enrichment or specific isolation of antibodies from serum (polyclonal antibodies), ascites fluid or cell culture supernatant of a hybridoma cell line (monoclonal antibodies). Purification methods range from very crude to highly specific and can be classified as follows:

Physicochemical fractionation-differential precipitation, size-exclusion or solid-phase binding of immunoglobulins based on size, charge or other shared chemical characteristics of antibodies in typical samples. This isolates a subset of sample proteins that includes the immunoglobulins.

Class-specific affinity-solid-phase binding of particular antibody classes (e.g., IgG) by immobilized biological ligands (proteins, lectins, etc.) that have specific affinity to immunoglobulins. This purifies all antibodies of the target class without regard to antigen specificity.

Antigen-specific affinity-affinity purification of only those antibodies in a sample that bind to a particular antigen molecule through their specific antigen-binding domains. This purifies all antibodies that bind the antigen without regard to antibody class or isotype.

Antibodies that were developed as monoclonal antibody hybridoma cell lines and produced as ascites fluid or cell culture supernatant can be fully purified without using an antigen-specific affinity method (third type) because the target antibody is (for most practical purposes) the only immunoglobulin in the production sample. By contrast, for polyclonal antibodies (serum samples), antigen-specific affinity purification is required to prevent co-purification of nonspecific immunoglobulins. For example, generally only 2-5% of total IgG in mouse serum is specific for the antigen used to immunize the animal. The type(s) and degree of purification that are necessary to obtain usable antibody depend upon the intended application(s) for the antibody.

Physicochemical fractionation antibody purification

The main classes of serum immunoglobulins (e.g., IgG, IgM) share the same general structure, including overall amino acid composition and solubility characteristics. These general properties are sufficiently different from most other abundant proteins in serum, such as albumin and transferrin, that the immunoglobulins can be selected and enriched on the basis of these differentiating physicochemical properties.

Size exclusion chromatography

Dialysis, desalting and diafiltration can be used to exchange antibodies into particular buffers and remove undesired low-molecular weight (MW) components. Dialysis membranes, size-exclusion resins, and diafiltration devices that feature high-molecular weight cut-offs (MWCO) can be used to separate immunoglobulins (>140kDa) from small proteins and peptides. However, except with specialized columns and equipment, these techniques alone cannot purify antibodies from other proteins and macromolecules that are present in typical antibody samples. More commonly, gel filtration and dialysis are used following other purification steps, such as ammonium sulfate precipitation (1).

Ammonium sulfate precipitation

Ammonium sulfate precipitation is frequently used to enrich and concentrate antibodies from serum, ascites fluid or cell culture supernatant. As the concentration of this lyotropic salt is increased in a sample, proteins and other macromolecules become progressively less soluble until they precipitate; the lyotropic effect is called "salting out." Antibodies precipitate at lower concentrations of ammonium sulfate than most other proteins and components of serum.

At 40 to 50% ammonium sulfate saturation (100% saturation equals 4.32M), immunoglobulins will precipitate while other proteins remain in solution (2).The usual method involves very slowly adding an equal volume of saturated ammonium sulfate solution to a neutralized antibody sample, followed by incubation for several hours at room temperature or 4°C. After centrifugation and removal of the supernatant, the antibody-pellet is dissolved in buffer, such as phosphate-buffered saline (PBS).

The selectivity, yield, purity and reproducibility of precipitation depends upon several factors, including time, temperature, pH and rate of salt addition (3). Ammonium sulfate precipitation provides sufficient purification for some antibody applications, but most often it is performed as a preliminary step before column chromatography or other purification method (e.g., Melon Gel Monoclonal IgG Purification Kit). Using partially-purified antibody samples can improve the performance and extend the life of affinity columns.

Other antibody precipitation reagents that have been used for special antibody purification situations include using octonoic acid, polyethylene glycol and ethacridine (3).

Ion exchange chromatography

Numerous chemically-based, solid-phase chromatography methods have been adapted and optimized to achieve antibody purification in particular situations.

Ion exchange chromatography (IEC) uses positively or negatively charged resins to bind proteins based on their net charges in a given buffer system (pH). Especially in commercial operations involving production of monoclonal antibodies, conditions for IEC can be determined that bind and release the target antibody with a high degree of specificity. Conversely, conditions can be found that bind nearly all other sample components except antibodies. Once so optimized, IEC is a cost-effective, gentle and reliable method for antibody purification.

Immobilized metal chelate chromatography

Immobilized metal chelate chromatography (IMAC) uses chelate-immobilized divalent metal ions (usually nickel, Ni2+) to bind proteins or peptides that contain clusters of three or more consecutive histidine residues. The strategy is most often used to purify recombinant proteins that have been engineered to contain a terminal 6xHis fusion tag. Interestingly, mammalian IgGs are one of the few abundant proteins in serum (or monoclonal hybridoma cell culture supernatant) that possess histidine clusters capable of being bound by immobilized nickel. As with IEC, IMAC conditions for binding and elution can be optimized for particular samples to provide gentle and reliable antibody purification (3). For example, the Pierce Conjugate Purification Kit uses this technique to separate AP- or HRP-labeled (enzyme-conjugated) antibody from excess, non-conjugated enzyme following a labeling procedure.

Thiophilic adsorption

Thiophilic adsorption is a highly selective type of protein-ligand interaction that combines the properties of hydrophobic interaction chromatography (HIC) and ammonium sulfate precipitation (the lyotropic effect). The interaction is termed thiophilic because it involves the binding of proteins to a sulfone group in close proximity to a thioether. In contrast to strict HIC, thiophilic adsorption depends upon a high concentration of lyotropic salt (e.g., potassium sulfate as opposed to sodium chloride). With typical antibody samples that have been equilibrated with potassium sulfate, binding is quite specific to antibodies. After non-bound components are washed away, the antibodies are easily recovered with gentle elution conditions (e.g., 50mM sodium phosphate buffer, pH 7 to 8). Thiophilic Adsorbent (also called T-Gel) is 6% beaded agarose modified to contain the sulfone-thioether ligand. The adsorbent has a high binding capacity and broad specificity toward imunoglobulins from various animal species. Notably, it is one of few affinity methods that is effective for chicken IgY purification.

Melon Gel chromatography

Melon Gel is a proprietary resin chemistry (and optimized buffer system) for purifying antibodies by chemical-based fractionation. In the specified mild buffer condition, Melon Gel resin binds most non-IgG proteins found in serum, ascites fluid and culture supernatants, while allowing purified IgG to be collected in the flow-through fraction.

The various Melon Gel kits are optimized for rapid, convenient and gentle purification of IgG. The Melon Gel kit for purification of monoclonal antibodies illustrates the benefits of combining two purification techniques. Ammonium sulfate precipitation is recommended for cell culture supernatant samples before performing the final Melon Gel purification. Treatment of ascites fluid samples with a conditioning reagent is recommended before Melon Gel purification to decrease the co-purification of transferrin with the antibody.

Because the Melon Gel system uses negative selection and requires no elution steps, it also provides a convenient and effective method for removing bovine serum albumin (BSA) or gelatin from antibody stock solutions so that these stabilizing proteins will not interfere with antibody labeling procedures. This is the basis of our Antibody Clean-Up Kit.

If the specific removal of one particular undesirable serum component can be considered a form of antibody purification, then it is appropriate here to mention albumin removal. Albumin accounts for approx. 60% of human serum protein. Cibacron* Blue Dye binds selectively to human serum albumin and can be used as an affinity ligand to prepare albumin-free serum samples for 2D electrophoretic analysis.

Class-specific affinity purification of antibodies

Because antibodies have an evolutionarily conserved overall structure, including relatively invariant domains, and their native function involves binding and defense against pathogens, it is no surprise that certain pathogenic bacteria have evolved proteins having specific antibody-binding functions. Several of these immunoglobulin-binding proteins have been identified and isolated from certain species of bacteria. In the same way that the native functions of antibodies are useful as target-specific probes for protein research, so also these native anti-Ig proteins are useful as affinity ligands for antibody purification.

Protein A, G and L antibody-binding ligands

Protein A, Protein G and Protein L are three bacterial proteins whose antibody-binding properties have been well characterized. These proteins have been produced recombinantly and used routinely for affinity purification of key antibody types from a variety of species. Most commercially-available, recombinant forms of these proteins have unnecessary sequences removed (including the HSA-binding domain from Protein G) and are therefore smaller than their native counterparts. A genetically-engineered recombinant form of Protein A and Protein G, called Protein A/G, is also widely available. All four recombinant Ig-binding proteins are used routinely by researchers in numerous immunodetection and immunoaffinity applications.

Sources and features of native Ig-binding proteins. Information in this table is gathered from various sources. See related "Learn more" pages for references and details about the recombinant forms used for immunoaffinity applications.

Protein A (SpA)

Protein G (SpG)

Protein L (SpL)

Human
Pathology
Component of human body flora; cause of "Staph" infections

Orig. isolated from pharyngitis patients (tonsils or blood)

Commensal and/or pathogenic anaerobic Gram-positive bacteria

Native
Size(s)
40 to 60kDa
(variable numbers of repeated domains)

40 to 65kDa
(variable numbers of repeated domains)

76kDa

Binding Domains
5 for IgG (most common form)

1 to 2 for IgG
0 to 2 for HSA

5 for Ig

Ig-binding Target
heavy chain constant region (Fc) of IgG (CH2-CH3 region)

heavy chain const. region (Fc) of IgG (CH2-CH3 region)

kappa light chains of Igs (VL-kappa)

Binding sites of antibody-binding proteins. Proteins used to immobilize antibodies to beaded support show specificity to different antibody domains. Protein A and G bind to the heavy chains of the antibody Fc region, while Protein L specifically binds the light chains of the two Fab regions of the F(ab')2 antibody fragment. † Protein G can also bind Fab fragments in certain conditions.
Antibody purification with Protein A, G and L

To accomplish antibody purification with Protein A, Protein G, Protein A/G or Protein L, they are covalently immobilized onto porous resins (such as beaded agarose) or magnetic beads. Because these proteins contain several antibody-binding domains, nearly every individual immobilized molecule, no matter its orientation maintains at least one functional and unhindered binding domain. Furthermore, because the proteins bind to antibodies at sites other than the antigen-binding domain, the immobilized forms of these proteins can be used in purification schemes, such as immunoprecipitation, in which antibody binding protein is used to purify an antigen from a sample by binding an antibody while it is bound to its antigen.

Protein A, G, A/G and L have different binding properties, which make each one suitable for different types of antibody targets (e.g., antibody subclass or animal species). It is important to realize that use of Protein A, G or L results in purification of general immunoglobulin from a crude sample. Depending on the sample source, antigen-specific antibody may account for only a small portion of the total immunoglobulin in the sample. For example, generally only 2-5% of total IgG in mouse serum is specific for the antigen used to immunize the animal.

Using a column of Protein A agarose resin and rabbit serum as the example, the general procedure for antibody purification with these ligands is as follows:

1. Bind: Add a clarified, physiologic-buffered (pH 7 to 8) sample of rabbit serum to the column and allow it to slowly pass through or mix with the Protein A resin to allow the IgG to bind to the immobilized ligand.

2. Wash: Add phosphate-buffered saline (PBS) and allow it to pass through the column to wash away nonbound serum components. Use a volume of wash buffer equivalent to 5 to 10 times the resin volume.

3. Elute: Add acidic elution buffer (e.g., 0.1M glycine-HCl, pH 2.8), and collect small fractions of solution that pass from the column. The low-pH condition dissociates the antibody from the immobilized Protein A, and the IgG is recovered in its purified state in one or several of the collected fractions.

4. Neutralize or exchange buffer: Use a protein assay or other means to identify and combine elution fractions that contain the purified antibody. Then add 1/10th volume of 1M Tris-HCl (pH 8.5) to neutralize the buffer. Alternatively, use a desalting column or dialysis to exchange the purified antibody into a more suitable buffer for long-term storage.

IgM purification

Protein A and Protein G bind IgM very poorly or not at all, in part because binding sites on the Fc regions of IgM are sterically hindered by its pentameric structure. For IgM (class M antibodies) that possess the appropriate type of light chains (VL-kappa), Protein L can be used for purification; however, IgGs having the same type of light chains will co-purify.

For commercial scale operations, IgM antibodies are usually purified by a combination of techniques, including ammonium sulfate precipitation followed by gel filtration, ion exchange chromatography or zone electrophoresis. With serum samples (polyclonal), a simple enrichment strategy is ammonium sulfate precipitation followed by removal of IgG with Protein A or G.

Nethery, et al. (4) developed an IgM affinity purification method using C1q, a 439kDa complement component that recognizes carbohydrate on cell surfaces. Nevens, et al. (5) extended and improved this approach by using as the ligand the similarly structured complement-activation protein called mannan binding protein (MBP). Our IgM Purification Kit uses immobilized MBP and is most effective for purifying mouse IgM from ascites. Purified IgM can be obtained from a single pass over an affinity column. Human IgM will bind to the support, albeit with slightly lower capacity, and yield a product at least 88% pure as assessed by HPLC. The purification of IgM from other species and mouse serum has not yet been optimized.

IgA purification

Jacalin is an a-D-galactose binding lectin extracted from jackfruit seeds (Artocarpus integrifolia). The lectin is a glycoprotein of approximately 40kDa composed of four identical subunits. Jacalin immobilized on supports such as agarose has been useful for the purification of human serum or secretory IgA1. The affinity ligand makes it possible to purify or remove IgA from the more abundant IgG and IgM in human serum or colostrum (6). IgD is reported to bind to jacalin (7).

Chicken IgY purification

Chickens produce a unique immunoglobulin molecule called IgY. There are several advantages to production and use of IgY over mammalian immunoglobulins. With regard to production, raising and immunizing chickens is relatively simple, chickens are more likely to produce an immune response to conserved mammalian protein antigens, and chickens produce 15- to 20-times more antibody than rabbits.

Most importantly, IgY is naturally packaged at high concentrations in egg yolks, making repeated collection of antibody from immunized hens noninvasive. A single egg yolk from an immunized chicken contains approximately 300mg of IgY. Whole eggs or separated egg yolks can be collected and stored frozen for later extraction of antibody.

Protein A, Protein G and other Fc-binding proteins do not bind IgY. Thiophilic adsorbent (see above) is effective for IgY purification from serum and other fluids. However, an effective thiophilic adsorbent procedure has not been developed for use with egg yolks, which have very high lipid concentrations. Instead, our Chicken IgY Purification Kit provides efficient purification of IgY from egg yolks by uses a variation of ammonium sulfate precipitation after first delipidating the egg yolk sample with a proprietary solution.

Antigen-specific affinity purification of antibodies

Although Proteins A, G, A/G and L are excellent ligands for purification of total IgG from a sample, purification of antigen-specific antibodies is often required. This can be accomplished by immobilizing the particular antigen used for immunization so that only those antibodies that bind specifically to the antigen are purified in the procedure. Activated affinity supports that can be used to immobilize peptides or other antigens for use in affinity purification are described in another article called Ligand Immobilization Methods for Affinity Purification (link coming soon). The present article summarizes particular concerns related to antigen immobilization for use in purifying antibodies.

Antigen immobilization and presentation

Successful affinity purification of antibody depends on effective presentation of the relevant epitopes on the antigen to binding sites of the antibody. If the antigen is small and immobilized directly to a solid support surface by multiple chemical bonds, important epitopes may be blocked or sterically hindered, prohibiting effective antibody binding. Therefore, it is best to immobilize peptide antigens using a unique functional group (e.g., sulfhydryl on a single terminal cysteine in a peptide) and to use an activated support whose reactive groups occur on spacer arms that are several atoms long. For large (e.g., protein) antigens, especially those with multiple sites of immobilization, the spacer arm length becomes less important since the antigen itself serves as an effective spacer between the support matrix and the epitope. Generally, if the antigen was crosslinked to a carrier protein to facilitate antibody production, best results are obtained when the antigen is immobilized for affinity purification using the same chemistry (e.g., reaction to primary amines, sulfhydryls, carboxylic acids or aldehydes). In this way, all epitopes will be available for antibody binding, allowing greater efficiency in purification and recovery of the specific immunoglobulin.

Peptide antigens and affinity ligands

Most antibodies are produced using peptide antigens that were synthesized and conjugated to an immunogenic carrier protein, such as KLH. Such antigens can be customized to contain a unique functional group (handle) for both conjugation and immobilization. A terminal cysteine is often added for this purpose; it provides a sulfhydryl group for efficient conjugation to maleimide-activated carrier protein and immobilization onto iodoacetyl-activated agarose resin (SulfoLink Coupling Resin).

Another common strategy is to use amine-functionalized resin and EDC crosslinker to immobilize peptides via their carboxyl (C-terminal) amino acids. Because peptides have both amine and carboxyl termini (as well as possibly internal lysines, aspartate or glutamate residues), the carboxyl-to-amine crosslinking caused by EDC boths polymerize and immobilizes the peptide so that it is presented to the antibody in a variety of orientations for affinity binding. This is the basis of the CarboxyLink Immobilization Kit.

Protein antigens and affinity ligands

Except in special situations, protein antigens are usually most easily immobilized for affinity purification by targeting primary amines, which typically occur in several locations at the outer surface of protein structures (i.e., wherever there are lysine groups or an N-terminus of a subunit). Several high-capacity, amine-reactive affinity supports are available for this sort of immobilization.

Alternatively, if the protein antigen is a purified glycoprotein and the carbohydrate groups are not the epitopes of interest, then the antigen can be covalently immobilized through the sugar groups after they have been oxidized with sodium periodate. This is the basis of the GlycoLink Immobilization Kit.

Binding and elution conditions

Little variation exists among typical binding and elution conditions for antigen-specific affinity purification of antibodies because they are based on native affinity interactions of antibodies with their respective antigens. That is, because antibodies are designed to recognize and bind antigens tightly under physiologic conditions, most affinity purification procedures use binding conditions that mimic physiologic pH and ionic strength. The most common binding buffers are phosphate-buffered saline (PBS) and Tris-buffered saline (TBS) at pH 7.2. Once the antibody has been bound to an immobilized antigen, additional binding buffer is used to wash unbound material from the support. To minimize nonspecific binding, many researchers use wash buffer containing additional salt or detergent to disrupt any weak interactions.

Specific, purified antibodies can be eluted from an affinity resin by sufficiently altering the pH or ionic strength of the buffer to disrupt the antigen-binding interaction. Most antibodies are moderately resilient proteins that tolerate a range of pH from 2.5 to 11.5 without permanent inactivation, and low-pH is by far the most common elution strategy. In some cases an antibody-antigen interaction is not efficiently disrupted by pH changes or is damaged by the pH, requiring that an alternate strategy be used.

References

1. Grodzki, A.C. and Berenstein, E. (2010). Antibody purification: ammonium sulfate fractionation or gel filtration. In: C. Oliver and M.C. Jamur (eds.), Immunocytochemical Methods and Protocols, Methods in Molecular Biology, Vol. 588:15-26. Humana Press.

2. Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.Gagnon, P. (1996).

3. Gagnon, P.S. (1996). Purification Tools for Monoclonal Antibodies. Validated Biosystems. Tuscon, AZ.

4. Nethery, A., et al. (1990). J. Immunol. Method 126, 57-60.

5. Nevens, J.R., et al. (1992). J. Chromatogr. 597, 247-256.

6. Roque-Barreira, M.C. and Campos-Neto, A. (1985). J. Immunol. Method 134(30), 1740-1743.

7. Aucouturier, P., et al. (1987). Mol. Immunol. 24(5), 503-511.

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The production and use of specific antibodies as detection probes and purification ligands-often called immunodetection or immunotechnology-has revolutionized bioresearch and diagnostic technologies. Animals immunized with prepared antigens will produce specific antibodies against the antigen.

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MabSelect PrismA

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GE Healthcare introduces a new Protein A chromatography resin, MabSelect PrismA, which will help biopharmaceutical manufacturers improve their monoclonal antibody (mAb) purification capacity by up to 40%. The resin is also significantly more alkaline-stable, meaning that MabSelect PrismA can be cleaned with a higher concentration of sodium hydroxide to better control cross-contamination and bioburden risks.

MabSelect PrismA addresses a number of key challenges, including the increased upstream titers. The new resin is highly efficient due to its excellent binding capacity, which is critical, because it determines how much resin is needed to purify a certain amount of protein.

"Biopharma companies are constantly looking for more ways to increase their output, and Protein A has a critical role to support these efforts. The thorough research and development work that we have carried out to create MabSelect PrismA sets a new standard for mAb purification. To put it simply, with this chromatography resin our customers will be able to produce more biopharmaceuticals out of their existing manufacturing equipment. Protein purification is no longer a bottleneck, but driving higher productivity and shorter manufacturing lead times for our customers," said Jan Makela, General Manager, BioProcess, GE Healthcare Life Sciences.

MabSelect PrismA has been developed at the GE Healthcare Life Sciences site in Uppsala, Sweden, where the resin is also manufactured. The factory in Uppsala is one of the largest manufacturing facilities for chromatography resins in the world, and between 2017 and 2022 GE is annually investing up to USD 70 million in the production facility to significantly increase its capacity.

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New Protein A Chromatography Resin Increases Monoclonal Antibody Purification by up to 40%

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GE Healthcare introduces a new Protein A chromatography resin, MabSelect PrismA, which will help biopharmaceutical manufacturers improve their monoclonal antibody (mAb) purification capacity by up to 40%. The resin is also significantly more alkaline-stable, meaning that MabSelect PrismA can be cleaned with a higher concentration of sodium hydroxide to better control cross-contamination and bioburden risks.

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https://www.creative-biostructure.com/custom-affinity-chromatography-service-257.htm

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Proceedings of the National Academy of Sciences

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Significance

Protein purification is a primary step and the basis for numerous biochemical and biomedical studies. It is particularly crucial for high-resolution structural analysis and industrial protein production, where it has to meet the high-yield, high-purity, and high-activity (HHH) requirement. However, the HHH purification of many proteins or protein complexes remains a difficult, target-dependent, and multistep process. The ultra-high-affinity (CL7/Im7) purification system described in this work allows for one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and DNA/RNA-binding proteins and complexes. It might emerge as an efficient, universal tool for high-throughput isolation of many significant biological systems to advance modern biological studies as well as manufacturing of therapeutic proteins.

Abstract

Protein purification is an essential primary step in numerous biological studies. It is particularly significant for the rapidly emerging high-throughput fields, such as proteomics, interactomics, and drug discovery. Moreover, purifications for structural and industrial applications should meet the requirement of high yield, high purity, and high activity (HHH). It is, therefore, highly desirable to have an efficient purification system with a potential to meet the HHH benchmark in a single step. Here, we report a chromatographic technology based on the ultra-high-affinity ( K d ∼ 10 −14-10 −17 M) complex between the Colicin E7 DNase (CE7) and its inhibitor, Immunity protein 7 (Im7). For this application, we mutated CE7 to create a CL7 tag, which retained the full binding affinity to Im7 but was inactivated as a DNase. To achieve high capacity, we developed a protocol for a large-scale production and highly specific immobilization of Im7 to a solid support. We demonstrated its utility with one-step HHH purification of a wide range of traditionally challenging biological molecules, including eukaryotic, membrane, toxic, and multisubunit DNA/RNA-binding proteins. The system is simple, reusable, and also applicable to pulldown and kinetic activity/binding assays.

Footnotes

Author contributions: H. Wang and D.G.V. designed research; M.N.V., S.K., A.D.V., H. Wesson, Z.Z., M.B.R., H. Wang, N.P.H., and D.G.V. performed research; D.G.V. contributed new reagents/analytic tools; M.N.V., M.B.R., H. Wang, N.P.H., L.T.C., and D.G.V. analyzed data; and L.T.C. and D.G.V. wrote the paper.

Reviewers: R.L., University of Wisconsin-Madison; and T.H.T., Nebraska Medical Center.

Conflict of interest statement: The CL7/Im7 purification technology is protected by a PCT patent (PCT/US2016/065843) filed by the UAB Research Foundation.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1704872114/-/DCSupplemental.

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Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins

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Protein purification is a primary step and the basis for numerous biochemical and biomedical studies. It is particularly crucial for high-resolution structural analysis and industrial protein production, where it has to meet the high-yield, high-purity, and high-activity (HHH) requirement. However, the HHH purification of many proteins or protein complexes remains a difficult, target-dependent, and multistep process.

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Features

Presents the latest research in the theory and practical use of affinity chromatography

Details common affinity methods such as bioaffinity, immunoaffinity, DNA, boronate, dye-ligand, biomimetic, and metal-ion affinity chromatography

Discusses techniques used in clinical, environmental, and pharmaceutical analyses

Illustrates the use of affinity chromatography for applications in biotechnology and molecular biology

Explains quantitative approaches for the analysis of biological interactions

Contains nearly 500 figures and tables while providing useful examples of affinity methods and their applications

Offers over 3000 references to original papers available on topics of interest

Summary

This essential handbook guides investigators in the theory, applications, and practical use of affinity chromatography in a variety of fields including biotechnology, biochemistry, molecular biology, analytical chemistry, proteomics, pharmaceutical science, environmental analysis, and clinical chemistry.

The Handbook of Affinity Chromatography reflects upon important factors to consider in the development of affinity methods, such as the choice of support material, immobilization methods, and application or elution conditions. It reviews common affinity methods and explores the latest preparative, analytical, and biophysical applications, including the use of affinity chromatography with other separation techniques and analytical systems. This basis seamlessly supports the discussion of recent developments in techniques including the use of affinity ligands in capillary electrophoresis, mass spectrometry, microanalytical systems, and optical biosensors. New chapters feature expanded discussions on molecularly imprinted polymers and biomimetic ligands, chromatographic immunoassays, affinity-based immunoassays, affinity-based chiral stationary phases, and affinity ligands in multidimensional systems.

Engaging the collaboration of 48 scientists and students from 23 laboratories and organizations to present the latest information on affinity methods, the Handbook of Affinity Chromatography illustrates a wide range of applications and theory for scientists, students, and laboratory workers throughout the fields of chemistry and biology.

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Handbook of Affinity Chromatography, Second Edition

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Presents the latest research in the theory and practical use of affinity chromatography Details common affinity methods such as bioaffinity, immunoaffinity, DNA, boronate, dye-ligand, biomimetic, and metal-ion affinity chromatography Discusses techniques used in clinical, environmental, and pharmaceutical analyses Illustrates the use of affinity chromatography for applications in biotechnology and molecular

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Chapter 2 - Monoclonal Antibodies pg 40-70

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Chapter 2 - Monoclonal Antibodies pg 40-70

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Practical Immunology, Fourth Edition - Hay - Wiley Online Library

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Practical Immunology is a basic text aimed at immunology students and researchers at all levels who need a comprehensive overview of the methodology of immunology. The rapid and startling innovations in immunology over the past two decades have their root in sound experimental practice and it has always been the aim of this book to educate researchers in the design and performance of complex techniques.