The affinity concept in bioseparation: evolving paradigms and expanding range of applications

The meaning of the word affinity in the context of protein separation has undergone evolutionary changes over the years. The exploitation of molecular recognition phenomenon is no longer limited to affinity chromatography modes. Affinity based separations today include precipitation, membrane based purification and two-phase/three-phase extractions. Apart from the affinity ligands, which have biological relationship (in vivo) with the target protein, a variety of other ligands are now used in the affinity based separations. These include dyes, chelated metal ions, peptides obtained by phage display technology, combinatorial synthesis, ribosome display methods and by systematic evolution of ligands by exponential enrichment (SELEX). Molecular modeling techniques have also facilitated the designing of biomimetic ligands. Fusion proteins obtained by recombinatorial methods have emerged as a powerful approach in bioseparation. Overexpression in E. coli often result in inactive and insoluble inclusion bodies. A number of interesting approaches are used for simultaneous refolding and purification in such cases. Proteomics also needs affinity chromatography to reduce the complexity of the system before analysis by electrophoresis and mass spectrometry are made. At industrial level, validation, biosafety and process hygiene are also important aspects. This overview looks at these evolving paradigms and various strategies which utilize affinity phenomenon for protein separations.     

Hybridization-based affinity partitioning of nucleic acids using PEG-coupled oligonucleotides.

Polyethylene glycol (PEG)-coupled oligonucleotides are partitioned in an aqueous two-phase system PEG/dextran. The affinity of the oligonucleotide for the PEG-rich phase increases proportionally to the length of the coupled PEG polymer. After hybridization, the PEG-coupled oligonucleotide is able to force a complementary nucleic acid strand into the PEG-rich phase. This property can be used for the sequence-specific isolation of nucleic acids through hybridization-based affinity partitioning. The dependence of the partition coefficient in this system on various parameters is described. The application of this principle to multistage chromatographic separations is demonstrated.     

Column chromatographic separation of cells using aqueous polymeric two-phase systems

Cell separation using aqueous polymeric two-phase systems is well established. For separations of cells having similar partition coefficients a multistep countercurrent distribution procedure has to be used. However, its operation is limited by time and apparatus constraints. As an alternative strategy we have developed a chromatographic technique in which the dextran-rich phase of a dextran/polyethylene glycol (PEG) phase system is immobilized onto derivatized agarose beads. The PEG-rich phase is used as the eluent. Inclusion of PEG-fatty acid affinity ligand gradients into the eluent produces separations of mammalian erythrocytes based on the differential interaction between the fatty acid and the erythrocyte membranes. A model separation of dog and human erythrocytes has been carried out.     

The solid phase in affinity chromatography: strategies for antibody attachment.

Antibodies (Ab) are commonly used in affinity chromatography (AC) as a versatile and specific means of isolating target molecules from complex mixtures. A number of procedures have been developed to immobilize antibodies on the solid matrix. Some of these methods couple the antibody via chemical groups that may be important for specific recognition of antigen, resulting in loss of functionality in a proportion of the antibodies. In other methods, the outcome of immobilization is coupling via unique sites in the Fc region of the antibody molecule, ensuring orientation of the antibody combining sites (Fab) towards the mobile phase. This review discusses the advantages and disadvantages of the various methods available for immobilization and outlines protocols for site-directed, covalent coupling of the antibody to the solid phase that essentially retains the activity of the antibody.     

Determination of antibody affinity by using antigenic and antibody immunosorbents

To determine the affinity of the active centers of antibodies, cellulose immunosorbents for antibodies and antigens have been used. The fixation of serum proteins on the sorbent, the interaction of fixed antibodies with a monovalent antigen and the graphic analysis of the results thus obtained allows one to assess not only the concentration of the effective active centers on the sorbent, but also all known characteristics of antibody affinity: the average association constant K0, the common association constant Kt, the geometric association constant Kg, the average association constants which determine the affinity of different antibody groups. The use of antigenic immunosorbent permits one to determine the value of the average internal association constant K0. The determination of antibody affinity in hyperimmune antiplague sera by means of immunosorbents and red blood cells coated with capsular antigen has resulted in obtaining similar values of affinity indices.     

Selection of proteins and peptides from libraries displayed on filamentous bacteriophage

This article attempts to review recent developments in the rapidly developing field of phage display libraries. The current state of peptide, antibody, and cDNA libraries, as well as current and future applications of phage display libraries are discussed. The main focus of the article is on the methods for selecting binding ligands against targets in a variety of different formats. These include solid phase and in-solution selection methods, and the strategies used to select for higher affinity, and binding ligands ampure and cellular target proteins.     

Protein based microarrays: A tool for probing the proteome of cancer cells and tissues

A novel proteomic approach for probing cell and tissue proteome, which combines liquid phase protein separations with microarray technology has been developed. Proteins in cell and tissue lysates or in cellular subfractions are separated using any one of a number of separation modes which may consist of ion exchange liquid chromatography (LC), reverse phase LC, carrier ampholyte based separations, e.g. the use of Rotofor, affinity based separations, or gel based separations. Each first-dimension fraction obtained using one separation mode can be further resolved using one or more of the other separation modes to yield either purified protein in solution or liquid fractions with substantially reduced protein complexity. The advantage of a liquid based separation system is that proteins in hundreds of individual fractions can be arrayed directly and used as targets for a variety of probes. Constituent proteins in reactive fractions are identified by mass spectrometry and may be further resolved to determine the nature of the reactive protein(s). We present in this report initial data based on microarray analysis of individual Rotofor fractions obtained from lung adenocarcinoma cell line A549 lysates which have been probed with antibodies against specific proteins.     

Na+/K+-dependent adenosinetriphosphatase in rabbit kidney. Separation by affinity chromatography.

We attempted separations by affinity chromatography of the Na+/K+-dependent adenosinetriphosphatase, using Sepharose 6B covalently coupled with ouabain, either on microsomal fractions or on preparations purified by discontinuous sucrose density gradient ultracentrifugation. The material, specifically eluted with ouabain, was tested to evaluate the recovery of ATPase activity and ouabain binding capacity. The results have shown the efficiency of this technique for (Na+/K+)-APTase isolation.     

Kinetic screening of antibodies from crude hybridoma samples using Biacore

Experimental and data analysis protocols were developed to screen antibodies from hybridoma culture supernatants using Biacore surface plasmon resonance biosensor platforms. The screening methods involved capturing antibodies from crude supernatants using Fc-specific antibody surfaces and monitoring antigen binding at a single concentration. After normalizing the antigen responses for the amount of antibody present, a simple interaction model was fit to all of the binding responses simultaneously. As a result, the kinetic rate constants (k(a) and k(d)) and affinity (K(D)) could be determined for each antibody interaction under identical conditions. Higher-resolution studies involving multiple concentrations of antigen were performed to validate the reliability of single-concentration measurements. The screening protocols can be used to characterize antigen binding kinetics to approximately 200 antibody supernatants per day using automated Biacore 2000 and 3000 instruments.     

Extension of the selection of protein chromatography and the rate model to affinity chromatography

The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.     

The differentiation of foot and mouth disease virus strains using an indirect sandwich enzyme-linked immunosorbent assay saturation model

Sigmoid saturation curves were fitted to the results of titrations of antiserum to foot and mouth disease virus against homologous and heterologous virus strains. Differentiation of strains was readily evident from the different levels of the homologous and heterologous curves. These differences could be quantified by comparison of the saturation curve parameters K and PRmax. Factors which affect variations in K and PRmax and their biological significance were investigated by varying the first phase antibody and the antigen used in the test. PRmax was found to represent an overall combining potential of the antigen with both sera used in the sandwich test. K, which was theoretically a measure of affinity, also reflected antibody titre. Relationships measured using this model were found to correlate with the reference test system--two-dimensional microneutralization.     

Separation of MBP fusion proteins through affinity membranes

Cellulose microporous membranes have been modified in order to obtain a stationary phase specific for the recovery of a class of fusion proteins containing the maltose binding protein domain, through affinity chromatography separations. The feasibility of a single step separation process for the recovery of large amounts of the desired product has been considered. To that purpose, a preparative scale module has been realized, suitable for flat sheet membranes. The affinity matrix used proved to be highly selective toward the fusion proteins examined. The binding capacity determined is comparable with the nominal binding capacity of commercially available supports. The influence of the relevant working parameters, such as flow rate, on the performances of the recovery process has been studied.     

Novel and simple ELISA-based method for antibody affinity determination

New coordinates for antibody affinity determination by ELISA are suggested. The suggested approach is very simple but at the same time it is more convenient and is deprived of the drawbacks inhered in the earlier suggested methods. The examples of antibody affinity determination by the suggested methods for both simulative and experimental binding curves are considered. It was demonstrated that the suggested methods allow getting more precise values of antibody affinity.     

Operational aspects of antibody affinity constants measured by liquid-phase and solid-phase assays.

The association constant of monoclonal antibodies (Mabs) to tobacco mosaic virus has been determined in solution and solid-phase binding assays. The ELISA equilibrium titration method developed by Friguet et al. (1985) was found to be suitable for large antigens such as viruses. In the case of intact IgG antibody, it gave equilibrium constant (K) values ca 30% lower than those obtained by classical solution-phase assay while in the case of Fab', the same values were obtained in both assays. Solid-phase binding assays gave higher K values than solution-phase assays by a factor which varied with the Mab tested (1.5- to 5.4-fold higher). Furthermore, in solution-phase assay, K values were found to depend on the antibody concentration used in the assay. These results confirm the operational nature of antibody affinity constants and indicate that in order to compare the affinity of different Mabs in a meaningful way, it is necessary to use a single technique under standardized conditions.     

Improvement of antibody affinity by introduction of basic amino acid residues into the framework region

Antibodies are widely used not only as therapeutic agents but also as research tools and diagnostic agents, and extensive efforts have been made to generate antibodies that have higher affinity. It was recently reported that introduction of charged residues into the framework region of an antibody improved its affinity; however, the underlying molecular mechanism has not been elucidated. In this study, we used kinetic and thermodynamic analyses of the antibody–antigen interaction to investigate the molecular mechanism by which an antibody with introduced charged residues recognizes its antigen with higher affinity. The introduction of basic amino acid residues resulted in improvement of the affinity whereas the introduction of acidic residues weakened the interaction. For two mutant antigen-binding fragments (Fabs) with improved affinity (named K5- and R5-mutants), the balance between the association rate constant k_on and the dissociation rate constant k_off was distinct despite each mutant having the same number of charged residues. Moreover, thermodynamic analysis of the interactions in the transition state revealed a difference between the K5- and R5-mutants in terms of enthalpic energy change following formation of the encounter complex with the antigen. These results suggest that the affinity of the K5- and R5-mutants is improved by distinct mechanisms. Although the mutations destabilize the Fab and necessitate further studies, our strategy is expected to become a versatile and simple means to improve the affinity of antibodies to their antigens.     

Traditional ELISA methods for antibody affinity determination fail to reveal the presence of low affinity antibodies in antisera: an alternative approach

Traditionally used methods of antibody affinity determination either by ELISA or by the surface plasmon resonance technique do not allow detection of the presence of low‐affinity antibodies in samples of high‐affinity antibodies. In this paper we demonstrate the possibility to reveal their presence and to determine the affinities of both categories of antibodies as well as the ratio of their concentrations. This is especially important since by using traditional methods for antibody affinity evaluation the admixture of low‐affinity antibodies in a sample diminishes the accuracy in determination of specific antibody affinity. In addition, the presence of an admixture of low‐affinity antibodies may be an important biological characteristic of the system under study; their revelation and the evaluation of their binding parameters may be valuable in many cases for obtaining a more complete characterization of the binding properties of the multiple antibodies generated in an immune response. Copyright © 2009 John Wiley & Sons, Ltd.     

Antigen-antibody interactions in capillary electrophoresis

Immunoreactions in combination with separations by capillary electrophoresis (CE) are increasingly being used to quantitate specific analytes in biological fluids. Both competitive and non-competitive approaches have been used for the purpose and, in selected cases, now compare favorably with conventional quantitative immunoassays with respect to concentration limits of detection. CE is also a useful method to evaluate antigen-antibody binding on-line and offers unique possibilities for binding constant estimates, also for weakly binding antibodies and antibody fragments. In this review we cover recent developments in the use of antigen-antibody interactions in conjunction with CE and conclude that continued development of miniaturization, on-line preconcentration and more sensitive detection schemes will contribute to the further dissemination of CE-based immunoassays building on already established affinity CE approaches.     

Phase Separations in Binary and Ternary Cholesterol-Phospholipid Mixtures

A pair of recent studies has reopened debate on the subject of phase separations in model bilayer mixtures of cholesterol (Chol) and dipalmitoyl-phosphatidylcholine (DPPC). Fluorescence microscopy methods have not been able to detect phase separations in binary DPPC-Chol mixtures that have been inferred from NMR studies. However, micron-scale phase-separated liquid domains are observed by fluorescence in ternary mixtures of DPPC, Chol, and diphytanoyl-phosphatidylcholine (DiPhyPC). Here, a model of condensed complexes of Chol and DPPC is used to account for these results. In particular, it is shown that the orientation of tie-lines in ternary mixtures of DiPhyPC/DPPC/Chol is compatible with phase separation in binary DPPC/Chol mixtures.     

Antibody fragment separations by capillary zone electrophoresis

This study investigated methods of improving the separation and identification of an IgA antibody, McPC603, and its pepsin fragments. The problem presented by purification of antibody fragments (Fabs) and the antibody light chain required accurate and informative analysis of highly hydrophobic proteins, which can polymerize and fold to form secondary structures. Capillary zone electrophoresis (CZE) permits the separation of peptides and small proteins by a method which is orthogonal to the traditional method of reversed-phase HPLC. To facilitate planned studies of the antibody's biological activity, our buffer composition was kept as simple as possible. During CZE analysis, if the buffer pH is below the isoelectric point of the protein, or the protein is large (with a heterogeneous distribution of surface charges), it can irreversibly bind to the capillary wall unless the capillary is coated. We found that C₁-coatings in RP-capillaries at pH 9.5 adequately prevented the antibody fragments from binding to the wall. However, the coating did not remain stable at such high pH, so different conditions were sought. We achieved adequate separations in several buffers at nearly physiological pH, in a bare silica capillary which had been coated once with a soluble cationic polymer coating (Micro-Coat applied during column conditioning). Antibody electropherograms changed depending on the type of inorganic buffer salt used in a separation. Phosphate binds to the antigen-binding site of the IgA with low affinity, and interesting effects were observed in separations using phosphate buffer. These effects will be discussed.