Affinity Chromatography for Biopharmaceutical Analysis: A Mini-Review of History, Principles, and Potential Applications

Biopharmaceuticals present a new set of challenges for bioanalytical chemistry and separation methods. Affinity chromatography is one approach that offers many attractive features for the selective analysis or isolation of these agents based on their structures or biological activities. Affinity chromatography uses an immobilized, biologically related binding agent as a stationary phase to capture or measure a set of specific target solutes. This review discusses the history and principles of affinity chromatography as a tool for bioanalysis, with an emphasis on its use with biopharmaceuticals and related compounds. Examples of methods that are considered include those using antibodies, antigens, and immunoglobulin-binding proteins as stationary phases, as well as other biological or nonbiological binding agents. Techniques ranging from direct or indirect biopharmaceutical analysis to sample pretreatment and the characterization of biopharmaceuticals are also discussed.

Biopharmaceuticals, or biologics, are therapeutic agents that are derived from biological systems or produced through biotechnology and protein engineering.^1,2 This class of agents consists of a range of products, including recombinant proteins and peptides, monoclonal antibodies (mAbs), vaccines, nucleic acid-based products, and cell-based therapies.)^1–3 Applications of biopharmaceuticals now include their use as replacement therapies (e.g., insulin and growth hormone), targeted therapeutics (e.g., mAbs and antibody-drug conjugates), vaccines, and diagnostic agents (e.g., antibody-based imaging agents).^4–7 These therapeutic agents have also been used to treat cancer, autoimmune and inflammatory disorders, cardiovascular disease, diabetes, hematologic and hormonal disorders, infectious diseases, and genetic diseases.^2,7–10

The analysis and characterization of biopharmaceuticals are currently areas of great interest. One tool that can be used for this purpose is affinity chromatography.^11,12 Affinity chromatography is a type of liquid chromatography in which specific analytes are retained by a complementary, biologically related binding agent that serves as the stationary phase.^12–18 This method typically involves strong but reversible interactions between the desired target and binding agent, which is also known as the affinity ligand.^11–17 The target is first bound to the affinity ligand under conditions that produce strong binding (i.e., in the presence of an application buffer). The target is later eluted by changing the mobile phase or column conditions (e.g., an alteration in pH or solvent polarity, as provided by an elution buffer) to weaken this binding or by adding a competing agent to displace the target from the affinity ligand (see Figure 1).^11,12 For targets with weak-to-moderate binding strengths with the affinity ligand, isocratic elution may also be used.^13,14 The result is a separation method that can provide both strong and selective capture and analysis of the target, even when this target may be present in a complex biological matrix.^11–14

The first known use of an affinity-based separation was by Emil Starkenstein in 1910, when he isolated the enzyme ⍺-amylase with a column containing insoluble starch as the support and stationary phase.^11,12,17,19 A few additional reports using adsorbed or “fixed partners” for the isolation of specific biological agents appeared over the next few decades, including work in the early 1950s that used antigens attached to cellulose for antibody purification.^12,17,18,20 However, the current form of affinity chromatography did not appear until the 1960s when new supports such as agarose and improved immobilization techniques became available for attaching biomolecules to these materials.^12,17,21 The term “affinity chromatography” originally appeared in a paper in 1968 that described the combination of these developments for the purification of selected enzymes using columns containing immobilized inhibitors.^12,17,22 This method was originally carried out with soft gels such as agarose but was combined with more efficient and rigid supports like silica by the late 1970s and early 1980s to create a method now known as high-performance affinity chromatography.^12,17,23,24 Over the last 50+ years, affinity chromatography has expanded to include a vast array of binding agents and applications,¹¹ including uses in green analytical chemistry and work with many biochemicals and biomaterials that are used in biopharmaceuticals.^11–14,25

This mini-review will discuss various types of affinity chromatography that have been used to analyze biopharmaceuticals or could be employed in this work in the future. This will include the categories of affinity chromatography that are listed in Table 1. The principles and history for each of these methods will be briefly considered, and some examples of their applications will be presented. The types of affinity chromatography that will be examined will include immunoaffinity chromatography, including the use of this approach for sample preparation, immunodepletion, and chromatographic immunoassays. The use of immunoglobulin-binding proteins, such as protein A and protein G, will then be discussed. Other forms of affinity chromatography that will be considered include those that use other biologically based binding agents, such as lectins and the avidin/streptavidin system, or additional types of binding agents, spanning boronates and metal ion chelates to biomimetic or synthetic ligands. Examples of applications that range from direct or indirect biopharmaceutical analysis to sample pretreatment and the characterization of biopharmaceuticals will also be discussed.

Immunoaffinity Chromatography

Immunoaffinity chromatography (IAC) is the most common type of affinity chromatography, based on a biological binding agent.^11,26–29 In this method, the stationary phase is an antibody or antibody-related agent, such as an antibody fragment or an antigen.^26–29 These interactions are highly selective and reversible, making IAC an important approach for modern biochemical analysis and biomedical research.^26–29 Modern IAC can be traced back to work by Campbell et al. in 1951, who used serum albumin immobilized on p-aminobenzylcellulose for antibody purification.^20,26 Since that time, IAC methods have been reported for a range of targets, spanning small molecules to peptides, proteins, antibodies, enzymes and receptors up to subcellular components and viruses.^26–38

Due to the high specificity of IAC, it has been widely used directly for the purification, separation, and analysis of various pharmaceuticals and biologics, including when they are present in complex samples.^{26–29,31,34–38} As an example, IAC has been used with short monolithic columns for the capture and purification of blood group antigens.³⁷ Erythropoietin has also been purified and concentrated from urine by using IAC.³⁸ IAC has also been used with streptavidin microcolumns and adsorbed biotinylated antigens for the activity-based analysis of therapeutic mAbs.³⁴ A similar format employing streptavidin microcolumns has recently been used with biotinylated antibodies to measure a high-to-moderate level antigen in production samples and the final formulation of a commercial vaccine.³⁵
Immunoextraction is another common application of IAC.^{11,26–29,39–42} In this method, an immobilized antibody or antibody-related agent is used to capture target analytes from complex samples prior to their further separation or analysis by other methods.^26–29 Immunoextraction can be combined either offline or online with a second method. In the offline mode, immobilized antibodies on a support (e.g., in a disposable syringe, in a solid-phase extraction cartridge, or on magnetic beads) are used to capture the desired targets from samples and to later elute these retained targets for use in the second method. ^{26–29,41,42} This approach can also be used for target enrichment and concentration if the capacity of the support is not exceeded.^27,39,40 The captured target can then be examined by various analytical methods. For example, this approach has been employed for the selective extraction of erythropoietin glycoforms before their analysis by capillary electrophoresis⁴¹ and the isolation plus enrichment of protein toxins prior to their analysis by high-resolution targeted mass spectrometry.⁴²

In online immunoextraction, the IAC support and column are directly integrated with a second separation or analysis method. ^26–29 Online immunoextraction has been applied to protein and peptide analysis in biological fluids, including human plasma proteins,⁴³ a type II collagen peptide biomarker,⁴⁴ and quantitative protein bioanalysis using online IAC coupled with protein digestion and liquid chromatography mass spectrometry (LC–MS).⁴⁵ Normal or modified preparations of human serum albumin (HSA) and samples of α₁-acid glycoprotein from serum have been captured by immunoextraction in microcolumns for use of the proteins in drug-binding studies.^46,47 An online immunoextraction/entrapment system has also been developed to isolate HSA from serum and entrap the isolated protein in affinity microcolumns for drug-binding studies.⁴⁸

IAC can further be combined with direct or indirect target detection to create chromatographic immunoassays.^{27–29,35,49–54} These techniques have been used in many formats, including those involving competitive binding assays, sandwich assays, and immunometric assays (see example in Figure 2).^{27–29,49–51} These methods can also be used alone or in combination with other methods, such as postcolumn immunodetection or two-dimensional (2D) chromatographic methods.^29,49,51 Chromatographic immunoassays have been reported for many biologically relevant molecules, such as serum proteins, protein biomarkers, immunoglobulins, drugs, and hormones.^{27–29,49,51} Specific examples include applications reported for HSA, granulocyte colony-stimulating factor, and α₁-acid glycoprotein (AGP).^29,49–53 This approach has been further used in recent work to analyze vaccine components.^35,54 These latter examples include the measurement of polysaccharides by IAC in multivalent pneumococcal conjugate vaccines based on the serotype specificity⁵⁴ and the use of IAC with both direct detection and a sandwich assay with fluorescent detection for the measurement of low-concentration antigens in vaccines.³⁵

Immunodepletion is similar to immunoextraction except that the goal for the former is to selectively remove an abundant or interfering component from a sample before analysis of some remaining component.^27,36,55 This method has been widely used in proteomics and biomarker discovery to eliminate highly abundant proteins (e.g., albumin and immunoglobulins) prior to the analysis of less abundant proteins, such as by 2D electrophoresis (2D E), reversed-phase LC (RPLC) or mass spectrometry (MS).^{27,36,55–61} Immunodepletion can also be adapted for biopharmaceutical analysis by selectively removing a therapeutic protein, drug-bound target, or possible interfering species.^62,63 This does, however, require that none of the desired targets be adsorbed along with the depleted species (e.g., through nonspecific or cross-reactive binding to the antibodies).

Affinity Methods Using Immunoglobulin-Binding Proteins and Related Agents

Immunoglobulin-binding proteins make up another set of binding agents that have been used for decades in affinity chromatography¹¹ and which have been recently applied to the purification and analysis of antibody-based biopharmaceuticals.^{11,26,64–69} Two common binding agents in this group are protein A and protein G.^64–69 Protein A is a cell wall protein found in Staphylococcus aureus, which binds to the fragment crystallizable (Fc) region of immunoglobulins from many species; this ability makes protein A a valuable ligand for antibody purification and analysis, and especially for IgG-class antibodies. ^{11,64–66,69–77} Protein G is from group C and G Streptococcus species and also binds strongly to the Fc region of many types of immunoglobulins.^{64,69,78–80} Protein A and protein G do differ in their binding to some types of subclasses and species of immunoglobulins, giving them complementary properties in some cases.^64,69 A fusion protein derived from these two agents, known as protein A/G, is also available for such applications.⁶⁹ 

Applications using protein A and protein G in chromatography and the analysis of antibodies and immunoglobulins in HPLC systems first appeared in the 1980s and 1990s.^64,69 Early work involved the use of these binding agents for the direct capture and measurement of immunoglobulins in both single- and multi-column systems.^{64,69,81–84} Recent work has combined columns that use these agents with methods such as mass spectrometry to capture, measure, and/or characterize therapeutic mAbs.^85-87 These binding agents have also been used with small-scale chromatographic systems as part of chromatographic immunoassays, such as with fluorescent tags or enzyme-amplified detection.^50,82,84 Part of this latter work also used these types of binding agents for the biospecific adsorption of antibodies within microscale affinity systems, as used in Figure 2.^50,88,89 These binding agents can also be used for the affinity depletion of selected sample components, as has been done by using protein A to remove antibody-based therapeutics from drug products prior to identification and analysis of the remaining components by reversed-phase liquid chromatography tandem mass spectrometry (LC–MS/MS).⁹⁰

Other immunoglobulin-binding proteins have also been used in affinity chromatography.^64,69 One example is protein L, which is produced by Peptostreptococcus magnus and can bind to light chains on human immunoglobulins.^64,69 Protein L has been used with protein A for the affinity depletion of therapeutic mAbs and removal of samples of residual host cell protein to allow analysis of the remaining contents of this preparation to be examined by LC–MS/MS.⁹¹ Other related binding agents have included recombinant or engineered affinity ligands that have been developed to specifically recognize other regions of antibodies, such as Fc, CH1, and light chain domains.^92–96 For example, a ligand that can bind to the CH₁ domain on the heavy chains and fragment antigen-binding (Fab) domains of human IgG has been reported, as derived from a 13-kDa llama heavy-chain antibody fragment.⁹²

Other ligands in this group are FcXL, KappaXL, and FcyRIIA. FcXL is a recombinant single-domain antibody designed to bind the Fc region, and particularly the CH₃ domain, of human IgG.^96,97 FcXL has been used in affinity columns for capture of antibody-drug conjugates and other antibody-derived agents for analysis based on native mass spectrometry.⁹⁴ KappaXL is an affinity ligand that binds to antibodies containing human kappa light chains, including antibody fragments.^98–102 KappaXL has been used for the analysis and purification of antibody light chains. ^98–101 Fcy-IIA is an immunoglobulin Fc receptor which has also been used as a binding agent for therapeutic antibodies.^103,104 This receptor has been used with affinity chromatography and mass spectrometry to separate and identify IgG1 proteoforms based on their changes in binding to this receptor due to glycosylation or stress-induced deamidation.^103,105

Affinity Methods using Other Biological Binding Agents

Other biological agents and ligands for affinity chromatography have also been employed for targets or applications related to biotherapeutics. Lectins are examples of such binding agents.^{11,64,106–109} Lectins are non-immune-system proteins that can bind to carbohydrate residues.^11,64 An example is concanavalin A (Con A), which can bind to glycans with high-mannose content or mannose branching.^11,64 The first uses of Con A and lectins in affinity chromatography occurred in the early 1970s when this approach was utilized to isolate glycoproteins and polysaccharides.¹¹⁰ Lectin affinity chromatography has since been used in glycomics and proteomics to examine cell membrane proteins, posttranslational modifications of glycoproteins, and glycoproteins as disease biomarkers.^108,109 For instance, lectin affinity chromatography has recently been used to identify novel biomarkers for prostate and breast cancer,^111,112 which may lead to new biopharmaceutical targets.

The avidin/biotin system is another biological interaction used in affinity chromatography.^11,64,117 Avidin is a glycoprotein in egg whites, and streptavidin is from Streptomyces avidinii.^64,113,114 Both of these agents bind strongly to biotin (i.e., vitamin H or B₇), with association constants of ~10¹³-10¹⁵ M^-1.^64,115 Immobilized avidin has been used since the mid-1970s to adsorb biotinylated compounds to supports.^64,115,116 Streptavidin has also been used for this purpose,^11,64,117 such as in recent work that employed small high-performance liquid chromatography (HPLC) affinity columns with immobilized streptavidin to adsorb biotinylated antigen for the capture and measurement of a biotherapeutic mAb (Figure 3).³⁴ The same approach has been used with streptavidin microcolumns to adsorb biotinylated mAbs against viral antigens, which were then utilized for direct detection or indirect detection of the antigen in vaccine-related samples and formulations through sandwich assays.³⁵

Affinity Methods Using Nonbiological Binding Agents

Many nonbiological agents have been used in affinity chromatography.¹¹ Important agents in this group are boronic acid and boronates, which were first used around 1970 to retain and study nucleosides and sugars.^118,119 This group of ligands can bind through the formation of a reversible covalent bond with cis-diol groups, as may occur in carbohydrates, glycoproteins, glycopeptides, nucleosides, catecholamines, and glycated proteins.^118,120,121 This ability has made boronate affinity chromatography an important tool in proteomics, glycomics, clinical testing, and biopharmaceutical characterization.^{120,122–148} A few examples have included the use of boronate affinity chromatography to analyze therapeutic mAbs such as adalimumab or rhuMab,^{123 –127} along with the measurement of various subclasses of IgG (e.g., IgG₁, IgG₂, and IgG₄),^128–134 and glycohemoglobin.¹³⁵ Examples of lower mass compounds that have been examined with boronate affinity chromatography are dopamine, levodopa, epinephrine, and norepinephrine.^149,150

Immobilized metal-ion affinity chromatography (IMAC) is another affinity-based separation method that makes use of a nonbiological binding agent.¹¹ In IMAC, retention is based on the interaction between immobilized metal ions and electron donor groups on the target analyte.^151–153 The binding agent is often based on a transition-metal ion (e.g., Ni2+, Zn2+, Cu2+, or Co2+) that is complexed with an immobilized chelating agent such as iminodiacetic acid, nitrilotriacetic acid, or carboxymethylated aspartic acid.^151–156 One common application of IMAC is the use of columns containing Ni²⁺ chelates for the purification of recombinant proteins that contain a histidine tag (i.e., His-tagged proteins).^{151,152,157,158} In addition, IMAC has been used to characterize and employ posttranslational modifications such as glycosylation and phosphorylation. For example, IMAC columns containing Ti⁴⁺ have been used to selectively enrich mannose-6-phosphate (M6P) glycopeptides in large-scale studies of M6P glycosylation.¹⁵⁹ IMAC has also been used to enrich and fractionate phosphorylated proteins by using their binding to trivalent and tetravalent cations, such as Fe³⁺, Ga³⁺, and Zr⁴⁺, prior to analysis of these proteins by mass spectrometry.¹⁵¹

Affinity Methods Using Biomimetic Binding Agents

Biomimetic ligands are another class of binding agents used in affinity chromatography for biotherapeutic applications. Dye-ligand chromatography is a good example of such an area. In this method, immobilized dyes are selected that can retain a biomolecule, such as a protein or enzyme, by mimicking the structure of a substrate or cofactor for these targets.^11,160–162 The first use of dye-ligand affinity chromatography was described in 1974; this method has since been used to purify hundreds of different targets.^11,163 The dye-ligand that is most often used for this purpose is Cibacron Blue F3GA or a related derivative, with other examples including Procion Blue MX-3G/MX-R and Procion Red HE-3B.^164–166 For example, Cibacron Blue F3GA has been used to isolate IgM mAbs from murine ascitic fluid,¹⁶⁶ and human IgG subclasses have been bound from aqueous solutions and human plasma using cryogels containing the dyes Reactive Green HE 4B or Reactive Red 120.¹⁶⁷ In another report, toluidine blue was used as a dye-ligand to extract a bioactive sulfated polysaccharide fucoidan from the marine brown macroalga Fucus vesiculosus.¹⁶⁸

Aptamers are another group of biomimetic ligands used in affinity chromatography.^{13–15,21,169,170} These ligands are typically created in a process known as the systematic evolution of ligands by exponential enrichment (SELEX), in which an oligonucleotide library is used to make single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) that binds to a given target.^{14, 169–172} Advantages of aptamers can include their relatively low cost, biospecificity, low immunogenicity, and high chemical stability,¹⁷¹ as well as their ability to be produced with both good affinity and selectivity for their targets.¹⁶⁹ Other useful features of aptamers as biomimetic ligands include their smaller size and lower complexity than antibodies, as well as their applicability to a variety of analytical and separation platforms.^170–172 Aptamers have been used in affinity chromatography for targets ranging from nucleic acid-binding proteins, antibodies, enzymes, and peptides to small molecules such as dyes, amino acids, and drugs.^170,173 In addition, aptamers have been used in affinity separations for applications such as sensing, target purification or capture, and chiral separations, among others.^172,173

Molecularly imprinted polymers (MIPs) are another set of biomimetic ligands that have been used in affinity separations.^174–178 MIPs are synthesized by the polymerization of functional monomers and crosslinkers using a template molecule. Once the polymerization is complete, the template is removed, leaving the target-specific recognition sites for binding.^174,176,177 MIPs have been used in pharmaceutical and biological research for the extraction, selective separation, and detection of various proteins, pharmaceuticals, and biomarkers.^{177,179–182} For example, MIP-based solid-phase microextraction has been used to extract interferon alpha-2a from plasma samples prior to HPLC analysis.¹⁸⁰ A pH-responsive polyacrylamide MIP has been developed to separate recombinant HSA from a mixture containing erythropoietin and to isolate normal HSA from human plasma.¹⁷⁹

Conclusions

In summary, it has been shown how many forms of affinity chromatography can be applied to the analysis and isolation of biopharmaceuticals and related biomolecules. Two important techniques in this group were immunoaffinity chromatography and methods that employ immunoglobulin-binding proteins, such as protein A, protein G, and related affinity ligands. The possible use of affinity separations based on other biological binding agents, such as lectins and biotin plus avidin or streptavidin, was also considered. Nonbiological or biomimetic affinity ligands that were discussed included boronates, immobilized metal-ion chelates (i.e., IMAC), dye-ligands, aptamers, and MIPs. The selectivity of these methods, as well as the wide range of binding agents available for affinity separations, is expected to lead to further growth in the use of affinity chromatography within the biopharmaceutical field. This, in turn, should lead to other applications of affinity chromatography in the isolation and analysis of new biological agents for disease treatment.

Acknowledgments

This work was supported, in part, by the National Science Foundation under grant CHE 2404209.

Conflicts of Interest

The authors have declared no conflicts of interest.

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