Alternative Affinity Ligands for Immunoglobulins - American Chemical

Review pubs.acs.org/bc

Alternative Affinity Ligands for Immunoglobulins Nika Kruljec*,† and Tomaž Bratkovič*,† †

Faculty of Pharmacy, University of Ljubljana, Aškerčeva 7, SI-1000 Ljubljana, Slovenia

ABSTRACT: The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond. content undergoes clarification via centrifugation and filtration through a series of depth filters.7,8 The clarified medium is then subjected to a series of chromatography steps starting with antibody capture, which usually represents protein A based affinity chromatography or cation exchange chromatography.9 The main objective of the subsequent polishing step is the final removal of remaining specific impurities such as host cell proteins and DNA, product-related impurities (e.g., aggregates and deamidated and oxidized product variants), viruses, and leached affinity chromatography ligands.10 Polishing typically encompasses several chromatographic steps, such as hydrophobicinteraction chromatography and size-exclusion chromatography. Finally, the drug product is filter-sterilized, concentrated along with buffer exchange using diafiltration, and the formulation is finalized by adding specific excipients.11 The final formulations of mAbs are sold as sterile solutions for parenteral administration or powders, which require lyophilization. Typical yields of monoclonal antibody purification processes are in the range of 60−80%, depending on the complexity of the specific downstream processing strategy.4 In addition to mAbs, Fc-fusion proteins represent another important group of immunoglobulin-derived products with great therapeutic potential. As the name implies, they represent a new

1. INTRODUCTION 1.1. Therapeutic Antibodies and Fc-Fusion Proteins. In recent decades, monoclonal antibodies (mAbs) and Fc-fusion proteins have emerged as dominant biopharmaceuticals for the treatment of various diseases. Representatives of both groups range from orphan drugs to those treating a large population of patients.1 At the current approval rate of approximately 4 new monoclonal antibodies per year, it is estimated that around 70 mAb-based products will be launched to the market by 2020, with combined worldwide sales of up to 125 billion dollars.2 The main reasons for the widespread use of mAbs are their high specificity and effectiveness, ability to recognize and neutralize specific pathogenic or mutated proteins and cells, and acceptable patient tolerability following administration. All currently approved therapeutic monoclonal antibodies and the majority of those in clinical trials are manufactured using mammalian cell cultures such as CHO, NS0, and SP2/0.3,4 Upon expression, mAbs are typically secreted into the cell culture medium.4 Antibody therapies generally rely on relatively large doses (e.g., a few milligrams of mAb per kg of body weight), necessitating high production rates.5 Increase in the number of therapeutic monoclonal antibodies and immunoglobulin-derived products in clinical practice has driven improvements in the upstream processing that, in turn, led to requirements for optimized downstream processing.6 Antibody purification processes generally follow the scheme in Figure 1. In the initial step, cells and cell debris are removed, whereby bioreactor © XXXX American Chemical Society

Received: June 14, 2017 Revised: July 10, 2017 Published: July 11, 2017 A

DOI: 10.1021/acs.bioconjchem.7b00335 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Review

Bioconjugate Chemistry

Figure 1. Schematic presentation of generic continuous downstream processing of monoclonal antibodies.

generation of engineered proteins in which the Fc region (consisting of the CH2 and CH3 domains and the hinge region) of an antibody is attached to a specific fusion partner such as receptor ectodomain, cytokine, peptide, or coagulation factor.12,13 By the fusing of therapeutic proteins to the Fc region, an extended half-life is achieved. This is attributed to slower sizedependent renal clearance and active recycling due to the interaction with the salvage neonatal Fc receptor (FcRn). Thus, prolonged therapeutic activity is achieved that consequently allows for less-frequent application of the drug.14,15 Furthermore, pharmacokinetic properties can be predicted with greater accuracy relative to small molecules or other biologics.16 From the technological perspective, the Fc region folds independently and may increase stability and solubility of the partner molecule, augmenting expression yield. Last but not least, Fc fusion enables the use of a well-established purification platform that includes the highly specific protein A affinity chromatography.17 1.2. Affinity Purification of Antibodies. Antibody affinity separation is a biochemical separation technique that relies on reversible noncovalent interactions between soluble antibodies and their ligands that are typically immobilized on a solid support packed in a chromatographic column. After the capture step, the unbound species and impurities from complex mixtures such as cell culture media are washed off, and antibodies are eluted either in a nonspecific manner (e.g., by pH shock or increasing ionic strength) or by using competitive ligands as specific eluents.18,19 Owing to high specificity, affinity chromatography is widely employed in initial steps of therapeutic antibody purification process. However, the use of natural immunoglobulin-binding ligands (such as bacterial proteins A (SpA) and G (SpG)) suffers from high production cost and poor column stability. In addition, antibody product release requires relatively harsh elution conditions with negative impact on activity and immunogenicity profile.20 Therefore, there is increasing interest in development of alternative antibody ligands to improve affinity chromatography methods.

rational design or identification by screening combinatorial libraries. A number of immunoglobulin ligands were evolved from natural immunoglobulin-binding proteins with the goal of increasing thermal stability and achieving higher expression yields or milder elution conditions. Moreover, several small synthetic ligands were designed based on molecular structural data of antibody-natural Ig-binding protein complexes. Finally, affinity ligands with unique structures were identified from chemical libraries and engineered using different molecular display methodologies. In the following sections, we briefly discuss diverse immunoglobulin affinity ligands and recapitulate their development and binding characteristics as well as review some of their technological applications focusing on affinity chromatography. 2.1. Natural Immunoglobulin-Binding Proteins. Several important pathogenic microorganisms are known to express immunoglobulin-binding proteins. These bacteria thus show selectivity for a range of Ig types and bind them in a manner not involving the hypervariable region, hence the name “nonimmune binding”. The strains and the Ig-binding proteins they produce are summarized in Table 1 and discussed in more detail below. Table 1. Bacterial Immunoglobulin-Binding Proteins protein

2. AFFINITY LIGANDS In the past 20 years, staphylococcal protein A and streptococcal protein G have been adopted as the capture step of choice by two-thirds of all commercial antibody manufacturers despite initial misgivings about its economic value and reusability and concern over the clearance of leached ligands. Considering the drawbacks, immunoglobulin affinity ligands with improved properties have been pursued with significant effort spent on

size (kDa)

source

binding site

protein A

54

Staphylococcus aureus

protein G

58−65

Streptococcal groups C and G

protein L

76−106

protein M protein MID protein H

40−65 200

Peptostreptococcus magnus Mycoplasma spp. Branhamella catarrhalis Group A streptococci

42.5

Fc region (CH2−CH3 interface) Fab region (VH domain)21 Fc region (CH2 and CH3 interface) Fab region (CH1)22 Fab (VL domain)23 Fab (VL domain)24 CH1 region of IgD25,26 Fc region27

A method for antibody purification based on bacterial immunoglobulin-binding proteins was first published in the 1970s.28,29 Immunoglobulin-binding proteins represent a powerful tool for detection and purification of antibodies ever since. In their native form, they are tethered to cellular surface and have extracellular regions composed of several repeating domains, which capture IgGs to help the bacteria evade the host’s immune response. The best known examples are protein A from B


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Figure 2. Molecular structures of antibody-binding domains of four major bacterial antibody-binding proteins and their binding modes. (A) Ig-binding domain of protein A (yellow) bound to the antibody Fc region (primary site) near the CH2−CH3 domain interface (PDB code 1FC2). (B) Ig-binding domain of protein L (blue) bound to VL of a Fab fragment (PDB code 1HEZ). (C) Ig-binding domain of protein G (pink) bound to antibody Fc region (primary site) near the CH2−CH3 domain interface (PDB code 1FCC). Molecular graphics were generated using PyMOL software.

elements.34 Whereas the binding between SpA and Fc is mediated predominantly by hydrophobic interactions,31 the binding of Fc to SpG primarily relies on polar interactions with multiple hydrogen bonds and salt bridges.34 Even though both proteins compete for the same binding site on Fc (i.e., the hinge region that links the second and third constant domains of the heavy chain), SpA and SpG share no sequence or structural homology.35 Nevertheless, several residues of Fc are involved in both complexes, explaining the competitive binding of these two proteins. SpG is also capable of binding antibody Fab region (specifically, the constant domain).22,36 Fab binding of SpG occurs through interactions with CH1 domain of IgG1,3,4, but it does not bind to Fab of IgG2.37 However, the binding strength of SpG is too low to support an efficient and robust purification process for human Fabs.38 One of the major drawbacks of native SpG is that it also interacts with albumin, α2 macroglobulin, and kininogen, which leads to reduced antibody purification efficiency from complex biological sources (e.g., cell-culture supernatants or blood plasma).39 However, recombinant versions with unwanted binding sites removed are available.40 Other limitations include low stability during relatively harsh cleaning in place (CIP) conditions and, in general, lower binding capacities compared to SpA. Therefore, there are only a few examples of the SpG usage for antibody isolation and purification on commercial scale. SpG affinity chromatography, however, is often the primary choice for antibody purification from serum or purification of human IgG3.41 2.1.3. Peptostreptococcal Protein L. Peptostreptococcal protein L (PpL) is a surface immunoglobulin-binding protein from Peptostreptococcus magnus that binds variable domains of IgG light chains. Although this binding occurs near the antigenbinding site of IgG, hypervariable regions of antibodies do not interact with the protein. Unlike IgG-binding proteins described thus far, PpL binds exclusively to the VL domain of the κ-chain. This unusual binding specificity allows for PpL to be used as a universal Ig ligand; it binds antibodies of any Ig class as well as single-chain variable fragments (scFv) and Fab fragments.42 PpL contains four or five highly homologous domains (the number of domains is strain-dependent), with a total molecular weight of 76−106 kDa. Structural studies have revealed that each (highly homological) domain consists of a β-sheet formed by four βstrands and a central α-helix (Figure 2B).43 The PpL domain appears to have two independent binding sites for VL, which interact with very similar areas on the light chain but with markedly different affinities (one site has a 25−55 fold higher

Staphylococcus aureus and protein G from the C/G Streptococcus strain. 2.1.1. Staphylococcal IgG-Binding Proteins. Staphylococcal protein A (SpA) was the first protein identified to bind human IgG in a nonimmune manner. The molecular weight of intact native molecule, anchored to the wall of S. aureus, is 54 kDa. It is composed of three distinct regions: the signal sequence that is cleaved off during secretion, five highly homologous Ig-binding domains (designated E, D, A, B, and C), and the C-terminal cell wall binding domain (designated X). Recombinant protein A used for IgG purification has been engineered to have the cell wall domain deleted, reducing its size to 42 kDa. Each of the five homologous domains is composed of three antiparallel α-helices of approximately 58 amino acid residues, and it is stabilized by a hydrophobic core.30 The Fc-binding site of the B domain was shown to involve 11 amino acid residues of helices 1 and 2 (see Figure 3A).30,31 Crystallographic studies showed that SpA binds at the junction between the CH2 and CH3 domains (Figure 2A). SpA also binds to a specific site on the Fab region of VH3 human antibodies.32 Each of the Ig-binding domains is capable of binding the Fc part of human IgG1, IgG2, and IgG4 with an estimated association constant (Ka) of 108 M−1, whereas interaction with IgG3 is practically absent.33 2.1.2. Streptococcal IgG-Binding Proteins. The first isolated immunoglobulin binding protein from streptococcal bacterial strain was protein G (from streptococcal groups C and G), which binds all four subclasses of human and animal IgG. However, there are also many other streptococcal bacterial strains that can also bind various immunoglobulins. Streptococcal protein G (SpG) consists of domains capable of binding serum albumin and immunoglobulins, located N- and Cterminally, respectively. The domain at the C terminus anchors the protein to the cell wall. The total number of IgG-binding domains varies from 2 to 3 depending on the strain from which the protein was isolated, and as a result, proteins have different molecular weights (58 to 65 kDa from the C and G strains, respectively). Each of the IgG-binding domains consists of approximately 55 amino acids residues packed as a β-sheet, which is built of two antiparallel and two parallel β-strands with an αhelix lying diagonally (Figure 2C). As a molecular tool, SpG offers some benefits over SpA, such as broader IgG subclass and species specificity. Another difference is the nature of interaction. Residues of SpG involved in Fc binding are situated within the Cterminal part of the α-helix, the N-terminal part of the third βstrand, and the loop region connecting these two structural C


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Bioconjugate Chemistry affinity for the κ-chain than that of the second site).23 The first observed binding site of PpL (with a Ka of ∼107 M−1) involves 12 residues mainly from its β-strand 2 and the α-helix. The lower affinity site involves 14 residues between 40 and 71 on the other side of PpL, i.e., β-strand 3 and part of the α-helix (Figure 2B).23 PpL also exhibits an avidity effect: native PpL binds the two VL domains of an IgG with up to five domains. 2.2. Engineered Variants of Natural IgG-Binding Proteins. To overcome specific drawbacks associated with bacterial immunoglobulin-binding proteins, a new group of affinity ligands has been engineered based on the structures of the natural counterparts. Some of these ligands outperform the natural ones in terms of chemical stability or specificity or display thermal-responsive behavior in Ig binding. Natural Ig-binding proteins often lack stability under extreme conditions such as low and high pH. This is one of the mostcommon issues in large-scale antibody purification because steps such as pyrogen removal and antiseptic management all require harsh conditions. CIP is a standard procedure for the regular removal of contaminants from chromatographic columns without having to dismantle them and is specifically designed based on the type of contaminants. For this purpose, sodium hydroxide in concentrations up to 0.5 M is the most commonly used cleaning agent. Because most proteins are sensitive to alkaline solutions, it is essential to use affinity ligands with ability to withstand such extreme conditions.44 2.2.1. Improved Protein A Variants. Several site-specific mutation studies were performed to improve IgG-binding domains of a protein A. An example of engineered variant of staphylococcal protein A is the Z domain, a mutated analogue of B domain.45 The Z domain contains two amino acid substitutions relative to the B domain at key positions affecting stability. The alanine residue at position 1 (located outside the helical structure) was changed to valine to furnish a nonpalindromic restriction site.45 By the introduction of the nonpalindromic site on both sites of the IgG-binding fragment, a gene structure allowing concatemerization was formed. A second exchange has been made at Gly29 (mutating it to Ala) to avoid the dipeptide sequence Asn−Gly,45 which is the cleavage site for hydroxylamine. Asparagine with succeeding glycine has also been found to be the most sensitive amino acid sequence to alkaline conditions.46 The resulting engineered variant is not susceptible to protease degradation and has greater alkaline stability, therefore allowing harsh elution or CIP conditions (discussed in chapter 2.3.1). The Z domain lacks the ability to bind to human VH3 domains and, as such, cannot be used for Fab purification. Additional site-directed mutagenesis studies of Z domain yielded novel versions with improved characteristics. The development progressed in two directions. In the first one, exchange of Phe30 for Ala or Asn23 for a Thr further increased tolerance to alkaline conditions (again, allowing harsh cleaning conditions of the corresponding columns).47,47,47 The second approach provided ligands enabling milder elution conditions.48 A single-mutation ligand, termed Z(H18S)4, and a doublemutation ligand, Z(H18S, N28A)4, support elution at higher pH (on average, >0.5 pH) and retain comparable dynamic binding capacity and selectivity.48 Also, the substitution of specific surface-exposed residues (Gln10, Asn11, Glu15, and Leu17) for histidine residues, which are protonated in a pH-sensitive manner, effectively increased the dissociation rate under acidic conditions. Double-mutation variant PAZ02 (Gln10His and Asn11His) exhibits higher pH sensitivity, enabling elution under milder conditions while maintaining affinity, thermal stability,

and alkaline tolerance.49,50 Z domain is also extensively used as an alternative-scaffold (i.e., non-antibody) binding protein (called the affibody) in a variety of affinity chromatographic systems and as detection or targeted delivery agent.51 Yet another example of modified protein A is the so-called “Thermal Responsive Protein A” (TRPA), in which mutations were made in the core structure to reduce thermal stability. The end result was a protein A mutant with native structure at 2−10 °C, which unfolds at 40 °C. Thereby, such mutein is capable of binding IgGs at low temperature and releasing them at a higher temperature. This allows for the entire process of antibody purification to occur at neutral pH. After elution at elevated temperature, TRPA refolds back as the temperature is decreased and is capable of binding antibodies again.52 2.2.2. Improved Protein G Variants. In addition to IgGs, protein G binds other naturally occurring proteins such as albumin, kininogen, and α2-macroglobulin. Recombinant versions of SpG were constructed by deletion of domains responsible for interaction with proteins other than IgGs.40 As with SpA, one of the goals of further development was to enhance SpG stability at alkaline pH. This was achieved by replacing critical amino acids (asparagine and glutamine) of the C2 immunoglobulin-binding domain with fewer alkali-susceptible residues. Alkaline sensitivity was analyzed using a singlepoint mutation strategy, revealing Asn36 as the most-sensitive amino acid residue.53 The most-stable version was found to be a double-mutant, termed C2N7,36A, where asparagines at positions 7 and 36 were exchanged for alanine.53 SpG was also engineered to display higher affinity for human IgG. Using the Rosetta protein modeling, a number of favorable polar residues at the SpG′s IgG binding interface were identified and predicted to enhance the binding interaction and increase the SpG mutein stability. A set of three mutations in SpG (Ala24Glu, Lys28Arg, and Val29His) enabled the formation of additional hydrogen bonds and salt bridges at the rim of an interface after interaction with an antibody. The SpG mutein showed 5-fold higher binding affinity for rabbit and human IgGs.54 Recently, an SpG mutein with eight point mutations (termed Protein-G-A1) was reported. This improved version of SpG was identified employing a soft randomization library design strategy (by diversifying 15 residues of SpG within 5 Å of complex interface). The affinity-matured protein-G-A1 has a 100-fold higher affinity compared to that of the parent domain of protein G (C2) and binds Fab fragments. Furthermore, this variant possesses pH-dependent affinity and enhanced stability in alkaline environment.55 Another example of SpG mutant with highly pH-sensitive IgG binding was reported by Watanabe and co-workers.49 They systematically generated a number of pHsensitive protein G mutants by rationally introducing histidine residues onto the binding surface of parent protein. This caused electrostatic repulsion upon protonation, resulting in decreased affinity at acidic conditions. The best engineered triple mutant BG0919 (Asp40His, Glu42His, and Gln32His) fully maintained the innate binding function but with improved pH sensitivity.49 2.2.3. Hybrid Proteins. Broad binding specificity can be achieved by combining IgG-binding domains of two different bacterial proteins (Table 2). One of the first designed hybrid proteins was protein A/G, which contains four Fc binding domains from SpA and two from SpG. The fusion protein A/G is less pH-dependent but capable of binding all subclasses of human IgG.56 A recently designed novel fusion protein consisting of SpA and SpG, labeled PAxPG, contains a flexible D


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acids, of which some 10−20 are randomized. The scaffold has a simple yet highly stable structure and good solubility. The small size of alternative-scaffold binding proteins enables multimerization, thereby enhancing avidity. Furthermore, the absence of post-translational modifications including disulfide bonds allows for high recombinant production yields in prokaryotic expression systems on the industrial scale. In this section, we briefly review alternative scaffold-binding proteins used as affinity ligands for immunoglobulins (depicted in Figure 3). 2.3.1. Affibody. The Z domain (Figure 3A), an engineered variant of B domain of Staphylococcal protein A, is one of the most widely used alternative scaffolds and represents the molecular origin of all affibody molecules.51 The first generation of affibody libraries were created by the randomization of 7 and 6 solvent-accessible residues in helices 1 and 2, respectively, using the NN(G/T) degenerate codons (Figure 3A).63,64 Because most of these positions are involved in interaction with the Fc region of human IgG, the original IgG-binding capability was typically lost. Display techniques such as phage display and ribosome display65,66 have been used to affinity select Z-domain muteins from vast combinatorial libraries. Diverse proteins were targeted, and affinities up to the mid-picomolar range were achieved.67 This methodology also yielded a number of affibodies showing specificity for the different classes of immunoglobulins. A total of five different affibody variants exhibited IgA binding with dissociation constants (Kd) in the low to submicromolar range68 with no detectable cross-reactivity for human IgG, IgM, IgD, or IgE. In addition, the affinity chromatographic recovery of IgA from a spiked bacterial lysate and unconditioned human plasma was demonstrated. Affibody molecules capable of binding human IgE with Kd of 0.4 μM have also been isolated.69 These affibodies, in monomeric and dimeric forms, have been successfully expressed on the cell surface of Staphylococcus carnosus, creating a staphylococcal bacterium capable of binding human IgE. Murine IgG 1 -specific binders were obtained from a combinatorial ribosome display library of affibody molecules

Table 2. Different Versions of Hybrid Bacterial Immunoglobulin-Binding Proteins hybrid protein A/G PAxPG protein L/G protein L/A

domain structure four SpA domains + two SpG domains SpA domain + SpG domain four PpL domains + two SpG domains four PpL domains + four SpA domains

binds all human IgG subclasses via Fc56 all human IgG subclasses via Fc57 all human IgG subclasses via Fc and Fab (κ-light chain)59 all human IgG subclasses via Fc and Fab (κ-light chain)60

linker (DDAKK) connecting Ig-binding domains D from SpA and C1 from SpG. Side-by-side comparison with individual parental domains revealed that the fusion protein with 6 tandem repeats of linker DDAKK possesses a 58-fold higher affinity for IgGs due to avidity and broader specificity.57 Useful hybrids are also protein L/G58,59 and L/A fusions.60 Protein L/G is a reagent for purification of a variety of Igs, while L/A provides a broad binding spectrum for naturally occurring IgGs as well as recombinant Ig fragments. 2.3. Affinity Ligands Based on Alternative Scaffolds. Regardless of the advances in tailoring natural Ig-binding protein properties, their use is still hampered by fairly high production costs associated with recombinant expression, and structural instability. Development of protein engineering and combinatorial libraries coupled with in vitro selection techniques led to the emergence of novel nonimmunoglobulin classes of affinity ligands. These new tailored-made affinity ligands, termed alternative scaffold proteins, are typically built from frameworks that are smaller and structurally less complex than immunoglobulins. The framework typically harbors altered amino acids or insertions, creating large libraries of variants, some of which have novel functions. Selection techniques based on bacterial, phage, yeast, or ribosome display can be used to isolate specific variants of a protein with desired features, typically new binding specificities. A non-Ig scaffold is usually built from a single relatively short polypeptide chain of between 40 and 100 amino

Figure 3. Molecular models of selected protein domains used as scaffolds for development of artificial ligands. The residues randomized in preparation of combinatorial libraries are shown in red. Details are given in the text. (A) Z domain (based on the B domain of SpA) used for the development of affibodies (PDB code 1Q2N). (B) Sac7d protein as the structural basis of affitins (PDB code 1AZP). (C) Sso7d protein as the structural basis of affitins (PDB code 1BNZ). (D) Designed ankyrin repeat protein (DARPin; PDB code 1MJ0).61 (E) Leucine-rich repeat for the design of Fc-specific Repebody (PDB code 3RFJ). (F) Fibronectin type III domain for development of immunoglobulin-specific monobodies (PDB code 3UYO).62 Molecular graphics were generated using PyMOL software. E


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Bioconjugate Chemistry with Kd in the nano- to low-micromolar range. One of the selected binders, termed Zmab25, competed with streptococcal protein G (SpG) for binding to mouse IgG1. Mapping of the binding sites for the Zmab25 affibody disclosed an epitope on the CH1 domain, overlapping with SpG binding sites. The narrow specificity of Zmab25 enabled the recovery of mouse IgG1 from complex samples resembling hybridoma cultured supernatant.65 2.3.2. Affitins. Affitins are artificial affinity ligands developed from extremophilic proteins from the “7 kDa DNA-binding” family found in Archaea.70 The main function of these proteins in Archaea is to prevent the melting of genomic DNA at high growth temperature. A pair of main proteins have been used as scaffold to design tailored highly stable artificial affinity proteins: Sac7d from Sulfolobus acidocaldarius (Figure 3B) and Sso7d from Sulfolobus solfataricus (Figure 3C). Both fold as an SH3-like fivestranded incomplete β-barrel capped by C-terminal α-helix and have closely related sequences. Unlike other protein scaffolds, affitins are extremely thermostable (Tm ≈ 80 °C) and withstand extreme pH (0 up to pH 12). Various libraries based on Sac7d and Sso7d scaffolds have been reported, typically involving the randomization of 10 to 14 amino acid residues located in the βsheet of the wild type proteins. The screening of such libraries led to specific binders of diverse set of targets, with novel ligands inheriting many advantageous properties of parental proteins.71−73 The first affitins with affinity for human IgG were derived from Sac7d protein by ribosome display applying affinity selection against the human Fc region as a target. A pair of them (C3 and D1) had binding affinity in mid-nanomolar range (74 and 34 nM, respectively).73 The binding of affitin C3 to human IgG was partially inhibited by protein A, which indicates an overlapping recognition site.73 The binding site of affitin D1 was grafted onto the thermally more stable scaffold of Sso7d.74 Interestingly, while the optimization of thermal stability required additional sitedirected mutagenesis, the new mutein D1sSso7d-DM turned out significantly more resistant to alkaline conditions, withstanding a pH up to 13. Affinity column with covalently immobilized D1sSso7d-DM was recently characterized.75 Specificity of affitinbased affinity matrix was comparable to that of protein A, allowing a single-step isolation of human IgG from cell culture media and ascites with a high degree of purity (∼95%) and recovery (∼100%). Of note is the fact that the affitin column withstood repetitive cycles of purification and CIP treatments with 0.25 M NaOH. pH-sensitive variants of affitins derived from Sso7d by yeast display were also explored for binding human Fc fragment.71,72 An Sso7d human Fc-binding variants were obtained using two different strategies: histidine scanning and random mutagenesis.71 Sso7d-hFc has a dissociation constant of about 400 nM and no pH sensitivity. Subsequently, systematic analysis of Sso7d-hFc variants with single histidine substitutions in the binding interface identified the Tyr40His mutant, Sso7d-his-hFc, as a pH sensitive hFc-binding protein. In parallel, a yeast-displaybased screening strategy was used to isolate another pH-sensitive binder, Sso7d-ev-hFc, from a library obtained by random mutagenesis of a pool of Sso7d-based hFc binders. Notably, unlike Sso7d-hFc, both Sso7d-his-hFc and Sso7d-ev-hFc have a higher binding affinity for hFc at pH 7.4 than at pH 4.5. Their dissociation constants for human IgG are in the low-micromolar range (1.6 and 5.3 μM, respectively) at pH 7.4. Both isolated proteins bind an epitope in the CH3 domain of hFc that has high sequence homology in all four hIgG isotypes (hIgG1−4), and recognize hIgG1−4 as well as deglycosylated hIgG in Western

blotting assays. pH-sensitive hFc binders are attractive candidates for use in affinity chromatography due to feasibility of IgG elution under milder pH conditions. 2.3.3. Designed Ankyrin Repeat Proteins. Designed ankyrin repeat proteins (DARPins) are one of the most widely studied artificial affinity proteins. They originate from ankyrin repeat protein (ARP), a protein composed of several repeat domains, each consisting of a β-turn followed by two antiparallel α-helices providing a stable structure (Figure 3D). Combinatorial libraries of DARPins have been created by randomizing 7 positions out of the 33 residues in each repeat domain. The randomization is to a large extent performed on secondary structure elements giving a rather flat binding surface (Figure 3D). Different numbers of the generated repeat modules can be connected together and flanked on each side by a capping module. The DARPin libraries are thus denoted NxC, where N and C stand for the N- and C-terminal capping units, and x denotes the number of library repeat domains, which is usually between two to four.76 High-affinity binders of various targets have been isolated from ribosomal or phage display DARPin libraries. DARPins have a molecular mass ranging from 14 to 21 kDa and are characterized by high thermodynamic stability, reversible folding behavior, and low aggregation tendencies.77,78 DARPins specific for the Fc fragment of human IgG1 with affinities in the mid- to low-nanomolar range were selected by the so-called signal recognition particle (SRP) phage display without additional affinity maturation.79 The SRP phage display employs co-translational translocation of displayed proteins to the periplasm, which allows efficient filamentous phage display of highly stable and fast folding proteins, such as DARPins. These binders were shown to be monomeric and able to interact with full-length human but not mouse IgG1 according to ELISA assays. The authors envision their anti-Fc DARPins to be used as analytical or biomedical tools rather than affinity chromatography ligands. Another group of DARPins were selected to bind human IgE; four were shown to recognize three different IgE, while they did not interact with any of the IgG subtypes, nor with IgA or IgM. Affinities of two monomeric and two bivalent DARPins against IgE were estimated at a low-nanomolar range (Kd = 0.9−6.8 nM). Bivalent DARPins were produced by joining two DARPins by a glycine-serine linker, leading to higher potency due to avidity effect.80 Anti-IgE-Fc-specific DARPins hold potential as drugs for the treatment of severe allergic asthma.80−82 DARPins’ rigid structure, concave shape, and incompletely randomized binding surface might hamper efficient recognition of certain epitopes. The latest generation of DARPins (the socalled “LoopDARPins”) overcomes this issue. In LoopDARPins the central β-turn is replaced by an enlogated loop with randomized positions, yielding a convex paratope, similar to antibodies.83 2.3.4. Carbohydrate-Binding Module. The carbohydrate binding module, CBM4−2, derived from xylanase of Rhodothermus marinus, has been developed as disulfide-free diversitycarrying alternative scaffold of extreme thermal stability (Tm = 89 °C).84 A small combinatorial phage display library was constructed by the randomization of 12 residues around the binding site of CBM4−2 and panned against human monoclonal IgG4. Selected CBM variants were specific for the protein structure of hIgG4 and did not interact with carbohydrates.84,85 2.3.5. Nanobodies. Nanobodies are recombinant camelid antibody fragments, consisting of a single variable domain (VHH).86 As one of the smallest antigen-binding domains (with a F


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Bioconjugate Chemistry Table 3. Short Peptides as Immunoglobulin Ligands peptide (ref)

origin

Kd

binding capacity [mg/mL]

PAM106 100 D-PAM 107 D-PAM-φ HWRGWV HYFKFD HFRRHL108 HWCitGWV Ac-HWMeCitGWMeV109 FGRLVSSIRY TWKTSRISIF110 FYWHCLDE FYCHWALE FYCHTIDE111−114 DWHW CEWW HEYW115 Fc-III116 FcBP-1 FcBP-2117 Fc-III-4C118 EPIHRSTLTALL119 D2AAG DAAG120 cyclo[link-M-WFRHY-K]121 APAR122 FcRM123

dendrimeric, synthetic combinatorial peptide libraries

0.3 μM NPa NP 3.8 μM 11 μM 26 μM 108 μM NP NP NP 2.4−3.6 μM 6.1 μM 5.7 μM 11 μM 12 μM 17 μM 70 nM NP 1.8 nM 2.45 nM NP 1.3 μM 384 nM 7.6 μM 94 μM 20 μM

25 NP NPb 91.6 27 33 72 NP NP NP 104 87 63 24.5 18.8 16.5 NP NP NP 28.9 NP 36 48 19.7 9.1 NP

a

synthetic solid-phase random hexamer peptide library

phage display library biomimetic design strategy

molecular simulation

phage display cyclic peptide library peptidomimetics of Fc-III rational design of Fc-III phage display library synthetic, combinatorial library mRNA display synthetic library combinatorial tetrapeptid library synthetic cyclic library

elution pH 3 3.5 4 4 4 4 4 4 NP NP 6

binding site CH2−CH3 interface

CH3 domain

Fc fragment

CH2−CH3 interface 3 or 9

Fc fragment

3.5 NP NP 3.5 NP 3.6

CH2−CH3 interface

Fc fragment CH2−CH3 interface

4 7.4c 2.7

Fc fragment NP Fc fragment

NP: not provided. b10-fold enhancement compared to D-PAM. cElution not pH-dependent.

molecular weight of ∼13 kDa), nanobodies offer several advantages: easy cloning, high stability and solubility, and low immunogenicity.87 Klooster and co-workers isolated two IgGspecific VHHs from the immune phage display library.88 These VHHs recognized all subclasses of human IgG but did not recognize antibodies from any other sources. The selection protocol was designed to drive the selection toward conserved epitopes between all human subclasses; the antigen used in sequential selection rounds was switched from one subclass to another.88 These VHHs were used as affinity ligands for IgG purification and were commercialized under the name CaptureSelect IgG-CH1 Affinity Matrix (Bac BV, Thermo Fischer Scientific). Resin’s binding capacity is 25−30 g IgG/L. As the name implies, these VHHs recognize the CH1 domain of human IgGs. CH1 domain is considered to be the mostconserved region among different IgG Fab fragments and therefore suitable for recognition of all human IgG subclasses. The unique affinity ligand enables the purification of all subclasses of human IgG and Fab fragments of both κ and λ isotypes.89 Another commercialized camelid derived antibody with antihuman IgG specificity was developed for IgG purification.88 Static binding capacity ranged from 3.4 to 15 mg/mL. Bound IgGs could be efficiently eluted at pH 5.0, which is advantageous in the case of pH-sensitive IgGs.90 Camelidbased affinity ligand directed against the constant domain of the human λ light chain (CLλ) was also developed, and the affinity matrix based on this VHH’s is commercialized by GE Healthcare Life Sciences under the name LambdaFabSelect. The results showed that LambdaFabSelect is as effective and robust for capturing λ Fabs as is SpA for mAbs.91

2.3.6. Repebodies (Leucine-Rich Repeat Modules). The repebody is a newly designed alternative scaffold based on variable lymphocyte receptors, which consist of highly diverse leucine-rich repeat (LRR) modules. Each module has 20−29 residues organized in a β-strand-turn α-helix fold forming a horseshoe-shaped fold (Figure 3E). These modular structures allow for the simple modulation of binding affinity in a moduleby-module manner.92 The scaffold was shown to be highly stable over a wide pH and temperature range; it allows a high level of soluble expression in bacteria (up to 80 mg/L in Escherichia coli).93 Repebody that binds the Fc fragment of human immunoglobulin G was selected from a phage display library and further optimized by module-by-module manner.94,95 A repebody library was constructed by introducing mutations into three variable sites (positions 8, 10, and 11) in two modules, LLRV 2 and LLRV 3. The library was panned against the human Fc fragment. The highest affinity repebody ligand, termed E10, Kd = 6.2 × 10−7 M, obtained from the primary phage library was used as the basis to construct a secondary phage library with the goal of affinity maturation. The main principle of constructing secondary phage library was based on modular architecture; each mutation on one module has minor effect on adjacent modules. Therefore, to construct a secondary library mutations were introduced into additional module LRRV5 in four variable sites (positions 6, 8, 10, and 11) (Figure 3E). The LRRV5 module was chosen over LRRV1 and LRRV4 to enlarge the interaction area between repebody and IgG, which led to higher binding affinity. Through additional selection rounds, the repebody F4 with enhanced affinity was identified. It displayed 3-fold improved binding affinity compared to E10 and was shown to completely dissociate IgG at pH 4. A resin-immobilized repebody as an G


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Figure 4. Schematic depiction of dendrimeric peptide ligands of antibody Fc fragment; (A) PAM, (B) D-PAM, and (C) D-PAM-φ. Lowercase letters indicate D amino acids, and circled φ represent a phenylacetyl group at free Nα position of arginine residue. (D) Crystal structure of human IgG1 Fc fragment in complex with dendrimer ligand D-PAM (in pink; PDB code 3D6G). (E) A closer look at the interface between D-PAM and a human Fc fragment. The first dendrimer arm binds through hydrophobic interactions via residues shown in red. The second dendrimer arm forms ionic bonds with negatively charged amino acids on antibody shown in green.125 Molecular graphics were generated using PyMOL software.

affinity matrix supported the isolation of antibodies in high yield and of high purity from CHO cell-cultured medium for more than 10 consecutive times without any loss of binding capacity.94 2.3.7. Monobody (Adnectin, Fibronectin Type III Domain). The 10th fibronectin type III domain (10Fn3) is a small monomeric protein (10 kDa) built of a β-sandwich structure connected by six loops (Figure 3F).96 The BC, DE, and FG loops of 10Fn3 are structurally analogous to the complementarydetermining regions of antibodies and were used to generate diversity by randomizing 10 to 21 positions in two or three surface loops using various mutagenesis schemes (Figure 3F).97 Artificial binding proteins based on 10Fn3 are referred to as monobodies and commercialized under the name Adnectins (Bristol-Myers Squibb). Diverse libraries containing either Ser− Tyr or the complete 20 amino acids on randomized positions were created and screened for binding to rabbit and goat immunoglobulin G.62 The highest affinity antigoat IgG monobody bound goat, bovine, and mouse IgGs with Kd values of 1.2 nM. However, the highest-affinity anti-rabbit IgG monobody displayed 51 pM affinity with a strict specificity for rabbit IgG. 2.4. Short Peptides as Immunoglobulin Ligands. In contrast to natural and alternative-scaffold IgG-binding proteins, simple peptides are more affordable and can be rapidly synthesized even on a relatively large scale. Moreover, peptides have favorable toxicity and immunogenicity profiles, while their stability is relatively high compared to larger proteins that rely on complex folding pattern for formation of binding sites. Furthermore, short peptide ligands usually bind to target proteins with only moderate affinity, which suggests that mild conditions will likely be required for breaking the interaction

with Ig molecule. However, short affinity peptides still possess many drawbacks; one of them is substantial degradation by proteases, consequently leading to a loss of binding capacity of peptide-coupled matrices. This problem occurs mainly during purifying antibodies from animal plasma or any other biological fluids containing proteolytic enzymes. The proteases trypsin and α-chymotrypsin cleave peptide chains at basic (arginine and lysine) and aromatic (tryptophan, phenylalanine, and tyrosine) residues, respectively.98,99 One of the options for overcoming the proteolysis susceptibility is to add protease inhibitors to the biological sample. However, these inhibitors are expensive and need to be removed from the final antibody product. A better solution to these issues is to produce protease-resistant variants of peptide ligands composed of non-natural amino acids (Damino acids) or synthesis of peptoids (protease resistant peptides with side chain groups attached to the nitrogen atom rather than at the α-carbon).100,101 However, non-natural amino acids are more costly and still prone to chemical degradation by acid and alkaline solutions used for protein elution and resin sanitization. Often, peptide ligands are developed by combining rational design (e.g., molecular modeling) and directed evolution (i.e., randomization of selected parts of peptide lead structure coupled with high-throughput screening techniques). Chemical combinatorial peptide libraries are created on solid supports through the “split synthesis” approach. Several library formats exist, either biological (such as phage-, yeast-, ribosome-, and mRNA display libraries) or synthetic in both the solid and the liquid phases.102 Detailed description of different display technologies is beyond the scope of this review; the reader is referred to recent reviews.103,104 Such approaches led to discovery of several small H


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Figure 5. Schematic presentation of work flow of designing peptide ligands of Fc fragment. (A) Original Fc-III cyclic peptide selected from a phage display library. (B, C) Peptidomimetics FcBP-1/2 made by transplanting 9 or 13 residues from Fc-III onto a D-Pro−L-Pro template. (D) Peptide Fc-III4C designed by replacing the D-Pro−L-Pro dipeptide with two cysteine residues to retain the double cyclic structure. (E) Crystal structure of peptide ligand Fc-III (blue) in complex with antibody Fc fragment (the binding site is located near the CH2−CH3 domain interface (PDB code 1DN2)). (F) Residues recognized by Fc-III are shown in red. Molecular graphics were generated using PyMOL software.

for antibody purification. The crystal structure of D-PAM− human IgG1 Fc fragment was solved (Figure 4D,E),125 providing the basis for further ligand improvement by rational design. Phenylacetly groups were introduced at the Nα positions of terminal D-PAM arginines (Figure 4C) to promote additional apolar interactions with the Fc domain.107 This third-generation dendrimer ligand (termed D-PAM-φ) efficiently captured polyclonal hIgG from serum and human recombinant IgG1 from crude feedstock. Moreover, the binding capacity was approximately 10-fold higher than D-PAM.107 A family of linear hexapeptides has been identified through a three step screening of a synthetic solid-phase random hexamer peptide library.108 These peptides share a common sequence homology of histidine on the N-terminus followed by aromatic amino acids and positively charged residue patch. A total of three hexapeptide ligands (HWRGWV, HYFKFD, and HFRRHL) have all been shown to bind human IgG.108,126 These peptides bind all human IgG subclasses as well as many animal IgGs (bovine, mouse, rabbit, goat, and llama) and IgY, and have been successfully used for purification of monoclonal and polyclonal antibodies from variety of sources. Owning to their moderate binding strength (Kd ≈ 10−5−10−6 M), they enable antibody elution under gentle conditions (pH 4.0−5.0), thus preventing aggregation and maintaining activity of the product.127−130 Moreover, there is no competition observed between HWRGWV and SpA nor SpG in binding to human IgGs, indicating that this affinity ligand does not bind at the CH2−CH3 interface. Mass spectrometry data and docking simulation

peptide Fc ligands (listed in Table 3), with the potential to replace Protein A in various applications.105 Dendrimeric protein A mimetic peptide, or PAM for short (also termed TG19318), is a tripeptide tetramer with chemical formula (Arg−Thr−Tyr)4−Lys2−Lys−Gly (Figure 4A). It is composed of four identical peptide chains that, when assembled together, are capable of mimicking the binding properties of protein A.106 It was isolated from a synthetic multimeric peptide library, prepared according to the principle of mix and split synthesis on a solid phase. This peptide was shown to bind the antibody Fc fragment with the dissociation constant of 0.3 μM. Compared to protein A, it displays broader specificity toward antibodies from different species (human, cow, horse, pig, mouse, rat, rabbit, goat, sheep, and chicken) and classes (IgG, IgY, IgM, IgA, and IgE).124 Optimum binding of PAM to antibodies occurs in neutral buffers of low ionic strength at room temperature. Elution of immunoglobulins adsorbed onto the PAM-coupled matrix was achieved simply by washing with acidic buffer (pH 3). Furthermore, PAM turned out to be a great lead compound as an affinity ligand for chromatographic purification of antibodies because a binding capacity up to 25 mg/mL of affinity matrix was achieved with PAM-based affinity columns. Further optimization yielded D-PAM, in which all the PAM amino acids were replaced with the corresponding D enantiomers (Figure 4B).100 Thus, D-PAM is not susceptible to protease degradation and has enhanced stability in biological environments.100 D-PAM, known under the name Kaptiv-GY (Interchim, Montlucon, France), is commercialized as a ligand I


Review


with a head-to-tail peptide bond replacing (FcBP-1; Figure 5B) or accompanying (FcBP −2; Figure 5C) the Cys−Cys disulfide bridge and resulting in a β-hairpin backbone with restricted conformational freedom.117 The two Cys residues represented an ideal place to insert a D-Pro−L-Pro template. The resulting peptide mimetic, FcBP-1, contained nine residues and interacted very weakly with the Fc fragment at pH 7.4 (∼65 μM). The reason for this was a backbone conformation with two consecutive cis peptide bonds, which pushed Trp9 (equivalent to Trp11 in Fc-III) to the “wrong face” of the SpA mimetic. The mimicry achieved with FcBP-2, however, was excellent. The double-ring structure of peptide FcBP-2 enhanced affinity toward IgG 80-fold compared to the parent peptide Fc-III (Kd ≈ 2.2 nM). However, head-to-tail cyclization is challenging for routine solid-phase peptide synthesis, and D-Pro cannot be easily incorporated into the nascent peptide. Recently, a novel version of the parent peptide Fc-III was designed by replacing the problematic D-Pro−L-Pro dipeptide with two terminal Cys residues,118 which facilitated the formation of a second disulfide bond (Figure 5D). This novel peptide, termed Fc-III-4C, thus retained a double cyclic structure required for high affinity binding to human IgG (Kd ≈ 2.45 nM), which is 30-fold higher compared to the parental peptide Fc-III. The newly designed FcIII-4C was conjugated to agarose beads to efficiently purify IgG molecules from serum and labeled with FITC for the immunofluorescence detection of proteins in cells.118 Short linear peptides often do not display enough structural rigidity to provide sufficient affinity for target molecules. The cyclization of linear peptide constrains flexibility leading to lower entropic barrier upon target binding, and can increase resistance to enzymatic degradation.132 By the screening of a synthetic dimeric tripeptide library for Fc binding a ligand with chemical formula (Cys−Phe−His−His)2−Lys−Gly, termed peptide H, was identified.133 Peptide H is cyclized by a disulfide bridge formed by two N-terminal cysteine residues. In affinity chromatography, the peptide was found useful for mouse and rat IgG purification but was less-selective for human and rabbit IgG.133 Cyclic peptides for the purification of IgGs from variety of mammalian species have also been obtained by mRNA display.121 mRNA display represents an attractive tool for the selection process because library construction does not rely on bacterial transformation, which is a common bottleneck in phage display; hence, extremely high library diversity can be achieved. In addition, non-natural amino acids can be incorporated in peptides, thereby further broadening library’s chemical space. Menegatti et al. 121 reported a number of homologous immunoglobulin ligands selected from an mRNA display library of cyclic pentapeptides. A novel design approach for the mRNA display library of cyclic pentapeptides was used wherein peptide synthesis and cyclization were carried out directly on the chromatographic support (without the need for purification, cyclization, and coupling, using a solid-phase reaction).121 This method composes a liquid-phase cross-linking reaction between two primary amino groups on both flanks of randomized peptides library.134 In brief, a mRNA−peptide hybrid was composed of parental mRNA; a poly-adenosine linker used for adsorption to oligo-dT resin; a puromycin linker; and a randomized peptide assembled of poly-His tag, Gly-Ser linker, and linear randomized peptide. After in vitro translation, the liquid-phase library of mRNA−peptide hybrids was adsorbed on the solid phase (oligodT-resin) via poly adenosine linker. Addition of homobifunc-

suggest that the peptide binds to the pFc (CH3 domain digested with pepsin) portion of human IgG and interacts with amino acids in the Ser383-Asn389 loop (SNGQPEN) located in the CH3 domain.131 The binding of the loop to HWRGWV is specific, involving several hydrogen bonds as well as hydrophobic and possibly electrostatic interactions. Upon prolonged exposure of the affinity adsorbent to serum, trypsin and α-chymotrypsin could cause substantial degradation of peptide ligand and consequential loss of binding capacity. Therefore, Carbonell et al.109 presented a strategy for developing different variants of their peptide ligands with enhanced biochemical stability. The peptides were modified with non-natural amino acids to gain resistance to proteolysis while maintaining target affinity and selectivity. A virtual library of variants was designed by replacing aromatic and basic amino acids with alkylated variants such as methylated tryptophan (WMe), methylated lysine (KMe), and dimethylated lysine (K(Me)2), while arginine was replaced with citrulline (Cit). Some analogues were designed using Nin-formyltryptophan (WFor) and 4-carbamoyl-phenylalanine; in others, glycine was substituted with aspartic acid. Using the docking software HADDOCK, the library was screened in silico for binding to the Ser283-Asn389 loop of human IgG. The mostpromising HWRGWV analogs (HW Me CitGW Me V and HFMeCitCitHL) were synthesized on chromatographic resin and tested for IgG binding and susceptibility to protease degradation. The variant HWCitGWVC showed slightly higher Kd (1.08 × 10−5 M) than did the parental sequence (Kd = 3.8 × 10−6 M), which indicates a small difference in binding strength. Due to electrical neutrality, the variants Ac-HWMeCitGWMeV displayed higher selectivity than the original sequence.109 Besides synthetic solid-phase peptide library screening, there are other approaches to identify peptide affinity ligands of antibodies. One example is a biomimetic design strategy based on SpA−human IgG complex with six hot-spot amino acids.114 To mimic the SpA affinity motif, the location of parental amino acids was determined to include similar amino acid residues in construction of novel peptide library. Based on this SpA IgGbinding motif, a library composed of 2173 peptides was built and screened using multiple docking and the root-mean-square deviation (RMSD) comparison and evaluation by molecular dynamics simulation. Among predicted peptide ligands, the top peptide FYWHCLDE was selected for experimental verification. This octapeptide was found to bind human IgG by electrostatic interaction at pH 5.0−6.0.114 The dissociation constant was 1.5 × 10−6 M, and the affinity ligand was successfully used for human IgG purification from complex feedstock.111 Further examples of successful peptide affinity ligand design based on molecular simulation between SpA and human IgG are the three small peptides DWHW, CEWW, and HEYW. Among them, DWHW displayed a dissociation constant of 1.1 × 10−5 M and could be used to purify IgG from different cell culture supernatants with purity of more than 95% under elution conditions of pH 9 or 3.115 Several peptide ligands of immunoglobulins developed using molecular display technologies were reported in recent years. One example is the 13-amino acid peptide Fc-III (DCAWHLGELVWCT) (Figure 5A), with high binding affinity for Fc at pH 7.4 (Kd ≈ 185 nM) obtained by bacteriophage display.116 The crystal structure of Fc-III peptide in a complex with antibody Fc fragment (Figure 5E) revealed that the peptide binds to the consensus binding site recognized by several natural bacterial immunoglobulin-binding proteins, including protein A. Further optimization of Fc-III led to the creation of peptidomimetics J


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Figure 6. Chemical structures and workflow of designing triazine-based ligands for immunoglobulin isolation and purification. MAbSorbent A1P and A2P are commercialized by Prometic Life Sciences (Laval, Quebec, Canada).

Figure 7. Chemical structure of (A) affinity ligand 8/7 and (B) AA18.

achieved under milder conditions (pH 4) compared to protein A affinity columns (pH 2.5); this was attributed to a relatively modest affinity of the cyclic peptide ligand (Kd ≈ 7.6 × 10−6 M).121 2.5. Synthetic Low-Molecular-Weight Affinity Ligands. With short peptide ligands, some of the limitations of natural immunoglobulin-binding proteins are surmounted; however, peptides are still susceptible to hydrolysis by chemicals and proteolytic enzymes. Therefore, attempts have been made to develop a new class of synthetic ligands of antibodies’ Fc region, either by screening chemical combinatorial libraries based on nonpeptide backbones or by rationally designing small functional mimetics of natural Ig-binding proteins.135 The general concept of the latter approach relies on the de novo design of a lead compound based on studying the molecular interaction of a natural ligand with cognate target. For example, synthetic affinity ligands mimicking SpA’s and PpL’s binding mode and containing

tional linker (disuccinimidyl glutarate) onto the resin caused cross-linking reaction between two primary amine groups. Crosslinking took place between fixed amino acids at flanked side of randomized pentapeptides; methionine on one side and lysine on the other side; in sequence MX1X2X3X4X5K, where X represents one of the 20 amino acids. After the peptide cyclization was achieved, the resin was washed, and the library was screened to isolate binders to the human Fc fragment. The selected sequences displayed an obvious similarity in amino acids configuration with other studies108 with the predominance of histidine, aromatic (F, Y, and W) and basic (R and K) residues. The study revealed the peptide cyclo[link-M-WFRHY-K] (where link denotes the glutarate cross-link) as the best candidate for further column development. This cyclic peptide-bound human immunoglobulins from complex mixtures as well as immunoglobulins from a variety of mammalian species. Notably, elution of IgGs from the matrix with the described peptide was K


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Figure 8. Chemical structures of affinity ligands based on a Ugi scaffold.

mimetic and was designed in a similar way as ligand 22/8.140 Initially, the interaction of PpL and the Fab fragment of IgG was mapped, and key amino acids involved in the interaction were identified. A total of 11 different, mainly hydrophilic amino acids on PpL were found to play a role in the interaction. A 169 member combinatorial library based on a triazine scaffold was constructed and screened for antibody Fab fragment binding. Ligand 8/7 was selected as the lead ligand for the purification of Fab, F(ab)2, and scFv fragments.141 PpL mimetic recognizes both κ and λ light chains of IgG with good selectivity. Moreover, it binds immunoglobulins from different classes and animal sources.140,141 A commercial adsorbent containing ligand 8/7 (termed Fabsorbent F1P HF) from ProMetic Bioscience (Quebec, Canada) binds human IgG with the estimated binding affinity Ka= 104 M−1. Many other synthetic nonpeptide immunoglobulin-binding ligands were reported in recent years. Further triazine-based ligands include ligand 8−6, synthesized for the purification of IgY from chicken, duck, and pigeon yolk with high binding efficiency,142 and artificial lectin ligands (8/10 and 11/11), described in section 3.6. Besides the well-defined triazine scaffold, ligands based on the products of the Ugi reaction were recently developed for purification of Fab and IgG. Ugi reaction is a four-component reaction involving an aldehyde (or a ketone), an amino- and an isonitrile-group-containing

a triazine ring scaffold that allows the attachment of natural or artificial amino acid side-chain residues have been developed.30,136 The first synthetic low-molecular weight affinity ligand of IgGs was the so-called artificial protein A (ApA; Figure 6B), which was the lead compound for many following synthetic affinity ligands. ApA is a nonpeptidic, triazine-based fully synthetic ligand generated by de novo design.30 Thorough analysis of the binding interface between protein A and IgG has revealed that the dipeptide motif on SpA (Phe132-Tyr133; Figure 6A) is essential for the interaction.31 These two residues coupled to a triazine scaffold (such as 1,3,5-trichloro-sym-triazine) mimic a hydrophobic core, exposed on a helical twist of the B domain of SpA.30 Based on the ApA’s lead structure, a library of 88 IgG-binding ligands coupled to agarose solid phase was constructed using combinatorial synthesis and screened for IgG binding.137 Thereby, an improved affinity ligand, termed 22/8, was identified. Ligand 22/8 is composed of two aromatic amines (3-aminophenol and 4-amino-1-naphthol) linked to a triazine scaffold (Figure 6C). It possesses high stability, high binding affinity (Ka = 1.4 × 105 M), and broad-range specificity.136 This work led to commercialization of two synthetic nonpeptidyl protein A mimetic adsorbents (Figure 6D,E).138,139 Another synthetic ligand for antibody purification is known under the name 8/7 (Figure 7A). 8/7 is a P. magnus protein L L


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Figure 9. Structure of the aptamer 8−2 in complex with human Fc fragment (PDB code 3AGV). The Apt8-2 is shown in orange, and the human Fc fragment is shown in gray. Ca2+ ions bound to Apt8-2 are indicated by yellow spheres. The loop CH2−CH3 is shown in red.

novel synthetic ligands as well as their successful commercialization. 2.6. Aptamers. Aptamers are short single-stranded nucleic acids that fold into complex structures, forming clefts and pockets for the binding of a wide variety of analytes with high specificity and affinity. They are developed by a process of in vitro molecular evolution, combining combinatorial synthesis and affinity selection from vast oligonucleotide libraries.149 Aptamers have found applications as affinity ligands in biosensors and separation techniques including affinity chromatography (see ref 150 for a recent review). Compared with ligands of protein nature, aptamers are produced by chemical synthesis rather than recombinant DNA technology, which significantly lowers the production costs of an affinity matrix. Moreover, a plethora of nucleotide modifications are available to increase stability of aptamers and broaden the chemical space.151 Perhaps the mostattractive feature of aptamers as affinity ligands is an alternative approach to elution under mild conditions. Aptamers often rely on “cofactors” such as divalent cations to attain or stabilize the folded structure preorganized to bind the analyte. Thus, instead of breaking the strong analyte−aptamer interactions, which would require harsh conditions, elution is achieved by aptamer unfolding through cofactor removal. There are a number of reports on immunoglobulin-specific aptamers developed for different applications, such as oriented IgG probe immobilization in optical biosensors,152 IgG quantification via qualitative polymerase chain reaction,153 secondary antibody surrogates for Western blotting,154,155 the blocking of the Fc receptor ε binding site on IgE with a goal to ameliorate allergy symptoms,156 IgE quantification using fluorescence polarization for allergy diagnosis,157 and IgG isolation and purification.155,158 In all of the cases, the aptamers displayed a remarkable degree of selectivity for the constant region of antibodies with virtually no cross-reactivity with cognate immunoglobulins of different species or different Ig classes of the same species. The most thoroughly characterized aptamer for human IgG affinity purification was reported by Miyakawa et al.158 They used an affinity selection from a library of 5 × 1014 RNA molecules randomized over 40 nucleotides, followed by the minimization and substitution of individual nucleotides for 2′-fluoro and deoxy analogues to develop an

compound, and a carboxylic acid that allows the combinatorial synthesis of bis-amides of high diversity. The Ugi reaction products are named AxCyIz, where “A” refers to the amine component, “C” the carboxylic acid, and “I” the isonitrile and x, y, and z are numbers denoting moieties from the combinatorial library list. The Ugi scaffold is formed directly within the matrix, thus considerably reducing the number of steps for production of an affinity matrix, saving time, reagent costs, and effort. Moreover, the Ugi scaffold can adopt more structural flexibility compared to the planar triazine scaffold, making it possible to develop branched, cyclized and 3D affinity ligands. 143 Furthermore, advantage of the Ugi ligands over triazine scaffold-based ones is the ability of the new ligand to accurately mimic a native dipeptide bond.143 To date, three affinity ligands based on the Ugi reaction have been developed for antibody purification (Figure 8): A3C1, a mimetic of a Fab-binding domain of protein L;144 A2C11I1, a mimetic of the Fc-binding domain of protein G;145 and A2C7I1, a mimetic of the Fabbinding domain of protein G.146 Ligand A2C11I1 can be used for the purification of all mammalian immunoglobulins, including camelid IgGs with Kd 4.78 μM.145 Another low-molecular-weight ligand of Fab fragment was developed by rational design. The interface between the light and the heavy chain (CH1 and CL domain) of human κ-Fab was identified as a conserved pocket.147 This conserved cavity was used as a target docking site for in silico screening and design. Virtual screening of 249 compounds revealed 84 hits, of which 46 were soluble. These were synthesized and further evaluated using the one-dimensional saturation transfer difference nuclear magnetic resonance (STD-NMR) method in combination with surface plasmon resonance (SPR). Structure−activity analysis suggested that a small hydrophobic side-chain in the shortened amino acids is essential for the interaction. Out of directed library, the best three compounds were selected and immobilized as chromatographic probes. In the best affinity ligand (termed AA18; Figure 7B), D-Pro was condensed to the N-methyl-N-(3,4dichlorophenyl)urea.148 The ligand is commercialized under the name KappaSelect (GE Healthcare Life Science). Synthetic ligands represent cheap, scalable, and stable versions of ligands for affinity chromatography. These characteristics are probably the main reasons for substantial growth in numbers of M


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Figure 10. Chemical structure of (A) HCIC ligand 4-mercapto-ethylpyridine; (B−E) selected commercial mixed-mode ligands for antibody polishing; (F) glycoprotein-binding ligand 11/11.

their use is associated with numerous limitations. In fact, chromatographic procedures relying on pseudospecific ligands require extensive optimizations and often necessitate the addition of lyotropic salts to chromatographic media. However, above all, because pseudoaffinity chromatography does not efficiently remove impurities, the entire downstream processing scheme needs to be tailored for the product to meet strict specifications. Typically, this means that several additional chromatographic steps are required, which leads to a lower final yield.162 3.1. Ligands for Hydrophobic Charge-Induction Chromatography. Hydrophobic charge-induction chromatography (HCIC) is based on the pH-dependent behavior of ligands.163,164 These dual-mode ionizable ligands, which combine the hydrophobic and electrostatic interactions, can be used as a potential alternative to protein A in purification of antibodies. Antibody binding typically occurs at physiological conditions and is based on hydrophobic interaction. With the reduction of pH in the mobile phase, both the ligand and the adsorbed antibody molecule take on a net-positive electrostatic charge, leading to desorption.165 High adsorption capacity, relatively low cost, and good stability are the major advantages of HCIC.166 However, the low pH needed for antibody desorption (although elution conditions are milder compared to protein A matrices) can be problematic in the view of antibody stability, especially at high product concentration and the presence of precipitating impurities. Moreover, HCIC may require extensive optimization due to nonspecific binding of other serum proteins, such as albumin.167 The most commonly used HCIC ligand for antibody purification is 4-mercaptoethyl-pyridine (MEP; Figure 10A) (pKa = 4.8), which is marketed under the commercial name MEP-HyperCel (Pall Corporation). In MEP-HyperCel resin, the ligand is attached to a hydrophilic matrix via a hydrophobic spacer arm, which provides enhanced selectivity for the adsorption of antibodies. With high ligand density, the dynamic binding capacities for IgGs can reach 25−35 mg/mL,166 and the purity of IgGs is comparable to the one accomplished by protein A chromatography.168,169 The elution of antibodies is achieved using low ionic strength buffers at slightly acidic conditions (e.g., 50 mM sodium acetate buffer with pH 4.0).168 Of note is the fact that an alternative elution strategy has recently been proposed,

aptamer (Apt8−2) consisting of merely 23 nucleotides with improved stability. Apt8−2 bound the Fc portion of human IgG1 with submicromolar affinity and the interaction withstood extreme conditions such as 1 M NaCl, 1 M urea, or pH 3 citrate buffer. Importantly, the interaction was susceptible to 10 mM EDTA and neutral buffers containing 0.2 M KCl, allowing gentle elution. Aptamer-based affinity matrix (at 1 μmol ligand/mg gel) enabled the purification of humanized IgG from cell culture fluid with purification factors equivalent to that of protein A resin. The crystal structure of Apt8−2 human Fc1 was solved (Figure 9),159 and there were no structural changes to the hFc1 induced by aptamer binding. The aptamer-binding site partially overlapped with that of protein A but mainly involved hydrogen bonding and van der Waals forces. In addition, calcium or magnesium ions were found essential for inducing the proper aptamer fold. The observed strict selectivity of anti-Fc aptamers (e.g., refs 155 and 158), despite the fact that no measures are undertaken to steer the affinity selection process toward highly selective Fc binders, is in contrast to the situation frequently seen with short peptide ligands, which bind IgGs of diverse species121,160 or even different Ig classes.124 This can be explained, at least in part, by the larger binding surface that aptamers typically occupy compared with peptides. For the purposes of purification of monoclonal antibodies by affinity chromatography or in immunotechniques in which the detection of primary antibody is warranted, strict selectivity is an undesirable characteristic because it limits aptamer applications. To avoid the overspecificity of aptamers, Ma et al.161 developed a modification of the affinity selection approach in which one sequentially replaces homologous targets every two selection rounds. In principle, this approach, applied to immunoglobulins of different species, might result in enrichment of aptamers with binding characteristics similar to those of protein A in terms of selectivity, yielding a universal (broadly specific) IgG ligand.

3. PSEUDOSPECIFIC IMMUNOGLOBULIN LIGANDS Pseudospecific affinity ligands of immunoglobulins can be classified on the basis of their structure or application type. Here, we have divided them into six groups. All pseudospecific ligands are generally robust, affordable, and compatible with CIP treatment. They typically possess good binding capacities but only moderate specificity for class G immunoglobulins. Thus, N


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and many commercial matrices are available (Figure 10B−E).186 Due to insufficient selectivity, mixed-mode chromatography is typically used in secondary polishing steps. 3.5. Single-Amino-Acid Affinity Chromatography. Amino acids are some of the simplest natural molecules used as pseudoaffinity ligands for antibody purification. They possess good chemical and physical stability and are inexpensive and nontoxic. Different types of amino acids have been evaluated as pseudospecific ligands for the purification of IgG, with L-histidine showing the most-favorable properties.187−189 Mild hydrophobicity, weak charge-transfer possibilities, asymmetric carbon atoms, and wide pKa values are some of the unique properties of 189 L-histidine. Other single amino acids reported as pseudospecific ligands are L-tryptophan and L-phenylalanine for bovine γglobulin purification.190 AbSep, L-tryptophan coupled to porous polymethacrylate matrix, has been developed based on the molecular docking and experimental screening studies. This rational approach was based on retrieving structural information on amino acid residues involved in IgG−protein A interaction. The dynamic binding capacity of AbSep is similar to SpA matrices (30 mg/mL of adsorbent), and the affinity of AbSep matrix for IgG is high (Kd = 5.13 × 10−6 M).191 AbSep was used to purify IgG from plasma and a monoclonal antibody from cell culture supernatant.191 3.6. Lectins. Lectins are proteins with the ability to recognize and specifically bind certain carbohydrate moieties of polysaccharides, glycolipids, glycopeptides, or glycoproteins.192 These nonimmune system proteins are found in plants, animals, and microorganisms and exhibit varying degrees of specificity based on the sugar content.193−195 Because immunoglobulins are glycoproteins, they can in principle be isolated, purified, or detected using recombinant lectins as ligands. Lectins are generally considered for IgD, IgA, and IgM purification, which are otherwise difficult to purify using conventional affinity chromatography.196 The binding of antibodies to lectins usually occurs at neutral pH. Elution can conveniently be carried out at neutral pH as well using competitive ligands (0.1−0.5 M sugar concentration).197 One of the lectins used in affinity chromatography for immunoglobulins is concanavalin A (ConA; isolated from the plant Canavalia ensiformis). The maximum binding capacity for IgGs of affinity cryogels was observed at pH 7.4. More than 90% of adsorbed IgGs were eluted using 2 M NaCl. The column continued to trap IgG antibodies, even after 10 cycles.198 Several synthetic carbohydrate ligands mimicking lectin binding modes were designed. Based on the structure of natural lectins, a synthetic library of 196 ligands was prepared on polymeric support. The ligand termed 11/11 (Figure 10F) with high binding affinity for monosaccharides, particularly for mannoside (Ka = 8.3 × 101 M−1), was based on a triazine scaffold and chosen as the lead ligand. Immunoglobulins, containing monosaccharides, were eluted using borate buffer.199,200 Borate forms a reversible charged complex with 1,2- or 1,3-diols in alkaline aqueous solutions and therefore interacts with terminal monosaccharides bound to immunoglobulins.200 The most-common lectin affinity chromatographic matrices for IgA and IgM purification are jacalin−agarose and mannanbinding protein lectin, respectively. Both are commercially available. Human IgA possesses a series of O-glycans, which are closely located within the hinge region of the molecule.201 The advantages of using lectin affinity chromatography are relatively simple immobilization and less-expensive production, with all steps executable at neutral pH. Nevertheless, lectins (as all

taking advantage of cyclodextrin competing with antibodies in binding to MEP, eliminating the need for acidic elution buffers altogether.170 Other HCIC ligands for Ig purification include 2-mercapto-1methyl-imidazole (MMI), 171 2-mercapto-benzimidazole (MBI),172 2-mercapto-imidazole (MI),173 and 5-aminobenzimidazol (ABI).174,175 To our knowledge, none of these ligands are commercialized yet. Apart from novel ligands, there has been some progress in stationary-phase development. A recent example are magnetic particles coated with HCIC ligand ABI, termed ABI-Mag. ABI-Mag showed high antibody adsorption capacity with 180 mg/mL and a robust performance, as antibodies can be purified directly from complex feedstock without clarification at pH 7.176 3.2. Ligands for Thiophilic Adsorption Chromatography. Thiophilic adsorption chromatography (TAC) for antibody purification177 is based on affinity sorbents with sulfone group that lie in close proximity to thioether groups. The main difference between TAC and HCIC is that in TAC hydrophobic association and ionic interaction do not occur with thiophilic sorbents because thio-ethylsulfone structures do not possess pronounced hydrophobicity and do not contain ionic charges. All known thiophilic solid sorbents base their interaction on electron donor and acceptor properties.178 A pair of structurally different sulfur containing ligands were developed to promote the adsorption of immunoglobulins. The first group is based on divinyl sulfone-activated agarose coupled with β-mercaptoethanol, 2-hydroxypyridine, or other mercaptoheterocyclic compounds. The other is based on epoxy-activated agarose coupled with 2-mercaptopyridine.178 These derivatives have shown good selective binding for immunoglobulins in the presence of high concentrations of lyotropic salts.179 Adsorption of antibodies is promoted by highly concentrated salts, and elution occurs when the salt concentration is lowered. The type of salt influences the strength of the interaction.178 3.3. Ligands for Boronate Affinity Chromatography. Boronate ligands are capable of forming a covalent bond with molecules containing cis-diols via reversible covalent ester bond. Molecules containing cis-diol groups are carbohydrates, glycoproteins, and many others. Importantly, antibodies are glycoproteins bearing an N-linked oligosaccharide at the asparagine residues of CH2 domain of the Fc region. These asparagine-linked oligosaccharides in antibodies typically consist of α-fucose, β-galactose, and mannose, all containing 1,2-cis-diol groups.180 Therefore, boronate ligands can be employed for antibody purification. The most widely used affinity ligand of boronate affinity matrix is phenylboronic acid (PBA). PBA as an affinity ligand can be used in different platforms; it can be immobilized to the chromatographic media (e.g., the no-longeravailable ProSep-PB, Milipore)181 or used as boronic acid magnetic particles.182 Both methods ensure a high clearance of process impurities and provide an alternative method for purification of mAb under gentle conditions, usually using competitive elution with sorbitol.183 3.4. Ligands for Mixed-Mode Chromatography. Mixedmode chromatography (MMC) can also be used for antibody purification. As the name suggests, ligands for MMC engage in diverse and complex interactions with antibodies.184 High capacity, salt tolerance, good stability, and relatively low cost are the major advantages of MMC.185 However, MMC resins usually involve complex mechanisms, and this type of chromatography therefore requires thorough optimization. MMC chromatography is not limited to antibody purification O


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currently used standard downstream processes include multiple purification steps, which usually lead to low yield and, consequently, substantial manufacturing costs. Among multiple purification steps, affinity chromatography still represents the cornerstone of antibody purification owing to high selectivity, rapid turnaround time, and simplicity. To date, most of the industrial affinity purification of whole antibodies and Fab fragments is still based on bacterial immunoglobulin binding proteins (SpA, SpG, and SpL). Manufacturers have focused on providing alternative affinity matrices (e.g., TRPA, Z domain, etc.) with improved characteristics and using additional chromatography polishing steps to achieve final product purity. New advanced computational tools for molecular design provide (bio)chemists and engineers with a better understanding of the molecular recognition phenomena and thereby enable the design of more-effective affinity ligands. Moreover, novel generations of libraries in combination with high-throughput screening present a powerful strategy that facilitates the identification of potent and selective affinity ligands. The progress of combinatorial protein engineering and selection techniques has led to the design of novel classes of affinity proteins with desired properties, termed “alternative scaffold proteins”. Innovative rational design approaches and combinatorial library-screening techniques are opening a new way to ligands with increased affinity, specificity, and stability at lower manufacturing costs.

proteins) suffer from instability under sanitization conditions usually encountered in the biopharmaceutical industry. Moreover, several lectins, including concanavalin A and wheat germ agglutinin, are toxic and mitogenic toward mammalian cells, and careful control of ligand leakage is required. Due to nonspecific interaction to immunoglobulins and other limitations, lectins are impractical for large-scale operations and general use for antibody purification.

4. OTHER APPLICATIONS OF IMMUNOGLOBULIN LIGANDS 4.1. Antibody Immobilization. One of the most-important applications of immunoglobulin ligands is immobilization of antibodies on a solid surface. Immobilization of captured antibodies is one of the key steps affecting the sensitivity and efficiency of target molecule detection in immunodiagnostics, immunoseparation, and more-complex techniques such as immunoarrays and biosensors.202 Natural antibody binding proteins such as SpA and SpG have been extensively used to capture antibodies onto the sensor surfaces with optimal and uniform orientation, which ensures their full functionality and reproducibility of an assay.203 Aforementioned proteins specifically bind to the intact and native Fc region of an antibody, and antibodies fully retain their antigen-binding activity.204 Nevertheless, as these proteins are susceptible to denaturation under harsh conditions,205 small peptide ligands may provide more stable and versatile surface platforms for antibody immobilization in applications such as SPR biosensors and enzyme immunoassays.205 4.2. Therapeutic Apheresis. Another application of immunoglobulin ligands is therapeutic plasmapheresis, which is often used to remove (excessive) antibodies from plasma. Immunoadsorption (IA) stands for the therapeutic removal of immunoglobulins from plasma in a nonspecific manner and has been commonly used to treat antibody-mediated autoimmune disorders and acute transplant rejections. In contrast to general plasma exchange, IA includes high-affinity columns containing bacterial protein A- or G-coupled adsorbent that deplete IgG (auto)antibodies from plasma.206,207 Because of potential risk of immunogenicity due to the leakage of the immunoglobulinbinding proteins, shorter synthetic peptides exhibit several advantages in comparison to native bacterial proteins as ligands for immunoadsorption. Several commercial adsorbents based on synthetic peptides for autoantibody elimination are already used in IA therapy.208−210 These newly developed specific synthetic peptide absorbers are typically covalently coupled to sepharose matrix and represent the basis for selective and effective elimination of immunoglobulins as well as immune complexes from plasma.211

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: nika.kruljec@ffa.uni-lj.si. Phone: +386-1-4769-570. Fax: +386-1-4258-031. *E-mail: tomaz.bratkovic@ffa.uni-lj.si. ORCID

Tomaž Bratkovič: 0000-0001-8367-5465 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Slovenian Research Agency (program P4-0127). The authors are grateful to Prof. Mojca Lunder for critical reading of the manuscript.

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ABBREVIATIONS ApA, artificial protein A; CBM, carbohydrate-binding module; CIP, cleaning in place; DARPins, designed ankyrin repeat proteins; Fab, antibody antigen-binding fragment; Fc, antibody fragment crystallizable; Fn3, fibronectin type III domain; HCIC, hydrophobic charge-induction chromatography; hIgG, human immunoglobulin class G; IA, immunoadsorption; Ig, immunoglobulin; LRR, leucine-rich repeat modules; mAbs, monoclonal antibodies; MEP, 4-mercaptoethyl-pyridine; MMC, mixed-mode chromatography; PAM, protein A mimetic; PpL, peptostreptococcal protein L; RMSD, root mean-square deviation; scFv, single-chain variable fragment; SpA, staphylococcal protein A; SpG, streptococcal protein G; TAC, thiophilic adsorption chromatography; TRPA, thermal responsive protein A; VHH, single-variable domain of heavy-chain antibody

5. CONCLUSIONS The increasing demand for affordable biopharmaceutics owing to higher life expectancy of world population simultaneously with lowering the production expenses is forcing a comprehensive rethinking of bioprocess technologies. Meeting the large demand for cost-effective products while still complying with increasingly stringent regulations requires constant improvements of manufacturing strategies. Downstream processing in particular is under the increasing pressure for radical changes to overcome the safety concerns of final product and to reduce the cost posed by growing economics on the global pharmaceutical market. To guarantee the high purity of the biopharmaceutical product,

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