key: cord-0707996-seqyoha7
authors: Chen, Serene W; Zhang, Wei
title: Current trends and challenges in the downstream purification of bispecific antibodies
date: 2021-05-07
journal: Antib Ther
DOI: 10.1093/abt/tbab007
sha: eb3c91e65d41f27007ff5e3cd214ab28e7dcc9fc
doc_id: 707996
cord_uid: seqyoha7

Bispecific antibodies (bsAbs) represent a highly promising class of biotherapeutic modality. The downstream processing of this class of antibodies is therefore of crucial importance in ensuring that these products can be obtained with high purity and yield. Due to the various fundamental structural similarities between bsAbs and monoclonal antibodies (mAbs), many of the current bsAb downstream purification methodologies are based on the established purification processes of mAbs, where affinity, charge, size, hydrophobicity and mixed-mode-based purification are frequently employed. Nevertheless, the downstream processing of bsAbs presents a unique set of challenges due to the presence of bsAb-specific byproducts, such as mispaired products, undesired fragments and higher levels of aggregates, that are otherwise absent or present in lower levels in mAb cell culture supernatants, thus often requiring the design of additional purification strategies in order to obtain products of high purity. Here, we outline the current major purification methods of bsAbs, highlighting the corresponding solutions that have been proposed to circumvent the unique challenges presented by this class of antibodies, including differential affinity chromatography, sequential affinity chromatography and the use of salt additives and pH gradients or multistep elutions in various modes of purification. Finally, a perspective towards future process development is offered.

Bispecific antibodies (bsAbs) demonstrate novel functionalities that yield remarkable promise in improving the drug therapeutic efficacy through the recognition and targeting of two different antigens. The enormous therapeutic potential of bsAbs has led to the development of over 50 different formats of recombinant bsAbs reported so far. Yet, in comparison with the numerous detailed reviews outlining the various different formats of bsAbs, along with the associated upstream platform technologies to generate them in order to minimize product-related impurities and their corresponding therapeutic applications [1] [2] [3] [4] [5] [6] , the for the purification of bsAbs, further optimization cannot be fully eliminated due to the differences in their intrinsic structural and concomitant physicochemical properties ( Fig. 1 ) as well as the presence of bsAb-related byproducts, an understanding of which will aid in the identification of potential challenges and therefore design of the optimal strategy for their downstream processing. To this end, this review aims to present the key structural properties of bsAbs and their associated byproducts, outlining the current major purification methods of bsAbs and highlighting the corresponding solutions that have been proposed to circumvent the challenges, as well as to offer a perspective towards future process development.

Here, we will consider three broad categories of bsAbs based on their structures, namely (i) asymmetric, (ii) symmetric and (iii) fragment-based bsAbs (Fig. 1) , as their structural properties have important implications for their downstream processing strategies. Although bsAbs may be bivalent with one binding site for each antigen (1 + 1), the number of valencies can be further increased by incorporating additional binding sites to the chains to achieve trivalency (e.g. 1 + 2 or 2 + 1) or tetravalency (e.g. 2 + 2), as illustrated in Fig. 1b -d.

Asymmetric bsAbs (Fig. 1b) typically possess heavy chains (HCs) and light chains (LCs) derived from two different parental mAbs, where parts or all of the LC and/or HC may also be designed to be equal on both arms where possible in order to minimize mispairing. Another example of an asymmetric bsAb is the antigenbinding fragment (Fab) × single-chain variable fragment (scFv) format, where a scFv from a different parental mAb replaces the Fab region on an immunoglobulin G. Like the asymmetric bsAbs, symmetric bsAbs also possess the Fc region (Fig. 1c) and maintains symmetry by having the same HC with additional antigen recognizing domains such as scFvs appended to the antibody. Fragment-based bsAbs (Fig. 1d) arguably consist of the most varied formats, which are made possible through different combinations of chains and linkers. The tandem scFv format illustrated here consists of two scFvs with a linker, each connecting the LC to the HC and another flexible linker connecting the two scFvs together. A similar format is the diabody, where the LC of one scFv is linked to the HC of the other scFv, rather than itself.

Although the increase in valency of bsAbs brings about various advantages for therapeutic purposes, bsAb-specific byproducts pose unique challenges to their downstream processing. Examples of such impurities include the HC and LC mispaired products as well as fragments and aggregates, as illustrated in Fig. 2 using an asymmetric bsAb as Figure 2 . Impurities formed as a result of HC mispairing (light blue dotted box), LC mispairing (light red dotted box), fragmentation (light green dotted box) and aggregation (light purple dotted box) are illustrated here for a representative desired asymmetric bsAb, with the proposed strategies to remove them shown in the corresponding dark colored non-dotted boxes. Not shown here: (1) for a Fab × scFv bsAb, differential KappaSelect affinity and CH1based chromatographic methods have proven to be useful [59, 60] ; (2) diabody-IgG mispaired products can be separated from scFv-IgG bsAb targets through the use of an alkaline pH near the pI of the bsAb using cation exchange chromatography [71] ; (3) hydrophobic and mixed-mode resins have been proposed to provide good separation for certain homodimer mispaired products from their bsAb targets [77, 78] . Examples listed here are not exhaustive. an example, although similar classes of impurities can be generated in other formats. Due to the presence of more than one different HC and LC that are often coexpressed in the producing cell, a myriad of undesired non-functional or monospecific antibodies of similar sizes and properties can be generated from mispaired HC and LC (Fig. 2 ). These mispaired products may account for up to 90% of the total mass if allowed to pair randomly [9] . One of the approaches employed for the generation of bsAbs in order to reduce mispaired products is to bias the formation of the desired bsAb, for instance, via the knob-intohole (KiH) approach [10] , whereas another approach is to employ molecular design techniques so as to build in the ability for an increased ease of mispaired byproducts removal during subsequent downstream processing steps, an example of which is the chimeric Fc sequence (Fc * ) that ablates Protein A binding [11] . The former does not provide a straightforward way to purify mispaired variants that do occur, whereas the latter incurs a drop in productivity as cellular energy is spent on making the variants in addition to the desired bsAb.

Undesired fragments make up another common group of undesired byproducts, with examples including 1 / 2 (antibodies with one less HC and one less LC) and 3 / 4 (antibodies with one less LC) antibodies (Fig. 2) . Another major group of byproducts is the presence of an overall higher level of aggregates, which has been reported for various classes of bsAbs (Fig. 2) . Although the absence of the Fc region in fragment-based bsAbs has been reported to render the antibody more aggregation prone compared with the parental conventional immunoglobulins [12, 13] , symmetric bsAbs have also been shown to possess significantly higher aggregation propensities [14] [15] [16] , with up to 50% of aggregates observed, which can be at least partially attributed in certain instances to the intermolecular domain swapping as a result of increased chain length and flexibility [17] . The increased aggregation propensity of these bsAbs is also reflected in their expression as inclusion bodies in some cases [18, 19] .

This section aims to provide a general overview of the common processes and methods currently employed for the purification of bsAbs, with general purification details summarized in Tables 1 and 2 and specific impurity removal approaches and strategies highlighted in greater details in the ensuing sections where appropriate. Due to the myriad of different bsAb formats reported, details of the purification methods of the different types of bsAbs listed here are by no means exhaustive; we seek to include studies where the purification of bsAbs is the main focus of the novel work, as well as methods that have been used for the purification of bsAb formats approved or in clinical trials [20] . Although it would be ideal to compare the efficiency of the different purification methods, the different loading amounts, culture quality, chromatography resins as well as methods of quantification render it particularly challenging to do so across each method in a meaningful manner.

Protein A and Protein G affinity chromatography.

As is the case for mAbs, Protein A affinity chromatography is arguably one of the most commonly employed affinitybased purification method in the downstream processing of bsAbs [11, 14, [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] . Such purification is made possible through the interaction between the Protein A and the Fc region of the target molecule as well as the heavychain variable domain (VH) region of the HC for targets belonging to the VH3 gene family [41, 42] . The details of the general purification processes of bsAbs using Protein A resins are outlined in Table 1 , where the sample is typically loaded after an equilibration (EQ) step at pH7.0-7.4, followed by a wash step often using EQ buffer, with a step or gradient elution performed at low pH<4.0 using acetate, citrate or glycine buffer [14, [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] . Through the recognition of the Fab and Fc antibody regions in a similar fashion to Protein A but with different binding specificities, Protein G has also been reported as a capture step for the purification of an anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV)/horseradish peroxidase (HRPO) quadroma [31] , with the polishing step performed using a specific affinity method of an maminophenylboronic acid (APBA)-agarose saturated with HRPO to eliminate the homodimeric monospecific anti-SARS-CoV (Table 1) . Protein A and Protein G affinity chromatography has proven to be particularly useful in the removal of mispaired products and fragments [11, [32] [33] [34] [35] [36] [37] [38] [39] [40] [43] [44] [45] , details of which are discussed below.

Removal of mispaired products Differential Protein A affinity chromatography is one of the downstream processing approaches adopted to remove homodimeric HC mispaired products, which often requires the bsAb to be generated with certain modifications made on one of the HCs of the bsAbs to alter the Protein A-binding affinity. This has been achieved through the construction of bsAbs consisting of one half from rat Abs, which cannot bind to Protein A, and the other from mouse Abs, which have a high affinity for Protein A [32, 33] , introduction of point mutations at the Fc and/or VH3 region to decrease and/or increase the binding affinity to Protein A [11, [34] [35] [36] [37] [38] or by having one Fc chain originating from human IgG3, which does not bind Protein A [39] . All of these methods will result in homodimer products possessing different Protein Abinding affinity as compared with the target bsAbs, thereby allowing for efficient separation and impurity removal. In contrast to the use of a step elution in typical Protein A chromatography methods (Table 1) , the differential Protein A affinity chromatography will require either a pH gradient or multistep pH elutions [34, 36, 37, 39] so as to separate the target from the undesired HC mispaired products. A similar concept has also been developed for Protein G, where a combination of three mutations in the Fc and CH1 region that disrupts Protein G binding was introduced so as to achieve differential elution between the homodimers and heterodimers [38] .

Notably, a detailed study of the utilization of differential Protein A chromatography on target bsAbs with one HC with a Fc * sequence has been performed by Tustian etal. [11] using a range of Protein A resins. Although Fc * Fc * homodimer should typically flow through the column with almost no binding interactions, the FcFc homodimer is expected to bind more strongly to the resin than the target bispecific Fc * Fc. Nevertheless, as HCs from the human VH3 gene family are also able to bind to Protein A [41, 42] , Fc * Fc * may be retained by VH binding to the recombinant Staphylococcal Protein A, with a reduced avidity difference between the bispecific and the FcFc homodimer. This can, however, be circumvented by using an engineered Protein A-based resin, such as MabSelect SuRe, where the affinity ligand is a modified non-Fab-binding Protein A domain [46] [47] [48] . The effect of salt additives was also investigated, and it was observed that the more chaotropic salts provided superior resolution between the bsAb and binding FcFc homodimer, resulting in an improved purity as well as yield, likely through the moderation of hydrophobic interactions between the antibody species and the Protein A ligand. Based on this study [11] , the ideal resin allowing for the best peak separation between the FcFc homodimer and bsAb target should possess a small particle size and a base matrix with large through pores and pore diameters for perfusive flow and mass transfer; an example of which is POROS MabCapture A. In addition, an engineered affinity ligand that lacks VH binding, such as that of MabSelect SuRe, will be ideal to allow for the Fc * Fc * homodimer to flow through. These properties were subsequently materialized in the MabSelect SuRe pcc Protein A resin, which combines an alkali stabilized non-VH region-binding ligand with a base matrix exhibiting excellent mass transfer properties [40] .

Removal of fragments In addition to mispaired products, Protein A chromatography has also been shown to be effective towards the separation of 1 / 2 antibodies from the target bsAbs by applying a pH gradient elution, the results of which may be dependent on the load density [43] . Comparing the results obtained from three resins-MabSelect PrismA, MabSelect SuRe LX and MabSelect SuRe pcc, it was observed that both MabSelect SuRe pcc and MabSelect PrismA offered improved resolution over MabSelect SuRe LX, with MabSelect PrismA displaying a higher capacity and alkaline stability than MabSelect SuRe pcc [43] . It was recently reported that the addition of 500mM NaCl or 300mM CaCl 2 to the elution buffer in Protein A chromatography results in a significant improvement in resolution between the 1 / 2 antibody and bsAb, using a gradient elution from pH5.5 without salt additives to pH ∼3 with salt additives [44] . Through the employment of three sequential optimal wash steps, it was demonstrated that the 1 / 2 antibodies can be removed more efficiently at the slight expense of the product yield, with a lower load density giving the highest yield with lower 1 / 2 antibody removal [44] .

Protein A ligand-binding affinity and selectivity for specific antibody domains has also been shown to be useful Table 2 . Overview of bsAb purification methods utilizing charge, size, hydrophobicity and mixed-mode-based separation techniques as polishing steps, with the exception of * * , which was used as the first capture step. The recovery of bsAbs from inclusion bodies or precipitation methods were also used as the first purification steps, except for ∧ , which was performed on post Protein A eluate that has undergone low pH (pH3.0) treatment 235mM NaCl, pH5.5 or 50mM His-HCl, 300mM NaCl, pH6.5 or 50mM NaAc-HAc, 386mM Arg-HCl, pH5.0 or50 mM Tris-HAc, 500mM Arg-HCl, pH8.0) [26] for the separation of fragments from the desired bsAb. Although the MabSelect SuRe ligand has greatly ablated binding to VH3 domains, which can be utilized for the removal of certain homodimers as described in the previous section, the greater affinity of MabSelect PrismA ligand for the VH3 domains has recently been shown to enhance the separation between the desired bsAb product from that which has lost one Fab arm [45] . This separation resolution was further improved when Fibro PrismA, which possess the same PrismA Protein A ligand on a cellulose fiber matrix, was utilized, illustrating the fact that increased resolution at much faster residence times may be obtained with emerging technologies such as FibroSelect as opposed to conventional bead-based chromatographic media.

Immobilized metal affinity chromatography. Although Protein A affinity chromatography may work well for symmetric and asymmetric bsAbs, fragment-based bsAbs that are not derived from the VH3 gene family [41, 42] and lack the Fc region typically utilize other affinity purification methods, with immobilized metal affinity chromatography (IMAC) being one of the most commonly employed methods for targets containing a polyhistidine tag as it allows for binding to immobilized metal ions on the resin [49] [50] [51] [52] [53] [54] [55] [56] . IMAC columns are typically charged with nickel, zinc or cobalt ions prior to EQ between pH7.2 and 8.0, followed by sample load and wash steps, with the elution performed with up to 500mM of imidazole ( Table 1) . As the generally low specificity of IMAC may lead to the binding and copurifying of non-specific proteins along with the target protein, low concentrations of imidazole may also be added to wash buffers to reduce non-specific binding [54, 56] .

An alternative, though less commonly employed, purification method for fragment-based bsAbs is the use of affinity ligands that are able to interact with the antibody's CH1 region or Kappa ( ) or Lambda (λ) LCs [57] [58] [59] [60] [61] [62] [63] , thus eliminating the need for the presence of a polyhistidine tag on the target molecule. Protein L affinity chromatography is an example of an affinity ligand that is able to interact with the variable region of the antibody's LC [64, 65] , with overall purification processes sharing similarities to that of Protein A and Protein G affinity chromatography (Table 1) . Although an overall low purity of 50-70% [57] was previously reported for a single Protein L affinity chromatographic step, with additional affinity chromatographic steps such as Protein A or Protein G chromatography required to achieve higher purity (Table 1) [58], we have recently reported a step recovery of 81% after a single Protein L affinity chromatographic step, achieving >90% monomeric purity. Further details of impurity removal using these affinity ligands will be discussed below ( Table 1) .

Removal of mispaired products KappaSelect affinity resin, in particular, has recently been demonstrated to be able to separate species possessing one LC constant region from that which consist of two LC constant regions, providing a convenient means by which mispaired homodimerized products can be separated from a Fab × scFv bsAb [59] . Using the same bsAb format, a CH1-based purification has also been shown to be effective at removing homodimers consisting of scFv segments [60] . In order to overcome LC mispairing, the use of sequential affinity chromatography has been demonstrated to be effective for bsAbs that have been coexpressed with two different LCs-and λ, where the CaptureSelect IgG-CH1 affinity resin was first employed to capture the antibodies, allowing contaminants and free LCs to flow through, followed by KappaSelect affinity resin, which removes λonly containing antibodies and finally LambdaFabSelect affinity resin to remove -only containing antibodies [61] . Such sequential affinity chromatography has also been explored through the raising of a panel of camelid antiidiotypic single-domain Ab fragments (VHH) that bind specifically to the correct HC/LC pairing of each arm. By coupling the respective VHHs to NHS-sepharose beads, the dual anti-idiotypic purification process was reported to be able to remove mispaired HC/LC products [66] . In a separate study [63] , the use of controlled conductivity at low pH, as demonstrated by varying NaCl concentrations, in Protein L chromatography has also been reported to be able to separate different antibody formats via their binding valency.

Removal of aggregates Although the bsAb aggregates have proven to be challenging to remove effectively using conventional methods such as Protein A affinity chromatography [14, 26] , we [62] and others [63] have recently found that Protein L affinity chromatography holds great promise in bsAb aggregate removal with the addition of salt additives. In particular, using an additive of Arg·HCl in the elution buffer at pH3.0, we reported that the aggregate amount can be reduced from 66.5% in the cell culture supernatant to 7.1% after Protein L affinity chromatography, with 81.3% recovery at a 10mg/mL load. Interestingly, we found that this occurs via a preferential strengthening effect of aggregate-Protein L interaction compared with the monomer-Protein L interaction, which is different from the widely reported chaotropic effect exerted by salt additives in Protein A chromatography [62] .

Charge-based purification is frequently employed in the form of ion exchange chromatography as polishing steps [14, 18, 19, 23, 26, 52, 67, 68] , and to a less extent as first capture steps [24, 54] (Table 2) . As a loading pH of 1-3units away from the isoelectric point of the target molecule is typically recommended for the use of ion exchange resins in the bind and elute mode, it is important to consider the isoelectric point (pI) of the target molecule before selecting the appropriate buffer pH due to the potentially huge variability of pI across different bsAb formats. bsAbs engineered to contain T cell receptor (TCR) constant regions, for instance, have relatively low pIs (6.1-6.5) [26] , and this is further reflected by the wide range of pH employed in the EQ buffer between pH5.0-8.1 and pH7.0-9.0 for cation exchange (CEX) and anion exchange, respectively, as shown in Table 2 . In comparison with mAbs, multiple peaks may be observed for bsAbs in the elution profile, which is typically obtained using increasing salt concentrations of up to 0.5M NaCl either in a step wise or gradient fashion, which may not correspond to aggregates or fragments; instead it may be a result of the multiple domain structure as well as the introduction of flexible linkers present in many of the bsAb formats that can lead to the capture of different conformational states [67] . Interestingly, while most of the methods employ an increasing salt concentration to elute the target and separate impurities (Table 2 ) and a pI difference of at least 0.5 between species is generally necessary for ion exchange chromatography [69] , it has been proposed that a highly linear pH gradient, achieved through the use of precise buffering solutions, is particularly useful in separating common LC bsAbs with differences in pI as little as 0.1 and differing in their sequence by only a single charged amino acid [68] . The utilization of ion exchange chromatography for the removal of mispaired products and fragments [24, 26, [69] [70] [71] [72] are discussed below.

Ion exchange chromatography has been reported to be able to separate homodimeric byproducts from target bsAbs through the introduction of mutations and/or by substituting certain regions of a mAb with that of a different subclass so as to increase their differences in pI [69, 70] . Non-TCRcontaining homodimeric byproducts, for instance, could be separated from the target bsAbs engineered to contain TCR constant regions in this manner as the latter tend to possess lower pIs of 6.1-6.5 [26] . Interestingly, using strong CEX resin, it has been demonstrated [71] that an alkaline pH near the pI of a scFv-IgG bsAb (pH8.1) increases the separation resolution between the bsAb and diabody-IgG mispaired product, which is a significant (>12%) byproduct in the generation of scFv-IgG due to mis-formed disulfide bonds. The enhanced separation resolution, which was attributed to the minimization of the protein net charge, therefore allowing the resin to better exploit subtle conformational or charge differences within the scFv domain resulting from the mis-formed disulfide bond, was demonstrated on both SP Sepharose HP and POROS HS50 resins.

CEX chromatography has also been reported to be effective for the removal of 3 / 4 antibodies through the use of a linear pH gradient with polyethylene glycol (PEG) in the mobile phase [72] . Fragments in the form of undesired diabodies, which have been reported to be expressed at levels up to 17% during the generation of symmetric bsAbs upon the dimerization of scFvs in a manner such that the variable domains pair with complementary domains of the other chain instead of the same chain, have been demonstrated to be reduced to <1% using CEX chromatography (salt gradient elution using 20mM bicine, pH8.1, 10-90mM NaCl over 20 column volume (CV)), as compared with <5% using Protein A chromatography (pH gradient elution from pH6.8 to pH2.5) [24] .

Size exclusion chromatography (SEC) is a frequently employed polishing method [18, 27-29, 49-54, 58, 73] that provides separation based on the hydrodynamic Antibody Therapeutics, 2021 85 radius, although the use of SEC is mostly limited to purification processes at the laboratory scale due to scalability issues. Phosphate-buffered saline (PBS) at physiological pH appears to be a commonly used size exclusion buffer (Table 2) , with the retention times and separation resolution between the target and impurities dependent on the differences between their molecular weights and thus hydrodynamic radius. Surprisingly, it was observed that bsAbs can be separated from their parental antibodies using the Zenix SEC-300 columns, despite similarities in their hydrodynamic radii [73] ; although the exact mechanism is unclear, it is likely to involve weak hydrophobic interactions in addition to size exclusion as the silica beads are coated with a proprietary chemically bonded stand-up monolayer. It should also be noted that besides purification purposes, size-based methods such as SEC is also commonly used for the analytical characterization of bsAb purity, providing valuable information on the presence of mispaired species, aggregates and truncated variants.

An alternative scalable size-based purification method is that of tangential flow filtration (TFF). High-performance TFF, which separates the target from impurities based on the sieving rates, was recently reported using a single-stage system consisting of a 50cm 2 C-screen regenerated cellulose Pellicon XL cassette with a 300kDa molecular weight cutoff [14] . Although this process provided good bsAb aggregate removal, it comes with additional challenges including large final product volumes as well as requiring large membrane areas, diafiltration buffers and long processing times.

Hydrophobic interaction chromatography (HIC) provides separation based on the overall differences in hydrophobicity of the product versus impurities, where a kosmotropic salt such as ammonium sulfate is typically used at high concentrations during the loading step to facilitate reversible interaction between the surface-exposed non-polar residues on the bsAb to the resin, with elution performed at lower salt concentration (Table 2) [74] [75] [76] [77] . HIC has been proposed in some cases to be able to separate homodimeric mispaired byproducts from the desired asymmetric bsAb product [76, 77] . Interestingly, a study of the chromatographic behavior of certain bsAbs on HIC revealed a multiple-peak elution behavior corresponding to different binding states on the chromatographic surface, with longer holding times enriching the late eluting peak and longer residence times and higher operating temperatures reducing the resolution of the peak elution profiles, indicating that the conformational changes occur on comparable time scales as that of the chromatographic separation [74] . It has also been reported that employing too high salt concentrations may lead to the formation of irreversible hydrophobic interactions between the resin and target, resulting in overall low yields [76] . In order to overcome potential difficulties in eluting hydrophobic proteins from the resin, mobile phase additives of up to 5% hexylene glycol and 1M arginine have been proposed to be effective in facilitating bsAb elution [75] .

As an alternative to hydrophobicity-based chromatographic methods, the use of crowding agents or high salts as a hydrophobicity-based precipitation method for bsAb purification have also been employed as the first purification step [58] or as a polishing step [14] . In cases where bsAbs formed inclusion bodies, the purification process begins with a series of wash steps, solubilization and refolding steps [18, 19] (Table 2) . Although this may be a multistep process in itself, an advantage of such treatment is that an affinity chromatography step is usually subsequently eliminated, as soluble host cell proteins and impurities will be removed during the isolation of the inclusion bodies, with further removal of insoluble impurities during the wash, solubilization and refolding steps.

Through the combination and utilization of more than one fundamental separation techniques, mixed-modebased purification methods demonstrate novel potential to further enhance the purification capabilities of existing purification platforms. Ceramic hydroxyapatite (CHT), for instance, is a mixed-mode chromatography method that provides separation based on CEX between the bsAb surface amines and the negatively charged CHT resin phosphate groups, as well as metal chelation between the bsAb carboxyl groups to the positively charged CHT resin calcium sites. As summarized in Table 2 , EQ is typically performed at near physiological pH in phosphate buffer, with elution of bsAbs performed with increasing concentrations of NaCl in EQ buffer [25, 30] . Other examples of mixed-mode resins include Toyopearl MX-Trp-650M, Capto adhere and Capto MMC, which are multimodal ion exchange resins that provide additional interactions such as hydrogen bonding and hydrophobic interactions. The salt-tolerant nature of some of these resins facilitates direct interfacing with post Protein A or CEX eluate, therefore allowing for further streamlining of the downstream process by obviating the need to remove chaotropic salts from the process stream [11] . These mixedmode resins have been proposed to be able to remove mispaired products, aggregates and fragments [11, 26, 30, 77, 78] , details of which are discussed below.

Toyopearl MX-Trp 650M mixed-mode resin has been shown to allow for the sequential separation of bsAbs containing both and λ LCs from homodimers [77] , with the and λ LCs containing bsAbs eluting at 75mM NaCl, whereas λ and homodimers were present in the flow through or eluted with 500mM NaCl, respectively. In addition, Capto MMC ImpRes and Capto adhere ImpRes have also both been proposed to be effective polishing steps [26, 78] , with Capto MMC ImpRes reported to be able to remove bsAb products such as hole-hole homodimer and half antibodies through the incorporation of additional pH and NaCl wash steps; this, however, occurs at a compromise of yield, with a reported yield of 40-50% at a load of 30mg/mL [78] . This, along with the different combinations of salts and pH that have been reported for elution [11, 26] , suggest that the careful optimization of conditions is of critical importance 86 Antibody Therapeutics, 2021 during the use of these mixed-mode resins in order to obtain products of both high purity and yield.

The mixed-mode CHT chromatography was also shown to be useful in separating 1 / 2 and 3 / 4 antibody fragments from a range of bsAbs [30] . Using post Protein A eluate containing ∼76% of 1 / 2 antibody in one case (CrossMab epidermal growth factor receptor (EGFR)-insulin-like growth factor (IGRF)), ∼16% of 1 / 2 antibody format, 77% bsAbs and∼6% aggregates in a second case (KiH angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF)) and 25% 1 / 2 antibody, 57% bsAb, 14% high molecular weight (HMW) species in a third case (KiH EGFR-IGFR), the 1 / 2 antibody fragments were observed to elute at a lower NaCl concentration, with the target bsAbs eluting at higher NaCl concentrations, yielding an overall purity of >80, >97 and∼97%, respectively, for all three cases. Similarly, when a post Protein A CrossMab Ang2/VEGF eluate containing 7.8% of 3 / 4 antibody fragment and 85% bsAb was loaded onto the CHT column, an enrichment of the 3 / 4 antibody in the side fractions of the elution peak was obtained with a NaCl gradient, with one fraction containing 95% bsAb and 1.5% 3 / 4 antibody and another fraction consisting of 78% bsAb and 9.1% 3 / 4 antibody.

For the separation of 5/4 antibodies (antibodies with an extra LC), CHT has also been proposed to be able to provide separation from the bsAb target [30] . Using a post Protein A KiH TWEAK/IL17 eluate containing ∼17% of 5/4 antibodies, the 5/4 antibodies were observed to elute at higher salt concentrations, whereas the bsAb eluted at lower salt concentrations.

The importance of obtaining a thorough understanding of the current bsAb downstream purification methods for the further development of optimal processes and novel technologies that can lead to an overall improvement of process productivity and product quality is accentuated by the massive clinical potential of bsAbs. In comparison with the substantial development in the area of upstream processing such as cell-line engineering and cell culture condition optimization, less has been reported in the development of novel downstream purification methods for bsAbs. As we have seen in this review, the wide variety of bsAb formats, along with the myriad of bsAb-specific byproducts generated, render it particularly challenging to develop effective processes that give products of high purity and yield within a limited number of purification steps.

Although Protein A and IMAC are the main affinitybased purification methods employed for the purification of bsAbs thus far, affinity chromatographic methods targeting other regions of the bsAb, such as the or λ LCs, present themselves as promising methods that may be utilized for the purification of bsAbs with or without the Fc region, with Protein L affinity chromatography in particular demonstrating superior aggregate removal properties. Decades of Protein A affinity purification optimization have led to the dramatic improvement in its performance and decrease in the cost of resins, with recent advances in fiber-based technology further pushing the limitations of conventional resin purification methods [79] , suggesting that the optimization of other forms of affinity purification that can overcome the specific challenges of bsAb purification may prove fruitful in the future towards increasing the overall productivity of bsAb purification.

Although purification modalities based on charge, size and/or hydrophobicity are frequently used as polishing methods, the use of these resins as capture steps for the purification of bsAbs have been reported to a lesser extent. Considering the fact that certain impurities such as diabodies can be removed from target symmetric bsAbs more effectively using CEX as compared with Protein A, the potential use of the former for impurity removal and as capture steps may be further investigated and optimized, especially in combination with novel continuous chromatographic technologies that can further improve the separation resolution between bsAb targets and impurities without compromising on product yield. Indeed, it has been demonstrated that the use of a multicolumn countercurrent solvent gradient purification resolved the conundrum of a trade-off between yield and purity by recycling bsAb target-containing side fractions to a separate column while collecting the pure bsAb fraction [80] . The ability of mixedmode resins to provide enhanced separation resolution over conventional methods also points towards the potential for greater utilization of them in future process development as well as encourages further development of other forms of mixed-mode resins.

Finally, the inclusion of additives such as salts and crowding agents have proven to lead to the enhanced separation of bsAbs from impurities in a wide variety of purification methods and may in some cases alter the interactions of the bsAbs with their respective ligands. As we have observed a different mechanism for the enhanced aggregate-monomer separation in the presence of salt additives in Protein L as compared with Protein A, it would be interesting to investigate the effect of different additives in other purification modalities as well so as to further exploit the different physicochemical properties and interactions between bsAbs and ligands for improved purification results.

This research is supported by the Agency for Science, Technology and Research (A * STAR).

Recombinant bispecific antibodies for cancer therapy

Bispecific T-cell engaging antibodies for cancer therapy

Bispecific antibodies for cancer therapy: the light at the end of the tunnel?

Dual targeting strategies with bispecific antibodies

The making of bispecific antibodies

Bispecific antibodies: a mechanistic review of the pipeline

A brief introduction of IgG-like bispecific antibody purification: methods for removing product-related impurities

Approaches to the Purification, Analysis and Characterization of Antibody-based Therapeutics

Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies

Knobs-into-holes' engineering of antibody CH3 domains for heavy chain heterodimerization

Development of purification processes for fully human bispecific antibodies based upon modification of protein A binding avidity

Bispecific antibodies rise again

A novel bispecific antibody against human CD3 and ephrin receptor A10 for breast cancer therapy

An integrated approach to aggregate control for therapeutic bispecific antibodies using an improved three column Mab platform-like purification process

Anti-tumor activity of stability-engineered IgG-like bispecific antibodies targeting TRAIL-R2 and LTbetaR

Development of tetravalent, bispecific CCR5 antibodies with antiviral activity against CCR5 monoclonal antibody-resistant HIV-1 strains

Evidence for intermolecular domain exchange in the Fab domains of dimer and oligomers of an IgG1 monoclonal antibody

T cell receptors

Therapeutic compounds and methods

Therapeutic bispecific antibody formats: a patent applications review

Il-17 binding compounds and medical uses thereof

Multifunctional antibodies binding to EGFR and MET

Chimeric protein composed of NGF antagonist domain and a TNFa antagonist domain

Anti-TNF-anti-IL-17 bispecific antibodies

Molecular engineering strategies and methods for the expression and purification of IgG1-based bispecific bivalent antibodies

A potential downstream platform approach for WuXiBody-based IgG-like bispecific antibodies

Bispecific, bivalent anti-VEGF/anti-ANG-2 antibodies

Bispecific antibodies specific for FAP and DR5, antibodies specific for DR5 and methods of use

Antibody that binds ERBB-2 and ERBB-3

Separation of bispecific antibodies and bispecific antibody production side products using hydroxyapatite chromatography

Sequential affinity purification of peroxidase tagged bispecific anti-SARS-CoV antibodies on phenylboronic acid agarose

Preferential species-restricted heavy/light chain pairing in rat/mouse quadromas. Implications for a single-step purification of bispecific antibodies

Simultaneous activation of T cells and accessory cells by a new class of intact bispecific antibody results in efficient tumor cell killing

A novel, native-format bispecific antibody triggering T-cell killing of B-cells is robustly active in mouse tumor models and cynomolgus monkeys

Fab-arm exchange combined with selective protein A purification results in a platform for rapid preparation of monovalent bispecific antibodies directly from culture media

Modulation of protein A binding allows single-step purification of mouse bispecific antibodies that retain FcRn binding

Rapid purification of human bispecific antibodies via selective modulation of protein A binding

Single-step Protein A and Protein G avidity purification methods to support bispecific antibody discovery and development

Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies

Development of a novel affinity chromatography resin for platform purification of bispecific antibodies with modified protein A binding avidity

Human IgA and IgG F(ab')2 that bind to staphylococcal protein A belong to the VHIII subgroup

Human IgM molecules that bind staphylococcal protein A contain VHIII H chains

Efficient protein A chromatography for bispecific antibodies

Removing half antibody byproduct by Protein A chromatography during the purification of a bispecific antibody

Removing a single-arm species by Fibro PrismA in purifying an asymmetric IgG-like bispecific antibody

Antibody variable region binding by staphylococcal protein A: thermodynamic analysis and location of the Fv binding site on E-domain

The interaction between different domains of staphylococcal protein A and human polyclonal IgG, IgA, IgM and F(ab')2: separation of affinity from specificity

VH3 family antibodies bind domain D of staphylococcal protein A

Binding molecules for BCMA and CD3

Nanocell targeting using engineered bispecific antibodies

Bispecific T cell engaging antibody constructs targeting a universally conserved part of the viral M2 ectodomain cure and prevent influenza A virus infection

MT110: a novel bispecific single-chain antibody construct with high efficacy in eradicating established tumors

Pharmaceutical compositions comprising bispecific anti-CD3, anti-CD-19 antibody constructs for the treatment of B-cell related disorders

CD19XCD3 specific polypeptides and uses thereof

A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity

Cloning, expression, purification and characterization of a bispecific single-chain diabody against fluoroquinolones and sulfonamides in Escherichia coli

Cloned transgenic farm animals produce a bispecific antibody for T cell-mediated tumor cell killing

Large-scale purification of r28M: a bispecific scFv antibody targeting human melanoma produced in transgenic cattle

Separating antibody species containing one and two kappa light chain constant region by KappaSelect affinity chromatography

High-throughput generation of bipod (Fab × scFv) bispecific antibodies exploits differential chain expression and affinity capture

Exploiting light chains for the scalable generation and platform purification of native human bispecific IgG

Investigation of the effect of salt additives in Protein L affinity chromatography for the purification of tandem single-chain variable fragment bispecific antibodies

Controlled conductivity at low pH in Protein L chromatography enables separation of bispecific and other antibody formats by their binding valency

A novel bacterial cell wall protein with affinity for Ig L chains

Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins

Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy

Chromatographic behavior of bivalent bispecific antibodies on cation exchange columns. II. Biomolecular perspectives

Purification of common light chain IgG-like bispecific antibodies using highly linear pH gradients

Methods of modifying antibodies for purification of bispecific antibodies

Identification and multidimensional optimization of an asymmetric bispecific IgG antibody mimicking the function of factor VIII cofactor activity

Alkaline cation-exchange chromatography for the reduction of aggregate and a mis-formed disulfide variant in a bispecific antibody purification process

Solvent modulated linear pH gradient elution for the purification of conventional and bispecific antibodies: modeling and application

Analysis and purification of IgG4 bispecific antibodies by a mixed-mode chromatography

Chromatographic behavior of bivalent bispecific antibodies on hydrophobic interaction chromatography columns

Use of mobile phase additives for the elution of bispecific and monoclonal antibodies from phenyl based hydrophobic interaction chromatography resins

Single-step purification of bispecific monoclonal antibodies for immunotherapeutic use by hydrophobic interaction chromatography

Methods of purifying bispecific antibodies

Removal of half antibody, hole-hole homodimer and aggregates during bispecific antibody purification using MMC ImpRes mixed-mode chromatography

HiTrap Fibro PrismA units HiScreen Fibro PrismA units

Purifying common light-chain bispecific antibodies

Both authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.

The data that support the findings of this study are openly available.Antibody Therapeutics, 2021 87