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REVIEW ARTICLE ISSN: 1389-2037 eISSN: 1875-5550

Multimodal Chromatography for Purification of Biotherapeutics – A Review

Impact Factor: 2.696

BENTHAM SCIENCE

Vivek Halan1, Sunit Maity1, Rahul Bhambure2 and Anurag S. Rathore3,* 1 3

Zumutor Biologics Private Limited, Yeshwanthpur, Bangalore, India; 2National Chemical Laboratory, Pune, India; Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, India

ARTICLE HISTORY Received: July 12, 2018 Revised: August 08, 2018 Accepted: September 05, 2018

Abstract: Process chromatography forms the core of purification of biotherapeutics. The unparalleled selectivity that it offers over other alternatives combined with the considerable robustness and scalability make it the unit operation of choice in downstream processing. It is typical to have three to five chromatography steps in a purification process for a biotherapeutic. Generally, these steps offer different modes of separation such as ion-exchange, reversed phase, size exclusion, and hydrophobic interaction. In the past decade, multimodal chromatography has emerged as an alternative to the traditional modes. It involves use of more than one mode of separation and typically combines ion-exchange and hydrophobic interactions to achieve selectivity and sensitivity. Over the last decade, numerous authors have demonstrated the significant potential that multimode chromatography offers as a protein purification tool. This review aims to present key recent developments that have occurred on this topic together with a perspective on future applications of multimodal chromatography.

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DOI: 10.2174/1389203718666171020103559

Keywords: Multimodal chromatography/mixed-mode chromatography, MMC, mAb, Fab, antibody fragments, Biopharmaceuticals, therapeutic proteins, host cell proteins, product related impurities, process related impurities. 1. INTRODUCTION

Downstream processing plays a critical role in biopharmaceuticals manufacturing. In most existing process platforms for production of biopharmaceuticals, overall time scale and economics of downstream processing is driven and is limited by the chromatographic separation. Chromatography acts as a workhorse for time and cost effective purification of biotherapeutics. Affinity, ion exchange, size exclusion, and hydrophobic interaction chromatography are some of the most commonly used chromatographic modes for therapeutic protein purification. Selection of a specific chromatography step is crucial for cost effective manufacturing as well as for achieving consistent product quality. Various process platforms have been developed for the different categories of therapeutic proteins such as monoclonal antibodies or antibody fragments. Such platforms use a combination of the different chromatography techniques to achieve high recovery as well as obtain elective clearance of the various process and product related impurities. Recently, the biopharmaceutical industry has seen advent of multimodal chromatography. As the name suggests, multimodal chromatography is based on the multiple modes/

*Address correspondence to this author at the Department of Chemical Engineering, Indian Institute of Technology, Hauz Khas, New Delhi, 110016, India; Tel: +91-9650770650; E-mail: [email protected]; Website: www.biotechcmz.com

1875-5550/19 $58.00+.00

types of the interaction between the analyte and resin ligand. Multiple interactions between different types of analytes can thus be effectively used to achieve selective clearance of the various types of impurities in a single step, thereby resulting in higher recovery and enhanced process throughput. This review focuses on applications of multimodal or mixed mode chromatography for purification of biotherapeutics including the various resins that are available in the market and their applications towards purification of different classes of biotherapeutics including monoclonal antibodies, antibody fragments, and fusion proteins. 2. NEED FOR THE MULTIMODAL CHROMATOGRAPHY

Recent advances in the microbial as well as mammalian cell culture technique has led to significant improvement in therapeutic protein titers. This has resulted in a focus on downstream processing for improving product recovery, purity, and yield [1]. Typical challenges involved in purification of therapeutic proteins include removal of various product related (aggregates, fragments, oxidized, reduced forms), host cell related impurities (host cell proteins, DNA), and process related impurities (colour components, media components). Existing downstream processing platforms for purification of different microbial and mammalian protein products rely on integration of various modes of chromatography techniques such as ion exchange chromatography, hydrophobic interaction chromatography, and affinity separation to achieve this goal. During each chromatography © 2019 Bentham Science Publishers

Multimodal Chromatography for Purification of Biotherapeutics

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the protein and C-sites on CHT support are two of the major types of multimodal interactions [7]. 4. APPLICATIONS OF MULTIMODAL CHROMATOGRAPHY FOR PURIFICATION OF BIOTHERAPEUTICS: 4.1. Monoclonal Antibodies Monoclonal antibodies have gradually emerged to become the largest entity amongst the various kinds of therapeutic protein drugs available commercially in the market [12]. Typical platform purification strategy for the monoclonal antibody therapeutics involves use of protein A affinity chromatography followed by an ion exchange chromatography purification for selective clearance of various process and product related impurities. High cost of the Protein A resin, limited life cycle, and Protein A leaching are some of the critical bottlenecks associated with the existing mAb purification platforms. Maintenance of the chromatography resin performance is also a major concern in large-scale manufacturing of monoclonal antibodies. Multimodal chromatography can offer a good alternative to overcome various bottlenecks at capture, intermediate, and polishing steps of mAb purification.

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step, selective interactions between target product/impurity and chromatography ligand in either “positive” or “negative” mode are used effectively to achieve selective impurity clearance. Due to the variable “nature” of the process and product related impurities, it is presently not possible to have a single step chromatographic purification platform for therapeutic protein manufacturing. Further, inability to load high conductivity feed materials is another critical drawback associated with conventional ion exchange chromatography. The idea of “multimodal chromatography ligand” originated from the necessity to obtain high throughput purification by exploring variable interactions between the analyte and chromatography matrix for achieving selective clearance of the impurities in a single step. Typically, cell culture harvest and Protein A chromatography outputs contain high amount of salt and this makes binding of the product to an ion exchange chromatography column difficult. The new generation multimodal ion exchange resins offer the required salt tolerance and thus a distinct advantage over conventional ion exchange resins.

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3. MULTIMODAL CHROMATOGRAPHY: TYPE OF LIGANDS AND MODE OF INTERACTION WITH ANALYTE

Multimodal or mixed-mode chromatography involves selective interactions between the chromatography ligand with the analyte molecule through different types of interactions which can be either ionic, hydrophobic or else may involve hydrogen bonding, or even Vander Waals interactions. The strength and mode of these interactions between the ligand and the analyte are driven by the resin chemistry, experimental conditions, and the target molecule. Ligands play a vital role in the development of mixed mode chromatographic resins and various types of mixed-mode ligands with different chemical structures have been developed over the years. In the last two decades, numerous multimodal resins have been introduced for recombinant protein production in the biopharmaceutical industry and (Fig. 1) represents some of the commercially available mixed mode ligands (resins) for protein purification. Table 1 enlists the existing multimodal ligands in the market and the nature of the interactions during binding of an analyte to the matrix, though more sophisticated computational and experimental methods in high throughput manner are required to develop novel mixedmode ligands [2]. With a proper balance between the hydrophobic and ionic moieties in the mixed-mode ligand, the adsorbent can offer salt-tolerant property, better separation and high binding capacity with which a wide range of therapeutic proteins can be purified. In the case of hydrophobic charge induction chromatography with selection of an appropriate pKa values, electrostatic repulsion between the analyte and the resin ligand can lead to selective elution. Capto series multimodal resins from GE healthcare offer binding of the analyte based on hydrophobic and ionic interactions [3-6]. Ceramic Hydroxyapatite (CHT) is a multimodal resin that is based on two functional groups: positive charged calcium ions (C-site) and negative charged phosphate groups (P-sites)(Fig. 2). The electrostatic interaction of both C- and P-sites with charged groups of protein surface and the metal affinity interaction between carboxyl groups of

Kaleas KA, et al. have evaluated the use of multimodal chromatography using Capto MMC resin for selective capture and purification of mAb from the harvested mammalian cell culture fluid. They have observed that Capto MMC can provide an efficient alternative to protein A chromatography with DBC ranging from 10-53 g/l observed for the various harvests. Different elution strategies like pH based elution gradient, salt based elution gradient, and sodium chloride gradient with urea can offer a product that is similar to the affinity capture process [13]. Various researchers have successfully demonstrated use of hydrophobic charge induction resins for effective capture of monoclonal antibody products. However, non-specific interactions and lower efficiency (than protein A resins) are some of the bottlenecks that have been associated with the use of hydrophobic charge induction chromatography (HCIC) [14]. Combining HCIC chromatography with other chromatography steps such as ion exchange chromatography or hydrophobic interaction chromatography has been successfully used to improve mAb capture. Phenyl boronic acid has also been demonstrated as a multimodal ligand for effective capturing of the mAb therapeutics from CHO harvest with recoveries as high as 99.00 % and a product purity of 81.00 % have been reported [15]. Multimodal chromatography can also be effectively used as a polishing step for removal of various product related impurities like aggregates. Researchers have evaluated the performance of Capto adhere and two different multimodal chromatography (benzylamine and butylamine) functionalities on aggregate clearance in mAb products. They concluded that simultaneous hydrophobic and electrostatic interactions in multimodal chromatography effectively improve process capability with respect to aggregate clearance for mAb products [16]. A group of four mAbs and two model proteins have also been used to study the binding mechanism on Capto MMC resin in presence of various mobile modulators such as ethylene glycol, arginine, sodium thiocyanate, urea, and ammonium sulphate [17]. The results show the

6 Current Protein and Peptide Science, 2019, Vol. 20, No. 1

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Fig. (1). Illustration of the different ligands that are available in various multimodal resins. Bestarose Diamond MMA and Bestarose Diamond MMC (www.bestchrom.com); MEP Hypercel, PPA Hypercel and HEA Hypercel(www.pall.com); Capto adhere and Capto MMC (www.gehealthcare.com); Nuvia C Prime (www.bio-rad.com) Toyopearl MX-Trp-650M (www.tosohbioscience.com); Eshmuno® HCX (www.emdmillipore.com).

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Multimodal Chromatography for Purification of Biotherapeutics

Table 1.

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Various commercially available multimodal resins and mechanism of interaction between the analyte and the resin.

Sr. No.

Resin

Type of interaction during binding

Elution technique

1

Capto MMCTM [2]

Hydrophobic and ion exchange

High salt and pH gradient

2

Capto AdhereTM [3]

Hydrophobic and ion exchange

High salt gradient

3

CHTTM/ CFTTM (Ceramic hydroxyapatite/ Ceramicfluorapatite) [6]

Affinity and ion exchange

Phosphate/sulphate based gradient elution

4

HEA HyperCelTM PPA HyperCelTM [8]

Hydrophobic and electrostatic repulsion

Changing the pH < pKa of the ligand

5

MEP HyperCelTM [9]

Hydrophobic and electrostatic repulsion

Changing the pH < pKa of the ligand

Hydrophobic and ion exchange

Product of interest in flow-through

TM

Capto Core 700

[4, 5]

7

Toyopearl MX-Trp-650M [10]

Hydrophobic and ion exchange

High salt gradient

8

Eshmuno® HCX [11]

Hydrophobic and ion exchange

High salt gradient

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significant decrease in HCP level when mobile phase modulators are used in Capto MMC chromatography for purification of mAbs as well as of model proteins. In addition to pH and salt, arginine has also been shown to be useful for eluting proteins from Capto MMC resin and has been compared to salt and urea for mAb purification. Despite a steady improvement in our understanding of how multimodal chromatography works, more needs to be done to understand the application of arginine as an eluent in multimodal chromatography [18]. Removal of heterogeneous charged aggregates during mAb purification has been shown to be significantly better in multimodal chromatography than conventional chromatography [19]. Structure based mixed mode adsorbents have also been designed and synthesized for mAb purification [20]. Another category of the multimodal resin (i.e., ceramic hydroxyapatite, CHT) has been effectively used as a polishing step for reduction of aggregates for mAb purification [21]. Chromatographic behavior of multimodal resin Capto adhere has been evaluated and compared with ion exchange and hydrophobic interaction chromatography resins for purification of several mAbs such as Cetuximab and Bevacizumab at different pH and salt concentrations [22]. It was observed that multimodal chromatography yielded superior performance when compared to other chromatography modes. Researchers have investigated performance of three multimodal resins namely Mercapto-Ethyl-Pyridine (MEP) HyperCel, Capto adhere, and ceramic hydroxyapatite/fluoroapatite (CHT/CFT) for mAb purification [23]. The results indicate that multimodal resins offer different selectivity than conventional chromatography modes with respect to removal of host cell proteins, high molecular weight species, and insoluble aggregates [23]. PPA HyperCel, HEA HyperCel, MEP HyperCel, and Capto adhere multimodal resins have been evaluated for mAb capture using the design of experiments and the effectiveness of multimodal chromatography has been demonstrated [24]. Bispecific IgG4 antibodies have also been separated by multimodal chromatography using a Sepax Zenix SEC-300 column, where bispecific hybrids have been shown to result in a more efficient separation than their parental monospecific antibodies [25].

A panel of four mAbs and two model proteins have been used to study the binding on mixed mode resins in presence of mobile phase modulators such as ethylene glycol, arginine, sodium thiocyanate, urea, and ammonium sulphate. The data exhibited a significant decrease in HCP level when mobile phase modulators are used. In another application, to determine optimal buffer conditions for capture and elution under static and dynamic adsorption conditions, pyridine based adsorbents have been screened in monoclonal antibody binding assays [26]. Capto MMC resin has been used for mAb purification from harvested mammalian cell culture fluid (HCCF) and the feasibility of purifying mAb from HCCF with purity that is comparable to the affinity capture process has been demonstrated [27]. Phenylboronic acid (PBA) has been used as a multimodal ligand in purification of immunoglobulin G (IgG) HCCF of CHO cells. Researchers have compared the quality of a mAb product that is manufactured using either a multimodal chromatography or hydrophobic interaction chromatography as a post-Pro A polishing step. The results show that both steps result in comparable product quality. Advantages of using MEP Hypercel mixed mode resins include the elimination of the buffer exchange step as the feed material can be loaded at low pH directly after eluting from Pro A column in addition to using MEP Hypercel as a capture step for CHO cell harvest [28]. Two mixed mode chromatography steps were used in monoclonal antibodies purification and compared with conventional purification process, where Pro A chromatography was used as a capture step followed by AEX chromatography and CEX chromatography or vice versa. The process flow was evaluated and compared by measuring the yield, purity and product related impurities such as HCP, HCD, aggregates. In another study, two mixed mode chromatography steps followed by a final polishing step using an AEX membrane adsorber was found to yield product quality that is comparable to what is obtained using a traditional purification platform [29]. Toyopearl MX-Trp-650M mixed mode resin has been evaluated for removal of mAb aggregates [30]. It has been observed that different mixtures of chaotropic and kosmotropic salts influence the binding and

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elution mechanisms of mixed mode chromatography and separation of aggregates. Developing new mixed mode ligands is gaining momentum in recent years. Researchers have immobilized four different ligands such as 4’-terpyridiny sulfanylethylamine(4’TerPSEA),5-bromo-2-pyridinylsulfanylethylamine(5-Br-2PSEA),quinolinylsulfanyl ethylamine (2-QSEA), and 4pyridinylsulfanylethylamine (4-PSEA) on Sepharose fast flow base matrix as a mixed mode resin for separation of IgG1, IgG2 and IgG4 mAbs. The data suggested that these pyridinyl based ligands allow for mAbs to elute at pH between 5 to 7, which is better from product quality perspective when compared to elution at acidic pH in conventional Protein A chromatography. Moreover, this chemical affinity mixed mode ligands have been found to offer effective clearance of HCPs from CHO cell culture supernatant, thereby offering an alternative to Protein A chromatography [31].

of 80 - 90% and reduction of endotoxin by 90% [36]. CHT type I ceramic hydroxyapatite and Capto MMC resins have also been evaluated for purification of anticancer minibody produced in mammalian cell culture [37]. Multimodal chromatography has the potential to emerge as a tool for achieving efficient purification of ScFv and fab fragments that are generated during phage display and yeast display libraries. Small bispecific antibody fragments such as BiTes (Bispecific T cell Engagers) and diabodies can also be readily purified using multimodal chromatography. 4.3. Peptides In recent years, peptides have also emerged as promising therapeutic agents for treatment of cancer, diabetes, cardiovascular diseases, and others. Several peptide based vaccines have undergone phase I and II clinical trials with promising results in immunological as well as in clinical responses [38]. A novel multimodal sorbent based on silica matrix with pentafluorophenyl (PFP) ligand has been shown to offer reversed-phase (RP) retention along with a relatively mild cation exchange interaction resulting in enhanced selectivity for phosphopeptides and sialylated glycopeptides produced in yeast and human serum tryptic digests [39].

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A novel ligand 4-(1H-imidazol-1-yl) aniline has been immobilized on Sepharose CL-6B base matrix for mAb purification. To elucidate the binding mechanism, separation of BSA and IgG was examined at various pH and salt concentrations. The results demonstrated the effectiveness of the mixed mode resin for mAb capture and purification [32]. A pyridine-based compound, 4′-terpyridinylsulfanylethylamine (4′-TerPSEA), has been used as a ligand in mixed mode chromatography for purification of an IgG1 mAb from CHO cell culture supernatant [33]. The novel resin offered performance that was comparable to that of Pro A affinity chromatography.

Halan et al.

Based on the findings reported above, it is clear that multimodal chromatography has become indispensable for mAb purification and is likely to find permanence in newer purification platforms. 4.2. Antibody Fragments

Antibody fragments is an emerging class of biotherapeutics that offers advantages including improved tumour/tissue penetration and more efficient manufacturing using E. coli as an expression host. Multimodal chromatography resins such as Capto MMC have been effectively used for capture of single chain variable antibody fragments and product recovery of 80-90% with endotoxin reduction by 90 % have been achieved [34]. Researchers have recently investigated protein selectivity in Capto MMC and NuviaTM cPrimeTM resins using in silico designed Fab fragment library [35]. Fab molecules from library were selected for the study, where the binding between the Fab fragment and the multimodal resin was achieved by the CDR region. In another Fab library, the binding between the Fab fragment and multimodal resin was facilitated by a different region. Single chain variable fragments against epidermal growth factor receptor (EGFR) expressed in periplasmic space of E. coli were captured by multimodal chromatography (Capto MMC resin) and the proteins were extracted from periplasm by osmotic shock and subjected to precipitation at pH 3.5 to achieve removal of host cell proteins. Advantages included the possibility of directly loading the elute form the multimodal column to the next chromatography step (ion exchange). The binding of the single chain variable fragment (scFv) on the resin at high conductivity has been demonstrated with a product recovery

Researchers have successfully modelled the retention of insulin on cation-exchange/reversed-phase (CIEX-RP) multimodal chromatography under diluted conditions [40]. Retention times were measured at various salt concentrations, pH, and temperatures. The data showed that an increase in temperature increases the binding strength and that pH and salt also affect the binding of tripeptides [41]. Glutathionebased HILIC/CEX mixed-mode stationary phase (Click TEGSH) has been successfully used for analysis of tryptic digest of human serum albumin (HSA) [42]. The resulting peptides were found to be well-resolved and as many as 86 peptides were identified. Binding and elution profiles of hexapeptides from the library was studied using ceramic fluorapatite (CFT) chromatography and compared with the chromatography behaviour with the traditional immobilized metal affinity chromatography [43]. Results suggest that CFT chromatography is a better alternative to IMAC. Overall, as in previous applications, multimodal chromatography has emerged as a better alternative for peptide purification as well. 4.4. Recombinant Proteins Recombinant proteins fused to maltose-binding protein (MBP) have been purified from E. coli cell lysates using two multimodal resins and the results compared to that using affinity resins [44]. The results indicated that the step recovery of purified proteins was significantly higher in multimodal resins while the purity was comparable. In another application, recombinant human VEGF165 has been expressed in E. coli periplasm as an inclusion body in a high cell density fermentation process and after refolding, the product has been captured by multimodal chromatography (Capto MMC resin) [45]. The product was loaded at pH ~ 8.7 (>pI of VEGF165) and eluted by high concentration of L-arginine-hydrochloride. Recombinant human serine proteinase inhibitor Kazal-type 6 (SPINK6) has been expressed in

Multimodal Chromatography for Purification of Biotherapeutics

Pichia pastoris and captured from culture supernatant by Capto MMC resin [46]. Mixed mode resins such as Capto MMC and Capto adhere have been used for purification of Fc fusion proteins, especially for the removal of misfolded proteins [47]. G-protein βγ subunit (Gβγ)-responsive phosphoinositide 3-kinase (PI3-kinase) and Rhodopsin kinase, a protein kinase, have been purified using hydroxyapatite chromatography [48, 49]. Capto adhere has been examined for intermediate purification of malaria vaccine D1M1 as an alternative to hydrophobic and ionic interaction chromatography [50]. Poly(ethylenimine) (PEI)-grafted Sepharose FF resins have been modified with hydrophobic benzoyl groups to create a multimodal resin and successfully used for purification of BSA and γ-globulin proteins [51].

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below. Mixed mode chromatography resin Capto MMC has been successfully used for intermediate purification of insulin. The salt tolerance of Capto MMC resulted in a higher binding capacity than the ion exchangers [59]. Oxidized, reduced, and fMet forms are critical quality attributes of granulocyte colony stimulating factor (GCSF) [60]. Mixed mode chromatography resins, namely HEA Hypercel, PPA Hypercel and Capto MMC resins, have been evaluated for GCSF purification and the results demonstrate highly efficient removal of the product variants using multimodal chromatography and very high product purity (> 99%) with high recovery (> 85%) was reported. Aliphatic based and aromatic based mixed mode resins, namely MEP Hypercel and PPA Hypercel, have been evaluated for purification of BSA, ovalbumin, ovotransferrin, IgG, catalase, lysozyme, and α-chymotrypsinogen-A [61]. It was observed that ionic interactions, hydrophobic interactions and hydrogen bonds play a critical role in protein separation along with the pH of the buffer and salt conditions.

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Displacer analogues with varying degrees of electrostatic, hydrophobic and hydrogen bonding moieties have been investigated for mixed mode property using model proteins such as Ribonuclease A (RNase A), Cytochrome C (CytC), and α-Chymotrypsinogen A (α-Chy A) [52]. The efficacy and selectivity of a library of potential displacers has been investigated and displacers with high positive charge and high hydrophobicity have been reported to show superior protein displacement [53]. Multiple interaction moieties have been found to be more efficient than single moieties and it has been observed that the selectivity of multimodal resins can be improved by use of a particular type of displacer under non-linear competitive binding conditions. Controlled pH gradients resulted in better selectivity in comparison to using salt gradients [53]. Recombinant human vascular endothelial growth factor (rhVEGF) expressed in E. coli as inclusion bodies has been successfully captured from lysates using Capto MMC at both pilot and manufacturing scales [54]. Hyper glycosylated rHRP expressed in Pichia pastoris has been captured by MEP Hypercel with about 90% recovery (better than other chromatography types) [55]. P-aminobenzamidine, a multimodal ligand immobilized on magnetic beads, has been used for purification of trypsin from bovine pancreas with 97% recovery and 86 % purity using a fluidized bed reactor [56]. Three mixed mode resins, namely MEP Hypercel, PPA Hypercel, and HEA Hypercel have been screened for capture of recombinant allergen rBet v 1a (major white birch -Betula verrucosa) using a high throughput platform and the results confirmed that no pre-treatment was required for crude sample loaded on PPA Hypercel and HEA Hypercel, while pre-treatment was required when crude extract is loaded on MEP Hypercel [57]. This strategy has been shown to significantly reduce process time as well as the cost of purification. Human recombinant F(ab’)2 has been expressed in sf9 insect cells and three mixed-mode sorbents (PPA Hypercel, HEA Hypercel, MEP Hypercel) have been examined along with two ion exchange sorbents (Q and S Ceramic HyperD) [58]. Surface-enhanced laser desorption/ionization - Mass Spectrometry was used to identify the optimal binding and elution conditions for capture of human recombinant F(ab’)2 from crude extract of Sf9 insect cells. Separation of human recombinant F(ab’)2 protein from host cell contaminants was found to be better in PPA Hypercel and HEA Hypercel than with MEP Hypercel. F(ab’)2 was captured without pre-treatment of the load sample and recovered in pH range between 4 to 5 in MEP Hypercel, whereas in PPA Hypercel proteins were recovered at pH 4 or

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More recently, multimodal chromatography has also been used for purification of plant based proteins [62]. The major bottleneck in downstream processing of expressed proteins from plants is the requirement of disruption of plant tissues and crude extraction of the product of interest. Once the product is isolated from the plants, there is a requirement of capturing the product from the crude sample with high recovery and purity and multimodal chromatography has been demonstrated to fit the bill successfully. Capto MMC and Capto adhere mixed mode resins have been used for purification of etanercept (Fc-fusion protein) and bovine serum albumin (BSA) [63]. Effect of pH, salt and arginine concentration on separation in multimodal chromatography was examined and it was found that misfolded or aggregated etanercept proteins separated well at higher concentrations of salt and arginine. Similarly, oligomers of BSA could be more effectively separated using an arginine gradient than the salt gradient as arginine hydrochloride weakened the hydrophobic interaction. Two agarose based mixed mode resins, namely AD agarose containing aminodecyl ligand and CPAD agarose containing N- (3 – carboxypropionylaminodecyl) ligand, have been reported to be used for purification of lipophilic proteins [64]. In another application, purification of recombinant Glutathione S Transferase (GST) from E. coli has been achieved using HEA HyperCel and PPA HyperCel [65]. In summary, separation of recombinant proteins is somewhat more challenging as the products offer significantly more diversity than the monoclonal antibodies. However, multimodal chromatography seems to offer similar success for purification of these compounds especially in capturing them from crude harvest samples. 5. OLIGONUCLEOTIDES Synthetic oligonucleotides have also seen increasing interest over the last 15 years as potential therapeutic drugs for treating various diseases such as cancer. In production of these products, product related impurities that are structurally quite similar to the product are a major challenge. Researchers have reported successful use of multimodal (re-

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versed-phase/weak anion exchanger) chromatography in separating entities differing by only 1, 2 or 3 nucleotides in length [66]. When compared to C18 HPLC, multimodal chromatography offered better selectivity and efficiency. A series of multimodal resins have been created by varying ratio of n-octyldimethylsilane and either 3chloropropyldimethylsilane or 4-chlorobutyldimethylsilane during silanization [67]. These resins have been demonstrated to offer excellent separation of single-stranded oligonucleotides and double-stranded DNA restriction fragments under relatively mild mobile phase conditions. In another study, change of mobile phase from ion-pair reversedphase liquid chromatography (IP-RPLC) to strong anionexchange liquid chromatography (SAX-LC), where NaCl or NaBr gradients are used, has shown to result in very different selectivity for purification of RNA oligonucleotides [68].

tion of pH, temperature, acetonitrile concentration, and various mobile phase buffers along with high selectivity. Literature suggests that there is significant potential in use of multimodal chromatography for analytical separation of a variety of compounds including chemical components, isoforms, and active pharmaceutical ingredients (APIs) where it offers better selectivity and shorter process time compared to the traditional methods [76]. In addition to above mentioned MMC resins, few other resins are widely being used in downstream processing of various proteins. Avantor has MMC resins of different functionalities which includes Bakerbond Poly PEI (Weak Anion Exchanger, amino groups with different pKa ), Bakerbond PolyABx (weak cation exchanger with weak anion exchange sites), Bakerbond Poly CSX (strong cation exchanger with weak cation and weak anion exchange sites), Bakerbond PolyQUAT (strong anion exchanger with weak anion exchange sites), Bakerbond Poly HI-Propyl resin containing Hydrophobic Interaction (HIC) with weak anion exchange sites [77]. Prometic Bioseparations Ltd has developed a multimode Mimetic Ligand™ library in a 96-micro column block format for screening the conditions in flow though mode [78]. JNC Corporation company developed cellulose based two mixed mode resins namely Cellufine MAX Amino Butyl and Cellufine Max IB for biopharmaceutical applications [79].

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Separation of single stranded DNA from double stranded DNA molecules has been attempted using Capto adhere multimodal resin with elution using a linear NaCl gradient [69]. It was observed that single-stranded DNA molecules could be separated from double-stranded DNAs due to the higher hydrophobicity of single strand DNA molecules. Plasmid DNA molecules have also been purified from E. coli cell lysate using Capto adhere resin, where a step NaCl gradient was used for separation of plasmid DNA from proteins and endotoxins. In another application multimodal chromatography involving use of two modes of separation (reversed-phase and ion-exchange) has been successfully used for HPLC based purification of RNA oligonucleotides [70]. NaCl and NaBr based gradients were used to achieve improved separation. Hydrophilic Interaction Liquid Chromatography/ Weak Cation Exchange (HILIC/WCX) mutimodal chromatography has been used for separation of chitooligosaccharides (COS) and it was observed that higher concentration of acetonitrile resulted in better separation [71]. Primesep SB, a multimodal resin, has been used to separate sugar phosphate, nucleoside mono, di- and triphosphates and nucleotide sugar without use of an ionpairing agent in the mobile phase and the separated product was subjected to mass spectrometry analysis. Effect of pH, buffers, percentage of organic solvents was studied in separation of nucleotide sugars [72]. Amplified DNAs from PCR mixture have been separated using Capto adhere resin in a single step resulting in improved recovery, selectivity, linearity, and accuracy [73].

Halan et al.

The success of multimodal chromatography in oligonucleotide separation demonstrates the versatility of the tool and the significant possibilities that it offers. 6. NON-BIOPHARMACEUTICAL APPLICATIONS Mixed mode HPLC coupled with charged aerosol detection (CAD) has been successfully used for separation of lithium metal ions [74]. The quantitative analysis of alkali metal showed better sensitivity than other analytical methods. Silica based mixed mode resin C18-DTT (dithiothreitol) silica(SiO2) has been developed and used for separation of nonsteroidal anti-inflammatory drugs, aromatic carboxylic acids, alkaloids, nucleo analytes and polycyclic aromatic hydrocarbons [75]. The results exhibited excellent stability as a func-

SUMMARY AND CONCLUSION This review presents the various applications of multimodal chromatography for purification of biotherapeutics. The significant advantages that this tool offers are evident from the diverse applications that we have covered in the review and the efficacy of this tool. These include the diversity in the type of ligands resulting in significantly higher selectivity, improved cost effectiveness and ligand stability, and wider operating window for conductivity and pH of the feed material. A key area of success is the ability of multimodal chromatography for capture of product from relatively crude process stream. We expect a continuation of success of this technology in times to come. New ligands and base matrix are likely to come to market resulting in more novel applications and further dominance of multimodal chromatography not only in the process but also in the analytical separations. We hope that this review serves as a useful compendium of information for those engaged in bioseparations. ABBREVIATIONS MMC

= Mixed mode Chromatography/Multimodal chromatography

mAb

= Monoclonal antibody

VEGF

= Vascular Endothelial growth factor

HPLC

= High Pressure Liquid Chromatography

FPLC

= Fast Protein Liquid Chromatography

CHO

= Chinese Hamster Ovary

Multimodal Chromatography for Purification of Biotherapeutics

CONSENT FOR PUBLICATION

Current Protein and Peptide Science, 2019, Vol. 20, No. 1 [17]

Not applicable. CONFLICT OF INTEREST The authors declare no conflict of interest, financial or otherwise.

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ACKNOWLEDGEMENTS Declared none.

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