Membrane Chromatography for Biomolecule Purification

CHAPTER 6

Membrane Chromatography for Biomolecule Purification Cristiana Boi Department of Civil, Chemical, Environmental and Materials Engineering, DICAM Alma Mater Studiorum-Università di Bologna, Bologna, Italy

Symbols DBC10% DBC100% m t

dynamic binding capacity at 10% breakthrough dynamic binding capacity at 100% breakthrough mass time

Abbreviations AEX CEX DNA D-PAM ELISA HIC HIS IEX IgG IMAC mAbs MCSGP MW pDNA PTFE PVC RNA USD VLP

anion exchange cation exchange deoxyribonucleic acid protein A mimetic with all amino acids in the D form enzyme-linked immunosorbent assay hydrophobic interaction chromatography histidine ion exchange immunoglobulin G immobilized metal affinity chromatography monoclonal antibodies multicolumn counter-current solvent gradient purification molecular weight plasmid DNA polytetrafluoroethylene polyvinyl chloride ribonucleic acid United States Dollars virus-like Particle

1 Membrane Chromatography Membrane chromatography was developed in the 1980s with the aim to overcome the drawbacks of packed-bed columns for which intraparticle diffusion is the limiting transport phenomenon (Klein, 1991; Brandt et al., 1988). Moreover, the high material and operational Current Trends and Future Developments on (Bio-) Membranes. https://doi.org/10.1016/B978-0-12-813606-5.00006-3 # 2019 Elsevier Inc. All rights reserved.

151

152 Chapter 6 Conventional bead Convective flow

Film diffusion

Pore diffusion

Membrane adsorber

Fig. 1 Transport phenomena involved in chromatography beads and membrane adsorbers. From Boi, C., 2007. Membrane adsorbers as purification tools for monoclonal antibodies purification. J. Chromatogr. B 848, 19–27.

costs and the difficulties associated with column packing and scale-up have been the drivers for a significant research effort to find optimal solutions for protein and biomolecule purification (Roper and Lightfoot, 1995; Charcosset, 1998; Zeng and Ruckenstein, 1999; Klein, 2000; Ghosh, 2002; Boi, 2007; Orr et al., 2013; Dimartino and Boi, 2017). The main advantage of membrane chromatography is related to the improved mass transfer, thanks to the membrane structure with through pores which minimizes the diffusional limitations associated with the use of beads (Brandt et al., 1988). Indeed, the ligand is immobilized in the membrane pores and convective flow brings the solute molecules very close to the active binding site as it is shown in the schematic illustration of Fig. 1 (Boi, 2007). Thus, the presence of convective transport reduces mass transfer resistance, and as a result, the binding capacity does not depend on flow rate in a wide range of operating conditions, indicating that binding kinetics controls the adsorption process (Briefs and Kula, 1992; Teeters et al., 2003; Knudsen et al., 2001; Wickramasinghe et al., 2006). In addition, the use of membranes eliminates packing requirements, avoids bed compaction, produces a decrease in the pressure drops due to the shorter bed height, lowers buffer consumption due to smaller unit dead volumes, and overall, reduces processing times (Knudsen et al., 2001; Etzel, 2003; Phillips et al., 2005), therefore decreasing the likelihood of

Membrane Chromatography for Biomolecule Purification 153 biomolecule inactivation (Roper and Lightfoot, 1995; Zeng and Ruckenstein, 1999; Klein, 2000). As for all adsorptive purification techniques, a plethora of applications are possible according to the functionality that is linked to the chromatographic support (Klein, 1991, 2000; Zeng and Ruckenstein, 1999; Ghosh, 2002). However, to describe the possibilities and limitations of membrane chromatography, it is rather useful to consider the operative mode of the process and to distinguish between capture and flow-through operations (Orr et al., 2013; Dimartino and Boi, 2017).

2 Membrane Chromatography for Biomolecule Capture Membrane chromatography for biomolecule capture has never gained the success foreseen in its early days, especially for affinity purification of proteins, mainly due to the limited binding capacity observed. However, binding capacity is strictly related to the size of the target molecule: for instance, for large molecules such as cells, viruses, mitochondria, plasmid DNA, extracellular vesicles, and large proteins with MW > 250 kDa, membrane chromatography has higher capacity than porous beads (Teeters et al., 2003; Etzel, 2003; Yang et al., 2002). Indeed, the surface area available for binding is larger for membranes than that of chromatographic media, because these molecules cannot enter the small resin pores and bind to the external surface of the beads (Ghosh, 2002; Etzel, 2003; Endres et al., 2003). Although two separate studies performed using surface plasmon resonance and confocal microscopy have shown that large biomolecules tend to bind to the membrane pore entrance, while smaller molecules such as proteins tend to bind inside the membrane pores (Wickramasinghe et al., 2006; Riordan et al., 2010). This is an important aspect to consider when designing and optimizing adsorptive membranes, since binding capacity depends not only on the target biomolecule size, but also on ligand density. In membrane affinity chromatography for IgG purification, it was shown that an increased ligand density was directly correlated with an increased binding capacity, indicating that ligand accessibility is not a limit when using small ligands and proteins as a target (Boi et al., 2011). However, the opposite happened when using ion exchange membrane chromatography for the purification of large biomolecules such as viruses, where an increased ligand density did not correspond to an increased binding capacity (Vicente et al., 2011). The different behavior could not be ascribed to the different membrane functionality, but rather to the pore accessibility and possible steric hindrance for large biomolecules. For smaller molecules, and for most proteins, including IgG, the dynamic binding capacity of membranes is lower than the binding capacity of chromatographic resins and still limits the widespread industrial application of membrane adsorbers for protein capture; even though a complete process evaluation should be based on a more comprehensive picture and not only on the basis of dynamic binding capacity.

154 Chapter 6 To this aim, two important parameters to consider are bed utilization and productivity: Bed utilization is defined as the amount of protein adsorbed at a given breakthrough point, with respect to the amount of protein adsorbed at complete saturation (Herigstad et al., 2015), in particular, bed utilization at 10% breakthrough can be written as: BED UTILIZATION ¼

DBC10% DBC100%

(1)

Productivity, P, is defined as the mass of the target biomolecule recovered in the elution step, with respect to the time of the complete chromatographic cycle, that is the sum of the equilibration, adsorption, washing, elution, and sanitization times. In symbols: P¼

meluted product

(2)

tchromatographic cycle

At low values of the residence time, bed utilization is higher for convective media, membranes, and monoliths, as it is shown in Fig. 2, in which bed utilization was plotted as a function of the residence time for three different protein A affinity chromatographic media that were tested for IgG adsorption (Dimartino et al., 2016). This indicates that the excellent flow properties of membranes and monoliths would compensate for the lower binding capacity with higher productivity even in the case of small target biomolecules, such as medium-sized proteins like mAbs (Bolton and Mehta, 2016), thus they could be ideally applied for manufacturing unstable biomolecules. A practical example is 100 90

Bed utilization (%)

80

Protein A membranes CIM Protein A monolith MabSelectXtra

70 60 50 40 30 20 10 0 0.01

0.1 1 10 Residence time (min)

100

Fig. 2 Comparison of bed utilization values for three different Protein A chromatographic supports: membranes, monoliths, and beads. From Dimartino, S., Herigstad, O.M., Boi, C., Lalli, E., Sarti, G., 2016. Experimental and theoretical analysis to assess the use of monolithic columns in process chromatography. Chem. Eng. Trans. 49, 25–30.

Membrane Chromatography for Biomolecule Purification 155 the commercial scale application of membrane chromatography for capture of recombinant blood coagulation factors which exploits the low residence time of AEX membrane adsorbers for the successful purification of these delicate biopharmaceuticals (Vogel et al., 2012). Even though membrane chromatography in bind and elute mode is an inherent discontinuous process, novel perspectives for its industrial implementation are given by continuous manufacturing (Zydney, 2016). Therefore, the use of multiple membrane adsorbers operated as to obtain a continuous product stream is an option that is worth investigating. Process schemes such as the ones that are in order for column chromatography, like simulating moving beds (SMB), Multicolumn Counter-current Solvent Gradient Purification (MCSGP), and periodic counter-current chromatography (PCC), can be considered by substituting the packed column with membrane adsorbers (Steinebach et al., 2016). A simpler approach, the straight-through processing (STP) that consists in the connection of two or more chromatographic media in series to reduce holding times, has been proposed for the purification of glycoproteins using a combination of membrane chromatography, monoliths, and resin (Hughson et al., 2017). However, additional studies are necessary to investigate the applicability of membrane adsorbers in continuous chromatography configurations (Zydney, 2016; Steinebach et al., 2016), since the continuous membrane chromatography model implemented in the sole study available in the literature is not convincing (Zobel-Roos et al., 2018). The process aspects, together with the progress in material science that has led to the development of novel membrane supports for chromatographic separation of biomolecules, have given an impulse to the field as testified by the increasing numbers of publications on membrane chromatography. Among the novel type of membrane proposed, electrospun nanofibers and nonwoven supports are worth mentioning due to their low manufacturing cost and the high surface area (Ma and Ramakrishna, 2008; Ma et al., 2005; Hardick et al., 2015; Heller et al., 2016), even though they are still at research stage. On the market side, membrane adsorbers with higher binding capacities are now commercially available with IEX and HIC and mixed mode functionalities (Orr et al., 2013; Kuczewski et al., 2011; Opitz et al., 2009a).

2.1 Affinity Membranes The word affinity is generally used to describe the specific and reversible interaction between a ligand and a ligate, such as those that occur between an enzyme and its substrate or between an antibody and its antigen. In both cases, the interaction is unique (Klein, 1991). However, affinity is also used to describe interactions that are less specific, but capable to isolate a group or a category of molecules that have similar properties. Affinity technology exploits these interactions for the purification of proteins, enzymes, antibodies, or other therapeutic agents. These products have very stringent purity requirements and any trace of ligand that may leak from the support must be removed from the final product (Lagasse et al., 2017). Therefore, an

156 Chapter 6 important requisite for affinity membrane manufacturing is that the ligands are coupled with a strong linkage in order to avoid ligand leakage (Boi, 2007). In addition, the immobilized ligand should maintain its activity and have the possibility to interact with the target molecules while being covalently attached to the support (Klein, 1991). While IEX and HIC chromatographic membranes are a consolidated commercial product, affinity membranes are subject of intense research, but so far, they did not reach commercial maturity. Nevertheless, ready-to-use affinity membranes are available from a limited number of suppliers and only for specific applications, i.e., with immobilized Protein A for IgG purification, or they can be customized for particular target molecules. However, it is possible to find in the market preactivated membranes, notably with epoxy or aldehyde functional groups that are ready for ligand coupling (Orr et al., 2013). The preparation of an affinity membrane follows procedures that have been adapted from those in use for chromatography beads (Hermanson et al., 1992). Starting from a specific problem in which the couple ligand-ligate has been identified, a three-step procedure is necessary to obtain an affinity membrane: • • •

Choice of an appropriate membrane support; Activation of the membrane; Immobilization of the ligand.

These steps are not independent of one another, but need to be integrated keeping in mind the target application (Klein, 1991). A good membrane support should have several properties that are difficult to find in a single membrane since many of them are contradictories. For instance, the membrane material needs to be chemically resistant in a wide range of pH values from the acidic conditions generally required for elution to the basic conditions used for cleaning and regeneration, but also easily derivatizable for covalent ligand coupling (Klein, 1991; Ghosh, 2002; Boi, 2007; Orr et al., 2013). The membrane should have a good mechanical resistance to withstand the pressure, but also a high internal surface area available for ligand immobilization that reflects its open pore structure. Moreover, the membrane material should be hydrophilic to avoid nonspecific adsorption of undesired proteins or contaminants; therefore, these materials should not favor van der Waals, or hydrophobic interactions since such adsorption leads to nonspecific retention of proteins (Klein, 1991, 2000; Charcosset, 1998; Zeng and Ruckenstein, 1999; Ghosh, 2002; Boi, 2007; Orr et al., 2013). Microporous membranes with pores in the microfiltration range, namely with a mean pore size between 0.45 μm and 3.0 μm, are selected as chromatographic supports. The materials most frequently used are regenerated cellulose, polysulfone, polyethersulfone, polyvinylidene fluoride, nylon, polyethylene, and polypropylene. The diverse chemical structures of these materials require very different modification procedures. Aromatic polymers, cellulose, and

Membrane Chromatography for Biomolecule Purification 157 polyamides possess functional end groups that can be modified by chemical activation for ligand immobilization, while aliphatic hydrocarbons with no end groups can be modified by coating or grafting. As possible alternatives, they can be prepared by copolymerization of two functional monomers or by other methods like cellulose derivative membranes, poly (etherurethane-urea) membranes, PVC and PTFE microporous composite sheets, macroporous chitin, and chitosan membranes (Zeng and Ruckenstein, 1999). Further details on matrix activation and coupling chemistry are beyond the scope of this chapter and can be found in the literature (Klein, 1991, 2000; Roper and Lightfoot, 1995; Charcosset, 1998; Zeng and Ruckenstein, 1999; Ghosh, 2002; Boi, 2007; Orr et al., 2013; Dimartino and Boi, 2017; Hermanson et al., 1992; Zou et al., 2001). When the ligand is a small molecule, it can be difficult for the active site of the target biomolecule, often located deep within the biomolecule structure, to make contact with the ligand itself due to steric hindrance. In addition, complexes that can easily form in solution may experience difficulties when one of the components is immobilized on a matrix (Klein, 1991; Boi et al., 2011). To facilitate effective binding, a spacer arm is interposed between the membrane matrix and the ligand; the spacer permits a rotational motion of the ligand and facilitates the correct orientation for the ligate-ligand complex formation. The length of the spacer molecule is a critical issue; a spacer too short is ineffective, while a spacer too long can lead to nonspecific binding. In most applications, spacers with 6–10 carbon atoms are chosen (Busini et al., 2006; Zamolo et al., 2010). The selection of the spacer is an aspect that is often disregarded, but it could be the key for an optimal performance of the affinity material. Several studies have been performed to select the optimal spacer arm, and the use of molecular dynamics simulation is an important tool that could be utilized to develop these materials (Boi et al., 2011, 2009; Zamolo et al., 2010). The degree of substitution of the membrane matrix, or ligand density, needs to be optimized since the membrane capacity is proportional to the number of active sites up to a certain limit of substitution. When the ligand density is too high, steric hindrance may be a problem, because not all the ligand molecules that are bound to the membrane are accessible for the target biomolecule, as it often happens with chromatography resins. Indeed, membrane chromatography is less critical and ligand utilization is more efficient for protein binding (Boi et al., 2011), whereas for large molecules such as viruses, an accurate optimization of ligand density is necessary to avoid these problems (Vicente et al., 2011). 2.1.1 Ligands The ligand is the molecule that binds reversibly to a specific molecule or group of molecules, enabling purification by affinity chromatography techniques. The selection of the ligand for membrane affinity chromatography is influenced by several factors: the ligand must exhibit specific and reversible binding affinity for the target substance, it must have chemically

158 Chapter 6 modifiable groups to be immobilized on a matrix without decreasing its binding activity, and it has to be stable and possibly economical (Klein, 1991; Roque et al., 2004, 2007). The ligands used for affinity membrane separations can be classified into four broad categories: (i) high affinity ligands, (ii) group-specific ligands, (iii) pseudo-biospecific ligands, (iv) synthetic ligands (Roque et al., 2007). (i) High affinity interactions are characterized by high values of the binding constant, or conversely by low values of the dissociation constant, KD that ranges from 1012 to 106 M. Pairs with high affinity are enzyme-inhibitor, protein-receptor, and antibodyantigen that are well-known for immunoaffinity chromatography. These class of ligands are not suitable for process chromatography, but immunoaffinity membranes have been developed and are successfully applied as diagnostic and analytical tools (i.e., ELISA assays) (Boi, 2007). (ii) Group-specific ligands are natural ligands with an inherent biological specificity for a class of molecules. They are based on biological recognition parameters, but not targeted to a particular sequence or conformation of the biomolecule. Lectins, protein A, and protein G fall in this category. Lectins, proteins of vegetable origin with therapeutic interest, bind selectively carbohydrates and glycoproteins; they have been immobilized onto membrane supports and used as affinity ligands to purify glycoproteins, but also the opposite was reported with the preparation of affinity membranes for the purification of lectins; however, the affinity interaction is somehow too strong to permit mild elution conditions that do not damage the target lectin (Boi et al., 2006). Protein A and protein G are ligands that exhibit high specificity for immunoglobulins and monoclonal antibodies; they both bind to the Fc fragment of the antibody. Protein A binds to many different antibodies with the exclusion of human IgG3 and murine IgG1. Protein G exhibits a stronger affinity than protein A and binds to a greater variety of immunoglobulins. However, the stronger is the interaction, the more difficult is the recovery from the stationary phase that requires low pH elution buffers. For this reason, protein A is the most important ligand for capture and isolation of monoclonal antibodies, but being a biotech product its production cost is high as testified by the prices of protein A resins that can exceed 10,000 USD per liter (Pathak et al., 2015). (iii) Pseudo-biospecific ligands generally include several categories such as hydrophobic and thiophilic ligands, metals and fusion tags, and mixed mode ligands. They are inexpensive and more resistant than natural ligands, although they do not exhibit an inherent biological specificity for a molecule and their performances must be optimized by varying the operating conditions for bind and elute. Immobilized metal affinity membranes exploit the interactions formed between metal ions and, mainly, the imidazole group of histidine and tryptophan on the protein surface; therefore, they have been studied and applied for the purification of proteins containing these amino or for HIS-tagged proteins (Porath et al., 1975). It is an economical and flexible technique that requires an additional step to couple the metal ion to the

Membrane Chromatography for Biomolecule Purification 159 membrane through chelating agents that have been previously immobilized to the membrane matrix itself (Gagnon, 2012; Borsoi Ribeiro et al., 2008). The metal ions, usually cations with charge 2+, interact in a specific manner with the electron pair of the amino acid forming a coordination complex. The separation properties of the resulting membranes depend on the membrane material, on the chelating agent, on the immobilization method, and on the nature of the metal ions and their concentration and an optimization in terms of metal choice and operating conditions is often required. However, as for IMAC chromatography, a drawback is the possible leakage of the metal ion from the support, requiring an additional step to remove the metal ion contamination from the final product (Gaberc-Porekar and Menart, 2001). For genetically engineered proteins, the use of affinity-tags is a very convenient purification method since it does not involve the development of a specific immobilization chemistry for every target protein. Systems like glutathione-Stransferase specific for glutathione, β-galactosidase that has affinity for p-aminophenil-βthiogalactoside, and maltose binding protein that binds amylose are used in chromatography purifications (Tripath, 2016). The system maltose binding proteinamylose has been successfully adapted to membranes in a one-step purification process with good results in terms of binding capacity and selectivity (Cattoli and Sarti, 2002; Cattoli et al., 2006). (iv) Group-specific synthetic ligands: The increased interest of the biotech industry towards therapeutic agents based on monoclonal antibodies has led to the development of new synthetic ligands with the aim to overcome the instability of natural ligands and to reduce production costs. These molecules, obtained through screening of combinatorial peptide libraries or by in-silico design, are synthesized to mimic the interaction between protein A, or a portion of it, with the constant fragment of the antibody (Li et al., 1998). Among those, Mimetic A2P and B14 (Roque et al., 2004; Palanisamy et al., 1999; Sproule et al., 2000; Teng et al., 2000), D-PAM (Fassina et al., 1998, 2001; Fassina, 2000; Verdoliva et al., 1995), CaptureSelect (ten Haaf et al., 2005), and peptide ligands (Yang et al., 2006; Menegatti et al., 2013, 2012, 2016) are worth to be mentioned. Since they bind antibodies of different classes, their performances are optimized for selectivity to the target antibody. Although they were developed for the purification of mAbs, the approaches that have been used for the design and synthesis of these ligands could be adapted to any biomolecule target and they are an important tool that is now available to the bioprocess industry. 2.1.2 Nonspecific binding When preparing an affinity membrane is of utmost importance that all the materials that compose the final membrane do not contribute to nonspecific protein binding, all other noncovalent interactions such as hydrogen bonding, ionic, Van der Waals, and hydrophobic interactions that may arise between any molecule in solution and any part of the derivatized

160 Chapter 6 affinity support should be minimized (Boi et al., 2011; Zamolo et al., 2010). Hydrophobic adsorption results from interactions between nonpolar side chains of proteins and the support material, the spacer arm, or from the ligand itself. The indiscriminate use of long spacer arms frequently leads to hydrophobic interference. Incomplete attachment of ligands, leaving free functional groups, is another source of hydrophobic interaction. Ionic interactions can arise from the matrix, the spacer arm, the ligand, or the coupling agent; the use of equilibration buffers with salt concentration in the range of 0.25–0.5 M NaCl/KCl can suppress ionic effects, although high concentration of salt will promote hydrophobic effects. Therefore, an optimization of buffers in this respect is necessary. Free functional groups can alternatively be blocked with hydrophilic molecules and the use of hydrophilic spacer arms will help to reduce hydrophobic interactions (Lalli et al., 2018). A careful investigation to evaluate nonspecific binding should take into account the membrane support, the spacer arm, and the chemicals used in the immobilization protocol, together with the endcapping methods. While control experiments are usually performed on the membrane support alone, the contribution to the spacer arm on nonspecific binding is often disregarded. This aspect is of particular relevance when screening for novel ligands since nonspecific binding of the target biomolecule to the spacer could be mistaken with uncomplete recovery in the elution step as it was demonstrated for the case of Mimetic A2P ligand (Lalli et al., 2018).

3 Ion Exchange, Hydrophobic Interactions, and Mixed Mode Membrane Chromatography Although ion exchange is not a high resolution purification technique, ion-exchange chromatography is more and more used for biomolecule capture due to its low cost and versatility. In particular, a combination of pore exclusion and anion exchange is exploited to capture cells, extracellular vesicles, and other biomolecules that are generally negatively charged, by carefully selecting the pH of the equilibration buffer and optimizing the elution method. This is particularly true for downstream processing of large biomolecules, such as plasmid DNA (pDNA), viruses, and virus like particles (VLPs) where membrane chromatography is one of the preferred methods together with monolithic chromatography (Podgornik et al., 2013). Since these molecules have a complex structure that should be preserved to maintain their biological activity, particular attention needs to be taken to limit the shear stress that may occur during processing and thus optimizing their purification process (Levy et al., 2000; Morenweiser, 2005). Anion-exchange membrane chromatography is the preferred method for the purification of pDNA (Chang et al., 2008; Guerrero-German et al., 2009; Syren et al., 2007; Zhong et al., 2011). The main difficulty for this separation is the removal of RNA and protein contaminants,

Membrane Chromatography for Biomolecule Purification 161 but successful pDNA purification was obtained by optimization of the washing steps at low salt concentrations, while nucleic acids are strongly bound to the AEX support and require high salts to be eluted (Zhong et al., 2011). However, to remove RNA contaminants, additional steps are necessary such as a precipitation with ammonium sulphate or/and hydrophobic interaction chromatography for complete separation (Guerrero-German et al., 2011). pDNA purification was also attempted with hydrophobic interaction membrane chromatography, but the results were not very satisfactory with respect to anion-exchange membrane chromatography (Pereira et al., 2010, 2012). Indeed, a good resolution was observed, but the removal of RNA was not complete and the overall pDNA yield was lower than with AEX. Although anion exchangers are still very common for the purification of viruses and VLPs, other functionalities, like the sulfated cellulose pseudo-affinity membrane adsorbers, have been successfully tested for several applications and are now commercially available (Opitz et al., 2009a). Virus purification using membrane chromatography has been extensively explored over the past decade. Purified viruses can be used for the production of attenuated vaccines and for gene therapy. Gene therapies, while still in commercial infancy, are expected to revolutionize the biopharmaceutical industry and how diseases are treated. Therefore, there is an increased need of scalable processes to produce these therapeutic products and membrane adsorbers are among the unit operations considered for downstream processing. Indeed, the applicative feasibility of membrane chromatography for clinical-grade manufacturing bioprocess has now been well-recognized and the increasing demand of virus-based products is a promise for its industrial implementation (Opitz et al., 2009a,b; Vicente et al., 2008).

4 Membrane Chromatography for Flow Through Applications The flow though mode of operation, also called negative chromatography, occurs when the chromatographic support does not adsorb the target molecule that flows through the column and retains process-related impurities. Indeed, this operating mode is an established technology for polishing steps of the bioprocess industry and, in particular, in antibody manufacturing where membrane adsorbers with ion exchange or hydrophobic interactions functionalities have become the preferred choice (Orr et al., 2013; Chang et al., 2008; Guerrero-German et al., 2009, 2011; Syren et al., 2007; Zhong et al., 2011; Pereira et al., 2010, 2012; Opitz et al., 2009a,b; Vicente et al., 2008, 2009; Gottschalk et al., 2008; Liu et al., 2011). This is due to the distinct flow advantages of membranes and to their sufficient capacity for binding trace level of impurities and contaminants, and to the excellent viral clearance properties. In addition, the use of disposable technology is another advantage of membrane adsorbers: they are modular and easily scalable, they provide several cost savings and benefits including a reduction in the number and volume of buffers used due to the elimination of resin storage, cleaning, sanitization, and revalidation as well as the elimination of the need for column hardware and

162 Chapter 6 packing (Shukla and Gottschalk, 2013). For polishing steps, IEX and HIC membrane chromatography modules are the most used functionalities, even though mixed mode ligands and other pseudo-biospecific ligands can be considered according to process needs (Knudsen et al., 2001; Phillips et al., 2005; Girard et al., 2015). Future perspectives for flow-through membrane chromatography are foreseen in the framework of a truly continuous manufacturing process that is more and more investigated by academic and industrial researchers. The underlying principle is the development of a process in which the target product flows continuously while the impurities are removed, thus the new high capacity membrane adsorbers are among the unit operations that are ideally suited for this type of processes.

5 Conclusions and Future Trends We are witnessing a very important growth in the number of biomolecules therapeutic that are being produced that need improved and more economical downstream processing. Within this framework, the development of novel membranes with higher surface area per unit volume and the improvement of surface modification techniques has opened new perspectives for membrane chromatography. Membranes could offer a valid alternative to packed-bed columns as they can be operated at higher flow rates and lower pressure drops, thus reducing both processing times and pumping requirements. More importantly, membranes do not require extensive costs associated with packing protocols and validation of the bed, a major burden in industrial scale chromatography processing. This is clearly supported by the current implementation of ion exchange membrane adsorbers as single-use or disposable units in the polishing steps. As a consequence, the research activity leading to improved membranes and membrane adsorbers is experiencing a significant increase, with great appreciation of the potentiality of membrane chromatography especially with novel ion exchange and hydrophobic interaction membranes that are endowed with capacities similar to that of chromatographic resins. Both these aspects become more and more relevant with the advent of continuous biomanufacturing. Indeed, membrane chromatography operation is not yet optimized, as far as materials and module design are concerned; nevertheless, the simulation techniques available allow to expect significant improvements in the near future. This could be possible if the advances in materials science and technology will lead to the production of low cost membrane supports, such as nonwovens and electrospun fibers, and of suitable modules to efficiently exploit the high surface area of these materials.

Acknowledgments The Italian Ministry of University and Research and the Alma Mater Studiorum-Università di Bologna for their financial support through RFO 2017 structural funds.

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Further Reading Boi, C., Dimartino, S., Sarti, G.C., 2008. Performance of a new Protein A affinity membrane for the primary recovery of antibodies. Biotechnol. Prog. 24, 640–647. Dimartino, S., Boi, C., Sarti, G.C., 2011a. A validated model for the simulation of protein purification through affinity membrane chromatography. J. Chromatogr. A 1218, 1677–1690. Dimartino, S., Boi, C., Sarti, G.C., 2011b. Influence of protein adsorption kinetics on breakthrough broadening in membrane affinity chromatography. J. Chromatogr. A 1218, 3966–3972.