Design And Preparation Of Monolithic Supports

Published Date: 02 Nov 2017

Antibodies have been technology drivers for over a century but never more so than now. In recent decades, the use of antibodies (Fig. 1), monoclonal antibodies (Mabs) and engineered antibody structures as effective therapeutics for cancer, autoimmune, inflammation and infectious diseases increased exponentially with an annual market worth tens of billions of US dollars. Additionally, innovative platforms for production and purification of larger scales of desired antibodies have been developed.

Several antibody purification procedures based on varying the pH, temperature and/or salt concentration were evaluated in the past but present research continues with the aim of developing more selective isolation methods than relying on chromatography. A chromatographic process can be defined as a separation process which allows the isolation of a target molecule from a complex mixture. This is enabled through the different chemical interactions between a specific ligand immobilized onto a chromatographic support and the target molecule. Presently, chromatographic methods such as hydrophobic interaction chromatography (HIC), ion exchange chromatography (IEC) and affinity chromatography (AC) dominate the manufacturing of biopharmaceuticals. The main reason for this pertains to the strict standard requirements concerning the stability and purity that must be satisfied for the process to be viable. The success of chromatographic methods as a first-choice process in antibody purification is associated with the accessibility to numerous biological molecules (proteins, antibodies, peptides, lectins and antigens) and non-biological ligands (synthetic dyes, ion exchangers, metal chelates) as well as to the different matrices (agarose beads, polymeric membranes and monoliths) available

Thus, a perfect support for an ideal separation and purification of biomolecules needs to fulfill the following criteria: (a) exhibit high selectivity as well as binding capacity, (b) possess good mechanical, morphological and chemical stability, (c) inhibit non-specific adsorptions (d) offer high stability at cleaning in place (CIP) and sterilization in place (SIP) conditions, and (e) allow short processing times for high volumes. Up to now, materials typically employed in chromatographic processes are beads or gels manufactured from such raw materials as agarose, and polymeric membranes. These materials, while readily available present certain shortcomings such as limitations with the mass transfer, gel compressibility and poor pore diffusion leading to high pressure drops and low flow rates – all of which incur time and cost. These weaknesses have led to an investment on alternative chromatographic supports which could maintain the efficiency of the established processes while improving their associated limitations. One of these new generation of alternatives are the recently described monolithic supports, hereon referred to as monoliths.

A monolithic chromatographic support can be defined as a porous, single-unit chromatographic material introduced into a chromatographic device. Individual monoliths are characterized by a network of large interconnected pores (or channels) which allow higher operational fluxes and consequently lead to faster processes. Due to their excellent morphological and mechanical properties, monoliths have attracted attention for antibody purification (Fig. 2) in research and at commercial level (Table 1). A vital requirement for industry is large-scale operation, while enabling high throughput at moderate pressure drops without sacrificing the product purity. In this respect, the main advantages associated with monoliths (transport based on convection, high porosity, low cost preparation and simple column filling) has encouraged different manufacturers to examine monoliths as potential supports, providing high impact in the bioseparation market for different purification requests. Nowadays there are different commercially available polymeric monoliths offering a wide range of pore diameters which allow the purification of a large number of biomolecules ranging in size and features in a simple and effective way (Fig. 2).

Monoliths were introduced in the early 1990’s. One of their main advantages is their rigid structure with a high degree of permeability due to the presence of large pores. The latter, permits the use of higher liquid flow rates (at low back pressures) even when processing different culture mediums, in contrast to traditional chromatographic columns packed with particles and porous membranes which are prone to fouling. The distinct porous architecture of monoliths brought them to prominence since in these devices the mass transport occurs by convection, thereby assuring a greater capacity and resolution independent of molecular size and the flow rate. Monoliths also avoid a high-shear fractionation atmosphere in the recovery of shear-sensitive molecules such as viruses, sensitive proteins (e.g. antibodies), DNA and cells. It is known that chromatographic processes involving porous beads or membranes configured in a chromatographic column are slower than using monoliths because the transfer of large biomolecules like cells or antibodies (in particular immunoglobulin M (IgM)) is limited by diffusion coefficients that are drastically smaller than those for lower molecular weight compounds. Consequently, this leads to a decrease in permeability accompanied by an increase in the back pressure of the chromatographic column. This effect is unfavorable when the velocity of mass transfer restricts the global rate, as happens in high-speed chromatography.

Monoliths employed in antibody purification have been prepared using inorganic materials as well as natural and synthetic polymers. Recently, Arrua et al. revised current developments and future possibilities for polymeric monolithic structures. Depending on the material, different processing routes can be followed including polymerization initiated by different stimuli (e.g. temperature or UV), sol-gel and cryogelation, creating porous networks with distinguished structural properties. Since these polymers and materials adopt the format of the mold used, monolithic materials can be prepared in different design formats such as large rod polymers (used in standard HPLC/capillary columns), monolithic disks, cylinders and flat sheet polymers. A classification according to the morphological features of different monolithic supports is indicated in Table 2 according to the commonly defined literature criteria.

Since the optimal performance of monoliths depends on the balance between morphological, mechanical and physicochemical properties, it is difficult to single out any particular parameter and set it as the ‘gold standard.’ Hence in general, a monolith for antibody purification must have an average of pore size diameter between 50 and 300 µm, a porosity of around 70 ± 10 %, a surface area within 10-400 m2 cm-3 together with a permeability and a binding capacity of up to 100 L m-2 h-1 atm-1 and 50 mg mL-1, respectively. This range of values can be fine-tuned according to the starting materials as well as the methods applied for the preparation of monoliths. Thus, different types of monoliths can be generated and customized to ensure maximum efficiency in the capture of the target antibody.

Organic based monolithic columns were introduced in the late 1980’s and the early 1990’s. Monolithic columns were prepared by radical polymerization of monovinyl monomer in the presence of a crosslinker, radical initiator and porogen (responsible for pore formation) (Fig. 3). Polymerization took place inside a mold that determined the monolith shape. Inspired by this straightforward strategy for monolith production, different monomers such as acrylamides-based (AAm-based), methacrylate-based and styrene-based were investigated to create rigid monoliths with desired morphological properties, and dimensions through different polymerization mechanisms. In particular, glycidyl methacrylate (GMA) and ethylene glycol dimetacrylate (EDMA) became the most commonly employed monomers for the preparation of organic-based monoliths. The great advantages of using these monomers is that GMA, already carrying the very reactive epoxy group, easily allows further functionalization according with the target molecule; while EDMA, as an excellent crosslinker, confers a high mechanical stability to the final monolith. As an example, Hahn et al. developed an affinity P(GMA-co-EDMA) monolith using a straightforward strategy for ligand immobilization. The model peptide (or ligand) was directly immobilized by reacting it with the epoxy group already available on the GMA chains incorporated in the matrix of monolith.

Ligand immobilization can be performed following different strategies to minimize the stereochemical hindrance promoting the accessibility of the ligand functional groups to the target biomolecule. One strategy to reduce stereochemical hindrance is to place a spacer arm between the support and the ligand. However, this approach does not always lead to the best performance of the purification systems. This is clearly demonstrated in the work of Luo et al. where reactive macroporous monoliths of P(GMA-co-EDMA) were prepared by "in-situ" copolymerization of GMA and EDMA in the presence of porogenic agents. Protein A and L-histidine were linked to monoliths either directly or through the use of a spacer arm. Both functionalized monoliths were used for the capture of immunoglobulin-G (IgG) from human serum. The adsorption capacity of the monolith functionalized with protein A was greatly increased with the introduction of the spacer. This strategy proved to be faster for human IgG collection and the monolith remained highly stable at mild elution conditions. An exception to this finding was observed with histidyl monoliths. These required additional chemical modifications, leading to decreasing amounts of histidine, thus resulting in a lower human IgG binding capacity.

Later, Wei et al. proposed the modification of the previous supports, GMA-EGMA monoliths, through graft polymerization of methacrylic acid (MAA), a pH-responsive polymer. The "in situ" grafting polymerization was achieved by pumping methacrylic acid through an acidic hydrolysis monolith with potassium peroxydisulfate as initiator. The modified monolith was used as a pH responsive stationary phase, showing an efficient separation of four different proteins; lysozyme (Lys), IgG, cytochrome C (Cyt C) and α-chymotrypsinogen A (α-Chy A) according to their isoelectric points led to a 98.8 % recovery of the total IgG retained.

More recently, Pecher et al. prepared a set of epoxide-based monolithic networks for affinity capture of IgG. Self-polymerization of polyglycerol polyglycidyl ether (PG-PGE) using methyl tert-butyl ether as a porogen agent resulted in the formation of a particularly rigid monolith where the epoxy groups served a dual purpose: first functional groups for the polymerization reaction, and secondly for direct functionalization of recombinant protein A for further extraction of IgG from rabbit serum. The monolithic capillary column thus produced allowed the isolation of IgG (5.3 ± 0.9 μg) and presented a capacity of 0.44 ± 0.08 mg mL-1, with a capillary volume of 12 μL. No information was given concerning the selectivity and the purity of the eluted IgG fractions.

P(GMA-co-EDMA) monoliths have also been also functionalized with Protein L and Protein G to amplify the number of fast and efficient platforms for antibodies purification. Promising results were obtained according to the concentration and molecular size of the compounds present in the samples to be processed. Therefore, the high impact of these monoliths as chromatographic supports led to their commercialization in BIA Separations with highly encouraging performance in downstream processes.

Styrene-based monoliths have also been shown to demonstrate desirable features. Walsh et al. reported for the first time a light initiated polymerization of poly(styrene-co-divinylbenzene) using a 470 nm light emitting diode array. The resulting capillary monolithic columns were used for protein separation with high resolution. For similar bioseparation purposes, monoliths prepared from poly(glycidil methacrylate-co-ethylene glycol dimethacrylate) and 2-methyl-4’-(methylthio)-2-morpholinopropiophenone were also photoinitiated using a wavelength of 660 and 350 nm, respectively.

The main features of monolithic columns are large pores and moderate surface areas serving as a good platform for the separation of large molecules. Further studies have extended the applicability of the monoliths to enable the isolations of small compounds and molecules. This has been achieved through the preparation of monoliths with larger surface areas. Urban et al. prepared poly(styrene-co-chloromethylstyrene-co-divinylbenzene), poly(Sty-co-CMS-co-DVB)], capillary columns (with a large surface area of 663 m2 g-1) able to separate uracil and alkylbenzenes in isocratic mobile phase mode and ribonuclease, cytochrome C, myoglobin, α-chymotrypsin A, and albumin in size exclusion mode. In order to improve monolithic devices for the efficient separation of large size range of biomolecules several studies were conducted to optimize the hydrodynamic properties of the matrix. Smirnov et al. prepared different poly(DVB-co-ethylvinylbenzene-co-2-hydroxyethyl methacrylate), poly(DVB-co-EVB-co-HEMA), monolithic columns for the separation of aromatic compounds evaluating the outcome of HEMA amount used during the polymerization on the permeability and the chromatographic performance of the monoliths prepared. They concluded that the increase of HEMA content in monolith’s composition allowed a significant improvement in the separation accomplished with a reduction of the hydrodynamic permeability. On the other hand and for the same purpose, Svobodová et al. recently prepared poly(Sty-co-DVB-co-methacrylic acid), poly(Sty-co-DVB-co-MAA), capillary columns varying the MAA ratio. The authors verified that the MAA addition to the monolith composition enhanced the permeability of the support which is crucial for an efficient purification process regardless the target molecule.

Various publications have reported on monolith preparation having different inner diameters (I.D.) as a means to develop bespoke monoliths of different pore size (from 75 µm to 0.8 mm) catering to different separation processes (for example synthetic and biological compounds).

To evaluate the application of organic-based monoliths for antibody purification several studies have been performed. Lokman et al. developed a novel porous monolithic system for an effective IgG purification from human plasma based on the preparation of porous monoliths through the bulk polymerization of HEMA and N-methacryloyl-(L)-histidinemethylester (MAH). An upper adsorption value (> 96.5 mg g-1) was achieved from human plasma with an associated purity value of 95.3%. Moreover, the authors verified that IgG could be reversibly adsorbed using poly(HEMA-MAH) monolith maintaining its adsorption capacity. Another strategy from the same group involved the preparation of imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-l-tyrosine methyl ester) particles using hepatitis B surface antibody as the template. These particles adsorbed hepatitis B surface antibody 18.3 times more than anti-hepatitis A antibody and 2-fold up than immunoglobulin E. Therefore hepatitis B surface antibody-imprinted particles ensure a great specificity towards their corresponding antibody. Other authors performed Mabs purification using a powerful anion-exchange methacrylate monolithic system constituted by a monolithic macroporous convective interaction media (CIM) with diethylaminoethyl (DEAE) and ethylene diamine (EDA). This system was tested and evaluated towards the isolation of anti-glycophorin-A IgG1 mouse mAbs from cell culture supernatant. The results indicated that a fast isolation and effective recovery of pure anti-glycophorin-A mAbs was achieved. Another plan for Mab’s purification was proposed by Rajak et al. who immobilized four different metal ions (Zn2+, Cu2+, Co2+ and Ni2+) on CIM- iminodiacetate (IDA) disks for the anti-human serum albumin IgG1 mouse MAbs isolation from cell culture supernatant, achieving maximum recovery of 85.4 % of highly pure antibodies.

Organic-based monoliths have also been employed in complex IgM purification systems. Recently, Neff and Jungbauer developed a method for IgM, IgG and Mab’s quantification straight from embryonic stem cells using an epoxy-activated monolith CIM disc that was further functionalized with an affinity peptide. With this approach it was possible to recover 67, 83 and 95 % of IgG, IgM and Mabs, respectively. In addition, they concluded that the binding capacity was reproducible over two thousand cycles. Still concerning the IgM purification, Gagnon et al. reported an efficient method to purify established complexes among genomic DNA and monoclonal IgM from mammalian cell culture. The authors showed that IgM-dominant complexes could be collected and eluted with IgM whereas DNA-dominant complexes were not retained. While a porous particle anion exchanger was incapable of dissociating DNA from IgM, the monolithic anion exchangers (CIM –QA (quaternary amine) and –EDA from Bia Separations), offering up to 15 -fold higher charge density, promoted a thorough detachment of the complex. Monoliths exhibited a dynamic binding capacity 2-fold higher dynamic binding than the porous beads anion exchanger owing the porous network availability and effective mass transfer. More recently, Kamalanathan et al. screened IgM antibodies from rheumatoid arthritis patients’ sera for peptide hydrolyzing activity. The removal of IgM antibodies in just one step was possible through an anion-exchange CIM-EDA disk. The recovered IgM antibodies hydrolyzed the Pro-Phe-Arg-4-methyl-coumaryl-7-amide (PFR-MCA) substrate in a similar manner to healthy donors. Moreover, IgM antibodies demonstrated ample proteolysis activity when compared to IgG. Hence, CIM-EDA system proved to be a good option for an efficient recovery of IgM molecules with an acceptable yield of 85%. Recently, high-throughput methods for isolating IgM from human plasma and for fractionating low abundance plasma proteins have been optimized. Purification of IgM (IgM isolation from blood sample of a patient) was achieved with high purity using a 0.34 mL CIM QA Disk Monolithic Column (and corresponding housing from BIA Separations) with high purity. Low abundance proteins were also successfully isolated and glycosylated proteins IgG and IgM were separated in sufficient amounts to allow further high-throughput analyses in a large number of patient samples.

Other approaches regarding IgM isolation have also been developed. Gautam and Loh recently reviewed all the challenges and perspectives related with the purification of this class of antibodies including the use of organic-based monoliths. Platonova et al. and Gunasena et al. revised and discussed carefully many more features, strategies and approaches related with organic-based monoliths namely their future perspective in the separation world. However, monolithic materials are not solely prepared from a homogeneous polymerization solutions using heat or radiation as initiator source.

There are other types of organic-based monoliths which present a different but still attractive porous network such as cryogels and hydrogels. These two subclasses of organic-based monoliths can be defined as supermacroporous gel networks formed, in case of cryogels, by the cryogelation of certain monomers (e.g. GMA, allyl glycidil ether AGE) at subzero temperatures using ammonium persulfate (APS) as an initiator and N,N,N’,N’-tetramethylene diamine (TEMED) as the catalyst (Fig. 4). Hydrogels are formed by the polymerization of acrylamide (AAm), N’,N’-methylenebisacrylamide (MBA) and AGE in an aqueous buffer which works as a porogen, just like acrylamide gels in SDS PAGE technique. As mentioned above, the use of GMA and AGE allows a directed introduction of epoxy groups making it much easier to functionalize cryogels and hydrogels with ligands or other synthetic and natural species.

The macroporous network of hydrogels and cryogels makes them very attractive for cells and antibodies separation. Therefore, during the last decade different cryogel approaches have been explored to take advantage of their higher porosity (up to 90%) and larger pore size (0.1-200 µm), for antibody capture. Unlike methacrylate or silica monoliths, cryogels and hydrogels have poor mechanical behavior which necessitates the use of alternative elution strategies based on monolith mechanical compression and shirking. Methods to overcome low rigidity include the preparation of physical blends or the addition of stiff polymers to the initial casting solution.

Over the last decade different polymeric cryogel systems such as AAm and MBA grafted with N,N-dimethylaminoethyl methacrylate (DMAEMA) and poly(MAA-co-polyethylene glycoldiacrylate) embedded with polystyrene or poly(EDMA) nanoparticles have been prepared at sub-zero temperatures. These systems have been successfully applied for the removal of pollutants and for the separation of target proteins from crude samples.

Since the main features of these supports are still the large pores, which allow high permeability fluxes and efficient separations, whole cells (or antibodies attached to cells) can also be purified using cryogels. An example is the efficient approach developed by Mattiasson et al. to separate different cell lines (Escherichia coli and Saccharomyces cerevisiae cells) using Con A-cryogel as an affinity monolith. It was possible to collect viable S. cerevisiae and E.coli cells with 95 and 100% of purity, respectively. The same group was able to capture viable antibodies from fermentation broth with high purity using an affinity supermacroporous monolithic cryogel functionalized with protein A. Mattiasson and co-workers published an extensive review evaluating the potential for different cryogel systems in the bioseparation field.

Concerning the use of cryogels in direct antibody purification, Babac et al. were able to capture human IgG from pure aqueous solutions and human plasma using poly( AAm-AGE) monolithic cryogel functionalized with concanavalin A (Con A). Due to the large porous network, the use of cryogel allowed the processing of crude samples such as blood without obstruction of the monolithic column. This approach yielded a high IgG capture capacity (up to 25.6 mg g-1) with an elution purity of 85%. Con A-poly(AAm-AGE) cryogels were used numerous times in adsorption/desorption runs of IgG maintaining their performance for at least 10 cycles. Similarly, Alkan and co-workers developed an approach to purify IgM using PHEMA cryogel activated with cyanogen bromide for further functionalization with Protein A. The blood biocompatibility of PHEMA cryogel was evaluated through the direct contact of blood with the cryogel. Moreover due to the large pore size of the cryogel, cells were easily processed. The authors also verified that the IgM antibody adsorption was reduced with the plasma flow rate intensification. The higher value obtained for the IgM antibody adsorption was 42.7 mg g-1 and the reproducibility of the PHEMA cryogel to adsorb IgM antibodies was assured. Recently, the same group extended the challenge and purified IgG from human plasma using a PHEMA cryogel modified with Protein A. The support was performed in a plastic syringe by cryo-polymerization. After Protein A immobilization the PHEMA cryogel was able to adsorb 83.2 mg of IgG g-1 at pH 7.4 from pure aqueous solutions. Using 0.1M glycine–HCl buffer (pH 3.5) it was possible to recover IgG with a purity of 85%. The cryogel reproduced its adsorption and desorption capacity at least up to ten runs. Related, Bereli et al. proposed the use of Cibacron Blue F3GA and (IDA)-Cu2+ covering PGMA particles incorporated into the PHEMA cryogel for IgG purification. The PGMA beads were functionalized with Cibacron Blue F3GA and (IDA)-Cu2+ to isolate albumin and IgG in one step. The adsorption capacity of albumin using the PHEMA/PGMACibacron Blue F3GA approach (PHEMA/PGMA-IDA-Cu2+) reached a value of 257 mg g-1. Furthermore, the authors concluded that the cryogels withdraw IgG and albumin from human serum with efficiency of 93.6 and 89.4%, respectively. The same group prepared a supermacroporous cryogel to achieve an effective and cost attractive isolation of IgG from human plasma. N-methacryloyl-(L)-histidine methyl ester (MAH) was selected to function as a pseudospecific ligand and as co-monomer simultaneously. Thus, the PHEMAH cryogel was produced through a free radical polymerization initiated by TEMED and APS at subzero conditions. The PHEMAH cryogel was able to adsorb 24.7 mg of IgG per g-1 of support providing considerable improvement in adsorption capacity compared to PHEMA cryogel. This enhancement was accomplished by the incorporation of the MAH to the support, which elevated the specific surface area up to one hundred times. In addition, the highest registered quantity of IgG adsorbed from human plasma was 97.3 mg g-1 of cryogel with an associated purity of 94.6%.

Silica-based monoliths as separation media in chromatography were first introduced in the 1990´s by Tanaka et al.. These supports are usually produced through the sol-gel method by consecutive hydrolysis and polycondensation of organo-silicium compounds, using silane precursors such as tetramethoxysilane, tetraethoxysilane and methyl(trimethoxy)silane. Silica-based monoliths possess great mechanical stability and high porosity, as well as providing a larger surface area, and better overall performance (enhanced selectivity and reproducibility). The preparation of silica-based monoliths is quite challenging due to the possible occurrence of structural shrinkage during the gelation process. This limits the operating range of pH during monolith preparation and utilization between 2-8. Despite this restriction and in view of the aforementioned desirable features offered by these supports, silica-based monoliths began to be commercialized by Chromolith-SiR Merck in 2000 and found applications especially in chiral and bioanalytical separation. In general, silica-based monoliths achieved better chromatographic resolutions and lower backpressures than those observed in traditional HPLC packed bed columns. Mallik et al. and Yoo et al. have been devising new methods based on silica monoliths to increase their competitiveness against traditional chromatographic processes.

Lin et al. advanced a new type of molecularly imprinted macroporous hybrid silica monolith for protein detection. The authors envisage such hybrid supports to exhibit higher specificity, increased rigidity and offer a longer life span than immobile phase imprinted silica alone.

A detailed review covering silica-based monoliths and their applications in drug and chiral separations is discussed by Cabrera et al. as well as the future perspectives of this type of monoliths. Kato et al. [14] published a detailed on silica sol-gel monolithic constituents and their application in numerous fields, highlighting the materials and methods for silica-based monolith production and their applications. Schiel et al. focused attention on the evaluation of the same supports as applied to affinity chromatography.

Societal, environmental, and regulatory drivers are pressing industry to design engineered products from "cradle to grave" and this has been a driving force for the use of natural and biodegrade polymers at an industrial level.

The most widespread natural polymers for the development of porous chromatographic supports are polysaccharides, such as cellulose, chitosan and agarose due to their well-defined chemical structure and the possibility for further chemical modification.

Cross-linked agarose beads present a weak mechanical behavior particularly for operation at high flow rates. The popularity of agarose beads as first-choice supports for traditional affinity chromatography is high due to their hydrophilicity and good chemical stability, even under extremes of pH. Thus it is not surprising that agarose beads have been used for monoliths preparation. Agarose gels are usually prepared following a sequence of four steps: (i) dissolution of agarose in water at 80 ºC (ii); decreasing the temperature to 60 ºC (iii); further mixing the agarose casting solution with an organic solvent containing a surfactant for the formation of an emulsion, and (iv) cooling the solution. Depending on the mold, different agarose-based monoliths can be prepared with different shapes. Unfortunately, agarose based monolith supports exhibit poor mechanical properties, and at the time of writing they are only known as porous particles confined in a mold or as a macroporous gel. None is known as a single rigid porous block.

Cellulose is a regular and complex macromolecule with a very poor solubility in water and in most organic solvents. It is the most abundant naturally occurring biopolymer on Earth. However, standard protocols for dissolving cellulose are unattractive from an economic and environmental point of view, since they involve the use of hazardous solvents which are difficult to recycle. Cellulose is typically employed in chromatographic procedures using cellulose derivatives in the form of discs/membranes retaining the possibility for further functionalization with different type of molecules for protein separation and evaluation of affinity interactions. To overcome the solubility issues a recent approach has employed the use of ionic liquids as effective and sustainable solvents. A major drawback of using ionic liquids is that while the approach improves the solubility of cellulose, the removal of the ionic liquid(s) at the end of the process is often laborious and costly. Thus, recently Barroso et al. prepared cellulose membranes/discs by a phase inversion method where the ionic liquid 1-butyl-3-methylimidazlium chloride ([BMIM][Cl]) acted as a solvent and water as a co-solvent. This alternative approach provided a facile route for cellulose processing enabling the generation of porous cellulose porous structures for different applications, namely that of human IgG purification. In this work, the cellulose casting solution was processed using a Teflon cap which has a diameter of 68 mm and a height of 1mm, leading to a flat membrane. This approach could be extended to monoliths’ preparation.

Chitosan is also a natural polymer obtained by deacetylation of chitin originated from exoskeleteon of crustaceans such as crabs, krill and shrimps. The backbone consists of a linear polymer of β (1-4) linked 2-amino-2-deoxy-D-glucopyranose. Chitosan has been extensively investigated in diverse fields of work due to its nontoxic, antimicrobial, biocompatible, and biodegradable properties and sensitivity towards changes in pH. Due to its high molecular weight, chitosan yields viscous solutions which can be utilized to produce porous gels and structures through different methodologies such as freeze drying and supercritical fluid technology. Interest in this polymer is widespread throughout scientific and industrial organizations and recently its use impacted in the bioseparation field. As an example, Mattiasson and co-workers developed a new macroporous monolith designed for enzyme immobilization by cross-linking chitosan with hen egg albumin (HEA) using glutaraldehyde (GA), which, as well as being the crosslinker, simultaneously acted as a spacer arm for further couplings, at subzero temperatures. Other authors developed a new cationic hydrophilic interaction monolithic stationary phase to perform capillary liquid chromatography. The authors modified the carboxymethyl chitosan (CMCH) to the monolithic silica skeleton employing carbodiimide coupling. This monolith exhibited a clear specificity for aromatic and aliphatic carboxylic acids as well as for nucleotides and nucleosides. Chitosan has also been blended with agarose and GA to create macroporous cryogels towards affinity purification of the two main egg white glycoproteins: ovotransferrin and ovalbumin. Owing the good morphological properties (10-100 µm) and moderate mechanical stability, chitosan blended cryogels showed a high specific recognition for glycoproteins with 90% recovery capacity. Similarly, Sun et al. prepared chitosan-agarose cryogels in-situ through cryo-polymerization to obtain a support with adequate adsorption capacity towards IgG purification. After linking the 2-mercaptopyridine onto divinylsulfone-activated matrix, the cryogels were used to purify IgG. Cryogels presented interconnected pores of 10– 100 μm size, a specific surface area of 350 m2 g-1 and a high adsorption and elution capacity for IgG 71.4 mg g-1 and 90 %, respectively. These supports proved to be stable and reusable for more 10 cycles without substantial loss in their performance. More recently, Barroso et al prepared chitosan-based monoliths for IgG purification, by combining freezing and lyophilization methods. The authors were able to improve the mechanical properties of chitosan through blending with polyvinyl alcohol (PVA) and by cryopolymerizing with GMA at sub-zero temperatures (Fig. 5). After exhaustive morphological and mechanical characterization, the supports were functionalized with a protein A biomimetic ligand, through a free solvent technique nominated plasma technology. This sustainable and faster approach allowed high binding capacities (150 ± 10 mg IgG g-1 support), and 90±5% recovery of the bound protein with 98% purity directly from cell-culture extracts.

Presently, the use of natural polymers for the preparation of chromatographic supports is still low, but this trend needs to be reversed in view of stricter chemical legislation regarding health and safety, thus pushing the industry towards green and sustainable processes. Table 3 below provides a summary of the key supports, targets and separation criteria for the processes discussed above.

Ensuring optimal performance in the applications for which monoliths are designed requires accurate characterization to determine whether the morphological properties fall within the range of desired values. Thus, depending on the application, the best balance between porosity, pore size and surface area must be attained. Larger pores decrease the available surface area and reduce the mechanical properties. Conversely, smaller pores allow for a larger surface area and impart better mechanical integrity, albeit at the expense of lower fluxes and slower processes. One of the most critical issues is the pore size distribution. Various authors allude to the difficulty in producing monoliths with an acceptable degree of heterogeneity. Therefore, a number of methods have been described for evaluating porous monolithic networks. These include scanning and transmission electron microscopy (SEM/TEM), mercury intrusion porosimetry (MIP), adsorption or desorption of nitrogen (AN/DN), and inverse size inclusion chromatography (ISEC). Each technique by itself is insufficient for a complete understanding of the complex porous monolithic network, necessitating the conjugation of more than one technique for an efficient structural analysis. On the other hand, a significant quantity (of the order of milligrams) of monolith sample is required to obtain representative results. Additionally, these analyses are often destructive. SEM/TEM, samples have to be coated with gold while MIP required samples to be impregnated with mercury. In case of AN or DN studies the samples can be destroyed through the degasification procedures and pressures employed during the analysis.

Developing non-invasive methods for characterizing monolith’s morphology became a great challenge for some researchers. Petter et al. utilized near-infrared spectroscopy (NIR) for determining pore size, pore volume, total porosity and surface area in a single analysis. Although this technique is not destructive, it still does not provide comprehensive morphological detail such as potential wall defects and the degree of radial heterogeneity, both which are particularly important in evaluating monoliths as chromatographic devices. A different example was provided by Gillespie et al. who developed scanning coupled contactless conductivity (sC4D) methods to evaluate porosity and axial variation of column functionality incorporated via photografting or surface coating. The main advantage of this approach is that it can be effortlessly applied to examine the length of a capillary column in a single analysis. However, the approach requires a high level of resolution to observe small nonconformities in structural morphology.

The introduction of other recent techniques such as confocal laser scanning microscopy, magnetic resonance imaging and small angle neutron scattering offer innovative options to complement the aforementioned techniques in order to attain a thorough understanding of monolith structural features, thereby matching a given porous monolith to its intended application.

As chromatographic supports for antibody purification, monoliths offer several advantages, such as tunable morphological characteristics, controlled mechanical behavior, low flow resistance, convective mass transport and stability under extremes of pH and variable temperature conditions. Important key parameters that must be studied when developing new monoliths include static and dynamic binding capacities, the scale up potential and resistance to cleaning and sterilization procedures.

An adsorption isotherm is a useful tool for estimating the maximum binding capacity to the target molecule as well as evaluating the level of non-specific adsorption. According to the porous network, material and surface area different adsorption isotherms can be applied to achieve the best fit to the experimental data obtained through the static studies.

To assess the monolith dynamic binding features and mass transfer, breakthrough curves obtained by frontal analysis are usually estimated. If breakthrough curves do not alter with feed concentration, or molecular dimension and velocity, it indicates that the adsorption is not restricted by mass transfer phenomena. Furthermore it also suggests the high interconnection of monolithic pores allowing a convective flow. Thus, to obtain an effective mass transfer, the pores have to be sufficiently wide. For this reason, monoliths became the greatest tool for antibodies and other biomolecules with diameters above ~5 nm since it is practically difficult to produce particles with a pore size wide enough to allow permeation of larger molecules. Moreover, when comparing nonporous and porous particles, membranes and monolithic materials, the capacity of monolithic materials increases with an increase in the size of the target molecule

Regarding the cleaning and regeneration issues of monoliths, different protocols can be adopted according to the stability of the immobilized ligand and of the polymeric composition. Thus, cleaning and regeneration regimes need to be optimized for individual situations. However, the most common procedures employed involve the use of alkaline (0.1-1 M, NaOH) and salt solutions (1-2M, NaCl) which contain competitor agents that force the removal of antibody and proteins from the monolithic supports. Alcohol solutions such as ethanol (up to 20%) and isopropyl alcohol (up to 30%) can also be used. Moreover, in particular feedstocks, the use of detergents (e.g. Tween 80) or organic solvents (acetone, ethanol, and isopropanol) might be required. For an efficient cleaning and regeneration control, different techniques such as X-ray photo electron spectroscopy (XPS) and mass spectroscopy can be performed in order to monitor the degree of residual contaminants remaining on chromatographic matrices.

Another encouraging factor in favor of employing monoliths in chromatographic processes is the pressure drop decline. Owing their attractive porous network, the monoliths pressure drop is inferior compared to traditional beads or membranes. Therefore, all monoliths used in biomolecules separation field should have porosity higher than 0.5 allowing a pressure drop reduction of 50% compared with beads or membranes.

An additional and also fundamental concern associated to the monolith efficiency at industrial separation level is the scale up capability. The preparation of monolithic devices with adequate dimensions is within the grasp of existing manufacturing processes. However, mechanical and chemical stabilities must be maintained as monolithic columns are projected to operate over multiple cycles without capacity loss. Moreover, the monolith attachment to the column wall is also challenging. Monoliths can be attached to a column with a flexible wall though this set-up would prove cumbersome when working at high pressure gradients and high flow rates. This issue probably justifies why silica monoliths are not yet available at industrial scale. In marked contrast, the scale–up of CIM disks and tubes made from polymethacrylate has been widespread since the preparation of these supports results in good mechanical behavior as well as conferring high stability at aggressive regeneration conditions (e.g. 1M, NaOH). Thus scale up is easier, straightforward and suitable for biopharmaceutical production. Monolithic supports with fragile immobilized ligands cannot be subjected to aggressive environments but in such cases there are specific regeneration procedures in place which can be fine-tuned to suit the ligand type.

At present, intermediate scales processes have been devised by linking columns in parallel, creating an array system with a volume capacity up to 1,000L. Effective scale-up from a 0.34-mL disk to 8-L radial columns and tubes is well established and shown to be able to maintain the efficacy of the system. In the near future it is expected that monoliths could increase processing capacity in direct competition with traditional chromatographic resins that are able in to process hundreds of liters with high resolution. At the time of writing, 8mL of a CIM monolith functionalized with Protein A (PA) is able to purify 10 mg IgG g-1 wet support, while 2mL of PA agarose resin purifies 20 mg IgG g-1 wet support. Thus, an improvement in monolith purification capacity is still required. Perhaps more so than size alone, the optimization of the monolith porous network is a key starting point. An understanding of the relationship between pore size, degree of overall porosity and surface area can be a useful indicator for generating a porous network with customizable morphological and mechanical features tuned towards the intended purpose.

During the last two decades, the publication frequency of studies involving different systems for antibody purification is nearly one paper per month. Interest for these high-value biomolecules in medicine, pharmacology, biochemistry and diagnostics is convincing researchers and industry to develop new alternative systems for antibody isolation and purification. Thus, monoliths provide an attractive alternative to traditional chromatographic methods, though this nascent technology requires further maturation before its full potential can be exploited. Concerning organic-based monoliths, the most widespread monomers are the acrylates, GMA, EDMA and HEMA. Due do their various advantages, summarized on Table 4, organic-based monoliths become a powerful antibody purification tool compared with the other monoliths types. Monomers such as AAm and allylglycidyl ether are sporadically employed to produce cryogels. A drawback for this kind of monoliths is the larger pore size (≥100µm) and porosities (≥90%) and hence the low surface area which can limit the amount of ligand that can be immobilized on the surface, thus affecting the efficiency of the support. However, the studies already published involving the use of cryogels in antibody isolation reveal better than expected performance since cryogels processed different crude samples considerably faster and with high level of purity. Thus, these cryogel features should encourage researchers to focus on optimization work in order to take advantage of this recent development.

In marked contrast, the use of silica-base monoliths in antibody purification is negligible. This could be related with the fact that the modification of silica-based monoliths is not straightforward requiring further modification steps than compared to other monolith types.

Recently, natural-based monoliths have been marking their potential in the separation field. Few communications involving the use of polysaccharides as starting material for monoliths production are available; however the government regulations concerning environment impact of hazardous substances have helped to encourage the scientific and industrial community to develop more sustainable chemistries, products and processes. In view of this legislature, with the promising performance already demonstrated by Mattiason et al. and Barroso et al. [94], the use of natural monoliths could be an excellent alternative to create competitive and greener biomolecule adsorbents with high impact on the market compared with other monoliths and traditional chromatographic supports. The potential for biodegradability of natural monoliths imparts a crucial advantage when considering disposable components in bioprocessing and safety issues for biopharmaceuticals manufacturing.

Multiple studies involving the use of different monolith types for antibody purification were discussed revealing the high potential of these emerging supports as strong platforms in bioseparation.

The technologic transition in purification processes has already begun. There is already substantial literature precedence highlighting the manifold virtues of monolithic supports. The commercial availability of various monolithic supports confirms their higher added value in different applications. Furthermore, the emergence of monoliths for the enrichment and purification of large analytes including antibodies of various formats, namely IgM, indicate that the field, though nascent, is crucial in paving the way towards finding multiple applications that are anticipated to uptake the new technology in the near future. Limitations still remain and some of the problems (including the need to prepare and to characterize each monolith separately), need to be addressed. Moreover, additional work is needed to expand the range of ligands and the immobilization strategies that have been adapted for monolith use. However, it is expected that these limitations will be circumvented and scale up will be possible and optimized. Monoliths may revolutionize the antibody bioprocessing paradigms.

Protein A was known to biochemists long before its true potential as an antibody purification ligand was fully realized.  This realization ushered in a new era for the separation of complex biomolecules.  However, recent advances in proteomics and the unraveling of entire animal and plant genomes has led to the identification and classification of multitudes of new proteins – each of which plays a vital role in living organisms. Hence the need to separate, purify and characterize proteins has never been greater. To meet this burgeoning demand, the time is ripe for the next leap in affinity supports. Monoliths may well prove to be the ideal bespoke chromatographic medium that takes complex bioseparations from the research bench to sustainable large-scale industrial processes.