revista mexicana de ngeniería uímica - scielo méxico · although upstream processing of mabs has...

Revista Mexicana de Ingeniería Química

CONTENIDO

Volumen 8, número 3, 2009 / Volume 8, number 3, 2009

213 Derivation and application of the Stefan-Maxwell equations

(Desarrollo y aplicación de las ecuaciones de Stefan-Maxwell)

Stephen Whitaker

Biotecnología / Biotechnology

245 Modelado de la biodegradación en biorreactores de lodos de hidrocarburos totales del petróleo

intemperizados en suelos y sedimentos

(Biodegradation modeling of sludge bioreactors of total petroleum hydrocarbons weathering in soil

and sediments)

S.A. Medina-Moreno, S. Huerta-Ochoa, C.A. Lucho-Constantino, L. Aguilera-Vázquez, A. Jiménez-

González y M. Gutiérrez-Rojas

259 Crecimiento, sobrevivencia y adaptación de Bifidobacterium infantis a condiciones ácidas

(Growth, survival and adaptation of Bifidobacterium infantis to acidic conditions)

L. Mayorga-Reyes, P. Bustamante-Camilo, A. Gutiérrez-Nava, E. Barranco-Florido y A. Azaola-

Espinosa

265 Statistical approach to optimization of ethanol fermentation by Saccharomyces cerevisiae in the

presence of Valfor® zeolite NaA

(Optimización estadística de la fermentación etanólica de Saccharomyces cerevisiae en presencia de

zeolita Valfor® zeolite NaA)

G. Inei-Shizukawa, H. A. Velasco-Bedrán, G. F. Gutiérrez-López and H. Hernández-Sánchez

Ingeniería de procesos / Process engineering

271 Localización de una planta industrial: Revisión crítica y adecuación de los criterios empleados en

esta decisión

(Plant site selection: Critical review and adequation criteria used in this decision)

J.R. Medina, R.L. Romero y G.A. Pérez

Vol. 13, No. 1 (2014) 349-357

INTEGRATED PLATFORMS FOR THE CLARIFICATION AND CAPTURE OFMONOCLONAL ANTIBODIES

PLATAFORMAS INTEGRALES PARA LA CLARIFICACION Y CAPTURA DEANTICUERPOS MONOCLONALES

S.C. Bernardo, A.M. Azevedo∗ and M.R Aires-Barros

Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Department ofBioengineering, Instituto Superior Tecnico, Technical University of Lisbon, Av. Rovisco Pais, 1049-001 Lisbon,

Portugal.

Received May 17, 2013; Accepted February 14, 2014

AbstractAntibodies play an important role in both medicine and analytical biotechnology. Recently, the demand for monoclonalantibodies (mAbs) has been increasing exponentially, since their potential for therapeutic use was disclosed and improvedtechniques to produce them at higher titers have become available. Although upstream processing of mAbs has suffered atremendous improvement, in the last decades, the downstream processing has been considered the bottleneck in providingantibodies at reliable costs, being, in fact, the major cost factor with 50 to 80% of total production costs. Therefore, alternativemethods for mAbs’s purification may be required. Aqueous two-phase systems (ATPS) have been described, for the last decade,as a viable alternative to the current establish platform, since clarification, concentration and purification can be achieved in justone step using a biocompatible environment. Therefore, the use of ATPS for integrative purification of mAbs it will be reviewed.

Keywords: antibodies, capture, purification, aqueous two-phase systems, integrated platform.

ResumenLos anticuerpos tienen un papel importante tanto en la biotecnologıa medica como en la analıtica. Desde que su potencial parauso terapeutico fue descubierto y las tecnicas para producirlos en concentraciones mas altas fueron mejoradas y se hicierondisponibles, la demanda de anticuerpos monoclonales (mAbs) ha estado incrementado exponencialmente. A pesar de que en lasultimas decadas los procesos de fermentacion de mAbs han mejorado notablemente, los procesos de recuperacion y purificacionhan sido considerados el cuello de botella para la produccion de anticuerpos a precios confiables. De hecho, es esta ultima etapala que involucra un mayor costo abarcando del 50 al 80% de los costos totales de produccion. Debido a esto, metodos alternativosde produccion de anticuerpos son requeridos. Los sistemas de dos fases acuosas (SDFA) han sido descritos en la ultima decada,como una alternativa viable a la plataforma actual establecida, dado que la clarificacion, concentracion y purificacion pueden serllevadas a cabo en un solo paso dentro de un ambiente biocompatible. Es por tanto que el uso de SDFA para una purificacionintegral sera revisado.

Palabras clave: anticuerpos, captura, purificacion, sistemas de dos fases acuosas, plataforma integral.

1 Introduction

Monoclonal antibodies (mAbs) are within themost important biopharmaceutical products of thepharmaceutical industry, with total sales expected toreach $58 billion by 2014 (Morrow, 2012). The

therapeutic mAbs market is growing fast and theirinnovative and lucrative offerings has thus resulted in apositive revenue growth rate even during an economicrecession (Frost and Sullivan, 2012).

∗Corresponding author. E-mail: [email protected]. +351218419065, Fax +351218419062

Publicado por la Academia Mexicana de Investigacion y Docencia en Ingenierıa Quımica A.C. 349

Bernardo et al./ Revista Mexicana de Ingenierıa Quımica Vol. 13, No. 1 (2014) 349-357

Figure 1. A typical monoclonal antibody recovery process.

Harvest/ Cell Removal

• Centrifuga-on • Microfiltra-on

Capture Step

• Protein A

Virus Inac7on

• Low pH hold

Polishing Steps

• IEX • HIC

Virus Clearance

• Ultrafiltra-on

Cencentra7on/ Formula7on

• UF/DF

Fig. 1. A typical monoclonal antibody recovery process.

Up to 2013, 33 mAbs have received regulatoryapproval by the FDA for therapeutic and diagnosticpurposes with the majority targeting diseases such ascancer and autoimmune disorders (PhRMA, 2013).According to the 2013 PhRMA’s report, there areover 907 new biotechnology medicines in currentdevelopment, targeting more than 100 diseases,including 338 monoclonal antibodies. Nevertheless,antibody therapeutics have some disadvantages,mainly their relatively expensive manufacture (Chanet al., 2009). Currently, research is being carried outon improving antibody efficacy, reducing productioncosts and improving affinity and specificity, withconsiderable success (Chan et al., 2009). Therefore, itis expected that the widespread use of antibodies willimpel the high costs down.

2 Production and purification ofmAbs

There are two critical issues in the processdevelopment of a biopharmaceutical product, suchas mAb. The first is to minimize the time to reach theclinical studies and the second is to develop a processwhich can deliver sufficient amounts of therapeuticproduct to meet market demands at an acceptable priceper dose (Birch and Racher, 2006).

Improvements in the upstream processing of mAbshave been responsible for an increase of 100 foldin cell productivity, in the last 15 years (Birch andRacher, 2006). For example, scientists working atPERCIVIA (PER. C6® technology platform) reacheda record level titer of 27 g/L at harvest for an antibodyproduct, which is a very important milestone in theproduction of monoclonal antibodies (DSM PressRelease, 2008). However, with increasing upstreamconcentrations, increases also the need to find betterdownstream recovery processes, which can become asignificant proportion of total manufacturing cost andcan as well limit the overall plant throughput (Birchand Racher, 2006). Therefore, the greatest constraintsof the current manufacturing platforms of mAbs arecurrentely found at the downstream level. Althoughthese high titers can be managed by increasing the

scale of the present purification platforms (e.g. usinglarger chromatography columns and filters), at somepoint, the physical limits of the existing facilities andscalability will be reached (Gottschalk, 2008). Thereis therefore a need to find better solutions, whichultimately can bring their prices down. In order tofind alternative downstream processing methodologiesto the traditional downstream processes (dsp), itis critical evaluate the traditional unit operationscurrently used by the biopharmaceutical companies tounderstand their limitations.

The downstream process of a biopharmaceuticalproduct aims to remove all the impurities fromthe feeding streams and obtain a purified productwith quality enough to meet the regulatory agenciesrequirements. Therefore, impurities such as hostcell protein, DNA, adventitious and endogenousviruses, endotoxin, aggregates and other speciesmust be removed while an acceptable yield ismaintained. In addition, impurities introduced duringthe purification process must be removed as well(Liu et al., 2010). The basic operations in a typicalplatform of downstream processing of mAbs, includeclarification, concentration, selective purification andvirus inactivation and removal, as illustrated in Fig. 1.

2.1 Cell harvesting

The first unit operation, in a mAb recovery process, isthe removal of cells and cell debris from the culturebroth and clarification of the cell culture supernatantthat contains the antibody product. This is generallyaccomplished using a centrifugation, depth filtration ortangential flow microfiltration. In particular, the disc-stack continuous centrifugation coupled with depthfiltration is the current preferred process (Liu et al.,2010).

2.2 MAb’s capture and virus inactivation

The next operation is a capture step, which is achievedby affinity chromatography. This is the most selectivetype of chromatography used in biotechnology, sinceit separates proteins based on reversible interactionsbetween a protein and a specific ligand covalently

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coupled to chromatography matrix (Liu et al., 2010).Specifically, for the purification of human mAbs, theaffinity ligand usually involves a recombinant ProteinA (Gottschalk, 2008), which has high affinity forthe Fc region of IgG-type antibodies, resulting in ahigh degree of purity and recovery in a single step(Liu et al., 2010). This chromatography involves thepassage of the clarified cell culture supernatant overthe column at pH 6-8. At these conditions, antibodiesbind to the column and the unwanted components flowthrough, these may include host cell proteins, nucleicacids, cell culture media components and, eventually,viruses. An optional intermediate wash step maybe carried out to remove non-specifically boundimpurities from the column, followed by elution of theproduct at pH 2.5-4, which allows also inactivation ofthe remaining virus (Liu et al., 2010).

2.3 Polishing steps and virus removal

For polishing steps, one or two additionalchromatography steps are commonly employed. MostmAb purification processes will include at leastone ion exchange chromatography step, generallya cation exchange column (CEX) for intermediatepurification and an anion exchange column (AEX)operating in flow-through mode to remove negativelycharged impurities, such as DNA, host cell proteins,endotoxins, and endogenous and adventitiousviruses. In addition, hydrophobic interactionchromatography (HIC), mixed mode chromatographyor hydroxyapatite chromatography may be chosen aswell (Liu et al., 2010). Membrane chromatographycan also be used instead the traditional packed-bedchromatography, especially for the polishing flow-through steps, as will be described in the next section.

Since mammalian cells used in the manufactureof mAbs produce endogenous retroviruses and areoccasionally infected with adventitious viruses duringthe upstream processing, a virus clearance step is alsorequired, prior to the final product formulation. Thisvirus removal is normally accomplished by a filtrationoperation using a membrane with a pore size adequateto the type of the virus. Finally, an ultrafiltration canbe used for protein concentration and buffer exchange(Liu et al., 2010).

2.4 Alternative capture steps

Currently, the downstream processing of mAb’s isfocused on chromatography, which is a critical andwidely used separation and purification technology

due to its high resolution (Gottschalk, 2008).However, chromatographic processes have somelimitations specially when are used at large scale.Thus, other alternatives have to be considered in orderto increase the manufacturing capacity and to decreasecost of goods, especially those that allow processintegration and intensification.

Other alternative unit operations may includeflocculation, precipitation, two-phase extraction,membrane chromatography and crystallization(Gottschalk, 2008). Flocculation and precipitationcan be used in combination with conventionalcell separation techniques, such as centrifugationand microfiltration, to remove residual particulatesand soluble impurities, which might otherwiseincrease the burden on polishing steps, allowing thereducing on the number of chromatographic steps.Crystallization, despite its limited application tomAb downstream process due to its low yields, isanother inexpensive technology that in some casescan simultaneously purify, concentrate, and stabilizethe protein. Membranes are already being used inmany bioprocesses, mostly for filtration rather thanchromatography. However there is a huge benefitof membrane chromatography over conventionalbead chromatography, mainly because pore diffusionis minimal. Membrane chromatography behavessimilarly to packed chromatography columns, butin the format of conventional filtration modules,which usually has multiple layers containingfunctional ligands attached to the internal pore surfacethroughout the membrane structure (Gottschalk, 2008;Liu et al., 2010).

Hence, there is a need to study innovativesolutions, which can include the review of simplerand less expensive separation technologies, suchas aqueous two-phase systems (ATPS), the use ofdisposable modules and the development of processintegration platforms.

3 Aqueous two-phase extraction(ATPE)

In the last years, liquid-liquid extraction using aqueoustwo-phase systems (ATPS), has been evaluated anddeveloped in order to obtain an alternative primarystage unit operation in downstream processing.However, despite the potentials of this technique,nowadays aqueous two-phase extraction (ATPE) isonly considered as a complementary technique and nota replacement of a unit operation in the dsp platform

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(Rosa et al., 2007). Nevertheless, ATPE has beendescribed as an effective method for the purificationof different biological products, such as cells, virus,RNA, plasmids and proteins (Azevedo et al., 2007).

ATPS are formed spontaneously when hydrophilicsolutes, such as two different polymers or a polymerand a certain salt, are mixed together in an aqueoussolution above a certain critical concentration, as aresult of mutual incompatibility (Azevedo et al., 2007;Azevedo et al., 2009a; Rosa et al., 2007; Azevedoet al., 2009b; Ferreira et al. 2008; Rito-Palomares,2004). Beijerinck, in 1896, upon mixing agar andgelatin, was the first to report this phenomenon.However, only sixty years later (in 1956) was shownthat proteins are suitable to be partitioned into ATPS(Albertsson, 1956).

Well-studied ATPSs include polyethylene glycol(PEG)/dextran and PEG/phosphate, where each phasegenerally contains 80-90% (w/w) water (Azevedoet al., 2009a). Therefore, since ATPS are mainlyconstituted by water, and most used polymershave a stabilizing effect on the protein tertiarystructure, these systems provide a biocompatibleenvironment for biomolecules, such as maps (Azevedoet al., 2009a; Azevedo et al., 2009b). Besidesproviding biocompatibility for recombinant proteins,this technique also allows integration of clarification,concentration and partial purification in just one step(Schugerl and Hubbuch, 2005). In addition, thisprocess is cost-effective and its scale-up is very easyand reliable (Azevedo et al., 2009b).

3.1 How to purify mAbs in ATPSs?

The partition of a target compound, mAbs forinstance, in ATPS depends on both intrinsic andextrinsic properties. Intrinsic properties include size,electrochemical properties, surface hydrophobicityand hydrophilicity, and conformational characteristics.While extrinsic properties include type, molecularweight and concentration of phase formingcomponents, ionic strength, pH and temperature,among others (Azevedo et al., 2009b). These extrinsicproperties can be manipulated in such way thatthe target mAb can be recovered in the phase thatcontains fewer impurities. Also, it is possible toincrease the predictability and selectivity of an ATPSextraction by attaching affinity-ligands covalently toone of the phase-forming components, thus the targetbiomolecule (mAb) can be partitioned to the phasecontaining the ligand (e.g. protein A) (Rosa et al.,2007).

Since proteins (including Abs) are usuallyhydrophilic molecules they will have higher affinityfor the more hydrophilic phase, which in polymer-polymer systems, such as PEG/dextran systems, isnormally the dextran-rich phase, and in a polymer-saltsystems, like PEG/phosphate, is the also the bottomphase (phosphate).

In polymer-polymer system, a typical strategy toenhance mAbs affinity towards the more hydrophobicphase (PEG-rich phase) is by introducing an affinityligand in the systems, i.e., in the PEG rich phase. Thiscan be achieved by adding the ligand to the systemwithout coupling (free ligand) or by modifying the twoterminal hydroxyl groups of PEG molecules with theaffinity ligand (Azevedo et al., 2009a).

The polymer-salt systems have been widelyused and are an attractive cost-effective alternativeto polymer-polymer systems, due to the use ofa relatively inexpensive phase component (salt)(Azevedo et al., 2009a). In this type of systemsthe selective extraction of antibodies can be achievedby using low polymer molecular weights, high Ionicstrengths through the addition of a neutral salt (e.g.NaCl), lowering the pH and even by introducingaffinity ligands. However, the high concentrationof salt usually masks the affinity or electrostaticinteraction between the ligand and biomolecule, whichlimits the applicability of this last methodology(Azevedo et al., 2009a).

3.2 ATPE vs chromatographic separation

At smaller scale (low mAbs titers), protein A (ProA)chromatography is very advantageous comparing withother processes, since it is possible to obtain more than95% of purity with a simpler process (Shukla et al.,2007). On the other hand, ProA chromatography alsoaccounts for the major costs in the whole productionprocess of mAbs (Rosa et al., 2010). The maindisadvantages of using ProA chromatography arethus essentially related to the high cost of ProAresins, which can be up to 10 times as expensive asconventional chromatographic supports (Shukla et al.,2007), and most importantly, to the possible leakage ofligand from the matrix and consequent contaminationof the final product with a Staphylococcus protein(Azevedo et al., 2008). In addition, higher-titerprocesses have been imposing practical limitations,namely the use of larger columns, which will makethe current technology platforms reach their limitin terms of throughput and scalability (Gottschalk,2008). Another key limitation of this type of

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chromatography is the need to carry out productelution at low pHs (Shukla et al., 2007), in order tobreak the interaction between protein A and Fc regionof Ab. Additionally, some antibodies demonstratea higher binding affinity than others, requiring alower elution pH (Liu et al., 2010). An exposureto low pH conditions can result in the formationof soluble high molecular weight aggregates and/orinsoluble precipitate formation during product elution.Insoluble aggregate formation can reduce the columnlifetime if precipitation occurs during elution, whichmight be threatening the product activity (Shukla etal., 2007).

ATPS on the other hand, constitutes an interestingalternative to the traditional chromatography sinceclarification, concentration and purification can becombined in only one operation using a non-toxicphase environment (Azevedo et al., 2008). In addition,it can overcome some of the technical drawbackscurrently encountered in chromatographic process(Azevedo et al., 2009a; Shukla et al., 2007). One ofthe major advantages of large-scale ATPE is its easyscale-up together with the possibility of continuousoperation using traditional liquid-liquid extractors.Besides, the equilibrium in ATPS is rapidly reachedand the recovery of proteins is facilitated, since thesystem operates with fewer theoretical plates thana chromatographic system (Azevedo et al., 2009a).Nevertheless, the handling, storage and disposal of thelarge amounts of raw materials required at a processscale may also constitute a disadvantage. AlthoughPEG is biodegradable and non-toxic, salt disposal(e.g. phosphate) can be an issue, which can beminimized by recycling the polymers and salts usedin the process. The recycled raw materials have,however, to fulfill the purity requirements to guaranteethe operating consistency and reproducibility of batch-to-batch ATPS (Rosa et al., 2010).

There are still some questions that still needto be answered until ATPE can be adopted by thebiopharmaceutical companies (Rosa et al., 2010).One concern is the maximum capacity of thesesystems, specifically, whether they can handle hightiter supernatants or whether the throughput willbe limited by solubility problems (Azevedo et al.,2009a). The other constrain is related to the limitedpredictive design of this process, due to the poorunderstanding of the responsible mechanisms for thebehavior of biomolecules in ATPS. The economicaland environmental sustainability of an ATPS-basedcapture process has been recently evaluated andcompared to the currently established platform and

it was shown that an ATPS alternative platform canprocess and purify continuously the same amount ofmAbs reducing significantly the costs, especially ifhigher titers of mAbs are used. The ATPS platformcan also be environmentally validated if the processcomprises the recycling of the phase forming phases(PEG and salt) and operates at high titer cell culturesupernatants (more than 2.5 g/L) (Rosa et al., 2011).

4 Integration and intensificationof process using ATPS

One of the major factors that have been interestingresearchers about ATPS is that it allows processintegration as the simultaneous separation andconcentration of the target protein can be achieved,with posterior removal and recycle of the polymer(Cunha and Aires-Barros, 2002). Furthermore, ATPScan also provide solutions for purification of mAbs atlarge scale in a continuous and multi-stage operation.

The productivity, yield and economy ofbioprocesses can be considerably improved by processintegration (Schugerl and Hubbuch, 2005). Therefore,the manufacturers of bioproducts, such as mAbs, havea considerable interest to achieve process integrationof the upstream operations of fermentation andespecially the downstream recovery processes, tofacilitate the development of scalable and efficientbioprocesses (Rito-Palomares, 2004). Thus, thereis nowadays a strong demand for intensification andintegration of process steps to increase yield, reducethe process time and cut down in running costsand capital expenditure. Besides these advantages,integrated processes still wait for broad industrialapplication, since it has a complex development, andthus there is a need for detailed process knowledge ofthe applicant (Schugerl and Hubbuch, 2005).

Process integration attempts to achieve specificobjectives not efficiently met by discrete processesby combining two operations into one. Thereare considered three major areas of research: theextractive bioconversion, the extractive fermentationand the integration of cell disruption and primarypurification step (Rito-Palomares, 2004). The latteris a key strategy to intensify the manufacturing ofbiologics, since it could enhance product yield andquality (Figure 2). The use of ATPS represents anatractive technlogy, since it is possible, by extractingthe proteins from crude feedstock, to concentrate andpurify the product in just one step (Schugerl andHubbuch, 2005).

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Figure 2. Integration of clarification and capture in an ATPS-based process.

Polishing

Viral clearance

Purification

Capture

Clarification

Harvest

Traditional DSP

Polishing

Viral clearance

Purification

ATPS Extraction

Harvest

ATPS-‐based process

Fig. 2. Integration of clarification and capture in anATPS-based process.

One of the first reports of using ATPS in anintegrative process of mAb production and purificationdates to 1996. Zijlstra et al. were able to design anATPS which support the long-term growth of animalcells, namely the mAb’s producing hybridoma cells,and concluded that with an ATPS constituted by PEG35,000 and dextran 40,000 hybridoma cells partitionedalmost completely to the lower phase. However,with this system the mAb also partitions into thelower phase. Nevertheless, preliminary work usingan affinity ligand coupled to PEG indicated that thepartition coefficient of the mAb could be considerablyimproved (Zijlstra et al., 1996), making this study veryimportant, since it opens the door for the integrationof both production and purification of bioproducts byanimal cell culture.

Process integration can also address a holisticapproach to process design in such a way that theoutlet streams from a certain unit operation can bedirectly used as inlets for the next unit operation,eliminating thus the need for feed preconditioning(Figure 3). In 2008, Azevedo et al. evaluatedan integrated process using ATPE, HIC and SECfor the purification of human IgG from a CHO cellsupernatant. These unit operations were designed toallow the removal of target impurities and also theintegration of different process units without the needfor any conditioning step between them. An ATPScomposed of 10% (w/w) PEG 3350 and 12% (w/w)citrate, at pH 6, allowed the recovery of IgG witha 97% yield, 41% HPLC purity and 72% proteinpurity. This bottom phase was then directly loadedon a phenyl-Sepharose HIC column to further improve

Figure 3. Integration strategies of ATPS with other unit operations in order to avoid pre-conditioning steps depending whether the product is recovered in a salt-rich phase or in a polymer-rich phase.

Final Product

Final Product

Final Product

ATPS Extraction

HIC

Polishing

ATPS Extraction

ATPS Back-‐

Extraction

HIC

ATPS Extraction

CEX

Polishing

Salt-‐rich Phase Polymer-‐rich Phase

Fig. 3. Integration strategies of ATPS with otherunit operations in order to avoid pre-conditioning stepsdepending whether the product is recovered in a salt-rich phase or in a polymer-rich phase.

the purification of IgG from the remaining proteinsand taking into account the high concentration ofthe citrate salt present in the bottom phase. Ingeneral, proteins bind to HIC columns at high saltconcentration followed by elution with a descendingsalt concentration. Thus, it was used a citratemobile phase and the antibody recovered in the elutionfraction with 99% of recovery yield, 86% HPLC purityand 91% protein purity. Finally, SEC allowed the finalpolishing by removing IgG aggregates and, at the sametime, to desalt the antibody solution. The fractionscollected in the HIC step were thus directly loadedin a Superose 6 size-exclusion column, without anykind of treatment and run in isocratic mode, leading a100% pure IgG solution with 90% yield (Azevedo etal., 2008).

Another important aspect to intensify a process isrelated to its scale-up. Until very recently, most of theprocess that involve ATPS were performed in agitatedvessels followed by centrifugation (Rosa et al.,2009a), however this equipment assembly (agitatedvessel + centrifuge) provides only one theoreticalstage and is expensive at large scale (Srinivas et al.,2002). From the total product recovery and economicview point, it is clear that the definition of one-stageATPS primary recovery processes are ideally preferred(Rito-Palomares, 2004). Nevertheless, more than onetheoretical stage are typically necessary to achieve thedesired yield and purity at large scale (Rosa et al.,2009a), since in order to achieve high recovery yields,higher volume ratios and larger amounts of phaseforming components and ligand will be necessary,

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leading to a highly diluted product with a low purity.Additionally, an extra concentration step may berequired, leading, as was discussed before, to a lesscost-effective process. Hence, it is necessary to finda suitable compromise between recovery yield andpurity. This problem can be solved using a multi-stage process. Actually, in a multi-stage approach, itis not necessary to select a system composition thatallows a very high recovery yield, since it can beachieved by increasing the number of stages, althoughit is important to select a system that allows a highpurification factor instead, in order to extract thelowest amount of contaminants along the differentstages. Thus, using a multi-stage process, it will bepossible to achieve a more effective process in termsof selectivity, target product enrichment, raw materialsconsumption and throughput (Rosa et al., 2009b).

The typically used multi-stage liquid-liquidextraction (LLE) equipments in chemical industryare known to provide more than one theoreticalstage, in which high recovery yields and puritiesmay be obtained. Conventional multi-stage extractionequipment such as spray columns, mixer-settlerbattery or column extractors, and non-conventionalextraction equipment such as pulsed plates column,agitated plates columns or pulsed flower columnscan be conveniently applied as an alternative totraditionally extraction equipment (agitated vessel +

centrifuge) may be employed (Rosa et al., 2009a).In fact, continuous extraction has been used in thepurification of proteins as this process increases thepartition of target proteins compared with the batchATPS process (Rito-Palomares, 2004). One exampleof an application of ATPS for a continuous operationin the recovery of a bioproduct is the work reportedby Biazus et al. (2007), in which α- and β-amylaseenzymes from Zea mays malt were recovered bycontinuous extraction using a PEG/CaCl2.

Recently, in 2009, Rosa et al. were able to evaluatethe feasibility of performing a multi-stage equilibriumATPE of IgG and respective purification from theCHO cells supernatant impurities by simulating across-flow multi-stage extraction in test tubes. Thisstudy was later validated in a packed column and in amixer-settler battery. In 2012, Rosa et al., evaluatedthe performance of a pilot scale packed differentialcontactor for the continuous counter-current ATPE ofhuman IgG from a CHO cell supernatant. An IgGrecovery yield of 85% was obtained with about 50% oftotal contaminants and more than 85% of contaminantproteins removal (Rosa et al., 2012). In 2013, the sameauthors showed the feasibility of using a mixer-settler

battery to successfully purify human antibodies froma CHO and PER.C6 cells supernatants. A full processincorporating 6 extraction stages, 1 back-extractionstage and 3 washing stages was proposed allowing theremoval of 97% of the impurities found in the CHOcell supernatant and 99% in the PER.C6 supernatant.

Final remarks

As a final conclusion, it is possible to state thatATPS represent an interesting alternative methodologyin the downstream processing of biopharmaceuticalproducts, mainly in mAbs, since they provide abiocompatible environment for biomolecules and it ispossible to achieve at large scale clarification, recoveryand purification of the target product in just onestep. However, it is required some additional studiesregarding the use of other polymers and conditionsand also to design better strategies that allows processintegration and intensification.

Acknowledgments

The authors acknowledge Edith Espitia for her helpin the translation of the abstract and title of thismanuscript to Spanish.

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