Mammalian, microbial (bacterial and yeast), and plant cellsare effective expression systems used commercially to producemass quantities of biological, pharmaceutical, or chemicalproducts of interest. However, there is the need to establishselective and scalable methods of product recovery thatintegrate effectively with upstream cell cultures to rapidlyyield products in a state suitable for validation operations. Thecurrent state of the art purification and recovery methods utilizewell-established multi-step processes (e.g. product release,solid-liquid, concentration and chromatography steps) thatusually result in low yield and high process cost. In this keynote address a series of case studies were presented wherean aqueous two-phase system (ATPS) extraction, an existingbioengineering strategy, was used to alleviate many of thepreviously mentioned existing process constraints. Specificcase studies utilizing ATPS were presented for the purificationof Rotavirus-like particles from insect cells, colorant proteins(i.e. C-Phycocyanin and B-Phycoerythrin) from microbialorigin, human granulocyte-colony stimulating factor (hG-CSF)
In Developments in Biotechnology and Bioprocessing; Kantardjieff, A., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2013.
from alfalfa, fractionation of PEGylated proteins, and stemcells. Conclusions will be drawn concerning the use of ATPSin downstream processes that can greatly simplify the currentway in which bioproducts are recovered.
1. Introduction
The recovery and purification of bioproducts (antibody, protein, chemicalcompound, etc…) produced in a host system requires separation of the productof interest from components of the host system, cellular debris, and othercontaminants after production. In general a bioprocess involves two major steps,production (fermentation) and bioseparation (primary recovery and purification).The process route in the bioseparation step depends upon the nature of the productof interest (extracellular or intratracelluar). Extracelluar products are locatedin the fermentation media culture and processed in a multi-step purificationprocess that includes a primary isolation procedure (usually involving solid-liquidseparation, product concentration and major contaminant removal operations), ahigh-resolution purification procedure (usually involving chromatographic steps),and a final polishing step (usually a drying step). Intracellular components areliberated from the cell after cell lysis, and the debris is removed, the remainingintracellular components are concentrated and fed through the mentionedpurification processes (Figure 1).
Figure 1. Schematic summary of the product production process with aqueoustwo-phase system (ATPS) added in primary recovery process.
In an effort to purify the target product more efficiently and effectively theprimary recovery of the product was achieved using an aqueous two-phase system(ATPS) (Figure 1). ATPS can be exploited to separate the product of interest frommajor contaminants. ATPS is a well-known liquid-liquid fractionation techniquethat exploits the difference in solubility (or affinity) of the product of interestand the contaminant in two different immiscible aqueous phases (1). The two
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immiscible aqueous phases are generated either by mixing two water-solubleimmiscible polymers (e.g. polyethylene glycol (PEG) and dextran) or a singlewater-soluble polymer and a buffer (e.g. PEG and phosphate) . As most biologicalproducts are water soluble, this allows a platform for extraction in an aqueousenvironment. Inserting ATPS as a unit operation integrates and intensifies aprimary recovery process in an easily scaled-up, economical process that can beused in continuous or in batch mode. Integration refers to the combination oftwo or more unit operations (processing stage) into one that achieves the samegoal. Intensification means more material can be loaded without altering alreadyestablished process capability. ATPS can be used on pilot or industrial scalerather economically.
Figure 2. Cartoon view of a simplified aqueous two-phase (ATP) extraction.Aqueous polymer phase (top phase) and aqueous salt/buffer phase (bottomphase) are immiscible. Exploiting the preferential solubility of the product ofinterest (red balls) versus contaminants (light blue balls) in opposite phases
allows for an efficient extraction and isolation of each component. The affinity ofboth components in the particular aqueous polymer phase or the buffer phase is
based on their partition coefficient, Kp.
In ATPS two immiscible aqueous solutions are carefully selected based uponthe thermodynamic partition coefficient (Kp) of each component in each phase(Figure 2) (1, 2). In a general sence (in the absence of solute-solute or solute-solvent interactions) the Kp can be defined according to the concentration of asingle molecular species in two phases if they are at equilibrium with one another,using equation (1) (1, 2).
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Where CT is the concentration of the un-ionized product of interest (or theun-ionized contaminant) in the top phase and CB is the concentration of the un-ionized product of interest (or the un-ionized contaminant) in the bottom phase.To concentrate a product of interest (or contaminant) in the top phase its partitioncoefficient must be greater than 1 (Kp >1) (3). The partition behavior dependsnot only on the system parameters (polymer molecular weight, tie line length(TLL), volume ratio (VR), pH, sample loading, and temperature), but also thephysiochemical properties of the product (or contaminant) such as its molecularweight, isoelectric point (pI) or pKa, and hydrophobicity (1). In an ATP extractionthe Kp of each component can be optimized by manipulating parameters such aspH, concentration of a certain reagent, and ionic strength such that the product ofinterest can be forced into a phase (in this case the upper aqueous polymer phase)while the contaminants are simultaneously forced into the opposite phase (in thiscase the aqueous salt/buffer bottom phase, Figure 2). This type of partitioningcan be seen graphically as mixture of two phases. The information in this binodalgraph is critical whenworking with ATPS. In this phase diagram twomajor regionsa monophasic region and a biphasic region are generated. The biphasic region isthe region of interest for ATPS, with the area above the binodal curve providingconditions that generate two-phase system. Depending on the system parametersthe composition of the coexisting phases and a tie line length (TLL) value can bedetermined. From the graph the tie line (TL) is the thermodynamic equilibriumbetween the two phases and is related to the concentration of polymer (or salt)forming phases (1). TLL is determined according equation (2) (1). In a particularTLL, different ATPS can be selected, these systems will have the same TLL butdifferent volume ratio (VR). The VR is defined according to equation (3):
Where ΔC12 and ΔC22 are squares of the differences (absolute) in the phaseforming constituents C1 and C2 in the top and bottom phases and VT is the volumeof the top phase and VB is the volume of bottom phase.
Taken together when optimized ATPS offers a potentially efficient extractionprotocol where one can simply select conditions where the target protein is, forexample, more soluble in an upper aqueous polymer phase, while the contaminantsare soluble in a lower salt/buffer phase. The current presentation presented asummary of case studies that utilize ATPS extraction as an in process step in theprimary recovery stage to simplify purification of Rotavirus-like particles frominsect cells (4), C-Phycocyanin (5) and B-Phycoerythrin (6) frommicrobes, humangranulocyte-colony stimulating factor (hG-CSF) from alfalfa (7) (and additionallytouched upon the potential use of combined 2D electrophoresis to identify processparameters for optimum ATP extraction - which will not be discussed here) (8),fractionation of PEGylated proteins (9), and stem cells (10).
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2. Case Studies: Application of ATPS2.1. ATPS in the Primary Recovery of Double-Layered Rotavirus-LikeParticles (dlRLP)
Virus-like particles (VLP) are viral mimics that contain the main structuralproteins of a virus of interest, but lack the genetic material to become infectious.Successful bioproduction of VLPs is achieved by expression of recombinantstructural proteins without expression of non-structural proteins and geneticmaterial in a host (such as in mammalian, yeast, plant, or insect cells) (11).Among other functions VLPs can be used in the development of vaccines (12)and nanomaterials (13).
Figure 3. Outline of the unit operations for the primary recovery of Rotavirus-likeparticles (dlRLP) from insect cells: (a) current process; (b) ATPS process.
Modified from reference (4).
In this particular project we studied the application of an ATP extractionsystem for the recovery of a double-layered Rotavirus like particle (dlRLP) thatwas produced on an 1000 ng/mL scale in the insect cell-baculovirus expressionsystem. Like most VLPs, dlRLPs, are difficult to purify (14). DlRLPs structurallycontain two protein layers an inner layer composed of VP2 protein and anouter layer composed of VP6 protein. Purification of dlRLP with ATPS wasthought to be an attractive alternative purification process to produce a vaccine
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against Rotovirus infection, which kills 500,000 children a year as a result ofacute gastroenterritus (15). Currently, the protocol in place for the isolation ofRotavirus-like particles is tedious and involves a five-step process (Figure 3,left). These steps include insect cell culture (Step 1), centrifugation (Step 2),a sucrose cushion (Step 3, where particle mixture is pelleted through a pad ofsucrose leaving particles intact), a Cesium Chloride (CsCl) gradient (Step 4, asedimentation method that separates particles by density), and ultrafiltration (Step5, Figure 3) (4, 7). DlRLP recovered from this process has high product purity(90%), but suffers from a very poor yield (<2%) (4, 7). To improve the processATPS was added as an in process step (Step 4, Figure 3), after centrifugation (Step2) and cell disruption (Step 3) (4). In this modified process, unlike in previousprotocols (4, 7), the intracellular and extracellular components were separatedafter centrifugation as previous studies revealed that approximately 60% of thetotal dlRLP from the insect cell culture are contained in the supernatant andthe remaining 40% are intracellular , and both were carried through to the end(Step 3-5). The overall process provided dlRLP in a high purity (90%) with adramatically improved recovery (85%) in comparison to previous non-ATPSmethods. The process is not only efficient, but in contrast with the previousmethod, this prototype process is suitable to be scaled-up (unpublished results).
2.2. Recovery of Natural Colorants from Microbial Origin
Not only can ATPS be used to purify virus-like particles, but it can alsobe applied to the recovery of natural colorants or cyanobacterial bioproducedin microbes (5, 6, 16, 17). In fact C-phycocyanin, a naturally occurringprotein-pigment complex was bioproduced in Spiruluna maxima and isolated ina compact process that included only five non-chromatographic unit operations(5). The chromophore that associates with C-phycocyanin is a phycocyanobilin,this chromophore is present in the light-harvesting phycobiliprotein familythat includes phycoerythrin (Figure 4) (18). The isolation of C-phycocyaninprocess (with its bound chromophore) included a step with ATPS, and providedC-phycocyanin in good yield (28%) and high purity (3.9, ratio of the absorbanceintensities at 620nm to 280nm) (5). Commercially C-phycocyanin is valued at$15,000 per gram of material. Similarly, B-phycoerythrin (BPE, Figure 4) aprotein-chromophore covalent complex (also with bound phycocyanobilin, Figure4) was also isolated using a modified ATPS procedure (6). Like C-phycocyanin,BPE also has various commercial applications as a colorant or marker in thefood, cosmetic, pharmaceutical and chemical industries . BPE was recoveredand purified from the red microalga Porphryidium cruentum after cell disruptionusing isoelectric precipitation (6). Subsequent polyethylene glycol (PEG) andphosphate buffer mediated ATPS extraction provided the pink colorant in highyield (72%) and purity (4.1) (6). The results were patented and have been scaledto a pilot plant process that is under evaluation and validation . ATPS extractioncan be applied for the recovery and purification of many other bioproducedproducts such as B-carotene, lutein, or recombinant proteins (16, 17).
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Figure 4. Phycocyanobilin is the typical chromophore present in thelight-harvesting phycobiliprotein family that includes phycoerythrin. The
[Phycocyanobilin + C-phycocyanin] complex absorbs and emits light at λabs/λem= 620/650 nm and the [Phycocyanobilin + Phycoerythrin] complex absorbs and
emits light at λabs/λem = 550 /575 nm (18).
2.3. Experience with ATPS Plant-Based Bioprocess Development
The ATP extraction system is a useful tool to isolate products from notonly animal and microbial cells, but also from transgenic plants. Transgenicplants are a cheaper, and thus more attractive, hosts to scale-up the production ofbioproducts (<$100/gram) versus production of similar products in mammaliancell culture ($10,000/gram) (19). As greater than 90% of the costs in a bioprocessare associated with the down-stream processes (costs associated initial captureand purification), to make plants an even more attractive bioproduction hostthe cost of downstream processes must also be economical (7, 20). The use ofplants as a bioproduct host is not a novel idea; transgenic plants have been usedas platform for the production of biopharmaceuticals from tobacco, maize, soybeans, and recently alfalfa (7, 20). Unfortunately, the bioproduction in plants doeshave its limitations. These limitations include a low yield of the target product,inconsistency in product quality, and a production system with a large amountof contaminant (7, 20). With these limitations in mind, and the added goal tolimit costs, we sought to apply our ATPS in the purification and recovery of abioproduct from a plant.
As a model system human granulocyte-colony stimulating factor (hG-CSF),a glycoprotein, was produced in alfalfa (7). Hg-CSF has been expressed inEscherichia coli, common yeasts, mammalian cells, and in tomato and tobacco(7). HG-CSF was chosen, as it is an important glycoprotein used in thetreatment of neutropenia in cancer therapy, in bone marrow transplants, and inHIV-associated neutrophil defects (21). Unfortunately, hG-CSF treatment isexpensive. The average cost per milligram is $800 and a single dose costs $250(21). To lower these costs and to provide an attractive alternative productionprocess, we investigated the production of hG-CSF in plants and utilized ourATPS technology during the recovery and purification process (7, 20).
Again the overall process strategy was simple, with three major steps inthe isolation of hG-CSF from alfalfa. The first step was to understand andcharacterize the behavior (i.e. partitioning in a particular aqueous solution) of themajor contaminants from the plants using ATPS. The second step was to identify
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and characterize the partition behavior of the model protein using ATPS. The laststep was to identify and characterize the aqueous partitioning behavior of themixture using the optimize ATP phase conditions.
Table 1. Summary of ATP extraction conditions screened for the recovery ofhost cell contaminants from the bioproduction of hG-CSF in alfalfa (7)
Ent-ry
PEG %TLL (p/p) K P % Recov. % Recov.
Size Top Phase Bottom Phase
1 600 32 ND 71 0
2 600 37 62.7 74 4
3 600 42 16.6 75 7
4 600 45 7.2 69 19
5 1450 27 5.0 54 14
6 1450 34 3.2 50 18
7 1450 42 3.0 44 17
8 1450 48 4.9 43 18
9 3350 42 1.7 19 17
10 3350 46 0.8 14 17
11 3350 51 1.1 21 18
12 3350 56 1.8 16 22
13 8000 21 ND 0 47
14 8000 36 0.1 7 65
15 8000 43 0.6 2 28
16 8000 48 0.6 18 23
Extraction of hG-CSF from its alfalfa host contaminants using an ATPSwas achieved, but was highly dependent on the size of the PEG polymer used togenerate the aqueous solution. For purposes of this discussion the term molecularweight (MW) refers to the size of the PEG polymer added to an aqueous solutionto generate different immiscible aqueous polymer solutions. Four PEG sizesor MWs were investigated PEG600, PEG1450, PEG3350, and PEG8000. Inaddition to investigating different sizes of the polymer added into the aqueousphase, the concentration of the polymer was also increased, as indicated withthe increasing %TLL (Table 1). Following the previous outlined steps we firstconcentrated the contaminant in the top phase using PEG600 and PEG1450, witha recovery ranging from 69% - 71% for the former and 43% - 55% for the latter.The contaminant could also be concentrated in bottom phase using PEG8000,
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all be it with a significantly wider range in recovery 23% - 65%, and suboptimalKp (Kp < 1). The results give two options for how to force the contaminants inone phase (top or bottom), with Entries 1, 6 and 14 (Table 1) standing out asthe highest recovery extraction conditions. Moving onto step 2 we were ableto force the recombinant hG-CSF (rhG-CSF) into the top phase of PEG600,PEG1450, or PEG8000 ATPS-phosphate at pH of 7 and VR = 1 with a percentrecovery of 72%, 98%, and 78%, respectively (Table 2). Quantification of proteinwas determined using a standard Bradford protein assay (22) and confirmed bySDS-Page electrophoresis (7). Based upon the combined results of step 1 andstep 2, PEG8000 with a %TLL of 35 (Entry 14, Table 1 and Entry 3, Table 2)presented the optimum profile where the plant contaminants were forced to residein the bottom phase (65% in the model) while a majority of the rhG-CSF wasforced to reside in the top phase (78% in the model) (7). In the step 3, the behaviorof the mixture (production contaminants + hg-CSF) was characterized using theoptimized ATPS (PEG8000, %TLL 35, VR = 1, pH 7, PEG 8000-Phosphate salts).The overall process was successful and rhG-CSF was extracted in the top phasefrom contaminants (7). SDS-Page of the two phases (layers) in comparison tocontaminants confirmed this result (7).
Table 2. Summary of ATP extraction conditions screened for the recovery ofhG-CSF from host cell contaminants after its bioproduction in alfalfa (7).
Not determined = ND, KP not determined
Entry PEG %TLL %PEG %K2PO4
% Recov. % Recov.
Size (p/p) (p/p) (p/p) Top Phase Bottom Phase
1 600 32 15 18 72 ND
2 1450 34 16 14 99 ND
3 8000 36 16 11 78 ND
2.4. Separation of PEGylated Therapeutic Proteins Using ATPS
PEGylation is the process of covalently attaching a polyethylene glycol(PEG, H(OCH2CH2)nOR) group onto the surface of a drug molecule or protein(where R = the drug molecule or protein, Figure 5) (9). The covalent attachmentis formed after the molecule of interest is treated with a methoxy-PEG (mPEG)that has only one reactive hydroxyl group (which avoids the formation of crossedlinked products) and has been approved for use in pharmaceutical preparations(23). PEGylation of a drug molecule or protein can hide it from degradationenzymes, reduce immunogenicity, enhance physical and thermal stability, increasesolubility or in vivo circulation time (24). PEGylated proteins have been approvedby the US Food and Drug Administration (FDA) (24).
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Figure 5. Cartoon view of PEGylation reaction (a) and its products (b).
In this case study ribionuclease A and α-lactalbumin were PEGylated (9).Unfortunately, because a protein possess multiple reactive sites a populationof over PEGylated conjugate species, such as mixture of native protein(non-PEGylated), mono- and di-PEGylated (Figure 5) that exhibit differencesin biological activity were produced in the PEGylation reaction (24). For ourpurposes the mono-PEGylated proteins were of interest, thus we sought to applyour ATPS technology to isolate the desired mono-PEGylated proteins. Thechallenge was to recover and purify these products from other reaction productsand then to sub-fraction the different PEGylated compounds. The separation ofthe mono- and di-PEGylated compounds was achieved chromatographically, incollaboration with researchers as Carnegie Mellon University (25).
The ATP partitioning behavior of the various PEGylated conjugates ofribonuclease A were investigated using four different aqueous PEG polymersizes (PEG400, PEG1000, PEG3350 and PEG8000) at different TLLs (15, 25,35, and 45 % w/w) and the results were plotted in a graph of lnKp as a functionof %TLL (% w/w, Figure 6). The results clearly demonstrate that the variousPEGylated ribonuclease A conjugates can be separated from the native proteinusing PEG8000, as demonstrated by their sub-fractionation in opposite phases ata %TLL of 35 (Figure 6). Similarly, sub-fractionation of native α-lactalbuminfrom its PEGylated conjugates was also possible, although to a lesser degree thanwith ribonuclease A, with aqueous PEG8000 using an ATPS as demonstratedby their sub-fractionation in opposite phases at a %TLL of 35. An interestingobservation in these experiments was that the native proteins tend to concentratein bottom phase of the ATPS and the PEGylated compounds in the upper phase,under the conditions investigated. Unfortunately, separation of the mono- fromthe di-PEGylated ribonuclease and α-lactalbumin has yet to be realized and isstill ongoing. However, we present the isolated yields for the various mixtures
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of the PEGylated conjugates of ribonuclease A and α-lactalbumin versus theirnative proteins isolated from these ATP extraction experiments are shown inFigure 7. The results do demonstrate that ATP extractions also have the potentialto separate native proteins from the PEGylated analogues (9).
2.5. Aqueous Two-Phase System Bioengineering Strategies for the PotentialRecovery and Purification of Stem Cells
Recent efforts in our group have extended the application of ATPS technologyinto an entirely new area, the recovery and purification of live and viable cells,specifically stem cells (26). Currently, the isolation process is performed on benchscale, and suffers frommany limitations (26, 27). Our goal in this area is to developa scalable, rapid, economic and novel bioengineering strategy for the recovery andpurification of stem cells exploiting non-conventional technologies, such as ATPS,that will allow for manipulation of high quantities of sample, reduce losses andprocessing times. Two strategies are currently being explored that include the useof a density gradient ATPS (Strategy 1) or the use of immunoaffinity ATPS, whichincludes free antibody or PEGylated antibody binding or immobilized antibodylabeled microbeads (Strategy 2).
Efforts have focused on the isolation of CD133+ stem cells from humanumbilical chord blood using first a well known procedure, lymphoprep (28).CD133+ are implicated in the treatment of many degenerative and chronicdiseases, and thus a scalable isolation process is necessary. After isolated bylymphoprep the behavior of the stem cells was analyzed by flow cytommetry and7AAD. Flow cytommetry and 7AAD steps are essential to ascertain cell viability,as all isolated stem cells must be alive and viable to be of any therapeutic use (29).
Efforts in the arena are ongoing and thus in progress. Briefly, novel ATPSexploiting the binding of antibodies and/or PEGylated antibodies to stem cells(Strategy 2) is the most promising way to direct (or force) stem cells into aparticular phase (Figure 8). PEGylation of an antibody (that selectively binds toCD133+ stem cells) forms a tightly associated complex with the stem cell andthus may influence the partition coefficient for complex in a particular phase of an
ATPS (Figure 8). Recently, we disclosed that PEGylation of Biotin (H2NPEG ,
where = Biotin) was achieved (30). Further, the PEGylate Biotin could bind
to Streptavidin ( ) and to another Biotin-CD133+ cell ( CD133) forming
a complex of all three components (H2NPEG(- - CD133, Figure 8) (30).Whether or not the PEGylated complex to influences or enhances solubility is asyet unclear.
In last part of Strategy 2 combining this immunoaffinity guided ATPextraction process onto solid support, with immobilized affinity labeledmicrobeads may provide an alternate strategy for the selective isolation of stemcells from contaminants in two-phase system. Finally there is a recent reviewcovers all uses of ATP technology in the purification of bioproduced products(26).
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Figure 6. Plots of lnKp versus %TLL (p/p) for the ATP extraction of ribonucleaseA. (a) PEG400 (top phase)-phosphate (bottom phase); (b) PEG1000 (top
phase)-phosphate (bottom phase); (c) PEG3350 (top phase)-phosphate (bottomphase); (d) PEG8000 (top phase)-phosphate (bottom phase). Kp = partitioncoefficient and TLL = tie line length. Legend: ♦ = native ribonuclease A, ▪ =mono-PEGylated ribonuclease A, ▴ = di-PEGylated ribonuclease A (9).
Figure 7. Recovered yields for the purification of ribunuclease A andα-Lactalbumin using an ATPS of PEG8000-phosphate. (a) mono- anddi-PEGylated ribonuclease A from native protein (b) and mono- and
di-PEGylated α-lactalbumin (9).
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Figure 8. Schematic look at the methods for isolating CD133+ stem cells usingATP according to Strategy 2. (a) Immunoaffinity based (CD133 antibody-stemcell) partitioning in ATPS; (b) PEGylated antibody ATPS based; and (c) Cartoon
representation of the association of PEGylated Biotin (H2NPEG , =Biotin) to Streptavidin ( ) and to another Biotin-CD133+ cell ( CD133)
forming a complex of all three components (30).
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3. ConclusionsOne of the major outcomes of all the strategies presented is that the product
and contaminant partition characterization in an ATPS is key to proposing initialrecovery conditions. An improved understanding of what drives ATP processeshave resulted in the rapid establishment of strategies for the recovery of biologicalproducts. Further ATPS strategies are easy, scalable, and economical and as suchare suitable vessels to help biotechnology engineers face the new challengesin downstream processing. ATPS can contribute with the existing techniquesto address current and new problems of the biotechnology industry. However,challenges such as handling high numbers of cells or high products concentrationstill needs to be addressed (Product >10g/L, Cell concentration > 40% w/w).
AcknowledgmentsThe authors wish to acknowledge the financial support of Tecnológico de
Monterrey, Bioprocess research chair (Grant CAT161), the Mexican ResearchCouncil (CONACyT) and the Zambrano-Hellion Foundation.
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