Design and Operation of Continuous ... - ACS Publications

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Design and Operation of Continuous Countercurrent Chromatography in Biotechnological Production Steffen Zobel, Christoph Helling, Reinhard Ditz, and Jochen Strube* Institute for Separation and Process Technology, Clausthal University of Technology, D-38678 Clausthal-Zellerfeld, Germany ABSTRACT: Continuous countercurrent chromatography has been established in industrial operations for over six decades and in fine chemical and pharmaceutical industry since the 1990s. In biotechnological processing, the area where chromatography plays a criticaland often multipleroles, implementation is lacking for various reasons. Options are shown to correct this and make continuous countercurrent chromatography a technologically and economically viable option in GMP-regulated processing. Current approaches, with the exception of MCSGP (multicolumn countercurrent solvent gradient purification), just operate a manifold of 2−6 columns in a sequential but batchwise scheduling. Modifications were made to reduce equipment complexity in GMP-regulated manufacturing and to exploit benefits of countercurrent operation for adsorbent and buffer reduction. A solution with only one column and a minimum of valves, pumps, and buffer vessels was developed. The approach also describes integration of two or more chromatography steps in continuous production, both GMP- and biocompatible.

1. INTRODUCTION Stratified and personalized medicine approaches are beginning to have an increasing impact on manufacturing scenarios of therapeutic drugs. The increasingly smaller and, in the future, even further decreasing number of blockbuster candidates justifying metric tons per year manufacturing capacities in large scale batch plants is no longer an efficient option to deal with the timely and cost-effective production of a manifold of 10− 100 kg scale stratified drug candidates. This will force a paradigm shift toward continuous operation of smaller scale dedicated plantswith or without disposable technology.1−4 Because of this, the time is now ripe to establish a fully continuous operation also in biologics manufacturing. Upstream fermentation technology has already created some solutions (e.g., with perfusion technology).5,6 However, the continuous mode on the upstream side is afterward switched back to batch, which of course gives up all the benefits gained before, like a lower titer and cell density with the corresponding lower product variability, which causes fewer side components and contaminants for more efficient and robust downstream processing.5,7 The existing continuous chromatography approaches all share the same drawback: they are not applicable in GMPregulated full continuous biologics manufacturing with more than one chromatography step. A way so reduce the number of orthogonal chromatography steps further, which are necessary to reduce typically heterogeneous side component spectra like DNA and HCPs, is still under regulatory discussion for product safety. Even when establishing new separation technologies like ATP-LLE (aqueous two phase liquid−liquid extraction)8,9 or MCSGP-IEX-chromatography instead of protein A capture steps10,11 or 3-PCC-chromatography with reduced protein A amounts,5,7 the better approach is capable of a consequent reduction of the number of steps and full continuous operation. Of course, any studies that point out protein A adsorbent amount reduction by three smaller columns in 3-PCC instead of one large batch column should consider the definition of adsorbent lifetime by number of batches, lifetime, or amount of © 2014 American Chemical Society

feed/adsorbent amount as well as an appropriate scheduling; if a batch column is operated only one day per week for 40 weeks per year, it is obvious that any process that utilizes the equipment longer than that number of days per week is more efficient, proportional to the utilization difference. As the product is stable in that time span, then any batch column could be operated as well within that time span and would generate similar good efficiency. No improvement is gained by a no-countercurrent mode. It is well known in chromatography operations that permanent operation of chromatography gives a more stable result than with some interruptions like in batch operation.12 General regulatory issues in any modifications of GMPregulated manufacturing, besides product safety and process robustness, is the stability of the product due to storage conditions. Here, any fully continuous operation mode does have benefits in definition.13 Another manufacturing issue is the lack of qualified personal who could not be recruited and trained for an upcoming manifold of batch-operated small scale plants otherwise needed for stratified medicine drug approaches. This bottleneck is reduced in the continuous manufacturing approach because fewer operators per plant are needed.14 Chromatography is one of the commonly used unit operations in downstream processing with outstanding importance in biopharmaceutical applications.15 Using fermentation to produce drugs (e.g., recombinant proteins or monoclonal antibodies (mAbs)) the biopharmaceutical downstream process deals with a large range of impurities such as host cell proteins (HCP) and DNA.16 Therefore, to meet the strict purity criteria, the downstream process is the major cost Special Issue: Massimo Morbidelli Festschrift Received: Revised: Accepted: Published: 9169

September 18, 2013 December 18, 2013 December 18, 2013 January 6, 2014 dx.doi.org/10.1021/ie403103c | Ind. Eng. Chem. Res. 2014, 53, 9169−9185

Industrial & Engineering Chemistry Research

Article

Figure 1. Comparison between batch-chromatography, true moving bed, and simulated moving bed (acc. to 20).

pumped in from the other end. This generates the effect that the lesser binding component moves along the with eluent stream, and the other component binds more strongly to the adsorbent and is transported along with it. That way, both components can be withdrawn at both respective ends of the column continuously. The main advantage of this countercurrent operation lies in the maximization of mass transfer.21 This leads to a better utilization of adsorbent capacity, a reduction of the eluent volume required, and thereby a reduced dilution of the components in the mobile phase.21 In many cases, a concentration increase of the lower-binding compounds within the column is possible, leading to a shift of the phase equilibrium toward higher loadings in accordance with the isotherms.22 A high utilization of adsorbent capacity leads to competition of the components for the free binding sites, leading to displacement effects, which enhances separation efficiency.22 The biggest disadvantage of the true moving bed technology lies in the fact that its technical realization goes along with particle abrasion and high axial backmixing.25 This problem is circumvented by the simulated moving bed process, where the chromatography column is subdivided into several segments (see Figure 1). Instead of circulating the adsorbent, the column segments are switched in such a way that they move against the direction of the eluent stream.21,23 For that at least four columns, are required. As can be seen in Figure 1, the four volume streams (feed, eluent, raffinate, extract) divide the SMB into four zones, each of which contains at least one, and sometimes several, columns.23 These columns pass each segment in sequence by periodically and simultaneously switching the inlet ports of the volume streams.23 Switching- or step-time is kept constant.24 Zone I acts as the desorption zone for the strong binding component by setting the highest elution strength, that way withdrawing a partial stream as the extract. In zone II, the lower binding compounds are desorbed at a lower flow rate and retention to enter zone III, mixed with fresh feed. In zone III, adsorption of the strong binding component occurs so that the partial stream of the lower binding component can be withdrawn as raffinate. At the lowest flow rate and highest retention time, zone III achieves adsorption of the weak binding component to recycle regenerated eluent back into zone I.23 Isocratically operated elution-SMB reaches almost ideal TMB (true moving bed) performance with relatively few (i.e., 4−5 columns).25 Nevertheless, in relation to batch chromatography the equipment and control demand for 5 pumps (or 3 and 2

factor in production, accounting 50 to 80% of the total manufacturing costs.10,16,17 The lion’s share of these costs is allotted to affinity chromatography, which is widely used because it provides relatively high yields and purities in a single chromatography step.16 Nevertheless, due to the high resin costs, replacement of affinity chromatography is a main goal of different research approaches. Up to now, the biopharmaceutical downstream process usually contains three buffer gradient batch chromatography steps. For economic, separation efficiency, and process variability reasons, the conversion of batch chromatography to continuous chromatography is highly valuable.18,19 Hence, during the last decades, various continuous chromatography concepts were established. In general, these can be classified in three groups: 1. Continuous countercurrent multicolumn elution chromatography. 2. Continuous multicolumn gradient chromatography. 3. Continuous countercurrent multicolumn gradient chromatography. All concepts mentioned above have in common that they operate with more than one column, often combined with complex interconnections requiring extensive measurement and control technology. Thereby, they all have to deal with manufacturing operators prejudices concerning handling and operation stability. To overcome these drawbacks, a single-column continuous countercurrent elution chromatography is presented (SC− CCC). Furthermore, the SC−CCC concept is used to establish a combination of two chromatography steps working as one continuous countercurrent gradient chromatography unit operation. This combination competes against the affinity chromatography as a benchmark to displace this cost-intensive step fully with one highly efficient process. 1.1. Continuous Countercurrent Multicolumn Elution Chromatography. If batch chromatography is operated in the isocratic mode, separation is based on the different migration velocities of the individual components through the stationary phase.21 Assuming sufficient separation efficiency, the components leave the separation column at different times. These differences in migration velocities are used by the “moving bed” processes. Figure 1 shows the schematic set up of a moving bed chromatography. In the true moving bed set up, the separation mixture is introduced in the middle of the column. Eluent is entering the column from one end, and the adsorbent is 9170

dx.doi.org/10.1021/ie403103c | Ind. Eng. Chem. Res. 2014, 53, 9169−9185


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Figure 2. Schematic visualization of Varicol-SMB switching times (acc. to 38).

Figure 3. Example for serial column set up.41

mass flow meters), 6−7 valves per column, and 1−3 detectors is higher, which is compensated by the lower operating costs.26 Gradient operation in SMB is theoretically possible, but difficult.27 However, operation with two different eluent concentrations is easier to realize.27 Several variants of the SMB-technology have developed over the years, eSMB,28 ModiCon,29,30 PowerFlow,30,31 TandemSMB,32 Multizone-SMB e.g.,33 open 3 zone SMB,34 Reactive SMB.35 A special variant of MSB technology that has penetrated production scale of enantiomers is the so-called variable column length (Varicol) technology.36,37 The apparative lay out of the Varicol-plant is in principal identical with the setup of a classical SMB system. The difference lies in the switching times of inlet and outlet streams (see Figure 2), which are not switched simultaneously but timely offset.38−40 This permits a better utilization of the individual columns and leads to a reduction of the total column number.38 1.2. Continuous Multicolumn Gradient Chromatography. Another often-employed concept for the implementation of continuous chromatography is the serial column arrangement (Figure 3). This set up only makes sense when in contrast to the classical SMB mode the columns are operated in the “bind and elute” mode. All elements of a gradient batch chromatography are executed starting with loading the feed onto the columns under conditions for reversible binding to the

stationary phase (load). In the next step, unbound materials are eluted from the column in a different buffer (wash) followed by another buffer change to remove the components from the column (elute). Last step in the sequence is the regeneration and equilibration of the column for the next cycle.42−44 Single column loading typically results in yield losses45 because dynamic binding capacity at high volume streams is a limiting factor resulting in shallow elution curves with early breakthrough. Though the adsorbent in the first part of the column can be completely loaded in accordance with phase equilibrium, during the loading phase in the last column segment, product breakthrough already occurs, although the stationary phase is not yet fully loaded.45 This leads to either loss of capacity due to incomplete loading or yield loss due to product breakthrough, both of which are prevented by the serial set up. Breakthrough from the first column is captured in the second column. When the first column is completely loaded, it is removed from the column sequence and goes through the wash, elution, regeneration, and equilibration off-line, these steps being identical with the classical batch chromatography in the “bind and elute” mode. After equilibration the column is recoupled at the end into the column sequence.41 A further advantage in the use of this column set up is that a gradient operation is possible, which allows better separation results.46 In addition, multicomponent mixtures can be 9171



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separated. Nevertheless, compared to the SMB-variants already presented, this operation mode does not provide the advantages resulting from a countercurrent operation. It is not possible to withdraw the desired components simultaneously and continuously. Also, the separation is not better than in a conventional batch-gradient-chromatography because no displacement effects can be utilized. Only the total column load is improved. This approach is, among others, implemented by the BioSMB of Tarpon [see 47], as well as the SMCC of NovaSep [see 48] and the PCC of GE [see 45], Though Tarpon uses two columns for the load phase, NovaSep uses three columns. 1.3. Continuous Countercurrent Multicolumn Gradient Chromatography. To combine the advantages of countercurrent processing with those of gradient elution is not possible in the operations presented so far. To the knowledge of the author, this becomes possible through the use of MCSGP (multicolumn countercurrent solvent gradient puri®cation) only. As pictured in Figure 4, in the six-column-

Figure 5. Two-column MCSGP.49

that, the columns switch their position and the sequence starts again.49 In the two-column-MCSGP mode, the maximum product yield is reached. In addition, by placing the feed entry point between the overlapping regions, the already-mentioned displacement effect is achieved. That way, the countercurrent character of the Six-Column-MCSGP is maintained.49

2. CONTINUOUS COUNTERCURRENT SINGLE-COLUMN ELUTION CHROMATOGRAPHY The possibility of reducing SMB to a one-column operation was already derived mathematically in the past.19 Consequent reduction shows the following: each column progresses sequentially through all four zones. In classical SMB, each zone has an entry and an exit each for the volume streams from the current to the next zone. Two inlets for eluent-stream (zone I) and feed-stream (zone III), plus two outlets for extract (zone I) and raffinate (zone III), have to be added. The minimal configuration of an SMB is one column per zone. This means that in one complete cycle, each of the columns is in contact with eight different volume streams. To operate a single column as if it was part of a four-column SMB requires a maximum of eight containers as shown in Figure 7. All four steps are visualized. The concentration profiles inside the column are displayed for the end of the step. It can be taken from Figure 6 that the outlet stream of zone III matches with the raffinate stream in its physicochemical properties apart from the volume stream. This means that both streams can be collected in one container. The same holds for the outlet of zone I and the extract stream. Operating a column in an SMB set up thereby requires six containers and two pumps, where the second pump is only used for zones I and III. Under certain conditions, further reduction of parts is possible (see Figure 8). As seen in Figure 7, the outlet of zone II and the feed mixture are fed into zone III. Therefore, it might be possible to leave out the container for zone II as it contains a mixture similar to the feed composition. However, the outlet from zone II has in the stationary mode of the SMB a higher concentration of both components than the feed. If the feed container is relatively large, then this concentration is diluted and the concentrationrelated displacement effect is lost. On the other hand, to ensure a continuous operation, the feed container has to be sufficiently large to contain the feed generated while the system goes

Figure 4. Six-column MCSGP (acc. to 49).

configuration, three columns each are connected and three are run independently from the others.50 The column switching occurs in such a way that each column is switched back into the recycling stream after a solo-run and then functions again in the single column mode.51 The column goes through the sequence of load, recycling stream, elution, recycling stream, wash/ regeneration, recycling stream. By using six pumps in total, it is possible to run different linear gradients in each column.52 This process flexibility is dearly paid for by the high investment costs. The MCSGP process has been developed further, starting from six, via four and three columns,53,54 down to the two column MCSGP.49 Figure 5 shows schematically the functional principle of a two-column MCSGP. Both columns are only linked in the phases I1 and I2, where the overlapping fraction between sideand target-components from column 1 is transferred to column 2. As column 1 at this point is in a gradient mode, the transferred medium has to be adjusted by a second buffer to the right buffer concentration. In step B1, pure product is withdrawn from column 1, and feed is provided to column 2. In step B2, the first column is washed with the highest buffer concentration, while at the same time, the gradient in column 2 starts and the lower binding component is withdrawn. After 9172



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cross section has to be increased 4-fold, which means doubling the diameter. The application of working diagrams and equilibrium theory allows the generation of starting values, which then can be optimized by aid of rigorous process modeling.23 2.2. Process Modeling. Process design via operation diagrams gives very limited process optimization potentials. A deeper insight into the critical elements is provided by rigorous process simulation. Here the physicochemical model of a batch column is linked in an equation-oriented flowsheet-process simulation program with the respective containers and hold-up tubes. The batch-column model uses the distributed plug flow model with linear driving force23,44 according to eq 1 for the mass balance of the mobile phase ∂ci (1 − εb) 3 ∂ 2c ∂c = Dax,I 2i − u int i − · ·k f (c i − c p,i|r = R p ) ∂t ∂z εb Rp ∂z (1)

Figure 6. Schematic representation of the SMB process.

Mass transfer resistance within the pores of the adsorbent is described by eq 2

through the other three zones. Therefore, the boundary conditions for defining the container dimensions depend on the equipment size. Zone I is supplied with fresh eluent. Also, recycled eluent from zone IV is added. Optionally, the eluent from zone IV can either be discarded or fed into the container for fresh eluent, both of which would save an additional container. In this case, one pump could be saved. The principal functionality of this set up has already been shown.19,55−58 It was found that the separation results were superior to conventional chromatography but were inferior to conventional SMB.55 This is due to the fact that concentration profiles are destroyed in conventional stirred tanks. A suggested solution was to capture the outlet streams of each zone in several containers in order to allow stepwise collection of the concentration front55 or to install a delay coil.19 2.1. Process Concept and Design Methods. The design of a one-column SMB can follow the classical SMB.24 The selection of columns and adsorbents depends on the mixture to be separated. Feed streams and switching times can be calculated on the basis of the “local equilibrium model” and the “solute movement” theory for the case of linear isotherms.55 For linear as well as nonlinear isotherms, the operating conditions can be laid out via operation diagrams (see Figure 9), wherein netto-flow rates m1, m2, m3, and m4 are put in relation to each other. In accordance with the equilibrium theory, in the operation diagram for the flow rates m2, m3 a region can be defined in which complete separation of the components is realized. The boundaries of this working range are only dependent on the equilibrium isotherms. Deductions, special cases, and applications have been comprehensively described by Morbidelli et al.59,60 and Mazzotti et al.61−65 To process the same throughputs with a one-column SMB as with comparable classical SMB requires a column of double the diameter than is required for the columns of the corresponding classical SMB. The single column is only every fourth step in the zone, in which it is loaded with the 4-fold SMB feed volume. During the other three steps, this volume is stored in a container. In order to match the flow rate of the one-column SMB with the calculated flow for the four-column SMB, the

εp

∂c p,i ∂t

+ (1 − εp)

∂qi

∂t ⎡ ⎛ ∂c p,i ∂q * ⎞⎤ 1 ∂ + (1 − εp)Ds,i i ⎟⎟⎥ = 2 ⎢r 2⎜⎜εp·Dp,i ∂r ⎠⎦⎥ ∂r r ∂r ⎣⎢ ⎝

(2)

Equilibrium can either be described by Langmuir isotherms or the “steric mass action” model. The fundamentals of the models used, as well as the description of the individual terms, have been comprehensively described in refs 42, 44, and 66. The experimental model parameter determination on the lab scale for complex mixtures can be found in, for example, refs 23 and 67. After the theoretical feasibility is proven, finally, an experimental validation in laboratory scale is necessary before scale up. 2.3. Simulation Studies of One Column CCC. Batch chromatography can be simulated for a wide variety of compounds, with or without gradients as well as SMB- and MCSGP-processes dynamically and rigorously.23,42,68 This capability is applied to the design of a one-column CCC. Figure 10 shows the concentration profile of a four-column SMB in comparison with the profiles of the one-column SMB composed into one diagram, based on the eight-container set up as presented in Figure 7. Feed loading always takes place in zone III. The differences in the profiles resulting from the backmixing of the concentration profiles within the containers shows specifically in the different concentrating and fronting forms. This shows quite clearly in the raffinate in zone III. The layout of the holding vessel as a stirred tank destroys the concentration profile of the preceding column. As a result, the following zone is loaded with a continuous, average concentration; therefore, no peak height increase occurs in the following zone (here, zone IV). However, under the given conditions a concentration increase within the zone takes place. With the parameters selected for this study, the one-column SMB gives a slight reduction in purity compared with the fourcolumn SMB (see Figure 11 and Table 1). At the same time, however, a higher concentration of the components is reached. For example, the average effluent concentration of the raffinate from zone III in the one-column SMB is approximately twice 9173



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Figure 7. Flowchart of one column with eight containers for all steps, with internal concentration profiles at the end of the step.

Figure 12 shows the operation diagram with the operating parameters for both simulation studies. In the operation range defined by the blue lines, no separation could be realized. In this operation range, kinetic effects have not been considered. The parameters used for the studies, however, reflect a kinetically controlled system with axial dispersion and internal equipment dead volumes. As shown in this chapter, with the aid of dynamic process simulation, operating parameters and process concepts can be developed that efficiently permit the operation of a lab scale unit for the validation of the process concept as well as setting the specifications for building a large scale production plant. Another advantage is the ability to design a complete process in silicowithout laboratory experimentsefficiently. On the

that of the four-column SMB. For the average effluent concentration of the extract from zone II, an increase of approximately 55% has occurred. Figure 11 shows that the yield to purity ratio for the raffinate is slightly higher for the one-column SMB. It can also be seen that 100% purity cannot be reached. For the extract, the lines for purity and yield of the one-column SMB are below those of the conventional SMB. Also in this case, a 100% purity is not reached. Nevertheless, the lines overall show a good comparability of the two processes. The differences are caused by the backmixing effects already discussed. The individual data points in the purity vs yield diagram (Figure 11) vary by the cycle times, which increase from left to right. The correlating operating parameters are presented in Figure 12. 9174



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Figure 8. Schematic representation of the one-column SMB with the possible simplifications.

for total process performance enhancement has already been validated.68 Apparently, sacrificing process efficiency in each single step for the sake of process integration in reality results in a total process enhancement. Figure 13 summarizes set up and phase equilibrium data. Although in the IEX step loading takes place at low ionic strength and elution at high ionic strength, in the HIC step loading occurs at high salt concentration and elution by reducing ionic strength. In many downstream operations, for instance, in the biopharmaceutical industry, several chromatographic steps are combined to achieve the required product purity. As the different operations rarely use the same buffer, a diafiltration or comparable step often is required to exchange the buffer. A suitable combination of two one-column SMB processes, linked similarly to the two-column MCSGP set up, could save this step. Figure 14 shows this set up. It is assumed that the sequence of components in the columns is the same. In case of changing sequences, analogous set ups can be realized. The goal of the iCCC is to capture and return all elution fractions containing target compound into their respective columns. In the IEX/HIC case, the mixed fractions with lower salt concentrations is fed back into the IEX column. For constant sequences, the following steps result: 1. The ion exchange column already preloaded in step 6 is loaded with feed. The loading is followed by a wash step prior to starting the gradient elution (step 2). 2. The ion exchanger is loaded with the fraction strong binder/target compound (S/P) of step 6 of the HICcolumn. At the same time, the salt gradient (E) starts. Product-free medium goes to waste (W). When target compound starts to elute, the now eluting mixed fraction (P/W) is caught in one container. The HIC column is

Figure 9. Operation diagram for the SMB design.65

same basis, continuous concepts for the partial or total process design can be drafted, including the economic aspects: Table 1 summarized the results of the comparison. One-column-SMB is capable of gaining the performance advantages of multicolumnSMB over batch chromatography but with less equipment

3. TOTAL PROCESS INTEGRATION: IEX AND HIC-COMBINATION A full implementation of a one-column CCC into a complete process has been undertaken, wherein the iCCC is operated in a full continuous and countercurrent mode. This needs, however, a couple of further process innovations. In principle, the elution strength of the preceding steps has to be matched with the loading conditions of the following unit operation. This is conceivable for several operations and here is implemented for the combination of IEX and HIC. The approach of matching the buffer strength of two unit operations 9175



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Figure 10. Four-column and one-column SMB chromatogram profiles in each zone.

3.

4.

5. 6.

In principle, for such a set up, two chromatography modes need to be selected that use the same buffer system but have opposite adsorption/desorption characteristics. This is the case in the combination of IEX and HIC. Also, coupling a normalphase and a reverse phase separation is possible. Further applications and process optimization studies need to be followed up. Detector combinations like DAAD, conductivity, and pH are under consideration, whereas different positions at the column outlet and vessel inlets are analyzed at the moment. In addition, an option to implement is the use of membrane adsorbers with the potential to replace some column operations. 3.1. Theoretical Process Feasibility. The simulation studies show the principal feasibility, including the relevant dependencies for the definition of the operating parameters. Figure 15 depicts the advantages of the IEX/HIC combination in a simulation. The first ten cycles are displayed, within which the stationary state is reached. Constant cut points of the product fraction are used for the calculation. As the recycling of the mixing regions is operated at a salt strength equivalent to the elution strength of the lower binding component, this peak shifts in the IEX step to the dead time of

preloaded in this step with the mixed fraction (P/W) from HIC-step 4. The ion exchanger column is further eluted. The now eluting product fraction is directly loaded onto the HIC column. The selection of the cut points depends on several factors with the main objective to find the compromise between purity of the directly transferred fraction and the volume of the mixed fractions. The salt gradient in the IEX step levels out. The mixed fraction of strong binding component and target compound (S/P) is also directly transferred to the HIC column, where the decreasing salt gradient starts. Medium free of the target compound is directed to waste (W). The mixed region of low binding and target compounds (P/W) is fractionated into a (separate) container. The IEX-column is washed and regenerated. From the HIC column, the target component is eluted with its target purity. The ion exchange column is loaded with the mixed fraction from IEX step 2. From the HIC column, the mixed fraction for the loading of IEX step 2 is taken. This ends the cycle. 9176



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Figure 11. Purity vs yield diagram of both one-column and four-column SMBs.

Thereby the simulation studies can verify that the basic concept of a countercurrent process with enhanced mass transfer and enrichment actually takes pace in comparison with the analogous batch process. In addition, Figure 15 shows also that the cut times can be chosen in such a way that the second side product, according to plan, is not enriched. If one compares the study conducted here with a simple, serial arrangement of an IEX and a HIC column operated in batch mode under otherwise the same conditions, the numbers from Table 2 result.

Table 1. Comparison of Characteristic Parameters

raffinate purity [%] raffinate yield [%] eluent consumption [LEluent/ gProduct] productivity [qProduct/kgSP/d]

four-column SMB

one-column SMB

batch

99.3 100 9.7

99 99 9.8

>99 >99 28.5

16.8

16.6

6.6

the system. The resulting depletion of this component in both columns is well visible.

Figure 12. Operation diagram for the simulated SMB-processes. 9177



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Figure 13. iCCC process set up of IEX and HIC.

It should be mentioned that process conditions are not yet fully optimized. Especially by adjusting volume flows, salt concentrations, and gradient conditions, the process can be designed more effectively. Also, the cut times still have optimization potential, but it must be recognized that these cannot be simply taken from the batch diagram. Instead, this is

For the batch simulation, the same parameters are assumed. The target compound is cut with a purity >99%, resulting in a yield of only 71.5%. By the column set up shown here, the yield can be increased to 97.8%, and in other simulation studies, even 97.8% can be reached. Other key parameters are enhanced accordingly. 9178



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Figure 14. Process scheme of the integration of IEX and HIC to iCCC (analogous to Figure 5).

column. The collected fractions are, therefore, reconstituted with the same amount of 1 M buffer. Adjustment of pH to smaller values can possibly delay elution, thereby avoiding additional buffer adjustment. Figure 17 shows several HIC cycles. The target component is the later eluting one; therefore, the recycle-fraction lies here between 18 and 21 min. The later cut point is chosen to ensure 100% purity. It can be seen that the product peak concentration is increased by a factor of 10 within six cycles, whereas the side component peaks only increase by a factor of 5. The overlap region contains more target compound than side component. It can be shown, that this set up is feasible at least for the HIC case. Also, a concentration increase of the target compound is reached, and the ratio target compound to side component is shifted from approximately 1/1 to 2/1, with the target compound purity reaching 100%.

a nontrivial optimization task in which, among others, the salt concentrations of the recycled media, the various loading capacities, and the isotherm parameters play a decisive role. This can most easily be seen at the concentration front of the product peak in the hydrophobic interaction chromatography. In order not to exceed the frame of this publication, the method developed for process optimization needs to be discussed in detail in a separate publication. 3.2. Experimental Feasibility Studies. Screening various proteins in the ion-exchange and the hydrophobic interaction mode show that various sets of operating conditions exist which proceed through both unit operations without changing elution sequence as depicted in Figure 16. However, just for the sake of a feasibility study, the fine tuning of the operating parameters, in particular pH values and salt concentrations, are too time consuming. With the HIC step considered the more complex one, a test system was selected for this study that allows the assessment of the HIC-loading steps. It is important in this case that the relevant side component is the stronger binding in the IEX and the earlier eluting one in the HIC step. This is achieved by the combination of IgG as target compound and chymotrypsinogen as side component. The same buffer system as described by Helling et al.68 is used (0−1 M ammonium sulfate at pH 7.0). As can be seen in Figure 16, the components elute at approximately 0.3 M salt concentration. The dead time of approximately five minutes is not taken into account for the visualization of the gradient profile. As the target compound (IgG) is eluted first, fractions between 6 and 11 min (product) and 11 to 13 min (mixture) are collected. The salt concentration is not sufficient for a direct loading of the HIC

4. EQUIPMENT IMPLEMENTATION Finally, a system design has to be developed that is GMPcompliant but also biocompatible to ensure product stability. The one-column SMB comes with an additional degree of freedom of one individual switching time per zone, as no synchronization between the zones is needed. Furthermore, unlike the conventional SMB, the different zones do not necessarily consist of the same column or vessel. On the contrary, different vessel types and dimensions are possible. Thus, a big separation optimization potential is given. The minimized equipment set up of four containers may be expanded by three simple delay coils as shown in Figure 18. This allows us to realize the mass transfer improvements characteristic for countercurrent operations by concentration of 9179



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Figure 15. Simulation studies of the IEX/HIC combination.

the recycled partial streams as in SMB processes. Thereby, the concentration profiles are kept and not destroyed as in stir vessels. Otherwise, a slight decrease in separation performance might result. Figure 19 shows a picture of the prototype built at the institute. Up to five pumps control gradients and mass flows in the range of 1−800 mL/min with a precision of ±0.1 mL/min. Between one and six columns can be operated in free configuration. Three UV detectors are positioned in the cycle stream plus two additional positions; in addition, conductivity and pH detection is available.

Table 2. Comparison of the IEX and HIC Combination in Batch and iCCC Mode purity [%] yield [%] eluent consumption [LEluent/gProduct] productivity [qProduct/kgSP/d]: IEX only: HIC only: overall:

batch

IEX/HIC combination

99.3 71.5 5.92

99.7 93.5 4.47

11.6 2.9 2.3

14.64 3.66 2.93

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Figure 16. Chromatogram IEX.

Figure 17. Multiple cycles of HIC reloading.

The extremely flexible prototype (Figure 19) is well suited for feasibility studies for SMB, MCSGP, one-column SMB, and also iCCC (integrated continuous countercurrent chromatography) on the basis of a process concept by simulation and its experimental validation. The subsequent transfer of individual elements like valves, pumps, columns, and tubing from classical batch-chromatog-

raphy units into GMP-compliant and biocompatible standard modules is an engineering task for which first solutions are available. A comprehensive package (concept) for continuous production has already been presented in earlier studies1,3,4,17 (see Figure 20), resulting in drafts for dedicated production 9181



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Figure 18. Process concept of CCC with delay coils (zone III).

Because of this, the time is now ripe to establish full continuous operation in biologics manufacturing. The paper summarizes an approach which enables to integrate a fully integrated continuous countercurrent chromatography (iCCC) process for total continuous manufacturing by operating two different separation mechanisms like, for example, IEX and HIC or NP and RP in continuous and sequential flow. Mass transfer is enhanced due to countercurrent flow operation. This should allow a reduction of the chromatography step involved or at least lesser adsorbent and buffer amounts. One of the most intense application fields for chromatographic operations is the biopharmaceutical field. In the past, up to five chromatographic operations in the downstream sequence were typical to achieve the necessary product purity for human use in spite of the costs incurred in the process. They were carried by the economy of scale, meaning the blockbusters were being brought to market until recently. More stringent patient safety requirements in combination with cost containment issues herald the end of the blockbuster age and the shift toward stratified or even personalized medicine. The result is drug variants tailored to the needs of smaller patient populations and the increase of generic drugs are causing paradigm changes, not least in the processing of such therapeutic products with much higher flexibility at smaller scale yet at acceptable costs. This paper presents a technical and economical contribution to this scenario, which is sustainable on one side and enables an integrated continuous countercurrent chromatography (iCCC) process for continuous manufacturing by operating two different separation mechanisms like (e.g. IEX and HIC or NP and RP) in continuous and sequential flow. Mass transfer is enhanced due to the countercurrent flow operation. This can allow a reduction of the number

Figure 19. Prototype of system capable of SMB, one-column-SMB, and iCCC at the institute.

systems in a container, into which the concept of iCCC as described before fits in perfectly.8−10,66,69,70

5. SUMMARY Personalized and stratified medicine approaches have changed the manufacturing scenarios recently. This caused a paradigm shift to continuous operation of small scale dedicated plants. 9182



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Figure 20. Dedicated system for continuous production.9,69

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DEDICATION Professor Massimo Morbidelli on behalf of his 60th birthday.

chromatography steps required or at least reduce adsorbent and buffer amounts used in the process. In combination with a stringent recycling strategy already published,2,3 this offers a significant contribution toward successful economic implementation of stratified medicine. The same approach can be beneficial in addressing resource and cost-effective purification strategies in natural products and adjacent fields. A challenge is the functional design of a suitably efficient process concept because of the many variables to be controlled. This is solved methodologically by process simulations with rigorous process models where the model parameters are determined experimentally in laboratory scale for the complex mixtures to be purified. An equipment prototype has been built and is in operation at the institute. For further applications, additional process optimization studies need further investigation. Configuration of GMP and biocompatible equipment pieces on a production scale are quite similar to classical batch chromatography. ICCC chromatography minimizes equipment parts, consequently, in order to open up innovative production technologies for transfer into production scale, which should generate some acceptance even with notoriously skeptical biologics operations.

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SYMBOLS

Symbols Name Dimension

c = Concentration [g/m3], [g/l] cp = Concentration pore [g/m3], [g/l] Dax = Parameter of axial dispersion [m] D = Diffusions/dispersion coefficient [cm2/s] keff = Mass transfer coefficient [cm/s] kf = Film transport coefficient [cm/s] kp = Mass transport coefficient pore diffusion [cm/s] K = Equilibrium phase constant [m3/g], [l/g] q = Load stationary phase [g/l] qmax = Maximal load [g/l] r = Radius [m], [cm] t = Time [s] uint = Interparticle velocity [m/s] X = Length in coordinate x-axis dimension [m] y = Load of solid surface [g/m3] Z = Length coordinate in z-axis diemnsion [m] Greek

ε = Porosity [-] εp = Pore porosity [-]

Indices

AUTHOR INFORMATION

Corresponding Author

*Prof. Dr.-Ing. Jochen Strube. E-mail: [email protected]. de.

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Notes

The authors declare no competing financial interest.

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i = Componente r = Radial s = Stationary phase r = Coordinate dimension r axis Z = Coordinate dimension z axis

REFERENCES

(1) Jornwitz, M. W.; Szarafinski, D.; Peuker, T. Single-Use Technology: Opportunities in Biopharmaceutical Process, in: Subramanian, G. Biopharmaceutical Production Technology, Vol. 2; Wiley-VCH: Weinheim, Germany, 2012. (2) Strube, J.; Grote, F.; Josch, J. P.; Ditz, R. Process Development and Design of Downstream Processes. Chem. Ing. Tech. 2011, 83 (7), 1044−1065. (3) Strube, J.; Grote, F.; Ditz, R. Bioprocess Design and Production Technology for the Future. In Biopharmaceutical Production Technology, Vol. 2; Subramanian, G., Ed.; Wiley-VCH: Weinheim, Germany, 2012.

ACKNOWLEDGMENTS The authors would like to acknowledge the fruitful work of Volker Recksiek for simulation studies and professional CEconform building of a prototype plant from the Institutes workshops by Frank Steinhäuser and Volker Strohmeyer with process control by Roland Mecke. JS would like to acknowledge Massimo Morbidelli for more than a decade of fruitful discussion, kind recommendations, and several successful collaborations. 9183



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