In fermentation-based processes, products are produced in diluted environments to prevent microbial growth inhibition by the products or the by-products. Consequently, downstream processing (DSP) is usually composed of several unit operations, which translate into higher costs for the overall process (e.g. for biopharmaceuticals DSP costs can represent 80 % of the total costs) and reduced overall yields. Therefore, there is a need to use process technologies that can improve process economics.
IN SITU PRODUCT REMOVAL
As bioprocessing is moving towards continuous modes of operation, in situ product removal (ISPR) is an interesting strategy to improve the DSP efficiency. ISPR enables the product separation during fermentation. However, ISPR requires unit operations that can perform primary product recovery.
PRIMARY RECOVERY STRATEGIES
Traditionally, centrifugation and membrane technologies are preferred for the primary recovery. Nevertheless, fermentation streams are sensitive to mechanical forces and complex to process smoothly using membrane operations. Consequently, with this strategy, there will be considerable amounts of product that degrades and cells that die before the broth is recirculated back into the fermenter. Therefore, a membrane filtration is not a viable option. Could chromatography be used instead then? The answer is yes.
Fermentation streams cannot be processed using packed bed chromatography, but they can be effectively processed using expanded bed adsorption (EBA) chromatography!
EXPANDED BED ADSORPTION (EBA)
EBA is a chromatography technology that combines cell removal, product capture and initial purification step in a single-unit operation. As a typical adsorption process, the adsorbent is kept inside the column where the product stream flows through. In EBA, the liquid stream flows upwards, enabling the adsorbent resin beads in the column to become fluidized (expanded). Thus, the particulate biomass can flow through the bed void .
IN SITU PRODUCT REMOVAL WITH EBA
The fermentation broth is directly fed into the EBA column, and, due to the selective interaction between the target compounds in the broth and the resin beads, a selective separation is achieved by capturing product and allowing the cells to flow through. The adsorbed product is collected later on, in an elution step.
This process does not result in any cell disruption. New media can be added to the flow through stream and recirculated back into the fermenter to improve the productivity using cell recycling.
Could this strategy be applied in your process? Then take a look at how XPure-E system could simplify the performance of EBA and contact us for more information. Let us discuss feasibility for your product.
 Pathapati et al. 2018. Expanded Bed Adsorption of γ-aminobutyric acid from E.coli broth by CS16GC and IRC747 resins. Chemical Engineering Technology. DOI: 10.1002/ceat.201800295
ECCE12/ECAB5 this year has been successfully held in Florence, the city of renaissance. The event was attended by more than 1,000 delegates (Professors, Scholars, Scientists and Graduate students) from across the globe. The event strongly resonated the fundamental objective to share state of the art developments in the field of chemical engineering and applied biotechnology. The keynote was vocal that it is the time for:
”Renaissance in Chemical Engineering”
Plenary Lectures: Some of the Pioneers in Chemical Engineering shared insights about some important elements that can help in achieving a Sustainable Chemical Industry. The lectures included some fundamental aspects like the need for a change in Chemical Engineering education and the use of Chemical Markup Language in writing, which can enable efficient use of reliable literature. Some other major topics included Process Intensification, Process Safety and Waste Recycling.
Process intensification has been addressed from both feedstock to product conversion and product purification aspects. Product conversion highlights mainly involved CO2 utilization and several chemical, catalytic and bioprocess alternatives were presented to convert CO2 to building blocks including industrial realization of Syngas Fermentation. An interesting approach was also presented as 5G biorefinery to use electricity and CO2 to produce building blocks. Purification highlights included recent developments in extraction, membrane separation and adsorption/chromatography technologies.
As XPure Team we are glad to have been part of ECCE12/ECAB5 as bronze sponsor. About 100 delegates participated in our talk on EBA-SMB Technology with very constructive questions after the talk. We were also able to exchange some interesting ideas to collaborate and become part of building Sustainable Chemical Industry.
Several organic and amino acids have found applications as specialty molecules in food, consumer goods and pharmaceutical industries. Itaconic Acid (IA) is a 5 carbon dicarboxylic acid produced through bacterial fermentation and used as an important building block for biopolymers. In case of IA production, downstream processing can account to 30-50 % of the overall cost of goods (COGs). Therefore, it requires effective and efficient operations to improve sustainability and cost competitiveness. In the current article, we will briefly discuss the role of expanded bed adsorption (EBA) and simulated moving bed (SMB) technologies in production of IA.
Figure 1: Purification of IA requires a downstream process to separate IA from suspended and dissolved impurities
The basis for selecting a specific unit-operation is defined by desired separation. Typical fermentation output stream contains 5-10 % IA, 5-10 % impurities and 80-90 % water. Impurities include both suspended solids like biomass and dissolved compounds that are produced as co-metabolites. Though precipitation is an easy method for IA purification, it results in formation of by-products and limits product purity. Therefore, instead of precipitation, adsorption using a chromatography resin is a preferred alternative. However, feed for packed bed chromatography columns need to be free of suspended solids and this require additional unit operations for clarification, resulting in yield losses and higher costs. To overcome these limitations, Expanded Bed Adsorption (EBA) can be applied. EBA is an integrated adsorption process, where the product is selectively captured by fluidized adsorbent beads from unclarified fermentation broth. Thereby, enabling removal of both suspended and soluble impurities in 1 step.
Successful EBA process can achieve desired product purity with high recovery yields using 30-60 % less number of unit-operations. In addition, it is easier to scale EBA for large-scale production processes with several kilotons annual capacity, due to no pressure drop limitations. However, it is important to note that application of EBA on large scale can lead to large side streams, which require processing before recycling or release into the environment. Additionally, fluid distribution and hydrodynamic performance of EBA may result in reduced dynamic binding capacity compared to packed bed. A prudent approach to overcome these limitations is by operating EBA in SMB mode.
This blog focusses on useful and valuable proteins in both milk and whey that can be recovered with chromatographic separation.
Milk and its derivatives contain various proteins that are useful as human nutrition. Milk proteins are especially rich in amino acids that stimulate muscle synthesis. In addition, some proteins and peptides in milk have positive health effect e.g. on blood pressure, inflammation, oxidation and tissue development . Some people suffer from deficiency of specific proteins, In order to to find a solution for this, manufacturers produce balanced, composed (semi-)synthetic milk products, e.g. baby food, by adding those proteins in the manufacturing process.
This blog describes a few examples where adsorption chromatography can serve as a separation technology to isolate useful milk and whey proteins.
Figure 1. Human versus bovine minor milk protein composition , , .
Above Figure 1 represents the average milk protein composition in both human and bovine milk (note that only the “high-value” minor proteins are represented in this figure, because the majority (i.e. > 80%) of cow milk proteins consists of different types of casein. For human milk approximately 40% of total protein consists of casein).
Total bovine milk proteins content amounts to 30-34 g/l.
Protein adsorption has been applied on batch scale utilising dedicated functionalised resins, e.g. agarose-based cation exchange resin .
In expanded bed mode, suited to direct the fermentation broth to the adsorption process, without having to clarify the crude broth first, it has been found that the feed flow rate can be substantially higher (~10 m/h or even higher) without significant backpressure .
Before loading milk on a chromatography column the fat (components) need to be skimmed first in order to prevent blockage of the adsorption phase, and reduce viscosity to attain higher bed flow rates.
Milk is a complex medium; in addition the composition of milk is subject to seasonal influences. and further pooling prior to processing is quite common in dairy industry. Volumes to be processed lie in the range up to 100’s of m3’s per day.
Since the proteins are utilised in food supplements, regulatory and hygiene rules apply.
Pasteurisation is a common step in milk processing, temperature largely affects the adsorption characteristics and may also pose additional demands on the adsorption materials in terms of thermosensitivity.
Figure 2. Part of flow scheme to recover milk proteins, including chromatographic adsorption
Figure 2 conveys part of a typical milk process where fat and protein are separated.
The Adsorption step comprises the capture of specific proteins to a specific, selective resin, and the elution with a proper salt buffer.
Depending on the magnitude of the process, as well as process control requirements, the adsorption step may be operated in a continuous mode. A well established continuous chromatography mode is based on simulated moving bed technology (SMB). This technology features a multiple (typically 4-16) column process that smartly accommodates all distinct process steps –running simultaneously- that are part of the bind and elute process, including wash/rinse/equilibration and regeneration.
XPure has developed a SMB system that can be operated both in fixed and expanded bed mode, so called Expanded Bed Adsorption (EBA). This EBA mode features all individual steps of the SMB process cycle, operated in upflow / expanded bed mode, refer to Figure 3.
Figure 3. Typical SMB flow scheme for protein isolation/purification from dairy
Note: Regeneration has not been included, every column can be read as a zone consisting multiple columns,, either in series or parallel
Process cycle time and specific residence time in certain zones may be adapted to changing input parameters, e.g. protein content and composition.
Case studies have been done both for lacto-peroxidase and lactoferrin. Based on packed bed mode for a daily milk production of 70 m3 a continuos process for lactoferrin would result in significant cost reduction in cost of goods, refer to Figure 4. A significant part of the cost reduction in SMB-mode in relation to batch-mode chromatography, can be related t0 consumables. It has further been found that skimming is necessary to reduce the residual fat content below 0.1 w-%, prior to applicable chromatography processes.
Figure 4: Cost of Goods for bovine-Lactoferrin from milk, batch versus SMB operation
XPure EBA-SMB has been successfully run on amino-acid containing fermentation broth without prior clarification or filtration. This process design can directly be translated to the milk protein application and could flexibly be fit in any process step that the skimmed milk goes through.
For large-scale dairy processes cost reduction can be attained by a continuous operating mode. For the adsorption process, simulated moving bed chromatography (SMB) is a state-of-the-art technology that contributes to a high level of process automation, product quality and yield.
 A. Haug, “Bovine milk in human nutrition – a review,” Lipids in Health and Disease, vol. 6, no. 25, 2007.
 M. Malacarne, F. Martuzzi, A. Summer and P. Mariani, “Protein and fat composition of mare's milk,” International Dairy Journal, vol. 12, no. 11, pp. 869-877, 2002.
 J. Billakanti, “Extraction of high-value minor proteins from milk,” University of Canterbury, Christchurch, 2016.
 S. Séverin and W. Xia, “Milk Biologically Active Components,” Critical Reviews in Food Science and Nutrition, vol. 45, pp. 645-656, 2007.
 General Electric Health Care - Pharmaceuticals, “GE Health Care - Pharmaceuticals,” 2019. [Online]. Available: www.ge-healthcare.com.
 R. Noël, “Industrial processing and biotherapeutics,” Filtration+Separation, vol. 44, no. 3, pp. 26-28, 2007.
Ascorbic acid was discovered in 1928 by Szent-Györgi. Commercially, ascorbic acid is mainly produced by a combination of synthetic organic steps and biotransformation. A well-known intermediate product is 2-keto-L-gulonic acid. This is produced from the sodium salt of the L-gulonic acid and has been acidified according to exact the same Ion Exchange route, as presented below for vitamin C.
In order to obtain the pure product, ion exchange is an attractive method for removing the salts.
The objective of this application note is to demonstrate the feasibility of the continuous ion exchange for removing Na+ from the sodium ascorbate. The feed flow consists of an aqueous solution containing Na-ascorbate. The sodium is exchanged by H+ on a strong cation resin in the H+ form. The resin is regenerated with HCl.
2-keto-L-gulonic acid (precursor to vit. C or ascorbic acid)
Ascorbic acid (vitamin C)
The sodium form associated with the Asc (ascorbic acid) would be exchanged for a H+ thus forming ascorbic acid on a strong acid cation resin with 1,8 eq/l (expressed in terms of equivalents per unit volume of packed bed) maximum capacity.
The entire operation is executed in a XPure carousel system with 20 to 30 columns.
The overall manufacturing process is schematically summarised in the next diagram Figure 1:
Figure 1. Flow diagram for vitamin C manufacturing
For the Ion Exchange step we focus on the acidification step to vitamin C. This is in fact a purification step, see below Figure 2.
Figure 2. Conceptual design of continuous IX purification for (Product) vitamin C. Each block may contain multiple columns. Total number of columns typically 20-30.
Note: yellow lines indicate water flows within the system
The regenerant involves hydrochloric acid implying that NaCl is the target compound to remove in the subsequent wash steps. Caustic is applied for intermittent regeneration and protein removal.
In an experimental study it has been found that residual Na-ascorbate is more difficult to remove (ads wash) than NaCl (regen rinse), the ascorbate obviously shows affinity to the resin surface.
Water consumption is an important process parameter. In order to save on water, a counter current contact concept has been applied. This can be accomplished by assigning multiple columns to the wash&rinse zones. The next Figure 3 shows the effect on water consumption –expressed in BedVolumes- for 4 different configurations. Please note ideally 0.3-0.5 BV (“interstitial volume” or void fraction) would be sufficient for 1 column displacement.
Figure 3. Wash efficiency with growing number of counter-current connected columns
A multi-column carousel system can easily, and relatively cost-effectively accommodate a counter-current contact zone.
The dilution in the vitamin C process typically is 20%-35%. This is significantly lower than fixed bed processes in which 2-fold dilutions are not exceptional.
The conversion of Na-ascorbate is at least 99.5%. . The water consumption, without using the possibilities to re-use water in the process is about 15 liter per kg Vitamin C. The hydrochloric acid (7 wt%) consumption is 5.4 liter per kg vitamin C, taking into account an excessive use of 20%. The caustic consumption should be fine-tuned to the protein content of the feed solution and can be minimised by optimising the intermittent caustic wash. Losses of product in the caustic wash and contamination of the product stream with chloride are minimised by carefully designing the adsorption wash and elution wash sections. Product dilution is minimised by applying an entrainment rejection zone.
One of the dominant factors in the operating expenses for ascorbate (and Na-2-KLG) acidification is related to the costs of the ion exchange resin. This can be related to the specific productivity of the system, which expresses the annual amount of product that can be purified per unit volume of resin. The productivity of a carousel system for the purification of KGA and vitamin C typically is higher than 800 tpa/m3. Productivities in fixed bed processes may be a factor 5-10 lower.
Platform molecules form the basis for several formulations in chemical, food and pharmaceutical industries. Lactic acid (LA), a 3 carbon carboxylic acid is one such platform molecule. LA has a broad application field ranging from
Figure 1: Fundamental aspects involved in industrial production of products (Orange: Involves XPure Technologies)
LA is produced by both chemical synthesis and fermentation, where fermentation has been the preferred choice due to enantio-selectivity and environmental sustainability factor. However,
Figure 2: Major sub-processes integrated to produce lactic acid
LA production process involves both cation and anion exchangers to remove positively-charged cationic impurities and to capture LA, respectively. These two steps use industrial resins, which are known for their robustness, high exchange capacity
Figure 3: Different separation principles applied in
This scenario leads to low productivity, less sustainable process and
In the traditional scenario of switching from batch to SMB, the IEX costs can be reduced by 30-50 %. For example, in a lactic acid purification process, where IEX step can contribute to about 20 % of overall DSP COGs, the SMB technology can reduce the DSP COGs by 5-10 %. This improvement in process economics can be critical for
Figure 4: Cash flow analysis with batch and SMB IEX steps for LA purification
Sugar is produced in many countries all over the world. The term “sugar production” relates to various products, primarily mono- and disaccharides.
There are two widely applied large-scale chromatographic carbohydrate (sugar) separations: the isolation of the disaccharide sucrose from molasses and the separation of the two monosaccharides fructose and glucose (figure 1).
Figure 1: The structure of fructose (left) and of glucose (right)
Monosaccharides like fructose and glucose are produced mainly from beet, cane or corn. After several refinery steps including milling, filtration
This mixture is separated by chromatographic separation into a fructose-rich and a glucose-rich component. The dry matter content of the feed flow varies up to 60%w/w.
The resin material used in fructose/glucose fractionation is a gel-type sulfonated polystyrene-DVB strong acid exchange resin in the calcium form. The separation is based on the preferential adsorption of fructose, which forms a complex with the calcium ions. This chromatographic method is called Ligand Exchange Chromatography (LEC). There are several commercially available resins for this application.
A secondary phenomenon is that larger molecules, i.e. higher oligosaccharides are not able to physically fit into the resin pores. The mechanism of separating large molecules from small molecules by preventing some of the large molecules from getting inside the stationary packing is called size exclusion chromatography. Size exclusion chromatography takes place simultaneously with ligand exchange chromatography in the purification of fructose. This results
Both components are recovered for over 90% and the purity of the product flows is above 90% as well. Glucose is recycled and re-isomerized; fructose is sold as a pure product or mixed with the equilibrium mixture mentioned above to yield
The chromatographic separation of fructose and glucose can be efficiently done in a simulated moving bed (SMB). Figure 2 shows experimental data from a glucose-fructose fractionation in
Figure 2.Comparison of production data of fructose/glucose fractionation on a 12-column system with calculations according to our process design model
The same fractionation mechanism applies for lots of other applications,
Figure 3. Representation of a 4-zone SMB system to separate Fructose (strongest chelating) in Extract Phase (yellow exit) from Glucose (weakest chelating) in Raffinate Phase (blue exit). Green input flow represents the feed mixture. The white influent represents the
Figure 3 conveys a generic projection of a 4 zone / 8 column fractionating SMB system., it is
The fractionating principle evidently can be applied in a large field of applications.
In conclusion, Fractionating Chromatography is a strong separation tool that perfectly suits continuous SMB operation.
This blog will present a new development regarding the application of Expanded Bed Adsorption (EBA) technology for small molecules.
Biotechnological fermentation processes are nowadays common in the industry to produce a broad range of biological molecules. Obviously, the bio-based industries need efficient,
The topic for this blog is the purification of small molecules like amino acids from unclarified bacterial broth using a combination of different technologies. To
Resin selection is one of the critical aspects of developing an EBA process. Relevant resin hydrodynamic properties include particle diameter and density. Typically, these beads are agarose-based and have a heavy core. Only a few specific EBA resin types are available, which are also quite expensive. These aspects are major hurdles for the application of the EBA technology in manufacturing of bulk chemicals where low production costs are key.
The current study focusses on the purification of ɣ-
Fig. 1 Text see
First, the GABA purification process using the gel-type strong acid cation exchange (SAC) resin was defined for a “single EBA column process”. The following process steps were identified:
Obviously, critical process requirement for EBA processing (e.g. maintaining the target bed expansion) should be fulfilled. Important parameters incl. buffer concentrations, target buffer volumes
For the integration of EBA and SMB process, a state-of-the-art 8-column lab scale system has been designed and built (see Figure 2).
Within the software, recipes can be generated that address
As mentioned, a critical aspect is to ensure optimal bed expansion. For this purpose, ultrasound sensors were installed at each column outlet that measured the expanded bed level. By this measurement, the software controlled the pump flow rate to maintain the desired bed expansion for the individual columns. The pH profile of the product stream exiting the different SMB columns passing through the elution zone was assessed to represent consistent product quality at a cyclic steady state of operation. The quality was defined by removal of biomass and other soluble impurities (Ref. 3).
Fig. 2. Text see
The promise of reduction of unit operations, increased product yield and productivity and reduced buffer consumption using EBA technology for complex feed streams
Ref. 1. Ping Li, Pedro Ferreira Gomes, José M. Loureiro
Ref. 2. Trinath Pathapati, Dennis N. Rutze, Piet den Boer, Pieter de Wit
Ref. 3. Trinath Pathapati, Dennis N. Rutze, Pieter de Wit, Piet den Boer
The use of Expanded Bed Adsorption (EBA) for the purification of biological molecules from unclarified harvest is well described for many applications in literature. The advantages are obvious; including shorter process time, higher yield, low buffer consumption, and no additional clarification step.
The next step to further increase the productivity within e.g. bio-based industries is
The main challenge is to prevent
A number of strategies can be applied to keep the level of the expanded resin bed constant during processing of unclarified broth.
The third option requires
Using well-characterized feed streams, it is worthwhile to anticipate
(a) Make an assessment of the number of settled resin bed volumes (BV) needed for the execution of the EBA process. This can be done based on a regular packed bed chromatography process. It must be recognized that the displacement of
(b) Establish the flow/expansion
(c) Establish the
Crucial is to know how the expanded bed responds to the change to a feed stream with a higher viscosity (for instance unclarified broth). Loss of resin from the column top may occur if the flow rates remain too high. This is particularly relevant in case the EBA process runs in SMB mode. At a worst case
Below, an example is given how the expanded bed level can be kept in a narrow range during the transition from an aqueous solution to a high viscosity solution just by changing flow rates in a smart manner.
Polyvinyl alcohol (PVA) is used as a model compound to mimic a high viscosity feed stream (i.e. “unclarified broth”).
First, the flow rates have been determined to obtain the target expansion of 1.85-fold for water and the viscous PVA solution (i.e. 610 and 344
Fig.1 (see text last page)
This is prevented by initially doing nothing i.e. just keep the flow rate unchanged upon the switch to PVA. As shown in Figure 2 there is a substantial delay of the bed expansion to respond to the PVA solution. By keeping the flow rate unchanged for 0.5 BV the collapse of the expanded resin bed has been prevented. Interestingly,
Fig.2 (see text last page)
A development strategy for processing unclarified harvest in EBA-SMB mode has been presented. By taking the delay of the expanded bed to respond to changes in viscosity into account, simple programming of flow rates and volumes for the individual process steps prevent significant changes in resin bed expansion. As a result, the resin bed expansion remains within a 90% – 110% range of the target bed expansion and enables the successful processing of complicated feed streams in EBA-SMB mode.
Impact of a 1-step decrease of flow rate on the resin bed expansion in case the flow rate is changed directly upon switching to the PVA solution (“unclarified broth”).
Impact of a 2-step decrease of flow rate on the resin bed expansion in case the flow rate is changed after 0.5 BV after switching to the PVA solution (“unclarified broth”).
XPure in conversation with Victor Koppejan, winner, ESBES award 2018, thoughts on EBA Technology
At XPure, we believe that the application of efficient downstream technologies like EBA leads to sustainable and profitable process industries. Here, we would like to share the thoughts of a young scientist from TU Delft, about his work on EBA Technology and award-winning talk at ESBES 2018.
How do you feel about winning ESBES (European Society of Biochemical Engineering Sciences) award for 2018
I’m really happy as a Ph.D. candidate in Bioprocess Engineering at Delft University of Technology, that I was able to participate and selected as one of the winners. At the previous ESBES in Dublin two years ago I saw two of my colleagues present during the award ceremony. Since then I’ve been looking forward to a change to participate and be able to share my story with a bigger audience.
What do you think made you stand out among the high-quality research presentations from different streams of biochemical engineering?
In the group of Dr. Marcel Ottens, I strongly feel that our approach in which we applied traditional chemical engineering techniques together with mathematical modelling to a high potential technology like expanded bed adsorption (EBA) helped me to stand out.
How do you see the future of EBA?
Here I must say that I’m really excited about the approach to EBA Xendo has within the European PRODIAS (PROcessing Diluted Aqueous Systems) project. So far the application of EBA in biopharma has been challenging, as investments on technologies which can have a small level of discrepancy can lead big risk factor. Also, cleanability (and with it the validation and compliance challenges) is a very critical aspect for pharma and one of the fundamental challenges. The fields of non-pharma proteins and small molecules have different drivers for process development and CAPEX/OPEX optimizations are more dominant. I think here EBA can be a competing separation technology. In turn, successful business cases based on EBA will help convince industries that might have become reluctant to implement it.
What do you see as challenges to be addressed that can prepare the technology for future?
In our research (which is part multi-disciplinary consortium) we focus on the fundamentals of fluidization in EBA columns. We try to approach it as a liquid-solid fluidized bed rather than a packed bed column “in disguise”. Since experimental research on hydrodynamics for such a system is highly challenging, we use advanced computer simulations. These allow us to investigate local resin particle environment as well as extract statistics for the overall expanded resin bed. Developing these models has proven to be a tough challenge but are now at a point where we can generate reliable data. Our aim is to use insights we get from the simulation data for the better design of column hardware and to provide a benchmark for ideal fluidization behavior. These benchmarks can then be used to detect early deviations in the process. We hope that this increases the rationale in process control, leading to a more robust equipment operation.
Development of EBA systems is a field dealing with multi-phase, multi-scale and multi-physics challenges. Typically, processes at various length and time scales will interact with each other in ways that may not seem obvious at first. These are challenges which, on a more abstract level, are not unique to EBA and I think lessons learned here can be applicable to a broader range of technologies. Continuing that line of thought I’d like to end with a quote from Sir Stanley Eddington I recently came across.
“we often think that we have completed our study of one we know all about two, because ‘two’ is ‘one and one’. We forget that we still have to make a study of ‘and’ ”
Innovation through hybridization/integration of technologies is a well-known approach. This approach has been effectively applied to achieve specific process objectives in chemical, biochemical, food and pharmaceutical industries. The approach can include either two separate units operating in tandem or as a single integrated unit. Membrane operations are usually found in tandem with other unit-operations for recycling cells during fermentation or buffers in case of downstream processing. In integrated operations, the overall efficiency is achieved by using two or more operating principles in parallel. Reactive distillation, pervaporation, fermentation with in-situ product removal through stripping etc. are some examples of integrated unit-operations. The current article briefly discusses one such integrated unit-operation called Simulated Moving Bed Reactors (SMBR).
SMB technology has proven to enhance the efficiency of chromatography/adsorption separation through continuous counter-current operation. This principle can have a similar impact on heterogeneous catalytic reactors with catalysts immobilized on support beads or adsorbent and packed as a fixed bed or fluidized bed. The countercurrent effect of SMB is known to enhance the performance of equilibrium driven reactions and mass transfer kinetics, whereas the continuous mode of operation helps in achieving improved productivity. Therefore, SMBR can find its application as a techno-economically viable process solution.
Fundamental phenomena during heterogeneous catalysis are mass transfer (MT), reaction and inhibition kinetics. MT is more critical in case of a catalyst on solid support and substrate dissolved in a liquid system. However, the difference in MT kinetics of a substrate and inhibitor can also be applied as a separation principle to maintain or improve catalytic efficiency. For example, consider a catalyst exhibits product inhibition at a specific product concentration and it is identified that the substrate and product have different characteristic times at which they flow through the column. If the characteristic time difference in a batch reactor is not sufficient to minimize product inhibition, an SMBR can help in increasing the difference through the countercurrent effect. Thereby enhancing step efficiency and process economics.
Schematic representation of modular fluid distribution system involving valves that enable SMB operation of catalysis columns/reactors
Blog by: Trinath Pathapati
Without experimental data to back up the resin costs, it's quite difficult to write a robust business case for chromatography; the XPure-R is our answer to this challenge. The system enables you to age up to 10ml resin under your process conditions in a fully automated and time-efficient manner and can be tested in both fixed and expanded bed (EBA) mode.
XPure-R allows you to focus on your other research and will age your resin because it was designed to operate without attendance. All you need to do is prepare the XPure-R by supplying all the required process liquids, programming the recipe, and press run.
It is also possible to outsource your resin aging studies to XPure, saving you even more time.
The cycle time for an investigated process was reduced from 1 hour and 33 minutes to 7,5 minutes; a 92% reduction.
For a resin aging experiment of 1000 cycles, the XPure-R reduced the experimental time from 64 days to only slightly over 5 days.
A shift from batch production to continuous production requires a different approach in terms of process control. Especially when the continuous chromatography is, in essence, a rapid sequence of “batch” operations with specific conditions for each process step. Combine this with
Simulated Moving Bed (SMB) Chromatography is a continuous form of chromatography that seeks to enhance the separation efficiency of chromatography while reducing operating expenses such as resin inventory and buffer consumption. There are 2 different types of SMB system available; the carrousel SMB and a valve matrix-based or static SMB. This blog is mostly aimed at valve matrix-based SMBs, but parts of this blog can also be applicable to carrousel SMBs.
Before running an SMB you have to decide how you want to control it: synchronously or asynchronously.
As the name implies, in a synchronous process all columns move through the process at the same pace. A carrousel SMB is a great example of
An asynchronous process gives a lot more opportunity to control the process. It is possible to break down the process into a series of positions, where each position has a detailed set of instructions and conditions. A column can only progress through the process as soon as it has completed these set conditions. This approach places more control and responsibility in the hands of the process engineer and the control strategy.
As with any continuous process, it is important that the process is controlled in such a way that the desired steady state is obtained and maintained. At steady state, the continuous process is performing at the intended efficiency and any deviation from this will impact overall process efficiency and economics. The process control and strategy should, therefore, be able to handle deviations that could disturb the cyclic steady state of an SMB.
For an SMB process, the progression of columns through the various positions within the process is conducive to the steady state of the process. The condition for a column to progress can be based on time but also on different parameters such as pH or conductivity. An example of such a step is an elution step. In the elution zone, it can be economically attractive to stop collecting liquid to the elution stream when the product concentration in the eluent becomes too low and would cause dilution of the product stream. When letting an inline parameter control the progression of columns, time becomes a secondary progression condition and thus variation in switch times could occur. The control strategy should be able to ensure steady state when this occurs.
Control strategy becomes even more important when SMB technology is hybridized with other technologies such as Expanded Bed Adsorption (EBA). As both technologies in a hybridization have their own specific set of requirements, the control strategy will need to be able to handle both technologies. To expand slightly on the EBA technology, this is a chromatography technique where the liquid flows in an upward fashion. This allows particulate matters (e.g. cells, cell debris) to flow through the bed without clogging. As the adsorbent is pushed up by the liquid flow, care needs to be taken that the bed level of the resin is monitored or controlled and that the adsorbent does not wash out of the column. An example of such a system can be seen in this article.
Figure A: the 5 levels of control within an asynchronous switching,
Figure B: example chromatographic process describing the various steps potentially
In processes where a certain set of conditions within a single column are needed to ensure the desired process efficiency, it is important that the control system can react to potentially detrimental effects down on a single column level.
Control of conditions within the column use sensors directly connected to a column to control a parameter within the column. To illustrate this, in an EBA setup a bed level sensor can be used to ensure that the resin bed expands appropriately but does not expand too much and overflow out of the column. The sensor would control the bed level by controlling the involved pump(s)
It is possible for an SMB to have multiple columns located in a zone. The column configuration can be both parallel and/or serial. In parallel the liquid stream is divided over the columns in parallel, effectively dividing the flow rate and increasing contact time. When the columns are in a serial configuration, the liquid is fed from the outlet of one column to the next.
When a zone has been configured with a parallel or serial configuration, the control strategy should ensure that the columns in the zone are only receiving fluid when the zone is populated with the required amount of columns. Therefore, it could be possible that two columns are “waiting” for a third in a zone requiring three columns. “Waiting” in this context would be that there is no liquid flow through the columns until the run condition of three columns is fulfilled.
Within a zone containing multiple columns, it should also be possible for a sensor to be able to influence the process. An example of this could be a zone with three columns linked in series. When product dilution in the product stream is undesirable, you would want to stop elution collection once a certain product concentration is reached. This means that a sensor connected to column three controls the outlet valve of column three, effectively controlling on a zone level
Little control is required in zones where time is the main controlling parameter. As discussed previously, it is possible for a zone to be controlled by different parameters such as time, pH, conductivity, bed expansion and UV absorption. In zones where other variables are the controlling parameter, time becomes a variable that could potentially affect system steady state if inappropriately controlled.
As the cycle time for the columns needs to be equal, the variation in position timing needs to be compensated. To compensate in a fixed bed SMB, a column can be parked in a waiting position. This means that the valves to the column are closed and that there is no liquid flow through the column. In an EBA-SMB, there is the added complication of having the expanded bed which potentially cannot recover if it collapses after, for example, the feed zone. In this case, the compensation positions are used to keep the bed expanded and are a prelude to the next zone.
Process optimization for both SMB and EBA-SMB systems is done in such a way to minimize the duration of any waiting position. The aim of these waiting positions is purely to ensure a cyclic steady state
The SMB as a singular entity also requires control. For a carousel SMB, this control is simple as only pump speeds and switch times need to be controlled.
As discussed above, a valve-based SMB allows for many more degrees of control enabling potentially higher levels of efficiency but requiring more rigorous process design. Depending on the requirements of control at column, intra-zone and inter-zone conditions, the SMB control can be simple time-based switching or integration of several levels of control
As the SMB is dependent on upstream and downstream unit operations to receive and discharge liquids, it is important that it is clear how the SMB should react to changes in the process. When upstream unit operations produce faster, the SMB may need to speed up its production speeds. The same holds true that it may be necessary to slow down the SMB production speed if downstream unit operations are disrupted. However, the range of flexibility to speed-up and slow-down the SMB process is defined by the permissible deviations
A comprehensive and holistic approach should be taken when designing an SMB control strategy. Single column experiments can yield significant information on how to control on a single column level and provide enough input for an initial SMB design. For further steps however SMB experiments would be required, to allow for fine tuning on higher levels of control.
Scenarios for inter-unit operation control for an SMB need to be determined from a good process understanding and then be tested to confirm appropriate SMB performance.
Blog by: Dennis Rütze
There are quite a few excellent textbooks that cover the design of separation processes, this also applies to adsorption chromatography processes, the subject of this blog. The majority of these books, however, deal with the design process from a rather academic approach. Most of the time, too little attention is paid to the specific product requirements and market or client specifications to be complied with.
Moreover, most often the design information is not readily available.
The reality is a bit more complicated and the majority of information required for designing an SMB operation has to be derived from column tests, estimated or correlated.
Any design procedure is an iterative process, it starts with some essential considerations
Next items to thoroughly think about are feed properties, process capacity, and product requirements.
Iterating to an optimal process design, it is important to look at:
For 2 and 3 most of the time good and reliable information can be obtained by column tests. Often an industrial scale batch chromatography process already is in place which data could be of tremendous value when it is considered to change to a continuous process.
A conceptual SMB configuration contains a number of zones. A zone is defined as a part of the overall process flow diagram in which the flows through all applicable columns are equal.
The conceptual configuration displays these zones in terms of a block diagram with the interconnections between the zones.
The figure below shows an elementary conceptual design for the most simple bind & elute chromatography system. It contains 4 zones:
In the conceptual design phase, the following zone may be considered
An SMB configuration displays how the sequence of process steps is included in the SMB process cycle. The concept doesn’t specify the number of columns (per zone) yet. This is one of the essential parameters that needs to be iterated in the detailed design process further on.
This iterative process concludes with a detailed design featuring:
To conclude the figure below shows a typical process flow diagram for a bind and elute IX-chromatography system. It features a diluted regeneration zone as well as additional upwash to remove any particulates that may be trapped in the feed adsorption zone.
Blog by: Pieter de Wit
PURIFICATION OF PROTEINS USING
(E.g. proteins from plant extract)
Expanded bed adsorption systems (EBA):
EBA chromatography effectively addresses the critical challenges of packed bed chromatography. It reduces e.g. clarification operations prior to the columns resulting in lower costs and improved yield. Minimal back pressure enables to achieve
Intensification of Process Development using a Design Based Turnkey Solution: a case from SMB Chromatography.
Rapid technology development and an increasing number of technologies entering the market are setting the bar high for their successful application. This is no different for separation technologies. How does a design based turnkey solution help in successful application of novel technologies? A case presented, based on SMB (simulated moving bed) technologies and intensification of SMB process development.
SMB is an industrially proven technology, which can improve the efficiency of adsorption/ chromatography based processes by enabling continuous processing, better resin utilization, reduced buffer requirement, improved yield, purity and productivity with a compact footprint of equipment. However, these benefits do not proportionally indicate the extent to which SMB technologies are currently applied or even being investigated at a process development stage. Major factors leading to this situation include limited availability of:
These limitations not only inhibit successful application of SMBs but also reduce the efficiency during process development stage.
It is clear from the problem background that it is essential to provide a turnkey solution that enables investigation of SMB technology under diverse process development scenarios overcoming the current limitations.
Stages of technology investigation and optimization during process development
Technology investigation and optimization as shown above consists of four major stages. Therefore, proposed turnkey solution needs to be flexible to aid at any of these stages.
The solution contains 4 parts, which form a package that can aid in SMB technology testing and implementation during process development:
All the four stages discussed, when integrated, can provide a turnkey DSP solution for SMB process development as it will allow you to design, validate and scale-up SMB systems for diverse process scenarios. Thereby enabling successful investigation and application of SMB technologies. This design-based approach, when implemented for other process technologies, can enhance the overall efficiency of process development in terms of time, process detailing and performance, without requiring major additional investments.
Blog by Trinath Pathapati
This blog will present a new development regarding Expanded Bed Adsorption (EBA) technology. A turnkey solution is presented that makes feasibility studies using EBA easy.
Biotechnological fermentation processes are widely used in industry to produce an abundant range of biological molecules (small molecules as well as large molecules, from amino acids to complicated monoclonal antibodies) which often need to be purified in order to meet high-quality standards. Examples of these fermentation feed streams include mammalian cell cultures, yeast
In the diagram, the initial phase of the purification of a biological molecule is depicted. Several technologies (or combination of technologies) are commonly used to remove particulate material and isolate the molecule from the biological fermentation broth. For clarification the following technologies are widely used:
In general, a packed bed chromatography step is applied after clarification of the broth. Different chromatography resins can be used (including cation exchange resins and affinity-type resins) for a typical bind (adsorption) and elute process.
This purification process can be significantly improved using the Expanded Bed Adsorption (EBA) technology as can be observed on the right-hand side of the diagram. Whereas traditional column chromatography uses a packed resin bed, EBA uses an expanded bed. Particles such as whole cells or cell debris, which would quickly clog a packed bed column, easily pass through the expanded bed. Therefore, EBA columns can be used directly on crude harvests or slurries of broken cells, thereby bypassing initial clarification steps such as centrifugation and filtration.
Xendo developed the
The XPure-E system has been designed and built to perform EBA chromatography operation in a smooth and controlled fashion.
The most important feature of the
In the figure below is illustrated how the expansion of the resin bed (orange line) is monitored when the flow rate is stepwise increased from approximately 500 up to 850
As mentioned the
Active control of the expanded level during the process can be established in 2 ways:
In addition, it is also possible to operate the system without active level control. This feature can be used in case all feed streams are well defined and result in a predictable resin bed expansion at target flow rate.
The software platform allows the user to perform experiments in an easy and automated manner.
It allows the user to define the process recipe including:
The software calculates the expected process time per step and for the whole process. The intuitive graphic user interface gives the analyst an insight into the current state of the EBA process through various overviews. The data generated can easily be processed in spreadsheets such as Excell.
Performing EBA feasibility studies with the
If you’d like to investigate what EBA could mean for your processes don’t hesitate to contact us.
Blog by: Piet den Boer
A comprehensive outlook on Industrial Biotechnology and its importance in
Fourth Industrial Revolution (Expoquimia 2017, Barcelona)
The XPure systems team was recently present at Expoquimia 2017, in the beautiful city of Barcelona. We presented our new product XPure-E, which has been specifically designed for expanded bed adsorption (EBA) as part of the EU subsidized horizon 2020 project PRODIAS. This exhibition was organized in collaboration with WCCE (World
The PRODIAS horizon 2020 EU subsidized project is developing technologies targeted at the above. Xendo is taking part in this project which is aimed at reducing the processing costs of compounds from renewable sources in diluted aqueous systems. PRODIAS targets on a holistic approach to
In view of the talks by professionals from various fields of Industrial Biotech and diverse organizational roles, it is concluded that the strong demand to balance sustainable products and processes with desired profitability makes it essential to simultaneously develop efficient upstream and downstream technologies. PRODIAS is working on these aspects and Xendo is happy to contribute to this with our XPure Systems.
Blog by: Trinath Pathapati
Ketogluconic, lactic, citric, succinic and many other organic acids are products that rely on a platform manufacturing technology. This means that most modern manufacturing facilities apply – to a large degree – the same processes. In both cases, the key step in the production process involves a fermentation followed by purification of products using at least one ion exchange step.
Application of a continuous countercurrent ion exchange technology for purification of these products result in significant advantages. In this memorandum, the effect of continuous ion exchange technology on purification of ascorbic acid will be elucidated.
L-Lysine is one of the essential amino acids, which cannot be synthesized in the body. The main application of Lysine is animal feed. The global market for food-grade L-Lysine is estimated at over 800,000 ton (2007 figure). The market price for bulk Lysine has dropped significantly over the past decade(s), forcing suppliers to apply more efficient manufacturing technologies.
This paper gives an overview of industrial SMB chromatography and focusses on the strategy how to develop a purification system either in the early development phase of a product or to assess whether a batch process can be optimised and scaled into a continuous process.
Biotechnological fermentation processes are widely used in industry to produce an abundant range of organic products which often need to be purified in order to meet high-quality standards.
Typical bioprocesses comprise:
Industrial Bioprocesses can be
Reducing downstream process steps can reduce capital investment and operational costs and reduce the overall energy consumption of an industrial process.
Conventional downstream processing involves biomass separation from the soluble fractions of the fermentation broth as a first step (e.g. filtration, centrifugation). Hereafter, other downstream process steps follow, depending on the required product purity and concentration. Decolorization is often necessary to remove the brownish
A typical process flow diagram is shown in the figure below.
Conventional bioprocesses can be summarised by the following steps:
In adsorption processes, the adsorbent is held in a (pressure) vessel, most often called a resin vessel. The stationary phase is referred to as a packed resin bed. As the process fluid flows through the vessel, the resin attains an equilibrium with the process fluid, resulting in a mass transfer zone that gradually moves through the bed. If the mass transfer zone has reached the exit of the resin bed, the bed is saturated and “breaks through”. The resin needs to be washed and regenerated before it can be loaded again. As a consequence, continuous processing of the liquid requires at least two fixed beds, but usually, three beds are installed.
In the previous century, the advantages of continuous countercurrent processing have been
In SMB technology, the chromatography material is kept inside columns or vessels. The transport of the chromatography material is obtained by periodically switching in- and outlet positions.
In the 1980’s, the SMB concept was originally developed for binary fractionation processes, where a stronger and weaker binding component
A state of the art example of such fractionating system is the production of High Fructose syrup fractions in the sugar industry. Here the Fructose is the monosaccharide with a stronger affinity towards the resin compared to Glucose.
At a somewhat later stage, the same concept has also been developed for bind and elute systems. Bind and elute systems typically comprise –at least- the following zones:
This zone distribution is not restricted to the four as mentioned, for instance, regeneration and cleaning in place have been frequently applied.
Bind and elute SMB systems are designed in carrousel configuration, featuring a central rotating fluid distribution valve, and a static vessel configuration featuring a valve block for each individual resin vessel. Each valve block is identical and comprises a number of valves accommodating all in- and outlet flows that have been defined for the chromatographic cycle.
The below figure represents a typical conceptual flow diagram for a bind and
Bind and elute IX chromatography systems based on the SMB principle has opened a huge field of applications where valuable products are recovered or purified on a continuous basis thereby saving substantial water consumption as well as elute and
Xendo has the experience and capability to design and build custom-made SMB continuous Ion Exchange and Chromatography systems under the product name XPure™.
In general, a process development study can be approached from different angles and started or
When developing a production process first the target objectives should be defined; what is the required yield and purity of the target compound; what is the composition of the starting material (feed); which recovery or purification process is most beneficial in terms of energy (including clean water) and material consumption and gives the least waste production; what is the scale of continuous operation.
If industrial (IX) chromatography could be
If literature cannot elucidate the case, based on the molecular structural or other adsorption relevant
Depending on the specific adsorption capacity of the target molecule onto the resin, further column tests – so-called breakthrough tests- will produce data on the resin capacity and information on how to elute (buffer composition, treatment ratio) the target molecule.
In the case, that potential resin candidates can be identified for the purification job the column tests can be elaborated with further break-through or pulse-tests at variable process conditions that cover the window of operation in a full-scale industrial setting. Typically this is conducted on one or two best performing resin candidates from the previous stage.
Here a Design of Experiment approach combined with the rationale of experienced chromatography engineering practice is used to define how many column tests will be conducted and what parameters will be varied at different levels.
Based on the data from the extended tests a preliminary process design and CAPEX/OPEX estimate can be made. Here we have developed our design tool where all relevant parameters can be put in and the outcome shows a full-scale SMB configuration and equipment dimensions. Dimensional data refer to a number of individual resin cells, dimensions of resin cells, line and valve sizing and pressure drop per distinct zone.
The design tool is based on the 2-film mass transfer kinetics model which is the principle for which we have created an algorithm. The design tool further features the (universal) Kremser equation for counter-current contacting.
A set of physical and mass flow-related variables have been accounted for. The most important parameters are:
Resin porosity, particle size (specific area) and evidently the most important -- specific adsorption capacity; diffusivity in both liquid and stationary phase; void fraction of resin bed; bed velocity; fluid viscosity and temperature.
The design tool could also be deployed if the adsorption system is a state of the art process, or close to this. In that case lab scale column tests could be skipped, and the specific feed characteristics need to be combined with the (specific) resin type that could do the purification/recovery job.
The output of the design tool can be used to do preliminary cost and value engineering. The outcome is essential to evaluate the purification/recovery process.
In the case of a positive decision, i.e.
Here we can enter two different scales for piloting.A
2. A large pilot SMB system featuring a bit larger resin cells from 1-4 inch column diameter on average 400-1000 mm bed height.
The selection merely depends on the availability of adequate feed and buffer volumes, any uncertainties that may not adequately be identified on an industrial scale, for example, impurities presence and identification, the presence of suspended solids or temperature variations.
The outcome of a pilot study will be a robust design of the industrial scale process also featuring chemical consumption figures, product
Simulated Moving Bed has distinct benefits over classical single column systems with significantly higher
This continuous production system is increasingly used on
If you’d like to investigate what SMB could mean for your production processes don’t hesitate to contact us or have a look at our currently available systems: XPure-C & XPure-S. We also have a wide variety of pilot studies available for those interested.
The static SMB system XPure-S that represents the by XPure patented valve system with a zero dead leg concept.
The carrousel SMB system XPure-C featuring a turn table that carries the sorption containers going through the distinct process steps of a typical IXchromatography cycle.
XPure offers SMB technology for product recovery and purification, comprising of
LAB SYSTEM FOR SEPARATION, PURIFICATION, AND RECOVERY
SYSTEM FOR SEPARATION, PURIFICATION, AND RECOVERY
The XPure-S SMB system offers a system for ion exchange processes with virtually unlimited scalability. The columns are not mounted on a
In addition to this, the modular design of the fluid distribution system allows extension in a later stage without significant extra investments, by connecting additional valve manifolds and extra columns (and resin volume). This allows a stage-wise capacity extension of the plant without having to build an entirely new system.