Simulated Moving Bed (SMB) chromatography:

Introduction:

A specific way of operating continous chromatography is called simulated moving bed (SMB) chromatography. In SMB all the chromatography columns are periodically and simultaneously moved in the direction opposite the sample flow. The movement of the columns is realized by appropriate redirections of inlet and outlet stream to/from the columns which requires a sophisticaed setup. Rose (WO 2017/140081)

The simulated moving bed (SMB) technology as been patetned about 40 years ago. it is a multi-column continous chromatographic binary separator involving a coutner-current between the liquid (eluent) and the separating medium (stationary pahse) packed in the columns. (Nicoud “Recent aspects in simulated moving bed” Aanlusis Magazien, 1998, 26, N 7). 

For large scale separations in actual productions continuous processes are much more economic than batch processes. The advantages of a continous process is for example high yield, less solvent consumption, less costly fractionation and analyses, better flexibility for the auntities to be purified. One way to realize a continuous chromatographic process is the so called simulated moving bed process (SMB). (Aumann, WO 2006/116886). 

In continous chromatography (SMB chromatography), several identical columns are connected in an arrangement that allows columns to be operated in series and/or in parallel, depending on the method requirements. Thus, all columns can be run in principle simultaneously, but slightly shifted in method steps. Compared to “conventional” chromatography, where a single chromatography cycle is based on consecutive steps of loading, washing, elution and regeneration, in continous chromatography based on multiple identical columns all these steps occur simulataentously but on different columns each. Continous chromatography operation results in a better utilization of chromatography resin, reduced prcoessing time and reduced buffer requriements. Rose (WO 2017/140081)

Aumann (US2011/0042310; US 2009/0050567; US8,496,836; US7,837,881) teaches a process for continuous purification of a mixture using columns that are grouped into at least four sections. After a switch time, the last column from the first section is moved to the first position of the second section, the last column of the second section is moved to the first position of the third seciton, the last column of the third section is moved to the first position of the fourth section and the last column of the fourth seciton is moved to become the first column of the first section. The column can be run in at least one batch mode step in which the outlet of one column is used to collect the desired intermediate product as well as in continous mode where the outlet of at least one column is fluidly connected with the inlet of at least one other column.

Bryntesson (WO2008/153472A1 and US2010/0176058) discloses a SMB method where a feed compriing a target compound is passed across a 1st adsorbent, the outflow passes to a 2nd adsorbent, the feed is redicted to the 2nd adsorbent, a wash liquid is passed across the 1st adorbent, this wash liquid outlfow is directed to a 3rd adsorbent and the second adsorbent outflow is directed to the 3rd adsorbent, the 1st adsorbent is regenerated, the feed is redicted to the 3rd adsorbent, wash liquid is passed across the 2nd adorbent, directed the wash liquid outflow to the 1st adsorbent, subsequently directly the outflow from the 3rd adsorbent to the 1st adsorbent, regenerating the 2nd adsorbent, redicteing the feed to the 1st adsorbent, washing the 3rd adsorbent and directly its outlfow to the 2nd adsorbent, directing the otuflow from the `st adsorbent to the 2nd adsorbent, regenerating the 3rd adsorbent and repeating all the steps.

Jeon (US 13/643146, published as US. 2013/0046080) discloses a chromatographic method for purifying an antibody where a sample is loaded on a 1st column, loading the outflow to a 2nd column, before washing the 1st column, stopping the loading of the sample and loading the sample on the 2nd column, loading the outflow from the 2nd column onto a third column and before washing of the 2nd column, stopping the loading of the sample on the 2nd column and loading the sample on the 3rd column and repeating the steps from the 1st to 3rd and 3rd to 1st columns.

Muller-Spath (US2014/0299547) teaches reducing the number of column to twofor the isolation of a product like an antibody comprising a first batch step, where said column are disconnected and a first column is loaded with feed and its outlet directed to waste and from a second column desired product is recovered and subsequently the second column is regnerated, a first interconnected step where the outlet of the first column is connect to the inlet of the second column, the first column is laoded beyond its DBC with feed and the outlet of the second column is directed to waste, a second batch step analogus to the first batch step but with exchanged column and a second interconnected step analogous to the first interconnected step but with exchanged column.

Thommes (US2004/0241878) teaches separating an antibody using a simulated moving bed (SMB) system where at certain points liquid streams may be introduced and at other points effluent streams may be withdrawn. Thommes (US 2007/0215534 and US Patent No: 8608960) also discloses a SMB system which incorporates a plurality of zones  such as an association zone, a wash zone, an elution zone. Suitable solid phases may include Protein A or G but can also include CEX. Use of continous chromatography in a continous counter-current mode where the columns are loaded to equilibrum capacity requires smaller column valumes compared to batch chromatgoraphy wehre the column are loaded to there DBC and requires using columns of 2-3 times the volume. In one embodiment, the feed is applied to 2 columns in parallel and makes a second pass through the 2 parallel columns. A carousel or similar apparatus may be used to move the columns relative to stationary valves and inlet and outlet streams. The columns moving to the left from the adsorption zone enter the adsorption wash zone. The effluent of the adsorption wash zone is fed back into the adsorption zone to minimize product loss. The adsorption was zone consists of 2 columns in series thus reducing the amount of buffer reuqired by about 40%. The product is leuted in an elution zone consisting of 2 columns in series. The eltuion zone is followed by an elution wash zone. 

Commercial SMB columns:

SMB systems have been designed by Novaept (Pompey, France), Tarpon (Worcester, Ma), Semba (Madison, Wi), Contichrom (Knauer, Germany) and GE Healthcare. (Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012)

Cature SMB:

In captureSMB process, two identical columns are used to recover the target compound form the feed mixture. After the columns have been equilibrated, they are interconnected by connecting the outlet of the first column to the inlet of the second column. For a certain time, the feed solution is loaded onto the first column, The fact that there is a second column behind the first one allows for longer feeding compared to the batch case, because the eventual breakthrough is cought by the second column instead of being lost. After this time, the first column enters the recovery and regeneration step, which is equivalent to the batch process. During this step, the now diconnected second column is further loaded with feed. As soon as the first column is re-equilibrated and ready to be loaded again, the columns swap roles and ocntinue with the next interconnected step.  (Baur, Daniel, “Design, modeling and optimization of multi-column chromatographic processes” Doctoral Thesis, 2017). 

Periodic Counter-Current Chromatography systems (PCCS or MPCC):

In multi-colunn periodic counter-current chromatography (MPCC) the breakthrough protein form the first column is captured by the second column. The first column stops laoding when a set protein breakthrough percentage is reached and the loading switches tothe second column. The first column is then eluted and regenerated and multiple columns are alternated to acheive continuous chromatogrpahic separation, thereby improving productivity, resin capacity utilization and reducing buffer consumption and equipemnt size. Thus for a three column MPCC, columns 1 and 2 are connected in series for laoding and column 3 is eluted and subjected to recovery and regenreation R-R). When column 3 is completely eluted and regeneration, column 1 also reaches the set protein brekathrough percetnage. Columns 1 and 3 a re connected and unadsorbed protein in column 1 is washed to column 3. At the same time, feed is swtiched to column 2. After column 1 is cwashed, it is disconencted form column 3 and column 2 is connected to column 3 for continous loading, while column 1 is subjected to R-R. When column 1 is completely eluted and regeenrated column 2 also reaches the set protein breakthrough percetnage . Column 2 and 1 are connected in series and the unadsorbed rptoein in column 2 is washed to column 1. At the same time, the feed is switched to column 3. After column 2 is completely washed, it is disconnected form column 1 and column 3 is connected to column 1 for continous laoded bhile column 2 is subjected to the R-R. When column 2 is completely eluted and regenreated, column 3 also reaches the set protein brekathrough percetnage. Column 3 and 2 are connected in series and the unadsorbed rptoein in column 3 is washed to column 2. At the same time, the feed is switched to column 1. Lin (US Patent Application No: 18/117,479, published as US 20230203092)

In PCC, multiple columns are used to perform the same steps in parallel such that continuous feed is acheived and all other process steps are discrete in time. For PCC, a cycle is defined when each one of the columns has completed equilibraiton, load, wash, elution and regeneration steps in sequence. To proceed from one step to another, an automated column switching algorithm is employed. There are essentailly two decision points required for every column cycle –one at the start of protein brekathrough and the other at column saturation. These switch points in PCC are detemriend by a UV based control strategy developed by GE Healthcare that is based on a difference between the feed and column outlet V. In principle, column switching can be determined by an process analytical technology tool capable of in-line measurement of product concentration. (Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012)

Konstantinov (US 2014/0255994, issued as US 9,650,412) discloses an integrated continous biomanufacturing process for producing a therapeutic protein such as an antibody which includes feeding a liguid culture medium into a first multi-column chromatogrpahy system (MCCS1) to capture the protein and then continously feeding the eluate which contains the protein into a second multi-column chromatgraphy ystem (MCCS2) to purify and polish the protein. The process can include the use of two or more mutli-column chromatography systems (MCCSs). A MCCS can include two or more chromatogrpahy columns or membranes or a combination of columns and membranes. The first and/or second MCCS can be a periodic counter current chromatogrpahy system (PCCS) which includes towo or more columns that are switched in order to allow for teh continous elution of the protein from the two or mroe columns. In PCCSs, multipe columns are used to run the same steps discretely and continously in a cyclic fasion. Since the columns are operated in series, the flow through and wash form one column is capture by another column. This unique feature allows for loading of the resin close to its static binding capacity instead of the DBC and is typical during batch mode chromatography. Once all the steps in the cycle are competed, the cycle is re-started. The system can include column switching based on teh UV absorbance difference betweenthe feed inlet and column outlet. During column laoding, the PCCC control system dtermiend the impurity baseline when the absorbance stabilizees. As the recombinant therapeutic prtoein breaks through, there is an increase in the outlet UV signal above the impurity baseline. At the point when deltaUV ahs reached a rpe-detemriend threshold (such as 3% breakthrough of the reocmbinatn therapetuic protein), the flow-through from column 1 is directed onto column 2 ineasted of to the wate. When column 1 is nearly saturated with recobmiannt threptuic protein adn the delta UV has reached a pre-detemriend vlue, the feed is switched to column 2. An important adantage of this deltaUV based column switching streegy is that it allows for uniform loading of the columns irrespective of the feed recobminant therapetuic prdcut concetnraiton and the oclumn capacity. In PCCCS, the resdience time (RT) of the protein on the column can be decreased without increasing the column size becasue the breakthrough form the frist column in the system can be captured on the second column in the system. 

3-column periodic counter-current chromatography (PCC) (3-C PCC):

The 3-column PCC process uses three identical columns. The first part of a switch consists of an interconnected wash step where the first, fully loaded column is washed and the wash is recylced into the third column, while the second column is loaded in a disconnected mode. Since the feed is applied to a disconnected column, this phase is denoted as athe batch pahse, with the duration tB and the feed flow rate BB. When the interconnected wash step is finished, the first and the third column are disconnected and the first column continues in the recvoery and regeneration procedure, with further wash steps, the elution, the CIP and the equilibraiton step. In the meantime, the two other columns are loaded in an interconnected manner using a flow rate QIc; therefore this part of the switch is called the interconnected phase, which last for the time tIC. After the interconnected phase is complete, all columns are moved one position upstream relative to the liquid phase flow, which denotes the beginning of a new switch. When three swithes have passed and the columns are back in the initial configuation, one cycle has passed. (Baur, Daniel, “Design, modeling and optimization of multi-column chromatographic processes” Doctoral Thesis, 2017). 

Gadgil (US 16/340,822, published as US 2019/0263855) discloses a multi-column chromatography system such as a periodic counter current chromatography system (PCC) which includes carboxypeptidase B immobilized on sepharose.  The C-terminal lysine residues on H chain can be truncated by passing a harvest recovered form perfusion cell culture on a column which has CPB on sepharose. The CPB preferentially acts upon the basic amino acids, such as arginine and lysine and thus the resin can be used for removal of charged isoforms belowing to any class of antibodies. In one embodiment, a continuous process for reducing heterogeneity of an antibody includes a CPB-Sepharose column connect to a Protein A column. The flow through from the CPB-Sepharose column is directly loaded ontto the protein A column for capture step. The third and fourth columns can be selected from AEX, CEX, HIC and MM chromatography. In one embodiment, the multicolumns can be run either in series or in parallel. In one embodimentm the prcoess was carried out on AKTA pcc (three column periodic counter curent chromatography, 3C PCC) GE Healthcare, where one CPB-CNBR activated Sepharose 4B column (column 1) and two prtoein A columns (column 2) are connected parallely. The residence time maintined on column 1 is equivalent to 1.7 min and column 2 was about 4.4 min (as resin used is MabSelect SuRE LX). 

4-column periodic counter-current chromatography (PCC) (4-C PCC):

The 4-column PCC process uses four identical columns that are loaded and eluted sequentially.  IIn the first part of the switch, the column in the first position, which has been washed in the previous switch, is eluted. Meanwhile, the second column undergoes the first part of the wash step, during which product from the liquid phase of the first column is directed to the cleaned and regenerated column in the last position. During this time, the column in the third position is disconnceted and loaded with feed. When the intercconencted part of the wash step is completed, the second column is disconnected and the recovery and regeneration continues. Product elution from the first column finishes during this phase, and CIP and regeneration follow. Meanwile, the other two column are interconnected and feed is applied to the third column. After this, one switch has passed and the columns are moved one position upstream. The 4-column PCC prcoess differs form the other processes in that there is more than one column in the recvoery and regeneration phase at any one time. If there is no constraint that the switching times in all positions must be the same, the recvoery and regeneraiton can be scheduled mroe efficiently in the 4-column PCC process. (Baur, Daniel, “Design, modeling and optimization of multi-column chromatographic processes” Doctoral Thesis, 2017). 

Gdawat (“Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012) discloses a 3 column PC cycle. At the begining of a cycle, the feed is laoded onto column 1 and the flow through goes to waste. (step 1) When the product start to breakthrough, the flow-through from column 1 is directed to column 2 to capture the unbound product. (step 2) Once column 1 is fully loaded, the feed is directly loaded onto column 2, while column 1 is washed onto column 3, eluted regenerated and requilibrated for the next cycle. (steps 3-4) Column 2 is now subjected to steps 3-6, which are identical to steps 1-4 for column 1. Finally, the same steps occur on column 3. 

–Commercial examples:

Gdawat (“Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012) discloses a 4 column PCC system which is a custom modified AKTA system (GE Healthcare). The system was equipped with five UV minotors, three pmumps, multiple vales and a pH and conductivity meter and was pwoered by a Unicorn based custom control strategy. MabSelect SuRe, iminodiacetate adn hydrophobic interaction chromatgoraphy HIC) media were packed into three of four 1.6 cm x 6 cm, 16.6 cm x 10 cm, or 0.66 cm x 6 cm columns. 

Purification of Target Proteins using Continous Multi-column chromatography  each with the same ligand for each of the Target proteins

Bataille (US Patent application 15/125483, published as US 2017/0073396) disclsoes purification of three separate plasma proteins, immunoglobulin, albumin and fibrinogen, where muticolumn affinity chromatography comprising several chromatogrpahy columns having the same chromatography support are linked in series, the chromatography in series being used to purify the threee separate proteins. For example, a first mutlicolum affinity chromatography step where each of the columns includes an affinity ligand that binds to immunoglobulins is first conducted, the flow through fraction is then subjected to a second muticuloumn IEX or mixed mode or HIC multicolumn step where the fraction containing albumin is collected and the flow through is usbjected to a third multicolumn affinity chromatography series having an affinity ligand that binds to fibrinogen, at the conclusion of which the fration containing fibrinogen is collected. Each of the multicolumn chormatrpahy steps can for example be 3 columns in series and performed such that once loading and washing of the first column is completed, the first column is separated from the other columns and undergoes elution while the second column becomes the first column in the series and once the equilibration is completed, the first column is replaced in series in last position and the bycle of steps is repeated. 

Optimization of PCC:

Protein adsorption to a chromatogrpahy resin is dependent on the bidning conditions, size, biochemical proeprties of the target protein, the resin bead morphology (bead size, pore structure and phase ratio) and the functional ligand properties (type and density). Collectively, these factors influence the thermodynamic and mass transfer kinetics of protein-resin interations. The thermodynamics govern the equilibrium binding properties, such as static binding capacity and the binding isotherm, while mass transfer kinetics govern the rate of protein adsorption onto the resin. The quantitative aspect of mass trasnfer kinetics in protein binding is demonstrated by the shape and nature of the protein breakthrough curve. Protein breakthrough form a column occurs before all the protein accessible binding sites are unitized on the resin due to mass transfer resistance. Since columns are operated at a finite flow rate with a finite protein residence time, only a poriton of the static binding capacity can be utilzied. This capacity is the dynamic binding capacity at a particular residence time. Chromatographers have traditionally deisgned batch processes with relatively long target protein residence times to increase resin capacity utilization. However, this approach also resutls in low throughput and subsequent oversizing of the chromatogrpahic column to aceive the desired target throughput. In contrast to batch chromatogrpahy, target protein breakthrough is laoded onto a second column in PCC until the first column is almost fully saturated. This leads to full capacity utilization irrespective o most cmmonly used residence times. This can also be acheived in batch mode in convective systems (perfusion media with large proes and membrane chromatography) at short residence times wehre the mass transfer effects are negligible.  (Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012)

(Gdawat “Periodic counter-current chromatogrpahy- design and oeprational considerations for integrated and continous purificaiotn of proteins” Biotechology Journal7, 1496-1508, 2012) isclsoes a mathematical framework for designing PCC for a given protein capture resin system. The scheduling required to perform continous captrue of a target protein using PCC is governed by the target prtoein breakthrough curve. For a given protien-capture resin system, the scheduling parameters can be detemriend from a mathematical transformation of the breakthrough curve. The optimal PCC operation can be defined using one of many potential bjective functions, including maximization of resin capacity, utlization, minimum recovery loss during was, resin lifetime usage or minimization of residence time. 

Countercurrent Tangential Chromatography:

Countercurrent trangential chromatography overcomes many of the limitations of conventional column chromatography by having the resin (in the form of a slurrry) flow through a series of static mixers and hollow fiber membrane modules. The microporous hollow fiber membranes retain the large resin particles while letting all disssolved species pass through the membrane and into the permeate. Thus, during the binding operation, the protein of interest stays bound to the resin while the impurites flow through the membrane and are removed as waste. The buffers used in the binding, washing, and elution steps flow countercurrent to the resin, enabling high resolution separation while reducing the amount of buffer needed for protein purification. For example, a concentrated slurry is pumped from slurry tank 1 into a first statis mixer (SM1) where it is mixed with recycled permeate form a second hollow fiber mrembrane module (HF2). The diluted slurry then passes into the frist hollow fiber module (HF1) where sufficient permeate is removed to return the slurry to its origianl concentration. This provides true countercurrent contacting, the slurry moves form stage 1 to stage 2 while the buffer (permeate) starts in stage 2 and moves to stage 1. For binding operations the concentrated slurry is then mixed with the protein mixture in the second statis mixer (SM2), pumped into HF2 and collected as a reconcentraed slurry in slurry tank 2. Permeate from the first module is sent to the permeate collection tank. For washing and elution, the concentrated slurry is mixed with an appropriate buffer in the second static mixer. (Zydner “Countercurrent tangential chromatography for large-scale protein purificaiton” Biotechnology and Bioengineering, 108(3), 2011), disclosing using a Macroprep 25Q strong anion exchange resin consisting of a methacryalte based modified with a quaternary amine functionality). 

The CCTX process utilizes the resin in the form of a slurry which flows thorugh a series of static mixers and hollow fiber membrane modules. The micro-porous hollow fiber membranes retain the large resin partciles while letting all dissolved species, including proteins and buffer components pass through the membrane an into the permeate. The buffers used in the binding, washing, elution, strippeing and equilbiraiton steps flow coutnercurrent to the resin slurry in a multi-stage confirguation, enabling hihg resolution separation while reducing the amoutn of ubffer need for protein purificaiton. (Shinkazh “Purificaiton of monoclonal antibodies form clarified cell culture fluid using Protein A capture continous coutnercurrent tangential chromatography” J Biotechnology. 213 (2015) 54-64). 

Shinkazh (US 2010/0193434; see also US 2017/0045483) discloses a module that consists of two or more interconnected tangential flow filters and static mixers. The chromatography resin flows thorugh this module in a single pass, while similar operations to a regular chromatographic process are performed on the resin (binding, washing, elution, regeneration and equilibration). The buffers for these operations are pumped into the module in a countercurrent direction to the flow of resin, and permeate solutiosn from latter stages are recyled back into previous stages. This creates concentration gradients in the permeate solution of the tangential flow filters in the countercurrent direction to resin flow, thus saving buffer volume and increasing process efficiency. The permeate solutions from binding, washing, equilibration and regeneraiton operations are put to waste. The permeate solution form the elution operation is the purified product stream which is collected in a separate product tank. In one embodiment, the module for CCTC includes a first input prot for receiving an input solutions, a first mixer for mixing the input solutions with a recyled solution from a second input port to produce a first mixer output, a stage 1 filter for concentrating the first mixer output to produce stage I retentate, wehrein stage I permeate exists the module from the stage 1 filter via a first output port, a second mixer for mixing the stage 1 retentate form teh stage I filter and an optional buffer solution from a second inpurt port and a stage 2 filter for concetnrating an output from the second mixer to produce stage 2 retnentate which exits the module form teh stage 2 filter via a second output port, wehrein stage 2 permeate exists the module form the stage 2 filter via a third output port. 

Shinkazh (US 2020/0030717) disclsoes replacing the hollow fiber membranes of CCTC systems above by spiral micro-fluidic partcile sorter devices. These dvices seaprate resin form the dissolved species using Dean Vortices at the curved outer wall of the device channel to selectively attract partciles to one wall of the device. The devices accordingly do not reuqire a physical barreir such as a membrane for separating resin partciles from dissolved speies.