See also simulated moving bed chromatography (this is a form of continuous chromatography) under moving bed. 

Definitions:

Binding capacity and mass transfer: The binding capacity strongly depends on the affinity ligand, its density on the surface and its accessibility. Due to the improvements in the modificaiton technologies, high binding capacities >30 g/L can be reached at residence times of >3 min independently form teh support matrix chemistry. On the other hand, mass transfer properites are quite variable, cuasing radical differences in the application. Film mass transfer and pore diffusion are two main parameters, which influence the target molecule diffusivity towards the adsorption sites. Film mass transfer is mainly influenced by the particle size and shape. The smaller the particle and the interparticle volume the great the mass transfer and the flow resistance. Thus, for the analytical scale applicaitons smaller particle size is used to assure a fast mass transfer and high efficiency. But due to the pressure resistance issues, the industrial scale operations are performed at <3 bar operation pressures. Bigger particules (60-120 um) are usually choses. Due to these issues, the pore diffusion is one of the most influential properties defining the mass transfer. It has been shown that some materials exhibit side pores (50-500 nm) to enable a fast mass transfer. But these materials show low binding capacities (about 20 g/L) due to the low surface area. To enhance binding capacity, higher surface area is required, susually acheived through smaller pore size. Materials exhibiting aobut 100 nm pore sizes have shown 40 g/L binding acpaicites, even more about 70 nm pore size exhibiting materials ahve shown 56 g/L binding capacities. Due to the smaller pore size, the diffusion of the target molecule is slower, requriing certain target molecule residence time to acheive binding capacities abbove 40 g/L. The usual range of used residence times is in the range 3-6 minutes to assure that the target molecules diffuses towards the binding sites. (Skudas, US 20130280788)

Breakthrough curve: a plot of product concetnraiton at the outlet of a column as the column is being laoded with fluid at its inlet (this can be plotted v. time, mass loaded or volumn loaded). (Gjoka (US 2017/0016864)

Breakthrough experiment: an experiment where a column is overlaoded with product in order to ensure product breakthrough at the outlet so that a plot of the breakthrough curve can be obtained (column is typically loaded to 100% of its dynamic binding capacity). (Gjoka (US 2017/0016864)

Cycle time: amount of time requried for one column to complet an entire set (load, wash, elution, reneration and equilibration) of chromatogrpahy unit operations. (Gjoka (US 2017/0016864)

Load residence time: the resident time condtiion at which feed material (containing product) is loaded onto the column. 

Operating binding capacity: amount of product that is loaded divided by the column volume. (Gjoka (US 2017/0016864)

Productivity: grams of product processed per liter of sorbent per hour (operating binding capacity/cycle time) (Gjoka (US 2017/0016864)

Residence time: the amount of time it takes for a non-interacitng particle in the mobile phase to pass through the volumn of stationary phase (sorbent volumn/flow rate). (Gjoka (US 2017/0016864)

Static binding capacity: amount of product htat stationary phase is capable of binding under the condition of no flow. (Gjoka (US 2017/0016864)

Introduction: 

In continous chromatography, several identical columns are connected in an arrangement that allows columns to be operated in series and/or in parallel. Comared to a single column or batch chromatogrpahy, wherein a single chromatogrpahy cycle is based on several consecutive steps, such as loading, wash, elution and regeneraiton, in continous chromatogrpahy based on multiple identical columns all these steps occur simultaneously but on different columnes each. Continous chromatogrpay operation results in a better utlization of chromatography resin, reduced proessing time and reuded buffer requirements, all of which beenfits process economy. Rose (WO 2017/140081).

In continous chromatography, several identical columns are connected in an arrangement that allows columns to be operated in eseries and/or in parallel, depending on the method requirements. Thus, all columns can be run in principle simultaneously, but slightly shifted in method steps. The rpcoedure can be reepated. so taht each column is laoded, eluted and regenerated several times in the process. Compared to conventional chromatogrpahy, wherein a single chromatogrpahy cycle is based on several consecutive steps, such as laoding, wash, elution and regeneration, in continous chromatography based on multiple identical columns all these steps occur simultaneously but on different columns each. Contuous chromatogrpahy oepration results in a better utilization of chromatogrpahy resin, reduced processing time and reduced buffer requirements, all of which benefits process economy. One example of continous chromataphy is simulated moving bed (SMB) chromatography. (Skudas, US 20130280788)

Multiple-column chromatogrpahy (MC) involves loading two or more columns connected in series, where feed sample is passed form the iinlet of a first column through the outlet and into the inlet of a second column, through the outlet of the second column and so on, depending on how many columns are connected. This allows the first column to be over-loaded and the target product passing form the first column (that would otherwise be lost to waste) is captured by a subsequent column. In MCC, one column can be loaded while another stage in the cycle can be carried out on another column. When a firt column is over loaded adn the target is passed to a second column, this can be referred to as a “second pass” as the over loaded target product is passing into the second column. Depending on the number of columns, there can be a third pass as the over-laoded target is passing into a third column and so on. (Gjoka (US 2017/0016864). 

Sequential multicolumn chromatography (SMCC): 

SMCC uses full automationa and flexible, asynchronous scheduling of multiple columns (up to six) in enabling processes to run at linerar flow rates over 1,000 cm/h and effectively make use of almost 100% of resin capacity. The end result of approaching the maxiumum loading capacity while increasing process flow rates is 1.5-40 fold improvement in productivity.  Furthermore, several chromatographic and tangential-flow filtration (TFF) steps can be integrated to make continous downstream processing possible. SMCC uses the same type of stationary phase and the same separation techniques as are used in any given batch chromatography prcoess. With SMCC, a single batch column is divided into four smaller columns. Three of those four columns are available for laoding in linear sequence while the fourth column is eluted, regenerated, and equilibrated. This happens out of sequence with continual laoding until each column is ready to return to the linear sequence in rotation. (Holzer, “multicolumn chromatography” Bio-Process Intl 2008, 6, 74-82).

Particular Ligands Used

(Skudas, US 20130280788) discloses pvoiding at least three separation units having the same matrix, preferably an affinity or ion exchange chromatogrpahy matrix, which are connected so that liquid can flow from one separation unit to the subsequent one and form the last to the frist separation unit, b) feeding the sample on the first separaiton unit so taht while the sample is loaded on this separation unit wherein the sample is at a pH and conductivity enabling the target molecuels to be bound to this separation unit, said separation unit is at least part of the loading time in fluid communication with the next separation unit so taht target molecuels not bound to the frist separation unit can bind to the next separation unit, at the same time at least eluting and reqequilibrating one separaiton unit different formt eh separaiton unit that is loaded and form the one that is in fluid communicaiton with the separation unit that is laoded. That measn that the other process steps like washing, eluting and reequilibrating that are needed in a chromatogrpahic seapration pcoress are perfomred on one or mroe of thsoe separaiton units that are not in fluid communication with the separation unit or units that are being loaded, c) switching the feed to the next separaiton unit. That means when the separation unit that has jsut been laoded is fully loaded, the sample feed to this separation unit is stopped and by simultaenous handling of the vlaves of the system without interruption directed to the separation unit that is next in the circle. The separation unit that is next in the circle is the one which was before at least part of the loading time connected to the outlet of the separation unit that has been laoded. Consequently, this separation unit is already partly loaded with those target molecuels that have not been bound to the frist seapraiton unit. d) feeding the sample on the next separation unit so that while the sample is laoded on said next seapration unit wehrein the sample is at a pH and condcutivity enabling the target molecuels to be bound to said net separation unit, said next separation unit is at least part of the loading time in fluid communication with the separation unit after the next so that target molcuels not bound to said next separation unit can bind to the separation unit after th enext, at the same time at least eluting and reequilbrating one separation unit different form the the separation unit that is laoded and from the one that is in fluid communciation with the separation unit that is laoded. 3) repeating steps c) and d) one ore more times. 

Protein A

Skudas (EP 2656892) discloses continous affinity chromatography such as Protein A to purify a target molecule such as an antibody. At least three separation units have the same chromatography matrix are connected so that liquid can flow from one separation unit to the subsequen one and form the last in the to the first separation unit. The sample is fed onto a first separation unit so that while the sample is loaded and the target molecule is bound to thsi separation, said separation unit is at least fluid communication with the next separation unit so that target molecuels not bound can bind to the next separation unit at the same time that washing, eluting and/or requilibrating one separation unit different from the separation unit that is being lodaed and switching the feed to the next seapration unit.  Skudas discloses using two seprations units A1 and A2 both having the same chromatography matrix such as an affinity chromatoraphy, a cation exchange, AEX or MM matrix and a separation unit B that has a CEX, ZEX or AEX matrix. In a preferred embodiment A1 and A2 have an affinity chramotgraphy matrix and unit B has a CEX matrix. In a preferred emodiment, the sample is continously loaded alternatively to either separation unit A1 or Ab and in another embodiment while loading the sample onto unit A1 the fluid outlet is at least partly in fluid communication with the fluid inlet of unit A2 to enable capture of the starting to leach target molecule from A1 to A2 and while loading the sample onto A2 the fluid outlet of A2 is at least partly in fluid communicaiton with the fluid inlet of A1 to enable the capture of the starting to leach target molecule form A2 to be bound to A1.

Hydrophobic Interaction Media:

Mattila (US 16/459,187, published as US 20200002373) discloses a method for preparing a target polypeptide form a mixture using a chromatography apparatus that includes a plurality of zones/chromatographic columns where the one or more columns include hydrophobic interaction media. Such chromatography apparatuses may include pre-manufactured apparatuses (e.g., Cadence TM BioSMB (Pall Biosciences), BioSCR (novadep), VaricolR (novasep), Octave (SembaR Biosciences) or mroely two or more standard batch chromatography apparatuses used in tandem. In some embodiments, the method includes passing the mixture to a first column, passing an effluent from the first column to a second column, passing mobilde phases to a thrid column wehrein each of the plurality of columns includes an outlet connectable to the other columns and a sum of residence times for teh mixture in the first and second column is substantially the same as the sum of the residence times in the third column. In some ebmodiments, passing mobile phase(s) to a column may include passing a wash buffer and after passing a wash buffer regenerating the column. 

Protein L:

Muller, “Intensification of Fab-fragment Purification, Multicolumn chromatography using prepacked prtoein L columns”, BioProcess international, June 2023, 21(6)) discloses a MCC prcoess using a protein L affinity chromatography (Tosoh Bioscience’s Toyopearl AF r-protein L-650F resin) where the Octave Bio system was equipped with five 1 ml SkillPak Bio prepacked columns (all from Tosoh Bioscience) containing the respective protein L resins. The Octave BIO system conssits of six pumps, a switching-valve block and a detector array. Each pump is designated for one buffer required in the process. Trhough the voalve block, up to eight columns can be addressed by different pumps in aprallel or connected to each other in series. The detector array provides precise control of up to four different process streams with regard to UV adsorption, conductivity, and pH. The method was used with Tosoh Bioscien’es ProComposer Method Wizard tool. When three of the columns are in the loading pahse, the remaining two go through the phases of wash and elute, then CIP and equilibration. Once the first column in the loading series is fully loaded, the column ports are switched into position against the flow stream. In MCC, product breakthrough is less of a risk druing laoding becasue of the secondary load columns. The columns were loaded to 85% of their previously determiend SBCs, resulting in loading masses of 45.3 mg/mL resin for the Tosoh resin. 

Mixed Mode Chromatography:

–HCIC:

Luo designed a swo step chromatographic separation method taht combined ABI-4FF and MMI-4FF resins for bioseparation. The ABI-4FF resin was first used to remove IgA at pH 4.5 and the flow through fraction from the frist step contained IgM and IgG. In order to capture IgM, the MMI-4FF resin was then used for purificaiton further at pH 4.5. After optimization, IgM purity of 65.2% and purificaiton factor of 28.3 were obtained. Heidebrechi also developed a scalable and cost efficient process that connected Capto MMC and MEP HyperCel to isolate bovine IgG from colostral whey.  (Yal “development and application of hydrophobic charge-induction chromatography for bioseparation” J. Chromatography 1134-11335 (2019). 

Optimization of Multi-Column Chromatography:

See also Tosoh Bioscience ProComposer Method Wizard tool  (converts batch method to MCC)

Gjoka (US 2017/0016864) disloses a method of determing an optimum operating binding capacity for a MCC where loading experiments are performed on a single column at different residence times and/or different flow rates to determine an optimum operating binding capacity for the MCC prcoess. Accurate prediction of the operating capacities enables estimation of many other multi-column process parameters such as productivity, cycle time, total number of columns and/or bufer ulitiziation. In one embodiment, the method includes (a) loading a target on a column at a first residence time and/or first flow rate, (b) loading the target on the column at a second residence time and/or flow rate wehre the first residence time and/or flow rate is different than the second residence time and/or flow rate, (c) generating breakthrough curves for the first reisdence time and/or first flwo rate and for the second residence time and/or second flow rate and (d) detemrining an optimum operating binding capacity fo the MCC process. Typically the second residence time is about double the first residence time and/or the second flow rate is about half the first flow rate. The inventors realized that if the columns are connected in series and loaded straight through to saturation (in the absence of valve switching to simulate countercurrent movement of the columns), the amount of target product bound by the first column immediately before breakthrough of the last column in the series is the ideal operating binding capacity. In other words, while one could masure the breakthrough curve by using a single column with a deterctor after it and the other breakthrough curve by  connecting two or mroe columns in series with a single detector at the end of the columns, the inventors found that the shape of the product brekathrough curve is almost completely dependent on the residence time applied whehn laoding a column. This enables generation of the two breakthrough curves with just a single column and single detector after the columns. To mimic the MMC one brekthrough curve is performed at a chosen residence time and to mimic two columns in series anouter breakthrough curve is perforemd at double the chosen residence time. The breakthrough curve at the chosen residne time is used to caclulate the amount bound to what would be the first column in series, where the area above the brekathrough curve is representative of the amount bound to the column. The break through at double the residence time is representative of the seocnd column in series and is necessary to inform how much product bould be laoed in MCC without incuring product low to the flow through. This brekathrough information is used to detemrine a limit as to how much proeduc is laoded and calculate a loading time or amount limit in the intial breathrough cruve. With these two breakthorugh curves it is possible to detemrine the amount bound ot the frist column before breathorugh of the seocnd column which is the oeprating binding capacity. This can be extned to three or more oclumns. For three columns, the seocnd breakthrough curve would be performed at three times the chosen residnece time and so on. 

Lin (US Patent Application No: 18/117,479, published as US 20230203092) discloses optimization for capturing proteins by multi-column continous chromatography (MCC) by performing a single time fo protein breakthrough experiment to obtain a protein breakthrough curve and integrating the breakthrough curve to obtain a single column loading capacity and establishing a linear relationship between the interconnected load time and the load residence time, solving for the optimal number of operation column for capaturing proteins and solving for the optimal load residence time for capturing proteins and solving for the maximum productivity of capturing proteins by MCC. In step 1, under the conditions of a set laoding protein concentration and arbitrary load residence time, a single time of protein breakthrough expeirment is performed to obtain a protein breakthroughcure. In step 2, under a set breathrough percentage (greater than or equal to 50%), integrating the breakthrough curce to obtain a singel column loading capacity of continuous chromatography and estalbishing a linear relationship between the interconnected load time and the load residence time through the single column loading cpacity. In step 3, using the linear relationship between the itnerconnected laod time and the load residence time to solve for the optimal number of operating columns for cpaturing proteins by MCC under the set laoding protien concetnraiton and protein breathrough perentage.