See also continous or SMB chromatography 

In General: 

Brown (WO2010/019148) discloses membrane chromatography run on either a standard chromatography system or a custom system like an AKTA exploer (GE Healthcare) equipped with pressure gauges, sensors, and pump plus pump controllers. Continous processing experiments were performed on the AKTA explore. The Q column flow through was pH adjusted in line and imediately loaded onto a Mustang S membrane. The Q column was packed with Q Sepharose Fast flow resin, the column outlet was attached to the inlet of a T connection and pH adjustment was accomplished in line by directly the B Pump to the opposite inlet of the T connection. The T connection provided adequate mixging and the pH adjsuted solution was directed to the inlet of the Mustang S membrane. Mustang S is a strong CEX and Mustang Q is a strong AEX. 

Coffman (US13/811178) teaches a method of protein purification using a column/resin that is arranged in tandem with the column of a second resin.

Vedadi (WO03/025156) discloses protein purification using one or more chromatography colun which are arranged so that teh flow through or eluate from one column loads directly onto another colun (e.g., the columns are arranged in tandem). In an exemplary embodiment, proteins are purified using a combination of an IEX, affinity and gel filtration column. In the embodiments, an automated process control system is included which includes various sensors, and pumps. 

Particular Schemes with In-Line Chromatography

AEX-Protein G Affinity:  

Qi (J. Biochem. Ciophys. Methods 49 (2001) 263-273) discloses injecting a diluted mouse serum onto a system whcih includes DEAE AEX and Protein G affinity columns coupled in series by a column switching technique. The advantage is that IgG and albumin could be separated and purified simultaneously. 

CEX-Mixed Mode:

Liu (US 2013/0079272) teaches loading a CEX and then a mixed mode material in continuous mode which is referred to as having the CEX and MM material eitehr direclty connected or other mechanisms which allows for continous flow.

Affinity Chromatography

–Protein A-AEX: 

Lundblad (WO2005/058452) discloses an automatic system in which a detector signal is monitored by a computer  comprising a plurality of chromatography column. Each column has an inlet end and an outlet end. In one bodmiment columns 1 and 2 are affinity columns each of which contains a ligand to which a protein of interest binds, column 3 is a desalting column and column 4 is an ion exchange column.

Shamashkin (Biotechnology and Bioengineering, 110(1) 2013) discloses a semi-continous downstream process where columsn and filters are linked and operated in tandem so as to eliminate the need for intermediate holding tanks. Shamashkin exemplifies a tandem process for the purificaiton of mAbs employing an Affinity Protein A capture step, followed by a flow through AEX step with the possibility of adding an in-line virus filtration step. All steps were linked sequentially and operated as one continous process using an AKTA FPLC equipped with two pumps and a system of valves and bypasses that allowed the components to be engaged at different stages of the process. The AEX was operated in a weak partitioning mode by a precise in-line titration of Protein A effluent. 

—-Protein A-AEX-CEX or CEX-AEX:

Duthe (US 14/889,397, published as US 2016/0083454) discloses a 3 step chromatography scheme of Protein A – CEX and AEX where each of the buffers used in the steps is Bis Tris. and the method is run in a closed system without buffer exchange or dilution.

Kulkarni (WO 2011/090719) discloses multiple chromatogrpahic steps wehre the low pH eluate from a protein A chromatogrpahy is further purified without the need of substantial pH adjustment. The eluate from the Protein A is loaded onto a CEX in Bind and elute mode without substantial adjustmnet of pH. A third purificaiton using AEX is performed in flow through mode without substantial adjustment of pH. 

Laursen (US7138120 and US2001/0051708; see also WO/1999/064462;  See also US 6,281,336) teaches a process for purifying IgG where AEX and CEX are preferably connected in series. Laursen teaches that the use of two serailly connected chromatography column makes the oerpation more practical because there is no need for an intermediary step of collecting the IgG containing fraction between the two IEX methods.

Mahajan (J Chromatography A, 1227 (2012) 154-162) discloses using Protein A and then loading the next column to capture the mAb. The continous operation is advantageous in that it can reduce both resin volumne and buffer consumption. However, the process is more complex than a batch process. The process utilizes a simulated moving bed concept where as the olumns are being used in series or parallel dpending on the process step. 

Soice (US2011/0065901 and WO2011/017514) discloses a method for purifying an Fc containing protein using the scheme of Affinity-CAEX-AEX which eliminates the need for a holding tank and/or a buffer exchange step. The purification process is referred to as a “connected process”. According to the prcoess, the sample is contacted with an affinity chromatography media, the Fc containing protein is eluted and contacted with CEX and eluted and then contacted with an AEX.

Williams (US2011/0073548) teaches a separation system comprising at least two sepration units such as a CEX and AEX unit which are connected in series outlet to inlet to form a line of separation units.

–Protein A – CEX:

Minakuchi (US15/022890, published as US 2016/0237113) discloses a method for purifying an antibody using a first carrier with an affinity ligand such as Protein A and a second carrier having a CEX group where the contacting occurs in a column having bothth carriers or where the columns are operated in-tandem and  where the affinit/Protein A chromatography and CEX are performed at one chromatography step such that and eluate is passed through the column where the pH of the eluate is 4.0 or less and the pKa of the CEX group is equal or greater than the pH of the eluate and the pKa of the CEX group is 4.0 or more. In one embodiment the CEX group is a carboxyl group. . 

Soice (WO2011/017514) discloses direct loading of an eluate from Protein A to CEX without the need for a holding tank. The chromatography column in effect becomes the “holding tank” storing the product while the buffer is switched to a buffer which works as an elution buffer for the loaded column and a loading buffer for the next column down stream. 

—-Protein A – low pH virus inactivation –HIC or CEX –UF/DF –AEX –NF

Ransohoff (US2013/0260419) teaches a continous process for the purification of antibody which includes clarification, Protein A, low pH viral inactivation, HIC, UF/DF, AEX, NF.

Ransohoff (WO 2012/078677) also teaches a continous process for the purificaiton of a mAB by conctating a product solution with an affinity ligand capable of complexing with the biological product such as protein A chromatography and continously transferring the purified product to a second unit operation comprising a low pH viral inactivation, CEX, UF/DF, AEX, NF. 

—-Protein A -AEX (flow through mode) – HIC (flow through mode)

Kremer (WO 2011/098526) discloses a method for purifying proteins such as antibodies using a single oeprational unit comprising both an AEX and HIC which are serially connected and which are operated in flow through mode. 

–Protein A-Multi-Mode (capto adhere)

Duthe (WO 2013/075849) disclsoes two chromatographic steps; one affinity and one multi-modal where all buffers can consist of Bis Tris in combination with NaCL, acetic acid and water. In one embodiment the affinity chromatography is Protein A. In one embodiment th eeluent obtained a the end of the first chromatographic step is passed over the second chromatography column with no treatment such as pH adjustment, buffer exchange or dilution.

—-Protein A — MM (capto adhere) — AEX

Duthe (US 14/889,397, published as US 2016/0083454) discloses a 3 step chromatography scheme of Protein A – MM (capto adhere) and AEX where each of the buffers used in the steps is Bis Tris. and the method is run in a closed system without buffer exchange or dilution. 

Mixed Mode – HA: Gagnon (US2009/0270596) discloses a method of purifying antibodies using mixed mode chromatography and HA where the first and second column tubes comprise inlets and outlets, wherein the outlet of the first column tube connects to the inlet of the second column tube such that the first column tube is in fluid communication with the second column tube. 

HIC-AEX: Urthaler (US2004/0002081) discloses a process for isolating a polynucleotide using a chromatographic secparation process characterized in that the process uses a combination of two steps selected from HIC and AEX and the two chromatographic steps may be carried out independently or, preferably in a single operation. To achieve a single operation, the device for the first step is directly linked to the chromatographic device for the second step by connecting the outlet of the first with the inlet of the second one. 

2 Unit Operation with Same or Similar Ligands (see also simulated moving bed chromatography)

Bryntessofn (WO 2008/153472) teach a simulated moving bed process wehrefor IgG adsorption to MabSelect affinity resin known as a 3C-PCC system where at least 3 substantially identically packed chromatography columns are operated in a semi-continous manner. Accordingy to the method, the absorbent is washed after binding of target compound and then the outlet of wash liquid from said adsorbent is subsequently passed onto another adsorbent for binding of target compound removed by said washing. Consequently, every time the feed is redicted to another adsorbent in the system, said adsorbent will have a small amount of target compound bound already. This allows for an efficient recovery of target compound. Chromatography resins may be Protein A based resins, IMAC resins, HIC resins, thiophilic resinds, IEX resins, multimodal resins. 

Mahajan (“improving affinity chromatography resin efficiency using semi-continous chromatography” J Chromatography A, 1227 (2012) 154-162 discloses operating Protein A affinity chromatography using several multiple smaller column in line. 3 Hi-Trap MabSelectu Sure columns were loaded individually at differen times until the 70% breakthrough point was achieves. The harvested cell culture fluid (HCCF) with unbound protein from the column was then laoded onoto the next column to capture the MAb, preventing any protein loss. 

Skudas (US 9,149,738) disclsoes a process of continous affinity chromatography by using at least three capture separation units have the same chromatography matrix such as Protein or IEX which are connected so that liquid can flow from one unit to the next and the last to the first, feeding the sample on a the first unit so that the first unit is at least part of the loading time in fluid communication with the next unit so that target molecules not bound to the frist unit can bind to the next unit and at the same time washing, eluting and/or reequilibreating one separation unit different from the separation unit that is being loaded and form the one in fluid communication, switching the feed to the enxt seapration unit and feeding the sample on the next unit so taht while the sample is being loaded on the next unit, this unit is at least part of the loading time in fluid communication with the unit after the next so taht target molecuels not bound can bind to the unit after the next and repeating the steps. In one embodim ent, the purified target molecuels that are lueted are further subjected to at least one flwo through purificaiton step.   See continous chromatography

Van Alstine (WO2011/035282) discloses the separation of complex mixtures of macromolecules such as proteins including polymer modified molecules using two or more columns in series whihc contain capture media with similar ligands such as IEX, IMAC, MM or Protein Affinity resin such that adsorption mobile phase flow through from the first column is used as adsorption mobile phase for the second or flow on column. For example, the macromolecules to be separated could be polymer modified proteins (e.g., PEGylated proteins). The two media used could have similar ligands and ligand density per unit area but different pore size distribution. The different proteins to be separated including modified proteins might differ in size such that the larger molcules are excluded from the first media  and thus flow through the first media. 

 Axial Flow Chromatography (AFC): Traditionally, chromatography systems use a cylindrically-shaped vessel. Buffers and samples containing products are pumped onto the top of the column and collected from the bottom. However, during processing, high pressure drops may occur, which sometimes prevent operation at high flow rates expecially for large pilot-scale or manufacturing-scale systems. (Demirci, 2012, 255-262, American J. of Biochemistry and Biotechnology).

Radial Flow Chromatography (RFC): was developed to reduce pressure drops in the system while maintaining high flow rates. In RFC, the column is cylindrical like AFC above, but the flow of the mobile phase passes from the outside of the cylindier through the resin bed to the inside of the cylinder. The packed bed is supported between two cylindrical fits and the gap between the frits represents the bed height or olumn lenght. The outer frit is the column inlet and thus the sample initially has a large area of stationary phase with witch to interact. The cross-sectional area of packing decreases progressively as the solute moves toward the center. The greatest advantage of RFC is oepration with less pressure drop under the same flow rates. (Demirci, 2012, 255-262, American J. of Biochemistry and Biotechnology).

Elimination of Intermediate Filtration Processes

Chen (WO2009/045897) teaches a protein purification system which includes one or more columns each with an adsorbent where a culture including a protein is flowed through the first column to provide a first eluate that is flowed the second conolumn without prior difiltration or ultra filtration of the first eluate. The process provides ebenefits for manufacture plant automation in that the process is capable of being operated on a high throughput and continous basis. The process is cpaable of handling high titer concentrations of about 5 g/L or even about 50 g/L.  One exemplified scheme is Pro-A-MEP (mercapto-ethylpyridine, which is a hydrophobic charge induction resin)-CHT (ceraminic hydroxyapatite)/AEX. For immobilization of the ligand an avidin/biotin chemsitry can be used.