Introduction:

In this mode of chromatography, the product of interest is loaded beyond the dynamic binding capacity of the chromatography material for the product, thus referred to as overload. 

In displacement mode chromatography which is covered in a different section on the site, the sample mixture in a carrier solvent that has a low affinity for the stationary pahse is loaded and the bound components are displaced by a solution of displacer which has a greater affintiy for the stationary pahse than any of the sample components. In “sample displacement mode” during loading of the sample there is competition among the sample components for the hydrophobic adsorption sites of the stationary phase. The more hydrophobic components compete more successfully for these sites than the more hydrophilic components, which are displaced and eluted from the column. Finally, the adsorbed components are eluted with an aqueous organic eluent (Veeraragavan J. Chromatography, 541 (1991) 207-220.

Particular Modes of Overload Chromatography:

Gradient mode elution in combination with Displacement Separation

Martin (WO2006/116064) discloses a method for separating a target comound by injecting an overloaded amount of the sample into a chromatographic conduit thereby forming a displacement train in which the target compound is substantially separated and then flowing a solvent causing an eluent of the at least one target compound.

Brown (WO 2010/019148) (see also below under IEX) discloses purifying a polypetpide from a contaminant which includes the steps of passing the composition through an ion exchange membrane where the polypeptide and the membrane have opposite charge at operating conditions comprised of a buffer having a pH sufficiently distinct form the pI of the polypetpide to enhance the carge of the polypeptide and a low ionic strengh effective to prevetn the shielding of charges by buffer ions which cause the membrane to bind the polypeptide and the at elast one contaminant and recoveirng the purified polypetpide form the effluent. In one emboidment the compoistion is passed thorugh a CEX membrane at operating conditions that include a buffer having a pH of about 1-5 pH units below the pI of the polypetpide and a conductivity of less than about 40 mS/cm which casues the membrane to bind the polypeptide and the at least one contaminant and recvoeirng the purifed polypeptide form the effluent. In anotehr alterrnative, the composition is passed through an AEX membrane at operating condtiions that include a buffer having a pH of aobut 1-5 pH units above the pI of the polypetpide and a condtuvitiy of less than or equal to about 40 mS/cm, which causes the membrane to bind the polypetpide and the at least one contaminant and recvovering the purifed polypetpide from the effluent. Accordingly, the purificaiton of plypeptide/antibody using the IEX membrane is in indigenous protein displacement mode. This is accomplished by operating at low inonic strengh and at a pH above the mAb pI during anion exchange and below the mAb pI during cation exchange. At these conditions, an attractive force is established between membrane and mAb resulting in product adsorption. Feedstream loading continues beyond the breakthrough capacity and the membrane effluent is collected in a purified form.

Particular Types of Media Run in Overload Mode:

In General:

Ayers (WO02/28194) discloses the separation of CMP (caseinomacropeptide, also known as GMP and CDP) which is rich in sialic acid by containing an anion exchanger with an excess of feedstock under conditions where the sialic acid rich glyco-CMP is selectively adsorbed and eluting the sialic acid rich glyco-CMP. Preferably the anion exchange is overloaded with respect to CMP so that non-sialic acid bearing aglyco-CMP which may initially bind to the exchanger is displaced by the more acidic sialic acid rich glyco-CMP.

Gilar (WO2006/116064) discloses separation of one or more compounds using displacement separation (overloaded sample condition) in combination with gradient mode elution. Additional embodiments also utilize a mass spectrometer or other suitable detector to provide a good resolution of eluting compounds to permit accurate observation of boundaries between the eluting sample compounds. In one embodiment, the method includes injecting an overload amount of the sample into a chromatographic conduit, causing an eluent including the at least one target compound to elute from an exit of the conduit.

Liu (US 2013/0079272) teaches overload chromatography using CEX, MM and IEX with a loading densitiy between about 150 g/L and about 2000 g/L

Nadarajah, US14/355818 (US2014/0301977)) discloses methods for purifying a polypeptide by a) loading the composition onto a chromatography material such a mixed mode material, an anion exchange material, a hydrophobic interaction material and an affintiy material in an amount in excess of the DBC of the material for the polypeptide, b) eluting the polypeptide from the chromatography material under conditions wherein the one or more contaminants remain bound to the material and pooling factions comprising the polypeptide in the chromatography eflluent form steps a and b. In some embodiments the partition coefficient of the chromatography material for the polypeptide is greater than 30 or greater than 100. 

Zarbis-Papastoitsis (WO/2012/030512) discloses a process for isolating a protein of interest from a sample which includes applying the sample to a chromatography material under conditions that highly bind the protein of interest but where the amount of protein of interest exceeds the binding capacity of the chromatography material such that a substantial amount of the protein of interest does not adsorb to the material and collecting at least a porition of the sample that does not adsorb to the material to thereby isolate the protein of interest.

Ion Exchange:

–AEX

Ahn (US2007/0264710) discloses separation of hTPO analogues according to their sialic acid content by loading eluted fractions obtained by reverse phased chromatography onto an AEX, washing the column and then eluting with 10 mM sodium phosphate buffer along with a 0-0.3 M sodium chloride gradient. The hTPO analogues with low sialic acid content were found at the fractions eluted with below 0.15 M sodium chloride whereas the analogues with high sialic acid content were found at the fractions eluted with 0.15 – 0.3 M sodium chloride. 

Ladiwala (US13/885446 and WO2012/068134) discloses an “overload bind and elute” mode of chromatography operation where a product such as an Fc-fusion protein is contacted with a chromatography medium at a concentration which exceeds the static of dynamic binding capacity of the medium. During the overlaod and bind step, the product having a selected characteristic (such as a high overall net-negative charge or a high sialic acid content) binds to the support while other impurities having less of the characteristic is excluded. Under an overloaded bind and elute process, higher sialytated glycoforms having higer net negative chage and binding affinity to TMAE HiCap compelted effectively for binding sites with the lower affinity lesser sialyated and non-sialyated glycoforms, thus displacing these lower affinity species.Subsequent to the overlaod and bind step, the target it eluted and recovered. In one embodiment, a strong anion exchange chromatography (TMAE HiCap) was used for separating Fc-fusion protein glycoforms. The more sialylated glycoforms were preferentially absorbed onto the TMAE HiCap, while the lower sialyated and non-sialyated glycoforms flowed through. Subsequently, the sialyated glycoforms were eluted form the column.

—-AEX-CEX (overloaded):

Pliura (US5,439,591 and 5,545,328) discloses a process for purifying hemoglobuilinwhere in the first stage the sample is applied to an anion exchange such that Hb and contaminants having lower affinity for the column are displaced such that certain contaminants which are more acidic than the Hb species are absorbed onto the solid phase and are thereby separated form the Hb which appears in the eluent along with the more acidic contaminants. Then, in a second stage, the eluent is applied to a CEX until saturation loading is execeeded so as to create an overload condition in the column so that at the overload condition, the Hb species is eluted from the column by displacement by the contaminants of greater affinity under the chosen conditions and the hb is thereby collected in the eluent at a very high state of purity.  A typical procedure starts with adult human red blood cells which are filtrated to remove leukocytes, then lysed to extract the hemoglobin, washed to remove plasma proteins and RBC proteins present, diafiltered and concentrated. The first stage of the process (anionic stage) is run at a high pH under conitions of low ionic concentration so that the desired Hb species will initially bind to the column medium along with all other speces but as the feed of impure solution continues, those species of greater acidity which show greater affinity than Hb gradually displace the Hb and more basic contaminants form the column. Eventually, an overload of the column is achieved so that all the Hb and more basic contaminants are displaced leaving the more acidic contaminant bound to the medium. The second stage is where the eluent is also run in overload mode. The desired Hb and other more basic impurities are bound in overloaded amounts. The contaminants of higher affinity (those more basic than Hb) displace the Hb from the column and Hb is thus eluted and recovered.

CEX-MM/HIC

Allen and Davies (WO2009135656) discloses cation exchange in displacement mode followed by by either a thiophilic interaction step or HIC step or mixed mode chroamtgoraphy step.

Liu (US2013/0079272; see also Liu, “Exploration of overloaded cation exchange chromatography for monoclonal antibody purification, 2011, 39, 1218) teaches methods for purifying a polypeptide from a composition using an overloaded CEX. For example, the methods comprise loading onto a CEX at a loading denstiy of greater than about 150 g/L of cation exchange material. The methods may further comprsies loading onto a mixed mode material.

Myers “out with the old and in with the new: Mixed-mode chromatography as an alternative to anion exchange as a polishing step”  BIOT, March 27, 2011) discloses a continous process of mixed mode chromatography in flow through mode with DEX chromatography in overlaoded mode. High throughput of over 300 g antibody/L of resin can be achieved.

Ion Exchange Membranes:

Brown, (14/365,449, published as US 10/364268; see also US Patent Application 16/433,763, published as US 2020/0102346) discloses overloading an ion exchange membrane such that at least one contaminant remains bound to the membrane while the polypeptide of interest is primarily in the effluent and collecting the effluent from the IEX compirsing the popypeptide of interest and subjecting this polypeptide to a purification step of similar charge as the previous step. For membrane Cation exchange chromatography run in overlaod mode, the pH of the load material is adjusted to aobut 1-5 pH units below the pI of the antibody and the conductivity of the load material is adjjusted to less than about 40 mS/cm. Becasue the pH of the load is less than the pI of the antibody, the antibody (which has becopositively charged) will not flow thorugh initially. Rather, the antibody will be electrostatically bound to the negative funcitonal groups of the cation exchanger. Since the pI of many contaminants such as host cell proteins that elute with the antibody during protien A affinity chromtogrpahy is only slightly different from the pI of the antibody, these contaminants liek the basic antibodies will also bind to the membrane. But when run in overlaod mode, the pH and conductivity conditions induce charge with minimal ionic shielding and competitive adsorption occurs with the contaminants preferentially binding and displacing the antibody form the membrane. For membrane anion exchange chromatogrpahy run in overload mode, the pH of the laod material is adjusted to about 1-5 pH units above the pI of the antibody and the conductivity of the laod mateiral is adjsuted to less than about 40 mS/cm. Becasue the pH of the laod is greater than the pI of the antibody, the antibody (which has become negatively charge) will not flow thorugh initially. Rather the antibody will electrostatically bind to the positive functional grups of the anion exchanger. This is becasue the antiobdy (negative) and membrane (positive) have opposite charge. Since the pI of many contaminants such as host cell proteins like CHOP that elute with the antibody druing protien A affinity chromatography is only slightly different from the pI fo the antibody, these contaminants, like the “acidic” antibodies, will also bind to the membrane. When the membrane AEX is run in overlaod mode at pH and conductivity conditions that induce charge with minimal ionic sielding, competitive adsorption occurs and the contaminants preferentially bind to the membrane and displace the antibody form the membrane. Brown teaches that unlike applications that use IEX membrane primarily as a sole purificaiton step ofr final polishing step, the membranes can be used to protect a similarly charged ion exchange column and thus reduce or eliminate the impurities going onto the column. The impurities can also displace the polypeptide/antibody so taht it eventually makes it way onto the column. The membranes can be used either continusouly or non-continueously with the column. 

Brown (Biotechnol. Appl. Biochem (2010) 56, 59-70) disclsoes overloading cation and anion exchange membranes in a normal flow mode resulting in retention of impurities and breakthrough of purified antibody. Althouh some amount of the product also binds to the membrane, yields of greater than 99% were acheived by loading more than 3 kg mAb/l membrane. 

Brown (WO 2010/019148) discloses purifying a polypetpide from a contaminant which includes the steps of passing the composition through an ion exchange membrane where the polypeptide and the membrane have opposite charge at operating conditions comprised of a buffer having a pH sufficiently distinct form the pI of the polypetpide to enhance the carge of the polypeptide and a low ionic strengh effective to prevetn the shielding of charges by buffer ions which cause the membrane to bind the polypeptide and the at elast one contaminant and recoveirng the purified polypetpide form the effluent. In one emboidment the compoistion is passed thorugh a CEX membrane at operating conditions that include a buffer having a pH of about 1-5 pH units below the pI of the polypetpide and a conductivity of less than about 40 mS/cm which casues the membrane to bind the polypeptide and the at least one contaminant and recvoeirng the purifed polypeptide form the effluent. In anotehr alterrnative, the composition is passed through an AEX membrane at operating condtiions that include a buffer having a pH of aobut 1-5 pH units above the pI of the polypetpide and a condtuvitiy of less than or equal to about 40 mS/cm, which causes the membrane to bind the polypetpide and the at least one contaminant and recvovering the purifed polypetpide from the effluent. Accordingly, the purificaiton of plypeptide/antibody using the IEX membrane is in indigenous protein displacement mode. This is accomplished by operating at low inonic strengh and at a pH above the mAb pI during anion exchange and below the mAb pI during cation exchange. At these conditions, an attractive force is established between membrane and mAb resulting in product adsorption. Feedstream loading continues beyond the breakthrough capacity and the membrane effluent is collected in a purified form. 

–AEX membrane:

Brown (US 10,927,144) discloses a method for purifying an antibody from CHO cell culture composition be subjecting the composition to AEX perforemd in idigenous protein displacement mode by laoding the composition onto an AEX membrane, wehrein the atnibody and the membrane have opposite charge as a result of the laoded composition haivng a pH of 1-5 pH units above the pI of the antibody and a conducitvity of less than 40 mS/cm which prevents the shelding of the charges by buffer ions and casue the membrane to bind both the antibody and CHOP and continuing to load the membrane beyond the breakthrough capacity to a load denstiy of at leats 1000 g/L wherein the boudn antibody is displaced form the membrane by the binding of CHOP. 

—-AEX Membrane-AEX:

Brown (14/365,449, published as US 10/364268; see also US Patent Application 16/433,763, published as US 2020/0102346) discloses that for membrane anion exchange chromatogrpahy run in overload mode, the pH of the load material is adusted to about 1-5 pH units above the pI of the antibody and the conductivity of the load material is adjusted to less than or equal to about 40 mS/cm. Becasue the pH of the load is greater than the pI of the antibody, the antibody (which has become negatively charged) will not flow through initially. Rather, the antibody will be electrostatically bound to the positive functional gruops of the anion exchanger. This is becaseu the antibody (negative) and membrane (positive) have opposite charge. Since the pI of many contamininants, eg., host cell prtoeins, aminoglycoside antibiotics sucha s gentamicine and ionic polymer additives such as polyethyleneimine (PEI) that elute with the antibody during protien A is only slightly different form the pI of the antibody, these contaminants like the acidic antibodies will also bind to the membrne. Accordingly, when the membrane AEX is run in overlaod mode, at pH and conductivity conditions that induce charge with minimal ionic shielding, competitive adsorption occurs and the contaminants preferentailly bind to the membrane or otehrwise effectively displace the antibody from the membrane, allowing the antibody to elute form the matrix or flwo through after binding and be recovered in the effluent.

–CEX Membrane:

Brown (US 10,927,144) discloses a method for purifying an antibody from CHO cell culture composition be subjecting the composition to CEX perforemd in indigenous protein displacement mode by loading the composition onto an CEX membrane, wehrein the antibody and the membrane have opposite charge as a result of the laoded composition haivng a pH of 1-5 pH units below the pI of the antibody and a conducitvity of less than 40 mS/cm which prevents the shelding of the charges by buffer ions and casue the membrane to bind both the antibody and CHOP and continuing to load the membrane beyond the breakthrough capacity to a load denstiy of at leats 2000 g/L wherein the boudn antibody is displaced form the membrane by the binding of CHOP.

–—CEX Membrane-CEX:

Brown (14/365,449, published as US 10/364268; see also US Patent Application 16/433,763, published as US 2020/0102346) discloses that for membrane cation exchange chromatogrpahy run in overlaod mode, the pH of the laod material is adjsuted to about 1-5 pH units below the pI of the antibody and the conductivity of the load material is adjusted to less than or equal to about 40 mS/cm. Becasue the pH of the load is less than the pI of the antibdoy, the antibody (which has become postivively charged) will not flow through initially. Rather, the antibody will be electrostatically bound to the negative functional gruops of the cation exchanger. This is becaseu the antibody (postive) and membrane (negative) have opposite charge. Since the pI of many contamininants, eg., host cell prtoeins, aminoglycoside antibiotics sucha s gentamicine and ionic polymer additives such as polyethyleneimine (PEI) that elute with the antibody during protien A is only slightly different form the pI of the antibody, these contaminants like the basic antibodies will also bind to the membrne. Accordingly, when the membrane CEX is run in overlaod mode, at pH and conductivity conditions that induce charge with minimal ionic shielding, competitive adsorption occurs and the contaminants preferentailly bind to the membrane or otehrwise effectively displace the antibody form the membrane, allowing the antibody to elute form the matrix or flwo through after binding and be recovered in the effluent.