A buffer is a solution that resists changes in pH by the action of its acid-base conjugate components. Buffers act by releasing hydrogen ions when a base is added and absorbing hydrogen ions when acid is added, with the overall effect of keeping [H+] relatively constant. The key buffer in human blood is an acid-base pair consisting of carbonic acid (acid) and bicarbonate (base). These two substances interact in a pair of reversible reactions. First, CO2 and H2O join to form carbonic acid (H2CO3), which in a second reaction dissociates to yield bicarbonate ion (HCO3-) and H+. If some acid or other substance adds H+ to blood, the HCO3- acts as a base and removes the excess H+ by forming H2CO3. Similarly, if a basic substance removes H+ form the blood, H2CO3 dissociates, releasing more H+ into the blood. The forward and reverse reactions that interconvert H2CO3 and HCO3- thus stabilize the pH of blood.

Buffers consist of a weak acid (HA) and its conjugate base (A-) or a weak base and its conjugate acid. Weak acids and bases do not completely dissociate in water, and instead exist in solution as a equilibrium of dissociated and undissociated species. Consider acetic acid. In solution, acetate ions, hydrogen ions and undissociated acetic acid exist in equilibrium. This system is capable of absorbing either H+ or OH- due to the reversible nature of the dissociation of acetic acid (HAc). HAc can release H+ to neutralize OH and form water. The conjugate base, A-, can react with H+ ions added to the system to produce acetic acid. In this way, pH is maintained as the three species constantly adjust to restore equilibrium. (Chapter 5, Buffers for biochemical Reactions” Protocls & Applications Guide, 2004-212, Promega Corporation).

All buffers have an optimal pH range over which they are able to moderate changes in hydrogen ion concentration. This range is a factor of the dissociation constant of the acid of the buffer (Ka) and is generally defined as the pKa (-logKa) value plus or minus one pH unit. pKa can be determined uising the Henderson-Hasselbalch equation. (Chapter 5, Buffers for biochemical Reactions” Protocls & Applications Guide, 2004-212, Promega Corporation).

Preparation of Buffers

In general, solvents are mixed in terms of their relative volumes (V/V) or relative weights (w/w). Buffer solutions are prepared by combining a weak acid and its sale (e.g., sodium salt) or a weak base and its salt. Commonly used preparation methods include 1) dripping the acid (or base) into an aqueous solution of the salt while measuring the pH value with a pH meter and 2) rendering the acid as an aqueous solution with the same concentration as the salt’s aqueous solution and mixing the two while measuring the pH value with a pH meters ((“Tips for practical HPLC analysis –Separation Know-how – Shimadzu LC World Talk Special Issue Volume 2, which also lists how to prepare many different buffer solutions).

Commonly Used Buffers

Bis -tris methane: (Bis-Tris or BTM): is an organic tertiary aminewhich is an effective buffer between pH 5.8-7.2. It has a chemical formuls C8H19NO5.

Tris (tris(hydroxymethyl)aminomethane: is an organic compound with the formula C4H11NO3 and commonlyly used in buffer solutions such as in TAE and TBE buffers. It contains a primary amineand thus undergoes the reactions assocaited with typical amines such as condensations with aldehydes.

Conductivity of Commonly Used Buffers

Sodium citrate:

(Falkenstein (US 14/934866 published as US 2107/006814) lists the conductivity for various concentrations of buffer containing sodium citrate in addition to sodium citrate and NaCL.

Sodium Free Buffers:

No Salts: (acid/base pair and sodium salt conjugate base)

Goklen (WO 2012/135415) discloses a method for purifying a protein using simplified sodium chloride-free buffer system that includes two components (acid and base pairs) for appropirate solution pH control and a thrid component for ionic strenght content where the thrid component is the sodium salt conjugate base. For edample, goklen discloses equilibrating a Protein A with a buffer that includes 55 mM tris base, 45 mM acetic acid at about pH 7.5, (b) adsorbing the protein from the contaminated solution to the Protein A and (c) removing contaminants by washing with a wash buffer that includes 55 mM Tris Base, 45 mM acetic acid, 300 mM sodium acetate at about pH 7.5 and recovering the protein with an elution buffer that includes 1.8 mM sodium acetate, 28.2 mM aetic acid, at about pH 3.6 wherein all the buffers are made without the addition of NaCl. Other chromatographies include AEX and CEX.

As to prokaryotic systems for the production of antibodies see Ypatent Health

Definitions:

Chaotropic agent: refers to a compound that in a sutiable concentraiton is capable of chaing the spatila configuration or conformation of polypetpides through alterations at the surface so as to render the polypeptide soluble in an aqueous medium. The concentraiton of chaotropic agent will directly affect its strength and effectiveness. A strongly denaturing chaotropic solution contains a chaotropic agent in large concentrations which will unfold a polypeptide present in the solution effectively eliminating the prtoeins secondary structure. The unfolding will be relatively extensive, but reversible. Examples of chaotropic agents include guanidine hydrochloride, urea and hydroxides such as sodium or potassium hydroxide. 

Reducing agent: refers to a compound that in a suitable concentraiton in aqueous solution maintins free sulfydryl gorups so that the intra or intermolecular disulfide bonds are chemically disrupted. Examples of reducing agents include dithiothreitol (DTT), dithioerythritol (DTE), beta-mercaptoethanol (BME), cystein, cysteamine, thioglycolate, glutathione and sodium borohydride. Pizarro (US 2008/0125580)

When the host cell is a Gram negative prokarytoic cell, newly synthesized protein is often isolated form the periplasmic space. Preferred Gram negative prokaryotic ccells used for periplasmic epxression are Escherichia coli strains. (Slough, US 15/538, 282, published as US 20170342105). Pizarro (US 2008/0125580)

Introduction:

Inclusion bodies are particles found in both the cytoplasmic and perioplasmic spaces of E. coli during high-level expression of heterologous protein. 

Inclusion bodies obtained by cytosolic microbial overexpression of a recombinant protein are large, spherical particles. Upon high-level expression, the inclusion bodies may span the entire diameter of an E. coli cell. Because of their refractile character, inclusion bodies can be observed directly in the living host cell by phase constrast microscopy. The term refractile body is therefore often used as a synoym for inclusion body. (Rudolkp “in vitro folding of inclusion body proteins” The FASEB J, 10(1)q, pp. 49-56, 2017). 

Inclusion body pellets consist of varying proportions of impurities (e.g., proteins, DNA and lipids) of host origin which may need to be purified prior to refolding the target protein. Purification of protein in the denautred state can be done as by RP-HPLC, gel filtration and ion exchange chromatography. (Mukhopadhyay, “inclusion Bodies and Purificaiton of Proteins in Biologically Active Forms” Advances in Biochemical Engineering/Biotechnology, 56, 1997).

The general strategy used to recover active protein from inclusion bodies involves three steps: inclusion body isolation and washing, solubiliation of the aggregated protein and refolding of the solubilized protein (Clark, “Protein refolding for industrial prcoesses” Current Opinion in Biotechnology, 2001, 12: 202-207). 

Isolation/Solubilization of Inclusion Bodies: 

Inclusion bodies must be separated from the host cells and solubilization of the inclusion bodies. There are many ways by which living organisms are disrupted to release intraceullar products. The outer membrane of E. coli si composed of lipoproteins, lipoolysaccharides, and proteins wehreas phospholipids and proteins are two components of the inner membrane. In addition, the periplasmic space contains peptidoglycan. These multilayer lipd protein glycan complexes contribute to the regidity of the E. coli cell wall and thus inclusion bodies produced in E. coli cannot be isolated satisfactrily by techniques like ultrasonication, osmatoic shock and eznymatic lysis. Although ultrasonic disintegration is the most widely used techniques, the disruption of bacterial cells is lower than 100%. The best technique for the disintegration of E. coli cells is high pressure homogeneization, although it suffers from the drawback of contamination of target protein with the cellular components.  (Mukhopadhyay, “inclusion Bodies and Purificaiton of Proteins in Biologically Active Forms” Advances in Biochemical Engineering/Biotechnology, 56, 1997). 

A variety of methods may be used to solubilize inclusion bodies; however, the choice of solubilizing agent can greatly impact the subsequent refolding step and the cost of the overall process. (Clark, “Protein refolding for industrial prcoesses” Current Opinion in Biotechnology, 2001, 12: 202-207).

Use of Chaotropic, Denaturing and Reducing agents:

After their isolation, inclusion bodies are commonly solubilized by high concentrations of chatropic agents such as guanidimium hydrochloride or urea. (Felipe, Microbial Cell Facotires, 2004, 3:11) 

–Guanidine+EDTA+DTE+Tris: 

Often functional heterologous proteins from E. coli or other bacteria are isolated form inclusion bodies and require solubiliation using strong denaturants. During the solubilization step, as is well known in the art, a reducing agent must be present to separate disulfide bonds. An exemplary buffer with a reducing agent is 0.1M Tris pH8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol). (Dimitrov, US2010/0316641). 

Buchner (Anal Biochem, 205(2), 1992, pp. 263-70) discloses solubiliation of an inclusion body pellet in the presence of strong denaturants and reducing agents in a buffer consisting of 0.1 M Tris, pH 8, 6 M guanidine, 2 mM EDTA, 0.3M DTE. The solution is incubated for at least 2 h at room temeprature. 

Refolding Buffers; Conditions: 

To obtain native conformation, the polypeptide chain has to be refolded into correct secondary and tertiary structures, which are further stabilized by the formation of intramolecular disulfide bonds. In order to facilitate this, the concentration of denaturant can be reduced to a level at which intramolecular stabilizing forms (e.g., hydrogen bonding, hdrophobic interactions) exist, buffers containing oxidizing agents are applied and the concentration of denatured protein in the refolding buffer is maintained low to avoid intermolecular aggregation. Dialysis and diafiltration membrane of defined MW cut-off are used for the exchange of buffer. In refolding by single stage dilution, the refolding buffer is typically composed of 50 mmol/l tris-HCL containing different redox systems such as reduced/oxidzed glutathione or atmospheric oxygen. Air oxidaition is catalyzed by incorporation 01.-1.0 umoll Cu+2 ions in the refolding buffer. The formation of disulfide bonds in air oxidation has been found more effective in the presence of trace amounts of thiol agents (e.g., 2-mercaptoethanol, DTT or cystein). In vitro refolding efficiencies have been imporved by incorporating various additives in the refolding buffer such as amino acids (e.g., arginine) , sugars, neutral surfactants, and polymers. (Mukhopadhyay, “inclusion Bodies and Purificaiton of Proteins in Biologically Active Forms” Advances in Biochemical Engineering/Biotechnology, 56, 1997). 

The formation of incorrectly folded species and in particular aggregates is usually the casue of decreased renaturation yields. A efficient strategy to suppress aggregation is the inhibition of the intermolecular interactions leading to aggregation by the use of low molecular weight additives. The most commonly used low molecular weight additives are L-arginine, low concentraitons of denaturants such as urea and detergents. (Clark, “Protein refolding for industrial prcoesses” Current Opinion in Biotechnology, 2001, 12: 202-207).

Use of Arginine, EDTA, Oxidized Glutathione (GSSG):

Atkinson (US2009/0291428) discloses that for protein refolding, solubilized inclusion body protein was added dropwise over 36 h into refolding buffer of 400 mM L-arginine-HCL, 2 mM EDTA, 0.02 M ethanolamine, 0.5 mM oxidized glutathione and 5 mM reduced glutathione 

Buchner (Anal Biochem, 205(2), 1992, pp. 263-70) discloses renaturation by a rapid 100 fold dilution of the denatured and reduced protein into refolding buffer consisting of 0.1M Tris, pH 8.0, 0.5 M L-arginine, 8 mM GSSG, 2 mM EDTA. The concentration of inclusion body protein during refolding was 30 ug/ml. The samples were incubated at 10C. 

Dechavanne (Protein Expression and Purificaiton 75 (2011) 192-203) discloses that a wide range of chemical additive have been described to prevetn or reduce misfolding of proteins during the refolding process. L-Arginine is one of the most widely used additives for refolding proteins and at a concentration between 0.2-1M fequently increases the renaturation yield. PEG is another additive frequently used to improve correct structure formation by inhibiting aggregation. A low concentration of EDTA is frequently recommended to prevetn metal-catalyzed air oxidation of cysteines, which could result in wrong disulfide bridge formation. Addition of a mixture of reduced (RS-) and oxidized (RSSR) forms of low MW thiol reagents such as glutathione (GSH/GSSG), cysteine/cystine, cystamine-cysteamine and (DTTODTT) usually provies the appropriate redox potential to allow formation and reshuffling of correct disilfide bridges. 

An exemplary refolding/renaturation buffer is 0.1MTris, pH 8, 0.5 M L-arginine, 8 mM oxidized glutathione (GSSG) and 2 mM EDTA. (Dimitrov, US2010/0316641) 

Linke (US2013/0202626) discloses the refolding of CAT-8015 initiated by a 10 fold dilution of the clarified and concentrated inclusion body filtrate into refolding buffer (50 mM ethanolamine, 1 M arginine, 2 mM EDTA, 0.91 mM oxidized glutathione, pH 9.5). The refold solution was maintained at 2-8C for 48-72 hours with continous mixing. 

O’Connor, (US13/880424, published as US2013/0273607) discloses refolding of an anti-CD22 antibody fragment using a refolding buffer having about 2070 mM ethanolamine, about 0.5-.2M arginine, aobut 0.5-3mM EDTA and about 0.5-1.5 mMGSSG. Surprisingly reducing the GSSG concentration from 9.1 mM to 0.91 mMreduced glutathione adduct i the product, while not impacting the yield of the antibody fragment. The refolding buffer is integrated into a purificaiton scheme of first solubilzing a mixture having the recombinant protein and inclusion bodies, clarifying the recombinant protein from the mixture with one or more depth filters, recovering the calirifed protein, concentrating the clarified recombinnt protein and refolding the clarified recombinant protein in the protein refolding buffer.

–Solubilization Buffer – Refolding Buffer:

Gal (US 2009/0042248) discloses preparation of MASP fragmetns which includes solubilizaiton of the inclusion bodies and tranfering the solubilized prtoein which contains a solubilization agent such as GuHCl or urea, prferably in a buffer comprising 6 M GuHCl. fragmetns into a refolding buffer at pH higher than 7, perferably between 8.5 and 10.5. 

Pizarro (US 2008/0125580) discloses a method for recovering reoflded recombinant prteoins from cell culture that includs solubilizng the protein in a first buffered solution with pH greater than 9 such as 9-10 using a first chatropic agent and then refolding the folubilized protein in a second buffer between pH 9 and 11 with a second chatropic agent, two or mroe reducing agents and addition of air or oxygen for such time and under such conditions that refolding of the recombinant protein occurs. In one emobdiment, the frist and second buffers also incues arginine and the chaotropic agent is 1 M urea. 

Sinner (US 2011/0237509) disclsoes purifying HGH from E. coli by obtaining the hGH containing includsion bodies by lysing (Tris/salt lysis buffer) to release the inclusion bodies, centrifugation, suspending them in a Tris buffer and solubilziing the inclusion body fraction with 8 mM followed by refolding the hGH containing granules in the presence of a phospahte buffer by dialysis at at pH about 80 and temeprature about 2-10C and then ioslating a precipitate of the refolded hGH udner appropirate pH of about 6.8-7.00. 

—-Alkaline pH (solubilization) – Lower but alkaline pH (refolding) + Glutathione

Hollander, (US 14/766,848, published as US 10,065,987; see also US 16/050,417, published as US 2019/0077828) discloses a method of isolating an Fc protein having at least one disulfide bond such as an antibody or fibronectin from inclusion bodies (IBs) using an alkaline pH whithout using a significant amount of denaturing and/or reducing  agent. The method includes suspending a denatured protein in a suspension solution and then using a solubilization buffer having a pH in the range of 10.5-13 (Arginine can be used to buffer the pH in this range) to obtain a solubilized denatured proteins and then using a refold buffer having a pH in the range of 9-11 to obtain the refolding protein. In other words, the method using a strongly alkline pH, followed by incubation at reduced pH. The refold buffer may contain Arginine or another positively charged amino acid to maintin the pH in the range of 9-11. In some embodiments in which the protein to be refolded includes one or more disulfide bonds, the refold buffer may also include an oxidizing agent such as glutatione to facilitate the formation of disulfide bonds. Following the refolding the pHmay be reduced to a lower value such as 6-8. 

Gonzalez-Villasenor (WO 03/102013) discloses a method of solubilizing and recovering a target peptide such as an antibody from a host organisms by (i) disrupting the host cell to produce a lystate, recovering the lysate precipitate containing the polypeptide (iii) resuspending the lysate precipitate in a denaturant free, non-buffered solubilzation solution. In one embodiment, the alkaline solubilizaiton solutions has a pH of about 11.2. The inclusion bodies are solubilized by stirring gently at room temperature (between about 20-25C) between about 20-40 minutes. After 20-30 min of stirring at room temperature, the pH of the preparation drops to pH between about 9.5-10.2s the protein solubilizes and interacts with the OH-ions of the NaOH in the solubilizaiton solution. The preparation is then centrifuged for about 15 minutes and the sueprnatanst provies the target peptide with a final pH of between about 9.5-10.3 

–Use of glutathione during chromatography process

Slough, US 15/538, 282, published as US 20170342105) discloses purifiction of proteins such as antibodies, and particularly a Fab bound to PEG, where throughout the chromatography scheme the protein/antibody is miantained in the presence of a reducing agent such as glutathione, beta-mercaptoethanol, beta-mercaptoehylamine, dithiothreitol, tris (2-carboxyethyl)phosphine, and cysteine. 

 Arginine, CHES, EDTA, Urea: 

Pizarro (US2008/0125580) discloses isolating a recombinant protein from a prokaryotic cell culture, solubilzing in a first buffered solution which can comprise 1M Urea, 300 M arginine, 10 mM CHES, 5 mM EDTA, pH 11, pH greater than 9, having a first chaotropic agent, refolding the solubilized protein in a second buffered solution pH 9-11 comprising 1 M Urea, 15 mM cysteine, 2 mMDTT, 100 mM arginine, 10 mM CHES, 5 mM EDTA, pH 10, having a second chaotropic agent, two or more reducing agents and addition of air or oxygen for such time and under such conditions that refolding of the recombinant protein occurs, and recovering the refolded protein. 

CuCl2+H2O: 

Burnie (US2007/02021160 discloses that for refolding the inclusion body solution was made with refolding buffer and CuCl2, 2H2O, The preparation was incubated for 48 h at 2-8C under strong agitation. The inclusion bodies were filtered and then filter sterilised with a depth filter. 

Purification of Non-antibody Proteins from Inclusion Bodies

Human Insulin:

Hartman (US 6,001,604) discloses a prcoess for the production of recombinant human insulin by folding a proinsulin hybrid polypeptide where the refolding includes incubating the polypeptide at about 4-37C for a period fo about 1-30 hours at a pH of about 8.5-12.

Human Growth Hormone:

Patra (Protein Expression and Purificaiton 18, 182-192 (2000) discloses large scale purification of r-hGH from inclusion bodies by solubilization in 16 ml of 100 mM Tris buffer, pH 12.5, containing 2 M urea. The oslubilized r-hGH was diluted 5 times with Milli-Q water and the pH was broght down to 8.5 by adding 1 N HCL. 

See also “purification schemes” under Antibody Purification.   See also “mixed mode chromatography” under Chromatography

Classification: 

HCIC is a type of mixed mode chromatographic process in which the protein of interest in the mixture binds to a dual mode (i.e., there is one mode for binding and another mode for elution), ionizable ligand through mild hydrophobic interactions in the absence of added salts (Arunakumari, US 2012/0165511 A1). Hydrophobic charge induction chromatography (HCIC) is a subset of HIC (EP2004052807). HIC is also a type of mixed mode chromatographic process in which the protein of interest in the mixture binds to a dual mode (i.e., there is one mode for binding and another mode for elution), ionizable ligand. HCI is useful for the separation of biological molecules such as proteins based on the pH dependent behavior of ionizable dual-mode ligands. At netural pH, the ligand is uncharged and binds a protein of interest via mild non-specific hydrophobic interaction. As pH is reduced during a buffer gradient, the ligand becomes positively charged and hydrophobic binding is disrupted by electrostatic charge repulsion.

HCIC was developed for protein separation based on the use of dual mode ligands so as to combine a molecular interaction supported by a mild hydrophobic association effect in the absence of salts. When environmental pH is changed, the ligad becomes ionically charged resulting in the deorption of the protein (Guerrier Bioseparation 9: 211-221, 2000). 

Types of resins: 

HCIC employs a hydrophobic charge induction chromatography material which comprises chromatographical function groups which can in one pH range form hydrophobic bonds to the substance to be separated and which are charged either positively or negatively in other pH ranges (i.e., HCIC uses ionizable hydrophobic groups as chromatographical functional group). A hydrophobic charge induction chromatogrophy resin contains a ligand which has the combined properties of thiophilic effect (i.e., utilizing the properties of thiophilic chromatography), hydrophbicity and an ionizable group for its separation capability. The term “thiophilic” refers to the selectivity that proteins have for sulfone groups that lie in close proximity to theioether groups. Thiophilic absorption chromatogrpahy is based on electron donor-acceptor properties and is distinct from chromatography based on hydrophobicity.

MEP-HyperCel®: Mercapto-ethyl-pyridine (MEP Hypercell) is a common HIC resin. MEP HyperCel, carries a ligand dervied from 4-mercaptoethylpyridine (4-MEP). Such nitrogen hererogycles have been shown to have particular selectivity for immunoglobulins. The pK, of 4-MEP is 4.8. Thus, under near neutral or alkaline conditions, the ligand is uncharged and behaves much like a phenyl group to bind antibody by hydrophobic interaction. However, the distinct structure of the ligand provides the antibody selective characteritics for which this sorbent was designed. The pKa of the ligand also provides sorbent characteristics that support antibody recovery under mild conditions. Desorption is achieved by reducing the pH of the mobile phase to confer a net positive charge on both ligand and antibody. At pH values in the range of 4-4.5, the ligand and most antibodies carry a predominant positive charge. Thus, desorption is achieved under conditions significantly milder than those typically employed during affintiy chromatorgpahy on protien A sorbents (Schwartz J. Chromatog, 908 (2001) 251-263).

Conditions/Parameters:

General Principles: 

Generally the polypeptide is bound to the hydrophobic charge induction material under neutral pH conditions and recovered afterwards by the generation of charge repulsion by a change of the pH value. Thus at one pH range, the ligand is predominantly uncharged and binds a protein of interest via mild non-specific hydrophobic interaction. As pH is reduced, the ligand acquires charge and hydrophobic binding is disrupted by electrostatic charge repulsion towards the solute due to the pH shift.

Thus HCIC is based on the pH dependent behavior of dual-mode ionizable ligands. When pH of the mobile phase is reduced, both the ionizalbe ligand and the target molecule take on a net positive electrostatic charge. The resulting electrostatic charge repulsion overcomes hydrophobic binding interactions, and desorption of the biomolecule occur (Schwartz, J. Chromatogr. 908 (2001) 251-263).

The pKa of the ligand for HCIC is carefully chosen so that the ligand will be uncharged at neutral pH where absorption can be achieved solely by hydrophobic interactions at physiological conditions. By adjustment of the mobile phase pH, the ligand can take on charges with the same polarity as the protein, and elution is achieved by electrostatic repulsion. Although the adsorption process in HCIC is similar to that in single mode hydrophobic interaction chromatography (HIC), the charge facilitated elution mode can offer extra advantages of salt independent adsorption and facile elution.

Burton (5,945,520) discloses mixed mode resins which have a hydrophobic character at the pH of binding of the target compound and a hydrophilic and/or electrostatic character at the pH of desorption of the target compound from the resin. These resins overcome the problem of typical mixed mode chromatographic resins of the prior art where binding efficiencies of less hydrophobic target compounds to the resin is not very high unless a high salt concentration is employed.The resins described comprise a solid support matrix and ionizable ligands that have an ionizable functionality and a spacer arm covalently attaching the ligand to the solid support matrix. The ionizable functionality is partically electrostatically charged at the pH of binding of the compound to the resin and is either further charged or charged at a different polarity at the pH of desorption of the compound from the resin.

Purification of particular Proteins

Antibodies:  See also Antibody purification –HCIC

MEP Hypercell was specifically designed to capture immunoglobulins. In contrast to HI chromatography, adsorption of antibodies form cell culture supernatants on HCIC resin is accomplished without the need of any pH or ionic strength adjustment. At neutral pH, hydrophobic capture of antibody occurs by both an aliphatic-hydrophobic spacer and a neutral (uncarged) pyridine ring. Once th pH is lowered from pH 7.2 to pH 4, the pyridine ring in the resin and the bound antibodybecome positively charged, resulting in charge repulsion. The immunoglobulins detaches and elutes form the column (WO2005/014621).

Non-immunoglobulin molecules:

Although MEP Hypercell is specifically designed for the purificaiton of antibodies, Schwarts-Burd (WO 2005/014621 A1) discloses that HCIC resins such as MEP Hypcercell can also be used for purifying non-immunoglobulin protines which have Ig-like domains such as IL-18BP. For washing out unbound contaminants, solutions which have the same pH as the loaded material (e.g., PBS 7.2) and/or neutral pH and/or even acidic (e.g., pH 3-6.8) can be used. Elution is carried out with organic solvent such as isopropyl alcohol, propylene glycols or polyalcohols such as glycerol, and polyethylene glycol (e.g., between about 25-50%). In contrast to immunogolbulins, IL-18BP is highly acidic (isoelectri point of about 3) and pH of 4.5 is not acidic enough to induce IL-18BP to become positively charged. Thus, in order to elute the IL-18BP from the column, an organix solvent such as propylene glycol, which can weaken hydrophobic interaction between proteins can be used.

In combination with other types of purification techniques

HCIC-AEX: 

Kinoshita (WO02/15927) discloses a method purifying a high mannose glucocerebrosidase (hmGCB) which includes HCIC in combination with one or more ion exchange steps such as AEX.

Daniel (US7,138,262) teaching a method of purifying high mannose glucocerbrosidase (hmGCB) by subjecting the hmGCB to HCIC such as MEP Hypercel and further purification by at least one ion exchange chromatography step such as AEX or CEX.

HCIC-CEX:

Arunakumari (US8,129,508) discloses a method of purifying a target protein from a mixure by subjecting the mixture to a CEX and a HCIC in either order wherein the elution buffer for the HCIC or CEX elutes the host cell proteins differentially in relation to the target protein and wherein the tailing of the ABS280 peak of the target protein is not collected at less than 20% agove the baseline as measured by ABS280 relative to the maximum of the peak height. 

Daniel (US7,138,262) teaching a method of purifying high mannose glucocerbrosidase (hmGCB) by subjecting the hmGCB to HCIC such as MEP Hypercel and further purification by at least one ion exchange chromatography step such as AEX or CEX.

Kinoshita (WO02/15927) discloses a method purifying a high mannose glucocerebrosidase (hmGCB) which includes HCIC in combination with one or more ion exchange steps such as CEX. 

See also bi-specific antibody production 

Bispecific antibodies can bind two different antigens. IgG type antibodies have two binding sites with different variable regions. An IgG variable region is made up of a variable light-chain sequence (VL) and a variable heavy chain sequence (VH). The light chains (LCs) of common LC antibodies are identical for both variable regions, leaving the heavy chain (HC) for generating different specificites. Thus, recombinant host cells for production of common LC bispecific antibodies carry genes for both hCs, with different specificites (A and B) along with one LC gene. A, B and the light cahins are expressed independently in those host cells, which then assemble them into three IgG types –AA, AB, and BB –for secretyion into a culture. By purefuly random assembly, the three types should be produced in a ratio of 1:2:1 (AA, AB, and BB). Genetic engineering methods can modify heavy cahin Fc regions such that A and B are assembled prereably, thus reducing the formation of AA and BB. (Bakker “purifying common light-chain bispecific antibodies: a twin-column, countercurrent chromatography platform process) BioProcess International 11(5): 36-44 (2013). 

Bispecific antibodies offer opportunities for increasing specificity, broadening potency, and utilizing novel mechanisms of action that cannot be acheived with traditional mAb. Cross-linking two different receptors using a bispecific antibody to inhibit a signalling pathway has shown utility and in one example a cell surface tyrosine phosphatase was recruited into an EgE receptor complex to decrease activity of the phosphorylated IgE receptor. This approach was more effective than blocking the ligand binding site because inhibition of signalting by the bispecific antibody occured even in the presence of high cocnetrations of ligand. The use of bispecific antibodies has also shown application in recruting cytotoxic T cells where T cell activation was acheived in proximity to tumor cells by the bispecific antibody binding receptors simultanteously on the two different cell types. (Scheer, US 2013/0017200)

Paticular Types of Bi-specific Antibodies that are Purified

kappa-lamda antibodies:

–Using KappaSelect and LambdaSelect Resins:

An appealing bispecific format for therapeuti use is an unmodified human IgG. This format would share stability, pharmacokinetic and other sought-after drug-like properties of therapeutic mAbs, while enabling novel modes of action. An approach previously treid was to co-express the H and L chains of two different antibodies in a single cell. However, the random assembly of the fourt chains resulted in a complex mixture of ten molecules, substantially challenging development from yield, cost and purity perspectives. A more selective approach is to use antibodies that share a common chain such that concomitant expression of two heavy and a common light chain in the same cell results in a mixture containing only two mAbs and one BiAb. However, the downstream purificaiton of the BiAb from this mixture is challening and relies of differences between the physicochemical properites (for example, overall charge or hydrophobicity) of teh BiAb and the two mAbs. A completely different approach is to create in vitro display libraries with a common heavy chain that are used to select against two different antigens. This allows the idolation of candidates with different target specificties that share the same heavy chain but carry either a kappa or lambda light chains. Three different chains (one heavy and two light) are then co-epxressed in a single cell to generate a mixutre containing two mAbs speies (one kappa and one lambda) and a BiAb constaining a kappa and lambda light chain. A BiAb assembled in this manner can then be efficiently purified form the mAb species using highly selective affinity resins binding to either human kappa or lambda constant domains. Base on its structure, this fully human BiAb format is referred to a s a “kappa-lambda” body. (Fischer, “exploting light chains for the scalable generation and platform puriicaiotn of native human bispecific IgG” Nature Communications, 2015). 

—-Protein A – Kappa Select –Lambda Select

Fischer (US2014/0179547; see also US Patent 16/042,889, published as 2019/0031714 and US 2012/0184716 (producing three or more monospecific antibodies and three or more bispecific antibodies). ) discloses a method of purifying a bispecific bivalent antibody that shares the same heavy chain (antibodies bearing both a kappa and a lambda light chain) by the use of affinity chromatography such as CaptureSelect Fab Kappa and CaptureSelect Fab Lambda that specifically interacts with the Kappa or Lambda light chain constant domains constant domains. The method uses a three step procedure: (1) Protein A to capture IgG (mono and bi), (2) Kappa select capture IgG containing a Kappa light chain(s) and (3) Lambda select to capture IgG containing a Lambda light chain.

Fischer “exploiting light chains for teh scalable generation and platform purificaiton of native human bispecific IgG” Nature communications, 2014) discloses assembly and purification of kappa/lambda-bodies using a three step affinity process. First, a non-distinguishing affinity capture step was perforemd using either a protein A or Capture Select CH1 risn that binds to the Fc or CH1 doamin of human IgG, respecitvely. The second affinity-capture purificaiton using KappaSelect binds to human kappa constant region and allows for recovery of the IgGk and the kappa/lambda body, while the IgGlambda is eliminated in the flow through. Third, the kappa/lambda body is purified by a LambdaFabSelect affinity rsin that binds selectively to human lambda constant region, eliminating the IgGkappa species. 

—-Protein A- Lambda Select –Kappa Select:

Hultberg (US 2012/0156206) disclsoes purification of camelid-derived bispecific cMET antibodies using the three step purificiaotn method of ProtA sepharose to select for only properly assembled Mabs containing two variable heavy and light chainsand then Labda-select and Kappa-select baes to separate the parental Mabs from the bispecific Mabs. 

—-Differential elution of bi-specific kappa/lambda antibody:

Elson (US13/655,955, published as US 2013/0317200) discloses purification of a kappa-lamda-antibody (termed a kappalamda-body) which consists of a common IgG1 heavy chain and two different light chains that drive specificity for two independent targets. One of the light chains contains a kappa constant region while the other contains a lamda constant region. Purification of the kappa-lamda antibody can be performed by sequential binding to KappaSelect and LambdaFabSelect affinity resins which have specificity and affinity for either the kappa or lamda constant region. The kappa-lamda body binds to either KappaSelect or LambdaFab Select resins with a weaker affinity than the monospecific kappa-Mab (for KappaSelect) or monospecific lamda-MAb (for LambdaFabSelect) due to the fact that it contains only one of each light chain rather than two for the monoclonal format. In one embodiment Elson discloses  using a higher pH step elution to preferentially elute a bispecific kappa-lamda-body from a KappaSelect affinity resin over monospecific kappa-MAB which elutes at a lower pH, as the monospecific MAb presumably has a higher affinity to the resin owing to the presence of two kappa chains in the monospecific format as opposed to a single kappa chain in the kappa-lampda body. For example, after column loaiding at 10 mg/mL and wash step with 50mM Sodium Phosphate, 250 mM Sodium Chloride, pH 7.0 (5 column volumes), a pH step-elution (pH 3.0 follwed by pH 2.5 and pH 2.0) was performed using a 50mM glycine buffer adjusted to the relevant pH. The bispecific kappa-lamda body product preferentially elutes at the ihger pH step. 

–Using Mixed Mode and HIC Resins

Elson (US13/655,955, published as US 2013/0317200; see also 16/032,873, published as US 2019/0040117) also disclose reducing free light chains from the kappa-lamda antibodies using Mep HyperCel mixed mode chromatography by applying a cell culture supernatant to the resin, eluting the mAb with an acetate buffered elution buffer at pH 5.0 (eluate) and removing free light cahins which are strongly bound to the resin at pH 2.1. 

Fourque (US15/068916, published as US 2016/02646485, now US Patent No: 10,457,749; see also US Patent Application 16/601,121, published as US 2020/0181287) discloses a method for purifying a bispecific antibody having a first light chain with a kappa constant region and a second light chain with a labda constant region (kappa/lamba-body) from a mixture using a mixed mode resin such as TOYOPEARL MX-Trp 60M and HIC such as a TOYOPEARL Butyl 600M resin. 

For different H and L antibody chains (“crossMab”):

–Cation Exchange:

Neumann (US 2014/0081000) discloses that recently a new bispecific bivalent antibody form called the “CrossMab” which is a bispecific antibody having four different antibody chains, i.e., two different light chains and two different heavy chains has been described (Proc. Natl. Acad. Sci, USA 108 (2011) 11187-11192). Neumann discloses that during the expression different product related impurities such as the 3/4 antibody, an antibody that is missing one light chain, displaying a MW of about 125 kDa compared to a MW of 150 kDa for a comlet four chain antibody is formed. Neumann discloses a method for purifying the full lenght four chain antibody with CEX by applying a solution comprising a non-ionic polymer and an additive to which the antibody has been adsorbed whereby the four chain full lenght antibody in monomeric form remains adsorbed to the CEX and then recoveirng the four chain full lenght antibody in monomeric form by applying a solution comprising a non-ionic polymer such as PEG, an additive such as zwitterions, and an elution compound (see also purificaiton of antibodies by CEX). 

VH3 domain containing bispecific antibody

A subset of antibodies bind Protien A in the variable region of the heavy chain. In particular, IgGs that contain H chains derived from the VH3 gene family have been shown to belong to this fmaily. Becasue of this, on SpA-based resins, many bsAbs exhibit poor resolution of the two bidning species as well as retention of the non-bnding Fc*Fc* parental antibody. This is becasue VH binding reduces the avidity difference between the bispecific and the FcFc parental and the Fc*Fc* parental is also retained by VH binding. This pehnomenon can be resolved through the use of the alkali stabilized MabSelect SuRe ligand. This is because whereas all five domains of SpA (E, D, A, B, and C) binding antibodies via the Fc region, only domains D and E exhibit significant Fab binding. As the MabSelect SuRe ligand is a tetramer of the Z-domain, a protein-engineered verison of thenative SpA B domain, it would be expected to lack significant fab binding, and this has been demonstrated in multiple studies.  (Tustian, Biotechnol. Prog., 2018, 34(3))

–Protein A chromatography:

Haywood (US 15/566,231, published as US 2018/0100007) discloses purification of a human VH3 domain containing antibdoy such as an Fab, Fab’, F(ab’)2, Fv and scFv as well as such types of antibodies that include more than one VH3 domain such as those in the format of diabodies, tetrabodies, minibodies, and domain antibodies. using Protein A chromatography where the VH3 domain containing antibody is recovered in monomeric form.

Types of Purification Techniques for Purification of Bi-specific Antibodies

Protein A/G or Combination:

–By modifying bi-specific antibody to create differential binding

Blein (US 14/431207, published as US 2015/0239991; see also US Patent Application 16/275821, published as US 20200010568) discloses a method for the purification of a hetro-dimeric immunoglobulin from a mixture where the immunoglobulin has a modification in a H1 and/or a CH2 and/or a CH3 region to reduce or eliminate binding to Protein G, applying the mixture to Protein G and eluting the hetero-dimeric immunoglobulin from the resin. In a second embodiment, the modification is in the VH3 region and the mixture is applied to Protein A and the hetero-dimeric immunoglobulin fragment is eluted from Protein A. In one embdoiment,t he amino acid substitution is 81E or 82aS. In a third embodiment, the modification is in a first heavy chain of the hetero-dimer that reduces or eliminates binding to a first affinity reagent and there is a modification in the second heavy chain that reduces or eliminates binding to a second affinity reagent, the mixture is applied to the first affinity reagent and hetero-dimers are eluted and this eluate is applied to a second column with a second affinity reagent and the hetero-dimers are eluted. When the first affinity reagetn is Protein A, the seond is Protein G or vice versa. 

Gramer (US 2014/0303356) discloses purifcation of a heterodimeric protein from at least a first and second homodimeric proteins using Protein A/G. In one embodiment, either the first or second homodimeric protein may have been engineered/ modified so taht it does not bind Protein A/G. This facilitates separation of the homodimeric protein which does not bind to ProteinA form the hterodimeric protein. 

Igawa (EP 2522724 A1) discloses methods for purifying multi specific antibodies by altering amino acid residues in the H chain constant and/or variable region so as to result in altered protein A binding which also exhibit plasma retention comparable or longer than that of human IgG1. In one embodiment the amino acid substtiution is from the the amino aicd residues of positions 250-255, 308-317 and 430-436 in the Fc domain or antibody have chain constant region.

Lindhofer (US 5,945,311) discloses a method for producing a bi-secific antibody by providing a quadroma fused from hybridomas of which one produces a first antibody which has an affinity to the binding domain of protein A where the antibodies are of the subclass IgG1, IgG2, IgG4 or rat antibodies of the subclass IgG2c and the other hybrodoma produces a second antibody which in comparision with the first antibody has a smaller or no affinity to the binding domain of the protein A the second antibodies being rat antibodies of the subclass IgG1, IgG2a, IgG2b, IgG3 or IgG3. The quadorma culture is applied to protein A, washed and the bi-specific antibodies are eluted in a pH range 5.6-6.01 which is at least 0.5 units above the pH at which the antibodies with greater affintiy to the binding domain of prtoein A are still bonded. 

Zhang (WO 2017/034770) discloses a multispecific heterodimeric antibody that includes a first and second H chain wherein the CH3 of the first H chain includes at least two amino acid modifications positively charged amino acids  and the CH3 domain of the second H chain includes at least 2 amino acid mutations negatively charged amino acids. The increased negative charges in the short H chain CH3 domain does not bind to protein A whereas the positivley charged long chain Fc is capable of binding to protein A. As a result, short chain homodimers will flow through the column as they no longer bind to protein, the homodimers haivng two long chain will bind strongly to protein wheras the ehterodimers will be to protien A with lower affinity due to the fact that only long cahin Fc provides bining to protein A. By eluting the column with an elution solution having a pH of 4 or above, the herodimer will be eluted out, but the long chain homidmers will not be eluted out due to their stronger binding to the column. 

Yeung (WO2010/075548) discloses variant antibodies with one or more amino acid modification in the VH region that have altered binding to Staphylococcus aureus protein A and methods of using such antibodies.

—-CH3 of IgG3 in second H chain or His435Arg subsitution in CH3 region in second H chain:

Examples of the Fc region include human IgG type Fc that may be of the IgG1, IgG2, IgG3 and IgG4 isotypes. The “Fc region” refers to the hinge porition, CH2 domain and CH3 domain in an antibody. According to EU numbering by Kabat, a Human IgG class Fc region refers to the region from cysteine at position 226 to teh C temrinus or from porline at position 230 to teh C terminus. The human CH2 domain refers to positions 231-340 and the CH3 domain refers to positions 341-447. Tanaka (Chugai, Tokyo, US Patent application 16/061,454, published as US 2019/0330268)

Davis (US 8,586,713 and 2010/0331527) discloses antigen binding proteins such as bispecific antibodies that have IgG CH2 and CH3 regions with different affinities for Protein A that allows rapid isolation by differential binding of the IgG regions to Protein A. In one embodiment, differentially modified heterodimeric human IgG2 and unmodified human homodimeric IgG2 were first enriched by a bind and wash process through a protein A column and then a step gradient elution was performed. In one embodiment, the bispecific antibody includes a first and second polypeptide, the first polypeptide having from N to C terminal a first antigen binding region that binds a first antigen followed by a constant region that includes a first CH3 region of human IgG selected from IgG1, IgG2, IgG4 anda combination thereof and a second ABP that seelectively binds a second antigen followed by a constant region that includes a second CH3 region of a human IgG selected form IgG1, IgG2, IgG4 and a combination thereof wherein the second CH3 region comprises a modificaiton such as a 435R modification that reduces or elimiantes binding of the second domain to Protein A. Davis disloses that the inability of IgG3 to bind Protein A is determined by a single amino acid residue, Arg435 (EU numbeirng: Arg95 by IMGT), which corresponding position in the other IgG subclasses is occupied by a histidine residue. It is therefore possible, instead of IgG3, to use an IgG1 sequence in which His435 is mutated to Arg. This single point mtuation in IgG1 should be sufificent to create the different binding affinities. The point mutation could, in theory, be potentially immunogenic. To avoid this pitfall, a dipeptide mutation H435R/Y436F (EU numbering) can be used. The resulting sequence in the vicinity of the alteration is identical to that of IgG3 and would thus be expected to be immunoglogically invisible because there would be non non-native short peptides available for presentation to T cells. It has been reported that this double mutant still does not bind Protein A. Finally, the dipeptide mutation does not include any of the residues that form the Fc dimer interface, so it is unlikely to interfere with the formation of heterodimers. 

Shitar, (US 2007/0148165) discloses that the binding activity to protein A is decreased when His 435 in the human Igg1 heavy chain constant region is replaced with Arg derived from IgG3. 

Tustian (US 14/808,171, published as US2106/0024147) discloses methods of purifying a heterodimeric protein such as a bispecific antibody by using affinity capture such as Protein A and elution processes at a particular pH range. The mixture of multimeric proteins contains (i) a first homodimer comprising two copies of a first polypeptide, (ii) a heterodimer comprising the first polypeptide and a second polypeptide and optionally (iii) a second homodimer comprising two compies of the second polypeptide. The first and second polypepides have different affinities for the affinity matrix such that the first homodimer, the heterodimer and second homidimer can be separated on the basis of differential binding to the affinity matrix. Differential binding can be manipulated by changing the pH and/or ionic strenght. In addition, a chaotropic agent such as CaCl2 or MgCl2 can be used to enhance the elution of each dimer species.   In one embodiment the bispecific antibody contains a single common light chain and two distinct heavy chains and one of the heavy chains contains a substituted Fc sequence that reduces or eliminates binding of the Fc* to Protein A such as an Fc* sequence that contains H435R/Y436F substitutions in the CH3 domains. As a result, three products are creates; two of which are homodimeric for the heavy chains and one of which is the desired heterodimeric bispecific produc. The Fc* sequence allows selective purification of the FcFc* bispecific product on the affinity column due to intermedite bindng affinity for Protein A compared to the high avidity FcFc heavy chain homodimer or the weakly binding Fc*Fc* homodimer. 

——For increasing the Dynamic Binding Activity of bi-specific antibody:

Binding capacities include statis binding capacity (SBC) and dynamic binding capacity (DBC). SBC refers to the upper limit of the amount of polypeptides that a resin can adsorb, and DBC refers to the degree to which polypeptides can be collected wehn a polypeptide containing solution is flowing through the column. A resin having a large dynamic binding capacity allows efficient polypeptide adsorption even under high linear flow rate, and polypeptide purification can be accomplished in a short time. DBC is determined by depicting the behavior in which a continuosuly loaded protein is discharged from the column as a breakthrough curve in a chromatogram by UV monitoring using a purification device connected to a UV detector. DBC can be determined by loading a column with a resin and allowing a polypeptide containing sample solution to flowthorugh the column at a specified linear flow rate. Then, the absorbance of the eluate is measured, and DBC is determined by identifying the mass of the added polypeptide when breakthrough (BT) for a specific proportion (for example, 5%) of absorbance of the added sample solution is measured. Specifically, a calculation method when using 5% BT is (1) the load fraction (IgG concentration: P g/L) is allowed to flow through the LC  apparatus without passing it thorugh the column and the value of OD280 nm for 100% leakage (= 100% BT) is confirmed. This value is denoted as “a”. (2) the value obtained by multiplying 0.05 to a is defined as the OD280nm at 5% BT. This value is denoted as “b5%”. (3) the load fraction is allowed to flow continously through a set amount of equilibrated resin (r L), and when the OD280 nm value reaches by 5%, teh volumen of the load fraction is read form the chromatogram. This valued is denoted as “C5% L”. (4) the value obtained by the equation (P x c5%)/r is calculate as DBC5% which is the DBC at 5% BT.  DBC5%=(PxC5%)/r (unit: g/L resin). When determining DBC10% the calculation is possible by determining c10% in a similar manner  (Tanaka, Chugai, Tokyo, US Patent application 16/061,454, published as US 2019/0330268)

Tanaka (Chugai, Tokyo, US Patent application 16/061,454, published as US 2019/0330268) discloses a method for increasing the dynamic binding capacity of a bi-specific antiody for a Protein A resin which includes preparing a first polypeptide chain that binds to the Protein A resin and a second polypeptide that does not bind to the resin or shows weaker binding. In one embodiment, the Fc region includes a CH3 of IgG1, IgG2 or IgG4 and the second polypeptide of the Fc region includes a CH3 of IgG3. In one embodiment, position 435 of the first polypetpide chain is modified to be His and position 435 of the second polypetpide chain is modified to be Arg. Examples of other modifications include modifying positions 435 and 436 of the first chain to be His(H) and Tyr and positions 435 and 436 of the second chain to be Arg (R) and Phe (F). The increase in the DBC of the Fc region containing polypeptide for a Protein A resin is at least 5, 15, 20 or even 25 g/L resin or mroe when taking 5% BT as the standard. 

–Using low conductivity wash

Falkenstein (US 15/900, 449, published as US 20180186866) discloses that HCP can be reduced if the conductivity of the aqueous solution used in the wash step is low (below 0.5 mS/cm). Advantageously futher process steps can be obviated before loading hte eluate to the next chromatographi material if a low conductivity aqueous solution wash step is ued in the preceeding affintiy chromatography step. 

–Protein A – Mixed Mode (AEX or CEX) – Mixed Mode (AEX or CEX):

Giese (US Patent Applicaiton 16/221,369, published as US 2019/0256556) discloses a method of purifying a bi-specific/heterodimeric polypeptice with a multi-step chromatography method which includes affinity chromatography such as Protein A followed by two different multimodal ion exchange chromatography steps., such as a multimodal AEX followed by a multimodal CEX or vice versa. In one embodiment, a bispecific antibody against target proteins X1 and Y1 (anti-X1/anti-Y1 bispecific antibody) was assembled as follows. Each half-antibody (aX1 (knob) and a Y1 (hole) was independently subject to an affinity chromatography step using prtoein A resin.The half antibody pools obtained from the protein A chromatography step were then combined in a 1:1 molar ration, ( the pH was adjusted to pH 82. L-gltathione buffer was added). The resulting assembled pool was cooled and pH adjusted to pH 5.5 and then subjected to a multimodal CEX using Capto MMC resin in a bind and elute mode. Eluate form the multimodal CEX was then subject to multimodal AEX using Capto Adhere resin in a flow-through mode which removed residual impuriteis like DNA, host cell protein and endotxoins as well as product variants including half-antibodies, homodimers and aggregates. 

IgG-CH1 Affinity Matrix

Fischer (WO/2013/136186) discloses using two immunoglobulin CH1 heavy chain constant domain sequences that differ by at least one amino acid in a bispecific antigen-binding protein. The amino acid difference results in an improved ability to isolate the biapecific protein form homodimers beause the difference results in a differentail ability of teh CH1 domain to bind the CaptureSelect® IgG-CH1 (BAC BV) affinity reagent. According to the invention, the bispecific antibody includes a first and a second polypeptide, the first polypetpide comprising from N-temrinal to C-temrinal a first antigen binding region that selectively binds a first antigen, followed by a constant region that includes a first CH1 region of a human IgG and a second polypeptide comprising from N-temrinal to C-terminal, a second antigen-binding region that selectively binds a second antigen, followed by a constant region that includes a second CH1 reigon of a human IgG wherein the second CH1 region includes a modificaiton that reduces or eliminates binding of the second CH1 domain to the CaptureSelect IgG-CH1 affinity reagent. 

Cation Exchange

Igawa (US 2009/0263392) discloses purificaiton of bipspecific antibodies that includes modifying the amino acids present on the variable framework regions of two types of polypeptides that constitute the bispecific antibody so that they may be separated as by CEX based on their differening isoelectric points of the H chains. 

Lebanon (US 15/565,494, published as US 2018/0079797) discloses a strong cation exchange for the separation of bispecific antibodies using various linear pH gradeints witha starting buffer A: CAPS, CHES, TAPS, HEPPSO, MOPSO, MES, acetic acid, hormic acid and NaCL, pH 4 and a final buffer composed of the same agents at pH 11. 

Scheer (US 2011/0287009) disclsoes purificaiton of a bispecific antibody by weak CEX using a carboxymethyl resin with a pH gradient elution from 4.5 to 9.2 The buffer A and B consisted of sodium citrate, MES, HEPES, imidizole, Tris, CAPS and NaCL where the A buffer is adjusted to pH 4.2 with HCl and the B buffer is adjusted to pH 9.2 using NaOH. 

Hydrophobic Interaction Chromatography:

Manzke (J Immunological Methods, 1992, 208(1), pp. 65-73) discloses a method for large scale production and single step purificaiton of bispecific antibodies using HIC. 

Hydroxapatite:

–Conditions:

—-Calcium ion (NaCL or KCL) Gradients:

Bertl (US 15/048308, published as US 2016/0376304) discloses a method of purfying a bispecific antibody using HA where the bispecific antibody is eluted with a buffer having chloride ions such as one having NaCL or KCL. By increasing concetnration of sodium chloride in the presence of phosphate, the bispecific antibody was separated from unwanted species. 

—-Phosphate gradients:

Ford (J Chromatography B , 754 (2001) 427-435) disclsoes purificaotn of bispecific antibodies recognising carcinoembryonic antigen and doxorubicin using Protein A affinity chromatography and HPLC hydroxyapatite affinity chromatography. Elution was with a 60-360 mM phosphate buffer gradient. 

Tarditi (J Chromatography 599 (1992) 13-20) disclsoes isloation of bispecific monoclonal antibodies (bi MAbs) using Protein A followed by a high performance hydroxyapatite (HPHT) column. Purification was performed on the HPHT at a low rate of 0.4 ml/min using 10 mM phosphate buffer (pH 6.8) as buffer A and 350 mM phosphate buffer (pH 6.8 as buffer B). Gradients were selected after evaluating the retention times of parental MAbs. The general gradient rage was 0-80% buffer B. The next step was a linear scale-up of selected gradients. The HPHT chromatography has unique selectivity for IgG idiotypes and separated IgGs according to their net charge. As the active bi MAb secreted by one hybrid hybridoma is formed of two IgG moieties, each corresponding to half of the parental IgG, this combination will have a total net charge intermedaite between those of the parental MAbs. Thus, the biMAb will be eluted form the HPHT column with an ionic strenght intermedaite between those of the parental MAbs. 

Reduction – Oxidation –Sequential Affinity:

Cariring (“A novel redox method for rapid production of functional bi-specific antibodies for use in early pilot studies” 6(7), 2011) discloses a chemical reduction-oxidation (redox) method for the production of purified bsAbs in a fraction of the time taken by traditional hybrid hybridoma technology by using the mild reducing agent 2-mercaptoethansulfonic acid sodium salt (MESNA) followed by dialysis under oxidizing conditions in order to allow the antibodies to reform. During this reaciton a mixture of antibodies is formed, including parental antibodies and bi-specific antibody. Bi-specific antibodies are purified over two sequential affinity columns. For example. bsAbs from different species (rat/mouse hybrid bsAbs) were purified first over an anti-rat IgG and secondonly an anti-mouse IgG affinity column. Cariring demonstrated that a similar schedule could be used to make bsAbs using two different antibodies form the same species and subclass. In order to purify these bsAbs the parental antibodies were conjugated to biotin or dinitrophenol prior to reduction. Purificaiton of the bsAbs was carried out by sequential purificaiton on anti-biotin and anti-DNP affinity columns. 

Twin-Column Multicolumn Countercurrent Solvent Gradient purificaiton (MCSGP): 

NCSGP is a chromatographyic process that allows for product isoaltion with high yeild and purity. In many cases (e.g., bispecific antibody isolation), the difficulty is caused by product-related ipurities taht elute closely to the product of interest. Often in preparative batch chromatography, the overlap remains even after reasonable elution-doncition optimization. The twin-column NCSGP process can be beneficial in through internal recylcing of the impure-side fractions. Those frations containing overlapping product and impurities are washed from one column inot the other, where the product is readsorbed and conserved. In phase I, the column are interconnected, and the chromatographic fraction containing the overlapping reigon of weakly adsorbing impurites and product elutes form the upstream into the downstream column. In between those two columns, the stream is diluted inline to ensure readsorption of eluted product on the downstream column. In phase 2, the columns are disconnected and pure product elutes form the upstream column. The overlapping region of product and strongly adsorbing impurity remains in the upstream column. In parallel, feed solution, is loaded onto the downstream column. In phase 3, the columns are again interconnected, and the chromatographic fraction containing the overlapping region is washed form the upstream into the downstream column. Again, product adsorption in the downstream column is ensured by inline dilution of the stream exiting the upstream column. Ini phase 4, the columns are disconnected again, and the strongly adsorbing impuriteies are washed out of the upstream column. Taht column is tripped, cleaned and equilibrated. In parallel, weakly adsorbing impurites elute form teh downstream column as elution is initated. After this last phase, the columns switch position so that the (equilibrated) upstream column becomes the downstream column and vice versa. Phases 1-4 repeat with the columns in this reverse order. On finishing phase 4 and another column-position switch, the columns have returend to their original position, so one cycle of the twin-column process is complete.  continues to run over multiple cycles. (Bakker “purifying common light-chain bispecific antibodies: a twin-column, countercurrent chromatography platform process) BioProcess International 11(5): 36-44 (2013).

Affinity separation relies on the specific recognition between the antibody molecule and a complementary ligand. As this binding is highly specific, the use of affinity purificaiton reduces non-specific interactions, increases operational yields and facilitates the elimination of undersirable containants. (J Chromatography A, 1160 (2007) 44-55). 

Affinity chromatography is a standard purificaiton option for some proteins such as antibodies. Protein A chromatography is typically the first step and anion exchange is employed frequently as the last chromatography step. The current industry norm is to have one additional fractionation step in the process (Gagnon, p. 498). The use of a cationic exchange material versus an anionic exchange material is based on the overall charge of the protein and thus one can employ an anionic exchange step prior to the use of a cationic exchange step, or a cationic exchange step prior to the use of an anionic exchange step. (WO 2010/048192, p. 30, lines 14-15). Almost all current industrial antibody purification platforms use protein A (Spitali, WO2012/013682) . 

Specific Ligands/Types of Affinity Chromatography  See outline

For the Purification of Specific Types of Antibodies

Monoclonal antibodies: 

Protin A chromatography exploits the fact that murine IgG binds to Protein A Sepharose at pH>8 but does not bind at pH<3.0. Typically, the pH of the MAb-containing solution is adjusted to 8.5 and passed over a column of Protein A. With the MAb bound to the protein A, contaminants are washed from the column with a pH 8.5 buffer, Finally, the purified MAb is eluted by passing a pH 3.0 buffer over the column. (Profy, EP0282308)

Hahn (J. Chromatography A, 1102 (2006) 224-231) teaches purification of a cell culture supernatant containing a monoclonal antibody (method for purifying an antibody compound from a suspension) brining the suspension into contact with MabSelect Sure (Protein A derivative), a washing step with an equilibrationbuffer and elution with 0.1M glycine pH 3.0 and finally collecting the eluate fractions. 

 For Separation by Fc region: US 7820799, issued 10/26/2012, describes methods of purifying polypeptides having a Fc region such as antibodies or antibody fusions by adsorbing the polypeptides to a Fc binding agent (pH 6-8) such as Protein A/G, followed by wash to remove impurities and subsequent elution).

Godavarti (WO 2006/138553 A2) teaches methods of purifying polypeptides having an Fc region such as antibodies or antibody fusions by adsorbing the polypeptides to Protein A or Protein G, followed by a wash with a divalent cation salt buffer to remove impurities and subsequent recovery (pH 2-4).

Nanobodies:  

Beirnaert (Wo 2006/122786A2) discloses using Protein A affinity chromatography for the purification of nanobodies using PBS as running buffer and glycine for elution. Beirnaert (WO2008/142164) also teaches purificaiton of nanobides via CEX as well as purificaiton of trivalent bispecitif nanobodies fused by a Gly/Ser linker via MabSelect Xtra and subsequent purificaiton by CEX. Also disclosed is the use of IMAC (see above). Hermans (WO2008/071447A2) also disclsoes purifiction of a fusion protein comprising a nanobody using protein A. 

Commercial Viral Filters: Viresolve®

See also Purification of plasma proteins, particularly virus inactivation. 

Operating Conditions

Addition of Amino Acids:

Hongo (US 13/260419) discloses a method of removing viruses from monoclonal antiboides uisng a virus removing membrane by adding a basic amino acid such as arginine, histidine, lysine or a derivative thereof. 

Lengsfeld (US 2003/0232969) disclsoes a method for separating viruses from a proteins such as fibrinogen and factor VIII by nanofiltration by adding to the protein solution chatropic substances choses from arginine, guanidine, citrulline, urea and derviatives and salts thereof or order to decrease or prevent aggregation of the protein molecules and then filtering the solution through a filter having a pore size ranging from 15-35 nm. The addition of the chatropic substances is disclosed as overcoming the protein of using nanofilters having a pore size of 15-35 nm which is regarded as too small to free larger proteins such as fibrinogen, von Willebrand factor and Factor VIII from viruses. 

Yamaguchi (US8741600) discloses a method of separating an immunoglobulin monomer comprising filtering an immunoglobulin solution containing the monomer and an aggregate having an immunoglobuiolin concentration of 1 to 150 g/L with cross-flow filtration using an UF membrane have a MWCO of 100k or more and less than 500k and further comprising a surfactant selected from the group of lysine, alanine, cysteine, glycine, serine, proline, arginine and derviatives thereof. 

Yang (WO2004/001007) discloses a method for producing a concentrated antibody preparation that includes adding a histidine or acetate buffer at a concentraiton in the range of from about 2-48 mM and then subjecting the antibody preparation to membrane filtration, which improves filtraiton flow rate. 

Addition of surfactant:

Surfactants are a class of industrially important amphiphilic substances (possess both hydrophilic and hydrophobic parts at the same time (i.e., water attracting and water repelling parts, respectively). One of the characteristic properties of amphiphilic substances is that they tend to assemble at interfaces. They are thus often referred to as surface active agents. A surfactant molecule consists normally of an alkyl chain and a hydrophilic head group. Surfactns are categoried into 4 groups depending on the charge of the head-group: nonionic (0), anionic (-), cationic (+) and zwitterionic (+). Ann-Sofi Jonsson (J. Membrane Science, 56 (1991) 49-76)

Ultrafiltration is a very efficient process for the separation of macromolecules such as proteins. A common problem in the application of UF membranes in separations is the decline of permeate flux. Fouling phenomena frequenly are observed due to solute accumulation at the membrane solution interfact and solute adsorption onto membrane pores. The use of surfactants such as nionic, anionic and their combinations has been proved very successful for membrane pretreatment. For example, fouling is reduced in UF of BSA using membranes pretreated with anionic and mixed anionic-nonionic surfactant solutions. Xiarchos “Polymeric ultrafiltration membranes and surfactants” Separation & amp; Purification Reviews, 2003. 

Ann-Sofi Jonsson (J. Membrane Science, 56 (1991) 49-76) discloses the influence of a nonionic (Tri-Ton X-100), two anionic (potassium oleate and sodium dodecylbenzensulphonate) and a cationic (hexadecyltrimethylammonium bromide) surfactant on untrafiltraiton membranes with respect to commercial membranes of polysulphone, poly(vinylidene fluoride) and cellulose acetate. The flu reductions of the hydrophobic membranes were found to be much more pronounced that the flux variations of the hydrophilic membranes. Both the material and the MWCO were found to influence the performance of the hydrophobic membranes. 

Brown, (US 14/007,610, published as US 2014/0309403) discloses a method of reducing fouling of an UF in the purification of a protein from virus particles by adding a non-ionic sufactant such as polysorbate 20, Triton X-100, Triton X-405, lauromacrogol and polysorbate or a surfantant such as polysorbate or a non-ionic agent such as polyethylene glycol, a cellulose derivative, arginine and a dextran.

Chugai (WO2002/013859 (in Chinese) discloses a method of inhibiting an antibody containing solution during UF from coagulating or becoming turbid by ading to the antibody containing solution a surfactant such as polysorbate 20/80. 

Rosenblatt US6,773,600 see also US 2003/0230532) discloses using a non-ionic surfacant during viral reduction using size exclusion nanofiltration for purificaiton of large proteinaceous biomolecules such as antibodies which allows efficient flowthorugh, minimal yield loss and no significant change in the immunoglobulin characterization aggregate level or stability.

Van Holten (US6,096,872) discloses purificaiton of immunoglobulins such as anti-D immunoglobulin substantially free of vrius using nanofiltration in a high ionic strenght buffer with an excipient such as nonionic detergents with polyoxyethlene or sugar head groups, lysopholipids and bile salts. Most preferred are nonionic polyoxyethlene detgernts such as polysorbates, PLURONICS, polyoxyethylene-polypropylene polymers or co-polymers, Brij, Sterox-AJ, Tritons and Tweens. Most preferred is polysorbate 80. 

Low concentration of acetate or histidine buffer (of from about 2 MM to about 48 mM) shown to stabilize antibody preparation during concentration by membrane filtration, lowering the viscosity of the antibody solution and suppressing aggregation (WO 2004/001007). 

 Ph and Conductivity

Takeda (US2006/0142549A1) discloses a method for removing impurities from a protein such as an antibody by forming the protein-containing sample into an aqueous solution of low conducitvity and a pH equal to or lower than the isoelectric poit of the protein and then removing the impurities by filtration. In some embodiments the low conductivity has an ionic strenght of 0 to 0.2 mM and a pH equal or greater than 2.0. 

Transmembrane pressure (TMP) or UF Pressure 

The term “transmembrane pressure (TMP)) denotes the pressure which drives the fluid to filtrate accross an ultrafiltration membrane. The value of TMP can be calculated as: TMP=Pfee + Pretentate)/2-permeate where the “feed pressure” denotes the pressue applied to the inlet of an UF, “retentate pressure” denotes the pressure applied to teh outlet of an UF and “permeate pressure” denotes the pressure applied to the permeate side of the UF. TMP is an average of the feed pressure and the retentate pressure in the case where the permeate side is open in the TFF equipment. The value of pressure is sually given in terms of “bar” or “MPa” or “psi”. Lau (US14/241567). 

In TFF, the driving force (transmembrane pressure or TMP) is the difference between the average of the membrane feed pressure (P1) and the retentate pressure (P2) minus the permeate pressure (P3).  TMP=(P1 + P2)/2 – P3. Schick (WO02/00331)

–Typical TMPs:

Antoniou discloses a process for removing protein aggregates using TFF at TMP of about 1 and about 10 psi, preferably between about 1 and about 4 psi (US 6,365,395 B1).

Couto (WO 2004/076695A1) discloses a method for separating molecules of interest such as antibodies using TFF. In a preferred embodiment, fat, casein miscelles and bacteria are removed from a transgenic milk feedstream. Couto further teaches that two important variable involved in TFF are TMP and CF.

Cui discloses using TFF with 0.3 bar (“removal of protein aggregates by ultrafiltration, Z.F. Cui).

–Devices for monitoring optimal TMP

Schick (WO02/00331) discloses a dvice that can be used to maintin a substantially constant TMP by varying filter inlet pressure in accordance with varying level of resistance to flow (increase in fluid viscosity). This is done using pressure sensors which read inlet pressure )P1 membrane feed pressure) , retentate outlet pressure (P2 retentate pressure) and filtrate pressure (P3 permeate pressure). Given that the TMP=(P1+P2)/2-P3, appropriate adjustments can be made using valves which are associated with each pressure sensor. For example, modifications can be made to modify pump speed and/or to modify the size of the vale openings so as to modify the TMP. P1 (inlet pressure) is a variable that is preimarily dependnt on the pump rate, viscosity of the lquid being pumped and the physical dimensions of the device. 

Schilog (WO 02/00331) discloses a filtration device which contain maintain a substantially constant trans-membrane pressure which includes pressure sensors to read the pressure at the inelt, retentate outlet and filtrate outlet. 

Temperature: 

–Variable TMPs and CFF:

—-Variable depending on Protein Concentration or Feed Pressure:

Bolton (WO2010/111378) discloses a method of generating a highly concentrated protein solution using an initial feed pressure at least about 5 psig and the maximum feed pressure limit of the device and recirculating the first protein solution thorugh the UF device. When oeprating at a fixed feed flow rate, the viscosity and feed pressure will increase as the proten concenrates. When the feed pressure reaches the limit of the device, the feed flow rate can be decreased to maintain the feed pressure near the maxium value. (#68). 

Hepbildikler discloses a method for removing immunoglobulin aggregates using TFF at constant delta p of 3.0 bar and constant TMP of 0.6 bar (US 12/744089). Hepbildikler also teaches a method for concentrating an immunoglobulin solution by TFF in that the transmembrane pressure and the cross-flow are variable and changed during the filtration process accoding to the concentration of the immunoglobulin. In some embodiments the transmembrane pressure and cross-flow are 1.5 bar and 80 m./min, 0.85 bar and 150 ml/min and 0.85 bar and 130 ml/min (US Patent application 12/668,661).

Hepbildikler also discloses a method for the concentration of solutions containing recombinantly produced immunoglobulins where the TMP and cross-flow vary based on the concentration of the antibody (WO 2009/010269A1).

Lau (US14/241567, published as US Patent 9630988; see also 15/461,874, published as US 2017/0204435) discloses a method where a feed flow rate is maintained at a high flow rate until an optimal protein concentration and is then reduced to a lower value to continue further concentration. For example, concentration is performed at a feed flow rate equal or greater than 200 LMH until the retentate solution is concentration to a protein concentration greater than 200 g/L, where a feed pressure builds up to 85-100% of the specified maximum feed pressure of an UF, then further concentration is continued at a feed flow rate equal or less than 120 LMH. 

(Rosenberg, J. Membrane Science 342 (2009) 50-59) discloses an optimized UF concentration method for the production of highly concentrated mAb solutions up to 140 mg/ml by varying TMP and cross-flow conditions systematically depending on the concentration in the retentate.

Schick (EP1623752) discloses a method and system for spearation of pharmaceutical liquies which involves automatically increasing pump rate over time until a maximum selected parameter such as pressure is achieved at which time the system automatically switches to for example a constant pressure filtration mode. For example, a constant pump rate of 15 ml/min was implemented until a backpressure of 20 psi was reached at which point the processor controlled pump unit switched automatically to a constant pressure delivery by modulating the pump ouput in order ot maintain the 20 psi pressure level. 

Winter (US2006/0051347, see also WO2006/031560A2) discloses a process for concentrating proteins including an ultrafiltering, diafiltering and second ultrafiltering sequence at elevated temperature such as above about 30C.

High Performance tangential flow filtration (HPTFF): is where the protein separations are carried out on the basis of both size and charge (US 2003/0229212). HPTFF is thus a two-dimensional purification method which can separate biomolecules with the same molecular weight. It is even possible to retain one biomolecule while passing a larger molecular weight species through the membrane. The degree of polarization increases with increasing concentration of retained solute in the feed, and can lead to a number of seemingly anomalous or unpredictable effects in real systems. For example, under highly polarized condtions, filtration rates may increase only slightly with increasing pressure, in contrast to unpolarized conditions, where filtration rates are usually linear with pressure. Use of a more open, higher flux membrane may not increase the filtration rate, because the polarized layer is providing the limiting resistance to filtration. The situation is further complicated by interactions between retained and eluted solutes. (WO 2005/091801).

HPTFF exploits a number of different stategies to achieve high resolution separations including (1) proper choice of pH and ionic strenght to maximize differences in the hydrodynamic volume of the product and impurity, (2) use of electrically charged membranes to enhance the rentation of like charge protines (3) operation in the pressure dependent regime to maximize the selectivity and (4) use of a diafiltration mode to wash impurities through the membrane (Cheang, J. Membrane Science 231 (2004) 159-167)

Parameters

The success of HPTFF is based on several important factors such as operating pressure, flux and selectivity (Szena “Advances in Colloid and Interface Science 145, 2009 1-22. 

Knowledge of the isoelectric point (pI) of the desired molecule of interest is the main factor in HPTFF. This will then dictate membrane setup and the intrinsic charge profile of the membrane, pore size and flow characteristics. For example, Perrault (WO 2005/091801) discloses that the isoelectric point of human alphafetoprotein is about 5 and the pI of the membrane is 7. In order to maximize the retention of human alphafetoprotein, the pH of the buffer solution was chosesn to be between 6-6.5. Under these conditions, both the membrane and recombinant alphafetoprotein will have a negative charge and repel each other.

Tangential Flow Filtration (TFF or crossflow filtration) refers to a filtration process where the feed stream passes parallel to the membrane face as one portion passes through the membrane (permeate) while the remainder (retentate) is recirculated back to the feed reservoir. In other words, the sample mixture circulates across the top of the membrane while applied pressure causes certain solutes and small molecules to pass through the membrane. The filtration membrane has a pore size with a certain cut off value.  In TFF, a pressure differential across the membrane causes fluid and filterable solutes (whose molecular weight is smaller than that of the membranes or behaves like so, such as globular proteins) to flow through the filter. This can be conducted as a continous flow process, since the solution is passed repeatedly over the membrane while the fluid that passes through the filter is continually drawn off into a separate circuit. The flow of solution accross the membrane during TFF helps prevent a gel of aggregated molecules form forming on the surface of the membrane. As a result, the flux rate for TFF drops off much more slowly as filtration proceeds than occurs during DFF. 

TFF is preferred when large amounts of polymer and molecule are to be recovered as TFF is less subject to clogging or fouling than NF methods (US 8,362,217).

 Types of TFF Filters

Examples of different filter modules include hollow fibre modules, spiral wound modules, tubular modules and plate modules. If desirable, TFF can be used to exchange the bufer in which the protein of interest is solubilized into another buffer that is more suitable for binding onto a chromatography resin.

System Confingurations:

Kevin (EP 2583744) disclsoes a TFF system that provides for a plurality of fluidly-interconnected filtration modules where each module is configured to route received feed material and deluent adjacent a filter to provide permeate and retentate. At least one of the modules has a permeate withdrawl flo line for withdrawing permeate from the syste. A plurality of the modules have a permeate flow line configured for returning permeate back to an inlet side of a same or a preceding module within the system. The system permits enhanced control of collections and purificaitons of premeate produces, retentate produces or both. Each filter can includes an ultrafiltration membrane and the system can be a tangential flow filtration system. 

Teshner (US 16/188,839, published as US 2019/0085064) discloses and UF/DF system )DF is performed withthe same membrane as UF) which includes where in order to recover the complete residual protein in the system, the post-wash of teh first larger UF ystem is done with at ealst 2 imtes teh dead volume in re-circulation mode to assure that all protein is washed out. Then the post-wash of the first UF system is concentrated to a protein concentraiton of at least 22% w/v iwth a seocnd UF/DF system equipped with the same type of membrane which is dimensioned a tenth or less of teh first one. The post-wash concetnrate is added to the bulk solution. 

 Operating Parameters (See also operating parameters for filtration)

There are two important variables involved in all tangential flow devices: the transmembrane pressure (TMP) and the crossflow velocity (CF). The TMP is the force that actually pushes molecules through the pores of the filter. The crossflow velocity is the flow rate of the solution across the membrane. It provides the force that sweeps away larger molecules that can clog the membrane thereby reducing the effectivenss of the process. In practice a fluid feedstream is pumped from the sample feed container source across the membrane surface (crossflow) in the filter and back into the sample feed container as the retentate. Backpressue appplied to the retentate tube by a clamp creates a transmembrane pressure which drives molecules smaller than the membrane pores through the filter and into the filtrate (or permeate) fraction. The crossflow sweeps larger molecules, which are retained on the surface of the membrane, back to the feed as retentate. The primary objective for the successful implementation of a TFF protocol is to optimize the TMP and CF so that the largest volumen of sample can be filtered without creating membrane clogging gell. A TMP is “substantially contast” if the TMP does not increase or decrease along the lenght of the membrane generally by more than about 10 psi of the average TMP and preferably by more than about 5 psi. As to the level of the TMP throught the filtration, the TMP is held constant or is lowered during the concentraiton step to retain selectivity at higher concentrations.(US 2005/091801).

Buffers: 

–Low concentration of acetate or histidine buffer (of from about 2 MM to about 48 mM) shown to stabilize antibody preparation during concentration by membrane filtration, lowering the viscosity of the antibody solution and suppressing aggregation (WO 2004/001007). 

–Adjustment of Concentration Prior to TFF: Hepbildikler (US13395893) discloses a TFF/UF method for concentrating an immunoglobulin solution by adjusting a first concentration of the buffer to a second concentration whereby the second concentration is calculated with one mathematical equation if the buffer is a cation/neutral pair such as histidine or with a second equation if the buffer is a neutral/anion pair such as acetate. The method is based on unequal partioning of compounds during diafiltration and concentration in excipient concentrations, pH and conducitvity values which are significantly different fro those of the diafiltration buffer used at the start of the prcoess. This influences stability of the final product. Although such changes which occur during TFF can be solved by restock/diluting with a buffer solution after concentration, the invention solves the problem by using a defined addition/reduction of solute concentration prior to the TFF in order to correct the concentration changes. For example, ine one embodiment in case of histidine buffer (solute) and adjustment at about pH 5.0 to 29.6 mM and 60 MM histidine, respectively, before TFF is required to achieve a predifined histine buffer concentration of 20 mM and 46 mM histidine respectively after TFF in the concentration of IgG1 and IgG4 to 215 mg/ml. 

The following are commonly used modes of filtration.

1. Direct Flow Filtration (DFF): or “dead-end” filtration is where the feed stream is applied perpendicular to the membrane face, attempting to pass 100% of the fluid through the membrane.

2. Tangential Flow Filtration (TFF or crossflow filtration): also known as crossflow filtration is where the feed stream passes parallel to the membrane face as one portion passes through the membrane (permeate) while the other remainder (retentate) is recirculated back to the feed resrvoire. See outline

TFF: has been used in the separation of blood components (US 4,888,115), beer solutions (US 4,644,056), immunoglobulins from milk or colostrum (US 4,420,398) and bacterial enzymes from cell debris (Quirk, 1984, Enzyme Microb. Technol, 6(5): 201.

(a) High Performance tangential flow filtration (HPTFF): See outline

Ultrafiltration is a form of membrane filtration in which hydrostatic pressure forces a liquid against a semipermeable membrane. Suspended solids and solutes of high molcular weight are retained, while water and low molecular weight solutes pass through the membrane. Ultrafiltration is typcially characterized by a membrane pore size between 1 and 1000 kDa and operating pressures between 0.101 and 10 bar. Membranes are characterized by their nominal molecular weight limit (NMWL) also referred to as their molecular weight cut-off (MWCO). This represents their ability to retain molecules larger than those of a given size. An ultrafiltration membrane’s MWCO or NMWL is expressed in kilodaltons and abbreviated as K (i.e., 10k, 30, etc).

Different types of UF membranes are commercially available made of ceramic, semi-conducting or polymeric materials, etc (WO 2009/139624). 

Ultrafiltration can be performed with tangential flow filtration (TFF mode) (US 2006/0051347). Ultrafiltration is normally carried out in TFF mode, in which fluid passes across the filer, tangential to the plane of the filter surface. (Liu, “Recovery and purification process development for monoclonal antibody production” mAbs,  2:5: 480-499 (2010)  See TFF right hand column. 

Selection of UF

UF membrane selection is based on several parameters such as the following:

(1) separation performance (retention): The most critical paramter here is determiend by NMWL (normal molecular weight limit). Membranes are available in numerous NMWLs.

(2) processing rate (flux): Membrane flux, as measured by flow rate per unit area determines how much membrane is required to process the feed stream in the desired time period. Higher flux results when the actual pore size closely resembers the desired NMLWL cut off. Maximizing membrane pore size allows process fluids to pass more quickly, thus increasing flux.

(3) Mechanical integrity: a Strong mechanically intact membrane will perform consistently for a long period of time.

Particular Schemes with UF (as to antibody purification using UF see antibody section)

Cordle (EP0363896A2) discloses an ultrafiltration process for enriching and concentrating a desired protein from a fluid by using metallic oxide ultrafiltration membranes and a pH shift. The process uses the same ultrafiltration membrane for two successive ultrafiltrations, shifting the pH between the two successive ultrafiltrations, in order to remove materials of lower molecular weight or higher molecular weight than the desired protein on the basis of charge as well as size. The process makes feasible the fractionation of immunoglobulins such as IgG and involves two factors; 1) the metallic membrane produces a high rejection of IgG at a pH of 5.8 or lower but a much lower rejection of IgG at a pH of 6.5 or greater and 2) the metallic membrane allows continual passage of smaller MW proteins at any pH level. In one embodment, a fluid is exposed to a first metallic oxide UF membrane at a pH which is below the isoelectric point of a slected protein whereby a retentate containing the protein is produced, then subjecting the retentate to a second UF at a pH above the isoelectric point of the protein whereby a permeate containing the selected protein is produced. 

Pre-filtration (reduces aggregation of the UF)

–Activated carbon filters:

Martin (EP1577319A1) teaches a prefilter positioned upstream of other purification systmes such as affinity chromatography and UF which is used to reduce the presence of non-specific binding (NSB) species that enter the system. Suitable agents for the prefilter include activated carbon, fumed slica, glass etc.

–Depth Filters:

Siwak (US2003/0201229; see also US7,118,675) discloses selectively removing plugging constitutions  from a biomolecule containing solution in a normal flow (NFF) filtration process before vrial filtration using UF. In the first step one or more layers of adsorptive depth filters are used to remove plugging constituents in a normal flow filtration mode of operation. The plugging constituent free stream can then be filed through one or more UF membranes to retain virus particles and allow passage of a plugging constituent free biomol;ecule.

–Negatively charged medium:

Kazlov (US2013/0056415) discloses using a negatively charged microporous filtraiton meidum for use as prefiltration membranes for slectively removing protein aggregates form a protein solution