Fluidized bed cchromatography involves the use of adsorbetn particles dispersed in a liquid medium. (Gagnon, J. Chromatogr. A 1221 (2012) 57-70).

Expanded bed chromatogrpahy (EBA): EBA is a type of stabilized fluidized bed chromatography. (Arpanaei, “improved expanded bed chromatography systems” thesis, 2007). EBA has found widespread appliations in the large scale purification of proteins from mammalian cell and microbial feedstocks in industrial bioprocessing. The technique is commonly employed in stirred-tank processes using particles with large particle diameters (0.5-mm) in order to separate adsorbent particles and biomass and to allow for a high capacity for a product. (Anspach, J. Chromatography A, 865 (1999) 129-144. Expanded bed chromatography differs from packed bed chromatoraphy in that  the upward flow of eluent provides that the adsorbent particles fall back down under gravity to form a stable bed whereas a downward flow of eluent in pack bed chromatography provides that the particles must be strong enough to support the entire column of adsorbent above them. Also, in expanded bed chromatography, the adsorbent is less crosslinked because it can be softer and more gel like which allows eluent to flow internally through the adsorbent as well as between the particles.

Expanded bed asorption reduces the number of operations in purificaiton processes by combining clarificaiton, concentration, and capture into one operation. It is based on stable fluidization and uses adsorbent particles with well-defined size and density distributions, otgether with columns designed to give even liquid flow distribution. The bed expands as the adsorbent particles are lifted by by an upward liquid flow through the column. The major benefit of using it is that adsorption can be carried out with unclarified feedstocks, there is no need for centrifugation or filtraiton to remove cells and debris. When the feedstock is applied, the target protein is captured by the adsorbent while cells and debris pass through the column unhindered. Washing is performed with the bed in an expanded mode, followed by elution of bound protein in a dedimented mode with downward flow. The choice of ligand is not limited to ion-exchangers. It is possible to sue hydrophobic, chelating and affintiy ligands. However, the feedstocks sued with expanded bed are usually much cruder than the feedstocks of traditional packed bed chromatography; the ligands must be abel to witstand a vigrous cleaning procedure. Frej (Biotech & Bioengineering 44 (1994) 922-929)

Chromatography Schemes Using EBA

Protein A EBA: There is significant increased in the dynamic binding capacity when Protein A EBA is operated at slower flow rates. Blank (Bioseparation 10: 65-71, 2001)

Breakthrough studies on streamine Protein a using purified antibody in packed-bed mode and unclarified cell culture fluid in the EBA showed very similar DBCs. Blank (Bioseparation 10: 65-71, 2001)

Fahrner (J. Biotechnology 75 (1999) 273-280) discloses that wehreas a packed bed protein A affinity column must have cells and cell debris removed from the cell culture fluid prior to loading, often by tangential flow filtration, the abiliyt to load unclarified cell culture fluid onto the protein A affinity column can eliminate a unit operation and thus could improve production efficiency and reduce production cost. Fahrner discloses that EBA protein A using Streamline rProtein A media is an efficient method for prufiying mAb from CHOPs comparible to packed bed. The DBC of the EBA is related to flow rate (measrued in column volues per hour) by a power function, which allows a high capacity at a low flw rate. At 250 cmh-1 with a 25 cm bed hight, teh DBC is 30 g/l

A 3 step process with Protein A EBA results in the same purity as the conventional prcoess. Blank (Bioseparation 10: 65-71, 2001)

–Protein A(EBA) -CEX – AEX: 

–Protein A EBA – CEX – AEX: 

CEX EBA: 

A pH of 4.5 and conductivity of 10 mS are reasonable parameters for manufacturing operations and given the accpetable DBC of 23 mg/ml. Blank (Bioseparation 10: 65-71, 2001)

Effect pH: At a pH to 4.75 at 10 mS improved the DBC to 15 mg/ml. At pH 4.5 the DBC was greater than 20 mg/ml. Blank (Bioseparation 10: 65-71, 2001)

Effect of conductivity: The DBC may also be increased by decreasing the conductivity. Blank (Bioseparation 10: 65-71, 2001)

–CEX EBA -Protein A – AEX: Blank (Bioseparation 10: 65-71, 2001) disclose that a 3 step process with CEX EBA as the capture step cannot remove antibody aggregate and leached Protein A to acceptable levels. 

Monolithic stationary pahses consist of a porous polymer block. To create the polymer, monomer and cross linker molecules as well as molecules beraing the interactive groups (e.g., an ionomer in the case of IEX) are polymerized in the presence of a porogen directly inside the column. The porogen is a good solvent for the molecules of te start mixture but a nonsolvent for the polymer. As the polyemr forms it “precipitates” into small coils, which subsequently aggregate into “nodules”. Due to the high cross linking, the nodules can be considered “non porous” from the viewpoing of biochromatography. The channels, which are formed between the aggregates, have average channel diameters between 1-5 um. Such columns are still porous enough to permit injection  and washing/regeneration by pressure. The fissured surface structure of the polymer provides a large surface area per unit volume and the capacity of such columns is comparable to that found for the conventional particle based ones. In monolith chromatography, the mobile phase can be considered to flow through the single “particle”. A monolith has more similarities to a bundle of capillaries than to a packed particle bed, and a recent treamtnet of the theory of monolith disk chromatogrpahy uses this approach to model the separations. Monolithis disks and monolithic columns have many similarities. A major difference is the lenght of the separation distance. For monolithic disks, this distance is only in the millimeter range. (UNO Q and UNO S respectively). (Ruth, “Novel approaches to the chomatography of proteins” Biotechnoly and Bioprocessing/Biotechnol. Bioprocess. 27, 2003 455-502)

A monolith is a continuous bed consisting of a single piece of a highly porous solid material where the void volume is decresased to a minimum. The most important feature of this medium is that all the mobile phase is forced to flow through the large pores of the medium. As a consequence, mass transport is enhanced by convection and has a positive effect on the seapration. Three types of monolithic supports are commercially avialble. (Urthaler (US2004/0002081).

 Specific Types:

Chromatographic monoliths have beomce commercially available in various forms. One example is the convective interaction media (CIM) disks produced by BIS d.o.o., Slowenia. These are flat (diameter in the centimeter range, thickness in the millimeter range) disks on the basis of a poly(glycidyl methacrylate-co-ethylene dimethacrylate) copolymer. Currently, the disks are available as strong and weak cation and anion exchanges. They can also be used directly to immobilize proteins and peptides in a very efficient way, since the base polymer in the underivatized state contains epoxy groups. The seoncd commercially avilalbe monolithic stationary phase is the UNO column by Bio-Rad. These monoliths have a typical column dimensions, with a lenght in the centimeter and diameter in the millimeter range. UNO columns are availabel as strong cation and anion exchangers (UNO Q and UNO S respectively). (Ruth, “Novel approaches to the chomatography of proteins” Biotechnoly and Bioprocessing/Biotechnol. Bioprocess. 27, 2003 455-502)

Silica gel based monolithic beds: are slid robs of slica monolith that have been preapred according to a sol-gel process. This process is based on the hydrolysis and polycondensation of alkoxysilanes in the present of water-soluble polmyers. The method leads to “rods” made of a single pieces of porous silica with a defined bimodal pore structure having macro (about 2 um) and mesopores (about 0.013 um) when smaller ordes intended for analytical purposes are preapred.

Polyacrlamide based monolithigc beds: are mode of swollen polyacrlamide gel compressed in the shape of columns. Their technology relies on the polymerization of advanced monomers and ionomers directly in the chromatographic column. In the presence of salt, the polymer chains form aggregates into large bundles by hydrophobic interaction, creating voids between the bundles large enough to permet a high hydrodynamic flow. (Urthaler (US2004/0002081).

Rigid organic gel based monolithic beds: These supports are preared by free radical polymerization of a mixture of a polymerizable monomer, optionally with functional groups, such as glycidyl methacrylate, ehtylene diemtacrylate, a crosslinking agent, a radical chain initaitor and porogenic solvents in barrels of an appropiate mold. (Urthaler (US2004/0002081).

Continuous (simulated moving bed) chromatography:  See outline    As to monoliths see outline

Companies:  Hosptial Supply Corporation

In general, chromatographic methods chromatography columns are employed which are essentially comprising a column housing with an upper and lower fitting which in turn comprises an inlet at the top of the column, an outlet at the bottom of the colum, a upper chromatography column firt, a lower chromatogrpahy column firt, an upper distributor plate and a chromatogrpahic material. Blaschyk (US2012/0123091).

In line or Tandem Chromatography Columns See outline

 Blood Purificaiton (see outline) 

Surge Tanks

Buffer or mixing or surge tanks are common in the industry and are installed between units to avoid propagation of disturbances for continous processes (Faanes et als. “Buffer Tank Design for Acceptable Control Performance” Ind. Eng. Chem. Res, April 4, 2003). 

Single Skids

Shukla (“Process scale Bioseparations for the Biopharmacetuical Industry” Taylor & Francis Group, LLC, 2007) teaches chromatography skids, ultrafiltraiton process skids, virus fitration skids and processing of a vareity of batch volumes in a single skid. 

Amphiphilic: contain both hydrophobic groups (“tails”) and hydrophilic groups (“heads”). Surfactants (detergents) are typically organic compounds that are amphiphilic which render surfactants soluble in both organic solvents and water. 

Aqueous solution: refers to a solution in which water is the dissolving medium or solvent. When a substance dissolves in a liquid, the mixture is called a solution. The dissolved substance is the solute, and the liquid that does the dissolving (in this case water) is the solvent (Junyan (US 13/536584). 

Batch mode: A single packed column is used and equilibration, load, wash, elution and regeneration/cleaning are performed sequentially. Thommes (US2004/0241878). Compare with “continuous chromatography” below.

Breakthrough:/Dynamic Binding Capacity is used in reference to situations in which compounds sought to be removed from a fluid are not removed. For example, breakthrough occurs during chromatography when the resin is incapable of binding the compound to be removed so that the compound remians in the fluid sample. Breakthrough usually starts when the resin becomes saturated with the compound. (US6,096,870).

The DBC of a chromatography media is the amount of target protein that will bind to an amount of media under flow conditions before there is a significant breakthrough of the product. It can be determeiend using either purified protein or preferably the starting feed stock. If pure protein is used, the breakthrough curve can be seen directly by monitoring UV absorbance. If starting feed stock is used, the DBC will be affected not only by the concentraiton of the target protein but also the presence of other interfering molecules and the presence other UV absorbing components will mask the breakthrough of teh target protein. This will require analysis of fractions using a target protein specific assay to determine the breakthrough properties. 

Dynamic binding capacity (DBC) of a chromatography resin represents the total amount of target protein that the resin will bind under actual flow conditions before significant breakthrough of unbound protein occurs. This is a useful parameter for predicting what the process performance of a resin will be in actual use. DBC affects the overall amount of resin that can be packed in a given column for a process –and the number of batches that can be processed cost-effectively in manufacturing each year. If resin DBC is low, then more gel must get packed inside a column, ultimately increasing the cost values, less can be packed inside a column, thereby decreasing the overall utility cost of each batch run. DBC can be determe3ind using either partly purified mAbs or cell culture feedstock. If the former are used, then the breakthrough curve can be measured directly by monitoring UV absorbance. Resin DBC also can be evaluated through spectrophotometric analysis at an optical density reading at 280 nm (OD280) and testing a sample of purified MAb eluted under binding conditions. (Gupte, “Dynamic Binding Capacities of Protein A Resins for Antibody Capture: A comparative evaluation” May 24, 2020, BioProcess International). 

Buffer: see outline

Chaotropic agent: refers to a compound that is capable of chaning the spatial configuation or conformation of polypeptides through alterations at the surface so as to render the polypeptide soluble in the aqueous medium. A strongly dentaturing chaotropic solution contains a chaotropic agent in large concentrations which in solution will unfold a polypeptide present in the solution effectively eliminating the proteins secondary structure. The unfolding will be relatively extensive, but reversible. Examples of chaotropic agents include guanidine hydrochloride, urea, and hdroxides. (Pizarro, US2008/0125580).

Column volume: refers to the volume of packed resin inside the column including any void volume. For example, if a 10 mL column is packed with 2 mL of resin, then one CV is 2 mL. Column volume is composed of three parts, the interparticle space known as the void volume, the volume within the pores known as the pore volume, and the volume occupied by the solid matrix of the particle frequently referred to as the matrix volume.  The void volume or inter-partical volume refers to the volume within a column not occupied by the particles themselves; the space between the particles. For roughly spherical particels of similar size, the void volume typically constitutes about 40% of a olumn pakced with those partciels wehn the particles are settled by gravity and not physically compressed by mechanical means. The pore volume refers to the volume within the pores of porous partciels. (Gagnon, US 14/555060). 

Continuous (simulated moving bed) chromatography: See “Columns used” right hand panel

Conductivity: refers to the ability of an aqueous solution to conduct an electric current between two electrodes. In solution, the current flows by ion transport. Thus, with an increasing amount of ions present in the aqueous solution, the solution will have a higher conductivity. The conductivity may be latered by chaning the concentration of ions as by altering the concnetration of a buffering agent and/or concentration of a soalt (e.g., NaCl or KCl). The unit of measurement is milliSeimens per centimeter (mS/cm or mS).

The conductivity of a solution can be altered by chaing the concentraiton of ions therein. For example, the concentration of a buffering agent and/or concentration of a slat (e.g., NaCl or KCl) in the solution may be altered in order to achieve the desired conductivity. (Arunakumari, WO2007/108955).

Detergents and Surfactants: A detergent refers to ionic, zwitterionic and nonionic surfactants which are useful for preventing aggregation of proteins and to prevent non-specific interaction or binding of contaminants to the protein of interest. A surfactantare molecules with well defined polar and non-polar regions that allow them to aggregate in solution to form micelles. Depending on the nature of the polar area, surfactants can be non-ionic, anioni, cationic, and Zwitterionic (contains a head with two oppositely charged groups). Some examples of common surfactnsts include anionic (based on sulfate, sulfonate or carboxylate anions): perfluorooctanoate (PFOA or PFO), perfluorooctanesulfonate (PFOS), sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, and other alky sulfate salts, sodium laureth sulfate (also known as sodium lauryl ether sulfate, or SLES), alkyl benzene sulfonate; cationic (based on quaternary ammounium cations): cetyl trimethylammonium bromide (CTAB) a.,.a hezadecyl trimethyl ammonium bromide, and other alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), polethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT); long chain fatty acids and their salts: including aprylate, caprylic acid, heptanoat, hexanoic acid, heptanoic acic, nanoic acid, decanoic acid, and the like; Zwitterionic (amphoteric): dodecyl betaine; cocamidopropyl betaine; coc ampho glycinate; nonionic: alkyl poly (ethylene oxide), alkylphenol poly(ethylene oxide), copolymers of poly(ethylene oxide) and poly(proylene oxide) (commercially known as Poloxamers or Poloxamine), alkyl polyglucosides, including oxtyl glucoside, decyl maltoside, fatty alchols (e.g., cteyl alcohol and oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates (Tween 20, Tween 80, etc), Triton detergents, and dodecyl dimethylamine oxide. Most parentally acceptable nonionic surfactants come from either the polysorbate or polyether groups. Polysorbate 20 and 80 are contemporary surfactant stabilizers in marketed protein formulations. 

–Nonionic Detergents

—-polyoxyethylene-sorbitain-monolaurate” (“TWEEN”): is a common non-ionic detergent (Dubrow, US 5932428).

—-polyvinylpyrrolidone (PVP) is a common non-ionic detergent (Dubrow, US 5932428).

Distribution coefficient: See partition coefficient below. 

Elution buffer: is used to elute the target protein form the solid phase. To “elute” a molecule from an ion exchange material is meant to remove the molecule by altering the ionic strength of the buffer surrounding the ion exchange material such that the buffer competes with the molecule for the charged sites on the ion exchange material.

Equilibration buffer: is used to adjust the pH and conductivity of the chromatography column prior to loading the column with the mixture containing the protein of interest for purification.

Fixed bed chromatography: continues to dominate the field, whether conductived wtih membranes, monoliths or porous particles packed in column. Most applications are run with a single bed. Bed dimensions can be increased if greater capacity is required, but chromaotgraphy media and buffer costs increase proportionally. Cycling can increase capacity without increasing media costs, but multiples buffer costs and process times. (Pete Gagnon, J. Chromatography A 1221 (2012) 57-70). Thommes (Recvoery of Biological Products, 2003) disclsoes feasibility for a continuous multicolumn system called simulated moving bed chromatography (SMB).

Fluidized bed chromatography: is a technique which by applying a flow in the reverse direction of gravity on chromatography beads with high density allows a space to be made between the latter thereby allowing a slightly clarified solution to pass into the chromatography column without causing any clogging of the solution flow. Fluidized bed chromatographies may be of the ion exchange type, affinity type or mixxed mode type  (Chtourou, US14/131944). 

Fluidized bed chromatography involves the use of adsorbetn particles dispersed in a liquid medium. The simplest example is batch chromatography butbut the format of industrial perference involves highly engineered up-flow columns that maintain the particles in an evenly dispersed state. One version is referred to as expanded bed chromatography. (Pete Gagnon, J. Chromatography A 1221 (2012) 57-70)

–Expanded bed chromatogrpahy (EBA): see outline

Flow rate: refers to the column volume divided by the residence time. For example, the flow rate for a column with 10 mL of resin at a residence time of 5 min would be 10mL/5min=2mL/min.

Gradient elution: The conditions for elution or recovery of a bound compound from a chromatography material is done continuously (i.e., sequence of small steps). Compare step elution below. 

High-performance liquid chromatography (HPLC): relies on the sue of rigid, small particle matrices at high oepration pressure. Unlike conventional chromatogrpahy, HPLC is most suitable for purifying low-microgram quantities of proteins collected in small fraction values with a short seapration time. Typically, HPLC is used in later stages of protein purificaiton after one or mroe conventional chromatographyic separations have been used tor educe the mass of contaminaitng protiens and to simplify a complex protein mixture. (Schwartz, WO 99/54355). 

Intermediate buffer: is used to elute one or more impurities from a chromatography resin, prior to eluting the molecule of interest.

Isoelectric point (pI): Protein are comprised of amino acids which include acidic and basic residues. At low pH (high H+ concentration) the carboxylic acid groups of proteins tend to be uncharged (-COOH) and their nitrogen containing basic groups fully charged (-NH3+) giving most proteins a net positive charge. At high pH the carboxylic acid groups are negatively charge (-COO-) and the basic groups tend to be uncharged (-NH2) giving most proteins a net negative charge. The isoelectric point (pI) of a protein is the pH at which that protein has no net charge since the positive and negative charges balance.  At a pH below its pI a protein will have an overall positive cahrge and thus bins to a negatively charged material such as CEX. At a pH above its pI aprotein will have an overall negative charge and thus will bbind to a positively charge material such as AEX. This is why in CEX load compositions and wash buffers of a relatively low pH are generaly used, in order that the protein or interest binds to the CEX matrix during the loading and wash steps and why in AEX load compositions sand wash buffers of a relatively high H are generally used, in order that the protein of itnerest binds to the AEX matrix during the loading and wash steps. 

In an aqueous solution, the amino and carboxyl groups of a protein are present in ionized states: NH3+ and COO- and the total charge of the protein in the aqueous solution depends on the pH. The total charge of the protein is zero at the pH of the isoelectric point (pI). The protein is made in a negatively charged state at a pH of more than pI, and in a positively charged state at a pH of less than pI. For the isoelectric point of various human immunoglobulins see “Agrisera Antibodies, “molecule weight and isoelectric point of various immunoglobulins”.

Monoliths: The term “monolith chromatography” or “monolith adsorbers” refers to a chromatography format wherein a continous volume of a porous polymer is housed in a container through which a feed stream is supplied and whose surfaces are affixed with resin or ligands which are materials which provide the physical and/or chemical properties that are employed for purificiton. Rose (WO 2017/140081)

Monoliths are fixed chromatography beds case as a single unit, characterized by a network of large highly interconnected channels. They offer the uniform flow distribution of packed particle beds and the convective mass transport efficiency of membranes. Monoliths easily accomommodate flow rates of 10 bed volumes per minute without loss of perforamance. Monoliths currently marketed for industrial applications have average 2 um channels optimized for purifciation of large solutes such as DNA plasmids and virus particles.  (Pete Gagnon, J. Chromatography A 1221 (2012) 57-70)

Loading buffer: is a buffer which is used to load the sample or composition comprising the target molecule of interest. The loading buffer may, for example, have a conductivity and/or pH such that the molecule of interest (and generally one or more impurities) is are bound to the chromatography resin or such that the protein of interest flows through the column while the impurities bind to the resin.

Loading density: refers to the amount (e.g., grams) of composition put in contact with a volume of chromatogrpahy material (e.g.,k liters). In some examples doading desntiy is expressed in g/L. (Nadarajah, 14/355,818).

partition coefficient (Kp) (also known as “distribution coefficient”): refers to the equilibrium ratio of the concentration of product absorbed to the resin to the concentration of product in the solution, under specified conditions of pH and solution composition (Kelly, US 8,067,182). 

The “partition coefficient” refers to the molar concentration of a product/ppolypeptide in the stationary pahse divided by the molar concentration of the product in the mobil phase (Nadarajah, US 14/355,818).

Organic salt: is a reactino product of an organic acid and an inorganic base, for example, sodium acetate from the reaciton of acetic acid and sodium hydroxide. In general, organic sals have much lower conductivity than inorganic salt; however, organic salt has buffering capacity due to its residues of organic acid.

pH: Typical CIEX loading conditions are above pH4.5, and generally pH 5-6.

Parts Per million (ppm): refers to a measure of purity of a molecule of interest.

Plasma: contain fibrinogen which is essnetial in blood clotting. This distibuishing plasma from serum (below) which remains after this fibrinogen is removed. Plasma is a clear and yellowish fluid part of the blood which is also found in lymph or in intramuscular fluids. Plasma makes up about 55% of the toal blood volumne (the main constituents of blood plasma is water). Blood plasma is preapred by spinning a test tube containing blood in a centrifuge until the blood cells are isolated. The plasma is then drawn off.

Reducing agent: refers to a compound that maintains free sulfydryl groups so that the intra or intermolecular disulfide bonds are chemically disrupted. Examples include dithiothreitol (DTT), dithioerythritol (DTE) beta-mercaptoethanol (BME), cysteine, cysteamine and thioglycolate (Pizarro, US2008/0125580).

Regeneration buffer: may be used to regenerate the chromatography resin such that it can be reused.

Serum: is the liuqid part of the blood after coagulation (when blood is extracted and left to clot). Serum contains a complex mixture of proteins. The most abundant serum proteins are albumins (60-80% of total serum protein). Albumin is a small protein produced by the liver and responsible for transport of small molecules such as calcium and also helps keep blood fluids from leaking out int o tissue. Globulins are a second form of protein found in large quantities and comprise 3 major types; alpha, beta and gamma. Alpha and beta globulins mainly carry various lipids, lipid soluble hormones and vitomains and other lipid like substances in the plasma. The alpha-1 fraction includes alpha-1 anti-tyrpsin and thryoxine binidng globuline. The alpha-2 fraction contains haptoglobin, cerfulosplasmin, HDL and alpha-2 macroglobulin. The beta fraction includes transferrin. The gamma globulins consist primarily of the immunoglobulin (i.e., IgG).

Static mixer: refers to a device for mixing tow fluid materials, typically liquids. The device generally consists of mixer elements contained in a cylindrical housing. As the streams move through the static mixer, the non-moving elements continously blend the materials. Bian (US2013/0197200).

Step elution: the conditions for causing elution (i.e., pH, ionic strenght, concentraiton of a salt) are changed all at once form a first starting value to a final value. Thus, the conditions are changed incrementally (i.e., stepwise) in contrast to a linear change. Compare gradient elution above. 

In a “step elution” one or more conditions such as pH, ionic strenght, concentration of a salt, etc can be changed all at once form a first, e.g., starting, value to a second, e.g., final, value. Thus the conditions are changed incrementally, i.e., stepwise, in contrast to a linear change. (Falkenstein, US 14/034866)

Surge tank: refers to a container which is used between process steps or within a process step (e.g., when a single process stp comprises more than one step) where the output from one step flows through the surge tank onto the next step. Thus, a surge tank is different from a pool tank in that it is not intended to hold or collect the entire volume of output from a step but instead enalbes continous flow of output from one step to the next. Bian (US2013/0197200). 

virus inactivation: refers to any process which may render a virus incapable of infecting a call or inhibit a virus fucntion through a physico-chemical means. Typical virus inactivation methods include low pH treatment (e.g., below pH4.5, 4.0 or 3.8), heat treatment, treatment with surfactants and radiation (e.g., ultraviolet light). Low pH condutions during or ater protein loading or binding onto a column can influence the overall target protein recovery. This can be availableby increasing the conductivity of a low pH buffer (US 12/851082).

Wash buffer (equilibration buffer): refers to a buffer used to wash or re-equilibrate the chromatography resin prior to eluting the molecule of interest. In some cases, the wash and loading buffer may be the same.

Yield: refers to the amount of product recovered divided by the amount of product loaded onto the column multiplied by 100. For example, a column loaded with a solution that contains 100 gms of product, but from which 80 gm of produce was recovered would have an 80% yield.

See also Helpful Books in Resources:  

The term chromatography refers to a process by which a solute of interest (e.g., protein of interest) in a mixture is separated from other solutes by percolation of the mixture through an adsorbent, which adsorbs or retains a solute more or less strongly due to the properties of the solute. The term embrances a family of closely related separation methods based on the contacting of two mutually immiscible phases, wherein one phase is stationary and the other phase is mobile. Once a clarified solution containing a protein of interest has been obtained, its separation is often performed using a combination of different chromatography steps, often based on different separation principles. Thus, such steps separate proteins on the basis of charge, degree of hydrophobicity, affinity properties, siez, etc. Several different chromatography matrices, such as matrices for ion exchange, hydrophobic interaction hcromatography (HIC), reverse phase chromatography (RPC), affinity chromatography and immobilized metal affinity chromatography (IMAC) are available for each of these techniques.

Chromatography is a method for fractionating a mixture to separate compoudns of the mixture, and at times is used for purification. In liquid chromatography, for example, a sample containing a number of compounds to be seaprated is injected into a fluid stream (i.e., a solvent) and directed through a chromatographic column. The column separates the mixture into its component species in response to differential retention of the compounent species in the column. Concentration peaks assocaited with the separated compoudns typically emerge in sequence from the column. The retention time of a peak is used to infer the identity of the eluting analyte based upon related analyses incorporating standards or calibrants. The presence of the seapratted species are often distinguished through use of a refractometer or an adsorbtometer utlizing ultraviolet (UV) light. Martin (WO/2006/116064).

Types of Chromatography

Column Chromatography can be used to fractionate proteins. The mixture of proteins in solution is passed through a column containing a porous solid matrix. The 3 types of matrices used are 1) Ion-exchange chromatography, 2) gel-filtration chromatography and 3) affinity chromatography

1) Ion-exchange chromatography includes a)cation exchange resin and b) anion exchange resin and c) mixed mode chromatography. 

2) Gel-Filtration Chromatography uses a porous matrix. Molecules that are small enough to penetrate into the matrix are delayed and travel more slowly through the column.

3) Affinity Chromatography 

Hydrophilic interaction chromatography (HILIC): is able to separate compounds by passing aqueous organic mobile phases across a polar stationary phase such as silica., causing solutes to elute in order of increasing hydrophilicity. This is the opposit of RPLC.

Hydrophobic Charge Induction Chromatography (HCIC): see mixed mode chromatography

Hydrophobic Interactive Chromatography (HIC): See Affinity Chromatography

High performance/Pressure Liquid Chromatography (HPLC): HPLC is distinguished from traditional liquid chromatography in that the operational pressures are much higher (50-350 bar) whereas ordinary liquid chromatography depends upon the force of gravity to pass the mobile phase through the column. HPTFF is unique among avaialbe separation technologies in that it can effect simultaneous purification, concentration, and buffer exchange, allowing several different separation steps to be combeind into a single scalable unit operation (Fahrner (WO03/102132).  

A typical high performance liquid chromatography (HPLC) system includes a pump for delivering a fluid (a “mobile phase”) at a controlled flow rate and composition, an injector to introduce a sample solution into the flowing mobile phase, a tubular column encasement containing a packing material or sorbent (a “stationary phase”), and a UV detector to register the presence and amount of the sample compounds in the mobile phase. The presence of a particular ocmpound in the mobile phase exiting the column is then detected by measuring changes in physical or chemical properties of the eluent. Response peaks corresponding to the presence of each of the compounds of the sample can be observed and recorded by tracking the detector’s signal over time. Martin (WO/2006/116064).

Metal ion affinity chromatography (IMAC):  See outline

Dye affinity chromatography: 

The adsorption of proteins to immobilized dyes generally consists of specific and non-specific interaction. Tthe specific interaction is known to be contributed by the nucleotide-specific interaction and biomimetic enzyme-coenzyme interaction. The non-speciifc interactions are contributed by their different functional groups where amine and sulfonate groups could contribute to electrostatic interaction while aromatic ring structure could contribute to hydrophobic interaction. Dye affinity chromatography has been applied as the primary capture step to minimize fouling and improve the separation efficiency of subsequent chromatogrpahy columns for enzyme purificaiton. Among the dye ligands used are Purple A, Navy HE-R, Scarlet MX-G, Green H-E4Bd and Red H-E3B. Because the itneraction of the dye with the enzyme is not clearly defined, emprical screening for a suitable dye is normally performed rather than simply adopting a commonly used dye. The trial and error steps can be time consuming. Commericalized dye screenign kits are available (e.g., PIKSI-M mimetic screen kit from Prometic Biosciences, Ltd). (Fu “Negative chromaotgraphy: Progress, applications and future perspectives, Process Biochemistry, 49(6), 2014, pp. 1005-1011). 

Immunoaffinity chromatography (see also immunoaffinity for antibodies): utilzies an antibody coupled to the adsorbent to interact selectively with biological compounds. In negative chromatogrpahy (flow through mode), it is mostly used to purify trace amounts of proteins and separate the subclasses of immunoglobin. The preparation of immunoaffintiy column is started from activaitng the surface of adsorbent particles and is followed by the incorporation of particular antibodies that are against the impurities on the activated adsorbent particle. (Fu “Negative chromaotgraphy: Progress, applications and future perspectives, Process Biochemistry, 49(6), 2014, pp. 1005-1011).

Reversed phase high-performance liquid chromatograph (RP-HPLC): see outline

Multidimensional Liquid Chromatography: A sample is fed through a HPLC using a particular type of separation method, and peaks are selected and fed into a second column using a different mode of separation.

To capture interacting proteins, a target protein is attached to polymer beads that are packed into a column. Cellular proteins are washed through the column and those proteins that interact with the target adhere to the affinity matrix. These proteins can then be eluted and their identity determined by mass spectrometry or another method.

Size Exclusion Chromatography (SEC) (AKA gel filtration or gel permeation chromatography): 

SEC separates on the basis of the size of proteins. It is among the most frequently used techniques for analysis and quality control of proteins (Arakawa “Solvent Modulation of Column Chromatography” Protein & Peptide Letters, 2008, 15, 544-555). But, SEC is slow, provides poor capacity, requires disproportionately large columns that require superior packing skills, and requires large buffer volumes. (Gagnon, “nonionic polymer enhancement of aggregate removal in ion exchange and hydroxyapatite chromatography” 12th Annual Waterside Conference, San Juan, Puerto Rico, April 23-25, 2007). 

In general, separation occurs when large molecules are excluded from entering the porous stationary phase and are carried straight trhough the column while progressively smaller molecules are increasinly able to enter the stationary phase and conequently have particularly longer elution times. It is the porosity of the stationary phase which thus determines the separation achieved. This technique is particularly good for determining levels of aggregate in the purified preparation. The stationary phase is a wide pore silica gel which may be modified with diol groups preferably a gel such as Zorbax GF450-GF250 (Dupont) or TSK gel G3000 SWXL or G4000 SWXL. The mobile phase is generally in the pH range 4-8 more preferably -7.5.

–Urea gradient size exlusion chromatography (SEC): is a proftein refolding method based on Protein folding liquid chromatography (PFLC) which is used for simultaneous refolidng and purificaiton of recombinant proteins in inclusion bodies. Wang (Biotechnol. Prog. 2008, 24, 209-213) used urea gradient SEX in the refolding of rhG-CSF expressed in E coli. A linear decreasing urea gradient from 8.0 (100% solution B which was a mixture of solution A; Tris, pH 8.0, EDTA, NaCl, glycerol, GSH and GSSG) and urea) to 2 mol.L-1 of urea (25% solution B (solution A containing urea) was used in the SEC column from top to bottom, then the denatured/reduced rhG-CSF was directly loaded into the column at a urea concentraiton of 8.0 mol.L-1. Due to its large effective MW the denatured rhG-CFS moved much faster than the urea gradeint formed by urea with a much small MW. Thus, the denatured rhG-CSF passed along the urea gradient gradually as it was eluted, the urea concentraiton around the denatured rhG-CSF molecuels would decrease gradually and linearly, adn rhG-CSF would refold step by step. 

Thiophilic chromatography: is a type of non-affinity chromatography in which a protien of interest, which contains thiophilic regions and aromatic amino residues, bind to a sulphur containing ligand for the isolation of the protein. (the term “thiophilic” refers to the selectivity that proteins have for sulfone groups that lie in close proximity to thioether groups) A thiophilic gel can be prepared by reducing divinylsulfone (coupled to Sepharose 4B) with beta-mercaptoethanol. Thiophilic adsorption chromatography is based on electron donor acceptor properties and is distinct from chromatography based on hydrophobicity. Hydrophobic associations and ionic interacts do not occur with thiophilic sorbents since theio-ethylsulfone strucutres do not possess pronounced hydrophobicity or ionic charges. Examples of commercially available thiophilic chromatogrpahy resins include Fractogel EMD TA, Uniflow and Superflow resin and T-Gel. (Arunakumari, WO2007/108955).

“Thiophilic” in reference to chromatography and smilar affinity-based methods, refers to affinity selection based on a sulfur containing ligand for a protein, in particular, for immunoglobulins, in high concentrations of certain salts. The protein can be eluted by removal of the salts. Thiophilic affinity chromatography (TAC) has a broad specificity for immunoglobulins of different classes and species, and offers advantages over Protein A or G purification because of the relatively mild conditions, reduced cost of materials, and broad range of specificity. (Relkin, US 2012/0183527). 

Conditions/Parameters Generally (See outline)

MAbs undergo several posttranslational modifications such as disulfide bond formation, oxidation, deamidation, truncation and cyclization of N terminal glutamine residues to pyroglutamate. The heterogeneity observed in mAbs is often moniteored by weak cation exchange (WCX)-high performance liquide chromatography (HPLC). Resolution on the WCX column depends on the surface charge of the molecule, which in turn determines the apparent isoelectripoint (pI) of the molecules. Regions of the chromatography before and after the main species are often referred to as the acidic and basic regions. Lowering of the apparent pI of the main molecule due to modification such as deamidation as asparagine residues, C terminal truncation of lysine residues and cyclization of glutamine residues will result in migration toward the acidic region wereas an increase in the pI of the molecule due to modification such as methionine oxidation and proline amidation will result in migration toward the basic region (Ouellette, Analytical Biochemistry, 397 (2010) 37-47). 

Acidic and basic species are commonly observed when recombinant mAbs are produced. Charge variants may substantially affect the in vitro and in vivo properties of antibodies. It has been demonstrated using chemically modified antibodies that charge variation can alter binding to proteins or cell membrane targets, thus affecting the tissue penetration, tissue distribution and pharmacokinetics (PK) of the antibodies. The impact on biological functions is highly dependent o the sites and levels of modificaitons. For modifications that are localized in teh Fc regions, even the presence of higher levels may not have direct effects on the binding affinity. Complete elimination of acidic and basic species is unrealistic and unnecessary. (Du, “Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies, mAbs 4-5, 578-585, 2012)

The extent of charge heterogeneity in Mabs also regulates their structure, stability, binding affinity, chemical properties and hence their biological reactivity. During manufacturing, various forms of microheterogeneities are formed with respoct to size, charge, and other attributes. The reason could be enzymatic processes, spontaneous degradation and other modificaitons. Common forms of charge hterogeneities that have ben obvserved include structural isoforms, glycosylation, glycation, degradation, deamidation, isomerization and peptide clevage. Charge heterogeneity resutls in modificaiton of the isoelectric point (pI) of the moelcule. It has been demonstrated that modifying the pI of an antibody by about one pH unit can lead to significant differences in the pharmacokinetcs of an intact Mab. (Joshi, “Avoiding antibody aggregation during processing; establishing hold times, Biotechnol. J. 2014, 9, 1195-1205)

Acidic variants: 

An acidic variants is more acidic than the main species antibody. An acidic variant has gained negative charge or lost positive charge relative to the main species antibody. Acidic variants of a main species antibody elute earlier than the main peak upon separation by cation exchagne chromatography (CEX) or later than the main peak from AEX. 

Sialylation and C-terminal lysine cleavage or deamidation increase net negative cahrge and produce acidic variants, which elute earlier during CEX. (Kim, Arch. Pharm. Res. (2016) 38: 1472-1481). 

Causes of Acidic Variants: Some of the main causes for the formation of acidic species are the following: 

Deamidated antibodies: is one in which one or more asparagine residues have been derivitized, e.g., to an aspartic acid, a succinimide, or an iso-aspartic acid. Deamidation occurs both in the variable domains as well as in the constant domains. Deamidation of Asn residues in the CDR region is almost guaranteed to result in the generation of acidic species. (Du, “Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies, mAbs 4-5, 578-585, 2012). 

Deamidation is a non-enzymatic chemcial reaction in which an amide functional group is removed. The reaction is important in the degradation of proteins because it alters the amide containing side chains of the maino acids asparagine and glutamine. In an example of the reaction, the side chain of an asparagine attacked the adjacent peptide group, forming a symmetric succinimide intermediate. The symmetry of the intermediate results in two hydrolysis products, either asparatate or isoaspartate. This process is considered a deamidation reaction becasue the amide in the asparagine side chain is replaced by a carboxylate group. A similar reaciton can occur in asparatate side chains, yielding a partical conversion to isoaspartate. In the case of glutamine, the rate of deamidation is generally ten fold less than asparagine, however, the mechanisms is essentially the same, requiring only water molecules to proceed. (Ram US 2011/0130544).

–Deamidation of asparagine (Asn or N):  The deamidation of asparagine residues in mAbs can be a major route of degradation, and it can limit shelf-life if appropriate formulation and storgage condtiions are not selected. In general, the Asn deamidation mechaisms has been well characterized. At alkaline pH, deamidation is a peredominantly base ctalyzed reaction that proceeds via a nucleophilic attack of the N+1 nitrogen of the protein backbone on the carbonyl carbon of the Asn side chain, forming a cyclic imide intermediate, succinimide. Spontaneous hydrolysis occurs at eitehr of the succinimide alpha or beta carbonyl groups to form isoaspartic acid or aspartic acid in a ratio of about 3:1. These deamidation producs result in the formation of antibody acidic variants because of the generation of additional carboxylic acid groups. (Pace “Asparagine deamidation depenence on buffer type, pH and temperature” J. Pharmaceutical Sciences, 10296) (2013)

–How measured:

Protein deamidation can be measured using the ProPac WCX-10 column (see Dionex, application Note 125 “Monitoring Protein Deamidation by Cation-Exchange Chromatography”).

Pyro-glutamate Variants:

Many of the human IgG1 or IgG2 types of antibodies contain a glutamic acid (Glu) and/or a glutamine (Gln) residue at the N-temrinus of either the light or heavy chain or both. Such N-temrinal glutamic acid and/or glutamine residues may undergo cyclization to form pyro-glutamate (pGlu) . During cyclization of glutamine, the N-temrinal primary amine (positively charged at a neutral pH) is converted to a neutral amide, resulting in a change of the net charge of the antibody. The lack of cyclization may be detected as basic variatns by CEX since the main peak is typical the fully cyclized species or as late-eluting peaks by reversed-phase HPLC tdo to the increased hydrophobicity after the loss of the N-terminal amine. Difffernt levels of completeness of the process are a common source of heterogeneity. (Van Schravendijk, US 14/780964, published as US 2016/0060349)

The role of N-terminal cyclization and the enzymes involved is not entirely known. However, it is speculated to provide a protective function against protein degradation from extracellular amino peptidases. Formation of pyroglutamate from glutamine results in the loss of mass of 17 Da due to the release of ammonia. The pyroglutamate variant is more acidic because of the loss of the primary amine and, thus elutes as a more acidic species. (Ouellette, Analytical Biochemistry, 397 (2010) 37-47).

–Methods to promote pyro-glutamate formation to reduce heterogeneity

—-Enzymatic conversion

Xu (Analytical Biochemistry 436 (2013) 10-12) discloses a method to reduce charge heterogeneity by adding glutaminyl-peptide cyclotransferase, which is specific in catalyzing protein N-terminal Gln to PyroGlu. Thus charge variants of terminal Gln and PyroGlu can be identified without fraction collection. In addition, treatment of mAb antibodies can assist fraction collection for the characterization of other antibody variants. 

—-Non-enzymatic conversion

Van Schravendijk, (US 14/780964, published as US 2016/0060349) discloses a method for the conversio f an N-temrinal glutamine and/or glutamic acid risude ofn antibody to pyro-glutamic acid within a purification process such as one including protien A chromatography and IEX by incubating the antibody under conditions to promote cyclization of the N-terminal glutamine and/or glutamic acid residue. In one embodiment, the conditions include a termpature of 20-45C with a pH in the range of 3.5-9.0 for 4-120 hours. 

—-Changing N-mterin Aln residues for other residues

Rasmussen (US 2008/0131882) discloses that charge heterogeneity may result from the N-tmerinal clockage by pyroglutamic acid (PyroGlu) resulting from cyclization of N-tmerinal glutamine residues (deamidation) which results in charge heterogeneity giving a coplex IEX pattern. The problem cannot be solved by the use of the specific enzyme, pyroglutamate aminopeptidase because deblocking has to be peormed on reduced and alklyated antibodies in order to obtain high yeidls of the deblocked antibodies not compatible with a subsequent IEX analysis and second because it is not possible to obtain a 100% cleavage for all the antibodies. Rasmussen discloses instead ensuring that no polypeptide chain contains a N-temrinal glutamine by chaning N-termianl glutamine residue to another amino acid. 

Production Methods to Reduce Acidic Variants:

Cell Culture Methods:

(Subramanian US9,150,645) discloses a method of producing adalimumab having less than 10% total acidc species by culturing a mammalian cell producing adalimumb with basic amino acids selected from arginine, lysine, ornithin and histidine.

Addition of stabilizers

–addition of acid:  Santora (US2004/0162414) discloses a method of identifying modifications of anti-TNF antibody such as acidic modification as detected using a weak cation exchange columnn (WCX-10) as well as a method for reducing modifications such as acidification by adding acid to a transgenic goat milk solution containing the antibody.

Glycated variants: See outline

Basic Variants

Basic species are variants with higher apparent pI when analyzed using isoelectric focusing methods.  Basic species elute later than the main peak from CEX or ealirer than the mean peak from AEX.

C-terminal lysines, glycine amidation, succinimide formation, amino acid oxidation, asparate isomerization and removal of sialic acid increase net positive charge and produce basic varitns, which elute latter. (Kim, Arch. Pharm. Res. (2016) 38: 1472-1481)

Similar to acidic species, the location s of modifications that form basic speceis are critical to whether or not the modifications have any effect on the structure, stability and biological functions. Modifications of either N termini or C termini of antibodies are not expected to have substantial effects on antibody structure, stability and functions because these regions are highly exposed and not part of any ligand binding sites. 

Some of the more common reasons for basic variants are the following:

C-terminal Lys: 

One major reasons for the formation of basic species is incomplete removal of C terminal Lys. MAbs with heavy chain C terminal Lys are more basic than the main species due to the additional positive charges.  (Du, “Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies, mAbs 4-5, 578-585, 2012). The removal of the carboxy-terminal lysine form the H chain is routinely observed upon the characterizaiton of mAbs and is cuased by intracellular enzymes. From a regulatory aspect, this “lysine clipping” is not regarded as critical under the condition that a potency assay is available that proofs the quality of the mAb. (Antes, J Chromatogr B Analyt Technol Biomed Life Sci, 852(-12), 2007, 250-6). 

Santora (Analytical Biochemistry 275, 98-108 (1999) discloses the use of cation exchange liquid chromatography (CIEX) (WCX-10 CEX0 and capillary isoelectric focusing (cIEF) for determination of heavy chain C terminal variants of D2E7, a human anti9-tumor necrosis factor monoclonal antibody. In addition, exveral enzymatic treatments were used to assist in determining the idnetity of isoforms; deglycosylation of the antiobdy with PNGaseF, which hydrolyzes all types of N-glycan chains and CPB which specifically cleaves Lys and Arg residues form the C termini.

Subramanian (US9,181,337) discloses a composition comprising human anti-TNF alpha (adalimumab) where less than 65% of the lysine variant speceis in the composition have zero C-terminal lysines and where the lysine variant species include the main peak and peaks that elute at a releative residence time later than the main peak as detected using weak cation-exchange chromatography. Since lysine can carry a positive charge, antibodies lakcing the basic C temrinal lysin(s) differ in their charge state from ones that contain the lysine, so that the distribution of lysine variants (% Lys 0, % Lys1, % lys2 of the total lysine sum) can be detected using IEX such as ProPac WCX-10 Weak CEX.

–Removal of basic isoforms with Carboxypeptidase B (CPB):

Carboxypeptidase B (CPB) is thought to specifically remove C-terminal basic amino acid residues. Therefore, the properotion of C-terminal basic amino acids in a Mab can be analyzed through teh change in the proportion of charge variants following CPB treatment. (Kim, Arch. Pharm. Res. (2016) 38: 1472-1481)

Gadgil (US 16/340,822, published as US 2019/0263855) discloses a multi-column chromatography system such as a periodic counter current chromatography system (PCC) which includes carboxypeptidase B immobilized on sepharose.  The C-terminal lysine residues on H chain can be truncated by passing a harvest recovered form perfusion cell culture on a column which has CPB on sepharose. The CPB preferentially acts upon the basic amino acids, such as arginine and lysine and thus the resin can be used for removal of charged isoforms belowing to any class of antibodies. In one embodiment, a continuous process for reducing heterogeneity of an antibody includes a CPB-Sepharose column connect to a Protein A column. The flow through from the CPB-Sepharose column is directly loaded ontto the protein A column for capture step. 

As to Particular Antibodies

HER2: (Harris, US2009/0202546) discloses a composition comprising a main species HER2 antibody comprising varialbe light and heavy sequence and acidic variants of the main species, as identified by methods like WCX-10 CEX chromatography. Rat pharmacokinetic dats showed that although acidic variants are chmically different from the main peak, they have equivalent pharmacokinetics.

TNF-alpha: Fraunhoffer (US21009/0291062) discloses an adalimumab solution after DF/UF processing having 2.26% acidic region 1 and 11.81% acidic Region 2. 

Particular Purification Methods Used  (just brief review from perspective of variants — see the particular schemes in outline)

AEX: Linke (WO2012/015912) discloses methods for purifying a polypetpide from a solution containing the polypetpide and an acidic variant thereof such as a deamidated species by contacting the immunoconjugate wtih an AEX and eluting with a high salt buffer thereby separating the active immunoconjugate form the deamidated varient.

CEX: With CEX which allows for charge based elution, mostly acidic negatively charged species elute first and basic positively chard species elute toward the tail end of the antibody peak. The early eluting aggregates are negatively charged acidic varitns while the later eluting aggregates are positively chard basic variants. (Nti-Gyabaah, US 14/355014)

HIC: Acidic variants will elute first from the HIC column as they are less hydrophobic than basic charged aggregates. (Nti-Gyabaah, US 14/355014, published as US 2014/0288278) )

Protein aggregation is a common phenomenon that can be encountered during various stages of a commercial antibody manufacturing process. For example, aggregates can form during the fermentation, purification, final formulation operations, or as a result of the storage of the drug substance or final drug product. Usually aggregation results from intermolecular associations of partially denatured protein chains, however it may also result from chemical degradation and subsequent exposure of hydrophobic surfaces, or from disulfide bond scrambling. Mono-dispersity of a therapeutic monolconal antibody is important in terms of both efficacy and safety. Molecular heterogeneity in size (e.g., aggregation) can compromise the biological activity of an antibody or result in a partial or total loss of its therapeutic properties. (Nti-Gyabaah, US 14/355014). 

Aggregation not only leads to loss of product but also has adverse effects such as reduced efficacy and immunological reactions. The role of aggregates in development of immune response to proteins is well reported. This can occur by cross-linked of the B-cell receptor, thus activating the B cells to proliferate and target proteins to the lysosomal pathway, eliciting the T cell response for antibody response. Aggregates can also present epitopes in an array form, evoking T cell independent antibody induction. Furthermroe, aggregates are readily taken up by antigen-prsenting cells and drive dendritic cell maturation, thus enhancing the thymus-dependent immune response. T (Joshi, “Avoiding antibody aggregation during processing; establishing hold times, Biotechnol. J. 2014, 9, 1195-1205). 

Protein aggregates can be classified in several ways, including soluble/insoluble, covalent/non-covalent/reversible/non-reversible and native denatured. Soluble aggregates are not visible and may not be removed by a 0.22 um filter. Conversely, insoluble aggregates may be removed by filtraiton and are often visible. (Nti-Gyabaah, US 14/355014).

What causes Aggregation of antibodies?

It has been suggested that IgG aggregation may be mediated primarily by unfolding of the CH2 domain or Fab domain, depending on which mAb is being considered. Haixia Ru “PH and temperature-dependent mechaisms of non-native aggregation of anti-CD40 IGG1” Dissertion, Fall 2015. 

In addition to aggregation prone domains known to occur in mAbs, both full length and Fab portions, aggregation domains are also found in antibody fusion molecules, such as, for example, the anti-VEGF fusion protein alfibercept, which is a fusion of the VEGF0binding portions form the extracellular domains of human VEGF receptors 1 and 2 to the Fc portion of a human IgG1 immunoglobulin as well as etanercept, which is a fusion of the TNF receptor to the Fc poriton of a human IgG1. Bevacizumab contains 4 aggregation prone domains in its light chains. (Kraft, US 2020/0031917). 

Effect of Buffer Conditions:

The underlying mechanism that results in the aggregation due to changes in buffers and salts is complex and not well understood. The complexity is compounded by the inability to definitively differentiate between the various protein stabilization mechanisms and the samll free energies of stabilization of globular proteins. T (Joshi, “Avoiding antibody aggregation during processing; establishing hold times, Biotechnol. J. 2014, 9, 1195-1205).

At both 4 and 30C, it has been shown that the increase in aggregate level is minimal even up to 7 days in buffers without salt. The only exception to this is the citrate buffer where appreciable amount of aggregation is observed even in the absence of salt. It can be concluded that the bomination of salt and high termpature results in a significant incrase in aggregation and that the citrate buffer is prone to higher aggregation as compared to acetate adn glycine buffers. T (Joshi, “Avoiding antibody aggregation during processing; establishing hold times, Biotechnol. J. 2014, 9, 1195-1205).

Low pH leads to Mab aggregation. At high pH (pH 8, close to pI of Mab), increase in charge variants of the Mab is observed. The decrease in main peak content is due to an increase in the acidic variants. Deamidation of asparigine residue is a common cause of increase in acidic variants leading to degradation and decrease in effiacy of Mabs. This is why this is more significant for CEX and AEX. T (Joshi, “Avoiding antibody aggregation during processing; establishing hold times, Biotechnol. J. 2014, 9, 1195-1205).

Aggregation in Fc Fusion Proteins

In addition to aggregation prone domains known to occur in mAbs, both full lenght and Fab portions, aggregation domains are also found in antibody fusion molecules, such as, for example, the anti-VEGF fusion protein alibercept, which is a fusion of the VEGF-binding poritions from teh extracellular domains of human VEGF receptors 1 and 2 to the Fc portion of a human IgG1 immunoglobulin as well as etanercept, which is a fusion of the TNF receptor to teh Fc potion of a human IgG1 immunoglobulin. Bevacizumab (AVASTIN) contains 4 aggregation prone domins in its light cahins. Ranibizumab (LUCENTIS), having the identical light chain, shares the same 4 aggregation prone domains in its light chains. Bevacizumab also cotnains another three aggregation prone doamins in its heavy chains past the inger (amino acid sequences SVFLFP, VVSVLTVL and GSFFL). The sequences are also found in the fusion protein aflibercept due to it including the Fc portion of a human IgG1 immunoglobulin. Aggregation prone domains are known to occr in bother biopharmaceuticals including albumin, somatotropin, insulin Factor VIII and glucagon. (Kraft, US 2020/0031917)

Wang (US 16/259,095, published as US 2019/0234959) disclsoes the Fc fusion protein Aflibercept haivng about 0.05% Fab2-Fab2 (Fab complexes). 

Particular Types of Purification Methods for Aggregates (just brief review from perspective of the aggregates — see particular schemes in outline for more detailed steps/parameters)

Protein A:

A few methods to reduce the risk of aggregation during protein A chroamtography have been disclosed. Most have centered on moderating the pH of elution for antibodies. Sodium chlorid (0-1M) has been disclosed as an elution buffer additive to increase the elution pH. Hydrophobic competitors such as ethylene glycol have been used to weaken hydrophobic interactions and thus increase elution pH from protein A columns. Urea in the concentration of 1-2M has been used as a mild denaturant to facilitate elution. A different approach has been to engineer the protein A ligan to allow for milder elution conditions.  (Shukla, “strategies to address aggregation during protein A chormotography” 36 BioProcess Technical. May 2005).

–With C Domain:

Bian (US 14/768,254, published as US 2016/0122305) discloses using the C domain of Protein A and either a pH gradient or pH step elution which elutes at least 30% of the protein aggregates (such as dimer, trimer, tetramer) prior to the elution of the Fc containing protein in addition to removal of protein aggregates after elution of the F containing protein. In one embodiment, the seris of pH changes steps are in the order of pH 5.0 to pH 3.0 or a pH gradient ranging from pH 7 to 3. 

Ion-Exchange:  See also CEX for the pufication of antibodies

The fact that aggregates will generally carry more charge than the product at the working pH range and thus bind more strongly to ion exchangers than the monomeric form of the can be exploited for purificaiton. (Nti-Gyabaah, US 14/355014)

Hydroxyapatite

HA and FA have been shown to be effective for removal of aggregates from many antibody preprations. Antibody aggregates usually elute after antibodies but may eoelute with antibodies to varying degrees. Aggregate removal is important because aggregates are known to contribute to nonspecific interactions that reduce the shelf stability, sensitivity, accuracy and reproducibility of analytical results in conjunction with in vitro diagnostic applications. (Gagnon, US 7,999,085). 

HIC

HIC is an established purification step for aggregate removal with the majority of aggregates eluting either on the tail portion of the native antibody peak or as a distinct peak. Aggregation increases the hydrophobicity of the resulting molecular form relative to the hydrophobicity of the monomeric mAb. Phenyl sepharose HP is usually the HIC resin of choice because of the particle size and known surface accessility of aromatic residues on the monoclonal antibody structure. (Nti-Gyabaah, US 14/355014, published as US 2014/0288278)

–bind & elute mode:

(Nti-Gyabaah US14/355014, published as US 2014/0288278) disclsoes showed using HIC in a bind and elute mode as a final polishing step to resolve heterogenous aggregates from anti TNF mAb by using Pehnyl Sepharose HP at pH 7 and conductivity 140 mS/cm. A linear elution gradient was employed with decreasing ammonium sulfate to separate the monomer from the aggregates by their hydrophbicity. The eluate was fractionated and tested for charge heterogeneity via HP-IEX and monomer purity via HP-SEC showing one main peak followed by a secondary small peak during the gradient elution. The main peak consisted entirely of high purity monomer while the secondary smaller peak contained increased levels of aggregate which included a mixture of both acidic and basic variatns. 

—-To separate three-light chain species:

Gentofte (US 15/114,655, published as 2016/0347833) discloses antibody purification using HIC where a “pre-monomer” that includes an antibody  monomer and a light chain non-valently bonded thereto is separated from the the main peak of the monomeric antibody when analysed by SEC-HPLC. A gradient with decreasing ammonium sulphate concentrations was used for the elution step on a Pehnyl Sepharose HP resin. Pre-monomer together with other aggregates elutes at the back end of the monomer peak. 

Wollacott, Monoclonal antibodies, 2013, 5(6), pp. 925-935) disclose a novel mAb species which eluted between the monomeric and dimeric species of a mAb as shown by size exclusion high performance liquid chromatography (SE-HPLC). The species, referred to as “shoulder” indicated that it was a mAb containig an extra light chain and had a MW of about 175 kDA. The extra light cahin was found to be non-covalently assocaited with the Fab portion of the protein. The species was idnetified as a mAb monomoer with an additional light chain covalently associated trhoguh a disulfide bond formed between a light cahin cysteine and an engineered cysteine in either the H or L chaims of the molecule. Wollacott discloses using HIC in place of CEX to exploit the subtle differences in the hydrophobicity between the monomer and shoulder. Using HIC, the shoulder species was reduced from 3% to less than 0.5% with good step recovery. More hydrophobic HIC resins such as GE Butly Sepharose HP were best at discrimiating between mAb-X monomer and shoulder, dimer and high MW species, resulting in product containing 99.7% monomer and less than 0.1% shoulder by the end of the elution by a lear gradient from 1.2-0 M ammonium sulfate in 20 mM citrate, pH 6. The Phenyl Sepharose resin also produced veyr pure monomer (99.2%), however, monomer did not bind as tightly to the resin. 

–Flow through mode:

(Nti-Gyabaah US14/355014, published as US 2014/0288278) discloses using HIC in a flowthorugh mode as a final polishing step to resolve heterogeneous aggreagates from TNF mAb. Anti-TNF mAb mixture was loaded onto a Phenyl Sepharose HP column at pH 7 and conductivity 110 mS/cml. After loading, the column was washed with an equilvaent pH and conductivity buffer to recvoer unbound anit-TNF mAb B. Fractions were taken at various loading points. Product pools in the flowthrough mode contained solely 100% pure monomter with an 85% yield. 

Size Exclusion Chromatography (SEC): 

Size exclusion chromatography (SEC): has been reproted to provide an effective process for reducing the level of aggregates in a partially purified builk product. The sucess of this type of chromatography exploites the fact that the monmeric mAb has a smaller size than aggregates and thus elutes earlier. (Nti-Gyabaah US14/355014, published as US 2014/0288278).

Thio-heterocylic cations:

Gagnon (US 14/766123; see also 14/769098) discloses a method of reducing aggregate content in a protein preparation having a target protein such as an antibody by contacting the preparation with a thio-heterocyclic cation such as methylene blue or methylene green. The cation is then removed by contacting the resulting mixture with a first functionalized solid such as a negatively charged solid or HIC.  In some embodiments, the thio-hterocyclic cation may be used in combination with allantoin. For example, allantoin can be added to a cell culture harvest in an amount of 1%, methylene blue is added to a concentration of 0.025%. Particles bearing an electropositive metal affinity ligand tris(2-aminoethyl)amine (TREN) are added in an amount of 2-5%.The mixture is incubated and stirred for 4 hours and the solids removed. (Gagnon 14/769098). 

As to charge variants see outline

Therapeutic proteins can undergo a variety of degradation processess, including aggregation, deamidation, isomerization, oxidation, disulfide bond scrambling and truncation. These events can occur during fermentation, purificaiton, formulation, manufacturing and storage. The product’s clinical utility or shelf life can be compromised if degradation adversely affects the biologcial activity of the moelcule. (Pan “Methionine oxidation in human IgG2 Fc decreases binding affinities to protein A and FcRn” Protein Science, 2008, 18, 424-433). 

The main species is the antibody that elutes as the major peak on chromatograms. The main species does not necessarily correspond to the unmodified or non-degraded antibody. In fact, the main peak often consists of species of antibodies with three types of typical postranslational modifications: (2) cyclization of the N-terminal glutamine (Gln) to pyroGlu; (2) removal of the heavy chain C terminal lysine (Lys) and (3) glycosylation of the conserved asparagine (Asn) residue in the CH2 domain with neutral oligosaccharides. (Du, “Chromatographic analysis of the acidic and basic species of recombinant monoclonal antibodies, mAbs 4-5, 578-585, 2012).

The US Code of Federal Regulations states that biologics must be free of extraneous material except that which is unavoidable. 25 CFR 610.33. 

Homodimeric impurities: (see also bispecific antibodies)

Homodimeric impurites are particularly challenging to evaluate with traditional antibody purity assays becasue the similarities between homidmer and heterodimer lead to difficult, if not impossible separations. Dixit “LC-MS characterization and purity assessment of a prototype bispecific antibody” mAbs 5-6, 711-722 (2013)

Low Molecular Weight (LMW) Species:

Examples of LMW protein drug product impruties includes precursors, degradation products, trancated speceis, proteolytic fragments including Fab, ligand or receptor fragments or H chain fragments, free L chain, half antibdoy, H2L (2 heavy chains and 1 light chain), H2 (2 heavy chains), HLeavy chain and 1 light chain), HC (1 heavy chain) and LC (1 light chain) .  (Wang, US 16/259095, published as US 2019/0234959)

LMW species of any therapeutic protein may result from host cell protease activity during production. LMW species often have low or substantially reduced activity relative  to the monomeric form of the antibody, while exposing novel epitopes that can lead to immunogenicity or potentially impact pharmacokinetic properties in vivo. (Wang, US 16/223,463, published as US 2019/0194298)

 Antibody Fragments

Antibody fragments are an impurity that is composed of part of a target protein that has a mass less than that of the target protein. The breakage of chemical bonds in the target protein results in the formation of one or more fragments. Fragments are common impurites requiring removal during the purificiaton of a taget prtoein. They are difficult impurities to separate form teh target prtoein becasue they often have properties very similar to the target protein, such as their hydrophobicity and isoelectric point. (Kozlov, US Patent No: 14/891,724, published as US 2016/0090399). 

Kashi (US 2015/0290325) disclsoes stable iquid formulations of human p75 TNF receptor fused to an Fc doamin of human immunoglobulin rpotein with percetnage of fragments ranging from 0 to over 10% fragments over a span of 0-6 weeks. The fragments were late eluting species in SEC. Kash teaches that size hterogeneity can be primarily attributed to fragmentation and aggregation. 

–Purification Methods used for ro reduce antibody fragments

—-Using Activated Carbon

Kozlov (US 14/891,724, published as (US2016/0090399) discloses that activated carbon can be used to absorbed and thereby reduce such fragments in antibody mixtures. For example, Kozlov demonstrate preparing a fragment spiked MAB solution from papain digested mAB, loading centrifuge tuabes with Nuchar HD activated carbon and loading the antibody fragment spiked solution, centrifugation and subsequent filtration. The amount of the MAB remaining int he samples is determeind using IG quantification by Protein A HPLC and the percentage of fragments in the samples is deteremind by size exclusion chromatography (SEC). 

—-Using Mixed Mode Chromatography

O’Connor (US 2016/0251441)  discloses a method for separating antibody fragments wehrein at least some of the antibody fragmentation product impurities are adsorbed to the mixed mode chromatography column and at least some of the target antibody is eluted from the column. 

Half Antibodies:

Birck-Wilson (US 2004/0092719) discloses methods for separation immunoglobulin half antibodies from whole antibodies by reducing the pH of the sample such that the half antibodies dissociate from one another and applying the resulting solution to a column that differentially retards mobility of the half antibodies and whole antibodies. In some embodiments the column is a cation exchange column, a size exclusion colum a hydrophobic interaction column or an affintiy column. In prefered embodiments that column binds to the half antibodies whereas in other embodimetns the column binds the whole antibodies present in the resulting solution. 

High Molecular Weight (HMW) impurities: (see outline, “aggregates”)

HMW impurities include mAb dimers and trimers. (Wang, US 16/259095, published as US 2019/0234959)

Intermediate HMW Species (HMW):

HMW include monomer with extra light chain (H2L3 and H2L4 species) (see outline), monomer plus Fab fragment complexes, Fab2-Fab2, Fc-Fc and Fab2-Fc. (Wang, US 16/259095, published as US 2019/0234959).

Free Fc:

Delvaille (US 2010/0249381) discloses purification of Fc fusion proteins via blue dye affinity chromatography for the reduction of free Fc moieties. The therapeutic moeity of the Fc fusion protein may be dervied from a receptor, preferably from the extracellular domain of a receptor. Examples include CD2, CD3, VEGF recetpor. The method results in elimination of free Fc moeities to less than 1%. Free Fc may contain dimers of the IgG hinge, CH2 and CH3 domains which are not linked to significant poritions of the therapeutic moiety. 

Eon-Duval (US 2010/0267932; see also (WO 2008/025748) discloses a process for the purification of an Fc-fusion protein which includes Protein A/G affinity chromatography, CEX, AEX and hydroxyapatite chromatogrpahy. The process was suitable for removal of free Fc, i.e., immunoglobulin H domains which are not fused to a complete therapeutic moeity such as a ligand binding extracellular porition of a member of the TNFR family. The two TNF receptors p55 (TNFR1) and p75 (TNFR (TNFR2) are examples of such members of the TNFR superfmaily. Ethanercept is an Fc fuion protein contianing the soluble part of the p75 TNR. OX40 is also a member of the TNFR superfamily. In particular, a ligand binding extracellular portion of a member of the tumor necrsos factor recetpor (TNFR) superfamily includes less than 0.1% of free Fc protein. The term “free Fc” encompasses any part of the Fc fusion protein which is dervied form the immunoglobulin part of the Fc fusion protein and does not contain a significant porition of the therapeutic moeity of the Fc-fusion protein. Thus, free Fc may contain dimers of the IgG hinge, CH2 and cH3 domains, which are not linked or boud to significant poritions of a therapeutic moeity. 

Nti-gyabaah (WO 2013/009526) discloses methods for purifying an Fc-fusion protein such as TNFR-Fc (etanercept) produced in eukaryotic systems using Protein A affinity chromatography and two ion exchange chromatography steps which are operated in the bind and elute mode resulting in highly purifed TNFR-Fc with an overall reduction of misfoled Fc fusion protein to less than 5% and reduction of fragments (including free Fc levels) to less tan 5%. Etanercept is a therapetuic recombinant fusion protein comprised of the extracellular ligand binding portion of the human 75 kDa (referred to as p75) human TNFalpha receptor linked to the constant region of human IgG1 (Fc region). 

Host Cell Proteins

Whether mammalina, bacterial or yeast derived, process related impurities can be host cell derived, including a myriad of proteins with various physicochemical characteristics, referred to as host cell proteins (HCPs). Originating from the production cell line, their level in the harvested cell culture fluid (HCCF) depends on both the cell culture process as well as harvest conditions. The potential that HCP has for eliciting immune reactions in patients has drawn significant interest, as the said adverse patient events can delay candidates and/or reduct drug efficacy. For this reason, it is not only important but also required to that porduct and process related impuriteties be removed to an acceptably low level during the manufacturing process. HCP levels can vary significantly for different mAbs. HCPs are one of the most challenging to remove impurities because of their varied physiochemical properties. The analytical method of choice used to monitor HCPs during the recovery process is a multiproduct immunoasaay. (Nogal Biotechnol. Prog. 28(2): 454-458 (2012). 

Chinese hamster ovary (CHO) cells are integral to the 125 billion biopharmaceutical market, which includes mAbs. Typically, therapeutic proteins are secreted into the extracellular media along with hundreds of endogenous host cell protoein (HCP) impurities, comprising both secreted proteins and intracellular proteins released during cell death. Putification processes for these extracellular CHO HCPs from the product becasue even low levels of HCP impurities have the potential to cause adverse patient reactions. A subset of these HCPs are difficult to remove during downstream purification becasue they exhibit product assocation with mAbs and have similar retention to mAbs on chromatographic media. (Valente, Biotechnology and Bioengineering 112(6): 1232-1242 (2015). 

Aboulaich “A novel approach to monitor clearance of host cell proteins associated with onoclonal antibodies”, Biotechnol Prog. 30(5) 1114-1124) discloses a method to identify HCPs (lists m HCPs accoiated with several antibodies) that specifically assocaite with mAb by immobilizing purified mAb onto chromatography resin via cross-linking, followed by incubated with HCPs obtained from supernatant of non-mAb producer cells that are representative of the expression systems used in mAb manufacturing. 

–-PLBL2: cathepsin L

Falkenstein (US 15/900, 449, published as US 20180186866) discloses that HCP, particularly PLBL2, 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.

Beigie (US 16/228,291, published as US 2019/0233468) discloes that using 0.5 M sodium benzoate pH 7.0 and benzyl alcohol in an intermeiate wash was very effective at removing HCPs such as PLBL2 during Portien A purification.

Dumetz (US 16/330,588, published as US 2021/0284686) discloses a method of purifying an antibody from HCPs such as Phospholipase B-like 2 protien and cathepsin L using a superantigen chromatography solid support such as Protein A with a wash buffer that includes about 50 mM caprylate and more than about 0.5 M arginine. 

Chromatin

Gagnon (US 14/769,098, published as US 2016/0009762) discloses adding allantoin to clarify chromatin from a cell culture harvest, then methylene blue or ethacridine or cetyl trimethyl ammonium bromide or chlorhexidine is added. Then particles bearing an electropositive metal affinity ligand tris (2-aminoethyl)amine (trEN) is added. In a separte embodiment allantoin is added to a cell culture and then heptanoic acid  or octanoic acid or pelargonic (nonanoic) or capric acid is added. The mixture is then stirred for 2 hours and the electropositive metal affinity ligand tris(2-aminoethyl)amine (TREN) are added. 

DNA and Endotoxins:

Protein A:

Morgan (J of Biotechnology, 4 (1986) 189-204) disclsoes purification of a mAb with a protein A-Sepharose column. The antibody absorbed to the colume at pH 8.4, followed by extensive washing with phosphate-buffered saline, pH 8.4 to remove endotoxins and other protein contaminants. The antibody was desorbed at pH 4.5. A 60-140 fold purificaiton of antibody with a 100-1000 fold reduction of endotoxins was acheived. 

Ion Exchange:

Since DNA and endotoxins are negatively charged over a wide pH interval, a CEX chromatography step at a pH below the isoelectric point of the antibody will bind the target protein and allow the negatively charged molecuels to wash through the colunm. Consequently, if AEX is used as the initial capture step, these contaminants will be removed at an early stage in purification. (GE Antibody Purificaton Handbook, (December 2007). 

Amino-terminal leader extension variants: 

Amino-terminal leader extension variants refers to a mian species antibody with one or more amino acid residues of the amino terminal leader sequence at the amino-terminus of any one or more heavy or light chains of the main species. (Harris, US2009/0202546)

Leachables/Extractables

Skudas (US 15/524824, published as US 2017/033741) teaches that activated carbon is usefuful in the removal of leacables/extractables which results from the use of disposible equipment in the purification of antibodies. 

Polyelectrolytes

Polyelectrolyte precipitation may require subsequent removal of the polyelectrolyte. Bozzano (J Membrane Science, volume 55, issues 1-2, pp. 181-198, 1991) discloses using ultrafiltraiton membranes to recover proteins in the permeate and polyelectroyte in the retentate. 

Proteases

Proteases can be subdivided into serine, cystein, aspartyle, and metalloprotease families, all of which can diminish protein drug performance. Proteolytic enzymes such as pepsin and papain often target unstructured hinge regions of proteins and antibodies. Becasue upstream and downstream processes generate myriad proteases, selection of suitalbe protease inhibitors is a common practice in the biopharmaceutical industry. Such inhibiotrs are classified by seze: e.g., small molecules (pheynylmethylsufonly fluoride), peptides (E64) and proteins (aprotinin). (perez, “Opporunities in the field of host-cell proteins” Bioprocess Technical, 2020). 

Clipped Masking Moieties in Cleavable moiety conjugates:

A “masking moeity” (MM) refers to a peptide that when positioned near an antigen binding domain (AB), interferes with binding of the AB to the biological target. The term “cleavable moeity” (CM) refers to a peptide that includes a substrate for at least one protease. Activated antibodies are designed to be selectively activated in dieased tissue by incorporated within the CM a substrate for a protease that is more prevalently found in the disease tissue. A “clipped impurity” or “clipped varaint” refers to a molecule that resutls after protease mediated clevage of an intact antibody. It contains the AB of the activated antibody but lacks all or a portion of the MM. It includes both “single-armed clipped” and “fully clipped” species. Removing clipped impurity from intact activatable antibody conompositions is challenging due to the relatively small differences between desired product and clipped impurity. Masking moeities are relatively short peptides such as less than 50 amino acids. Single-arm clipped variants are hte predominant clipped variant such that the entire difference between an intact activatable antibody and a clipped variant is the absence of a portion othat lacks a MM and a protion of a CM. Patrick (US Patent Application No: 17/227,029, published as US 20210317188)

By Hydrophobic Chromatography:

Patrick (US Patent Application No: 17/227,029, published as US 20210317188) discloses a process for purifying intact activatable antibody that includes a MM, a CM and a AB from clipped variant as determined by SDS-cGE which includes loading an aqueous feedstock that includes water, the intact activatable antibody and the variant and a first salt onto a support matrix that inclues hydrophobic substituents and eluting with water and a second salt. Exemplary hydrophobic substituents include hydrophobic itneraciton chromatogrpahy (HIC) and mixed mode chromotography (MM).  Salts for use as a first salt in the aqueous feedstock may be any salt that promotes binding of intact activatable antibody and clipped impurity to the column. Illustrative first salts include arignine chloride, arginine hydrochloride, NACl and the like. The salt may be introduced into the aqueous composition prior to the hydorphobic chromatography step or it may be introduced in connection with a process step upstream of the hydorphobic chromatogrpahy process step. When the column includes a HIC stationary pahse, the first salt typically exhibits kismotropic (salting out) behavior. Such salts can be identified for example form the hofmeister series of ions. In the case of HIC, the eluent is generally less porlar than the aqueous feedstock. Patrick further discloses that amounts of an activatable antibody and a clipped masking moiety can also be determine by subjecting a sample composition containing activatable antibody and clipped variants to a gel capillary electrophoresis. 

By Protein A chromatography:

Acoba (WO2020247574) discloses purifying masked antibody with protein A chromatography under conditions suitable for binding the masked antibody, washing the column with the bound masked antibody once with an acidic wash buffer and eluteing at pH 2.5-4 to form an eluate that includes the masked antibody. 

Biosimilars

A biosimilar product can have minor difference in clinically inactive components, provided that there are no clinically meaninful differences between the biological product and the reference product in terms of the safety, purity, and potency of the product. 42 USC 262(i)(1). 

Different Molecular Forms (i.e., monomeric, dimeric, tetrameric forms)

Aoyama (J. Immunological Methods, 162 (1993) 201-210) discloses HPLC on spherical beads of hydroxyapatite (HA) to separate different molecualr forms of mouse IgG and IgM mAbs. Monomeric, dimeric, trimeric and tetrameric forms of an IgA mAb were eluted from the column separately with appreciable differences in retention volume by a 20 ml gradient of pohsphate buffer (pH 6.8) of concentraiton from 10-400mM. IN addition, a monomeric form was resolved from a pentameric form of IgM mAbs under the same conditions. 

Introduction

Small genetically engineered immuoglobical constructs are being developed industry wide for a growing range of in vivo applications. Examples include Fab, F(ab’)2, single-chain (sc) Fv, bis-scFV, diabodies, minibodies and single domain antibodies. Their small size potentially tives them access to tissues that are poorly accessible by intact antibodies; rpaid clearance form blood and neotargeted tissues, lower immunogenic response and eye-drop inhalent or oral administration. (Gagnon, “Minibodies and Multimodal chromatography methods” BioProcess Interational Feb 2010).

Domain antibodies are characterized by formation of the antigen binding site by a single antibody domain, which does not require interaction with a futher domain (e.g., in the form of VH/VL interaction) for antigen recognition. Produciton of nanobodies, as one specific example of a domain antibody, in lower eukaryotic hosts such as Pichia pastoris is described in WO94/25591. In contrast to difficulties observed with conventional four chain antibodies or their fragments, including Fab and scFv, domain antibodies can be readily expressed and secreted in a correctly folded, fully funcitonal form from hosts like E. coli or P. pastoris at a sufficient rate and level. Schotte (US13/266503 and US2012/0157664)

Fab fragments:

Fab fragmebts are classically generated by treating immunoglobuilin (Ig) with the protease papain and isolating a 50-kDa disulfide-linked intact clevage product composed of a heavy + light chain fragment that contains only one antigen binding site. Unlke their bivalent Ig precursor, monovalent Fab fragments can bind their targets without cross-linking and thus possess significant potentail as reagents that can block receptors and signaling pathways. However, few Fab fragments are used in clinical applications because Fabs display a relatively short serum half-life, although streategies exist to address this issue such as conjugating Fabs to PEG or engineering Fabs as fusion proteins. The second limigation involves the common observation that Fab prepatations may aggregate as a function of time, concentraiton, temrpature or salt. If a Fab prepration is composed mostly of monovlaent species but contains a small contaminant of prtoein aggregate, the few multivalent complexes can display disproportionate activity becasue they intrinsically possess higher potential for antigen binding avidity and cross-oinking and thus may out-compete the potentail clocking effects of the more numerous monovalent Fabs. (Nelson, J. Biological Chemistry, 287(51) pp. 42936-42950). 

Particular Schemes for Purification of Domain Antibodies

Difficulties with purifying domain antibodies/Importance of conditions:

Gagnon, “Minibodies and Multimodal chromatography methods” BioProcess Interational Feb 2010) discloses that due to their architecture in lacking bidning sties for protein A, the principal enabling too for IgG platform purificaiton is inapplicable for mibodies. With respect to purificaiton of an antiprostate stem-cell antigen )PSCA minibody, the minibody did not bind to protein A and coeluted with BSA to a substantial degree on CEX, AEX, hydorphobic-interaction and hydroxyapatite (eluted by phosphate gradient chromatographic methods).

Affinity Chromatography:

–Protein A:

Beirnaert (US 2010/0297111) teaches nanobodies which includes variable domains present in naturally occurring heavy chain antibodies (referred to as VVHH domains) against TNF alpha using Protein A affinity purification including separation of PEGlyated and non PEGlyated nanobides using cation exchange chromatography.

Basran (WO/2009/074634) discloses that although Protein A based chromatography resins have been extensively used to purify VH dAbs from microbial culture supernatants in a single step, for some molecules the low pH elution conditions can result in the formation of aggregates. There is alo the issue of the limited capactiy of affintiy resins for dAbs. Mixed modal charge induction resin on Capto MMC from GE Healthcare was thus used as the primary capture step follows by anion exchange. Column equilibration was performed using 50 mM sodium phosphate pH 6. After washing the protein is eluted by pH gradient using 50 mM Tris pH 9.0.

Brown (US2010/0172894) discloses purifying single domain antigen binding (SDAB) molecules using Protein A based affintiy chromatography. In one embodiment, the elution and load buffers include sodium phosphate, sodium chloride. In other embodiments, the equilibration and wash buffer include sodium phosphate, sodium chloride and arginine.

Casterman (WO/1994/004678) discloses an isolated immunoglobulin comprising two heavy polypeptide chains sufficient for the formation of a complete antigen binding site which are devoid of light polypeptide chains (camelid antibodies) which are purified by Protein A or G chromatography.

Frenken (WO/1994/025591) discloses the production of antibody fragments derived from heavy chain immunoglobulins of Camelidae by eukaryoties such as yeast and fungi using Protein A chromatography.

—-Protein A – CEX –AEX

Stals (US 2012/0141460) discloses purifying a bivalent nanobody using Protein A affintity chromatography and eluting the nanbody using 100 mM Glycine pH 2.5. After neutralization the nanbody was further purified using CEX and an additional AEX step. 

—-VH3 Domain Antibodies

The term “VH3 domain” refers to the framework subgroup 3 of the human heavy chain variable region of an immunoglobulin. The heavy chain variable domains of antibodies are classified into distinct subfamilies (VH1 to VH6) on the basis of DNA sequence and protein homologies. (Slough, US Patent Application No: 15/566231, published as US 2018/010007)

Boein (US 2015/0239991) discloses  the purification of a hetero-dimeric antibody such as scFv using Protein A chromatography such that one of the H chains is modified to reduct or eliminate binding to the Protein A and the unmodified H chain retain binding to Protein A. 

Hagai (WO 02/059264) discloses the pruificaiton of an scFv blonging to the VH3 family by Protein A where the bound scFvs were recovered form the column by acid elution (0.1 M glycine, pH 3.0)

Haywood (US 15/566,231, published as US 2018/0100007) discloses purification of a human VH3 domain containing antibody 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 chromatogrpahy where the VH3 domain containing antibody is recovered in monomeric form. 

–Protein G (see affinity chromatography and “Protein G”)

Ion Exchange:

Cho (US 2006/0153860) teaches purification of antigen dbinding polypeptides which can include the light chain or variable region of a heavy chain or at least one CDR of a light chain or heavy chain or Fab or diabody or llama antibody using anion or cation chromatography, HIC hydroxylapatite chromatography, etc.

–Anion Exchange

–Cation Exchange

Beirnaert (US 2010/0297111) teaches nanobides which includes variable domains present in naturally occurring heavy chain antibodies (referred to as VVHH domains) against TNF alpha using using cation exchange chromatography.

Brown (US 12/608964, published as US 2010/0172894 and US 10,118,962) disclsoes purifying single domain antigen binding molecules by adjusting the cell culture medium containg the antigen-binding fusion polypeptide to a conductivity between about 12 and 9 mS/cm and a pH equal to or less than about 4.5 and contacting the medium with a CEX and selectively eluting the nanobody from the support by washing teh support to remove contaminant(s) and eluting the antigeng binding fusion polypeptide wiht an elution buffer. Further chromatography such as hydroxyapatite can then be performed. In one embodiment the antigen-binding fusion polyeptide is a singel cchain polypeptide that includes at least one immunoglobulin variable domain from an antibody naturally devoid of light chains. .

Jonniaux (US2011/0183861) discloses purification of nanobides via CEX with a wash buffer of 10 mM citric acid, pH 4.0 and elution buffer of 10 mM citric acid/1M NaCL, pH4 followed by size exclusion chromatography. The nanobdy was produced intra cellularly or from an inclusion body in a bacterial cell or produced extracellularly and isolated form the medi7um in which the host cell is cultivated.

Silence (US2006/0115470) discloses purification of VHH on a cation exchange column which had been equilibrated in 25 mM citric acid pH 4.0 and then eluted with 1M NaCL.

Mixed-Mode:

Gagnon, “Minibodies and Multimodal chromatography methods” BioProcess Interational Feb 2010) discloses that Capto MMC media provides adequate capture and significant dimer removal for  purifying a stem-cell antigen (PSCA) minibody. 

Structural Variants of Domain Antibodies:

Lack of Disulfide Bonds:

Schotte (US13/266503 and US2012/0157664) discloses that despite the high yield and functionality of domain antibodies produced in non-coli hosts, in particular yeast, there is a quantitatively significant fraction of product that represents a structural variant. In particular, a fraction of the product lacks at least one disulfide bond. It is consistently reported that conventional antibodies or fragments lacking at least one disulfide bond are characterized by a loss of function. Schotte provides a method of counteracting this. Schotte teaches a method of producing a domain antibody in yeast by applying conditions such as addition of oxidizing agents, preferably oxidizing metal ions such as Cu2+, Fe2+, Fe3+ and Zn2+ that promote the formation of disulfide bridges in the domain antibodies and/or removing domain antibodies lacking at least one disulfide bride. The method results in the production of domain antibodies wherein the quality of the domain antibodies is improved with a reduced level of free thiol or its absence.

See also purification of IgA from plasma

Immunoglobulin A is the dominant immunoglobulin in human secretions including breast milk. Due to the low levels of IgA normally present in cows’ milk, the established method to increase the yield of IgA on a commercial scale is by immunization regimes to boost the levels in the milk. An inherent problem with this procedure is that it generally takes up to 3 months for the imminization regime to generate a hyperimmune response in the cows and then a further month to harvest the hpperimmune milk in sufficinet quantities. (WO2006/119560).

IgA is the most abundant of the five classes of immunoglobulin and is primarily found in the nucosa of the gastrointestinal tract and the respiratory tract. There are two types of IgA, secretory IgA (sIgA) and serum IgA. Serum IgA is generally a monomer found in the serum wehre it functions as a seocnd line of defense whereas sIgA is dimeric or polymeric and contains a scretroy component in addition to IgA. SIgA is a common constituent of breast milk. Sand (US 15/249811, published as (US 2016/0362477)

Starting Sources/Samples for IgA

Isolation of IgA from Seminal and Vaginal Fluids

(donadoni (“setting of methods for analysis of mucosal antibodies in seminal and vaginal fluids of HIV seropositive subjects from Cambodian and Italian Cohorts” 2010, 5(3)) discloses techniques for isolation of samples from vaginal fluid and semen. The IgA was subsequently isolated from the samples using a scheme of 1st IgG  purificaiton by Protein G affinity chromatography (Protein G binds to IgG fc region but not IgA and IgM), 2nd: IgM-IgA residual fractions from the IgG purificaiton underwent an AEX step where the fractions were applied to a HiTrap Q HP column, washed and eluted with buffer B (340 mM NaCl, 20 mM TrisHCL, pH7.2), 3rd: IgM and IgG were separated using gel filtration chromatography. 

Starting Sources/Samples for sIgA

See also isolation of sIgA from plasma

Intestinal luminal fluid/mucosal of pigs and cows:

Sand (US 15/249811, published as (US 2016/0362477) discloses methods for the purificaiton of sIgA from the intestinal luminal fluid or mucosa of pigs and cows. The lumen is the interior caviy of the intestine wehre digested food passes through and nutrients are absorbed. In one emboidment, the prcoess includes treating the intestinal luminal fluid with polyethylene glycol (PEG) having a molecular weight of 3-30k and isoalting the precipitated sIgA. The sIgA may also be pastuerized. 

Purification Schemes

Affinity Chromatography:

Jacalin: Ttotal IgA1 fraction can be purified from the precipitate using the well established jacalin affinity chromatography method for the separation of IgA1 from IgA2, IgG and IgM from human sera. Agarose bound jaclin with a binding capability of 1-3 mg IgA/mL gel slurry is obtained commercially (perice). Briefly, the Cohn Fraction III precipitate is resuspended in phosphate buffered saline (PBS) and applied to a bed of jaclin-agarose. The column is washed with PBS until protein in the flow through portion is no longer detected. The jacalin bound portion cotaining the IgA1 is eluted with 0.1 M Galactose. (Simon, US2008/0260822). 

Affinity purification methods have a long established history isoalating various specific ligand binding macromolecules form complex biofluid and tissue extracts.Preparations of IgA and IgM dervied form human plasma or serum are enriched in C. difficle toxin A and toxin B specific antibodies by affinity purificaiton methods.  In this case, C. diffcile toxins A and B are immobilized to an appropriate solid-phase support with which re-suspended Cohn fraction II precipitate or jacalin-purified IgA solutions will be incubated, then washed. Proteins binding only C. diffcile toxins A and B will bthen be eluted. (Simon, US 2008/0260822)

–Affinity Tagged Secretory Component as the ligand: 

Brown (US 14.476,559, published as US 2014/0371431; see also US 16/401,322, published as US 20190256577) discloses a method of islating IgA by application of an IgA source to secretory component that is modified to contain an affinity tag so as to form secretory IgA containg the affinity tag which can then be captured by a solid support resin. Purification of secretory IgA is advantageous because the secretory componant tag protects IgA proteins from digestion in the gastrointestinal tract. Affinity tags which can be used include peptide tags such as AviTag which allows biotinylation by the enzyme BirA so the protein can be silated by streptavidine, a calmodulin tag, and a FLAG tag. In some embodiments plasma dimeric IgA in the naturally occurring monomer-dimer mixture is convalently bound to the recombinant peptide tagged secretory component in vitro. In other embodiments, native secretory component is covalently bound to one or more amino acid residues through conventional synthetic techniques. As an example using a histidine tag, a single histidine residue or a poly histidine is added to secretory components, regardless of whether produced by recombinant, synthetic addition or other techniques. The secretory IgA that is now tagged can then be purified by affinity binding of the tag to a binding moiety immobilized on a resin. In the case of a FLAG tag, an antibody to the FLAG peptide is immobilized on the support as described in US 4,703,004. 

Corthesy (WO 2013/132052) discloses that it is possible to combine plasma-derived J chain-containing immunolgoublin, in particular IgA and/or IgM, with secretory component without the need to first purify the J chain containing immunoglobulin. The method includes the steps of obtaining a bood dervied protein composition that includes J chain containing IgA and/or IgM and admixing the composition with secretory component. The SC binds to the J-chain containing immunoglobulin. The SC may also contain a tag such as a hexa-histidne tag, which can aid in the purificaiton of the resulting protein. If such a tag is attached via a cleavalbe linker, the tag may be cleaved off prior to use in the invention. 

Jones (J of Immunoglogical Methods, 104 (1987) 237-243) discloses humans secretory component bound covalently to Sepharose 4B as an affinity adsorbent to isolate and purify polymeric immunoglobulin from cell culture sueprnatants. The method was used to isolate murine IgM isotype. Gel filtration of the eluted antibodies followed by enzyme immunoassay showed that all recovered IgM was of pentameric molecular size. Murine IgA were also isolated form cell culture supernatants. Most of the IgA recovered following affinity adsorption with SC was greater molecular size than dimer. 

Cation Exchange: 

IgA has an acidic isoelectic point in the range of about 4.5-6.5 due to variable glycosylation and is generally considered not to be able to adsorb to cation exchange resins in an amount that would be of commercial value. However, Brown (WO2006/119560A1) discloses fractionation of IgA from milk products such as skim milk by cation exchange by modifying the loading and elution conditions.

Hydroxyapatite (HA): 

(Luellau, J. Chromatography A, 796 (1998) 165-175 disclsoes using hydroxyapatite (HA) elution chromatography for the separation of molecular variants of monoclonal IgA from cell culture supernatants.

Metal chelate chromotography: 

Bertonlini (WO00/41721) discloses a method for separating IgA from other proteins such as IgG and IgM by metal chelate chromotography where a metal ion is attached via a chelate forming ligand to a matrix. The metal ion include ions such as zinc, copper, nickel, iron, manganese etc. The starting fluid can be any material containing IgA such as serum plasma, plasma fractions or mucosal secretions (e.g., milk, tears, saliva).

Ion Exchange:

–AEX Exchange:

(Lullau, Biotechnology techniques, 12(6), 1998, pp.; 425-430) dislsoes mIgA produced under serum free conditions by a murine hybridoma cell lineina stirred tank reactor, binding the IgA to DEAE Sepharose FF under low salt conditions and performing salt elution steps of 100-500 mM NaCl. . To separate dimeric IgA form monomeric and polymeric forms, the igA containing fractions from the DEAE IEX were applied to type I cereamic hydrozapatite (CHT). 

Microfiltration: 

Couto (US 2004/01673200 disclsoes a prcoess for seaprating moelcules of interest such as antibodies which emplys three filtraiton unit operations, clarification using microfiltration, concentration using UF. 

Hensgens (US 2011/0130545) disclsoes a a process for separating secretory IgA which includes separating the fat from the mild such as by skimming followed by subjecting the low-fat milk to microfiltration yeilding an S-IgA containing prempeate and a casein rich retentat. 

–acidifying skim milk first: Gregory (US5670196) teaches a method of preparing a whey product with immunoglobulins such as IgG by first seprating cream or fat, then acidifying (lower pH from about 6.0 to about 4.5-4.7) to precipitate casein and then smicrofiltering the supernantant milk or milk serum using a charged depth filter. 

Salt solutions: Uemura (US5258177) discloses using low inoic strenght salt solutions such as sodium caprylate or zinc caprylate to provide an IgA rich preparation. The starting solution can be a Cohn’s Fraction III or Fraction II + III.