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.