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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)