See also antibody purification

Definitions

Ion-exchange chromatography: retention is based on the attraction between solute ions and charged sites bound to the stationary phase. 

High-perfromance liquid chromatography (HPLC): is a technique in analytical chemistry used to separate, identify and quantify each component in a mixture. It relies on pumps to pass a pressurized liquid solvent containing the sample mixture through a column filled with a solid adsorbent material. Each component in the sample interacts slightly differently with the adsorbent material, causig different flow rates and leading to the separation of the components as they flow out of the column. HPLC is distingusihed from traditional low pressure liquid chromatography becasue operational pressures are significantly higher whereas ordinary liquid chromatography typically relies on the force of gravity to pass the mobile phase through the column. 

–Normal phase HPLC (NP-HPLC): separates analytes based on their affintiy for a polar stationary phase such as silica. It is based on analyte ability to egnage in polar interations (such as hydrogen-bonding or dipole-dipole types of itneractions) with the sorbent surface. 

–RP-HPLC: oeprations on the principale of hydrophobic interactions. 

Reversed-phase chromatography (RPC): has a non-polar stationary phase and an aqueous, moderately polar mobile phase. One common stationary pahse is a slica. With such a sationary phase, retention time is longer for molecuels whihch are less polar, while polar molecuels elute earlier.

Size-exclusion chromatography: also known as gel permeation chromatography separates particles based on their molecular size. It is generally a low resolution chromatogrpahy and thus often reservef for the final “polishing” step of the purificaiton. 

Modes of Operation

In complete flow through chromatography, the partition coefficient (Kp) is less than 0.1 and there is no protein binding to the resin. In weak partitioning chromatography, Kp is 0.1-20 and there is weak partitioning between the product and the chromatogrpahy media. In a bind and elute mode, product is tightly bound to the resin, and the Kp is greater than 100 but the load desnity is limited to the product binding capacity. However, in an overload and elute mode of chromatogpahy, load conditions are such that the product and the impurities Kp are greater than 100 and although the product flows through after reaching its binding capacity, the impurities keep binding to the resin and does not break through until they reach their binidng capacity, which could be higher than the product binding capacity. (Nadarajah, US 14/355,818).

Bind-elute Mode: Under B/E chromatography the product is usually laoded to maximiz DBC to the chromatography material and then wash and elution conditions are identified such that maximum product purity is attahed in the eluate. A limitation of B/E chromatography is the restriction of the load density to the actual resin DBC. (Nadarajah, US2014/0301977)

Flow through mode:  Using F/T chromatography, load conditions are identified where impurities strongly bind to the chromatography material while the product flows through. F/T chromatography allows high load density for for standard MAbs but may not be implementable for non-platform MAbs or the solution conditions that enable F/T operation for these non-platform MAbs may be such that they are not implementable in existing manufacturing plants (Nadarajah, US2014/0301977). 

–Recyling of flow-through: 

Rose (WO 2017/140081) disloses a method for the purificaiton of a protein of interest which includes loading a first mixture containg the protein onto a chromatography materix such that the protein binds until 40-100% of the maximum static binding capacity of the matrix is reached (the extend of overloading material need not be to the point of saturation) , collecting flow-through containig unboudn protein of interst and recyling the collected flow-through by re-loading it and a second volume of the protein of interest mixture. 

Weak partitioning mode: See outline