The removal of pigment via chromatography method
In the production of bio-reagents, it is inevitable for pigment to exist in protein intermediate or finished protein solutions as a result of being in contract with culture media or metal instruments. This issue is particular common in case of high concentrated protein solutions. The appearance of protein constitutes a vital parameter for quality control. Colour is an easily-spotted indicator showing the possibility of contamination or degradation of the products, therefore the control and removal of pigment constitutes a crucial step in the production process of proteins.
Pigment removal in purification process of protein
Effective control and removal of pigment are usually applied in the production of proteins, including molecular construction, fermentation &culture, sample preprocessing and chromatography purification. Specifically, chromatography resin can effectively remove pigment in samples especially in purification process.
Affinity chromatography
Affinity chromatography is usually applied in the capture step in protein purification. Pigment can be effectively removed at the same process of protein accumulation by damaging ionic adsorption and non-specific adsorption between pigment and resin via optimized buffer system.
Ionic exchange chromatography
In neutral condition, most pigments carry negative charges. Under anion exchange flow-through mode, pigment can bind with resin and then being eluted by high salinity or alkali; alternatively, target protein can bind with cation exchange resin while undesirable pigments flow through. Solution pH and ionic strength play vital roles in the purification process, which means suitable sample loading condition is the key for effective removal of pigment.
Hydrophobic interaction chromatography (HIC)
When applying HIC method, removal of pigment is obtained by using the surface hydrophobic disparity in pigment and target protein.
Size exclusion chromatography (SEC)
In case of significant molecular weight disparity between pigment and target protein, SEC resin can be used for the removal of pigment. For instance, in the process of buffer exchange by desalting column or impurities removal via high resolution resin in polishing step, pigment can be removed or adsorbed to a certain extend.
The removal of pigment from chromatography resin
Pigments possess complex properties. It is ideal to design CIP plan according to the tolerance of resins. NaOH is the most commonly used reagent for cleaning. Apart from that, high salinity solution, organic reagents (20% ethanol or 30% isopropanol), urea, GuHCl or acids are also widely chosen for cleaning approach. It is also possible to process resin with mixed reagents or other more targeted cleaning methods.
However, as an increasing frequency of resin being used, accumulated pigment will inevitably cause issues such as darkening of resin color and decrease in DBC. In more severe cases, resin bed can collapse or harden and form lumps, compromising performance and life cycle of resins.
Conclusion
Pigments enjoy great diversity. Despite being one of the most effective method for pigment removal, chromatography resin performance will be impacted by pigment property. Thus, filtration and clarification in the pre-processing of feedstock can dramatically mitigate purification pressure in subsequent steps as well as extend life cycle of resins.
Recommended related content