Source and Removal Methods Optimization of the Pigment Adsorption by Resins

The Impact of Adsorbed Pigments on Chromatography Resins

Pigment deposition in the chromatography column not only affects its resolution and yield but also severely impacts its service life.

Manifestations of Chromatography Resins Contaminated by Pigments

The specific manifestations of pigment contamination in chromatography resins are as follows: During the loading process of intermediates containing pigments on the chromatography system, the pigments will first bind to the upper layer of the resin, causing the resin to change from creamy white to the corresponding color of the pigment. As the loading amount and the number of resin cycles increase, the pigments will gradually diffuse to the middle and lower layers of the resin. In severe cases, the contamination caused by pigments and other impurities can cause the collapse of the resin within the chromatography column.

Excessive accumulation of pigments will reduce the service life of the resin, specifically manifested by a significant decrease in the loading capacity of the resin and a darker appearance color. Normally, the pigments adsorbed by these resins will be removed by the regeneration solution in the subsequent regeneration process, thereby restoring the resin performance and preventing impurities adsorbed on the resin from contaminating the next process cycle. This minimizes the residual impurities in the protein stock solution, resulting in a lighter color of the protein intermediate and achieving better quality of the stock solution.

Sources of Pigment Adsorption by Resins

The pigments adsorbed by resins mainly originate from proteins, and the pigments that affect the appearance of protein solutions and are difficult to remove are mainly formed by the interaction between medium fractions and protein molecules during the cultivation and harvesting processes.

It’s reported that pigments are widely present in protein intermediates and final stock solutions, manifested specifically by the different colors displayed by protein intermediates and stock solutions, especially in high-concentration protein solutions. For example, in mammalian cell culture media, the conversion of vitamin B12 to hydroxycobalamin and its subsequent binding to antibody molecules results in a pink color. The high oxidability of iron ions and other elements generates highly reactive oxygen species (ROS) under oxidative stress, which oxidizes the tryptophan residues on the surface of antibody molecules, leading to yellow or brownish-yellow discoloration. During the mid-to-late stages of Pichia pastoris induction, the release of AOX1 alcohol oxidase into the fermentation broth combines with flavin adenine dinucleotide (FAD) cofactor to form an octamer, resulting in a green color. Additionally, natural interacting proteins and small molecule pigments may also contribute to coloration.

Removal Methods of Pigments from Resins

Pigments are a diverse group of substances, and the cleaning processes for pigments from different sources can vary significantly. Cleaning can be performed based on the properties of the medium and the pigments themselves, with alkaline cleaning, acid cleaning, organic solvents, and denaturants all being potential options. Depending on the different properties of the pigments, different detergents can also be tried, with care taken to avoid generating bubbles during the operation. If the pigments cannot be removed, the resin in the contaminated section can be periodically removed, or it can be verified whether the contamination affects the sample purification.

Methods for Pigment Removal:

• Yellow brown removal caused by residual iron ions: 0.1M HCl+2M NaCl; 0.5M phosphoric acid.

• Pink removal caused by vitamin B12: 1M acetic acid+4% cysteine; 0.3M histidine+0.5M NaCl.

• Removal of pigments by hydrophobic interaction: 0.5-1M NaOH.

• Lipid pigments: 0.5M non-ionic detergent; 70% ethanol; 30% isopropanol.

• Stubborn pigments deposited: 1M acetic acid; 1M NaOH+2M NaCl； 6M guanidine hydrochloride; 8M urea.

Precautions for Pigment Cleaning Process:

• The type of contaminants and the type of medium must be considered comprehensively during the cleaning process, and the damage to the medium should be minimized while cleaning.

• The use of sodium hydroxide or other extreme conditions for cleaning can significantly impact the lifespan and performance of the resin. The exposure time to extreme conditions should be controlled within 30 minutes during cleaning, and within 3CV during CIP (Clean-In-Place).

• In addition to a single cleaning method, a mixed reagent can also be considered for treatment. The cleaning process is not fixed and can be adjusted as needed.

• High-purity reagents should be selected for cleaning to avoid secondary contamination.

• Immediately after cleaning, the resin should be stored in a neutral environment.

• The determination of the cleaning status and method should be integrated with the entire process.

• After cleaning, the cleaning process should be validated to ensure that the cleaning objectives are achieved. This can be done by measuring the performance indicators of the medium, such as loading capacity and flow rate.

Case Share of Pigment Removal from Resins

Regenerative cleaning of MaXtar^® ARPA resin with residual pink pigment after CIP cleaning was performed using 1M Acetic Acid + 4% Cysteine. After cleaning, the appearance indicated that the pigment residue was completely removed.

The regeneration solution from the chromatography column after cleaning was collected and sent for VB12 content determination. The test report also indicated no VB12 present. A comparison was made between the conditions before and after the regenerative cleaning.

Left: Before cleaning; Right: After cleaning

BioLink VB12 Content Test Report:

References:

[1]Weng Zhibing, Wang Penghong, Zhao Li, et al. Study on a new method for removing stubborn pigments in chromatographic packing [J]. Journal of Food Science and Biotechnology, 2022(002):041.