The primary function of the solution selected for chromatography medium preservation is to inhibit the growth of microorganisms. Particularly after use, organic components such as protein may remain, and the risk of microbial growth will be significantly increased compared with the new medium that has not been used. Effective cleaning during the CIP and disinfection stages after media use, and the antibacterial ability of the preservation solution are equally important for bioburden control in biopharmaceutical processes.

Selection Principles for Chromatographic Media Preservation Solutions

It is not that a solution with stronger antibacterial effect is more suitable as a preservation solution for the chromatography medium. When choosing a preservation solution in the biopharmaceutical process, it is also necessary to consider: the chemical stability of the chromatography medium, the risk of preservation solution residue, the compatibility with equipment and containers, the safety and cost of the preservation solution, etc.

Common Preservation Solutions and Characteristics for Chromatographic Media

The most common preservation solution for chromatography media is 20% ethanol, which has good compatibility with almost all chromatography media and does not affect meida stability. The antibacterial effect is significant and it is easy to remove, and the 20% ethanol solution can be reduced to an acceptable range through a simple solution change. Therefore, it is the most widely used and serve as the default preservation solution for Bio-Link chromatography media upon shipment.

The biggest concern about the preservation of 20% ethanol comes from its safety. Ethanol is an organic reagent with a low explosion limit. As a medium manufacturer that meets the safety requirements of the chemical industry, Bio-Link can safely and widely use it. However, for most biopharmaceutical companies, the explosion-proof problems caused by ethanol limit its application as a medium preservation solution in production. 2% benzyl alcohol solution is a good alternative for 20% ethanol. It does not pose explosion-proof concerns. At the same time, it has good antibacterial ability and medium compatibility, and is easy to be effectively removed by liquid change.

Table 1. Stability data of a chromatographic medium stored in 20% ethanol at 40 ± 2 ℃

Figure 1. Study on the removal of 20% ethanol and 2% benzyl alcohol preservation solution in a chromatographic medium of Bio-Link

2% benzyl alcohol solution has been widely used in the preservation of protein A affinity media, but the main reason why it is less used in ion exchange and hydrophobic interaction chromatography media is the cost. In contrast, NaOH solution is a cheaper and more efficient preservation solution, and 10mM NaOH solution can effectively inhibit the growth of microorganisms.

When affinity media such as protein A are exposed to alkali for a long time, the stability of ligand will be affected, resulting in the reduction of binding capacity and shortening of life. Chromatography media types such as ion exchange and hydrophobic interaction have ligands that are simple chemical groups with good tolerance to alkali, so it is suitable to choose NaOH as the preservation solution. Bio-Link has conducted media stability research on NaOH preservation to provide users with data support for preservation.

Table 2. Research data on preservation stability of a Bio-Link chromatography medium in 0.1M NaOH

Table 3. Research data on preservation stability of a Bio-Link chromatography medium in 0.01M NaOH

Preservation Considerations for Special Media

Apart from the fact that the alkali tolerance of affinity media influences the choice of preservation solution, a category of medium primarily consisting of cation exchange media also adopt slightly different preservation solutions due to stability considerations. In addition to using 20% ethanol to inhibit bacteria, this type of medium is additionally added with different concentrations of sodium acetate. Many users have asked whether it can be stored with only 20% ethanol without adding sodium acetate.

Agarose tends to decompose at low pH. Generally, the pH stability range of agarose medium is above 2. When the pH decreases to 2, an obvious increase in Total Organic Carbon (TOC) can be detected in the supernatant. The cation exchange medium uses negatively charged sulfonic acid groups or carboxyl groups as ligands. When only water or 20% ethanol is used to preserve the medium, a large amount of hydrogen ions will accumulate at the group positions close to agarose due to the need to keep the charge neutral, resulting in a local low pH environment, thus affecting the stability of the medium.

The addition of sodium acetate can effectively buffer the local acidic environment, which is necessary for cation exchange media, Heparin affinity with negatively charged ligand, virus affinity (MaXtar VirsCap) and other media. Using NaOH as the preservation solution can also avoid local overacidity, so NaOH solution alone can be used to preserve such media without adding additional sodium acetate.

Figure 2. Research data on pH stability of a Bio-Link agarose medium

Figure 3. Schematic diagram of the principle of local overacidity in a cation exchange media

Supplementary Points for The Use of Preservation Solutions

At present, the above solutions are mostly selected for industrial-scale media preservation. Commonly used preservation solutions such as sodium azide in labrotories especially for analyze, basically do not appear in biopharmaceutical processes due to the possible impact of their residues.

Another point that needs to be noted is the potential for preservation solution to lose effectiveness. Ethanol and benzyl alcohol can volatilize after long-term storage, and the antibacterial effect can be greatly reduced when the concentration is reduced; NaOH solution can react with CO ₂ in the air, and even if the concentration is reduced, it cannot effectively inhibit the growth of microorganisms. Therefore, after selecting the storage solution, it is also necessary to verify the preservation and determine the replacement cycle of the preservation solution.

In summary, many factors need to be considered when choosing the preservation solution of chromatography media, and Bio-Link provides comprehensive data support to support the preservation of chromatography media.