Removal Solutions for Endotoxins in Bioproducts Via Chromatography

Endotoxins are the primary pyrogenic contaminants in bioproducts. Even trace amounts of endotoxin entering the human body can cause fever, diarrhea, vasodilation, and even lead to fainting or death.

To minimize endotoxin contamination and maximize its removal, it is essential to optimize the preparation processes of bioproducts.

Endotoxins exhibit significant variability depending on their source, and even within the same sample, different endotoxin molecules can vary greatly.

This variability poses significant challenges in endotoxin removal. Effective endotoxin removal methods should consider the specific characteristics of both the bioproduct and the endotoxin.

Chromatography is currently one of the most effective means for removing endotoxins.

In developing chromatography processes, different additives can be added to bioproducts that interact with endotoxins to dissociate or reduce these interactions, enhancing endotoxin removal.

Endotoxin Properties

Endotoxins are lipopolysaccharides consisting of three structural regions: lipid A, the core oligosaccharide, and the specific polysaccharide chain. The monomer molecular weight ranges from 10 kDa to 20 kDa, forming aggregates in different aqueous solutions that can reach up to 1000 kDa. The complex and heterogeneous structure of endotoxins enables multiple interactions with proteins in solution, significantly complicating their removal.

Chromatography Techniques for Endotoxin Removal

Based on different properties of endotoxins, we can use various chromatography resins such as ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and multi-model chromatography to reduce and eliminate endotoxins in biological products.

1. Ion Exchange Chromatography

Endotoxins have an isoelectric point (PI) of around 2, resulting in a high negative charge in buffer systems with pH 4–8. They usually bind to positively charged groups on anion exchange resins, which can be removed at high salt concentrations. At pH 4, endotoxins retain their negative charge and cannot bind to cation exchange resins. However, in some cases, cation exchange resins can be used to allow the target product to bind while letting endotoxins flow through.

Non-ionic surfactants like Triton X-100, Triton X-114, and additives such as arginine and histidine help dissociate the interaction between endotoxins and sample molecules. For bioproducts where endotoxins interact with sample molecules, resulting in poor removal efficiency during ion exchange chromatography, adding non-ionic surfactants and amino acids during the wash step can help reduce endotoxin levels in the eluted product.

MaXtar® Q HR chromatography resin from BioLink, when combined with arginine during the wash step in a recombinant protein project, significantly reduced endotoxin levels in the bioproduct (see Table 1).

Table 1: Endotoxin Removal Efficiency of MaXtar® Q HR Chromatography

2. Hydrophobic Interaction Chromatography (HIC)

Due to their hydrophobic properties, endotoxins tend to aggregate at high salt concentrations (greater than 1.5 M ammonium sulfate). Aggregated endotoxins do not bind to hydrophobic resins, allowing some unaggregated endotoxins to bind tightly and become difficult to elute. During loading, aggregated endotoxins can flow through, while unaggregated endotoxins remain strongly retained and can be removed using a salt gradient for elution.

Adding non-ionic surfactants, arginine, and histidine aids in reducing the interaction between endotoxins and proteins. By utilizing the differing hydrophobicities of endotoxins and proteins, HIC can effectively remove endotoxins through gradient elution. MaXtar® Butyl HR hydrophobic resin from BioLink demonstrated effective endotoxin removal in a viral project (see Table 2).

Table 2: Endotoxin Removal Effectiveness of MaXtar® Butyl HR Chromatography

3. Gel Filtration Chromatography

Endotoxins can form aggregates with a molecular weight of up to 1000 kDa in certain aqueous solutions, which differ significantly from protein molecular weights. Gel filtration can separate these aggregates from proteins based on size, effectively removing endotoxins.

4. Multi-model Chromatography

The multi-model chromatography resin MaXtar® MMA and MaXtar® MMC exhibit multifunctional properties, including electrostatic interactions, hydrogen bonding, and hydrophobic effects. This dual action of ion exchange and hydrophobic interactions allows for a broader range of charge binding and selectivity in the ion exchange chromatography mode. Proteins are loaded under specific salt concentrations (0–0.5M NaCl) and can be eluted using both salt and pH gradients. In the hydrophobic chromatography mode, the binding is stronger compared to single hydrophobic media, providing enhanced selectivity for the removal of endotoxins.

Combined Chromatography Techniques for Endotoxin Removal in Bioproducts

While individual chromatographic methods can optimize endotoxin removal, they may not meet the stringent control requirements for endotoxins in bioproducts. Therefore, a combination of multiple chromatography techniques is often necessary.

To remove endotoxins using combined chromatography, it's essential to consider the characteristics of the endotoxins and select suitable chromatography methods that complement each other effectively. In a recombinant protein project, we optimized the process route using four chromatographic methods, focusing on controlling purity and endotoxin levels. Each step improved purity while further removing endotoxins, resulting in a comprehensive control strategy (see Table 3).

Table 3: Purification Process Data for a Recombinant Protein Project

Endotoxins in Bioproducts

Endotoxins are a key quality attribute for bioproducts. To control and remove endotoxins during chromatography purification, appropriate cleaning methods should be employed for relevant materials:

Cleaning and regeneration of chromatography resins: Typically performed using 0.5M NaOH and 1-2M NaCl for cleaning in place (CIP).

Removal of endotoxins from tubing or containers: For materials like glass, plastic, and silicone tubing, it is recommended to soak in 0.1-0.5M NaOH for over 4 hours.

Treatment of glass and metal products: Generally, heating at 180°C for 3-4 hours or at 250°C for 1-2 hours is used.

Table 4: Characteristics of Resin for Endotoxin Removal Based on Different Principles