Endotoxin Control in Gelatin and Collagen Peptides

Gelatin and collagen peptides are used across a wide range of industries, including food, pharmaceuticals, medical materials, cosmetics, and biotechnology/regenerative medicine. Their functionality—such as gel formation, water retention, and film-forming properties derived from collagen—makes them highly versatile materials.

At the same time, as natural proteins derived from animal-based raw materials (e.g., porcine skin, bovine bone/skin, fish skin), they carry a risk of contamination and residual endotoxins originating from Gram-negative bacteria present in raw materials and manufacturing environments.

In particular, collagen peptides tend to be used at higher concentrations in final formulations due to their low molecular weight and high solubility. As a result, the total endotoxin load originating from the raw material can become relatively high.

Although endotoxins may not pose a concern in some applications, depending on the use environment and route of human exposure, they can impact safety, functionality, and overall product reliability. 

For example, concerns related to endotoxins may arise in the following areas:

Particularly in life science applications, the use of low-endotoxin gelatin and collagen peptides is essential.

Reducing endotoxins in gelatin and collagen peptides is difficult to achieve through conventional processes, such as washing, filtration, and sterilization, alone. Instead, it is essential to combine dedicated removal steps depending on the intended application (e.g., pharmaceutical, medical, or biotechnology use).

In particular, gelatin is a high-molecular-weight, high-viscosity material. Endotoxins can form aggregates or bind to proteins; therefore, removal efficiency and reproducibility are highly dependent on process conditions. 

Ultrafiltration (UF) is a membrane-based separation method that relies on size exclusion. Under suitable conditions, it can retain gelatin while reducing endotoxins.

Because LPS can form micelle-like aggregates in aqueous systems, selecting an appropriate membrane (e.g., ~100 kDa cut-off) can enable partial removal.

However, several technical challenges arise when processing gelatin solutions:

• Membrane fouling due to high viscosity 
  High-concentration or low-purity gelatin solutions tend to form a gel layer on the membrane surface, leading to fouling. This reduces process efficiency and increases cleaning frequency, resulting in higher operational costs. 

• Dissociation and membrane permeation of lipopolysaccharides 
  Changes in pH, salt concentration, or temperature may cause LPS aggregates to dissociate into smaller monomers (~10–20 kDa), which can pass through the membrane and remain in the final product. 

• Interaction between gelatin and lipopolysaccharides 
  Gelatin, as a protein, can interact with LPS through hydrophobic and electrostatic interactions. Strong binding may trap LPS within gelatin molecules, preventing separation even when size differences exist.

• Material degradation due to shear stress 
  High pressure and high flow rates required for filtering viscous solutions can introduce shear stress, potentially affecting gelatin’s higher-order structure and altering its properties, such as gel strength. 

In addition, large-scale processing requires extensive membrane area, frequent cleaning, and strict control of operating conditions, all of which increase cost.

For these reasons, UF is typically not used as a standalone solution but rather in combination with pretreatment (e.g., pH, salt, temperature control) or other purification methods.

Ion exchange removes negatively charged LPS by adsorption onto anion exchange media (resins or membranes). It is a well-established industrial method with high removal potential. In some cases, endotoxin levels have been reduced to injection-grade water levels (<0.25 EU/mL).

However, in gelatin and collagen peptide systems, improper condition design can lead to several issues:

• Adsorption and yield loss depending on the isoelectric point (pI) 
  Gelatin properties vary depending on processing type (Type A: pI 7–9, Type B: ~pI 5). When operating conditions cause gelatin to carry a negative charge, it may adsorb onto the ion exchange medium along with LPS, substantially reducing yield. 

• Scale-up challenges due to high viscosity and polydispersity 
  Gelatin solutions exhibit high viscosity and broad molecular weight distribution, leading to pressure drops and uneven flow in columns, which limits industrial scalability. 

• Process complexity depending on raw materials 
  Lipopolysaccharides removal behavior depends strongly on raw material origin, salt concentration, and pH. This requires precise optimization for each batch, increasing process complexity and reducing reproducibility. 

To address these issues, process design often includes controlled salt addition (e.g., NaCl) to suppress excessive gelatin adsorption while maintaining LPS removal.

Although endotoxins are highly heat-stable, extreme conditions can partially degrade or reduce them. For example, dry heat depyrogenation (e.g., 250°C for 30 minutes) is used for equipment sterilization.

However, applying such conditions to gelatin leads to the following: 

• Hydrolysis and molecular degradation 
  Excessive heat or extreme pH conditions break down the gelatin backbone, reducing gel strength and viscosity. 

• Irreversible changes in physicochemical properties 
  Denaturation and chemical modification can alter pI, color, and transparency, substantially impacting product performance. 

As a result, this method is generally limited in practical use. 

This approach uses nonionic surfactants to dissociate protein–LPS complexes, allowing LPS to form micelles that can be captured and removed using adsorbents.

Although effective in principle, several challenges limit industrial application:

• Residual surfactant risk 
  Complete removal of surfactants from high-viscosity gelatin solutions is extremely difficult. Residual surfactants pose major safety concerns, particularly in pharmaceutical and cell culture applications. 

• Process variability and scale-up challenges 
  Phase separation requires precise temperature control and uniform mixing. In high-viscosity systems, uneven heat transfer can lead to variability in endotoxin removal efficiency. 

• Separation and yield issues 
  Handling and filtering high-viscosity slurries containing adsorbents increases equipment load, reduces throughput, and causes product loss. 

Despite its high removal potential, this method requires careful process design and strict quality control.

Activated carbon can remove endotoxins through hydrophobic interactions with the lipid A component.

However, there are some drawbacks:

• Non-specific adsorption and yield loss 
  Gelatin and collagen peptides also contain hydrophobic residues and may be co-adsorbed, reducing yield and causing variability in material properties. 

• Limited adsorption capacity 
  Activated carbon has a finite capacity and may be insufficient for materials with high initial endotoxin levels. 

• Process burden 
  Additional solid–liquid separation steps increase processing complexity. 

Therefore, this method is often used as a supplementary step rather than a primary solution.

Summary of Purification Methods and Key Challenges

Nagase ChemteX has developed a ligand-specific adsorption technology that enables the targeted removal of endotoxins while maintaining the functional integrity of the protein matrix.

This adsorption technology makes it possible to remove endotoxins from gelatin and collagen peptides efficiently. Because the process is less affected by raw material lot variation or material properties, such as molecular weight distribution and viscosity, Nagase ChemteX offers multiple grades of low-endotoxin gelatin (Arcofeliz™ GE series) and low-endotoxin collagen peptides (Arcofeliz™ CP series), allowing proposals tailored to specific applications, such as pharmaceutical, medical, and biotechnology use.

Nagase ChemteX’s low-endotoxin gelatin, produced using this proprietary selective adsorption method, offers the following advantages. In addition to very low endotoxin content and high purity, gel strength and molecular weight can also be tailored to customer requirements.

• Preservation of gelatin properties (e.g., gel strength) 
  The process does not require harsh heating, extreme pH adjustment, or high-pressure filtration. As a result, the original molecular weight distribution, gel strength (Bloom value), and viscosity characteristics of gelatin can be maintained. 

• No surfactants or antibiotics used 
  Because the process does not rely on hard-to-remove surfactants or polymyxin B, the risk of residual components causing cytotoxicity or immunogenicity is minimized. This makes the materials suitable even for applications requiring a high level of safety, such as regenerative medicine and injectable formulations. 

• Stable process independent of raw material lot variation 
  The adsorption design is less affected by gelatin-specific factors, such as isoelectric point (pI) and molecular size, helping to suppress lot-to-lot variation and maintain stable low-endotoxin quality.