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EXCLUSIVE WHITEPAPER: Next-generation Triton™ X-100 replacements for pharmaceutical bioprocessing

Triton™ X-100 (tert-octyl phenyl ethoxylate) is used extensively in the manufacture of biologics, plasma-derived products and cell and gene therapies (CGTs). This non-ionic detergent is used because it readily disrupts biologic membranes while leaving proteins unharmed. As a result, it is widely used for cell lysis applications and to inactivate adventitious lipid-enveloped viruses that may contaminate biologic manufacturing processes that employ human and animal cell lines. However, degradation of Triton™ X-100 in the environment results in the formation of endocrine disrupting by-products that are toxic to aquatic organisms. 
  
As a result of its harm to the environment, the European Chemicals Agency (ECHA) added Triton™ X-100 to the list of Substances of Very High Concern (SVHC) in 2017, leading to a complete ban, except for a small number of publicly listed exemptions on the use of this chemical in the European Union as of 4th January 2021. The ban of Triton™ X-100 is anticipated to be adopted by other jurisdictions in the near future. Consequentially, there is now an urgent need for new safe, sustainable and effective alternatives to Triton™ X-100.

Explore some snippets from our whitepaper...

Virodex™ as a solution to the ban of Triton™ X-100 

To address the needs of the biopharmaceutical industry, we developed two chemically distinct detergents, Virodex™ TXR-1 and Virodex™ TXR-2. To facilitate their adoption in biopharmaceutical manufacturing processes, the Virodex™ detergents are compendial-grade materials manufactured to cGMP EXCiPACT standard. Both Virodex™ detergents show equivalent or better cell lysis and virus inactivation (VI) activity than Triton™ X-100. The Virodex™ detergents were also compared to three competitor products in VI tests and were shown to outperform the competitors in this important application (Fig.1). 

XmuLV VI by three competitors compared to Virodex™ TXR-1 and TXR-2 and Triton™ X-100. Competitor products, Virodex™ detergents and Triton™ X-100 were tested at 22 °C at a concentration of 0.1 % (v/v), unless indicated otherwise. The dotted red line indicates the LRF at which no additional virus can be detected. Figure 1 VI by three competitors compared to Virodex™ TXR-1 and TXR-2 and Triton™ X-100. All detergents were tested at 22 °C at a concentration of 0.1 % (v/v), unless indicated otherwise. The dotted red line indicates the log-reduction factor (LRF) at which no additional virus can be detected. 

Virodex™ and biologic product integrity 

The Virodex™ detergents are non-denaturing detergents that do not affect the structure of proteins, making them ideal reagents for processes that generate protein-based therapeutics or non-enveloped viral vectors used in CGT products. 

Figure 2. XmuLV VI by three competitors compared to Virodex™ TXR-1 and TXR-2 and Triton™ X-100. Competitor products, Virodex™ detergents and (THERE SHOULD BE A SPACE BETWEEN “detergents” and “and”)Triton™ X-100 were tested at 22 °C at a concentration of 0.1 % (v/v), unless indicated otherwise. The dotted red line indicates the LRF at which no additional virus can be detected.
 

Virodex™ TXR-1 and TXR-2 do not affect the enzymatic activity of secreted embryonic alkaline phosphate (SEAP) activity at concentrations up to 2.5 %, and their protein compatibility is equivalent to that of Triton™ X-100. Good protein compatibility is consistent with the non-ionic nature of the Virodex™ detergents. The anionic detergent sodium dodecyl sulphate (SDS) shows strong enzyme inactivation at concentrations > 0.3 % (Fig.2).

Figure 2 Activity of SEAP after detergent treatment: Expression of SEAP by HEK-293T cells was induced by treatment with tumor necrosis factor alpha (TNFa) and 100 % activity was defined as SEAP activity in untreated controls (pink dashed line). Base-line SEAP activity (0 %) was determined using cells that were not induced with TNFa (pink dotted line). Following TNFa activation (24 h) cells were treated with detergents for 2 h at 37 °C and then an aliquot of the cell culture medium was mixed with HEK-Blue Detection medium (Invivogen) and incubated for 24 h at 37 °C and 5 % CO2.

Enzymatic activity was monitored by measuring the absorbance at 620 nm using a Cytation 5 plate reader (BioTek). 

Compatibility with traditional Protein A chromatography for detergent removal from final drug product  

Analytical detection methods were utilised to assess the affinity of the Virodex products to protein A resin, wherein no binding was seen demonstrating the ease of depletion in standard downstream processing (Fig.3).

Protein A column affinity of Virodex™ TXR-1 and TXR-2. Protein A resin affinity was assessed using MabCaptureC™ MiniChrom columns (ThermoFisher). Virodex™ detergents in chromatography eluates was measured by HPLC-ELSD. The limit of quantification was 5 ppm for Virodex™ TXR1 and 10 ppm for Virodex™ TXR-2. One CV was 1 mL.

Figure 3 Protein A column affinity of Virodex™ TXR-1 and TXR-2. Protein A resin affinity was assessed using MabCaptureC™; MiniChrom columns (ThermoFisher1). Virodex™ detergents in chromatography eluates was measured by HPLC-ELSD. The limit of quantification was 5 ppm for Virodex™ TXR-1 and 10 ppm for Virodex™ TXR-2. One column volume (CV) was 1 mL. 

Choose Virodex™: Effective, sustainable, REACH compliant alternatives to Triton™ X-100 

The Virodex™ chemistries are not on the ECHA list of SVHC and have several positive sustainability characteristics that set them apart from Triton™ X-100. The results presented here clearly demonstrate that Virodex™ TXR-1 and TXR-2 are high performance, safe and sustainable replacements for Triton™ X-100. Read our full Technical Whitepaper to learn more about our Virodex™ range: sustainable, compendial, cGMP EXCiPACT-manufactured and REACH compliant replacements for Triton™ X-100. 

 

1. https://www.fishersci.fi/shop/products/mabcapturec-minichrom-column/30019344 

Triton is a trademark of The Dow Chemical Company or an affiliated company of Dow.

Next-generation Triton™ X-100 replacements for pharmaceutical bioprocessing

Download Croda Pharmas latest whitepaper: Next-generation Triton™ X-100 replacements for pharmaceutical bioprocessing
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Virodex TXR-1

Virodex™ TXR-1

Virodex™ TXR-1 é um surfactante não iônico de biodegradação rápida para inativação viral e lise celular, atendendo às necessidades de aplicações biofarmacêuticas como um substituto eficaz e seguro...
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Virodex TXR-2

Virodex™ TXR-2

Virodex™ TXR-2 é um surfactante não iônico com 40% de base biológica para inativação viral e lise celular, atendendo às necessidades de aplicações biofarmacêuticas como um substituto eficaz e seguro...
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