Next-generation Triton™ X-100 replacements for pharmaceutical bioprocessing
Abstract
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. 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, 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.
As a result, there is an urgent need for new safe, sustainable and effective alternatives to Triton™ X-100. To address the needs of the biopharmaceutical industry we developed two chemically distinct detergents, Virodex™ TXR-1 and TXR-2, that 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. 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.
To facilitate their adoption in biopharmaceutical manufacturing processes, the Virodex™ detergents are compendial-grade materials manufactured to cGMP EXCiPACT standard. Functional assessment of three batches of each detergent in VI tests demonstrated excellent batch-to-batch consistency for both products. To facilitate the detection and quantification of the Virodex™ detergents in finished products and waste streams, we developed liquid chromatographic analytical methods with universal detectors capable of detecting and quantifying low parts-per-million or billion concentrations of the detergents.
Analytical detection methods were utilised to assess the affinity of the Virodex products to protein A resin, no binding was seen demonstrating the ease of depletion in standard downstream processing. 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.
Introduction
Biological therapeutics, commonly referred to as “biologics” are a class of medicines that are manufactured via large-scale cultivation of bacteria, yeast, plant or animal cells. This diverse group of medicines includes vaccines, antibodies, immune modulators and growth factors, as well as products derived from human blood and plasma. Typically, biologics are purified proteins and are differentiated from conventional modalities which are small molecule chemicals produced synthetically or purified from plants or microorganisms.1
Manufacturing of biologics is a complex process that utilises living production systems which present challenges not encountered in the manufacturing of conventional medicines. Due to the extensive use of human and animal cell lines in biologic manufacturing, these products are susceptible to adventitious microbial contaminants. Blood and plasmaderived therapies may contain blood-borne pathogens and are also susceptible to contamination. Due to their small size and difficulty to detect, viral contaminants are of particular concern. Unfortunately, there are several examples in the twentieth century of viral contamination of released vaccines and blood-derived plasma which resulted in the infection of patients receiving these treatments2.
These lessons resulted in increased regulation and the development of integrated strategies to ensure product safety. Current best practice dictates the use of three complimentary strategies (the “three pillars” approach) to prevent the release of products contaminated with viral pathogens and involves 1) section of raw materials with a low risk of viral pathogen content, 2) testing of cell banks and in-process samples for viral contaminants and 3) inactivation and removal of undetected adventitious or endogenous virus during downstream processing steps.2
Downstream processing (DSP) for biologic products can vary significantly but typically utilise one or more virus removal steps to mitigate the risk of viral contamination and ensure product safety. DSP protocols employ multiple orthogonal techniques to remove and inactivate viruses and viral particles through chromatographic purification and/or nanofiltration and virus inactivation (VI) by exposure to heat, low pH or detergent treatment (Figure 1). For biologics that are labile when exposed to heat or low pH conditions, treatment with non-ionic detergents is the preferred VI approach as non-ionic detergents do not denature proteins and can be removed from the finished product using commonly used chromatographic purification steps.3
Cell and gene therapy (CGT) products can utilise viral vectors to deliver altered genes to cells either in vivo or ex vivo to treat conditions attributed to genetic disorders. The viral vectors used for these therapies are manufactured using production cell lines, and as a result are susceptible to endogenous and adventitious viral contamination. Like protein based biologics, the risk of viral contamination is mitigated in CGT products using the three pilar strategy where detergent-based VI is an important strategy for virus removal from non-enveloped viral vectors that are not sensitive to mild detergent treatment, such as adeno-associated viruses (AAVs). AAVs are produced in the cell and detergent treatment is used to both lyse the cells to release the AAV and inactivate adventitious enveloped viruses.4
The detergent most frequently used for VI and cell lysis in biologic and CGT manufacturing is tert-octyl phenol ethoxylate, which is more commonly known by the Dow Chemical Company trade name Triton™ X-100. Triton™ X 100 is a non-ionic surfactant with a hydrophilic polyethylene oxide chain and a hydrophobic alkylated aromatic group (Figure 2).5 This detergent has found widespread use in pharmaceutical industries with diverse applications including endotoxin removal, VI, cell lysis, and membrane permeation.6
However, as research on Triton™ X-100 advanced, concerns emerged regarding the safety of its degradation products. Like other alkyl phenolic compounds, the degradation products from Triton™ X-100 exhibit longlasting estrogenic properties in fish, birds, and mammals, causing disruptions to their endocrine systems.7 In response to these environmental and health concerns, the European Chemicals Agency (ECHA) took regulatory action and included Triton™ X-100 in the Authorisation List (Annex XIV) in 2017 under the EU REACH regulation, leading to a total ban from 2021 on its use in products manufactured in Europe and defined limits on those manufactured elsewhere but exported to Europe that contain Triton™ X-100.8
Despite these regulatory developments, some pharmaceutical manufacturers outside Europe continue to employ Triton™ X-100 in biologic and CGT manufacturing processes. However the latest version of the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Humans Use (ICH) Guideline Q5A(R2) for the Viral Safety Evaluation of Biotechnology Products derived from Cell Lines of Human or Animal Origin.9 clearly highlights concerns about the hazards posed by Triton™ X-100’s degradation products and recommends the adoption of other commercially available detergent alternatives possessing similar physicochemical properties and equivalent VI capabilities.9 Given that ICH guidelines are recognized by health authorities in Europe (EMA), the United States (FDA), Japan (PMDA), and other countries, phasing out of Triton™ X-100 is anticipated in regions beyond Europe, likely leading to a global phasing out of Triton™ X-100 and potential ban in many key regions. To avoid timeconsuming and costly modifications to approved manufacturing processes it is imperative that both global and regional biopharmaceutical and CGT manufacturers proactively adopt alternative detergents for VI and cell lysis to avoid filing for process changes in the future.
To enable the transition away from Triton™ X-100, alternative detergents should provide equivalent or better performance in DSP operations (cell lysis and VI), while possessing similar physiochemical properties to Triton™ X 100. Alternative detergents should be easily handled (i.e., not highly viscous) and readily water soluble under normal processing temperatures, be compatible with biopharmaceutical products and be readily removable by common DSP techniques. Sensitive analytical detection methods should also exist to quantify residues in finished products and waste streams. To facilitate their rapid adoption in cGMP manufacturing processes Triton™ X-100 alternatives should be available as compendial and cGMP grades. Detergent alternatives should also be sustainable and free of potential safety or environmental concerns. This is the basis on which we have developed our Virodex™ range of detergents for viral inactivation and cell lysis: sustainable, compendial, cGMP EXCiPACTmanufactured, and REACH-compliant materials.
See the full technical brochure on “Next-generation Triton™ X-100” here
(click the picture to download the brochure)
Source: Croda technical brochure “Next-generation Triton™ X-100”