SubscribeThe following article as published by Pharmaceutical International concerns Therapeutic Protein Substances.
Detergents
Detergents are organic molecules with a long, lipophilic hydrocarbon chain and a terminal hydrophilic residue, and thus exhibit surface-active properties. Detergents are categorized according to the charge of their respective hydrophilic residues as anionic, cationic, hybrid-ionic, or non-ionic detergents.
Detergents in the Field of Biotechnology
In the field of biotechnology, the applications for detergents are far-reaching. Their hybrid properties are used to solubilize, to stabilize, and, in some cases, to denature proteins. Thus detergents are used as anti-foaming agents, for cell lysis to break up bipolar lipid membranes, and in downstream processing to inactivate viruses.
Non-ionic detergents are considered «mild» detergents. In low concentration, they are used to stabilize proteins in solutions. Proteins have the tendency to aggregate at phase interfaces (liquid-air or liquid-liquid). The addition of surface-active substances such as detergents reduces surface tension, thereby inhibiting the unfolding of the protein at the interface.
Tween
As a result of their hybrid properties, detergents are also used in the formulations of therapeutic proteins. Polysorbate, better known as Tween, is most commonly utilized. Tween is a mixture of polyethoxylated sorbitol fatty acid esters with approximately 20 moles of ethylene oxide equivalent for each mole of sorbitol. Tweens are categorized as 20, 40, 60 or 80, depending on their respective fatty-acid composition. The main component of Tween 80 is oleate. In addition, the following fatty acids can be identified as impurities: myristic acid, palmitic acid, palmitolic acid, stearic acid, linoleic acid, and linolenic acid.
The chain length of the polyethoxoxylate is variable, which contributes significantly to the complexity of the molecule.
If Tween is used as an excipient in the formulation of protein therapeutics government regulations require a quantitative analysis of this excipient in the formulation as part of the release testing of the drug product. The heterogeneous composition of the polymer on the one hand, and the tendency to form micelles of various sizes on the other, make this analysis a challenge.
The ability of Tween to interact with both the protein and surfaces exacerbates this problem. Another issue requiring analysis for Tween arises when Tween is used in downstream processing, in which case Tween must then be detected as a trace impurity. Here too, the heterogeneous composition, micelle formation, and its «binding» properties complicate the analysis procedure.
Practical Solutions to Complex Problems: Determination of Tween - our Solution
Due to significant market demand for the determination of Tween in trace and low concentrations, Solvias has developed methods for Tween quantification in protein formulations that can be validated. Depending on the sensitivity requirements, LC or GC are used to quantify the principal carboxylic acid component, which is separated from the sorbitol poly-oxylate by alkaline hydrolysis during sample preparation. The quantitative analysis of Tween for a particular drug formulation under GMP quality standards typically proceeds as follows. In a feasibility study, the Solvias method is to be tested on the product-specific matrix. Thereafter, a product-specific SOP is established and the method validated.
In projects with multiple issues, such as the quantitative analysis of highly concentrated antibody formulations, the selective detection of various Tween species in a single method, and the quantitative analysis of Tween as a trace impurity, Solvias' method has proven its reliability in terms of reproducibility, robustness, selectivity and sensitivity.
Are you interested? We would be delighted to give you more detailed information about the detection of Tween, other detergents such as Triton 100®, or of anti-foaming agents like Pluronic®.
Tween 20
Tween 80