Bacterial endotoxins (acidic lipopolysaccharides (LPS), fever producing endotoxin pyrogens) are approximately 10 kDa in size (with potential aggregates ~10-fold larger in certain chemistries), located in the cell walls of gram-negative bacteria, and can be shed by the bacteria in their active/growing state or upon cell death. Endotoxins do not act directly against cells or organs but through activation of the immune system, especially monocytes and macrophages, thereby enhancing immune responses1. Their extremely heat- and pH-stable structure consists of hydrophobic fatty acids surrounded by a moiety of hydrophilic polymeric carbohydrate molecules that make endotoxins difficult to remove or inactivate2,3. Divalent cation (Ca2+, Mg2+, Ba2+) salts help stabilize aggregates4.
In laboratories, endotoxins can originate in test samples, media, water, saline, and buffers. High contamination levels can increase the likelihood of unwanted false readings, especially in cell-based assays. Many researchers in drug discovery are finding ways to lower endotoxin levels in their protein purification schemes and cell-based assays using detergents (Triton X-114), filtration, or bead-based chromatography to separate LPS from protein5. For ultrafiltration (UF) devices, depyrogenation pre-cleansing techniques consisting of strong sodium hydroxide (NaOH, inorganic base) treatments can be utilized, provided the device has compatibility with the base.
This focus of this review is the compatibility of Microcon® polyether sulfone (PES) ultrafiltration devices using a 1 N NaOH depyrogenation technique with a 1-hour soaking method and subsequent 2-minute washes. Device performance was evaluated by protein retention after processing.
The performance of Microcon® PES 10K and 30K ultrafiltration devices was evaluated after a standard depyrogenation technique using a base solution of 1 N NaOH with 60 minutes of exposure to the internal upstream filter device. The input and flow through of the primary spin and subsequent washes were measured by pH. The data showed ~10- to 15-fold reduction in alkalinity after washing. The treated filters had retention values >98% and recoveries >92%, indicating that the device maintained its function to concentrate a 67 kDa bovine serum albumin protein.
For applications in which the removal or reduction of endotoxins is essential, we describe a method for fast and reliable depyrogenation of Microcon® PES devices using a sodium hydroxide soaking method. Additionally, it could be shown that 1 N NaOH-treated Microcon® PES devices displayed equivalent performance to untreated devices.
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