Chemical compatibility of 1 N sodium hydroxide
with Microcon® PES filters

Bacterial Endotoxins and Endotoxin Removal

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.

 

Evaluation of Sodium Hydroxide Chemical Compatibility of Microcon® PES Filter Devices

Materials:

  • Microcon® PES 10K and 30K filter devices (MilliporeSigma #MPE010025 and #MPE030025)
  • Sodium Hydroxide (NaOH), 1 N (MilliporeSigma, #SX0607H)
  • Milli-Q® water, cell culture grade (MilliporeSigma)
  • Probumin® bovine serum albumin (BSA) (MilliporeSigma #821001)
  • Spectrophotometer (BioTek)
  • UV microplates (MilliporeSigma #CLS3679/Costar #3635)
  • Microcentrifuge with rotor for 1.5 mL tubes (Heraeus™ Pico™)
  • Standard pipettes and tips
  • pH meter with appropriate standards
  • Timer

Protocol:

  1. Measure initial pH of 1 N NaOH.
  2. Apply 0.5 mL 1 N NaOH to Microcon® PES devices. Incubate for 1 hour at room temperature.
  3. Centrifuge at 14,000 x g for 2 minutes. Analyze pooled (n = 4) filtrate for pH.
  4. Wash and centrifuge 2X, measuring pH in filtrates after each wash.
  5. Add 0.5 mL of 1 mg/mL BSA in PBS, pH 7.0 (~67 kDa).
  6. Centrifuge at 14,000 x g (7 minutes for Microcon® PES 10K device; 5 minutes for Microcon® PES 30K device).
  7. Analyze filtrate and retentate for BSA concentration and calculate recovery based on input material (interpolated from standard curve measuring absorbance at 280 nm).

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.

 

Step
Filtration device
Microcon® PES 10K device Microcon® PES 30K device
No treatment 1 N NaOH No treatment 1 N NaOH
Time (min) Time (min) pH Time (min) Time (min) pH
Incubation with 1 N NaOH x 60 13.28 x 60 13.28
Primary spin x 2 13.28 x 2 13.28
Wash 1: Water x 2 13.17 x 2 12.92
Wash 2: Water x 2 12.31 x 2 11.60
Challenge: 1 mg/mL BSA 7 7 7.01 5 5 7.01
Total process time (min) 7 73 x 5 73 x

Table 1: Device filtrates were pooled (n = 4) and assayed for pH during each wash cycle to demonstrate that, even when the pH was lowered, there were no negative effects of NaOH on the flow rate of Microcon® PES devices. The baseline pH of NaOH was measured to be 13.28, and pH of the filtrates decreased with subsequent washes.

 

Result
Performance
Microcon® PES 10K device Microcon® PES 30K device
No treatment 1 N NaOH No treatment 1 N NaOH
Avg. value Std. Dev. Avg. value Std. Dev. Avg. value Std. Dev. Avg. value Std. Dev.
Input volume (mL) 0.500 0.000 0.500 0.000 0.500 0.000 0.500 0.000
Final concentrate volume (mL) 0.056 0.007 0.043 0.007 0.080 0.002 0.065 0.001
Concentration Factor (X) 9.0 1.0 12.0 2.1 6.0 0.2 8.0 0.2
Retention efficiency (%) 98.8% 0.0% 98.0% 0.0% 98.8% 0.0% 99.6% 0.0%
Recovery (%) 94.7% 2.0% 92.7% 1.9% 95.2% 1.7% 95.2% 1.2%

Table 2: Performance of Microcon® PES filter devices pre-treated with 1 N NaOH or untreated.

 

Use of Sodium Hydroxide for Cleaning and Sanitizing Microcon® PES Filter Devices

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.

 

References

  1. Pérola O. Magalhães, A. M.-Y. Methods of endotoxin removal from biological preparations: a review. J Pharm Pharmaceut Sci. 2007;10(3):388-404.
  2. SK., S. Endotoxin detection and elimination in biotechnology. Biotechnol. Appl. Biochem. 1986;1:5-22.
  3. Reichelt P, S. C. Single step protocol to purify recombinant proteins with low endotoxin contents. Protein Expr. Purif. 2006;46:483-488.
  4. Garidel, P. R. Divalent cations affect chain mobility and aggregate structure of lipopolysaccharide from Salmonella minnesota reflected in a decrease of its biological activity. Biochimica et Biophysica Acta. 2005, Oct;1715(2):122-31.
  5. Adam O, V. A. A non-degradative route for the removal of endotoxin from exopolysaccharides. Anal. Biochem. 1995;225:321– 327.