**9. Experimental study to determine endotoxin LRV of a protein-free membrane under online-priming test conditions**

Test articles were high flux dialyzers Polyflux 210H and Revaclear 500, as well as medium cut-off dialyzer Theranova 500. The products were taken from regular manufacturing with standard sterilization and within their specified shelf-life. For each type of dialyzer, the test items were taken from three separate production lots. Revaclear 500, Theranova 500, and Polyflux 210H are the products with the largest membrane area of their respective product family. In this study, the product with the largest membrane area in each product family was chosen because it provides the largest interface between the dialysate and the blood side compartment. An experimental pre-study under online-priming test conditions did not indicate an impact by the membrane area between 1.4 m2 and 2.1 m2 on endotoxin retention properties and, if adsorptive retention was assumed, a saturation of adsorptive capacity was not observed for smaller and larger membrane areas. Under this presumption, the items selected for testing in this study can be considered to be representative under onlinepriming conditions for versions with smaller membrane area down to 1.4 m2 in their respective product family.

The sample number was defined to be six (6). The sample number definition was not based on a formal statistical approach. Previous studies had shown 95% confidence intervals within a range of ± 10% of the mean LRV for a similar experimental design using six samples.

The test system had two parts. The first part comprised sample generation, and the second part the sample analysis.

Sample generation was done in a benchtop experiment of filtration (**Figure 4**). Fluid from a challenge solution made of endotoxin-contaminated bicarbonate-based dialysis fluid was pumped by a peristaltic pump across a dialyzer membrane. The fluid flow direction in the dialyzer was from dialysate to blood side. The filtrate was collected on the blood side for subsequent analysis.

#### **Figure 4.**

*Schematic drawing of the filtration setup. A feed solution containing a defined amount of endotoxin is pumped through the dialyzer using a peristaltic pump. Flow direction is from dialysate to blood compartment. Unused ports of the dialyzer were blocked. The filtrate was collected on the blood side.*


*1 Volumetric measurement accuracy range within the accuracy range of the scale of suitable measurement cylinders.*

#### **Table 1.**

*Sample generation test settings and justification.*


#### *The Dialyzer as the Last Line of Protection against Endotoxins DOI: http://dx.doi.org/10.5772/intechopen.109358*

#### **Table 2.**

*Results of the experimental study. LRV was measured using the protein-free pristine membrane under onlinepriming conditions. LRV is shown for Pseudomonas aeruginosa (P. aer) and Escherichia coli (E. coli). When LRV was calculated using the lower limit of detection of the LAL assay (0.005 EU/ml) values are shown as " > ."*

**Table 1** identifies and specifies the critical settings and provides justification for the selected value.

The challenge solution was filtered directly through the dry dialyzer to simulate the clinical priming process.

A portion of each feed sample was diluted 10x with bicarbonate-based dialysis fluid. A diluted sample was needed in certain cases to bring the endotoxin concentration within the working range of the endotoxin assay. The endotoxin concentration was determined using Limulus amebocyte lysate (LAL) assay in accordance with the manufacturer's instruction and validated using local work instruction

The results obtained are shown in **Table 2**.

In conclusion, all dialyzers tested met the proposed LRV requirement of a minimal LRV of 1, for both types of endotoxin, LPS from *Pseudomonas aeruginosa* and *Escherichia coli* under conditions of online priming with dialysate infusion.
