The R&D Notebook
RD006
Qualification study of a point-of-use ultrafiltration device for the production
of pyrogen-free ultrapure water
Stéphane Mabic, Daniel Darbouret and Ichiro Kano
Research & Development, Laboratory Water Division, Millipore S.A., Saint-Quentin-en-Yvelines, France.
Abstract: Sensitive biological experiments, such as cell culture, require ultrapure water, free of
contaminants like pyrogens. To obtain pyrogen-free water, Pyrogard TM D, a polysulfone hollow
fiber ultrafilter embedded in an ABS housing, was designed. Qualification and validation of the
ultrafiltration device are presented in this study. No significant amounts of extractables from the
Pyrogard D cartridge were detected using ICP-MS and ion chromatography methods. Analysis
of TOC also showed very good performances regarding organic leaching. Data obtained from
the air diffusion test were correlated to the removal of endotoxins over a wide range of pyrogen
concentrations. The Pyrogard D ultrafilter was fully efficient over a five-week period, and endotoxin
concentrations in the filtrate were consistently below the detection limits (0.001 EU/mL).
Sequential treatment of water using activated carbon,
electrodeionization, UV-photooxidation and microfiltration
affords sufficiently pure water for many uses.
Specific applications, however, require an additional
purification step. Among these water contaminants are
endotoxins, which need to be removed with special
care in order to perform biological tests under optimum
conditions. The major and most significant components
of endotoxins are lipopolysaccharides (LPS) from Gram
negative bacteria walls.1 The LPS can be formally
divided into three substructures: a hydrophobic and
highly conservative structure formed by Lipid A
(non-polar), a core oligosaccharide conservative part,
and a variable heteropolysaccharide surface structure,
the O-antigen (Figure 1).2,3 The two glycosidic parts
can vary largely in structure and size, depending on the
bacterial strain and species. This leads to a wide range
of molecular weights, 3,000 to 25,000 Da (average
10-12 kDa), and a variety of biological activities.
Introduction of LPS into the blood or spinal fluid causes
toxic responses and induces the development of fever
(i.e. a “pyrogenic” effect). The words endotoxin and
pyrogen have become used in a wider sense to
designate the active LPS molecules. Due to their
molecular structure, LPS are particularly stable molecules,
withstanding autoclaving procedures (3 h at 121 °C).
Exposure at 180 °C for four hours reduces the pyrogen
concentration by a factor of 1,000 (Log reduction
value, LRV, is 3). Owing to the branching of negatively
charged phosphate groups on glycosidic residues, LPS
pair readily with bivalent cations (Ca+2, Mg+2). This
results in aggregation of the LPS and in the formation of
micelles and vesicles, generating superstructures of
100,000 to 1,000,000 Da. Removal of these superstructures
with an integrated filtration device reduces
pyrogen levels to < 0.02 endotoxin units (EU)/mL. This
type of built-in filter, however, requires careful
maintenance on a regular basis. It was particularly
challenging, therefore, to design a cartridge that could
be easily connected at the point-of-use of a water
A publication of the Laboratory Water Division of Millipore
Introduction
