The removal of endotoxins, a potential contaminant of dialysis water and dialysate, is a very difficult task. The endotoxin removal capacity of commercial ceramic membranes with a nominal molecular weight cut-off of < 1,000 and adsorber membranes was investigated. The dead-end filtration results showed that all investigated ceramic membranes produce water meeting the European standards when challenged with low endotoxin concentrations, but only one membrane type succeeded at high endotoxin concentrations. In addition, we present preliminary analysis of the factors determining bacterial fragment removal from water of different ceramic and polymeric adsorptive membranes

Catapano, Gerardo

Czermak, Peter

Ebrahimi, Mehrdad

Hackel, Rene

K-State Research Exchange

FEASIBILITY OF CERAMIC ULTRA- AND NANOFILTRATION MEMBRANES FOR REMOVAL OF ENDOTOXINS   Peter Czermak1,2, Mehrdad Ebrahimi1, Rene Hackel1,3, Gerardo Catapano3 1University of Applied Sciences Giessen-Friedberg Wiesenstrasse 14, 35390 Giessen, Germany 2Kansas State University, Dept. of Chemical Engineering, Manhattan KS, USA 3Department of Chemical Engineering and Materials, University of Calabria, Italy   ABSTRACT  The removal of endotoxins, a potential contaminant of dialysis water and dialysate, is a very difficult task. The endotoxin removal capacity of commercial ceramic membranes with a nominal molecular weight cut-off of < 1,000 and adsorber membranes was investigated. The dead-end filtration results showed that all investigated ceramic membranes produce water meeting the European standards when challenged with low endotoxin concentrations, but only one membrane type succeeded at high endotoxin concentrations. In addition, we present preliminary analysis of the factors determining bacterial fragment removal from water of different ceramic and polymeric adsorptive membranes.   KEYWORDS ceramic membrane, charged membrane, retention efficiencies, dialysis,  water treatment   1. Introduction  The quality of water in dialysis fluid varies considerably and as a result, dialysate is often contaminated by large amounts of bacteria and endotoxins.  The latter may cross the dialysis membrane and so endotoxins are a potential contaminant of dialysis water and dialysate.  Endotoxins are lipopolysaccharides (LPS) derived from the cell membranes of Gram-negative bacteria and are responsible for organization and stability of cell membrane.  The molecular weight of endotoxin is about 10 kDa [1]. They can indirectly cause fever because they stimulate synthesis in host cells and release endogenous proteins.  Membrane properties and operating pressures can cause the bacteriological potential to favor passage of endotoxin fragments from the dialysate into the blood side and to trigger monocyte activation and cytokine production during high-flux dialysis with bicarbonate and during membrane re-use.  For these reasons, a sterile dialysate free of endotoxins and other cytokine inducing factors will likely become a standard.  Ultrafiltration across membranes made of polysulphone, polyamide or polyethylene has been shown to remove bacteria, endotoxins and their fragments and other cytokine inducing factors from water by a combined effect of filtration and adsorption.  Also, the removal of bacterial endotoxins from membrane adsorbers was shown [2].  However, use of these modules is limited by the operation time which is dependent upon polymer type and device.  Repeated Volume_II_M_Sessions.pdf   487 17.03.2008   22:02:3210th World Filtration Congress Page II - 487cleansing with aggressive agents may damage the membrane and may change the performance of the filter [3, 4].  Ceramic membranes are generally more resistant to harsh operating conditions (extremes of pH, high temperatures and pressure etc.) than polymeric membranes, and may represent an alternative for endotoxin removal.  They can be re-used many times and are easily regenerated.  Membranes will be used to reduce endotoxin content of product streams in the downstream processing of biopharmaceutical products.  Additionally, ceramic membrane ultrafiltration has been validated for the production of water meeting the requirements of pyrogen-free water for injection standards [5].  This ability can be put to immediate use in other pharmaceutical water systems that require pyrogen removal but are not limited by USP definitions of production methods—bulk active pharmaceutical production is one example.   In this paper, the adsorption capacities of different membranes for bacterial fragments such as LPS are investigated.  Additionally, we present results for the capacity of different membranes to retain endotoxins from E. coli (Serotype 055:B5)in long-term runs.  2. Materials and Methods  MembranesDifferent types of commercial tubular ceramic membranes for ultra- and nanofiltration (Table 1, Fig. 1) and different types of commercial adsorber membranes (Tab.2, Fig. 2) were investigated for endotoxin removal.  Table 1: Properties of the ceramic membranes used in this investigation  Ceramic Membranes Identity Membrane #1 Membrane #2 Membrane #3 Geometry Single channel Single channel Multiple channels Number of channels 1 1 3 Material Al2O3/TiO2/ZrO2 Al2O3/TiO2/ZrO2 Al2O3/TiO2/ZrO2 Length 250 mm 250 mm 250 mm Nominal MW Cut-Off 20 nm1,2 5 kD1 1 kD1 1 as indicated by the manufacturer; 2 average pore size   Fig.1: Module and ceramic membranes (SEM-micrograph) used. Volume_II_M_Sessions.pdf   488 17.03.2008   22:02:3210th World Filtration Congress Page II - 488To determine adsorption capacity, defined volumes of the membranes were challenged in a static experiment with endotoxin-containing solutions at different concentrations ranging from 30 to 1000 EU/mL (i.e., endotoxin unit/mL). In all experiments endotoxins from E. coli (Serotype 055:B5) were used.   To investigate the recovery capacity during long-term runs, the membranes were challenged with endotoxin-containing solutions at concentrations ranging from 0 to 1000 EU/mL (i.e., endotoxin unit/mL) over a period of at least 24 h. In all experiments endotoxins from E. coli were used (E. coli Serotype 055:B5).  Analytical method  Permeate samples were collected at intervals and assayed for endotoxins. Endotoxin concentration was measured with the kinetic turbidimetric Limulus Amoebocyte Lysate (LAL) Test. This method ensures sensitive detection of bacterial endotoxins, down to 0.125 EU/mL. In this investigation, reference shall be made to the upper limit of 0.25 EU/mL set for endotoxin concentration in dialysis water by the European Pharmacopoeia. Table 2: Properties of the adsorber membranes used (as indicated by the manufacturer)  Adsorber Membranes Identity Membrane #1 Membrane #2 Functional group R- CH2-N+- (CH3)3 R- CH2-N+- (CH3)3 Membrane height (mm) 0.08 0.06 Membrane volume with a diameter of the flat membrane of 9 cm (ml) 0.515 0.39 Average pore size (m) 0.2 0.8 Average binding capacity - 60 mg/ml (membrane volume) - BSA              Fig.2: Module and adsorber membranes (SEM-micrograph) used in this investigation.  Volume_II_M_Sessions.pdf   489 17.03.2008   22:02:3210th World Filtration Congress Page II - 4893. Results and conclusion  In static experiments LPS adsorption was significant on polymeric adsorbtive membranes, but minimal on ceramic membranes (Table 3).  Table 3: Adsorbed endotoxin in units per ml membrane volume (EU/ml) after 24 h.   Adsorber membranes Ceramic membranes Endotoxin Membrane #1 Membrane #2 Membrane #1 Membrane #2 Membrane #3 EU/ml 5600 2600 15 8 11  In dead-end filtration, significant LPS amounts permeated across ceramic membranes in the short term but were completely rejected in the long term (Table 4).  In cross-flow filtration, none of the permeate produced with ceramic membranes meets the European standard, as shown earlier [4].  Table 4: Endotoxin concentrations in the permeate (EU/ml) after 1 h filtration across the membranes. NA: not available.  Endotoxin Ceramic membranes Adsorber membranes challenge [EU/ml] Membrane #1 Membrane #2 Membrane #3 Membrane #1 Membrane #2 50 NA NA NA 1,5 0,75 100 <0,05 <0,25 <0,25 5 NA 1000 <0,05 <0,5 NA NA NA   01020304050600 100 200 300 400Endotoxin concentration [EU/ml]time[min] Fig.3: Breakthrough of one layer of adsorber membrane # 1 (membrane diameter= 9 cm, membrane volume=0.515 ml) challenged with low LPS concentration in dead-end mode (50 EU/ml LPS).   Volume_II_M_Sessions.pdf   490 17.03.2008   22:02:3210th World Filtration Congress Page II - 490Adsorber polymeric membranes are not able to keep the limit of 0,25 EU/ml even when challenged with low LPS concentrations, which may possibly be caused by binding site saturation (Figure 3).   The results show that different factors contribute to the endotoxin retention capacity of a membrane.  Adsorption to charged moieties and hydrophobic interactions with the hydrophobic membrane backbone seem to dominate in ion-exchange polymeric membranes.  However, pore size exclusion appears to play an important role in endotoxin retention by more chemically inert membranes. Acknowledgements  Work performed with a MIUR-PRIN 2004 and a VDI BVMHE research grant.   References  1. Ken-ichiro Yamamoto, Masato Matsuda , Masayo Hayama et al., Evaluation of the activity of endotoxin trapped by a hollow-fiber dialysis membrane, J Membr Sci 272 (2006) 211–216 2. Zhang R., T. Bouamama, P. Tabur and G. Zapata, Q membrane chromatography application for human antibody purification process, BioProduction, Oct. (2004) 26–27. 3. Bender H., A. Pflänzel, N. Saunders, P. Czermak, G. Catapano and J. Vienken, Membranes for endotoxin removal from dialysate: considerationson feasibility of commercial ceramic membranes, Artificial Organs 24(10) (2000) 826–829. 4. Czermak P., M. Ebrahimi and G. Catapano, New generation ceramic membranes have the potential of removing endotoxins from dialysis water and dialysate, Int J Artificial Organs 28 (2005) 694–700. 5. Reinholtz W, Making water for injection with ceramic membrane ultrafiltration, Pharmaceut Technol 9 (1995) 84–96. 6. Ebrahimi, M., R. Hackel, G. Catapano, P. Czermak: Preliminary Analysis of the Factors Determining Bacterial Fragment Removal from Water of Different Commercial Ceramic and Polymeric Membranes, Desalination 200 (2006) 496-497 Volume_II_M_Sessions.pdf   491 17.03.2008   22:02:3210th World Filtration Congress Page II - 491WFC1010th World Filtration Congress April 14–18, 2008 Leipzig, GermanyCONGRESS PROCEEDINGSVOLUME II – M-SessionsCONTENT VOLUME II M-SessionsScientific Committee II -2 – 3Session Survey II -4 – 5Congress Programme II -6 – 18Invited Lecture 2 II-19 – 29Invited Lecture 5 II-30 – 39Papers M-Sessions II-40 – 563Keyword List (Page Indicator) II -564hosted bysupported byVenue:Congress Center Leipzig · Messeallee 1 · 04356 Leipzig · GermanyCongress Secretariat:Filtech Exhibitions Germany PO Box 1225 · 40637 Meerbusch – Germanyphone: +49 (0) 2132 93 57 60fax: +49 (0) 2132 93 57 62e-mail: Info@wfc10.comweb: www.wfc10.comDiscover the Future ofFiltration & Separation10th World Filtration Congress Page II - 1

Feasibility Of Ceramic Ultra- And Nanofiltration Membranes For Removal Of Endotoxins

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Feasibility Of Ceramic Ultra- And Nanofiltration Membranes For Removal Of Endotoxins

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