Method for qualifying microbial removal performance of 0.1 micron rated filters. Part I: characterization of water isolates for potential use as standard challenge organisms to qualify 0.1 micron rated filters.

Although 0.1 microm rated filters intended for pharmaceutical sterilization applications have been commercially available for at least 15 years, there is no industry-wide standard for qualifying the microbial removal performance of these filters. In this article, we report on the bacterial challenge methodology used to screen four bacterial species for potential utility as a standard challenge organism to qualify 0.1 microm rated filters. These isolates were, in their natural state, demonstrated to penetrate 0.2/0.22 microm rated filters in prior studies. In the screening challenges described in this study, three out of these four candidates tested demonstrated consistent penetration of one 0.22 microm rated filter type tested (when cultured in a low nutrient medium under standard laboratory conditions). These included 6204-22 (FAME ID Acidovorax avenae citrulli), 6266-15 (FAME ID Comamonas acidovorans), and 6266-34 (FAME ID Hydrogenophaga pseudoflava). Of these, H. pseudoflava (6266-34) was chosen for additional experiments with other 0.2 microm rated filter membranes. In total, seventeen 0.2 and 0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from three different filter manufacturers were tested. H. pseudoflava penetration was observed for every filter tested. Under the same challenge conditions, H. pseudoflava was consistently retained by a 0.1 microm rated hydrophilic PVDF (polyvinylidenefluoride) filter with a specified high titer reduction claim for Acholeplasma laidlawii. In order to ensure selection of the most stable penetrative phenotype (i.e., select for nonrevertants), H. pseudoflava was subjected to three rounds of "filter cloning," and these results are described herein. The advantages of using H. pseudoflava for qualifying the microbial removal performance of 0.1 microm rated filters are also discussed.     

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part II: preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for use as a standard challenge organism to qualify 0.1 micron rated filters.

In this article, we report on the preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for potential use as a standard challenge organism to qualify 0.1 microm rated filters. Filter-cloned H. pseudoflava (ATCC 700892) was easily cultured in a low nutrient broth (R2A broth) under standard laboratory conditions, reaching high titers of 10(8)-10(9) cfu/mL within 48-65 hours of incubation at 25+/-5 degrees C. Under these conditions, H. pseudoflava is a rod-shaped bacterium, averaging 0.25+/-0.03 microm by 1.65+/-0.35 microm, and appears to be smaller than Brevundimonas diminuta in width (0.31+/-0.03 microm), but somewhat longer in length (0.88+/-0.19 microm), which may partly explain the observed penetration. In total, thirty-five 0.2/0.22 microm rated filter discs, spanning five different "sterilizing grade" filter types from two different filter manufacturers were challenged with H. pseudoflava. In all cases, H. pseudoflava was shown to consistently penetrate every 0.2/0.22 microm rated filter disc tested. These tests also spanned three different challenge durations, including short-term challenges (30-40 minutes), and two different challenge fluids. The use of serial (double) 0.22 mm rated filters, which is a common industry practice to reduce the prefiltration bioburden to the final "sterilizing" filter, was also shown to be inadequate to fully retain H. pseudoflava under the challenge condition used. In contrast, two different 0.1 microm rated filter types functionally qualified with a specified high titer reduction claim for Acholeplasma laidlawii, were shown to consistently and fully retain H. pseudoflava, and retention by these two filter types was shown to be robust and independent of the challenge duration.     

Retention of water-borne bacteria by membrane filters. Part I: Bacterial challenge tests on 0.2 and 0.22 micron rated filters

The results of bacterial challenge tests conducted on several 0.2 and 0.22 micron rated "sterilizing grade" filter cartridge types with bacteria from a natural water source are presented. Eight different 0.2/0.22 micron rated "sterilizing grade" filter types from four different filter manufacturers, claimed to be capable of retaining Brevundimonas diminuta at a challenge level of 10(7) CFU/cm2, were tested. The filters tested included nylon 6.6 and polyamide filters from two manufacturers, modified or hydrophilic PVDF filters from two manufacturers, modified or asymmetric PES filters from three manufacturers, and cellulose acetate filters from a single manufacturer. Consistent bacterial penetration was observed, over the 18-24 h challenge period, for all twenty-five integral 0.2 and 0.22 micron rated filter cartridges tested, at challenge levels of about 10(1)-10(4) CFU/cm2, indicating that natural waterborne bacteria were more penetrative than B. diminuta. The observed penetration was thus qualitatively independent of filter media type or manufacturer. These results add to the growing body of evidence that shows 0.2 and 0.22 micron rated filters may not remove all microorganisms under all conditions. These results further establish that bacterial penetration of 0.2/0.22 micron rated filters is not limited just to (1) specific membrane types, or (2) extended duration challenges (> 24 h), or (3) extremely high challenge levels, or (4) bacteria that can only exist in a penetrative state in an artificial laboratory setting.     

Method for qualifying microbial removal performance of 0.1 micron rated filters. Part III: bacterial challenge tests on 0.2/0.22 and 0.1 micron rated filter cartridges with Hydrogenophaga (formerly Pseudomonas) pseudoflava.

We have previously reported on the preliminary characterization of Hydrogenophaga (formerly Pseudomonas) pseudoflava for potential use as a standard challenge organism to qualify 0.1 microm rated filters. This article reports on the retention efficiencies of a large panel of 0.2/0.22 microm and 0.1 microm rated filter cartridges for H. pseudoflava (ATCC 700892) versus the retention capabilities of the same filters for Brevundimonas diminuta (ATCC 19146). A total of thirty-two 0.2/0.22 microm rated filter cartridges, spanning nine different "sterilizing grade" filter types from four different filter manufacturers, were challenged with H. pseudoflava at challenge levels exceeding 10(7) cfu/cm2. H. pseudoflava was shown to penetrate every 0.2/0.22 microm rated filter tested, with log titer reduction (LTR) values ranging from 3.5 to 7.7 logs. H. pseudoflava was shown to be more penetrative than B. diminuta under the same challenge conditions. B. diminuta was fully retained by nineteen of the twenty 0.2/0.22 microm rated filters that were challenged with both organisms. In the case of 0.1 microm rated filters, eighteen filter cartridges, spanning five different filter types from three manufacturers were tested. H. pseudoflava was consistently retained by four out of the five filter types tested, with LTR values in excess of 11.5 to 12.2 logs. The 0.1 microm rated filter type that was penetrated by H. pseudoflava has been previously demonstrated to be not fully retentive for naturally occurring bacteria. The data show that H. pseudoflava penetrates 0.2/0.22 microm rated filters just as readily as B. diminuta penetrates 0.45 microm rated filters. In addition, titer reductions provided by 0.2/0.22 microm rated filters for H. pseudoflava are comparable to those reported for A. laidlawii mycoplasma, albeit under different conditions. This study demonstrates that H. pseudoflava meets all criteria for use as a standard organism for qualifying the microbial removal performance of 0.1 microm rated filters for enhanced sterility assurance.     

Retention of water-borne bacteria by membrane filters. Part II: Scanning electron microscopy (SEM) and fatty acid methyl ester (FAME) characterization of bacterial species recovered downstream of 0.2/0.22 micron rated filters

The results of scanning electron microscopic (SEM) and fatty acid methyl ester (FAME) characterization of the bacterial species shown to penetrate conventional 0.2/0.22 micron rated "sterilizing grade" filters are presented. SEM data suggest that retention of bacteria by these filters appears to be strongly influenced by the morphology, and especially the width of bacteria and less so by length. When the bacterial cell width is small, less than 0.3 micron or so, the cell length does not appear to limit the ability to penetrate 0.2/0.22 micron rated filters. As the bacterial width increases, there is also a strong, almost exponential, decrease in the allowable length for penetration, with most penetrative cells tending to be coccoid beyond a width of 0.5 micron. Significant percentages of the bacteria (40-50%) that were observed downstream of these filters were larger than B. diminuta, the standard organism used to qualify 0.2/0.22 micron rated filters. The average sizes of natural waterborne bacteria that penetrated the filters tested were 20-40% larger in width, and 40-70% larger in length, compared to B. diminuta. These results indicate that size exclusion is not the sole mechanism governing bacterial retention. All isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp. and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems. Most of the bacteria recovered downstream of 0.2/0.22 micron rated filters were gram negative, oxidase positive, motile, nonfermentors.     

Application of membrane filtration for removal of diminutive bioburden organisms in pharmaceutical products and processes

In this report, we present results of a recent investigation in our laboratories demonstrated the effect of process conditions and/or drug product composition on the ability of 0.2 micron and 0.22 micron sterilizing grade filters to fully retain Ralstonia (formerly Burkholderia, formerly Pseudomonas) pickettii. R. pickettii is a opportunistic pathogen widely distributed in nature as well as clinical specimens and there have been several reports of nosocomial infections due to intrinsic manufacture-related R. pickettii contamination in filter-sterilized parenteral fluids. This study documents the penetration of 0.2 micron nylon 66 and 0.22 micron modified PVDF sterilizing grade filters by R. pickettii (grown and challenged) in a drug solution under conditions that simulated a pharmaceutical filling operation. Penetration was not observed for every filter disc tested, and this may be explained, in part, by the stochastic nature (i.e., governed by the rules of probability) of the retention mechanisms involved. Scanning electron microscopy revealed significant changes in the microorganism's size and morphology as a result of exposure to the drug solution; these changes are consistent with those reported for bacteria subjected to nutrient deprivation. The SEM analyses of R. pickettii challenge suspensions in the drug solution showed that the average cell length decreased from 1.25 +/- 0.27 microns to 0.84 +/- 0.17 micron between zero and 24 hours. In addition, significant changes were observed in the size (length) distributions, with approximately 35% of the cells at 24 hours being smaller than any cell observed at the start of the challenge. These data suggest that the significant reduction in bioburden size and morphology that occurred as a result of exposure to the drug solution may play a role in the reduced ability of the 0.2 micron and 0.22 micron filters tested in this study to retain these organisms. Under the same test conditions where penetration of 0.2/0.22 micron filters was observed, 0.1 micron rated membrane filters qualified with both B. diminuta and Acholeplasma laidlawii mycoplasma consistently provided sterile effluent. Bacterial penetration of 0.2 (or 0.22) micron sterilizing grade filters was not observed under identical test conditions with either R. pickettii in a standardized solution (saline lactose broth) routinely used in challenge testing filters, or with the standard test organism, B. diminuta, in the drug solution. This study thus supports the renewed emphasis on both product- and process specific validation as well as routine bioburden monitoring expressed by regulatory agencies, and the use of enhanced bacterial removal efficiency 0.1 micron rated filters to provide enhanced sterility assurance in pharmaceutical processes.     

Retention of water-borne bacteria by membrane filters. Part III: Bacterial challenge tests on 0.1 micron rated filters

Clear performance differences were observed between different 0.1 micron rated filters in terms of their microbial removal efficiency when challenged with naturally occurring waterborne bacteria from a water source. Penetration occurred with three 0.1 micron rated "sterilizing grade" filter types tested, from three different filter manufacturers, that did not have a specific high titer reduction claim for Acholeplasma laidlawii. Bacteria shown to penetrate these 0.1 micron rated filters were quite similar to those recovered downstream of 0.2.0.22 micron rated filters (described in Part II). All of the isolates identified via FAME analyses were common environmental or ubiquitous organisms, and some, such as Acidovorax sp. and Hydrogenophaga pseudoflava, have also been isolated from pharmaceutical water systems. In contrast, four different 0.1 micron rated "sterilizing grade" filter types from two different manufacturers, which had been qualified with both B. diminuta and A. laidlawii, consistently produced sterile effluents under similar test conditions. This study thus highlights the need for an industry or regulatory standard method of defining the microbial removal performance of 0.1 micron rated filters, and supports the use of functionally qualified 0.1 micron rated filters as sterilizing grade filters in pharmaceutical operations for enhanced sterility assurance.     

Changes in the cell size of Brevundimonas diminuta using different growth agitation rates

Brevundimonas diminuta (ATCC 19146) is a standard organism for validation of sterilizing-grade membrane filters. Cell size is critical for the determination of retention characteristics of 0.2 micron rated membrane filters. In this study, cell size changes of B. diminuta cultured under different physiologic states and variable agitations at 50, 100 and 200 rpm were measured by a particle size analyzer and scanning electron microscope (SEM). The smallest cells were obtained at initial stationary phase in saline lactose broth (SLB) as a shaking culture at 50 rpm. Cells grown under agitation at 50, 100 and 200 rpm showed an increase of specific growth rate (mu), about 2.9, 3.6 and 3.6 fold, respectively, compared to the non-agitated cells in SLB media. These results suggested that the cell size decreased proportionally with increase of the specific growth rate (mu) in SLB. These size changes were associated with penetration through a 0.2 micron rated cellulose acetate filter. A scale-down filtration system was developed and performed bacterial challenge test and bubble point test with cells cultured in SLB. Cells grown under agitation conditions in SLB were not retained by 0.2 micron rated membrane filter.     

Microbial retention characteristics of sterilizing-grade membrane filters with alginate substituted for oil-based products

For oil-based products, FDA recommends substitution of the oil with a compound which has similar viscosity and physical characteristics. In this study, a substitute for oil-based products was screened by measuring the viscosity and filterability, and examined for the presence of cell clumps in the various test fluids using an optical microscopy. The viscosity of the test fluids measured in the range of about 60-75 cP. Brevundimonas diminuta (formerly Pseudomonas diminuta), a standard challenge test organism for validation of 0.2 micron rated membrane filters, formed clumps in oils (corn, olive, sesame, and soybean) and polyethylene glycol (PEG, Molecular Weight (MW) = 400 and 1,000). During the viability test, cells suspended in 80% glycerol showed a ten-fold mortality rate after an exposure for 6 hours, but there was no significant change in viability in alginate (low, medium, and high viscosity) for 24 hours. These results suggested that alginate is better suited as a substitute for oil-based products than 80% glycerol. Since high viscosity fluids take longer to filter, the glycerol mortality rate would influence the challenge test negatively. A scaled-down filtration system has been developed for the described trials, and the bacterial challenge and bubble point tests have been performed in 1.6% alginate (66.7 cP), which was the choice of carrier fluid.     

Scaling from discs to pleated devices

Membrane discs offer a convenient format for evaluating membrane performance in normal flow filtration. However, while pleated devices of different sizes tend to scale in close proportion to their contained areas, they do not necessarily scale in direct proportion from flat discs. The objectives of this study are to quantify differences in performance among sterilizing-grade membrane devices as a function of device type and size, to develop an understanding of the factors that affect device scalability, and to develop a mathematical model to predict a cartridge-to-disc scalability factor based on membrane properties and porous support properties and dimensions. Measured and predicted normalized water permeability scalability factors for seven types of pleated cartridges, including 0.1-micro and 0.2-micro rated PES, and 0.2-micro rated polyvinylidene fluoride (PVDF) sterilizing-grade filters in nominal 1-inch to 5-inch lengths, were determined. The results of this study indicate that pleated cartridge performance can be closely predicted based on 47-mm disc performance provided that a number of measured device parameters are properly accounted for, most importantly parasitic pressure losses in the filter device and plumbing connections, intrinsic membrane variability, true effective device filtration area, and the hydraulic properties of all porous support materials. Throughput scalability factors (discs to devices) tend to converge towards unity, especially for highly plugging streams. As the membrane fouls, the resistance through the membrane dominates other resistances, so the flux scales more linearly with membrane area and the overall scaling factor becomes close to one. The results of throughput tests on seven different cartridge types and five different challenge streams (with widely varying fouling characteristics) show that most of the throughput scaling factors were within +/-10% of 1.0. As part of this study, the effects of pressure and temperature were also evaluated. Neither of these factors was found to have a significant effect on scalability.     

Characterization of filter extractables by proton NMR spectroscopy: studies on intact filters with process buffers

Studies were conducted to characterize potential extractables from sterilizing grade filters. The focus of this report is the 0.22 micron Durapore (hydrophilic modified PVDF) filter which is used throughout our recovery processes. The objectives of this study are (1) to identify potential filter extractables from the hydrophilic PVDF filters; (2) to show that NMR spectroscopy may be used to detect filter extractables in the presence of product and excipients; and (3) to establish levels of filter extractables obtained by extraction with a variety of buffers. The data show that the primary source of filter extractables is the hydrophilic modification of the PVDF membrane surface. Extractables from the modified hydrophilic PVDF filter include propylene glycol (PG) and soluble oligomers of the hydroxypropyl acrylate and cross-linker. Propylene glycol, arising from the hydrolysis of the hydroxypropyl acrylate, appears to be the primary extractable in buffers above pH 11. Since the 1H-NMR method can easily detect the methyl proton signals of PG, an NMR assay was developed to detect PG in the presence of buffer excipients and final product. Propylene glycol can be used as a marker for the extractables from Durapore hydrophilic PVDF filters. Although numerous buffers were used to generate extractables from the PVDF filter, significant extractables (PG and soluble oligomers) were found only in high pH extraction buffers. As a result of this finding, only a limited number of new buffers or new PVDF filters will require testing for future validation studies. Process validation studies have shown that neither PG nor soluble oligomers are at levels that impact the quality or safety of the product.     

The importance of accurate microorganism identification in microbial challenge tests of membrane filters--part I

Microbial challenge testing is a common procedure to determine the retention efficiency, performance, and validity of a sterilizing-grade filter. The ASTM 838-05 standard describes a bacteria challenge test procedure based on Brevundimonas diminuta (ATCC 19146), routinely used to verify a 0.2 μm rated sterilizing-grade filter. Process validation procedures most often also utilize B. diminuta (ATCC 19146), but instead of the standard procedures and fluids, process, and product parameters are employed to determine whether these parameters influence the retentivity of the filter or changes to the challenge organism, which might result in the penetration of the filter. In certain instances, the native bioburden within the drug manufacturing process is used to perform such process validation challenge tests. Filter penetrations can happen and cause concern; therefore, it is essential to identify the organism species with accuracy to avoid unnecessary confusion. This paper and its follow-up will describe such imprecision and the resulting misconceptions. It will clarify past determinations and put perspective on the findings. LAY ABSTRACT: Sterilizing-grade filters are used to remove microorganisms from biopharmaceutical solutions. To determine the retention performance of such filters, bacteria challenge tests are utilized, often with a standard challenge organism (Brevundimonas diminuta), in instances with native bioburden. The accuracy of the microorganism identification is of importance to avoid flawed results and misinterpretation of the filter's performance.     

In line final filters for removing particles from amphotericin B infusions.

The feasibility of using membrane filters to remove particles from intravenous infusions of amphotericin B in dextrose 5% (a colloidal solution) was studied. Six types of commercial membrane filters, ranging in pore size from 0.45-1.0 mum, were used. Because of the effect of pH on the particle size of colloidal solutions, each filter was tested at solution pH 4.7, 5.6 and 6.5. Samples of filtrate were analyzed spectrophotometrically for amphotericin content. All filters of pore size 0.22 mum removed amphotericin B from solution and were inappropriate for use with this product. Solutions at pH 4.7 were turbid, filtered slowly and were generally unacceptable for clinical use. At pH 5.6, only filters with pore sizes of 1.0 mum or greater showed acceptable results. At pH 6.5, filters with pore sizes of 0.45 mum or greater gave acceptable results; the use of a filter with a pore size of not less than 1.0 mum would provide a margin for error to compensate for variations in the colloidal particle size of amphotericin B.     

Impact of tubing material on the failure of product-specific bubble points of sterilizing-grade filters

The following study was conducted to determine the effect of different preservatives commonly used in the biopharmaceutical industry on the product-specific bubble point of sterilizing-grade filters when used to filter product processed with different types of tubing. The preservatives tested were 0.25% phenol, m-cresol, and benzyl alcohol. The tubing tested was Sani-Pure (platinum-cured silicone tubing), Versilic (peroxide-cured silicone tubing), C-Flex, Pharmed, and Cole-Parmer (BioPharm silicone tubing). The product-specific bubble point values of sterilizing grade filters were measured after the recirculation of product through the filter and tubing of different types of materials for a total contact time of 15 h. When silicone tubing was used, the post-recirculation product-specific bubble point was suppressed on average 13 psig when compared to the pre- recirculation product-specific bubble point. Suppression was also observed with C-Flex, but to a much lesser extent than with silicone tubing. Suppression was not observed with Pharmed or BioPharm tubing. Alcohol extractions performed on the filters that experienced suppressed bubble points followed by Fourier transform infrared spectroscopy analysis indicated the filters contained poly(dimethylsiloxane). Direct addition of poly(dimethlysiloxane) to solutions filtered through sterilizing-grade filters suppressed the filter bubble points when tested for integrity. Silicone oils most likely reduced the surface tension of the pores in the membrane, resulting in the ability of air (or nitrogen) to pass more freely through the membrane, causing suppressed bubble point test values. The results of these studies indicate that product-specific bubble point of a filter determined with only product may not reflect the true bubble point for preservative-containing products that are recirculated or contacted with certain tubing for 15 h or greater. In addition, tubing material placed in contact with products containing preservatives should be evaluated for impact to the product-specific bubble point when being utilized with sterilizing-grade filters.     

Accuracy of automated instruments used in the pharmaceutical industry for integrity testing sterilizing filters

In the pharmaceutical industry, the integrity of sterile filters is critical to ensure sterility of filtered products. Filter integrity is frequently tested by measuring gas diffusion across water-contacting hydrophobic or hydrophilic membranes with the same automated test devices. Constant device accuracy over the whole range of possible operating conditions is an especially important requirement, as set by the GMP regulations for product critical devices. In this paper, we investigate the accuracy of gas diffusion rate and water intrusion rate estimates provided by a batch-operated and a refilling, continuous-flow commercial automated test device used both for diffusive flow tests and water intrusion tests. Tests were performed on custom-designed model filter systems and full-scale filters over a broad range of gas diffusive flow rates and upstream gas volumes. Neither tested device provided accurate measurements of gas diffusion rate when a small gas diffusion flow was measured out of a very large upstream volume. The batch-operated device provided measurements of gas diffusion rates (either gas diffusion or water intrusion rate) with an accuracy that strongly depends on the gas diffusion rate and on the gas volume upstream from the membrane. Gas diffusion rate measurements were particularly biased in diffusive flow tests of filters with less than 500 mL gas upstream volume. Gas diffusion rates were underestimated by as much as -14.5% in diffusive flow tests and -25% in water intrusion tests. The refilling, continuous flow device generally provided consistent and accurate gas diffusion rate and water intrusion rate measurements within less than 5% of the reference value, practically independent of the gas diffusion flow rate and upstream volume value. A serious bias was only noted in diffusion flow tests at very high upstream volumes and low gas diffusion rate. The results reported in this paper show the importance of qualifying the automated test devices used to assess sterile filter integrity.     

Comparison of LTB4- and C5a-stimulated chemotaxis of isolated human neutrophils: difference revealed by cell migration in thick filters using the multiwell cap procedure

Under comparable conditions (90 min incubation in 2% albumin buffer) using 3 micron pore cellulose nitrate filters and the multiwell cap procedure (Evans et al., Bioscience Reports 6:1041, 1986), C5a was more potent than LTB4 as a chemoattractant (EC'50 s = 0.5 and 4 nM) and caused 5 times as many cells to completely traverse the filter. During the 90 min incubation, no cells traversed the filter in the absence of chemoattractant. The cap assay was modified to study migration of cells within the filter. During the 30 min incubation, EC50 values for C5a and LTB4 were comparable (1.2 and 1.6 nM) in causing cells to enter the filters but C5a was superior in causing the cells to move as measured by the mean distance of migration (EC'50 s = 0.25 and 1.8 nM). Our studies support the view that LTB4 acts primarily to cause cell adhesion (and penetration) of the endothelium and that C5a plays a major role in cell migration (McMillan and Foster, Agents and Actions 24: 114, 1988).     

Membrane filters and membrane-filtration processes for health care

The development of membrane-filtration processes is reviewed, and current types and uses of membrane filtration in health care is discussed. Development of adequate support structures for filters and of disposable filtration devices has facilitated development of filtration processes for pharmaceutical industry, manufacturing in hospital pharmacies, and direct patient care. Hydrophobic filters have also been developed; aqueous solutions cannot wet the pore structures of these filters and therefore cannot pass. Sterility-testing systems have also been developed. There are two types of filters: depth (constructed of compacted fibers) and membrane (which have a homogeneous internal structure). Depth filters retain only a portion of particles in a particular size range and are generally not acceptable for use in health care. Membrane filters retain all particles of a given size. Types of membrane filters are selected for specific uses based on needed flow rates, particulate load, and retention capability. Membrane filters may be validated using bacterial-passage, bubble-point, and diffusion tests. Most membrane filters used in health care are microporous filters that retain particles in the 0.1-10-micron size range. Applications are currently being developed for ultrafilters, which retain both particles and substances with large molecular structures such as proteins, and reverse-osmosis filter membranes, which allow only water or water-miscible solvents of very low molecular weights to pass. Experience in engineering designs, quality assurance, and test procedures has led to the development of many safe, reliable, and effective membrane products for health care.     

Effects of sterilizing-grade filters on the physico-chemical properties of onion-like vesicles

Spherulites are new promising multilamellar vesicles that we study in a drug delivery context. The sterilization of spherulites suspensions is a necessary step before biological tests and later, before pharmaceutical applications (for example, parenteral or local injections). Among all sterilizing operations, the filtration through 0.22 microm sterilizing-grade filters (of the type Millex (? 4 mm) by Millipore) is easy and rapid, and we decided to study it as a mean to obtain sterile suspensions. The spherulites diameter is usually comprised between 0.2 and 0.5 microm but bigger vesicles occur and reach ? 1 microm. The effects of such filters on vesicles' size and lipids' concentration were then compromised. After examination of this challenging operation, results proved that the sterilizing filtration had no effect on these two parameters whatever the formulation chosen. Then, the possible release of amaranth, an encapsulated hydrophilic dye was followed. With the formulations and in spite of a filter diameter inferior to that of the vesicles, the encapsulation yields were not significantly different before and after the filtration and no leakage could be detected. Finally, the spherulites' functionality after sterilizing filtration was studied under the chemical angle: vesicles containing an amphiphilic reactive anchor (CholE3ONH2) were still able to bind covalently a peptidic molecular recognition pattern. The ligation was quantified by fluorimetry as high as for non-filtrated suspensions. Thus, though spherulites can present a diameter superior to that of the sterilizing filters, their passage through them do not alter the physico-chemical properties of these vesicles.     

Particles shed from syringe filters and their effects on agitation-induced protein aggregation

We tested the hypothesis that foreign particles shed from filters can accelerate the rate of protein aggregation and particle formation during agitation stress. Various types and brands of syringe filters were tested. Particle counts and size distribution (≥1 μm) in buffer alone or in solutions of keratinocyte growth factor 2 (KGF-2) were determined with a micro-flow imaging. Submicron particle populations were characterized by dynamic light scattering. Loss of soluble protein during filtration or postfiltration incubation was determined by ultraviolet spectroscopy and bicinchoninic acid protein assay. There was a wide range (from essentially none to >100,000/mL) in the counts for at least 1 μm particles shed into buffer or KGF-2 solution from the different syringe filters (with or without borosilicate glass microfibers). Filtration of KGF-2 with units containing glass microfibers above the membrane resulted in 20%-80% loss of protein due to adsorption to filter components. Filtration with systems containing a membrane alone resulted in 0%-20% loss of KGF-2. Effects of 24-h postfiltration incubation were tested on KGF-2 solution filtered with polyether sulfone membrane filters. Loss of soluble protein and formation of particles during agitation were much greater than that in control, unfiltered KGF-2 solutions. Similar acceleration of protein aggregation and particle formation was observed when unfiltered KGF-2 solution was mixed with filtered buffer and agitated. Particle shedding from syringe filters--and the resulting acceleration of protein aggregation during agitation--varied greatly among the different syringe filters and individual units of a given filter type. Our results demonstrate that nanoparticles and microparticles shed from the filters can accelerate protein aggregation and particle formation, especially during agitation.     

除菌过滤器验证(四):兼容性验证

兼容性验证是过滤器工艺验证项目之一,以证实工艺及工艺条件对过滤器的特定性能没有产生负面影响。文中介绍了兼容性的概念、兼容性验证的目的和除菌过滤器兼容性验证实验的方法,同时介绍了重复使用过滤器的兼容性验证和同系列药品在兼容性验证中的分组原则。