Sterility assurance is critically important for liquid parenteral products. For pharmaceuticals that are not heat-labile, terminal sterilization may be performed. However, in most cases, sterilizing-grade filters must be used. Following their use in manufacturing, these filters are tested to ensure that the membrane remained integral. A method referred to as the bubble-point integrity test is commonly performed. This test may be completed manually or with an automated bubble-point integrity tester. More than 85% of companies use the bubble point as the test for filter integrity when manufacturing sterile products (1). In addition, the US Food and Drug Administration guideline states that the bubble-point test is mandatory to ensure that filter leaks or perforations did not occur during the filtration process (2). 

 Figure 1: Apparatus used to recirculate the antimicrobial preservative-containing solution through the test filter. (IMAGE IS COURTESY OF MERCK & CO.)  

Before using a sterilizing-grade filter to manufacture a parenteral formulation, a bubble-point value is determined with the filter and the product in the laboratory. A procedure for performing and determining the product-specific bubble point has been described in the literature (3). In this procedure, the product is first pumped briefly through tubing connected to a filter. Once the filter has been wetted with the product, it is removed, and the bubble point is determined, typically using an automated bubble-point integrity machine. Three replicates are performed, and the average product-specific bubble point is established. The bubble-point value is then transferred to a batch document that is used during the manufacturing process. If the filter fails the minimum product-specific bubble point following the manufacture of a product, a series of tests may be performed on the filter to determine whether a true leak is present or whether a false failure occurred (3). 

Table I: Preservatives used in filter bubble-point studies  and typical amounts found in pharmaceutical products. (IMAGE IS COURTESY OF MERCK & CO.)  

The authors demonstrated that in some cases the failure of the product-specific bubble point may not result from a leak or loss in membrane integrity, but from factors such as interaction between the product and materials used during the formulation and filling process (4). The selection of tubing was shown to be critically important when filling parenteral liquid products that contain antimicrobial preservatives such as phenol, 

-cresol, and benzyl alcohol (4). In these studies, the interaction of these preservatives with the tubing resulted in the release of polydimethyl siloxane (silicone oil), which lowered the product-specific bubble point (4). The lowering of the bubble point resulted from a reduction of the liquid surface tension of the wetted membrane pores and was caused by the adsorption of polydimethyl siloxane to the filter membrane. This article will review these findings and discuss factors to consider when defining a product-specific bubble point. 

Table II: Tubing types evaluated in filter bubble-point studies. (IMAGE IS COURTESY OF MERCK & CO.)  

The equipment that was used to evaluate the effect on filters with various types of tubing and antimicrobial solutions is depicted in Figure 1 (4). In this procedure, the solution was recirculated at flow rates of 160–200 mL/min for a minimum of 15 h. The bubble point was measured using an automatic integrity tester (Sartocheck 4, Sartorius, New York). 

Table III: Corrected product-wetted bubble-point (CPBP)âpreservative solutions. (IMAGE IS COURTESY OF MERCK & CO.)  

A series of tests was performed using various types of tubing and antimicrobial preservative-containing solutions. Table I lists some of the antimicrobial preservatives that are used in parenteral products and the typical amounts found in these products (5). The levels of antimicrobial preservatives ranged from 0.0715% to 1.9% (5). Solutions containing 0.25% of each preservative in saline solution were used in previous filtration studies (4). Each of these preservative-containing solutions was recirculated through several types of tubing (listed in Table II) with a sterilizing-grade, 0.22-µm filter (Millipak 20, Millipore, Bedford, MA).  

Table IV: Effect of preservative-containing solutions and platinum-cured silicone tubing on the bubble point. (IMAGE IS COURTESY OF MERCK & CO.)  

The experiments were performed by first determining the corrected product-wetted bubble point (CPBP) for the preservative solutions tested. Each filter was flushed with water, and the bubble point was determined. These tests were performed with replicates according to the procedures outlined by the Parenteral Drug Association (3). The results of these tests are summarized in Table III.  

Table V: Effect of preservative-containing solutions and C-Flex tubing on the bubble point. (IMAGE IS COURTESY OF MERCK & CO.)  

Before the 15-h recirculation experiments were initiated, the filters were wetted with the preservative-containing solutions (see Tables IV–VII), and the bubble point at time zero (i.e., preuse bubble point) was measured (4). The solution was then recirculated for 15 h, then a postuse bubble-point test of the filter was performed (see Tables IV–VII). The results indicated that the bubble point of the filter was affected the most when using platinum-cured silicone tubing (see Table IV). The smallest effects on the bubble point occurred when using C-Flex, PharMed, and Biopharm tubing (Cole-Parmer, Vernon Hills, IL) (see Tables V–VII). 

Table VI: Effect of preservative-containing solutions and PharMed tubing on the bubble point. (IMAGE IS COURTESY OF MERCK & CO.)  

These studies demonstrated that the type of tubing selected for filter-sterilizing an antimicrobial preservative-containing formulation may affect the filter's postuse product bubble point. In the case of platinum-cured silicone tubing, bubble-point failures were caused by the presence of poly(dimethyl) siloxane, or silicone oil, trapped on the filter membrane (4). The presence of poly(dimethyl) siloxane was detected using Fourier transform infrared spectroscopy (4). The lowering of the bubble point was confirmed by readdition of poly(dimethyl) siloxane in a separate recirculation study (4). The lowering of the bubble points resulted from a reduction in the surface tension of the liquid that was wetting the membrane pores. For example, it has been previously demonstrated that silicone oils have low surface tensions (6). The surface-tension reduction was caused by the adsorption of polydimethyl siloxane to the filter membrane. The lower surface tension caused the gas to pass through the pores of the filter more easily, resulting in lower bubble-point values. The following items must be considered when determining the product-specific bubble point of a filter for sterilizing parenteral drugs:  

Materials that have product contact, specifically those upstream of the filter, should be tested to determine if they affect the bubble point. If the materials are found to suppress the bubble point, a material screening-procedure should be used to identify those that have the least effect on the bubble point. 

When selecting tubing material that will be in contact with product that contains preservatives, consider the length of time that the product will be in contact with the different materials, including the filter, during the manufacturing process. Incorporate the processing time into laboratory studies for determining the product-specific bubble point (7). 

Table VII: Effect of preservative-containing solutions and Biopharm tubing on the bubble point. (IMAGE IS COURTESY OF MERCK & CO.)   

s a senior engineer, both at Merck & Co., 770 Sumneytown Pike, West Point, PA 19486, tel. 215.652.3992, fax 215.652.5299, 

*To whom all correspondence should be addressed. 

 1. M. Russell and T.H. Meltzer, "An Interpretation of the Pharmaceutical Industry Survey of Current Sterile Filtration Practices," 

Guideline on Sterile Drug Products Produced by Aseptic Processing

 3. Parenteral Drug Association, "Technical Report No. 26: Sterilizing Filtration of Liquids," 

 4. B.K. Meyer and D. Vargas, "Impact of Tubing Material on the Failure of Product-Specific Bubble Points of Sterilizing-Grade Filters," 

 5. B.K. Meyer et al., "Antimicrobial Preservative Use in Parenteral Products: Past and Present," 

 6. T. Svitova et al., "Wetting Behavior of Silicone Oils on Solid Substrates Immersed in Aqueous Electrolyte Solutions," 

7. B.K. Meyer and D. Vargas, "Impact of Tubing Material on the Failure of Product-Specific Bubble Points of Sterilizing-Grade Filters," presented at the National PDA Conference, Colorado Springs, CO, April 2008.  

Articles

The authors explain the factors that can cause a failure in a bubble-point integrity test and what to consider when a product-specific bubble point must be defined.

Establishing Material Compatibility, Process Conditions, and Bubble Points of Filters

Isotechniques, or techniques of adjacent volumes, are a processing method for pharmaceutical preparations containing high-potency active pharmaceutical ingredients (APIs) (e.g., cytostatics, hormones, and antibiotics) in absolute secure conditions to ensure the safety of the operator and the quality of the finished product. Manufacturing using isotechniques takes place in isolators.  

Isotechniques offer certain advantages for aseptic production of injectable, high-potency drugs. In the sanitized areas of cleanrooms, the operator brings in contamination in a limited manner. A completely biodecontaminated environment exists inside the isolator, where only the drug and material for manufacturing come in direct contact with the processing system. In such operative circumstances, contamination by microbes is more limited than in cleanroom conditions because the absence of direct human intervention allows for better cleaning and complete biodecontamination of the entire working environment. Complete biodecontamination with vaporized hydrogen peroxide of the working area inside isolators allows for operation in a biodecontaminated environment. Cleanrooms, however, are exclusively sanitized and operate under conditions of controlled contamination.   

The automatic cycle of biodecontamination guarantees the reproducibility of the process. Manual disinfection is a time-consuming activity that may not be consistently performed and is dangerous because of the continuous exposure of operators to sanitizing agents. Biodecontaminating agents such as vaporized hydrogen peroxide achieve sterilization by the dispersion of gas. The gas can reach all surfaces exposed to its contact, even the hidden ones. Its products are oxygen and water, which are harmless. Isotechniques also exceed the onerous barrier created by the modality of clothing and microbiological control. The US Food and Drug Administration has expressed a favorable opinion for increasing the use of isotechniques for drug production in asepsis, especially for the production of high-potency APIs.  

Some aspects to consider when using isolators while processing injectable, high-potency drugs are:  

Containment of contamination, particularly airborne contamination 

Aseptic processing and sterility of handled material and product

Efficiency of barriers and their integrity 

Management of the environmental impact as a result of the process (i.e., refluent, industrial waste, liquids, gas, and air).

A practical demonstration of the productions of liquids or lyophilized injectables in glass vials is shown in Figure 1. The operating line is set up by several isolators partly connected in a row, where all manufacturing operations are done. These operations are: weighing of raw materials, their volatilization in tanks, transfer of the solution to the dosage systems, washing and depyrogenation of vials, filling of vials, capping (complete if liquid; partially, if liquids are to lyophilize), eventual freeze-drying, sealing, external washing, and drying of filled and sealed vials. 

Figure 1: Production flow for liquids in vials. (ALL FIGURES AND IMAGES ARE COURTESY OF THE AUTHOR.) 

The process is made in a production line with isolators. The following are phases of the main production process:    

Phase I: Preparation of the distribution circuit (distributor, needles, and tubes)

Phase II: Preparation of isolators (cleaning and biodecontamination) 

Phase III: Preparation of various materials (vials, stoppers, and seals) 

Phase IV: Preparation of the solution and its filtration. 

Phase II is typically for a production process using isotechniques. Phase IV is strongly affected by the kind of API (i.e., high-potency) used in the solution preparation, and this aspect considerably changes the manipulation modalities of the API.   

The use of a production line for manufacturing injectable, high-potency drugs in asepsis has risks. It is important to determine the risk levels of the critical aspects of the processing. The principal areas that should be taken into consideration when evaluating risk areas and modalities to control are as follows:  

Comparison of different aseptic techniques used in manufacturing high-potency drugs.

Isolators are an up-to-date and suitable solution for manufacturing injectable high-potency drugs as they guarantee elevated risk control. Isolators have two unique features that make them suited to this type of manufacturing: containment of contamination, particularly airborne contamination, and protection of personnel. These features avoid dispersion of high active material to the environment and guarantee the protection of operators to exposure from these materials. 

Isolators (ALL FIGURES AND IMAGES ARE COURTESY OF THE AUTHOR.) 

The intermediate phases of the production process do not pose significant risk because the liquid product is confined inside the isolators or the distributor system (tanks, transfer tubes, and needles). From a containment perspective, it is much easier to manage liquid solutions than powder because of the following:  

Liquids have more limited characteristics of dispersion and diffusion   

Solutions are significantly more diluted than an active principle in powder 

The principal risks to diffuse contamination are tied to dispersion in air and are  present when liquids are in  an aerosol phase.

The risky points of the production process are:   

Preparation of the solution (i.e., the handling of high-active powders)

Product vials (i.e., the potential that a solution drops on the surface of sealed vials or vials break inside the freeze-dryer)

Washing of soiled process components (e.g., tanks, tubes, needles, and the freeze-dryer)

Transfer of various materials potentially contaminated on their surface. 

The approaches to mitigate risk in each of these areas are detailed below.  

 Adoption of isolators that operate with negative-pressure conditions and the integrity control of the isolators, particularly the gloves, shield operators from exposure to powders. The direct transfer of the powders to the predissolution tank, integrated in the isolator, avoids any risk of environmental dispersion. The cleaning of the closed isolator and the gathering of washing water in such a tank guarantee that the product of manufacturing remains confined, in liquid form, inside the tank. The periodical substitution of the filters, mainly exhaust ones, is executed by a trained staff, which is aware of the risks, and through a system of high containment (i.e., bag-in/bag-out). 

 The last phase of the production process involves the external washing and drying of the sealed vials, which eliminate eventual traces of contamination by the product solution. The unloading of the vials from the freeze-dryer is another extremely risky point for the operators' exposure to the powder because the product is turning to its solid state. Because of the high incidence of vials breaking in the freeze-dryer, this phase is conducted within isolators both during loading and unloading. These operations further use an automatic system (i.e., pusher) and an adequate condition of loading (i.e., "pizza door"), which noticeably reduce human intervention during the process and favor the maintenance of aseptic conditions.   

 The cleaning procedures are mainly handled automatically. Where manual intervention is necessary, however, gloves are used. All manual washing activities are conducted on soiled solution components and never on powder ones.    

 Because of previous handling (e.g., the supplier's container that holds the API, the container holding the API that is returned from production to the warehouse, and samples of material), all materials potentially contaminated by traces of high-active powders on their surfaces are handled with protective gloves and individual protective equipment such as masks and glasses. As a precautionary measure, all containers are wrapped in new plastic covers that collect traces of powder and avoid dispersion into the  environment. All critical manipulations are performed under the safety hood or inside the isolators by trained personnel who are aware of and trained to control the risk.     

Processing schemes help to individualize and graphically identify the main areas of risk for cross-contamination and the related precautions that need to be taken. Adopting dedicated components is the principal precaution needed to avoid cross-contamination.  

During solution preparation, gloves, containers, equipment for pouring, transfer tubes, and filters are dedicated to the product, which limits common use to only the tanks. This dedication reduces to a minimum the cleaning-validation studies that show the absence of cross-contamination between different products. The use of disposable tanks completely eliminates the risk of cross-contamination, which allows related studies to be avoided.  

During the repartition phase of the solution, gloves, transfer tubes, distributors, connections, filters and needles are completely dedicated to the product. The adoption of push systems for the liquids such as peristaltic pumps with harmless gas or with pressure through harmless gas mitigates the risk of cross-contamination in all production steps where these push systems are used  (e.g., from tank to tank or from tank to needles).    

During post-lyophilization, cross-contamination has to be evaluated by studies that verify the efficiency of automatic processing of cleaning in place.  

Aseptic processing and sterility assurance is essential when producing injectable drugs with isolators. This process is connected with the efficiency of the biodecontamination cycles of isolators and barriers and their integrity.  The risky points for the sterility assurance are as follows:   

Sterilization of material in contact with solution to fill 

The author discusses the key issues to consider when using isolators such as containment, protection of personnel, the efficiency of biodecontamination cycles, sterility assurance levels, barriers and their integrity, and environmental impact.

Manufacturing High-Potency Drugs Using Isolators

Integrity testing multiround filter housings is difficult and inaccurate because of  the multitude of filter elements involved. The diffusive flow sum of all of the filter elements can make it difficult to detect a single flawed filter or determine the bubble point which is bulk flow point. 

The single-point diffusion test reflects the overall diffusion rate of the cartridges being tested, thus increasing the risk for a minor failure (e.g., one single filter element with a loss in integrity) being potentially masked by the overall low diffusion values of the surrounding cartridges. A statistical approach with a reduced maximum allowable diffusion test limit would reduce this potential risk. 

The bubble point will reflect the biggest pore-size area. Its accuracy depends on the capacity of the integrity test unit to detect the over-proportional increase of gas flow when passing from the diffusive flow region to the bulk flow region. For a large filtration area this transition phase potentially will be masked because of the overall high diffusion rate. In addition, the bulk flow through possible flaws does not increase exponentially as the test pressure increases, thus making bubble-point determination difficult. 

The multipoint diffusion test will create a diffusive flow profile (diffusion versus applied pressure) of the filter system over a broad range of test pressures all the way up to the characteristic bulk flow of the bubble point. The combination of multipoint diffusion test and bubble point leverages the advantages of the combination and increases a higher accuracy of detection. 

Some automated multipoint diffusion-test systems allow programming of a specific maximum allowable diffusion value for every pressure step, thus setting a maximum diffusion profile. 

The following materials were used: automated integrity-test unit (Sartocheck 4, 

); five pieces of 30-in. PESU 0.2-µm cartridges (Sartopore 2, Sartorius Stedim Biotech GmbH), referred to as A, B, C, D, and E, respectively; one piece of 20-in. PESU 0.2-µm cartridge (Sartopore 2), referred to as F; one piece of 10-in. PESU 0.2-µm cartridge (Sartopore 2) failing cartridge (low bubble point), referred to as G; one piece of 10-in. PESU 0.2-µm cartridge (Sartopore 2) failing cartridge (high diffusion), referred to as H; one multiround (5 x 30 in.) housing; one single-round 30-in. housing; and one single-round 10-in. housing. 

All filter cartridges were individually tested three times with a multipoint diffusion test over a range of pressure points. The lowest point was at 2500 mbar (36.25 psi), which is the traditional single-point diffusion test pressure for the filter used. The highest point was 3250 mbar (47.13 psi), which is 50 mbar (0.73 psi) above the specified minimum allowable bubble point of the filter tested. The filter cartridges were thoroughly rinsed with demineralized water at room temperature before each integrity test. 

After the diffusion profile curves (from the multipoint diffusion test) and the individual bubble point values were generated, the data were analyzed. The individual cartridge values were used to mathematically simulate different filter combinations in a multiround housing (see Figure 1). 

 Figure 1: Individual values of cartridges A, B, C, D, F, and H and their sum. (ALL FIGURES ARE COURTESY OF THE AUTHORS.)  

To verify the mathematically simulated test results, the 30-in. filter elements (i.e., A, B, C, D, and E) with no break in integrity were installed into the multiround housing and tested with the bubble point and multipoint diffusion tests. All cartridges were tested three times and rinsed between each test (see Figure 2). 

Figure 2: Multiround housing 5 x 30-in. cartridges A, B, C, D, and E.  (ALL FIGURES ARE COURTESY OF THE AUTHORS.)  

One 30-in. cartridge (cartridge E) was then replaced by a 20-in. cartridge. A 10-in. filter housing, containing the bubble-point failing cartridge, was connected in parallel with the multiround housing, consequently representing a five-round 30-in. setup in terms of membrane surface. The system containing cartridges A, B, C, D, F, and G was tested with bubble-point and multipoint diffusion tests. All cartridges were rinsed between each of the three tests (see Figure 3). 

Figure 3: Multiround housing and 10-in. housing, 4 x 30-in. cartridges A, B, C, and D; 1 x 20-in. cartridge F; and 1 x 10-in. cartridge G or H. ( (ALL FIGURES ARE COURTESY OF THE AUTHORS.)  

The bubble-point failing cartridge (cartridge G) in the 10-in. housing was then replaced by the high-diffusion cartridge (cartridge H), and the same tests and rinses were performed. Instead of performing a separate single-point diffusion test at 2500 mbar (36.25 psi), the authors considered the diffusion value at the same pressure during the multipoint diffusion test was representative. 

The expected difference in diffusion between five 30-in. cartridges (i.e., A, B, C, D, and E) (137.9 mL/min) without loss of integrity and an equivalent setup including the bubble-point failing 10-in. cartridge (i.e., A, B, C, D, F, and G) (139.4 mL/min), at the standard test pressure of 2500 mbar (36.25 psi) was <2 mL/min, thus being not possible to detect with a high degree of confidence. 

An equivalent setup including the failing 10-in. high-diffusion cartridge (i.e., A, B, C, D, F, and H) would require the maximum allowable diffusion value to be lowered by more than 40% as compared with the cumulated individual maximum diffusion values to be detected at all. Using such a high safety margin creates a high potential risk of getting false-failure test results with filters that otherwise have no break of integrity.  

Based on the typical diffusion profile of a cartridge with no loss of integrity, it was possible to define a maximum allowable diffusion curve, which made it possible to get a clear multipoint-diffusion test failure when the bubble-point failing 10-in. cartridge was included in a multiround setup (cartridges A, B, C, D, F, and G). The same approach showed that it should be possible to get a clear test failure when having the 10-in. high-diffusion cartridge in a multiround set-up (cartridges A, B, C, D, F, and H). 

To simulate the expected bubble-point value of various multiround setups the automated integrity test bubble-point test algorithm was programmed in an Excel (Microsoft) spreadsheet, and the diffusion profiles of the individual cartridges were entered. The authors found that it should be possible to get a clear bubble-point test failure involving the 10-in. bubble-point failing cartridge in a multiround setup (cartridges A, B, C, D, F, and G). In addition, the authors found that it should not be possible to get a bubble-point test failure when including the failing 10-in. high-diffusion cartridge in a multiround set-up (cartridges A, B, C, D, F, and H), because no exponential increase would be expected to take place before the minimum bubble point. 

The diffusion profile of a filter cartridge can be plotted in two distinct areas, with the applied differential pressure on the 

-axis and the resulting diffusion on the 

-axis.The first area is the so called diffusive flow region, where the diffusion rate can be considered linear as a function of the applied test pressure. In this test-pressure region, the pores are still filled with the wetting liquid. 

The second region is the so-called bulk flow region where the applied pressure gradually blows out an increasing number of pores, giving a free-gas flow through the membrane. The flow graph turns exponential. 

Between the linear and exponential area is the so-called inflection point or transition phase. The sharpness of the inflection point is directly related to pore-size distribution, pore design, and the size of the filter surface. The smaller the filter surface and the more homogeneous the pore-size distribution, the sharper the inflection point (see Figure 4). 

Figure 4: Small filter surface inflection point versus large filter surface inflection point. (ALL FIGURES ARE COURTESY OF THE AUTHORS.)  

The multipoint diffusion limit curve is based on the diffusive flow and the bulk flow and is defined by three set points. It is a straight line from the first test pressure point, which is the common single-point diffusion-test pressure (in this case 2500 mbar or 36.25 psi), up to a point at 100 mbar before the minimum bubble point (in this case 3100 mbar or 44.95 psi). The last point at 50 mbar after the minimum bubble point (in this case 3250 mbar or 47.15 psi). This much higher maximum allowable diffusion rate allows one cartridge to reach the bubble point at the minimum bubble point without giving a false failed test result (see Figure 5). 

Figure 5: Multipoint diffusion test limit curve. (ALL FIGURES ARE COURTESY OF THE AUTHORS.)  

The maximum allowable diffusion value for the two first pressure steps is calculated taking into account the number of cartridges, their individual maximum allowable diffusion value, the applied test pressure, and a safety margin. The maximum allowable diffusion value for the last test-pressure point is calculated taking into account the same variables as the two previous set points but allowing one cartridge to reach bubble point. This is achieved by adding a cartridge size-related diffusion value representing the over-proportional increase generated by the bulk flow at the bubble point. In the integrity-test unit bubble point test algorithm, this value is called the 

2-value (c.f. Functional Design Specification of Sartocheck 3, Sartocheck 3+, Sartocheck 4). The set point equations thus become: 

 is the number of cartridges in the multiround housing, 

minus the cartridge eventually reaching the bubble point, Diff

 is the validated maximum allowable diffusion value for the concerned cartridge type (here 54 mL/min), α = (1 – safety margin), 

1 is the standard diffusion test pressure (here 2500 mbar or 36.25 psi), 

2 is the second set point (here 3100 mbar or 44.95 psi), 

3 is the third and last set point (here 3250 mbar or 47.15 psi), and 

2 is the cartridge size–related diffusion value (here 240 mL/min). 

During out-of-box trials using new 30-in. Sartopore 2 filter cartridges, the typical diffusion value was only slightly higher than half of the maximum allowable diffusion value given by the manufacturer. During the simulation trials, a maximum allowable diffusive flow limit one-third lower than the standard manufacturer's limit created a robust test result without causing false failures. The a-value was thus set to two-thirds, and the programmed multipoint diffusion limit line became: 

  5 x 54 x (2/3) x (3100/2500) = 223.2 mL/min  

(5-1) x 54 x (2/3) x (3250/2500) + 240 = 427.2 mL/min 

The trials showed that the traditional single-point diffusion test was not accurate enough for multiround housing integrity testing. Subtracting more than 40% from the cumulative individual maximum diffusion values would have detected the failing high-diffusion cartridge (cartridge H), but it also would have increased the risk for getting false-failure test results. Single-point diffusion testing also was incapable of detecting the bubble-point failing cartridge even when the maximum value was reduced by almost 50%. 

Figure 6: Multipoint diffusion test on cartridges A, B, C, D, and E. (ALL FIGURES ARE COURTESY OF THE AUTHORS.)  

The bubble-point test, however, was capable of detecting the bubble-point failing cartridge. If the multiround setup contained the high-diffusion cartridge as the only failing cartridge, the bubble point was incapable of detecting this flawed filter because the high diffusion cartridge did not have a clearly defined bubble-point value below the minimum bubble point. The difference between the expected bubble-point value and the measured bubble point was ≤50 mbar (0.73 psi). 

Figure 7: Multipoint diffusion test on cartridges A, B, C, D, F, and G. (ALL FIGURES ARE COURTESY OF THE AUTHORS.)  

The multipoint diffusion test was capable of detecting the flawed high diffusive flow filter element respectively low bubble-point filter element. The difference between expected diffusion values and measured diffusion values on the multiround housing was less than ±5% (see Figures 6–8). 

Figure 8: Multipoint diffusion test on cartridges A,B, C, D, F, and H. (ALL FIGURES ARE COURTESY OF THE AUTHORS.)  

The authors describe a novel approach for the integrity testing of large sterile filter systems such as multiround housings and describe a multipoint diffusion test capable of detecting minor failures.

Improving the Integrity Test Assurance of Multiround Housings Assessments

Normal flow filters are used widely in biopharmaceutical operations to remove colloidal material, bacteria, and viruses from growth media, buffers, and process intermediates. A modern biopharmaceutical process typically contains 40-50 normal flow filtration operations from seed culture propagation to final vial filling. As shown in Figure 1, normal flow filtration accounts for approximately one-fourth of the total cost for downstream processing. Therefore, the choice of normal flow filter(s) has a potentially large impact on the total production cost for a biotherapeutic. 

 Figure 1: The relative cost of biopharmaceutical separations. (CREDIT_PHOTO) 

The variety of filter materials available to process development scientists is large—from depth media containing nominally-rated micron-sized filtration-matrices to validated sterile filtration membranes containing submicron-sized pores. The criteria by which one chooses the optimal filter is commonly application-specific, and it is therefore important to understand these criteria when designing experiments, analyzing data, and comparing product attributes. 

Quick guide to the selection of normal flow filters 

In general, normal flow filtration operations can be divided into three main categories:  

Figure 2 shows a typical biopharmaceutical process and highlights where each of these filteration steps occurs. 

Figure 2: Normal flow filtration in biopharmaceutical production. 

One of the first unit operations in any biopharmaceutical operation is the preparation and sterilization of cell culture media. Cell culture media are nutrient-rich, buffered solutions containing amino acids, salts, vitamins, and energy sources (e.g., glucose)—all of which are essential components for the culture of healthy cells. Over the past several decades, formulations have evolved from generic basal media supplemented with animal-derived sera, to more cell-line specific formulations that are serum-free, animal-derived component-free and chemically defined. The sterilization of these media is critical to successful cell growth and protein expression. 

There are many characteristics one should look for when selecting a cell culture media filter. The following paragraphs describe some of the most important features.  

The term "sterilizing-grade filter" is defined in the FDA's document 

Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing 

(1), which describes a sterilizing-grade filter as a filter that is "validated to re-producibly remove viable microorganisms from the process stream, producing a sterile effluent." The validation of sterilizing-grade membranes is commonly performed using the procedure documented in ASTM F838-05, "Standard Test Method for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration" (2). To be labeled "sterilizing-grade," a filter must produce a sterile effluent when challenged with 

  colony forming units (CFU) per square centimeter (cm

 ) of membrane area. Sterile filters are nearly always constructed of one or more sheets of polymeric membrane, either in pleated or flat-sheet form. 

 Media filters must not only retain contaminants, but they must also be chemically and biologically compatible with the cell culture media. This means that the filter must be constructed of components that are proven to be safe and that the materials that extract from the filter during normal operation have been quantified and characterized. Filter manufacturers will typically provide flushing recommendations for their particular filter products and also have standard specifications for many contaminants, including (but not limited to): total organic carbon (TOC), oxidizable substances, toxic compounds, particulates, and fibers. Some manufacturers also offer application-specific testing of extractables. 

 Potential interactions between filters and culture media must be assessed carefully to ensure no inhibition of cell growth or protein expression. Cell cultures are highly sensitive to growth media composition; hence, the materials of construction for a sterilizing filter must be proven inert. Membranes used in media sterilization operations should have low nonspecific binding to ensure that key media ingredients are not removed during the filtration process. 

Physical robustness is important in all filtration applications, but it is critical in media sterilization applications because of the stress which the filters undergo before, during, and after use. A typical media filtration process consists of the following steps:  

Many of these steps result in physical, thermal, and/or chemical stress on the membrane and other filter components. Nonetheless, every component must retain its functionality for the media sterilization operation to be considered successful. 

The term "permeability" refers to the flux rate achieved through a filter, normalized with respect to differential pressure. Permeability is typically reported with units of liters per square meter per hour per psi. Permeability is important because most media sterilization processes have relatively short batch times (1-2 h) and filters with low permeability may drive the required filter area to be larger than required based on filter capacity alone. (This results in a filter train that is underutilized with respect to capacity.) Media sterilization filters with high permeability result in a system that maximizes the filter throughput. 

The term "capacity" refers to the volume of feed that can be processed by a given membrane area before the membrane's resistance to flow becomes unacceptably high. Sterile media filters are expected to have high capacity (thousands of liters per square meter of membrane area). This is an important characteristic for media sterilization filters because of process economics, ease-of-use considerations, and the minimization of nonspecific binding. For  sterilizing-grade filters that are designed for cell culture media sterilization, high capacity is achieved by the addition of an on-board prefilter layer—typically having a pore size rating of 0.4 - 0.8 µm. 

GE Healthcare (West Borough, MA) performed a study to evaluate the most common filters that are designed for media sterilization. In this study, five commercially-available cell culture media were prepared from dry powder per the manufacturer instructions. The tested media are as follows: 

Invitrogen RPMI-1640 basal media supplemented with Sigma Type IV soy peptone

Each of the media was tested on a panel of sterilizing-grade membranes from Pall (East Hill, NY), Millipore (Billerica, MA), Sartorius (Goettingen, Germany), and GE Healthcare. Solutions were filtered at a constant pressure of 10 psid, and the volume filtered as a function of time was recorded until the flow rate had decayed by at least 50% or until the solution was exhausted. Based on the test results, estimations of the required number of equivalent 10-in. filter cartridges were made for a 12,000 L batch of media filtered in 2 h. Results are presented in Figure 3. 

Figure 3: Comparative performance of multilayer sterilizing-grade filters for cell culture media filtration (shorter bars represent better performance). {{ART: x axis should be labeled "Media type"}} 

Buffer solutions are used widely in nearly every step of bio-pharmaceutical production processes. In fact, buffer filtration is the most commonly performed filtration operation in any biopharmaceutical process. During operation of the bioreactor, buffers are used to control pH and osmolality of the cell culture media. At cell harvest, buffers are used to precondition filters and to assist in product recovery operations. Chromatography steps use numerous buffers for such operations as column conditioning, column elution, and column regeneration. Once a biopharmaceutical is ready for formulation, buffers become a key ingredient in the bulk drug substance. Finally, buffers are used throughout the process for clean-in-place operations. 

Biological and particle contaminants present in buffers can have a large impact on process efficiencies and final product quality. Therefore, normal flow filtration is one of the first steps (after dissolution) in any buffer preparation process. Buffer filtration is key to protect chromatography columns and ultrafiltration operations and to produce an endotoxin-free final product. 

The following paragraphs describe the key characteristics of a buffer filter. 

Buffer filtration is commonly done with 0.2-µm membrane filters to reduce bioburden or to achieve sterility of the buffer and to remove particulate contaminants. The choice between a sterilizing-grade and a bioburden-reduction filter* often depends on the final use of the buffer. For example, sterility is a requirement for buffers used as additions to the bioreactor to prevent contamination of the cell broth, while bioburden reduction may be sufficient for buffers used in chromatography operations, which are often not aseptic processes. Bioburden reduction filters are generally less expensive (per unit membrane area) and require less filtration area for a given batch size (thereby improving their economics even further). In either case, it is important for regulatory purposes that the membrane is validated for retention of bacteria and that the retention can be correlated to an in-process integrity test. 

Buffer filters must have broad chemical compatibility, because buffers used in biopharmaceuti-cal production span a wide range of pH levels (1-14), and must withstand exposure to alcohols and (occasionally) other organic chemicals. 

Filters used in buffer preparation must withstand the rigors of steam-in-place and/or autoclaving. They must also be validated for multiple sterilization cycles since buffer preparation areas may be designed to re-use filters. Buffer filters should remain integral within a wide range of operating conditions in order to avoid filter failures which can lead to batch reprocessing, lost product and/or costly regulatory investigations. 

Buffer filtration is a high-volume, short-time operation. Because buffers are generally fluids with low particle loading, they do not tend to plug membrane filters. Therefore, permeability (rather than capacity) becomes the key determining characteristic in the size of the filtration system. Using high permeability membranes can significantly reduce the amount of filter area needed for buffer preparation. Small filtration footprints are desirable because they are not only cheaper in terms of consumables, but they also require smaller capital investments and reduce the risk of filter integrity failures. 

Survey of filters for buffer filtration. 

GE Healthcare performed a study to evaluate the most common filters that are used for buffer filtration. In this study, eight common buffers spanning a range of concentration, pH, and organic content were prepared and tested on a panel of buffer filtration membranes from Pall, Millipore, Sartorius, and GE Healthcare. The tested buffers are shown below:  

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Proper selection of normal flow filters leads to increased process efficiency from early phase product development through to full-scale biopharmaceutical production.

Guidelines for Selecting Normal Flow Filters

Thilly and coauthors introduced closed-vial filling systems, called "Crystal" technology, for aseptic filling of liquid products in 

's 2005 Aseptic Processing supplement (1). Aseptic Technologies (Gembloux, Belgium) has now taken closed-vial technology a step further by developing a process using the closed vial for lyophilization. The technology consists of using ready-to-fill, gamma-sterilized, closed vials and filling them using a needle passing through the stoppper, followed by a laser resealing to restore the closure integrity. The vial body, made of cyclo-olefin copolymer (COC), rather than glass, and the stopper, made of thermoplastic elastomer (TPE), are injection molded and assembled by robots in an ISO 5 (Class 100/Grade A) environment.  

The process involves securing the closure integrity with a top ring, and then gamma-irradiating the vial to ensure sterility. The closed vial is then determined to be clean on the inside and sterile and therefore ready for filling. Plastic molding of vial elements allows the vial to have specific functionalities such as an optimized stopper shape and fully secured closure integrity (2).   

In the filling line, the TPE stopper is pierced by a special filling needle. Immediate self-reclosing takes place at the needle withdrawal and the piercing trace is resealed with a noncontact laser beam, restoring a continuous upper surface of the stopper. Overpressure inside the vial during filling is eliminated by venting grooves in the outer surface of the needle shaft. A sterile plastic cap is then snapfitted on top of the stopper, which is still inside the barrier. The special cap design protects the piercing target until use by a healthcare professional (1, 2).  

Because the container is permanently closed, the entire process from vial manufacturing to liquid filling benefits patients by optimizing sterility assurance (see Figure 1). Pharmaceutical manufacturers also benefit from the simplicity of the filling operation compared with the classical but complex glass-vial filling process. This article describes the concept and process of closed-vial, freeze-drying technology in more detail. 

Figure 1: Closed vial process: Vial bodies and stopper are molded in ISO 5 (Class 100/Grade A) environment and immediately assembled by robot before the top and bottom rings are added; after packaging, the vials are gamma-irradiated; at the pharmaceutical site, vials are filled with a needle passing through the stopper, which is then laser resealed; and finally, capping is completed. (ALL FIGURES ARE COURTESY OF THE AUTHORS.) 

To maintain optimal sterility assurance, an opening in the vial is required but has been limited in this particular process. The vial actually remains closed most of time, especially between the filling station and the freeze dryer's shelf. The opening and closing of the vial is generated by rearranging the movements of the freeze-dryer shelves. When the cycle is finished, the vial closes again before the freeze dryer's door is opened and stays closed until the vial is resealed with a laser and capped. This process has several  advantages over traditional glass-vial processing:  

The closed vial content is not exposed during its movement from the sterilization tunnel to the freeze dryer (no half-seated stopper).

There is no risk of product spillage on the vial track to the freeze dryer or on the shelves themselves. Moreover, the plastic vials are shock resistant and nearly unbreakable. 

The stoppers do not stick to the upper shelf.

There is no risk of incomplete reseating of the stopper or stopper pop-up after the stoppering step; closure integrity is maintained throughout the process. 

When required, this process has the capability of inspecting for particles of the liquid before freeze-drying, provided that the inspection machine is compliant with a Class 100 environment. The vial's bottom ring enables it to be held from the bottom only for the rotation, without an upper spindle above the vial. Its slightly elevated bottom allows for a perfect view on the critical bottom part of the content. 

 A small, disposable device called the penetrator reopens the piercing trace made by the filling needle. This penetrator is installed on top of a regular closed vial by a light snapfit over its existing top ring. Made of medical-grade, high-density polyethylene (HDPE), the penetrator is gamma-irradiated to avoid contamination during lyophilization. The penetrator has an internal cone with an open tip located above the piercing spot.  

Vertical downward movement of the freeze-dryer shelf, carried out before the freezing phase, pushes the penetrators down. At the same time, the front ring of the open tip pushes (but does not pierce) the surface surrounding the piercing trace. This push reopens the piercing trace and generates a passage for the water vapor during freeze-drying. The size of the cone's pushing front ring on the penetrator is large enough and tolerant enough to accommodate the needle piercing. 

Shelves are kept down during the three lyophilization phases (freezing, sublimation, and secondary drying) to keep the stopper open and allow gas to exit. At the end of the secondary drying phase, when temperature is back in the 25–35 °C range, the shelves are lifted to their initial position. The resilience of the deformed stopper push up the penetrators while the stopper opening recloses and reseals itself. 

The entire process can be separated into three steps: vial-filling, lyophilization, and vial-closing and capping (see Figure 1). Each is described below.  

The vial-filling step is identical to the classical filling process described by Verjans et al. and Thilly et al. (1, 2). The same vial is loaded on the filling equipment and the vial is filled by a needle piercing through the stopper. The only modification is that the needle has a slightly larger diameter (11G gauge instead of 13G) and its tip configuration is trocar, giving a precise cut in the elastomer, with the shape of a three-branch star. These two features facilitate future reopening of the stopper. As with liquid-filling needles, the stopper's elasticity allows it to immediately self-reclose after filling. After filling, the process changes radically as the immediate laser resealing and capping are eliminated and replaced by the positioning of the penetrator on top of the vial with a placement tool (see Figure 2, positions 1 and 2). The vial, still closed, is then conveyed to the freeze dryer's loading unit and onto the shelves (see Figure 2, position 3). 

Figure 2: Lyophilization process: (1) A vial and the penetrator, (2) penetrators are placed on the vial with a placement tool, (3) vials are introduced inside the lyophilization unit and the shelves are moved down to break the bridges, (4) the shelf reopens the piercing trace to allow lyophilization, (5) the shelf is moved up, and (6) the penetrator is withdrawn.  (ALL FIGURES ARE COURTESY OF THE AUTHORS.) 

After classical freeze-dryer loading and once the door is closed, the shelves move down, pushing on the penetrators located above the closed stoppers. The penetrator is molded as one piece but has two subcomponents linked with six breakable bridges (see Figure 3). The subcomponents are: 

Figure 3: Penetrator with cone attached to the base with small breakable bridges. Once these bridges are broken by the vertical movement of the shelves, the cone slides inside the base. (ALL FIGURES ARE COURTESY OF THE AUTHORS.) 

A cylinder base that ensures the holding (by light snapfit) on the top ring and its adequate centering.

A cone that can slide inside the base when the shelf is pushing. Three large side openings in the upper part of the cone enable gas passage between the cone and shelf. 

As the shelf moves down at constant speed, the six bridges are broken (taking about 25 to 30 N/vial), releasing the cone for further pushing work on the stopper. At the end of the downward stroke of the shelves, the front tip of the cone pushes on the stopper around the pierced spot, without penetrating the stopper. By radial elastomeric expansion, the cone reopens the piercing trace and the lyophilization cycle can begin (see Figure 2, position 4). The opening cross-section of the stopper is adequate to allow vapor exit (as tested in fast cycles and with sucrose 10%). The reopening of the piercing trace requires about 45 N/vial, slightly below the classical stoppering pressure needed for glass vials (industrial freeze dryers are classically calculated for a minimum of 10 tons/m

  of stoppering pressure). During the entire cycle, shelves are kept in that low position, keeping the stoppers open. 

At the end of the cycle, as the temperature rises above 25 °C, the stopper fully recovers its elastomeric resilience. When the shelves are lifted, the stopper takes its original shape, lifts the penetrator cone back on top of itself and self-reseals (see Figure 2, position 5). This happens even during long lyophilization cycles (e.g., three days).  

After exiting the freeze dryer's chamber under barrier conditions, the penetrators need to be removed. This step is easily achieved because the penetrators are linked to the vial with three very light snapfits. A passage of the vial along a fixed side-cam finger is sufficient to withdraw the penetrator (see Figure 2, position 6). At that time, the vial is ready for laser and capping.  

Laser resealing is performed using a 6 mm-diameter laser spot which ensures proper cover of the piercing area. Finally, capping is completed as usual. These last three steps are performed under an ISO 5 (Class 100/Grade A) environment to avoid contamination before laser-resealing. This environment also ensures asepsis of the stopper top surface as it remains protected against contamination until use.  

It is recognized that water still present in washed, steam-sterilized stoppers after a lyophilization cycle can cause a 1% elevation of moisture content during shelf-life in glass vials. However, with closed vials, this problem does not occur because the stopper processing does use water. 

Despite the fact that the plastics used in the Crystal vial (COC and TPE) have low water-vapor transmission rate (WVTR), they do not warrant the high protection required for keeping freeze-dried cakes in a low-percentage humidity range during full shelf-life (e.g., as recommended by World Health Organization). Therefore, additional barriers against moisture ingress should be obtained and can be easily achieved with adequate secondary packaging (e.g., an aluminum pouch [pinhole-free Al thickness of 20 µm, within a multilayer film] or a blister cavity with aluminum lidding and a low WVTR transparent thermoformable film [polyvinylidene chloride multilayers have very low WVTR]). 

To ensure that Crystal technology is suitable for lyophilization, several tests have been conducted using various products, quantities, and conditions. For example, the authors:  

Used various excipients to investigate lyophilization feasibility: qualification started with an easily lyophilized excipient (lactose 5%) and finished with a complex one (glass transition point below –38 °C).

Increased vial quantities per lyophilization cycle: starting with a few vials and using glass vials as a control, lyophilization cycles were made with up to 720 vials placed on two shelves (see Figure 4). 

Used sensitive products to assess the quality of the lyophilization process for the product. (Living viruses that are particularly sensitive to lyophilization have been used to assess the recovery after lyophilization).

Figure 4: Lyophilization unit loaded with closed vials; lyophilized products inside closed vials. (ALL FIGURES ARE COURTESY OF THE AUTHORS.)  

All tests were performed in a Christ Epsilon 2.25 D freeze dryer with seven shelves and an industrial controlling system, using 2-mL closed vials (22.25-mm diameter) and penetrators molded in high-density polyethylene, from a two-cavity mold.  

Closure integrity after laser resealing has been tested, as has effective protection of the plastic cap. The following aspects were observed and analyzed for most cycles:  

Correct lift-back of pushers at time of shelf's upward movement at the end of the cycle.

Quality of the closure integrity after resealing.

Humidity content of cakes (target is 1%, +/- 0.2%). 

For viruses, content titer was measured at cycle end and after accelerated aging (seven days at 37 °C), compared with lyophilized glass vials. In some cycles, temperature was recorded with probes installed in plastic vials and in control glass vials to analyze temperature curves.

Closed vials and their closure integrity remain unaffected after undergoing temperature and pressure cycles of a freeze-drying cycle. 

Plastic material and elevated bottom of the vial are not deterrent to effective energy transfers. It is likely that the combination of a continuous pressure over the vial top applied by the upper shelf during the complete cycle and a perfectly flat vial bottom help the contact between the vial and the shelves and therefore the conductive part of the heat exchange process. 

Cycle is extended by 20 min during the first phase of freezing, right after the change of phase and temperature uplift. However, the same terminal temperature (–55 °C) is reached in the same manner at the end of the freezing phase. 

All tests are successful even for most complex excipients when similar cycles are used compared with a glass vial (2-mL vial).

Highly sensitive viruses are recovered with similar titers in Crystal and glass vials when both are being processed together in the same cycle. 

The authors present an aseptic-filling process for freeze-dried liquids using the closed-vial technology.

Pharmaceutical Technology-11-01-2008

Establishing Material Compatibility, Process Conditions, and Bubble Points of Filters

Manufacturing High-Potency Drugs Using Isolators

Improving the Integrity Test Assurance of Multiround Housings Assessments

Guidelines for Selecting Normal Flow Filters

Freeze-Drying with Closed Vials

Comparison of Conventional Cleanrooms, Restricted Access Barrier Systems, and Isolators

USP <1117> "Microbial Best Laboratory Practices": An Interview with Scott Sutton

Considerations on Re-Use of Sterilizing-Grade Filters