Sterile Filtration of Liquids and Gases

Kerry Roche Lentine

Richard V. Levy

Sterile filtration is used successfully in a variety of applications at a variety of scales. Medical practitioners employ sterilizing grade syringe filters or in-line large volume parenteral filters as an extra measure of safety for their patients at the 1 mL to 1 to 2 L range. Laboratory scientists employ sterilizing grade filters to safeguard media and reagents to ensure robust and consistent experimental results. These laboratory scale processes can range from self-contained, disposable vacuum-driven filtration devices in the 50 to 500 mL range to pressure-driven filter capsules that can process several liters of feed. In pharmaceutical manufacturing for plasma, vaccines, biotherapeutics, and small molecule drugs, sterile filtration is the portion of a process that includes the installation, sterilization, system preparation, integrity testing, and filtering of a liquid or gas through a sterilizing-grade filter with the intention of removing microorganisms. Sterilizing grade filters are used at multiple points throughout the pharmaceutical manufacturing process from media and gas filtration for bioreactor protection to process intermediate and bulk drug substance bioburden reduction to final sterile drug product filtration at scales from 10 L to greater than 10 000 L.

From a regulatory standpoint, sterile filtration is defined as the complete removal of microorganisms and particles within a specific size range from liquids or gases to produce a drug product that meets the current aseptic processing guidelines set by each country such as the US Food and Drug Administration (FDA)¹ or the European Medicines Agency (EMA).²,³ In the spirit of harmonization, sterile filtration may also be defined by other organizations, including the Pharmaceutical Inspection Co-operation Scheme (PIC/S),⁴ World Health Organization (WHO),⁵ the International Organization for Standardization (ISO),⁶ or by a compendium such as the United States Pharmacopeia (USP).⁷

The intent of this chapter is to provide the reader a general overview of sterile filtration of liquids and gases with an emphasis on their application in the pharmaceutical industry.

CONTAMINATION AND THE ROLE OF FILTRATION

Types of Contaminants

Three broad classes of contaminants are found in biological and pharmaceutical solutions: (1) dissolved impurities, (2) suspended particulate matter, and (3) suspended microorganisms.

Water is a major source of dissolved impurities. Dissolved contaminants can be subdivided into inorganic (ionic) and organic impurities. Inorganic salts find their way into water supplies from soil, rocks, tanks, pipes, and other sources. Organic compounds include lignins, tannins, detergents, polysaccharides, proteins, and other biodecomposition products that are commonly associated with surface water supplies. Suspended particulate matter includes colloidal solids, metal, cotton, dust, lint, and an endless variety of other solid contaminants. When used generically, the term is assumed to include microorganisms.

Microorganisms can be a problem whether alive or dead. Alive, they can multiply at logarithmic rates, overburden prefilters and final filters, and overpower preservative systems. Dead, they are a cause of premature filter plugging and the source of certain types of endotoxins, which may prove to be pyrogenic when introduced with drugs into humans and animals.⁸ Because of the low absorption characteristics of many membrane filters, pyrogens in solution are not removed by simple size exclusion alone. There are filters that supplement sieving removal with charge (usually cationic) to capture endotoxin. These must be carefully validated because removal by charge is in general less robust. Pyrogenic activity may be attributed
to the presence of bacterial cells. Intact cells may be removed quantitatively, but no assurance can be given that cell lysis, and thus release of the soluble pyrogenic constituents into the solution, has not taken place.

Controlling Exogenous Contamination

Some of the major exogenous contamination sources that need to be recognized and addressed are related to the design of the work area, raw materials entering the facility, equipment used for processing intermediates, drug substance, and final drug product as well as the people associated with the process. Specifically, the following are points of consideration when performing a sterile filtration:

The manufacturing area, including its physical layout, location, traffic patterns, and use, influences the particulate burden in the air. Most airborne microscopic contaminants are associated with dust or other particles that are dispersed by the movement of people.
Raw materials: All raw materials pose a risk of contaminating the drug manufacturing process such as cell culture media, buffer salts, surfactants, solvents, and excipients are potential sources of contaminating debris, dissolved impurities, and microorganisms. Dead bacteria and mycotic spores left over from sterilization processes such as autoclaving or ultraviolet radiation remains a source of particles and pyrogens. Chemical incompatibility of raw materials with processing equipment may lead to insoluble precipitates, which fail to go entirely into solution.
Equipment, piping, vessels, tubing, and nearly every surface to which the fluid is exposed can contribute contaminants. Particles are inevitable wherever there are moving parts, valves, bungs, O-ring seals, threaded connectors, tubing, or any surface subject to abrasion or wear, as in even the best-designed filling machines and apparatus. Furthermore, improper cleaning can result in formation of biofilm (see chapter 67). Biofilms can be attached to internal surfaces of process systems or detached and suspended within the fluid to be filtered. Bacteria are the most common type of organism present in biofilm or bioburden in fluid system being sterile filtered. Although prefiltration systems are designed to remove physically intact bacteria and parts of bacteria cells, microbial by-products produce many compounds that can cause immune responses. For further understanding of both biofilm and bioburden in this context, consult Parenteral Drug Association (PDA) Technical Report 69, entitled Bioburden and Biofilm Management in Pharmaceutical Manufacturing Operations.⁸
People are a well-known source of both microbial contamination as well as fomites that contaminate the pharmaceutical environment such as skin cells, hair, clothing fibers, and sputum.
Postfinal filtration particle generation from containers and closure systems is another concern. Both glass and elastomeric particles are shed when a stopper is inserted into a bottle or a plunger is inserted into a syringe.⁹ Glass vial delamination can occur from vial handling or product container interactions.¹⁰ When detected, this type of contamination can result in discarding the drug product batch or in product recalls. A recall in 2018 described a sterile injectable drug contaminated with particulate matter believed to be from the manufacturing process. The recall discussed concern that particles, even sterile particles may be harmful to the patient. “In the absence of in-line filtration, these particles may cause local vein irritation, inflammatory reaction, aggravation of preexisting infections, allergic reactions, phlebitis, pulmonary emboli, pulmonary granulomas, immune system dysfunction, pulmonary dysfunction, pulmonary infarction, and systemic embolization.”¹¹ Although there is some debate concerning the clinical impact of particle contamination in injectables, point of use sterile filtration in the form of syringe filters or in-line infusion filters can provide the patient an extra measure of safety.¹²

HISTORY OF STERILIZING FILTRATION

Filtration is used as a means of stabilizing pharmaceutical and biological fluids. The Pasteur-Chamberlin filter was one of the first filters designed for the removal of bacteria from solutions and was patented in 1894.¹³ These porous unglazed porcelain tubes were originally designed to purify drinking water. Also, in the 1890s Theobald Seitz, seeking to remove spoilage microorganisms in wine, developed a fibrous filter structure. In 1909, he filed a patent in the United States where he described the layering of asbestos, cellulose, and/or cotton fibers in a graded density to remove successively finer particles from a liquid stream. This patent also described the use of support materials to strengthen the structure of the filter material.¹⁴ In 1918, his brother Georg Seitz and Friedrich Schmitthenner¹⁵ were awarded a patent for a depth filter specifically designed to sterilize liquids. This filter material combined diatomaceous earth or clay with asbestos, cotton, and flax fibers for higher throughput in a presterilized, easy to replace format that eliminated filter cleaning. Although asbestos was a highly effective filter material, in 1973 the FDA proposed that parenteral manufacturers eliminate asbestos or other fiber-releasing filters from their manufacturing process.¹⁶

Retention of bacteria by these media depends on absorption to the inner filter structure and random entrapment throughout the filter matrix. Starting in 1919, Zsigmondy and Buchmann¹⁷,¹⁸ patented true membrane filters for the removal of bacteria from solutions that were
larger than the pore size at or near their surface. These membranes were screen-like cellulose ester-based media produced by phase inversion—a technique still used for most of microporous membranes to this day. In 1927, Membranefiltergesellschaft m.b.n. was founded with Zsigmondy and Wilhelm Sartorius of Sartorius-Werke A. G. among group of owners and produced these membranes with laboratory scale operations until the 1960s.¹⁹ The primary use of this technology in Germany during World War II was to determine the degree of bacterial contamination of potable water supplies. After the war, Goetz, under contract with the US military, acquired the membrane technology and worked from 1947 to 1950 with the California Institute of Technology to refine these membranes to increase porosity and uniformity while eliminating the need to boil filters prior to use.²⁰ These developments led to the analytical membrane filters for bacterial recovery and enumeration referenced in industry standards today.²¹ In 1950, the Lovell Chemical Company won a contract to further develop Goetz’s work for large-scale membrane manufacturing. In 1954, Jack Bush acquired the rights to this membrane technology and founded the Millipore Filter Company to expand the applications for these membranes.

Many of the mid-century cellulosic microporous membrane applications focused on laboratory scale bacterial recovery for enumeration; however, as researchers began to use polymers such as polyamides, polyvinylidene fluoride (PVDF), polyethersulfone, and polytetrafluoroethylene, they developed more robust membrane filters for particle removal, bioburden reduction, and sterilization of gas, liquids, water, and certain types of drug products.²²,²³,²⁴,²⁵,²⁶,²⁷ These removal applications required more surface area than that used in laboratory settings resulting in the development of various sanitary filtration device formats such as tube filters, stacked disks, and pleated cartridge filters.²⁸,²⁹,³⁰,³¹,³²,³³

Because many sterile filter applications in a pharmaceutical process are for aqueous fluids and most of the membrane polymers used in these applications are natively hydrophobic, surface modification methods such as grafting of hydrophilic coatings, radiation polymerization, and other techniques are employed to hydrophilize these membranes. Surface modification can also be designed to reduce nonspecific protein binding.³⁴

During the 1960s, 0.45-µm rated membrane was quickly applied to the removal of bacteria, yeast, and molds from biological and pharmaceutical fluids as well as for sterility testing of drug products.³⁵,³⁶ But in the late 1960s, a pseudomonad-like organism, now known as Brevundimonas diminuta, associated with a proteinaceous solution, was isolated, which could pass through that membrane during the filtration process.³⁷ Membranes with a “tighter” pore size rating of 0.2 (or 0.22-µm) were then introduced for critical sterilizing filtration applications.

Although filter media are used to affect separations such as solid/liquid, chromatographic, electrophoretic, and molecular separations, this chapter is limited to the removal of microorganisms other than viruses, which have been considered elsewhere and, in general, are not expected to be removed to any degree by sterilizing-grade filters.³⁸ In addition to many other commercial and scientific applications, sterile filtration is used extensively in the production of sterile and particle-free biopharmaceutical fluids that cannot be sterilized by any other means. Because terminal sterilization methods (eg, autoclaving, ionizing radiation) are preferred by worldwide regulatory agencies, pharmaceutical manufacturers must demonstrate that terminal sterilization cannot be used. The filters then must be validated to ensure that they perform effectively under process conditions. Adherence to rigid Good Manufacturing Practice (GMP) performance criteria imposed on filtration media and filtration systems, particularly those used to produce parenteral drug products, is required and enforced by regulatory inspection.³⁹,⁴⁰ Even higher standards for sterile filtration are being established as the complexity and duration of processing has increased. Simultaneously, there has been an increase in the physicochemical and biological complexity of the drugs themselves, further complicating the filtration equation.

FILTER TYPES

The filtration process consists of passing a mixture of fluid and solids through a porous medium that retains the solids on the surface, entraps them in the matrix, or both. Filters may be broadly categorized as primarily depth, surface, or screen filters, depending on their composition and construction characteristics, particle-fluid-filter interactions, and mechanisms of filtration. The distinction between depth, surface (prefilters), and screen filters is of considerable importance, particularly to pharmaceutical process filtration.

Filtration devices and systems typically operate by two different modes: normal flow (also known as dead ended) and tangential flow (also known as cross flow). Normal flow filtration (NFF) occurs when the flow of the feedstock is driven perpendicular to the filter medium with the objective of filtering all the feed through the filter. Tangential flow filtration (TFF) occurs when the feed material flows parallel to the filter medium and a portion of the feedstream permeates through the filter medium by the effect of transmembrane pressure and the bulk of the feedstream moves across the membrane and is recirculated for multiple passes to accomplish the separation. In TFF devices, the cross flow provided by a feed pump helps in reducing buildup of the retained products on the filter surface. This chapter focuses on NFF as it relates to sterile filtration. The topic of TFF in biopharmaceutical processing has been comprehensively reviewed by Lutz.⁴¹

When used independently, no one type of filter is cost-effective for most high-volume pharmaceutical filtration applications that require complete removal of microorganisms and particles down to a specific size. High-volume microfiltration usually calls for removing particles in a precise, definable way and at an optimal cost. The most effective way to accomplish this is to capitalize on the complementary properties of depth, surface (prefilters), and screen filters. Depth and surface filtration are the most economical ways to remove the bulk of the particulate burden from a fluid.⁴² Prefilters can be depth, surface, or screen filters depending on the application and are necessary for most sterilizing applications. The optimum choice of filters involves a careful weighing of two factors: filtration efficiency and particle and colloid loading capacity. These are discussed later in this chapter.

Depth Filters

Depth Filters in Liquid Applications

Depth filters are used in bioprocessing for the clarification of centrate or direct harvest from the bioreactor to remove large particles and colloids as well as host cell proteins. In plasma processes, they can be used for clarification of plasma fractions and precipitate removal. In small molecule processes, depth filters can be used for color and haze removal, catalyst recovery, as well as removal of carbon fines and undissolved intermediates and excipients. Depth filters can be combined with prefilters and sterilizing-grade filters for economical process trains, which are discussed later in this chapter.

A depth filter consists of fibrous, granular, or sintered materials pressed, wound, fired, or otherwise bonded into a tortuous maze of flow channels. Particles in a fluid that pass through the irregular channels defined by the tortuous orientation are principally retained by a combination of mechanical entrapment and adsorption that occur throughout the depth of the filter matrix. The depth of the filter bed can range from 3 to 30 mm providing longer residence time for the capture of smaller particles through adsorptive mechanisms. Figure 30.1 illustrates the role of small particle adsorption inherent with depth filters. Materials commonly selected for pharmaceutical processes include cellulose, cellulose esters, polymeric fibers such as polypropylene, and inorganic fibers and may include filter aids such as diatomaceous earth and carbon. Surface modification, such as a cationic treatment, can be used to remove negative species. These filters are typically used in lenticular pads installed in stainless steel housings or in enclosed single-use pods or capsules.

Because there are many permutations of materials and fabrication methods, depth filters cannot realistically be assigned an absolute particle retention rating. Instead, they are assigned a nominal rating, that is, some particle size above, which a certain percentage of contaminants is retained. The nominal rating can be determined experimentally after fabrication by passing a test fluid through the depth filter. This fluid, having a known concentration of suspended particles and a known size distribution, is assayed with membrane filters (screen-type filters) before and after filtration. Particle size counts are made on the pre- and postfiltration assay filters, and the percentage retained (in various size ranges) by the depth filter is calculated. The nominal rating of a depth filter is valid only under a strictly defined set of conditions (flow, temperature, pressure, and viscosity). Changes in any one of these parameters may affect the retention mechanisms and may have an important bearing on critical filtration. For instance, when a solid contaminant in a fluid moves through a depth filter, it follows the path of least resistance until it becomes trapped or is adsorbed. As the pressure differential increases, which often results from filter plugging or an increase in operating pressure, particles and microorganisms are driven deeper into the matrix, eventually “breaking through” to enter the downstream process.

FIGURE 30.1 Scanning electron micrograph of Brevundimonas diminuta ATCC 19146 on a glass fiber depth filter. Arrows indicate examples of bacterial cells adsorbed to fibers.

Depth Filters in Air and Gas Applications

Of the two types of filter classifications (depth and membrane) used in air and gas filtration, the fibrous depth type
is the most common when very large volumes of air must be handled. Glass fibers are commonly used for this application such as those used in high-efficiency particulate air (HEPA) filters for cleanroom air. The gas passing through the matrix of such filters follows a tortuous path, and the microorganisms present are trapped both on the surface and in the depth of the filter.

Advantages of Depth Filters

The advantages of depth filters may be summarized as follows: (1) Depth filters normally exhibit a highest particle capacity (because depth filters can collect contaminants throughout their thicknesses and because they have relatively large spaces between the interstices compared with surface or screen filters) and (2) depth filters retain a substantial percentage of contaminants smaller than their normal size rating because of adsorption.

Disadvantages of Depth Filters

The following are considered disadvantages of depth filters:

Media migration, or the tendency of the filter media (filter fragments) to slough off during filtration, is a severe drawback peculiar to all depth filters. Although continuous throughout the life of the filter, media migration becomes more pronounced when a hydraulic surge or continuous flexing of the filter matrix takes place.
Release of microorganisms initially trapped in the matrix downstream also presents a problem, particularly during long filtration runs. If adsorption has played a role in the removal of certain particles, even minute changes in the fluid-particle-filter matrix interactions may trigger a release of those particles. Under certain conditions, microorganisms may reproduce within the filter matrix, penetrate deeper into the matrix, and emerge on the downstream side to contaminate the filtrate.⁴³ This phenomenon is often called grow-through.
Because they have no meaningful pore size, depth filters impose no definite limitation on the size of particles that may pass through the filter bed.
The relatively large amount of liquid retained by depth filters (holdup volume) can be a serious shortcoming with valuable feedstreams.

Selection of Depth Filters in Pharmaceutical Processing

Selection of depth filters requires (1) clearly defining objectives of the filtration step such as product quality, yield, and impurity removal; (2) specifying batch size; (3) identifying the desired process endpoint such as turbidity, pressure, or process time; (4) understanding the properties of the feed material, including feed lot to lot variability; (5) understanding how material from this filtration step can affect subsequent steps in the purification process; and (6) describing any microbiological needs for the filtrate such as low bioburden or low endotoxins, which would necessitate pre-use sterilization or sanitization of the filter and system. Finally, establishment of the desired capacity and surface area to achieve the processing goals of this step requires bench-scale testing.

Surface Filters

A surface filter, in the context of pharmaceutical filtration, is composed of multiple layers of nonwoven media, usually glass or polymeric microfibers, or web-supported cellulosic membranes. When a fluid is passed through a surface filter, particles larger than the interstices in the microfiber matrix are retained on the surface, whereas smaller particles may be trapped within the matrix, giving a surface filter the advantages of both depth filters and screen filters.

Surface filters can be constructed of microfibers of glass, cellulose, or a variety of polymers or nonwoven supported cellulose membranes. These materials can be used singly or combined to create the filter structure. In addition to traditional fiber laid processes, polymeric microfibers can be melt blown to develop an integral structure that does not require binders. Surface filters are often rolled or pleated and supported by nonwoven base materials for incorporation into a variety of device formats.

Selection of Prefilters in Pharmaceutical Processing

Prefiltration is an effective method to reduce production costs by extending the throughput of downstream membrane filters. Selection of the appropriate prefilter requires (1) clear definition of the objectives of the filtration step, including the objectives of the other filters in the train; (2) specifying batch size; (3) specification of the desired process endpoint such as pressure rise or process time; (4) determination of compatibility with the feedstream, cleaning, sanitizing, or sterilization agents such as steam or gamma radiation; (5) understanding of extractable and leachable contributions; and (6) assessment of impact of protein, preservative, or formulation binding. As with depth filters, establishment of the desired capacity and surface area to achieve the processing goals of this step requires bench-scale testing, which is discussed later in this chapter.

Prefilter Applications

Typical applications that use prefilters include filtration of IVs and small volume parenterals (SVP), ophthalmics, topicals, oral liquids, rinse water such as for vial and stopper washers, solvents, and excipients as well as prefiltration of buffers, cell culture media, and serum.

Screen Filters

Screen filters or sieve filters are used to achieve controlled and predictable particle removal, particularly for final filtration applications that produce a sterile effluent. A screen filter is a highly uniform, rigid, and continuous structure with regularly spaced uniform meshes or irregularly formed spaces or pores. When a fluid is passed through a screen filter, all particles and microorganisms larger than the pore size are retained predominantly on its surface. Particles that penetrate the surface are captured if they are large enough to be retained by the pores within the filter matrix. Examples of screen filters are stainless steel; polymeric mesh; and microporous, polymeric membrane filters, which are the focus of this chapter.

Advantages of Microporous Membrane Filters

The following may be considered advantages of using microporous membrane filters:

Filter efficiency is independent of flow rate and pressure differential for particles larger than the pore size.
There is no media migration with membrane filters because of their homogeneous structure or do such filters permit passage of particles or organisms larger than the most open pore size, even at very high-pressure differentials. For example, hyaluronic acid filtration can reach pressure differentials approaching 500 psid. Because sterilizing filter cartridge construction does not withstand these excessive pressures, this type of filtration is conducted with large areas of flat sheets in specialized high-pressure housings.
Large, inflexible particles tend to form a filter cake on the membrane filter surface. Although interfering little with flow, this porous mat acts as a depth filter by retaining particles smaller than the membrane pore size, thereby increasing the efficiency of the membrane filter.
Membrane filters are thin (<150-µm), and because there is little void volume within the filter structure, there is minimal feedstream loss.

Disadvantages of Membrane Filters

There are two main disadvantages to using microporous membrane filters. First, because of their surface-retention mechanism, membrane filters have a relatively low capacity, particularly if the particles approximate the pore size of the filter surface. Such particles can plug the pores and prevent fluid flow. Second, not all particles smaller than its pore size pass through the membrane filter. Some of these particles are collected on the membrane surface, and some are trapped in the tortuous interstices themselves. If there are enough of these smaller particles, a rapid buildup in pressure will occur.

RETENTION MECHANISMS

Retention Mechanisms in Air and Gas Filtration

The interstices or pores within a gas filter may be large compared with the size of the particles or microorganisms being trapped, but the mechanism of filtration is such that efficiency in removing small particles is high. A filter having an effective diameter on the order of 5 to 100-µm removes more than 99% of submicron particles.⁴⁴ One of the primary ways of increasing the efficiency of a fibrous filter is to reduce the spacing among the fibers. Filters constructed of fine rather than coarse fibers tend to decrease the gap. The removal of particles or microorganisms from air and other gases may be attributed to the following five basic mechanisms.⁴⁴ Figure 30.2 schematically depicts these mechanisms.

Interception

Interception is the contact of the surface of the microorganism or particle with the surface of the filter. An assumption is made and can be readily demonstrated that particles adhere to the filter surface once they make contact. Interception would be favored, then, by a small fiber diameter of the filter matrix.

Sedimentation

The sedimentation mechanism is of secondary practical importance because the settling rate of microorganisms is low. If a stream of gas is flowing through a filter matrix, eventually, the microorganisms settle on the surfaces of the filter fibers.

Impaction

The momentum of microorganisms traveling in a gas stream does not allow them to make sharp changes of direction, so they impact on the fiber surface. The efficiency of impaction is favored by a higher gas velocity through the filter and by the small fiber diameter of the filter matrix, which gives rise to a more abrupt change in the flow path of the gas.

Diffusion

Low velocities of air or gas through the filter matrix favor diffusion. Bacteria undergo Brownian movement to some extent and can diffuse to the surfaces of the fiber matrix where van der Waals forces contribute to particle capture.

Electrostatic Attraction

Gas flowing through a filter matrix causes a triboelectric effect, resulting in the charging of the filter fibers. The charge
acquired depends on the nature of the fiber. Microorganisms carrying a charge become attracted to the surfaces of the filter. Coating the fibers of a filter with a good electrical insulator was shown to increase the efficiency of the filter.⁴⁵ In a sterile filtration application, the filters should not become moist or the retention effect caused by the electrostatic charge could dissipate. Thus, hydrophobicity is an important characteristic of air filters.

FIGURE 30.2 Mechanisms of filtration in gas streams.

These particle-capture mechanisms lead to a most penetrating particle size and dictate construction of a filter with a surface area as large as is practical. The most penetrating particles for fibrous filter media and for membrane filters have been measured to be 0.15- and 0.05-µm, respectively.⁴⁶ Retention ratings for HEPA fiberglass filters have been determined to be 99.99%, and for typical membrane filter, 99.9999999%. These mechanisms dictate that it is desirable to construct a filter with a surface area as large as is practical. A large surface area maintains a low differential pressure across the filter and maintains filter efficiency by decreasing the translational velocity of the gas. The large surface areas of HEPA filters in use today are obtained by pleating the filter material back and forth around fluted separator plates.

Retention Mechanisms in Liquid Streams

Size Exclusion

The primary filtration mechanism of a microporous membrane filter in liquids is physical sieving or size exclusion, whereby all particles larger than the surface opening or pore size are retained at or near its surface (Figure 30.3). Uniformity of pore size permits well-defined limits of particle retention to be determined by appropriate testing (ie, high bacterial challenge levels at a defined number of microorganisms per square centimeter of effective filtration area equals no passage detected).

Adsorption

Adsorption is also a contributing factor in the capture of particles smaller than the pores they encounter. Charge differentials between sites on a particle and within the membrane structure is one of many adsorptive mechanisms. Process conditions and physicochemical properties of the feedstream can affect both size exclusion and adsorption mechanisms. The physicochemical properties of the feedstream could overcome adsorptive forces and allow
particles to be released into the downstream. Deformable particles retained by size exclusion could be forced through the filter structure by high pressure or flow rates, whereas feedstream properties such as pH or a high salt concentration could decrease the size of microorganisms retained by size exclusion allowing them to be released into the downstream. This is one reason that process-specific bacterial challenge testing of sterilizing-grade membranes is required for critical applications.

FIGURE 30.3 Cross-sectional scanning electron micrograph of a microporous membrane filter with microorganisms retained on the membrane surface. The cross section highlights the irregular torturous nature of the membrane.

FILTER RATINGS

Filter pore ratings can be a confusing topic in pharmaceutical filtration. In theory, pore size relates to a filter’s ability to retain particles, including microorganisms, larger than the rated pore size. In practice, the pore rating may have very little to do with the actual relevant performance of the filter. For example, a 0.2-µm rated, sterilizing-grade filter will allow passage of 0.2-µm particles into the effluent, this grade of filter is characterized for the removal of standard bacteria, not for the removal of 0.2-µm particles. The special case of sterilizing-grade filters is discussed in detail later.

Another confusion arises from the use of absolute and nominal pore ratings. Nominal is intended to mean that the filter will remove a certain amount, but not all particles, whereas the term absolute is intended to indicate that all particles above a specified size will be removed by the filter. These are both functional terms based on the particle challenge test used. Without information on the test parameters, one might assume that an absolute filter will remove everything “absolutely.” The method used by one manufacturer to determine the pore rating and provide an absolute claim can be very different than the method and claim used by another, particularly among different filter structures. Even though manufacturers establish their removal claims under well-controlled laboratory conditions, particle removal in an end user’s process will be impacted by feed quality and process conditions; these need to be carefully understood prior to implementation.

Additionally, filters from various manufacturers with the same pore size rating and same polymer components may structurally be very different. This could lead an end user to assume that the filters are equivalent or that one filter will perform better than another based solely on the filter rating. Therefore, pore size rating alone is unreliable selection criterion because these ratings vary from manufacturer to manufacturer and from product to product. The responsibility squarely rests with the user to understand the manufacturer’s filter claims to select the appropriate filter for the intended use.

Approaches to Filter Ratings

Approaches to establish pore ratings are varied and depend on the filter type (depth, surface, or screen) and whether it will be used for a liquid or gas application. Methods include gas-liquid or liquid-liquid intrusion tests, flow rate, particle challenge tests, aerosolized oil tests, and microscopy.

Microscopy

In scanning electron microscopy, a sample of membrane is evaluated, using appropriate imaging software to determine pore size and distribution. Track etched membranes can be reasonably sized using this technique owing to their flat surface punctuated by cylindrical pores.

Gas-Liquid or Liquid-Liquid Intrusion Tests

Porosimetry is a physical method where liquid is forced into the membrane under pressure from a gas or other liquid and the penetration profile is analyzed mathematically to determine pore size, porosity, and pore distribution. Porosimetry is based on the assumption that the pores are straight cylindrical pores and flow is directed vertically from upstream to downstream. Microporous membrane filters are complex structures with irregular torturous pathways that move multidirectionally and even dead-end throughout the structure. Correction factors are employed to account for this.

Bubble point is simple method for determining the largest pores in a membrane structure. When the pores of a microporous membrane are full of a wetting liquid (eg, water in a hydrophilic membrane), a gas (eg, air) only displaces the liquid from the pores if the gas pressure is high enough to overcome the local liquid surface tension-induced capillary forces. This method is not practical for depth filters where interstices are so large that the force required to displace liquid is too low be impractical. For membrane filters, the bubble point test can be used for membrane characterization as well as a nondestructive in-process test for filter system integrity during pharmaceutical processing. The principles of the bubble point test are discussed in detail later.

Particle Challenges

Manufacturers use a variety of particles of defined sizes to determine the minimum size that can be retained by the filter. The choice of challenge conditions is dependent on the filter type, desired claims and application and is often used for filters claimed to have a nominal pore size rating.

A filter efficiency rating should indicate the particle size or the microorganism being used for the test and is calculated as described in equation 1 (Particle Removal Efficiency):

Where

E_ps = particle removal efficiency at a specified test particle size

C_u = downstream particle count

C_d = upstream particle count

A pore rating for a filter can be helpful as a high-level classification. Regardless of the claimed particle removal efficiency, in actual use, particle removal can be different than product claims due to the interactions of the filter material and structure with the infinite permutations of end user feed compositions and processing conditions.

Sterilizing-Grade Membrane Filter Ratings

Sterile filtration is the process of removing microorganisms from a fluid stream without adversely affecting the feedstream. A sterilizing-grade designation is not pore size dependent despite the common industry use of the 0.2-µm descriptor. It is a functional definition whereby membrane filter demonstrates removal of a standard test organism B diminuta (formerly classified in the genus Pseudomonas) at a minimum challenge concentration of 1 × 10⁷ colony-forming units (CFUs)/cm² under a specific flow rate or pressure. Those filters carrying a sterilizing-grade claim will have data to support that claim. If the manufacturer of a 0.2-µm does not provide a sterilizing-grade filter claim or data to support such a claim, the filter cannot be considered sterilizing grade. To be qualified as sterilizing grade, filter manufacturers will typically perform studies based on American Society for Testing and Materials (ASTM) F838-15a.⁴⁷ In addition to membrane filter qualification, manufacturers also perform lot release bacterial challenge tests on samples of membrane and devices.

Functionality of sterilizing-grade membrane filter performance is further demonstrated by the end user for critical operations such as aseptically processed final drug products. It is a regulatory requirement for the end user to conduct process-specific bacterial challenge test validation using the actual drug product under actual (downscaled) processing conditions.¹,²,³,⁴,⁵,⁶ The PDA Technical Reports 26 and 40 provide the end user guidance on best practices related to filter validation of liquids and gases.⁴⁸,⁴⁹

FILTRATION APPLICATIONS OF LIQUIDS AND GAS IN PHARMACEUTICAL APPLICATIONS

Filter Sterilization of Liquids

Sterilizing-grade filters are used throughout pharmaceutical and biopharmaceutical processes where either bioburden reduction or sterilizing performance is needed, particularly for feedstreams that cannot be terminally sterilized by heat. Common applications include media and feed filtration into bioreactors, buffer filtration, chromatography column protection, process intermediates, aggregate removal prior to virus filtration, bulk drug substance as well as critical final formulated drug product in both terminal sterilized, and aseptically processed applications.

End users should define the objectives of each filtration step to establish the process claims and determine the data needed from the manufacturer, the type of testing required to justify the filter selection, and any in-process testing to demonstrate fitness for use. For example, a user states the objective for a 0.2-µm filter used for aseptic processing of a heat labile drug product into vials is that the filtrate will be sterile. The manufacturer of a 0.2-µm filter with a sterilizing claim will need to supply data supporting the sterilizing claim. The user will need to meet regulatory expectations and validate that the filter will exhibit sterilizing performance in their product under their usage conditions and will need to perform integrity after use to demonstrate the filter is fit for use. In certain countries, there may also be a regulatory requirement to conduct a pre-use integrity test. On the other hand, a user may select the same 0.2-µm filter for protection of a chromatography column and describes the objective of the filtration as controlling bioburden. In this less critical application, the end user could perform a risk assessment to determine what type of testing is appropriate to indicate the filter is suitable for use.

Filter Sterilization of Air and Other Gasses

Filters are by far the most commonly used devices for the sterilization of air and other gases. The relative ease of handling, the low cost per unit volume of air sterilized, and the efficiency in removing not only microorganisms but also other submicron particles all account for the extensive use of filters. Filtration is the method used for the sterilization of both small and large volumes of air and gas. The mechanics are such that large volumes may be processed economically. For efficient and economic operation, however, the aerosol content of the gas to be filtered must be low; otherwise, a prefilter may be necessary. This is of importance with microporous membrane filters in use today for the removal of bacteria, yeasts, and molds.

Microorganisms, particles, or droplets of liquid dispersed in a gas are referred to as aerosols. The behavior of aerosols is of concern in many scientific and industrial applications. Dwyer⁴³ reports that particles suspended in a gaseous medium behave according to the
classic laws of mechanics. Complications are involved, however, because the mass of these particles is so small that they have an extremely small inertia but high viscous drag properties. Dwyer points out that this is not surprising given that for a spherical particle, inertial effects are a function of the cube of the radius, whereas the viscous drag effects are a manifestation of surface area and are a function of the square of the radius. It is expected, then, that as particle size decreases, the viscous effects become predominant.

Many viable microorganisms exist in aerosols. In fact, the air around us serves as the vehicle for many species of bacteria, molds, yeasts, and their spores. The constant presence of microorganisms is of concern to the food and beverage processing industries, the pharmaceutical industry, hospitals, and the many smaller laboratories requiring sterile environments.

Selection of the appropriate filter for critical gassterilizing applications is important. Due to the varied retention mechanisms operating in gas filtration, microorganisms smaller than the specified pore size are retained. A filter having an average pore diameter of 0.8-µm was demonstrated to retain particulate material as small as 0.05-µm.⁵⁰ Electrostatic charge plays a greater role in removal than any other mechanism in gas streams.

Hydrophobic membrane filters are ideally adaptable to the filtration of sterile air of venting air for sterile tanks or containers from which sterile pharmaceuticals are being filled, stored, or withdrawn. Sparging for bioreactor applications is ideally suited to membrane filtration because an entire filtration element may be sterilized in place. Vents on fermenters protect not only the influent into the tank but also the environment from the exhaust material. Sterile air filters protect the environment in a blow-fill-seal filling area, and parison support air provides robust and reliable sterility assurance. Autoclave and lyophilizer vacuum breaks require sterilizing-grade hydrophobic filters to ensure the contents remain sterile. In these cases, vent filter material should be hydrophobic; otherwise, moisture droplets, often found in pressurized gas systems, would wet a hydrophilic membrane, essentially reducing the effective filtration area (EFA) by blocking the pores to bulk flow of gas to the extent that a pressure above the bubble point (described later) of the membrane would be required to force air through. In addition, wetting of an air filter may decrease its retention efficiency.

Unlike conventional depth-type filter, hydrophobic microporous membranes (if properly qualified) will not allow bacterial passage when challenged with copious amounts of moisture that may arise in certain applications such as hot water-for-injection (WFI) tanks and fermenter/bioreactor vent applications; however, the addition of coalescing prefilters prior to the sterilizing filter, jacketed filter housings, and proper system design can mitigate the deleterious effects of filter wetting.

FILTER INTEGRITY TESTING OF STERILIZING FILTERS

Filter Integrity Tests

A key feature of an integrity test is its ability to predict membrane performance. In sterilizing filtration, the removal of microorganisms to high levels of efficiency is the most critical performance characteristic. Sterilizing filters must perform to total removal levels of 10¹⁰ to 10¹¹ total microorganisms. Expressed in percentage of efficiency (equation 1), the removal ability equates to greater than 99.999999999%, a high level indeed. This high efficiency is needed because even one organism emerging from a sterilizing filter renders the filtrate nonsterile.

The process of sterilization is often characterized by both physical and biological parameters. For example, the physical measurement of a moist heat process such as steam-in-place (SIP) or autoclaving includes the time/temperature profile of the sterilization cycle (see chapter 28). Examples of physical measurement on sterilizing filters include gas-liquid integrity tests such as diffusional flow or bubble point. These physical parameters are used during each sterilization process to monitor and assess performance. A biological parameter would use a biological indicator (BI) as a measure, expressed in quantitative terms, of the microbial kill for a moist heat process or microbial removal for a filtration process. A BI is a standardized preparation of a specific microorganism at a specific concentration that is relevant to the sterilization process. For example, Geobacillus stearothermophilus spores are used as BIs for moist heat processes because the spores are resistant to heat. But these spores would be too large to pass through a 0.2-µm sterilizing-grade filter. B diminuta, described in the next section, is the analogous BI for sterilizing filtration.

Biological parameters are used during characterization and validation of the sterilization process and are not employed during routine processing; therefore, a relationship or correlation between the physical and biological parameters is a critical feature. The physical measurement should predict the biological performance of the sterilization process. In an analogous manner, the most useful integrity test of filtration is one that provides information on its ability to remove microorganisms.

Bubble Point

An important advantage of a membrane filter is its ability to be nondestructively tested for integrity before and after filtration. Commonly used integrity tests are the bubble point, diffusive airflow, and pressure hold tests. Test theory has been thoroughly reviewed by others.⁵¹,⁵²,⁵³

When the pores of a membrane filter structure are full of a wetting liquid (eg, water in a hydrophilic membrane),
a gas (eg, air) displaces the liquid from the pores only if the gas pressure is high enough to overcome the local liquid surface tension-induced capillary forces. The critical pressure at which this occurs is controlled by the size of the pores and the surface tension of the wetting liquid (Figure 30.4) (equation 2, Bubble Point):

Where:

BP = bubble point pressure

K = shape correlation factor

γ = surface tension

θ is the liquid-solid contact angle

d is the pore diameter

FIGURE 30.4 Bubble point pressure determination. Schematic of a pore cross section. γ is the surface tension, θ is the liquid-solid contact angle, and d is the pore diameter as shown in equation 2. As the pore diameter increases, the force required to overcome the capillary forces decreases resulting in a lower bubble point value. The surface tension of the wetting fluid will also impact the bubble point value. Using a lower surface tension fluid than water, such as alcohol, will reduce the liquid-solid contact angle. In this case, less force will be required to displace the liquid in the pore resulting in a lower bubble point value than for the identical filter tested with water as the wetting fluid.

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