Guidelines for Selecting Normal Flow Filters

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:

• Cell culture media sterilization

• Buffer filtration

• Product-stream filtration.

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

Figure 2: Normal flow filtration in biopharmaceutical production.

Cell culture media sterilization

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.

Sterilizing-grade membranes. The term "sterilizing-grade filter" is defined in the FDA's document Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice (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 Brevundimonas diminuta (B. diminuta) at a minimum concentration of 10⁷ colony forming units (CFU) per square centimeter (cm² ) of membrane area. Sterile filters are nearly always constructed of one or more sheets of polymeric membrane, either in pleated or flat-sheet form.

Low extractables. 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.

Low nonspecific binding. 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. 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:

• Filter installation and wet-out

• Pre-use filter integrity test

• Steam-in-place

• Media filtration

• Filter flush

• Post-use filter integrity test

• Clean-in-place

• Filter disposal.

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.

High permeability. 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.

High capacity. 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.

Survey of media sterilization filters. 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:

• DIFCO Miller LB broth for E. coli

• DIFCO YPD broth for yeast

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

• Hyclone SFM4CHO utility

• SAFC Biosciences EX-CELL 302.

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 filtration

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.

Validated 0.2-µm membranes. 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.

Chemical compatibility. 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.

Physical robustness. 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.

High permeability. 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:

• 1 M hydrochloric acid (pH = 1)

• 1 M sodium hydroxide (ph = 13)

• 50 mM Tris-buffered saline (pH = 8)

• 10 mM phosphate-buffered saline (pH = 7)

• 50 mM phosphate-citrate buffer (pH = 5)

• 0.1 M acetic acid

• 10% ethanol

• 6M urea.

Solutions were filtered at a constant pressure of 10 psid, and the volume filtered as a function of time was recorded until the solution was exhausted. Based on the test results, membrane permeability for each filter tested was calculated and estimations of the required number of equivalent 10-in, filter cartridges were made for a 12,000-L batch of buffer filtered in 1 h. Results are presented in Figure 4.

Figure 4: Comparative performance of sterilizing-grade and bioburden reduction filters for buffer filtration (shorter bars represent better performance).

Product-stream filtration

Biopharmaceutical products are filtered numerous times in the course of their manufacture to control bioburden, remove precipitates and separate solid contaminants (e.g.. fines from chromatography resins or diatomaceous earth flushed from depth filters). In most cases, sterilizing-grade filters are used for product-stream filtration, although growing concerns about cost-of-goods is resulting in increased use of bioburden reduction filters for these steps.

The following paragraphs describe the key characteristics one should look for when selecting a product-stream filter.

Validated 0.2 µm membranes. As is the case with buffer filtration, the choice between a sterilizing-grade and a bio-burden-reduction filter often depends on the unit operation to which the filtration is coupled. For example, sterilizing-grade membranes are required for the filtration of bulk drug substance and are usually desired for filtration steps performed before any product hold. However, bioburden reduction membranes maybe sufficient and more economical for many intermediate filtration steps (e.g., before a chromatography column) and therefore their use can result in significant cost savings. As mentioned previously, regardless of the choice of filter, it is important to choose a membrane that is validated for retention of bacteria and that the retention can be correlated to an in-process integrity test.

Physical robustness. Product-stream filtration is the highest value normal flow filtration operation in any biopharmaceutical process. Filter failures which occur during product-stream filtration require time-consuming and costly investigations and may result in lost product. As a result, normal flow filters used for product-stream filtration must

be constructed to withstand a broad range of operating conditions with respect to temperature, pressure and pH. Furthermore, product-stream filters should be 100% tested by the manufacturer and should include instructions for integrity testing at the point-of-use.

High capacity. Product streams are some of the most challenging filtration steps and filter performance can vary widely because of the strong effects of filter-fluid interactions. Therefore, it is important to select a filter that is optimized to provide high capacity, regardless of the protein type, concentration, and formulation buffer. Furthermore, many product-stream filtrations are coupled to relatively low-flow unit operations (e.g.. centrifugation, cell harvest or chromatography column loading), thereby making differences in membrane permeability less important when determining the required filtration area.

Low extractables. Product streams contain a drug substance that will eventually be administered to a human patient. Therefore, product-stream filters must be constructed of components that are proven to be biologically safe. Biological safety is demonstrated by the performance of the USP (88) Class VI Plastics Test for Biological Reactivity and the burden of obtaining this information rests with the filter manufacturer who should include test results as part of a validation package. In addition, vendors should be able to provide information regarding filter effluent quality in terms of total organic carbon, buffering capacity, non-volatile residue, bacterial endotoxins, and particle or fiber shedding.

Low protein binding. Membranes and other materials used to construct filter cartridges and capsules should not bind proteins or preservatives that are in the fluid because this may lead to product-loss or decreased shelf-life. Figure 5 shows the amount of several proteins bound by microporous membranes cast from several common polymers. Most modern membranes are cast from PES or PVDF to ensure minimal product loss.

Figure 5: Protein binding on various membrane materials.

Survey of filters for product-stream filtration. GE Healthcare performed a study to evaluate the most common filters that are used for product-stream filtration. In this study, two protein-containing feedstreams (1% bovine serum albumin and a monoclonal antibody purified on Protein-A chromatography and subjected to low pH viral inactivation) were used to challenge a panel of membrane filters from Pall, Millipore, Sartorius, and GE Healthcare. The specifics of the feedstreams are shown in Table 1.

Table I: Feed characteristics for product-stream filtration experiments.

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 number of equivalent 10-in. filter cartridges were made for a 2000-L batch filtered in 1 h. Results are presented in Figure 6.

Figure 6: Comparative performance of multilayer sterilizing-grade filters for product stream (shorter bars represent better performance).

In addition, experiments were run to compare the performance of ULTA Pure HC (sterilizing-grade) and ULTA Prime CG (bio-burden reduction). As shown in Figure7,ULTA Prime CG provides equal or better filter capacity which, when coupled with a lower per-filter cost, can translate to significant cost savings for applications where sterile-effluent is not required.

FIgure 7: Comparative performance ULTA Pure HC and ULTA Prime CG for product-stream filtration (shorter bars represent better performance).

Summary

The selection of the optimal normal flow filter is highly process dependent because of the varied performance requirements demanded by each process step. When considering the three main applications of normal flow filters, one should consider filters with the following characteristics:

• Media sterilization: Filters incorporating high capacity/high flow membrane formats minimize filtration system sizes while meeting full validation and integrity test requirements.

• Buffer filtration: Sterilizing-grade and bioburden reduction filters providing high permeability and wide chemical compatibility.

• Product stream filtration: Bioburden reduction filters reduce or eliminate bioburden and particulates immediately before chromatography columns and UF/DF operations. Sterilizing-grade filters provide the highest level of assurance before product hold steps and during bulk drug substance formulation and final vial filling. The quick guide to selection of normal flow filters (inset) provides additional details.

Regardless of the application, proper selection of normal flow filters leads to increased process efficiency from early phase product development through to full-scale biopharmaceutical production.

Acknowledgments

The authors would like to thank our colleagues Jozsef Vasi, Daniel Calnan, Helena Skoglar, Alisa Liten, Olivier Boizet, and Kristian Nilsson for their valuable contributions to this work.

Jonathan Royce is a senior application scientist and Jeffrey Carter, PhD, is director of filtration R&D, both at GE Healthcare, 14 Walkup Drive, Westborough, MA, Jonathan.Royce@ge.com Jakob Liderfelt is a scientist at GE Healthcare, Björkgatan 30, 751 84 Uppsala, Sweden.

References

1. US Department of Health and Human Services, Food and Drug Administration, Guidance for Industry Sterile Drug Products Produced by Aseptic Processing — Current Good Manufacturing Practice U.S. Department of Health and Human Services, Food and Drug Administration, September 2004.

2. ASTM Standard F838 - 05, "Standard Test Method for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration," ASTM International, West Conshohocken, PA, www.astm.org.

*Bioburden reduction filters are not defined by an industry standard. The term "bioburden reduction" is a designation used to describe a class of filters which provide a high level of microorganism retention (i.e., LRV4-6), but do not yield a sterile effluent under the high bacterial load called for in the ASTM F838-05 test method. Manufacturer claims on bioburden reduction filters vary from "typical" retention data to full validation of a minimum LRV. In practice, most bioburden reduction filters are of a 0.45 or 0.2 urn rating and may yield a sterile fluid in common usage, where bacterial loads are much lower than those used in the ASTM challenge. Nevertheless, process-specific claims of fluid sterilization through the use of a bioburden reduction filter are generally not appropriate.