Jeffrey Carter, PhD, is director of filtration R&D, at GE Healthcare, 14 Walkup Drive, Westborough, MA

Jeffrey Carter

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:  

Articles

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