Filtration is the process of separating various components in the feed material. In bioprocessing, it can be categorized into two main types based on the movement of the liquid relative to the filter media:

1. Normal Flow Filtration (NFF): Also known as dead-end filtration. In this method, the feed liquid flows perpendicular to the filter medium, causing particles to be trapped on the filter. Common filter elements used in this process include syringe filters.

2. Tangential Flow Filtration (TFF): Also referred to as crossflow filtration (CFF). In this method, the sample liquid flows tangentially (parallel) to the surface of the filter media. A portion of the fluid passes through the filter media, while particles that are trapped or deposited on the surface are swept away by the liquid flow parallel to the media. This action helps to mitigate membrane clogging.

Key parameters in tangential flow filtration

Pressure Drop (ΔP): The pressure decay along the membrane channel can be calculated using the formula ΔP = Pf - Pr, where Pf is the feed pressure and Pr is the retentate pressure.

Transmembrane Pressure (TMP): The pressure difference across the two sides of the filtration membrane serves as the driving force that allows the solvent and components smaller than the membrane's molecular weight cut-off (MWCO) or pore size to pass through the membrane and enter the filtrate. This pressure difference can be calculated using the formula TMP = (Pf + Pr) / 2 - Pp, where Pf is the feed pressure, Pr is the retentate pressure, and Pp is the permeate pressure.

Molecular Weight Cut-off (MWCO): MWCO is defined as the lowest molecular weight of a solute that can be retained by the membrane. For instance, a 30 kD membrane can reject 90% of solutes with a particle size of 30 kD in the solution.

Shear Rate: When the liquid material flows through the filter channel, it exerts a shearing effect on the product due to its interaction with the membrane wall of the flat sheet cassette or the inner wall of the hollow fiber lumen, as well as the turbulence within the liquid. For unstable or active products, shear is a critical factor in process operations.

Flux: The permeate flow rate, standardized by the filter membrane area, is measured in liters per square meter per hour (L/m2/h).

Cross-Flow Rate: refers to the flow rate of the liquid moving parallel to the surface of the membrane medium, which is typically calculated based on the flow rate at the retentate end.

Membrane Polarization (Fouling): Membrane polarization, also known as fouling, refers to the accumulation of solutes on the surface of a membrane. This process can result in the formation of a barrier layer that obstructs permeation.

Volume Concentration Factor (VCF): Concentration factor calculated as the ratio of the starting volume to the final volume, expressed as VCF = starting volume / final volume.

Classification of Tangential Flow Filtration

Microfiltration: In TFF systems, microfiltration typically refers to membranes with pore sizes greater than 0.1 μm. This process is commonly employed for the filtration or clarification of suspended particles, such as in the harvesting and clarification of cell culture fluids to collect cells and/or remove impurities, including cell debris.

Ultrafiltration: Ultrafiltration typically refers to membranes with pore sizes ranging from 0.001 μm to 0.1 μm. In ultrafiltration applications, MWCO is commonly used to specify the membrane's cutoff characteristics, typically ranging from 1 kDa to 750 kDa. Ultrafiltration is frequently employed for the separation of small molecules.

Guidelines for Selecting Membrane MWCO and Pore Size: For most applications, it is advisable to select a pore size or molecular weight cut-off (MWCO) that is 1/3 to 1/5 the size of the molecules to be retained, or 3 to 5 times the size of the molecules to be permeated. However, this selection depends on the specific purpose of the application. For instance, if TFF is employed for buffer exchange, the significant difference in particle sizes between the molecules to be retained and those to be removed allows for the selection of the lower end of the range to maximize yield. Conversely, if the application requires the removal of impurities with a smaller size difference, it is necessary to choose the higher end of the range to enhance purity as much as possible.

Basic Steps for Tangential Flow Filtration

Successful TFF operations typically involve several fundamental steps:

Rinse: The rinsing step is essential for removing the preservation or storage fluid from the filter, which typically contains glycerol. This process is generally performed using purified water. To achieve a specific backflow and/or filtrate volume as the endpoint of the rinsing step, one should adhere to the manufacturer's recommendations for the filter.

Disinfect: For new membrane filters and/or before each processing run, the filters must be disinfected. The purpose of disinfection is to eliminate residual storage fluid and to monitor and control bioburden. Filter manufacturers typically recommend compatible disinfecting solutions, which usually include sodium hydroxide (NaOH), sodium hypochlorite (NaClO), and specific acids.

Integrity Test: Integrity testing is conducted to verify the system setup and ensure the integrity of the filter both before and after the use of the membrane.

Normalized Water Permeability (NWP): Measuring the permeability of purified water through a membrane under standard pressure and temperature conditions, Normalized Water Permeability (NWP) serves as an indicator of a new filter's permeability and a means to assess the cleaning effectiveness of the membrane upon reuse. NWP is typically measured under consistent conditions, including feed flow rate, TMP, conductivity, and temperature. The normalized water permeability should be standardized based on the TMP. NWP can be calculated using the following formula: NWP = (flow rate in L/h) * (Temperature Correction Factor, TCF) / TMP / membrane area.

Buffer Rinse: The primary purpose of a buffer rinse is to eliminate the wash solution and to hydrate the membrane with a buffer that is compatible with the feed solution.

Operation: The operational modes of the TFF system can be categorized into two primary types: 1) concentration and 2) diafiltration.

Concentration: Concentration is a straightforward process. During operation, the liquid components in the solution are removed, while components larger than the membrane's molecular weight cut-off (MWCO) or pore size are retained. As the liquid components are extracted, the volume of the feed liquid decreases, leading to an increase in the concentration of the retained components. In essence, concentration refers to the process of increasing the amount of solute in a given volume of solvent.

Diafiltration: Diafiltration is a fractionation process in which components smaller than the membrane MWCO/pore size are removed along with the liquid and enter the filtrate. Conversely, components larger than the membrane MWCO/pore size are retained, while diafiltration buffer is simultaneously added. This technique is commonly employed to remove salt ions or to exchange buffers. Based on the method of adding the diafiltration buffer, diafiltration can be categorized into two types:

Discontinuous Diafiltration: There are two modes of operation for discontinuous diafiltration.

1) First add a specific volume of diafiltration buffer to the feed solution. Next, concentrate the solution to the initial volume, then add more diafiltration buffer, and repeat the process.

2) First concentrate the feed solution to a specific volume. Next, add diafiltration buffer to reach the initial volume, then concentrate again. Repeat this process as necessary.

Continuous Diafiltration: In continuous diafiltration, the diafiltration buffer is added to the feed vessel at the same rate that the filtrate is removed through the filter, ensuring that the volume in the feed vessel remains constant. Typically, approximately five diafiltration volumes (DV, where one Diafiltration Volume is equal to the volume of the feed liquid at the beginning of the diafiltration process) can reduce the ionic strength of the solution by about 99%. In recent years, advancements in the automation of TFF systems and the increased efficiency of continuous diafiltration have established it as the primary method for diafiltration.

Product Recovery: During TFF operation, in addition to the feed liquid that returns to the feed vessel, a portion of the feed liquid will remain in the system's flow path and filter. For straightforward system configurations and/or stable products, the retained feed volume can be recovered through air purging. In more complex settings, the product can be retrieved by flushing with a buffer, or a low discharge point can be established on the pipeline for recovery.

Cleaning in Place (CIP): The purpose of cleaning is to eliminate product residues, prevent cross-contamination, remove bioburden and endotoxins, restore process performance, and extend service life. The most commonly used cleaning agent is 0.1-1N NaOH. Other cleaning agents include sodium hypochlorite (NaClO), phosphoric acid (H3PO4), and specific detergents. In addition to evaluating the cleaning efficacy, the selection of cleaning agents must also consider the tolerance of the membrane. Furthermore, it is essential to establish standards for assessing the cleaning effectiveness and to conduct cleaning validation. For certain applications, a sterile TFF process may be necessary. For instance, when the final product cannot be sterile filtered to ensure sterility, but most membrane products cannot endure stringent steam-in-place conditions, one may consider using system SIP in conjunction with pre-irradiated sterilized filter module or employing a pre-irradiated sterilized closed TFF flow path.

Integrity Testing

NWP Testing

Storage: Filters should be stored in a designated storage solution as recommended by the manufacturer. The primary purpose of this is to maintain the moisture of the membrane and inhibit the growth of bacteria, fungi, and other microorganisms.

To enhance operational efficiency, single-use tangential flow filtration products can be utilized to eliminate or simplify cleaning and cleaning validation processes. Modern biopharmaceutical manufacturing facilities also favor the use of fully single-use production lines to increase flexibility and expedite turnaround times. Additionally, single-use products are a more suitable option for multi-product facilities.

Duoning Biotech provides a complete range of hollow fiber tangential flow filtration modules, including modified polyethersulfone (mPES) and polyvinylidene fluoride (PVDF) materials, 5 kD to 0.2 μm MWCO/pore size specifications, 1 mm and 0.5 mm fiber inner diameters, and 16 cm2 – 14.2 m2 surface area options to meet different applications and scale requirements.