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Membranes and filtration:

Membranefiltration in the biopharm industry

andar Dixit of Sartorius Stedim explains the usage and importance of membranes as a sterile form of filter media in the biopharmaceutical industries, with an overview of the general process and a look at recent developments in this sector of the filtration industry.

Introduction

Normal flow or dead-end filters using micro-porous membranes of synthetic polymers are used extensively in a wide variety of biopharmaceutical liquid filtration applications. A typical bioprocess consists of various unit operations beginning with media preparation and sterile media addition into a bioreactor or a fermenter followed by several stages of clarification of cell harvest post-fermentation, followed by three to four stages of purification using a series of chromatography columns and ultrafiltration/diafiltration (UF/DF) steps for concentration and buffer exchange, prior to formulation, fill and finish. The processing steps prior to the first chromatography column in the purification suite are generally referred to as “upstream” processes, whereas the

unit operations thereafter are termed as “downstream” processes. See Figure 1 for a generic block diagram of the process for better appreciation of the various stages in upstream and downstream processing.

Media filtration typically involves membranes with retention ratings ranging from 0.45-µm for prefilters and 0.2-µm and 0.1-µm as final filters. The membrane filters are also used in cell harvest clarification post-cellulosic DE clarification filters. The primary goal of these filters is to reduce bioburden in the filtrate, thereby reducing the chance of bacterial contamination in the product. In downstream processing, sterilising grade 0.2-µm rated membrane filters are commonly used for reducing bioburden as well as maintaining sterility of pooled downstream purified intermediates. A sterilising grade 0.2/0.22-µm

rated filter is also used to sterilise the final purified bulk drug substance into vials.

For a filter to qualify as sterilising grade, it needs to provide a “sterile” filtrate when challenged with Brevundimonas Diminutabacteria at a minimum challenge level of 1 x107 CFU/cm2 of filter area. This Bacterial Challenge Testing (BCT) has to be performed in strict accordance with ASTM F838-05 standard. Most major filter manufactures perform this testing in-house and correlate the BCT to a non-destructive Integrity Test with limit values of diffusive flow and bubble point reported, within which the membrane filter can be construed as “integral” for bacterial retention. These Integrity Test values are reported in the validation literature of the filter manufacturer and the customers routinely use these non-destructive Integrity

M

Figure 1: Typical bioprocess block diagram showing various stages of upstream and downstream processes.

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Tests to ensure filter integrity pre- and post-use. Automated Integrity Testers are used to carry out diffusive flow and bubble point tests.

Membrane filtration is also routinely used in traditional pharmaceutical applications such as blood fractionation, processing of sera and Large Volume Parenterals (LVP). The goal here is the same as in biopharmaceutical processes, i.e. reduce the potential of bacterial contamination of the product as well as provide a method for cold sterilisation. A right choice of the prefilter and final membrane filter combination strikes an optimum balance between flow rates, filtration time and overall filtration costs.

Important membrane filter characteristics

Since the primary goal for membrane filtration is to reduce bacterial contamination in the filtrate and provide “sterile” filtrate for 0.2-µm rated filters; they are evaluated based on the following criteria:

• Effective retention of bacteria to significantly reduce bacterial contamination risk;

• High total throughput performance resulting in the low filtration area and hence reduced filtration costs;

• Acceptable flow rate range to ensure that the entire batch is filtered in a reasonable time-frame;

• Low degree of absorption/adsorption of target protein, especially as one moves

downstream and the concentration of target protein increases.

Typical comparison charts of various membrane materials in terms of flow rate, total throughput and adsorption/yield loss are seen in Figures 2, 3 and 4, which highlight significant differences between various membranes in performance including the same membrane material composites (PESU and PVDF) from different manufacturers.

A variety of tests are usually conducted with the fluid of interest on a bench scale using small-scale 47 mm disc filters to determine what type and grade of membrane filters work well for flow rate and throughput performance. If the flow rate and throughput for the membrane filters yield reasonable sizes for the full-scale, only a membrane filter is specified in the process. If the throughput is not high enough from different grades of membrane composites selected; a range of prefilters are tested for throughputs as well as their ability to protect the final filter If the final filter throughput is also in an acceptable range; such a combination is recommended for further larger scale testing. The flat disc trials only provide an indication of filter train performance. It is absolutely essential to verify the filter train selection by running trials on a larger scale using pleated capsules. Sometimes, a confirmation filterability trial is also conducted using all the filters in a train at the full, process scale. Such a test provides a definitive assurance of sizing of each filter in the train.

The goal of membrane filtration is to reduce the potential of bacterial contamination of the product as well as provide a method for cold sterilisation.

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Most of the membrane filters are offered in a variety of sizes beginning with 150 cm2 effective filtration area (EFA) through 18,000 cm2 EFA. Availability of such a wide range of pleated devices is critical, especially with the recent move to completely disposable bag plus filter assemblies becoming quite common in small-to-

medium scale applications. The filter capsules are designed such that they can be sterilised using Gamma Irradiation. These capsules are assembled with tubing on to the inlet ports of the bags with the filter capsule attached and the entire assembly is then subjected to Gamma Irradiation. This “sterilised” assembly is then

ready-to-use by the customer. It is critical to choose a membrane as well as all other filter capsule components that are compatible with Gamma Irradiation.

Types of membrane filters

A variety of polymers are used by different manufacturers for casting membranes. Cellulose Acetate, Polyethersulfone (PES/PESU), Polyvinylidene Fluoride (PVDF) and Nylon are some of the common polymers used in manufacturing pleated membrane filters for liquid service. These membranes are typically 100-150-µm thick and consist of a highly porous structure with a narrow particle size distribution. The retention of particles and microorganisms is mainly achieved on the “surface” of the filter matrix by sieve retention. Particles and microorganisms larger than the actual pore size of the membrane are effectively removed. Membrane filters are predominantly used in the final filtration step because they ensure the most reliable retention of particles and specific microorganisms, sacrificing throughput capability for most applications.

The main advantage of these filters is their high degree of bacterial retention. There are also more open (> 0.2 µm) membranes available, which are used as an in-built prefilter layer, providing enhanced throughput performance. Many of the commercially available membrane filter devices are generally pleated from two heterogeneous membrane layers. The upstream membrane layer is coarser for larger particulate removal, whereas the downstream membrane is finer to reduce colloidal content and bioburden. Such 0.45- µm rated membrane Prefilters are usually used as chromatography column protection filters or as a bioburden reduction step in downstream processing. Many manufacturers also pleat together the micro-glass fibre fleece as the upstream layer in combination with a membrane as the downstream layer. The Micro-GF provides depth filtration as well as high clarification capabilities, while the membrane provides finer particulate removal and higher bacterial retention. Such micro-glass(GF)/membrane combination filters are popular in serum-containing media filtration, small-scale cell harvest and diagnostic solutions.

Chemical compatibility of membranes is another important aspect that needs to be considered for some applications. Most of the biologic solutions are aqueous-based and are at near-neutral pH conditions. However, caustics as well as acid solutions are routinely used during Cleaning-in-Place (CIP) processes. The filters thus get exposed to a very wide pH range from 1 to 14. Polypropylene works best for such applications, which is why it is widely used for manufacturing all accessories of the cartridge or capsule filters including core, cage, adapters and support and drainage layers. In terms of membrane materials themselves,

Figure 2: Flow rate comparison for various membranes per 10” element at 15 psig ΔP.

Figure 3: Total throughput comparison per 10” element at 15 psig ΔP constant pressure.

Figure 4: IgG unspecific adsorption comparison (mg./10” element).

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except PES which is good over the entire pH range, all other liquid-service membranes have pH limitation. As an example, cellulose acetate can be used in the pH range of 4-8, while PVDF is not recommended for solutions with very high pH values.

Sterilisation is also commonly performed in biotech applications with saturated steam with temperatures reaching as high as 134 °C for 30 minutes minimum. Complete filter cartridge construction (outer cage, inner core, and adapter) needs to withstand these harsh process conditions for short time intervals. Once again, polypropylene is an optimal choice for the materials of construction. Filter manufacturers also have to test a filter’s ability to withstand multiple steaming cycles.

There are also new, innovative application specific approaches in membrane filtration like the Sartopore® 2 XLG 0.8/0.2 and Sartopore® 2 XLI 0.35/0.2 PES filters from Sartorius Stedim Biotech. Both of these filters have different prefilter membrane retention ratings designed for specific particle size distribution characteristics in fluids commonly encountered in biotech as well as pharmaceutical industries. The Prefilter layers in XLG and XLI are effective in different applications, achieving very high throughputs combined with optimised pleat pack construction, resulting in 30% higher effective filtration area per 10”

element. The 0.2-µm final filter layer is the same as the current Sartopore 2 0.45/0.2 combination, providing highly reliable bacterial retention. This new development promises a quantum leap in membrane filtration compared to the current commercially available double layer membrane filters. These filters have reduced total membrane filters surface area by more than half in some large-scale applications while eliminating the need for prefilters.

Summary

Dead-end nembrane filtration has wide use in bioprocessing and they are primarily relied upon for reducing bacterial contamination as well as ensuring sterility of the process fluids. An optimally designed membrane filter has to provide excellent balance in all of the following criteria:

• Highest total throughput performance, directly resulting in lower filtration costs;

• High Flow rate;

• High degree of bacterial retention;

• Low unspecific adsorption of target protein.

A wide range of membrane materials are available from all the major suppliers.

Some of the widely used polymers are: Cellulose Acetate, PES, PVDF and Nylon.

Most of these filters have heterogeneous double layer construction with an in-built more open membrane prefilter layer. Each membrane material has its strengths and is selected appropriately for a certain stage in a bioprocess.

Membrane filtration is absolutely necessary in many biopharmaceutical applications for bioburden reduction as well as a method for cold sterilisation. Some of the latest developments in membrane filters include application-specific filter composites manufactured out of asymmetric PES membranes exhibiting very high flow rates and total throughputs, coupled with optimised pleat pack designs. •Contact:Mandar Dixit is the Product Manager for Sterilising Grade Filters at Sartorius Stedim North America Inc. located in Edgewood, NY. He has over 14 years of experience in filtration and separation technologies. He currently supports the North American Biopharmaceutical market for Filtration Technologies with special focus on Sterilising Grade Filters and Prefilters. He is a member of PDA and ISPE. Mr. Dixit has co-authored papers in trade journals on Prefiltration as well as Filter Optimisation and Scale-up studies. He received his Bachelor’s Degree in Chemical Engineering from IIT-Bombay in India and his Master’s Degree in Chemical Engineering from Louisiana State University in Baton Rouge, Louisiana.