Sterile Filtration of Serum-free Mammalian Cell Culture Media

Transcription

1 Sterile Filtration of Serum-free Mammalian Cell Culture Media Abstract Cell culture media is a complex mixture of various synthetic and biological components that, in an aqueous solution, provide the proper chemical environment and necessary nutrients for healthy cell propagation and high-protein expression. Although mammalian cell culture has a relatively long history, growth media remains an evolving technology where change is driven not only by the expanding variety of production cell lines but also by concerns over product safety and purity. Careful preparation of cell culture media is of key importance to ensuring sterility of the cell culture batch and subsequent purification processing. This application note evaluates and compares three different Millipore Express PES and other sterilizing-grade filters for different media filtration applications, across a range of serum-free media formulations. Table of Contents Mammalian Cell Culture Media Overview 1 Classical Media 2 Serum-free Media 2 Chemically-defined Media 2 Media Preparation 3 Experimental Method and Data Analysis 4 Results of Millipore Express SHC and SHF Membranes in Serum-free Media 5 Soy Peptone-based Media 5 Commercial Growth Media 6 CHO Media 6 NSO Media 7 Results of Millipore Express SHR with Prefilter and Other Sterilizing-grade.1 µm Membranes in Serum-free Media 8 Experimental Method and Data Analysis 8 Commercial Growth Media 9 CHO Media 9 NSO Media 1 Soy Peptone-based Media 1 Conclusions 11 Mammalian Cell Culture Media Overview Cell culture media is commercially available in a great number of types and variations to suit the particular needs of specific cell lines and genetically-modified variants. In many cases, cell biologists have developed an even wider variety of proprietary formulations to satisfy their specific culture requirements. Although mammalian cell culture has a relatively long history, growth media remains an evolving technology where change is driven not only by the expanding variety of production cell lines but also by concerns over product safety and purity. Chief among those concerns are adventitious biological contaminants (bacteria, viruses, prions) introduced with animal products, principally sera, that are often used as supplements in cell culture media. Of secondary concern are the complications, brought on by the bulk addition of undefined proteins contained in the growth media. A P P L I C A T I O N N O T E

2 Classical Media Classical or basal media, such as Eagle s media, refer to the essential chemical components required to sustain mammalian cells in vitro. The composition mimicks in vivo conditions of ph, osmolarity and basic nutrients. The main components of basal media are: sugar (glucose) salts (electrolytes) amino acids vitamins trace elements Basal media are often supplemented with sera to provide the essential growth factors, hormones and carbohydrates necessary for cell proliferation and high protein production. Serum, generally fetal calf or newborn calf, is added to basal media at concentrations of 1 1%. Serum-free Media To minimize the risk of serum-borne pathogens, a number of serum substitutes are used in mammalian cell culture including: bovine pituitary extract (BPE) bovine or porcine tissue hydrolysates (peptones) soy bean hydrolysates (peptones) The term "peptone" is used to define a water-soluble product produced by the hydrolysis of a particular protein or proteins. Hydrolysis is done by various methods based on acid, alkaline or enzymatic processes. The hydrolysate contains a distribution of free amino acids and peptides of various molecular weights. In general, the proteins used in the production of peptones are of two types, animal proteins (casein, gelatin meat) and vegetal proteins (soy). Soy peptones in particular have become increasingly popular in tissue culture processes. Apart from amino acids, these peptones contain other constituents that stimulate cell growth, such as nucleic acids, minerals, vitamins and carbohydrates. In a number of cases, serum-free media are supplemented with serum proteins, such as albumin and transferrin which, although low, still carry some risk of pathogen contamination. Serum-free media is also available in protein-free formulations which further mitigate, but do not eliminate, the risk of biocontamination. Aside from contamination, the cell culture developer must consider other concerns related to the use of these serum substitutes. Both extracts and peptones are poorly defined products of variable quality. Since they are derived from natural sources, their chemical as well as physical properties are dependent on environmental, process, and storage conditions. Given this uncontrolled variability, the filterability of these materials, once incorporated into the cell culture media, can also be expected to vary significantly. As a result, the robustness of the sterile filtration process for these variable feed properties is critical for proper production control. Chemically-defined Media The optimal media in terms of minimum risk of bio-contamination and maximum process control are those in which every chemical component is precisely defined and quantified. Such media types are available only in limited varieties and have not yet found their way into large-scale tissue culture operations. Still the risk of contamination is not completely eliminated in these products since some components (principally amino acids) are generally of animal origin. The next generation of chemically-defined media (which are now beginning to appear on the market) are truly animal-derived and component free. These products represent the highest attainable quality in cell culture media from both a regulatory and a process perspective. Nevertheless, the validation of animal-free origin remains an issue. 2

3 Media Preparation Cell culture media must be sterilized before it can be delivered to an aseptic bioreactor or fermenter to prevent the growth of adventitious bacteria or other organisms. This sterilization is generally accomplished by normal flow or dead-end filtration using a validated, sterilizing-grade microporous membrane, typically carrying a nominal pore size rating of either.2 or.1 micron. Careful preparation of cell culture media is important to ensure sterility of the cell culture batch and subsequent purification processing. Mycoplasma has become widely recognized as the smallest bacterium to thrive in cell culture that cannot be retained by.2 µm sterilizing-grade filters. Mycoplasma is the generic name assigned to microorganisms in the class Mollicutes. They lack a cell wall and are bound together by a trilaminar plasma membrane. Due to their size and deformability, mycoplasma can penetrate a.2 µm rated filter. Frequently, methods for media preparations are being revised to replace.2 µm sterilizing-grade filters with.1 µm sterilizing-grade filters for sufficient log removal of mycoplasma. Media is typically mixed in bulk and then aseptically transferred to the bioreactor. Prefiltration is used to remove the bulk of particular and colloidal contaminants from media, in order to reduce process bioburden and extend the service life of the filter train. As cell culture media spans many media types among several cell lines, there exists a variety of constituents at a wide range of concentrations. Prefilters should be sized appropriately to handle batch-to-batch media variability and ensure that the sterile media fill in the bioreactor is completed successfully and on time. Final sterilizing-grade filtration should remove bacteria (and mycoplasma) to preserve the aseptic barrier around the bioreactor. Currently, media products are sold both as dry powdered formulations as well as pre-sterilized liquid solutions. Small-scale operations (less than a few liters) may opt for prepared liquid formulations that are ready to use. Cost considerations, however, dictate that media preparation for production-scale processes start with dry ingredients dispersed into pharmaceutical-grade water. In media preparation, process time, temperature and degree of agitation all contribute to some degree of variability in the properties of the media, particularly with regard to the amount and size of insoluble particulates that in turn, impact filterability. Pasteurization (short duration) is sometimes used prior to sterile filtration as a means of reducing pathogens other than bacteria, such as viruses and prions. Experience has shown that pasteurization processes typically cause no substantial changes in media quality and filterability. Sterile filtration is generally a batch operation. Prepared media is transferred under pneumatic pressure from a mix tank to the bioreactor or intermediate hold tank through the sterilizing-grade filter(s), while transfer pressures typically range from 1 3 psig. Sterilizing-grade cartridge filters are first steamed in place and often integrity tested prior to use. Smaller scale operations generally use gammasterilizable or sterile capsules that are aseptically connected to the process system. This application note evaluates and compares three different Millipore Express PES filters and other sterilizing-grade filters for different media filtration applications. The filters were evaluated on the basis of filter throughput or volume processed per unit area. In this Application Note, Millipore Express SHC filters, designed for high-fouling fluids, and Millipore Express SHF filters, used in flux-based filtration applications, were compared to other.2 µm sterilizing-grade filters using a variety of growth media types (Table 1). Millipore Express SHR filters, which are designed to provide a high level of mycoplasma removal, were compared to other.1 µm sterilizing-grade filters with different types of serum-free media. Table 1: Millipore Express Filter Selection SHC (sterilizing grade, high capacity) High fouling media.5/.2 µm SHF (sterilizing grade, high flux) Prefiltered media.2 µm SHR with Prefilter (sterilizing grade, Mycoplasma protection/removal high retention).5/.1 µm 3

4 Experimental Method and Data Analysis Millipore Express nominal.1 and.2 µm rated membranes were evaluated for the sterilization of serum-free cell culture media using a variety of growth media types. The Millipore Express SHF filters consist of a sterilizing-grade, asymmetric composite polyethersulfone membrane which is nominally rated at.2 µm. In Millipore Express SHC, the sterilizing-grade Millipore Express SHF.2 µm membrane is coupled with a similarly constructed membrane prefilter of slightly larger pore size (nominally rated at.5 µm) for enhanced particle-holding capability. These membranes were compared in side-by-side tests to a selection of.2 µm sterilizinggrade filters, including Durapore filters (as a single membrane and with various prefilter configurations) and several other commercially available products as described in Table 2. Millipore Express SHR high-retention PES filters provide sterilizing-grade performance and a high level of mycoplasma removal. An on-board polyethersulfone membrane prefilter provides extended filter capacity. Millipore Express SHR filters were compared to other.1 µm sterilizing-grade filters with a range of media types, including CHO serum-free media, NSO serum-free media and soy peptone-based serum-free media. As noted in Table 3, membranes tested also include: Supplier A PES.2/.1 µm and Supplier B PVDF.2/.1 µm. Since all membranes tested are validated by their respective manufacturer as sterilizing-grade filters, they were judged only on the basis of filter throughput, defined as flux or accumulated filtrate volume, for set process times. To mimic actual process conditions, all tests were conducted under constant feed pressure conditions. All tests were conducted on 47 mm cut disks housed in standard stainless steel holders (Millipore XX44 47 ) in duplicate sets. The 47 mm membrane discs were wetted with 3% isopropyl alcohol and rinsed with water. Once assembled in the holder, they were integrity tested to ensure proper membrane installation. To accomplish this, the holders were submerged in a water bath with filtrate open. At an air inlet pressure of 5 psi, no visual air bubbles from the holder were noticed. The membranes were water flux tested at a fixed Table 2: Filter Construction:.2 µm-rated membranes Filter Millipore Express SHF.2 µm Millipore Express SHC.5/.2 µm Durapore (Millipore).2 µm Construction single-layer polyethersulfone dual-layer polyethersulfone single-layer polyvinylidene difluoride Multilayer Durapore (Millipore) dual-layer polyvinylidene.45/.2 µm difluoride Multimedia Durapore (Millipore) Multimedia Durapore (Millipore) Supplier A1.2/.2 µm Supplier A2.8/.2 µm Supplier A3.45/.2 µm Supplier B.45/.2 µm single-layer polyvinylidene difluoride with single-layer mixed cellulose esters prefilter single-layer polyvinylidene difluoride with dual-layer mixed cellulose esters prefilter dual-layer polyvinylidene difluoride dual-layer polyethersulfone dual-layer polyethersulfone dual-layer polyethersulfone Table 3: Filter Construction:.1 µm-rated membranes Millipore Express SHR with Prefilter.5/.1 µm Supplier A.2/.1 µm Supplier B.2/.1 µm dual-layer polyethersulfone dual-layer polyethersulfone 1 psi differential pressure for three minutes to ensure adequate wetting. Up to five membrane disk samples were tested concurrently using a single-feed supply to minimize extraneous variability. Feed solution temperatures were C. Filter pressures were fixed at 1 psi differential, and the feed solution was stirred for test duration. For each membrane sample, the accumulated filtrate volume was weighed on a calibrated load cell every 4 5 seconds and the time/weight recorded using a PC- based data acquisition system. The tests were continued until 9% reduction in initial membrane flux was reached or the feed solution was exhausted. The various growth media used in these tests were chosen to represent each of the major serumfree media types. Special consideration was given to those most commonly used in the industry. dual-layer polyvinylidene difluoride 4

5 Figure 1 depicts two hypothetical constant-pressure tests. In one case, the growth media causes pronounced membrane plugging and the rate of flow steadily declines until the volume of fluid processed reaches a set maximum (V max ). In the second case, the feed is virtually free of plugging solids, and the membrane is capable of sustaining a steady flow rate almost indefinitely (or over the course of the test period). For tests in which membrane plugging was evident, performance was measured by the throughput achieved (filtrate volume per unit area in L/m 2 ) at one or more set process times. Throughput was measured at 3 -, 6 - and/or 12-minute process times. As most membranes did attain greater than a 9% reduction of initial flow (V9) within 6 minutes, the plugging profile or membrane performance limit was effectively captured within this time interval. Comparison of filtrate volumes at the 3-minute mark highlight those membranes with high initial flux. In feeds where very little to no plugging occurred (no measurable flow decay), performance was measured according to filtrate volume per unit area per unit of time, typically referred to as flux (in L/m 2 /hr or LMH). Membrane rankings differed with the time intervals, due to variation in membrane plugging profiles with respect to time. Results of Millipore Express SHC and SHF Membranes in Serum-free Media Soy Peptone-based Media Given the growing popularity of soy peptone as a serum substitute, a synthetic growth medium was prepared in our laboratory where peptone was added to a common basal medium. The feedstock for this study was a solution of 3 g/l soy peptone [peptone from glycine max (soybean), type IV, powder, Sigma-Aldrich Co., C/N P521] dispersed in Dulbecco s Modified Eagles Medium (DMEM, Sigma-Aldrich Co., C/N D-7777). Constant-pressure tests were run in duplicate for each membrane sample. Throughput results are shown in Figure 2. As evident from the general lack of gain in throughput between 3 6 minutes, this peptonebased medium causes relatively rapid plugging in all membranes tested, regardless of membrane polymer material, membrane structure or number of layers. In spite of the severe fouling nature of the peptone, Figure 1. Hypothetical Performance Curves for Constant-pressure Filter Test 1,6 1,4 1,2 1, Nonplugging feed, flux-driven throughput Time (minutes) Comparison intervals at 3 and 6 minutes Figure 2. Soy Peptone in DMEM 2,2 2, 1,8 1,6 1,4 1,2 1, Millipore Express SHC filters demonstrated remarkable process capability, outperforming the nearest competitor two fold in throughput. The on-board membrane prefilter of the Millipore Express SHC filters efficiently captured undissolved peptone particles, raising the throughput of the underlying Millipore Express.2 µm membrane (SHF) nearly nine fold. Given the poor performance of the other sterilizing-grade membranes in this media type, prefiltration would normally be required to raise throughput levels to a range that is considered economically practical, typically above 1, L/m 2. Plugging feed, capacitydriven throughput 1 psid at 3 min at 6 min 5

6 To assess the potential benefit of prefiltration, two prefiltration schemes were evaluated to note the impact on sterile membrane performance. The prefilters chosen were Milligard CWSS membranes (Opticap 4 in. capsules, C/N KWSS4TC3) and Polysep II CGW1 prefilters (cartridge 4 in., C/N CGW1M4S3). Milligard prefilters mixed two ester cellulose membranes [RW6 (.5 µm) and RW3 (.2 µm)]. Polysep II CGW1 prefilters are comprised of a nonwoven glass material (1 µm) atop two layers of mixed esters of cellulose RW3 and RW1 membrane (.1 µm). With a tighter final membrane (RW1 versus RW3), the Polysep II prefilter is expected to yield a more highly clarified filtrate and to have a higher sterile filter capacity than Milligard prefilter. Following prefiltration, membrane throughput for the soy peptone media was predominantly flux driven, with very little plugging detected in any tested membrane. Overall, throughput increased several fold with prefiltration in some cases more than 3x. Flux data for these tests are provided in Figure 3. On average, Millipore Express SHF membrane yielded approximately 25% higher flux (or throughput per unit time) than its closest competitor with either prefiltration scheme. The benefit of an on-board prefilter membrane as in the Millipore Express SHC and multi-layer Durapore formats is reduced since the Milligard and Polysep prefilters appear equally capable of protecting the.2 µm-rated, sterilizinggrade membrane. CHO Media Chinese hamster ovary (CHO) cells are by far the most common expression system for the production of recombinant proteins and, in particular, monoclonal antibodies. JRH Ex-Cell 32 CHO (powder, C/N 24324), a serum-free medium, was chosen as a representative growth medium with respect to the current industry focus on MAb production and away from serum-based media. The medium was prepared by mixing the packaged powder with high-quality water (Milli-Q ) and sodium bicarbonate, for buffering capacity. Sterile filtration tests were run at constant pressure to compare the performance of Millipore Express SHF and SHC membranes relative to other.2 µm sterilizing-grade membranes. Figure 4 shows the throughput results. For this moderately plugging feed, throughput was measured at 3- and 6-minute intervals. Flux (LMH) Figure 3. Flux Performance Data for Prefiltered Peptone Streams 12, 1, 8, 6, 4, 2, 1 psid Polysep II prefiltered Milligard prefiltered Commercial Growth Media Due to cost and time considerations, cell culture developers often rely on commercially available growth Figure 4. Serum-free CHO Media media rather than custom formulations. Large commercial suppliers include JRH Biosciences, Hyclone, Cambrex and others. Their product offerings span a broad range of formulations covering all major media types (serum-based, serum-free, protein-free, etc.). Millipore Express membranes were evaluated with 6, 5, 4, 3, 2, 1 psid at 3 min at 6 min two of the more commonly used media formulations 1, available from JRH Biosciences (Lenexa, KS). In keeping with industry trends the media chosen were serum-free. 6

7 Despite some variance between test runs, the performance rankings were maintained. On average, Millipore Express SHC membranes demonstrated roughly 4% higher throughput than its closest competitor at 3-min and 6-min time intervals, indicating superior performance in both flux and particle-holding capability. The advantage of the on-board prefilter membrane in SHC is apparent in these tests where throughput is increased nearly three-fold over Millipore Express SHF membranes. The performance differences noted between test runs is attributed to lot-to-lot medium variability. All membrane samples performed better on the first batch of media than the second. Although medium composition, solution temperature, mixing times, and degree of agitation were tightly controlled, subtle differences between media lots in particle size distribution translated to pronounced differences in sterile filter performance. Despite relatively high throughput levels achieved with these media (1, 4, L/m 2 ), some process developers may consider the use of a prefilter before sterile filtration, particularly in light of the variability of media properties. As a result, membrane filtration tests were conducted using this same CHO medium (JRH Ex-Cell media 32) after prefiltration through Polysep II CGW1 and Milligard CWSS prefilter, respectively. Of the two tested prefilters, Polysep II CGW1 prefilter proved to be the better prefilter for this application. Prefiltration with CWSS provided no significant increase in.2 µm membrane throughput; whereas CGW1 prefiltration virtually eliminated.2 µm membrane plugging over the one-hour test period. Figure 5 displays the test results in terms of membrane flux for the CGW1-prefiltered CHO media. As expected, the superior permeability of the Millipore Express SHF membrane yields the highest flux values of all membranes tested, 45% higher than the nearest competitor. NSO Media The second most popular mammalian cell expression system (after CHO) for large-scale production of monoclonal antibodies is NSO, a mouse myeloma cell line. Here again, serum-free media is preferred and a variety of commercial NSO products are available. For these tests, serum-free, protein-free, JRH Ex-Cell NSO media was selected (powder, C/N 2465 with cholesterol 5X supplement). One unique feature of NSO cell cultures is the special nutritional requirement for cholesterol. Animal cells require cholesterol for a number of metabolic functions, particularly maintenance of cell membrane structure. Although most cell lines can generate endogenous cholesterol, the NSO cell line, after its long history of mutations, has lost the ability for cholesterol synthesis. As a result, NSO media is commonly supplemented with cholesterol at concentrations of 2 6 mg/l. The relatively high lipid content for NSO media has been perceived as potentially detrimental to sterile filtration where there is increased risk of fouling membranes that bear some oleophilic (hydrophobic) surface characteristics. The test results for the NSO media are shown in Figure 6. Throughput for each membrane was determined by the rate of plugging which, although relatively low, was evident in these one-hour tests. Millipore Express SHC membrane was the best performer overall, demonstrating, at both 3- and 6-minute run times, a 3 4% throughput advantage over its nearest competitor. Millipore Express SHC membrane achieved the greatest net increase in throughput between 3 and 6 minutes, a consequence of the efficient particle-holding capability of the on-board prefilter membrane. Other membranes expired at the 3-minute mark. Fluxt (LMH) Figure 5. CHO Media Prefiltered with Polysep II (CGW1) Prefilter 14, 12, 1, 8, 6, 4, 2, 1 psid 7

8 The throughput results for all membranes tested with NSO media were consistent with those generated on CHO media in absolute as well as relative terms. This finding suggests that cholesterol is not a particular foulant for sterilizing-grade membranes, regardless of its structure, base polymer or surface chemistry. Results of Millipore Express SHR Membrane with Prefilter and Other Sterilizing-grade.1 µm Membranes in Serum-free Media Recent industry trends have seen a shift from the use of.2-micron filters to.1-micron filters as a way to reduce the risk of bioreactor contamination. Mycoplasma is widely recognized as the smallest bacterium thriving in cell culture and cannot be retained by.2 µm sterilizing-grade filters. Mycoplasma lack a cell wall and are bound together by a trilaminar plasma membrane. Due to their small size and deformability, mycoplasma can penetrate a.2 µm rated filter. As a result, cell culture developers are increasingly revising their media preparation methods to replace.2 µm sterilizinggrade filters with.1 µm sterilizing-grade filters for sufficient log removal of mycoplasma. In response to this growing market trend, Millipore developed and introduced the Millipore Express SHR with Prefilter sterilizing-grade.1µm filter. Filters containing the Millipore Express high-retention (SHR) sterilizing-grade polyethersulfone membrane offer a high level of mycoplasma removal and high throughput for cell culture media and other process intermediates. Millipore Express SHR.1 µm membrane-containing filters are designed with an on-board polyethersulfone membrane prefilter that protects the sterilizing-grade.1 µm-rated membrane from premature plugging for enhanced throughput in high fouling streams. To illustrate the high throughput performance of filters containing the Millipore Express SHR membrane in cell culture media, these filters were tested against other.1 µm sterilizing-grade filters with a range of media types, including CHO serum-free media, NSO serum-free media and soy peptonebased serum-free media. Other membranes tested included Supplier A PES.2/.1 µm and Supplier B PVDF.2/.1 µm. Water Flux (LMH at 1 psi) Figure 6. Serum-free, Protein-free NSO Media , 6, 5, 4, 3, 2, 1, Millipore Express SHR with Prefilter 1 psid Figure 7. Water Flux of.1 µm Sterilizing-grade Membranes Experimental Method and Data Analysis The 47 mm membrane disks were wetted with 3 percent isopropyl alcohol and rinsed with water. Once assembled in the stainless steel holder, they were integrity tested to ensure proper membrane installation. To accomplish this, the holders were submerged in a water bath with filtrate open. At an air inlet pressure of 5 psi, no visual air bubbles from the holders were noticed. The membranes were then water flux tested at a fixed 1 psi differential pressure for three minutes to ensure adequate wetting. Figure 7 shows water flux data for all.1 µm membranes tested. Millipore Express SHR membranes demonstrated at least 6% greater flux than their closest competitor. This flux advantage can contribute greatly to media throughput, particularly for flux-driven filtration. n4 at 3 min at 6 min 8

9 Media feed solution temperatures were C. Filter pressures were fixed at 1 psi Figure 8. Serum-free CHO Media differential and the feed solution was stirred for test duration. A run consisted of five membrane samples tested simultaneously from a single feed tank. For each membrane sample, the accumulated filtrate 2,5 2, 1 psid at 3 min at 6 min volume was weighed on a calibrated load cell every 4 5 seconds and the time/weight recorded, using a PC-based data acquisition system. The tests were continued until 9 percent reduction in initial 1,5 1, membrane flux was reached or the feed solution 5 was exhausted. Time and volume raw data were analyzed with a combined pore plugging model to estimate final throughput volumes at time infinity for Millipore Express SHR with Prefilter V max for each filter. Commercial Growth Media CHO Media Figure 8 displays throughput results of two trials for Figure 9. Serum-free CHO Media non-prefiltered JRH CHO media. As seen in Figure 8, Millipore Express SHR membrane with Prefilter yielded the greatest throughput by at least 2x. At filtration times in excess of one hour, V max membrane values were 9 1k L/m 2 for Millipore Express SHR membrane with Prefilter. These values were at least 5x greater compared to Supplier A and Supplier B. The 4,5 4, 3,5 3, 2,5 2, 1,5 1, 1 psid at 3 min at 12 min on-board prefilter layer of Millipore Express SHR 5 membrane provided adequate protection of the.1 µm layer resulting in extended flux-based Millipore Express SHR with Prefilter filtration times relative to other membranes. Figure 9 presents throughput results of CHO media after filtration through a Polysep-II prefilter. Following Polysep-II filtration, Millipore Express SHR membrane with Prefilter achieved the greatest throughput at 2 hours due to sufficient protection of the.1 µm filter for flux-driven filtration. Millipore Express SHR with Prefilter demonstrated 2 3x V max improvement over other.1 µm filters. Feed solution was exhausted after 3 minutes in trial 1. 9

10 NSO Media Figure 1 presents throughput results of two trials for Figure 1. Serum-free NSO Media nonprefiltered JRH NSO media. At a two-hour filtration time, Millipore Express SHR membrane with Prefilter demonstrated 6% greater throughput than its closest competitor, 2,5 2, 1 psid at 3 min at 12 min Supplier B. Longer filtration times widen this performance gap, as V max was 2x greater for Millipore Express SHR membrane with Prefilter than Supplier A. 1,5 1, 5 Soy Peptone-based Media Filtration of non-prefiltered media,with a soy peptone concentration of 2 g/l, is presented in Millipore Express SHR with Prefilter Figure 11. For all membranes, except Millipore Express SHR with Prefilter, at least 8% of the final throughput on V max was reached after 3 minutes of filtration time. The resulting low flow rates contributed very Figure 11. Peptone-based Media little to filtrate volume beyond 3 minutes. Millipore Express SHR with Prefilter demonstrated a 3 4x V max improvement over.1 µm filters. 1,4 1,2 1 psid at 3 min at 6 min 1, Millipore Express SHR with Prefilter 1

11 Conclusions The Millipore Express filter family provides cost-effective solutions for the sterile filtration of serum-free media. For production-scale sterile filtration of serumfree tissue culture media, Millipore Express SHC membrane offers high throughput and high flow rates for superior filtration economy. Millipore Express SHC filters combine a sterilizing-grade, asymmetric composite Millipore Express SHF membrane with an on-board asymmetric composite prefilter membrane for superior particle-holding capacity and process robustness. Millipore Express SHC filters provide consistent, high performance across a wide range of serum-free media formulations. For applications using a dedicated prefiltration system, high permeability, sterilizing-grade Millipore Express SHF filters offers superior flow rates with prefiltered serum-free media. Where sterilizing-grade performance and a high degree of protection against potential mycoplasma contamination is needed, Millipore Express SHR (sterilizing-grade, high-retention) filters are recommended. Millipore Express SHR.1 µm filters offer the highest throughput and lowest area requirements for a majority of serum-free media types. 11

12 To Place an Order or Receive Technical Assistance In the U.S. and Canada, call toll-free 1-8-MILLIPORE ( ) In the U.S., Canada and Puerto Rico, fax orders to 1-8-MILLIFX ( ) Outside of North America contact your local office. To find the office nearest you visit Internet: Technical Service: Millipore, Durapore, Milligard, Millipore Express, Milli-Q, Opticap and Polysep are trademarks of Millipore Corporation. Ex-Cell is a trademark of JHR Biosciences Inc. Lit. No. AN1EN Rev. A Printed in U.S.A. 5/ , Millipore Corporation, Billerica, MA 1821 U.S.A. All rights reserved.