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BioProcess International FEBRUARY 2005 M embrane fouling is a process that causes a marked decrease in the performance of ultrafilter membranes. Fouling of ultrafilters has been inextricably linked to protein processing. By understanding the fouling phenomenon, we can minimize or eliminate this problem through proper selection of currently available modified (stabilized) regenerated cellulose (M-RC) ultrafilters. ULTRAFILTER MEMBRANE POLYMERS Ultrafilter membranes are made from a variety of polymers. Some have surface modifications intended to improve their performance. Today in biotechnology two main polymer families are in use: polysulfones and cellulosics. The polysulfones include hydrophilic polysulfone and polyethersulfone (PES), which are most widely used mainly because they have been on the market for quite some time. These membranes gained acceptance for use in validated processes partly because of their robustness and the fact that they can withstand one-normal sodium hydroxide (1N NaOH) exposure. NaOH is commonly used throughout industry for chemically cleaning, depyrogenating, and sanitizing process equipment. Natural polysulfones are hydrophobic. In the casting phase of membrane manufacture, the polymer is treated with hydrophilizing agents that render it hydrophilic. Depending on the agent and process used, the resulting membrane has a greater or lesser lipophilic profile. Cellulosic membranes have also been available for many years in the form of cellulose acetate, cellulose nitrate, mixed esters of cellulose, and regenerated cellulose. They have two shortcomings in common, individual chemistries notwithstanding: a limited pH range (4–8) and a general lack of robustness. Because of those limitations, these membranes are more difficult to clean in a manner acceptable to the biopharmaceutical industry and its regulators. Over recent years, however, stabilized regenerated cellulose ultrafilters have been introduced that overcome the pH limitation by extending their usable range to pH 2–14. PROTEIN–MEMBRANE CHEMISTRY Polymer chemistry plays a crucial role in the interaction of a membrane and the product being filtered. Polysulfones are rich in conjugated benzene rings, which serve as sites for hydrophobic– hydrophobic interactions. Likewise, the nitrate groups on cellulose nitrate and mixed esters of cellulose interact strongly with proteins and other biomolecules. When such interactions occur, the result is adsorption and denaturation of proteins at the membrane surface. Such effects have been well documented with protein-containing solutions. Truskey et al. measured protein adsorption, circular dichroism, and biological activity of several protein solutions (insulin, IgG, and alkaline phosphatase) before and after passing through a variety of membranes (1). Observed shifts in circular dichroism and decreases in biological activity were determined to be the result of conformational changes in protein structure. Truskey’s study showed clearly that the most hydrophobic membranes had the greatest effect on protein adsorption and deformation. B IO P ROCESS TECHNICAL PRODUCT FOCUS: ALL PROTEIN PRODUCTS PROCESS FOCUS: DOWNSTREAM PROCESSING WHO SHOULD READ: PROCESS DEVELOPERS, QA/QC KEYWORDS: MEMBRANE FILTERS, FOULING, CELLULOSE, ULTRAFILTRATION LEVEL: BASIC Could Membrane Fouling Be a Thing of the Past? Michael Dosmar SARTORIUS AG (WWW.SARTORIUS.COM) 62

Could Membrane Fouling Be a Thing of the Past?

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Page 1: Could Membrane Fouling Be a Thing of the Past?

BioProcess International FEBRUARY 2005

Membrane fouling is a process that causes a marked decrease in the performance of ultrafilter

membranes. Fouling of ultrafilters has been inextricably linked to protein processing. By understanding the fouling phenomenon, we can minimize or eliminate this problem through proper selection of currently available modified (stabilized) regenerated cellulose (M-RC) ultrafilters.

ULTRAFILTER MEMBRANE POLYMERS

Ultrafilter membranes are made from a variety of polymers. Some have surface modifications intended to improve their performance. Today in biotechnology two main polymer families are in use: polysulfones and cellulosics.

The polysulfones include hydrophilic polysulfone and polyethersulfone (PES), which are most widely used mainly because they have been on the market for quite some time. These

membranes gained acceptance for use in validated processes partly because of their robustness and the fact that they can withstand one-normal sodium hydroxide (1N NaOH) exposure. NaOH is commonly used throughout industry for chemically cleaning, depyrogenating, and sanitizing process equipment. Natural polysulfones are hydrophobic. In the casting phase of membrane manufacture, the polymer is treated with hydrophilizing agents that render it hydrophilic. Depending on the agent and process used, the resulting membrane has a greater or lesser lipophilic profile.

Cellulosic membranes have also been available for many years in the form of cellulose acetate, cellulose nitrate, mixed esters of cellulose, and regenerated cellulose. They have two shortcomings in common, individual chemistries notwithstanding: a limited pH range (4–8) and a general lack of robustness. Because of those limitations, these membranes are more difficult to clean in a manner acceptable to the biopharmaceutical industry and its regulators. Over recent years, however, stabilized regenerated cellulose ultrafilters have been introduced that overcome the pH limitation by extending their usable range to pH 2–14.

PROTEIN–MEMBRANE CHEMISTRY

Polymer chemistry plays a crucial role in the interaction of a membrane and the product being filtered. Polysulfones are rich in

conjugated benzene rings, which serve as sites for hydrophobic–hydrophobic interactions. Likewise, the nitrate groups on cellulose nitrate and mixed esters of cellulose interact strongly with proteins and other biomolecules. When such interactions occur, the result is adsorption and denaturation of proteins at the membrane surface. Such effects have been well documented with protein-containing solutions. Truskey et al. measured protein adsorption, circular dichroism, and biological activity of several protein solutions (insulin, IgG, and alkaline phosphatase) before and after passing through a variety of membranes (1). Observed shifts in circular dichroism and decreases in biological activity were determined to be the result of conformational changes in protein structure. Truskey’s study showed clearly that the most hydrophobic membranes had the greatest effect on protein adsorption and deformation.

B I O P R O C E S S TECHNICAL

PRODUCT FOCUS: ALL PROTEIN PRODUCTS

PROCESS FOCUS: DOWNSTREAM PROCESSING

WHO SHOULD READ: PROCESS DEVELOPERS, QA/QC

KEYWORDS: MEMBRANE FILTERS, FOULING, CELLULOSE, ULTRAFILTRATION

LEVEL: BASIC

Could Membrane Fouling Be a Thing of the Past?Michael Dosmar

SARTORIUS AG (WWW.SARTORIUS.COM)

62

Page 2: Could Membrane Fouling Be a Thing of the Past?

FEBRUARY 2005 BioProcess International

The result of such interactions is that the internal hydrophobic sites of a protein become exposed. These exposed hydrophobic groups then serve as sites for membrane–protein binding, protein–protein binding, and protein denaturation. Such protein–membrane interactions ultimately increase the overall rate of membrane clogging and fouling, reduce performance, and lower product yields.

MEMBRANE FOULING

One consequence of protein adsorption is a corresponding decrease in permeability and increase in fouling of membranes (2, 3). It has been shown that protein adsorption is greater on hydrophobic membranes than on hydrophilic ones. And protein conformation influences membrane performance. Globular or spherical deposits decrease flux less than deposition of protein sheets does. Freeze-fracture and deep-etching techniques have illustrated that BSA, for example, deposits spherically onto hydrophilic regenerated cellulose membranes. The size of those deposited spheres is consistent with the size of the BSA molecule. However, deposition of BSA onto hydrophilic polysulfone membranes is filamentous, which suggests that the tertiary structure of the protein has been disrupted through interaction with the membrane (2).

When fouling occurs, composition of the feed stream can dramatically affect the

membrane’s retentive properties. Retention of large–molecular-weight components can increase retention of smaller components. Blatt et al. demonstrated that human serum albumin (67 kDa) retention on a 100-kDa MWCO (molecular-weight cut-off) membrane was nearly zero (4). However when gamma-globulin (160 kDa) was added to the feed stream, the albumin retention rose. In fact, it showed a linearly increasing correlation to increasing concentrations of the -globulin (4). Porter similarly showed that retention of ovalbumin, chymotrypsin, and cytochrome C increased when a 1% solution of albumin was added to the feed mixture (5).

Rejection of IgM on 100-kDa MWCO polyethersulfone membranes and stabilized regenerated cellulose membrane show divergently different results even though the mixed dextran rejection profiles are similar. These differences can be attributed to membrane fouling and the nature of the resultant protein cake that forms as described above.

MATERIALS AND PROCEDURE

All experiments are conducted using a Sartorius Sartocon Benchtop Crossflow System configured with magnetic flowmeters. The system includes a positive displacement pump, three pressure gauges, a 20-L jacketed vessel, a Sartocon 2 cassette filter holder, and sanitary diaphragm valves. Filter cassettes are Sartorius Hydrosart

30-kDa, Hydrosart (Modified Regenerated Cellulose, 10-kDa), and Sartorius Polyethersulfone 30-kDa and 10-kDa.

Flux measurements were made at crossflow rates of 5 and/or 15 L/min and at TMP 15, 25, 35, and 45 psi respectively with four solutions: 0.9% saline, 0.1% lysozyme, 0.1% lysozyme with 0.2% albumin, and skim milk diluted to 0.2% protein. Cassettes were cleaned with 1N NaOH, rinsed with water, and equilibrated with 0.9% saline prior to each experiment. All tests were conducted at 15 °C ± 0.5 °C. BSA and lysozyme samples were tested for protein concentration (absorbance at 280 nm) and lysozyme activity (enzyme kinetics assay).

DATA AND DISCUSSION

Figure 1 compares flux with transmembrane pressures (TMP) for saline, 0.1% lysozyme, and 0.1% lysozyme with 0.2% BSA on 30-kDa M-RC membrane filters. Flux determinations were made at low and high recirculation rates. The profiles for lysozyme/BSA solution and lysozyme alone at a low recirculation rate (5 L/min) showed a traditional flux-limited, TMP-independent performance resulting from membrane fouling and protein polarization. At a higher recirculation rate (15 L/min), the lysozyme flux increased linearly with increasing TMP.

Figures 2 and 3 are the flux/TMP curves for 10-kD and 30-kD membrane filters before and after protein (1:20 diluted skim milk)

Figure 1: Flux/TMP profiles for saline, lysozyme and lysozyme/BSA solutions at low and high recirculation rates.

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Figure 2: Saline flux on 10-kD polyethersulfone (PES) and modified regenerated cellulose (M-RC) membranes before and after the 10X concentration of skim milk diluted 1:20 with saline

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BioProcess International FEBRUARY 2005

concentration and 0.1% lysozyme concentration. For the 10-kD filters, the membrane flux was completely recovered through dilution, whereas flux remained irreversibly depressed on PES. After concentration of the lysozyme, flux on 10-kD M-RC was not recovered through dilution. Figures 4–8 examine saline flux and TMP through the different membranes tested when new and then again after 2–3 cycle of use followed by cleaning.

Differences in channel spacing between cassettes was accounted for throughout all testing. Test conditions were adjusted to hold shear rate constant as TMPs were varied. That allowed for direct comparison of gel layer formation on each membrane surface while standardizing for manufacturing differences. Liquid viscosity was kept constant by maintaining the temperature at 15 ± 1 °C. The membranes were challenged with a solution containing a protein above

their MWCO. Bovine serum albumin is a 67-kDa protein retained by the 30-kDa MWCO membranes used in this study. Less hydrophilic membranes are expected to adsorb more BSA and form a more substantial gel layer.

Flux was completely recovered after filtration of diluted skim milk on a 10-kD M-RC membrane — in contrast with the permanent loss with PES. In this instance, it can be concluded that although the 10-kD PES membranes were fouled by the milk, the M-RC membranes were not. Likewise, lysozme at high recirculation on the 30-kD M-RC membrane did not foul, but at low recirculation the M-RC membranes did foul with lysozyme, as they did with the lysozyme/BSA solution at both high and low recirculation rates. In all experiments, the PES membranes fouled in the presence of protein.

Most notable is the difference in flux recovery after filter use and cleaning with

NaOH. PES membranes lost 60–76% of their flux after use, whereas the M-RC membranes lost 0–31%. After cleaning, however, the M-RC membranes’ saline flux recovery was 89–100%, compared with the PES membranes’ permanent flux loss as high as 48%.

A NEW ERA IN FILTRATION?Although product rejection and membrane fouling have long been part of the ultrafiltration process, stabilized regenerated cellulose ultrafilters may make the problem a thing of the past. Increased hydrophilicity limits the fouling process, which in turn minimizes the effect of a polarizing protein layer on the membrane surface. Rather than forming a “secondary membrane” (the protein gel) that eclipses the intrinsic properties of the filter membrane, chemical properties and effects of the

Figure 3: Saline flux and TMP on 30-kD M-RC membranes before and after the 10X concentration of 0.1% lysozyme

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Figure 4: Saline flux on 10-kD M-RC membranes before and after three cycles of 10X-concentrated lysozyme followed by cleaning with 1NaOH

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Figure 5: Saline flux on 30-kD polyethersulfone membranes before and after three cycles of 10X-concentrated lysozyme followed by cleaning with 1NaOH

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Figure 6: Saline flux on 30-kD PES membranes before and after three cycles of 10X-concentrated lysozyme followed by cleaning with 1NaOH 10kD modified regenerated cellulose flux after two protein concentration and cleaning cycles

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BioProcess International FEBRUARY 2005

stabilized regenerated cellulose membrane are preserved. The result should improve overall performance and facilitate cleaning to substantially better recovery of flux.

REFERENCES1 Truskey G, et al. The Effect of Membrane Filtration Upon

Protein Conformation. J. Parenteral Sci. Technol. 41(6) 1987: 180–193.2 Marshall AD, Munro PA, Trägårdh G. Effect of Protein

Fouling in Microfiltration and Ultrafiltration on Permeate Flux, Protein Retention, and Selectivity: A Literature Review. Desalination 91, 1993: 65–108.

3 Levy P, Shehan J. Performance Characteristics of Polysulfone and Cellulose Membranes for the Ultrafiltration of Biological Process Streams. BioPharm 5(4) April 1991: 24–33.

4 Blatt WF, et al. Solute Polarization and Cake Formation in Membrane Ultrafilters: Causes, Consequences, and Control Techniques. Membrane Processes in Industry and Biomedicine. Beir M, ed. Plenum Press: Blatt, NY, 1971; 65–68.

5 Porter MC. The Effect of Fluid Management on Membrane Filtration. Synthetic Membranes Vol. 1. Turbak A, ed. ACS Symposium Series #153, 1981: 407.

Michael Dosmar is product manager in crossflow filtration for Sartorius Corporation, biotechnology division, 131 Heartland Boulevard, Edgewood, NY 11717; 1-800-368-7178, ext. 8360, fax 1-773-327-0568; [email protected]; www.sartorius.com.

Figure 7: Saline flux on 10-kD M-RC membranes before and after three cycles of 10X-concentrated 1:20 diluted skim milk followed by cleaning with 1NaOH

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Figure 8: Saline flux on 10-kD PES membranes before and after three cycles of 10X-concentrated 1:20 diluted skim milk followed by cleaning with 1NaOH

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