Preparation and Characterization of Cellulose− Stainless Steel

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Preparation and Characterization of Cellulose-Stainless Steel Powder Composite Particles Customized for Expanded Bed Application Zhi-Jun Miao, Dong-Qiang Lin, and Shan-Jing Yao* Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, People’s Republic of China

Expanded bed adsorption (EBA) is an integrated technology for capturing target proteins directly from unclarified feedstock. The specially designed adsorbent is essential for the formation of a stable expanded bed. Using the method of water-in-oil suspension thermal regeneration, a novel type of composite porous particles for expanded bed application was prepared with stainless steel powder (SSP) as the densifier and cellulose as the skeleton. The preparation conditions were explored, and two size fractions with different SSP contents were obtained with mean particle sizes of 100 and 170 µm. The composite particles showed a spherical appearance, suitable size and size distribution, appropriate wet density of 1.2-1.8 g/mL, water content of 50-80%, porosity of 80-90%, pore radius of 30-40 nm, and specific surface area of about 50 m2/mL of wet particles, and the stable expanded bed with an expansion factor of 2-3 could be achieved under varying flow rates. In addition, the influences of SSP addition on the physical properties were analyzed, which provided some useful information on the design of EBA adsorbents. A linear relationship between the particle wet density and the SSP/cellulose viscose ratio was found. The addition of SSP into the cellulose skeleton showed little effect on the porosity, specific surface area, and pore radius in the particle, which verified the prepared composite particles are potentially suitable for expanded bed application of biomolecule separation. 1. Introduction Expanded bed adsorption (EBA) is an innovative chromatography technology that allows the adsorption of target proteins directly from unclarified feedstock, e.g., culture suspensions, cell homogenates, or crude extracts. EBA technology combines solid-liquid separation with an adsorption step in a single-unit operation aimed at increased overall yield, reduced operational time, and less requirements for capital investment and consumables.1-3 The most important property of an expanded bed is the perfectly classified fluidization of the adsorbent.4 On one hand, the increased void fraction between adsorbents allows the introduction of particlecontaining feedstock without the risk of blocking the bed. On the other hand, the movement of adsorbent particles is limited in a small range, and the bed stability is maintained even in the presence of a turbid feedstock with a low extent of axial mixing, which ensures a perfect sorption performance comparable to that of packed beds. To form a perfectly classified fluidization in the column, the adsorbent is the key pillar except the particular fluid distribution unit in the column inlet.5 Unlike the adsorbents used in normal chromatography that approach single size for an efficient separation factor, the adsorbents specially designed for EBA have an obvious size and/or density distribution.6 The EBA adsorbents named Streamline were introduced to the market in the early 1990s and were developed on the basis of the demands from the biotechnology industry in the late 1980s. Till now there have been quite a few new commercial adsorbents going * To whom correspondence should be addressed. Tel.: +86571-87951982.Fax: +86-571-87951015.E-mail: [email protected].

into the EBA family. Compared with those 10 years ago, however, new host cells, the expression level of the target product, and the culture volume have increased significantly today. Improved adsorbents are, therefore, needed to match the fast development of biotechnology.7 The Streamline series of adsorbents are based on 6% cross-linked agarose containing a crystalline quartz core as the densifier. The particle size is in the range of 100300 µm, and the mean density is about 1.2 g/mL. The physical properties of Streamline adsorbent indicate that it can be used under a fluid velocity of 200-400 cm/h in water corresponding to a suitable expansion factor of 2-3.8 For the real application the suitable fluid velocity should be lower due to the higher density and viscosity of bioparticle-containing feedstock than those of buffers. The perfect adsorbents for EBA processes should be designed to give maximal productivity, which could be gained with the operation at high fluid velocity, have high dynamic capacity, and allow minimal feedstock dilution. There are two ways to increase the suitable operation fluid velocity of adsorbents in an expanded bed: larger particle size and higher density. The increase of particle size would certainly reduce the mass transfer and decrease the dynamic adsorption capacity. Therefore, increasing the density should be the best way. Some research groups have been developing new EBA adsorbents based on stainless steel-agarose,9 Nd-Fe-B alloy-agarose,10 cellulose,11-13 titanium oxide-cellulose,14-17 etc. Some companies are also focusing on exceeding the limitation of exiting Streamline adsorbents. Prototype adsorbents which are based on 4% cross-linked agarose containing a stainless steel alloy as the densifier are being developed at Amershem Biosciences AB (Uppsala, Sweden) to substitute for the normal Streamline series. The density of particles varies

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from 1.6 to 2.0 g/mL, which ensures that a feedstock containing 12% dry weight Saccharomyces cerviseae could be run at 300 cm/h without exceeding 2.5-fold expansion.7 A new series of adsorbents for EBA which combine cross-linked agarose weighted by incorporating high-density particulates of tungsten carbide have also been developed by UpFront Chromatography A/S (Denmark).18 The density is about 2.5-3.5 g/mL, which can be used at about 800 cm/mL for 2.5-fold expansion. However, since the lack of all-sided understanding on the rule of density and size distribution for EBA performance, it is quite difficult to develop the perfect EBA adsorbents. For the directed design of EBA adsorbents, a series of adsorbents with different densities and sizes is be needed first to match the demand of detailed research. Unfortunately, few reports concerned with this are filed in the literature. In the present work a novel cellulose-stainless steel powder composite adsorbent is prepared with the method of water-in-oil suspension thermal regeneration as published previously by Lei et al.14 Cellulose, one of the most abundant and low-cost natural polymers, which has been successfully manufactured in porous bead form and widely used as column packing material for liquid chromatography,20-23 is chosen as the skeleton of the adsorbent. Fine stainless steel powder (SSP) with a size of 20 µm and density of 7.9 g/mL is used as the densifier. A series of adsorbents are obtained successfully with a density gradient and two particle sizes. The physical properties of the resulting adsorbent are studied, analyzed, and compared with those of the commercial Streamline quartz base matrix. The influence of densifier addition on the adsorbent density, water content, volume shrinkage percentage, porosity, pore volume, pore radius, specific surface area, and mechanical strength are discussed to gain some useful information for the design of EBA adsorbents. The expansion property and adsorption ability are also analyzed for potential EBA application.

were washed in turn with boiling water, methanol, and sulfuric acid (10% w/w). After sieving, composite particles were obtained with two diameter ranges, 60-125 and 125-300 µm, and were named Cell-SSP-S and CellSSP-L, respectively. On the basis of the amount of stainless steel powder in the composite particles, the smaller fractions were named Cell-SSP-S0 to Cell-SSPS8 and the larger fractions Cell-SSP-L0 to Cell-SSPL8. 2.3. Physical Properties. The size distribution of the prepared composite particles was determined with a Mastersizer laser particle size analyzer (Malvern Instruments Ltd., Worcestershire, U.K.). An XS-200 biologic microscope (Jiangnan Optics and Electronics Ltd., Nanjing, China) was used to observe the shape of the particles. Several physical properties were determined or calculated as follows. After a slurry of prepared composite particles was sucked up, the wet density Fp (g/mL of wet particles) of particles was determined by water replacement in a 10 mL gravity bottle. The water content ω was obtained by dehydration at 120 °C to a constant mass. Presuming that all pores in the particles were full of water, the porosity P (%) expressing the pore volume per volume of wet particles and pore volume V (mL/g of dry particles) expressing the pore volume per gram of dry particles can be roughly estimated as follows, in which Fw represents the density of water:23

2. Experimental Section

where Vw and Vd represent the volumes of the wet and dried matrixes, respectively. The specific surface area S (m2/mL of wet particles) was obtained by adsorption of methylene blue solution and calculated as follows:24

2.1. Materials. Degreasing cotton was purchased from League Health Material Ltd. (Jiaozuo, China). Stainless steel powder with a density of 7.9 g/mL and a mean particle diameter of 20 µm was ordered from Feida Powder Metallurgy Ltd. (Haining, China). Vacuum pump oil was provided by Sifang Special Oil Factory (Beijing, China). The Streamline quartz base matrix was purchased from Amersham Biosciences (Uppsala, Sweden). All other reagents were of analytical reagent grade and purchased commercially. 2.2. Preparation of the Composite Particles. The cellulose xanthate viscose was prepared by reacting 45 g of alkali-treated and aged degreasing cotton with 20 mL of CS2 and then dissolving the resulting mixture into 6% w/w NaOH solution as described previously.14 Composite particles based on SSP-densified cellulose beads were prepared through the method of water-inoil suspension thermal regeneration as described in the previous paper.14 Briefly, a series of different amounts (0, 3, 6, 9, 12, 15, 18, 21, 24 g) of stainless steel powder were mixed with 60 g of viscose (containing 6-9% w/w cellulose). The mixture was dispersed in 360 mL of pump oil in a 500 mL flask with agitation at 500 rpm for 0.5 h at room temperature. The suspension was heated to 95 °C, kept for 2 h under continuous stirring, and then cooled and filtered. The resulting particles

P)

Fpω × 100 Fw

(1)

V)

ω (1 - ω)Fw

(2)

The shrinking behavior was characterized as the volume shrinkage percentage Sr (%) as

Sr )

S)

Vw - Vd × 100 Vd

(C0 - C)GFp × 2.45 mp

(3)

(4)

where C0 and C represent the initial and equilibrium concentrations of methylene blue solution and G and mp represent the masses of methylene blue added and sample particles, respectively. The constant of 2.45 (m2/ mg of methylene blue) means that 1 mg of methylene blue could cover an area of 2.45 m2 for the assumption of mono-molecule-layer adsorption. The pore radius R (nm) can be estimated as follows:25

R ) 2 × 1000 ×

Vp(1 - ω)Fp S

(5)

To characterize the mechanical strength of the matrix prepared, the pressure drop ∆P of the packed bed was determined by means of column chromatography. 3. Results and Discussion 3.1. Preparation of the Composite Particles. As the adsorbents used for the normal chromatography

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Figure 2. Size distribution of the matrixes: (a, top) Cell-SSP-S; (b, bottom) Cell-SSP-L.

Figure 1. Photographs of particle appearance: (a, top) Cell-SSP matrixes with a small size and relatively low content of SSP; (b, bottom) Cell-SSP matrixes with a large size and relatively high content of SSP.

process, a fine spherical shape is the prime property of the matrix prepared. In addition, for the EBA process, a suitable density and size distribution are most important to form a perfectly classified fluidized bed. In the present work the obvious density difference between cellulose viscose and stainless steel powder increases the difficulty of combining the densifier into the cellulose skeleton while the spherical cellulose matrixes are formed. Hereby, the preparation process would be optimized by altering the viscosity of viscose and the stirring speed. Generally, the viscosity of viscose is influenced by the aging time and aging temperature during the preparation of cellulose xanthate viscose. Too high a viscosity causes poor dispersion, while too low a viscosity results in a quite small particle size and loss of SSP densifier. The results indicate that viscose with a viscosity of 5000-8000 cSt is suitable for the preparation of a cellulose-SSP composite matrix. It was found that the stirring speed also affects the particle size and size distribution. Normally increasing the stirring speed is favorable to the formation of smaller particles. In the present work, the stirring speed was optimized as 500600 rpm. One typical batch gave about 20 mL of the small size fraction Cell-SSP-S (60-125 µm) and about 30 mL of the large size fraction Cell-SSP-L (125-300 µm). A fraction of larger particles (>300 µm) was also

obtained (about 10 mL), which was wasted in the present work. 3.2. Particle Appearance and Size Distribution. With the optimized conditions mentioned above, the SSP was embedded perfectly into the cellulose skeleton, and regular sphericity of the composite particles could be obtained. Figure 1 gives two samples of particle appearance. Figure 1a shows Cell-SSP composite particles with a small size and relatively low content of SSP, while Figure 1b shows composite particles with a large size and relatively high content of SSP. In addition, cracking and adhesion of the particles were not found during the preparation. The size distributions of composite particles prepared were measured. Figure 2 shows the size distributions with different SSP contents of two size fractions. The two size factions revealed the same tendency; with an increase of the SSP content, the composite particles showed a poorer symmetrical distribution. Under low SPP content (0.3 w/w SSP/viscose) was used, an obvious unsymmetrical distribution was found. The results indicate that the higher content of SSP in the composite particles, the more nonhomogeneous the distribution during the dispersion of the water-in-oil suspension. In other words, it seems to be more difficult to prepare composite particles with a higher density and homogeneous distribution of the densifier. The mean sizes of two particle fractions are shown in Figure 3. Although the size distribution was affected by the SSP content in the composite particles, the mean particle size was kept at the same level for the same particle fraction. The small size fraction has an average

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Figure 3. Effect of the SSP content on the mean particle diameter for two size fractions, Cell-SSP-S and Cell-SSP-L.

Figure 5. Effect of the SSP content on the water content for two size fractions, Cell-SSP-S and Cell-SSP-L.

Figure 4. Effect of the SSP content on the particle wet density for two size fractions, Cell-SSP-S and Cell-SSP-L.

Figure 6. Effect of the SSP content on the SSP volume percentage for two size fractions, Cell-SSP-S and Cell-SSP-L.

value of 104 ( 10 µm, and the large one an average value of 174 ( 11 µm. 3.3. Particle Wet Density and Water Content. The particle wet density determines the operation velocity of the fluidized bed, and a higher particle density means a higher productivity of the separation process. Figure 4 shows the influence of the SSP content on the mean wet density of the prepared composite particles. A good linear relation between the mean wet density and the SSP/viscose ratio was found for the small size fraction Cell-SSP-L, while an approximately linear relationship was also obtained for the large size fraction Cell-SSPL. The results indicate that the SSP as a densifier was successfully entrapped within the cellulose skeleton to increase gradually the wet density of the composite particles. Therefore, it is possible to predict, design, and control the wet density of the composite particles prepared with the appropriate SSP/viscose ratio. The water content, which reflects the hydrophilicity and space capacity in the particles, is one of most important properties of the matrixes for protein separation application. From Figure 5, it is obvious that the water content in the composite particles decreases gradually with an increase of the SSP content. Although there are significant differences in the sizes between Cell-SSP-L and Cell-SSP-S, the change in the water content with the SSP/viscose ratio follows nearly the same tendency. The water content decreased from 80% without SSP addition to about 50% with an SSP/viscose ratio of 0.4. The relatively high values of the water content in the composite particles would certainly provide a perfect hydrophilic environment for the separation of biomolecules. On the basis of the data of particle density and water content, the mean SSP volume content (%) in the

composite particles can be calculated with the mass balance of materials added as follows:

Fp RSV SSP volume content ) (1 - ω) × 100 FSSP C + RSV (6) where FSSP is the mean density of stainless steel powder, C is the cellulose mass concentration in the viscose, and RSV is the SSP/viscose ratio during the preparation. Figure 6 gives the results for two size fractions. It was found that the SSP volume content was less than 10% even at an SSP/viscose ratio of 0.4. This result indicates that the addition of high-density SSP has a slight influence on the efficient volume of the particle and the composite particles are suitable to use as the matrixes for biomolecule adsorption. 3.4. Shrinking Behavior and Mechanical Strength. As shown in Figure 7, the volume shrinkage percentage decreased with an increase of the SSP/ viscose ratio, which means that the embedding of SSP into the cellulose skeleton could play a supporting role in the particles and sequentially enhance the structure stability of the cellulose bead. Therefore, the composite particles prepared in the present work would be as suitable for biochromatography processes as cellulose beads. The mechanical strength of the matrixes was also investigated with the pressure drop test in a packed bed (data not shown). It was found that the pressure drops increased linearly with increasing fluid velocities for all matrixes tested, which indicates that the mechanical strength of the composite particles prepared is high enough for use under a high fluid velocity up to near 1000 cm/h. This certainly benefits the demand of high productivity for the EBA process.

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Figure 7. Effect of the SSP content on the shrinkage for two size fractions, Cell-SSP-S and Cell-SSP-L.

Figure 9. Effect of the SSP content on the specific surface area for two size fractions, Cell-SSP-S and Cell-SSP-L.

Figure 8. Effect of the SSP content on the porosity for two size fractions, Cell-SSP-S and Cell-SSP-L.

Figure 10. Effect of the SSP content on the pore radius for two size fractions, Cell-SSP-S and Cell-SSP-L.

3.5. Porosity and Pore Volume. The effects of SSP content on the pore structure of different fractions are shown in Figure 8. The results indicate that the addition of SSP in the particle hardly influences the porosity property. The composite particles maintained a porosity of 80-90% even for a high addition of SSP, while the porosities of Cell-SSP-L were slightly higher than those of Cell-SSP-S. The average porosity of Cell-SSP-L was 89.8%, and that of Cell-SSP-S was 84.3%. The pore volume per gram of dry particle weight decreases with an increase of the SSP/viscose ratio, from 3.8 to about 1.0 mL/g of dry particle weight (data no shown). The high values of porosity and pore volume in the cellulose particles were still maintained after the addition of SSP as the densifier, which demonstrated that the prepared composite particles would keep the perfect properties of cellulose beads as chromatography adsorbents. 3.6. Specific Surface Area and Pore Radius. To reveal the ability of composite particles prepared as the matrixes for EBA adsorbents, the specific surface area and pore radius in the particle were investigated too. The results are shown in Figures 9 and 10, respectively. It was found that the addition of SSP increased slightly the specific surface area in the particle for two size fractions tested, which indicates that the SSP not only acts as a densifier in the composite particles but also improves the porous structure of cellulose beads. The reason might be the change from crystal to amorphous form cellulose with SSP addition, which loosens the compact structure of the cellulose bead.26 Similar results were reported for using TiO2 superfine powder as the densifier to prepare TiO2-cellulose composite particles.16,27 The values of the specific surface area of CellSSP-L were slightly lower than those of Cell-SSP-S. The average specific surface area of Cell-SSP-L was 48.3 m2/

mL of beads, and that of Cell-SSP-S was 59.2 m2/mL of beads, which was higher than that of the Streamline adsorbent, about 46 m2/mL of beads, measured with the same method.14 The mean pore radii in the matrixes were estimated in the range of 30-40 nm, while the pore radii of Cell-SSP-L were slightly larger than those of Cell-SSP-S. Those values were obviously higher than that of the Streamline adsorbent, about 22 nm. The high value of the specific surface area means the composite particles have abundant space to bind the functional group for target adsorption, and the relatively large pore radius would benefit the passage of biomolecules with a high molecular weight and reduce the transfer limitation. 3.7. Bed Expansion and Adsorption Characteristics. The bed expansion characteristics of the prepared composite particles were measured in the column under the normal operation conditions for EBA. The results are shown in Figure 11. It was found that the expansion factors under the same flow rate decreased obviously with an increase of the particle density for two size fractions. The perfect operation expansion factor of EBA is in the range of 2-3, and Cell-SSP-S could be used for a low fluid velocity of 100-250 cm/h and Cell-SSP-L for a high fluid velocity of 300-1000 cm/h. Therefore, Cell-SSP-S is favorable for the process with mass transfer limitation, which needs a short transfer route and relatively low flow rate, and CellSSP-L is beneficial for the separation with fast adsorption and high productivity. The results of the bed stability test indicate that a stable expanded bed could be obtained for all composite particles prepared in the present work (data not shown). In previous papers,14-17,27,28 a spherical TiO2-densified type of cellulose composite particles was prepared with

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Acknowledgment This work was financially supported by the National Natural Science Foundation of China (Grant No. 20206029). Literature Cited

Figure 11. Effect of the bed expansion factor on the fluid velocity for two size fractions of composite particles, Cell-SSP-S and CellSSP-L.

a similar method, and the composite particles were further derived to function as an anion exchanger (CellTi DEAHP) and a cation exchanger (Cell-Ti CM). The adsorption capacities were comparable to those of the well-known commercial Streamline series adsorbents, with 61 mg of BSA/mL of wet gel for Cell-Ti DEAHP17 and 98.7 mg of lysozyme/mL of wet gel for Cell-Ti CM.28 Therefore, the adsorption abilities of the Cell-SSP composite particles prepared in the present work are hoped to be practical. The composite particles have been modified with Procion Red HE-3B for use as a dyeaffinity adsorbent. The new absorbent showed a high affinity for NADH-dependent lactate dehydrogenase with an adsorption capacity of 1600 U/g of wet gel. Other works on functionalizing the Cell-SSP composite particles as ion exchangers and mixed-mode adsorbents are being carried out in the laboratory to expand the applications for the EBA process. 4. Conclusions A novel type of composite particles was prepared with SSP as the densifier and cellulose as the skeleton using the method of water-in-oil suspension thermal regeneration. The preparation conditions were optimized, and two size fractions with different SSP contents were obtained with mean particle sizes of 100 and 170 µm, respectively. The composite particles showed a spherical appearance, suitable size and size distribution, appropriate wet density of 1.2-1.8 g/mL, water content of 50-80%, porosity of 80-90%, pore radius of 30-40 nm, and specific surface area of about 50 m2/mL of wet particles, and a stable expanded bed with an expansion factor of 2-3 could be obtained under varying flow rates. The results demonstrate that the prepared composite particles have a potential to be used as the matrixes of EBA adsorbents. The results of bed expansion indicate that Cell-SSP-S is favorable for a relatively low flow rate and the process with mass transfer limitation, while Cell-SSP-L is beneficial for the separation with fast adsorption and high productivity. In addition, a linear relationship between the particle wet density and the SSP/cellulose viscose ratio was found, which would be quite useful for directed design of EBA adsorbents. Since the desired matrix was developed for EBA processes, more information about the influence of the SSP content on the properties of bed expansion, fluid mixing, and hydrodynamics in the column will be studied in the coming work.

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Received for review July 30, 2004 Revised manuscript received August 10, 2005 Accepted August 16, 2005 IE049317O