Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases

Analytica Chimica Acta 564 (2006) 106–115

Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases Travis R. Besanger, Richard J. Hodgson, James R.A. Green, John D. Brennan ∗ Department of Chemistry, McMaster University, 1280 Main St. West, Hamilton, Ont. L8S 4M1, Canada Received 20 July 2005; received in revised form 19 December 2005; accepted 29 December 2005 Available online 3 February 2006

Abstract Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol–gel processing route. While such columns proved to be useful for applications such as screening of protein–ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low ␮L/min range. Using the enzyme ␥-glutamyl transpeptidase (␥-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in kcat and decreases in KM , switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography. © 2006 Elsevier B.V. All rights reserved. Keywords: Immobilized enzyme reactor chromatography; Protein retention; Enzyme activity

1. Introduction Chromatographic stationary phases that are used as supports to immobilize proteins have been employed for a variety of applications including purification and cleanup [1–6], chiral separations [7–10], on-line proteolytic digestion of proteins [11–16], biocatalysis [17] and screening of protein-small molecule interactions via frontal affinity chromatography [18–23]. The latter application is of particular importance for understanding the function of proteins, since small molecules can often be used as modulators of protein function both in vitro and in vivo [24]. The predominant methods used to prepare protein-loaded columns have been based on covalent or affinity coupling of proteins

∗

Corresponding author. Tel.: +1 905 525 9140x27033; fax: +1 905 527 9950. E-mail address: [email protected] (J.D. Brennan). URL: http://www.chemistry.mcmaster.ca/faculty/brennan.

0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2005.12.066

to solid supports. However, such methods have several limitations, including loss of activity upon coupling, low surface area, potentially high backpressure (which may alter protein structure and function [25,26]), difficulty in miniaturization and poor versatility, particularly when membrane-bound proteins are used [20–23]. In the past few years it has been shown that a biocompatible sol–gel processing method can be used for entrapping a wide variety of soluble [27–31] and membrane bound [32–35] proteins within the matrix of an inorganic silica network, allowing the formation of sol–gel based bioaffinity columns. For example, Niessner’s group reported the development of immunoaffinity columns based on packing of crushed antibodydoped silica materials into columns [36–39]. Zusman’s group has developed monolithic columns using glass fibers coated with antibody-doped sol–gel glass as a support to analyze tumor-associated antigens [40]. Toyo’oka’s group used capillary electrochromatography to both prepare and elute compounds

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from monolithic protein-doped silica columns [41–46]. Our group has utilized a sol–gel method based on poly(ethylene glycol) (PEG)-doped diglycerylsilane (DGS) [47] to form proteindoped meso/macroporous monolithic silica stationary phases for frontal affinity chromatography/mass spectrometry (FAC/MS), immunoextraction and immobilized enzyme reactor chromatography/mass spectrometry (ER/MS) [48–51]. Such columns have a bimodal pore distribution that provides both micrometer size flow channels (macropores) and nanometer size pores (mesopores) that encapsulate the protein. Monolithic bioaffinity columns can be used for pressuredriven liquid chromatography and possess many of the same advantages of their monolithic reverse-phase counterparts [52–62]. These self-supporting structures do not require frits and have additional advantages, such as the ability to operate at low backpressures and high flow rates. However, the presence of a significant fraction of macropores with diameters of >50 nm within the materials results in significant leaching of entrapped proteins, which can range from 75 to 95% [48,50], as compared to leaching of <5% from mesoporous materials with pore diameters in the range of 3–5 nm [63]. The loss of protein results in low binding capacities (Bt ) when applied to frontal affinity chromatography/mass spectrometry (FAC–MS) analysis [48], or low turnover rates (Vmax ) if applied to on-line bioreactor studies [51]. While formation of purely mesoporous silica can minimize leaching of entrapped proteins, we have observed that the small pores within such materials (3–5 nm) lead to very high backpressure (>3500 psi at flowrates of 1 ␮L/min) and thus these materials cannot be used for pressure-driven LC. To create bioaffinity columns that showed high retention of protein and sufficient porosity to achieve pressure-driven flow of eluents, we systematically investigated how sol formulation and processing conditions could be adjusted to produce largely mesoporous materials. We show that both the amount of PEG and the pH used in forming the monolithic silica alter the morphology of the material, and that under specific conditions, materials with relatively large mesopore diameters can be prepared that show high retention and activity of an entrapped enzyme and reasonable flow properties. The kinetic properties of the model enzyme ␥-glutamyl transpeptidase (␥-GT) were examined in these materials using an immobilized enzyme reactor chromatography method [51]. 2. Experimental 2.1. Materials Diglycerylsilane (DGS) was prepared by methods described elsewhere [47]. l-Glutamic acid p-nitroanilide (GPN), pnitroaniline (pNA), ␥-glutamyl transpeptidase (EC 2.3.2.2, 10 unit/mg solid where 1 unit is equivalent to turnover of 1.0 ␮mol/min of l-3-glutamyl-p-nitroanilide at pH 8.5, 25 ◦ C), 10 kDa poly(ethylene glycol), glycylglycine and sodium hydroxide were obtained from Sigma–Aldrich (Oakville, ON). Cy5–maleimide was purchased from Amersham Biosceinces (Piscataway, NJ). Fused silica tubing (250 ␮m i.d., 360 ␮m o.d.) was purchased from Polymicro Technologies (Phoenix, AZ). CostarTM clear 96-well polystyrene microtiter plates were pur-

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chased from Nalge Nunc International (Rochester, NY). HPLC grade water (double distilled and deionized) was purchased from Caledon Laboratory Chemicals (Georgetown, ON). 2.2. Procedures 2.2.1. Preparation of silica materials A total of 35 unique silica compositions were prepared using seven different pH values (5.3, 6.1, 6.6, 6.9, 7.4, 8.0 and 8.5) and five different final concentrations of 10 kDa PEG (0, 1.25, 2.5, 5 and 10% (w/v)) with final concentrations of 0.5 g/mL DGS and 50 mM HEPES. We note that the optimal pH range for HEPES buffer is 6.8–8.2, and thus the buffer capacity of samples outside of this range will be low. However, our experience has shown that buffer type can have a significant effect on the gelation of silica sols, and hence this parameter was not altered. The pH of all samples in the range of 5.3 and 8.5 were checked for accuracy of their pH prior to formation of silica. Other parameters, such as silica:water ratio, buffer type and ionic strength can also affect the gelation kinetics, and hence the morphology, of the silica. Therefore, we chose to keep these latter parameters constant in order to better evaluate the effects of pH and PEG concentration. We note that high silica:water ratios led to rapid gelation, while low silica:water ratios led to materials that were not mechanically robust. The value used in this study provided a robust silica material, but still allowed for significant control over gelation and phase separation time by varying pH and PEG concentrations. Samples used for leaching studies and bioreactor chromatography contained a final concentration of 106 ␮g/mL of ␥-GT, while those used for enzyme activity studies in microwell plates contained 3.2 ␮g/mL of ␥-GT. In all cases, the ␥-GT samples were prepared at an initial concentration of 1.0 mg/mL in 5 mM HEPES·NaOH pH 7.4 and dialyzed against this buffer at 4 ◦ C using a 3000 MWCO membrane prior to entrapment. DGS based sols were prepared by sonicating DGS with water (1 g + 1 mL) at 0 ◦ C for 15 min to hydrolyze the monomer, followed by filtration through a 0.2 ␮m filter. Protein-doped silica monoliths were formed by first mixing 48 ␮g/mL ␥-GT with an equal volume of 400 mM HEPES that had been adjusted to various pH values ranging from 5.3 to 8.5 using 1.0 M NaOH. The resulting buffered protein solution was then mixed with an equal volume of an aqueous solution of 40, 20, 10, 5 or 0% (w/v) 10 kDa PEG. 20 ␮L of the protein–PEG mixtures were then dispensed into the well of a microwell plate followed by 20 ␮L of the hydrolyzed DGS sol solution. The samples were mixed on a microwell plate shaker for 20 s and allowed to gel. After gelation samples were and aged at room temperature for 1 h and then for an additional 48 h at 4 ◦ C. 2.2.2. Characterization of silica materials Bulk silica monoliths were prepared to a final volume of 2 mL using the protocols described above but with no entrapped protein. Separate samples were made for gelation, phase separation and porosimetry studies and for SEM imaging. Gelation times were assessed by determining the point when solutions lost flow relative to the time when all components were mixed.

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Phase separation times were determined visually by noting the time when samples became translucent or opaque. Following gelation, materials were sealed and aged for 48 h in air and then aged for an additional 7 days in buffer (50 mM HEPES pH 7.4), with the buffer replaced twice daily to remove glycerol and PEG. For porosimetry and SEM studies, the samples were then aged an additional 7 days with the buffer exchanged for HPLC grade H2 O that was also changed twice daily. Samples were then desiccated for 2 weeks at room temperature followed by drying at 115 ◦ C to remove solvent. The use of high temperature drying (>300 ◦ C) was avoided as this process may lead to partial collapse of the mesopores [64], although macropores are less susceptible to capillary stresses during drying and thus are not likely to collapse even at high temperature [65]. A temperature of 115 ◦ C was thus chosen to allow drying with minimal alterations to pore morphology. The degree of material shrinkage was assessed by measuring the diameter of the silica monoliths in the hydrated and desiccated states. The degree of shrinkage was normalized to the inner diameter of the scintillation vial in which they were initially formed. The morphology of all materials was assessed by Hg intrusion porosimetry measurements, using a Quantachrome Poremaster® GT mercury intrusion porisimeter. The contact angle used was 140◦ , running a fixed speed pressure gradient from 20 to 59,658 psia (10.6 ␮m to 3.57 nm pore diameter range). Median pore diameters and percentage of meso of macroporosity were determined as a function of intruded volume using the PoreMasterTM software provided by Quantachrome, assuming a cylindrical pore shape. In certain cases, nitrogen porosimetry of completely dried monoliths was also performed using a Quantachrome Nova 2000 surface area/pore size analyzer. Samples were washed as noted above, crushed to a fine powder, freeze–dried and outgassed at 120 ◦ C for 4 h to remove air and bound water from the surface of the powder. The pressure was measured as nitrogen was adsorbed and desorbed at a constant temperature of −196 ◦ C. Using the desorption branch of the resulting isotherm, the average pore size and distribution of pore sizes was determined using the BJH (Barrett, Joyner and Halenda) calculation [66]. Scanning electron microscopy images were obtained for the bulk silica monoliths using a JOEL 840 scanning electron microscope. Silica samples were first coated with a thin gold film to improve conductivity during imaging. 2.2.3. Leaching of entrapped enzyme by diffusion Leaching was assessed by monitoring the fluorescence of Cy5-labelled ␥-GT present in a wash buffer used to incubate the monoliths. ␥-GT was labelled by mixing equal volumes of 1.0 ␮g/mL Cy5–maleimide and 1.0 mg/mL ␥-GT and allowing the reaction to proceed for 30 min at room temperature and an additional 18 h at 4 ◦ C. Unreacted dye was removed from the labeled enzyme by gel filtration through Sephadex G25® . The 1 mg/mL stock solution of Cy5–␥-GT was then used to form silica monoliths as described above. After aging, 100 ␮L of buffer was added to the tops of the enzyme-doped samples and incubated overnight to allow equilibration. The samples were then incubated for an additional 1 h with gentle mixing using a microwell plate shaker. A 75 ␮L aliquot of solution was then

taken from the tops of the samples and transferred into a separate microwell plate. The fluorescence of Cy5 was monitored using a TECAN Safire microwell plate reader with excitation and emission wavelengths of 649 and 670 nm, respectively. The fluorescence from the leached samples was normalized to the initial fluorescence obtained from a concentration of enzyme equivalent to that which would be obtained for 100% leaching. 2.2.4. Activity of entrapped γ-GT Monolithic samples containing unlabelled enzyme were prepared as described above using a 48 ␮g/mL stock solution of enzyme. Monoliths were aged and washed four times as described for the leaching study above. The activity of retained ␥-GT was assessed after washing the silica monoliths three additional times with 100 ␮L of 50 mM HEPES·NaOH pH 7.4 (wash buffer). The buffer was then carefully removed from the tops of the protein-doped monoliths and 200 ␮L of reaction buffer (4.69 mM l-glutamic acid p-nitroanilide (GPN), 200 mM glycylglycine in 50 mM HEPES·NaOH pH 7.4) was added. Because the materials had different optical clarity at 410 nm the kinetic data was determined by stop-time analysis. In this case, the reaction was allowed to proceed for 45 min, after which 140 ␮L of the reaction mixture was drawn from the tops of the monoliths and immediately transferred to a separate microwell plate where the concentration of pNA was determined by measuring absorbance at 410 nm using a TECAN Safire® microwell plate reader. 2.2.5. Formation of monolithic columns Columns were prepared from each of the 35 different silica compositions. Following mixing of all components, the resulting solutions were rapidly infused into a 110 cm length of clean, 250 ␮m i.d., 360 ␮m o.d., polyimide coated fused silica tubing. After gelation the capillaries were looped such that both ends could be submerged in Eppendorf tubes containing 50 mM HEPES·NaOH pH 7.4 and aged for a minimum of 3 days to achieve a stable internal structure. Columns were cut to a length of 10 cm prior to use to provide columns with acceptable levels of enzyme that were amenable to rapid assays with suitable backpressures. End pieces were generally discarded (∼15 cm from each end) owing to non-uniform drying of the end segments. A total of 4–5 columns could be obtained from the central region of each capillary. 2.2.6. Flowrate studies Silica columns were interfaced to a two-channel Eksigent nanoLC pump using the appropriate micro-adapters and 75 ␮m i.d., 360 ␮m o.d. fused silica tubing. The Eksigent pump was used to deliver eluent at flowrates from 0.25 to 10 ␮L/min and concurrently measure the backpressures produced (up to 3500 psi). Note that the Eksigent pump has a maximum flowrate of 10 ␮L/min per channel, or 20 ␮L/min in total. For some materials maximum flow rates exceeded 20 ␮L/min, and thus could not be measured. 2.2.7. Performance of γ-GT-doped bioreactor columns Based on the results of the activity, leaching and flowrate studies, enzyme-doped bioreactor columns were fabricated using

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only a subset of the initial 35 silica compositions with a final concentration of 125 ␮g/mL ␥-GT, 2.5% (w/v) of 10 kDa PEG and pH values of 6.9, 7.3 or 7.6. A separate set of columns were also made to assess pressure-induced leaching of ␥-GT using Cy5-labeled ␥-GT in place of unlabeled ␥-GT. Columns were interfaced to the Eksigent pump as described above which was interfaced to a GL Sciences Inc. Model 701 UV–vis detector containing a 6 nL flow-cell (40 ␮m i.d., 4 mm path length). Columns were first equilibrated off-line from the detector with 50 mM HEPES pH 7.4 to remove the residual glycerol and PEG that were used for column fabrication. After thorough equilibration the apparent activity of ␥-GT in the various columns was determined by measuring the production of pNA from GPN at 410 nm under the same buffering conditions used for the microplate assays described above. The raw signal (in mV) obtained from the UV–vis detector was calibrated using the reaction product pNA in order to determine the concentration of pNA. The resulting catalysis rates were calculated as a function of pNA concentration, per contact time at a given flow rate, per column. Catalytic constants for immobilized ␥-GT were determined by infusing different concentrations of GPN through the bioreactor column at flow rates of 250, 500, and 1000 nL/min. To determine the total amount of pressure-driven protein leaching, 25 bed-volumes of 50 mM HEPES pH 7.4 were passed through the various Cy5-labeled ␥-GT columns and collected into a 96-well microwell plate. The fluorescence intensity of the labelled protein was determined as described above and normalized as a percentage of the total amount of Cy5-labeled ␥-GT present in the column. 3. Results and discussion 3.1. Properties of silica materials 3.1.1. Gelation and phase separation times The structural properties and processing methods used to prepare monolithic reverse-phase chromatographic supports from phase separated silica–PEG materials have been studied in detail [65]. However, such materials are processed using alkoxysilane precursors, high levels of alcohol and low pH values, and are treated at high pH to etch out the mesopores. A final high tem-

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perature step is also used to drive the condensation reaction to completion. Columns containing entrapped biomolecules are processed from glyceroxysilanes at neutral pH using buffered aqueous solutions at room temperature. Therefore, it was necessary to establish how factors such as pH and the inclusion of buffer affected the ability of PEG to control the final morphology of the biocompatible material. The morphology of silica materials derived by the spinodal decomposition process depends mainly on the time available for formation of large domains (the coarsening time), which is defined as the difference in phase separation and gelation times (tg − tps ). Fig. 1A shows the gelation times of the 35 compositions used to form monolithic silica materials. Gelation times ranged from 1 to 37 min, and generally increased with decreased pH regardless of PEG concentration. Alterations in PEG concentration at constant pH generally led to only minor changes in gelation times, with gelation time decreasing up to about 2.5% (w/v) of PEG and then increasing slowly at higher PEG concentrations. Fig. 1B shows the coarsening times for the various materials, which ranged from 0 min (i.e. no phase separation) to ∼13 min. At a constant PEG concentration coarsening times decreased with increasing pH, but for a given pH the coarsening times increased up to about 2.5% (w/v) of PEG and then decreased slowly at higher PEG concentrations, opposite to the trend observed for gelation times. The effect of pH on the processes of gelation and phase separation is consistent with the reaction kinetics for a two-step sol–gel process, where tg decreases with increased pH [65]. The decrease in tg causes a decrease in coarsening times since the gel network forms before a significant amount of phase separation can occur. PEG controls the gelation and phase separation times through interactions with the silica surface [65]. At low PEG concentrations ≤2.5% (w/v) the phase separation tendency of the system is greatest, which causes the local concentration of silica to increase thereby increasing the condensation rates and decreasing the gel times. As PEG concentrations increase the polymer adsorbs to the silica surface, which reduces the interaction between sol particles, reducing condensation rates and increasing gel times. Of the two factors studied, pH provided much larger alterations in gelation time, indicating that control over pH was critical for optimizing morphology.

Fig. 1. Gelation times (panel A) and coarsening times (panel B) for various diglycerylsilane derived silica materials. Materials were formed at pHs ranging from 8.5 to 5.25 and contained a final composition consisting of 0–10% (w/v) 10 kDa PEG, 0.5 g/mL DGS and 50 mM HEPES.

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Fig. 2. The amount of shrinkage incurred by the silica materials following condensation for wet and dried materials.

3.1.2. Material shrinkage The fabrication of capillary columns requires that the materials shrink as little as possible to prevent pullaway of the column from the surface of the capillary, which would result in flow channeling. The linear shrinkage in the hydrated state (the state in which enzyme reactor studies are done) was compared to that of the dried material to assess how drying influenced shrinkage. Fig. 2 shows the relative shrinkage of the various hydrated and dried materials relative to the original diameter of the material immediately after preparation. Translucent (non-phase separated) materials underwent only minor shrinkage when aged in a hydrated state, while those that were opaque (phase separated) shrunk by as much as 20% shortly after condensation (i.e. samples surrounding 2.5% (w/v) PEG, low pH). On the contrary, upon drying the translucent materials showed up to 60% shrinkage, while opaque materials underwent very little additional shrinkage beyond that obtained in the hydrated state. The data show that while formation of opaque, macroporous materials might be problematic owing to shrinkage and pullaway, formation of hydrated, translucent mesoporous materials should show minimal shrinkage, and may result in the ability to produce wide-bore columns without pullaway, which in turn could allow entrapment of higher amounts of protein per length of column.

3.1.3. Pore morphology The pore size distribution of the silica material will affect the performance of a protein-doped column since a high proportion of macropores can lead to significant protein leaching, while a predominantly mesoporous material is likely to show high backpressure. The pore morphology and distribution of phase separated silica is largely a result of changes in coarsening times. Materials with long coarsening times were generally translucent or opaque, suggesting a significant degree of macropore formation, while those with a coarsening time of <50 s were slightly translucent or transparent, suggesting a mainly mesoporous structure. Mercury porosimetry studies were performed to examine the effect of coarsening times on the morphology of the silica materials. While previous reports have suggested that this method can lead to erroneous porosity values due to compressibility of silica materials, this is only relevant for low density aerogels [67,68]. The highly connected nature of our materials and their relatively high density relative to aerogels lead to materials that are not compressible [69], and thus it is likely that the Hg porosimetry data accurately reflect the porosity of the materials. Fig. 3A shows the Hg intrusion profiles for materials made with 5.0% (w/v) PEG at various pH values. In general, increases in pH led to decreases in both the average pore diameter and the total intruded volume. Similar trends were observed for materials prepared at other PEG concentrations. In cases where samples were prepared at low pH (pH 5.25), the intrusion curve showed a pronounced double inflection, which is indicative of a bimodal pore distribution. A similar bimodal pore distribution was obtained for materials created at this pH over the PEG concentration range of 1.25–10% (w/v). The decrease in pore diameters as a result of increasing pH is most likely due to the reduction in the gelation time, which thus reduces the coarsening time, as noted above. The decrease in total intruded volumes is a result of a significant degree of shrinkage that occurred during the drying process, as noted in the previous section. Decreases in total intruded volume are also a byproduct of the reduced pore diameter, where a lower fraction of pores are accessible to the mercury during the intrusion experiment. Fig. 3B shows the median pore diameters of the various materials that were determined from the intruded volume statistics. Average pore diameters ranged from ∼6 nm up to 5 ␮m, and

Fig. 3. Typical Hg intrusion profiles for DGS derived materials. Panel A shows the intrusion of Hg as a function of silica pore diameter for materials made with 5% PEG and at various pH values. Panels B and C depict the median pore diameter and percent macropore volume, respectively, as determined from Hg intrusion for the various materials fabricated with different concentrations of 10 kDa PEG and at various pHs.

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tended to increase with the coarsening time, being largest for materials prepared at low pH in the range of 2.5–5.0% (w/v) PEG. Variation of pH over the range of 5.3–7.3 resulted in the ability to access a relatively wide range of pore diameters when operating in the range of 2.5–5% (w/v) PEG, indicating that a range of macroporous columns could be obtained under biocompatible processing conditions. Fig. 3C shows the percentage of total intruded volume attributed to macropores (pores with diameters >50 nm). It can be seen that most samples fabricated at pH 6.75 or less had 45–90% of the intruded volume attributed to macropores, with the largest macropore volumes correlating well to the largest pore diameters (r2 = 0.875). 3.2. Enzyme activity and leaching in monoliths An optimal silica material should provide the maximum amount of active enzyme in an accessible form with minimal leaching of enzyme. Previous studies of enzymes and antibodies entrapped in macroporous silica indicated that only 6–25% of initially entrapped protein remains both active and accessible in column format, with up to 65% of the lost activity being due to protein that had leached from the macroporous material [48,50]. Activity and diffusion-based leaching were initially examined in monoliths. Fig. 4A shows the relative amount of Cy5-labeled ␥GT that leached from the various materials by diffusion, which ranged from 4% for mesoporous materials containing no PEG up to ∼17% for macroporous materials containing the higher levels of PEG. Above 2.5% PEG, a relatively broad range of similar values are obtained for enzyme leaching; the largest changes in leaching occur in the range of 0–2.5% PEG. It should be noted that this is the amount of leaching from a single incubation with buffer. Additional buffer washes would likely lead to higher levels of diffusive leaching [50], but were not employed in this study. Fig. 4B shows the enzymatic activity of ␥-GT that was measured in monoliths after thorough washing and equilibration with buffer. In general, maximum activity was obtained for mesoporous materials, and increased slightly with increasing pH, likely owing to the inherent dependence of ␥-GT activity on pH. Addition of PEG caused significant losses in the amount of activity retained in the monoliths, with the minimum activity being obtained for materials formed at low pH with 2.5% PEG, the same materials that showed the largest pores. These results suggest that the major determinant of low activity is leaching of protein from macroporous materials.

Fig. 4. Dependence of entrapped enzyme activity on DGS derived silica composition. Silica samples were formed at pHs ranging from 8.5 to 5.25 and contained a final composition consisting of 0–10% (w/v) 10 kDa PEG, 0.5 g/mL DGS and 50 mM HEPES. Panel A shows the enzymatic activity of ␥-GT retained in the various silica monoliths following thorough washing with buffer. Panel B shows the relative leaching of Cy5-labeled ␥-GT from the various silica compositions.

250 ␮m i.d. fused silica tubing. As shown in Fig. 5 the maximal flowrate increases with the macropore volume and the average pore diameter, as expected. The broad maximum in the lower right quadrant of the graph is due to the fact that the maximum flowrate that was accessible was 20 ␮L/min, thus all materials

3.3. Column studies 3.3.1. Flowrates Based on the studies presented above it was apparent that formation of columns with low leaching and high enzymatic activity would require fabrication of materials at intermediate pH values (i.e. pH 6.5–8) with low levels of PEG (2.5% or less). To assess the range of compositions that could be used to form columns suitable for pressure-driven LC, each of the 35 compositions tested above were used to create columns within

Fig. 5. Maximal flow-rates achieved through DGS derived silica materials made with various concentrations of 10 kDa PEG and different pHs.

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that could attain this flowrate appear on this plateau. An unexpected finding was that a flowrate of as much as 5 ␮L/min could be obtained from materials that were essentially mesoporous, i.e. materials formed at pH 6.9 or higher with 2.5% PEG, which had mean pore diameters of <50 nm. Although the hydrated pore diameters were probably significantly larger than those measured for dried materials, it is noteworthy that even materials with median pore diameters <15 nm in the desiccated form still showed flowrates of at least 1 ␮L/min, making such column suitable for nanoflow LC applications. 3.3.2. Properties of γ-GT bioreactor columns Table 1 shows the structural characteristics and enzymatic properties of ␥-GT bioreactor columns made with 2.5% (w/v) PEG at pH 6.9, 7.3 and 7.6. This selection of materials showed adequate flowrates, low diffusion-based leaching and good enzyme activity in monolith-based assays. To gain a relative measure of the catalytic rates of the ␥-GT columns, GPN was

infused at a concentration of 2.0 mM at a constant flowrate of 1 ␮L/min in the presence of 200 mM GlyGly and the catalytic properties of the enzyme were calculated based on the concentration of pNA produced as a function of time in a 10 cm column. It is clearly seen that as the pH used to fabricate the column increases from 6.9 to 7.6, the relative activity increases by nearly 10-fold. Slightly reduced activity was observed at increased pH in monolithic samples, ruling out direct pH effects as the cause of the change in activity. Inspection of Table 1 indicates two factors which could be responsible for the higher activity obtained in the columns created at higher pH values. The first point to note is that the amount of pressure-driven leaching drops substantially from 84% to only 15% as the pH used for column fabrication increases. Thus, columns formed at pH 7.6 retain almost six-fold more enzyme than those formed at pH 6.9. It is clear that the leaching obtained by diffusion is significantly lower that that obtained under higher pressure flow. Under diffusion, protein that is in partially buried

Table 1 Combined data for ␥-GT bioreactor columns made with 0.5 g/mL DGS, 2.5% (w/v) PEG, 50 mM HEPES at various pH values

activitya

Column (nM s−1 column−1 ) Monolith activity (nM s−1 ) Diffusive leaching (% total) Pressure-driven leaching (% total) Backpressure (psi) Surface area (m2 /g) (multipoint BET) Bulk particle density (g/cm3 ) Total intruded volume (cm3 /g) Total porosity (%) Macropore volume (% total volume) Mesopore volume (% total volume) Macropore diameter (nm) Mesopore diameter (nm)b

pH 6.9

pH 7.3

pH 7.6

1.05 ± 0.23

1.97 ± 0.31

9.79 ± 0.42

8.09 ± 1.71

7.28 ± 1.42

6.43 ± 1.19

14 ± 1

16 ± 2

15 ± 1

84 ± 2

76 ± 2

16 ± 8

9 402.9

120 288.9

2100 312.5

0.300

0.770

0.820

2.63

0.81

0.44

78.8 74

53.1 6

40.7 1

5

47

39

690

–

–

(14.8)

15/(14.6)

5/(25.1)

SEM image (bar = 10 ␮m) a b

10 cm column, infused GPN concentration = 2.5 mM. Bracketed values obtained from N2 porosimetry.

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pores or strongly adsorbed to the silica may not be removed. However, under higher pressure flow such species would likely be removed. This highlights the issues with diffusion based leaching studies, and indicates that such studies can underestimate leaching that can occur during operation of columns. A second issue to note from Table 1 is that the backpressure of the column at pH 7.6 is ∼200-fold higher than was obtained for the column formed at pH 6.9, owing to the smaller average pore size in the column formed at higher pH (see below). The higher backpressure will also cause more rapid diffusion and force the substrate into regions of the column that might be inaccessible at lower pressures, leading to higher catalytic efficiency. It is also possible that at very high pressures there will be a transition from diffusion-controlled to reaction-controlled kinetics for substrate conversion, as discussed below. The structural features of the three column materials were more carefully evaluated using both mercury and nitrogen porosimetry in conjunction with SEM imaging. Table 1 indicates that the material formed at pH 6.9 had a high total porosity of 79%, with 74% of the total porosity attributed to macropores with an average diameter of 690 nm and 5% of the pore volume attributed to mesopores with an average diameter of 15 nm. SEM images clearly show a macroporous morpohology, with domain sizes on the order of 1–3 ␮m. The high degree of macroporosity correlates well to the high amount of leaching and the low backpressure obtained for such columns. In contrast, the material fabricated at pH 7.3 was less porous (53% pore volume), and had only 6% of its total volume attributed to macropores. On the other hand, the average mesopore diameter obtained by Hg porosimetry was relatively large (15 nm), and the SEM image clearly shows a grainy structure, indicative of the presence of relatively large pores within the material. The intrusion curve obtained by Hg porosimetry showed a tail extending out beyond 200 nm (see Fig. 3A, pH 7.4 intrusion curve for typical intrusion data) suggesting a relatively porous structure. This may explain both the moderate backpressure (120 psi) and the relatively high amount of protein leaching (76%) obtained for this material. It is also possible that the drying process led to significant pore shrinkage, and hence a high volume of macropores may have existed in the hydrated state, but collapsed during drying. The material formed at pH 7.6 was completely mesoporous (<1% macropore volume) and had an average mesopore size of only 5 nm (based on Hg porosimetry). BET data suggested a higher mesopore size of 25 nm, which is likely related to the differences in models used to assess pore diameters for the two methods. SEM images clearly show a lack of any large pores, confirming that the material formed at pH 7.6 was fully mesoporous. This material showed much higher backpressure (∼2100 psi at a flowrate of 1 ␮L/min) and significantly less protein leaching in the hydrated state, consistent with the mesoporous morphology being present in both the hydrated and dry states. Overall, the data clearly show the adjustment of a single parameter, fabrication pH, can lead to a range of morphologies with good control over the relative proportion of meso and macropores, and further demonstrate the importance of morphology in optimizing enzyme reactor column performance.

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Fig. 6. Enzymatic activity and backpressures generated from DGS derived monolithic silica capillary bioreactor columns as a function of flow rate. Panel A illustrates the enzymatic activity of materials made with 2.5% (w/v) PEG, at pH 6.9 (䊉); 7.3 (); and 7.6 (
). Panel B shows the backpressures generated from the same bioreactor columns at the various flow rates.

3.3.3. Catalytic performance of γ-GT-doped bioreactor columns Fig. 6A shows the rate of pNA production as a function of flow rate for the various ␥-GT bioreactor columns, indicating that apparent enzyme activity increased with flowrate. In all cases the backpressure scaled as a linear function for the flow rate, as shown in Fig. 6B, suggesting that the increased backpressure was likely the origin of the increased enzyme activity. Comparing data from panels A and B allows for a comparison of activity and backpressure. However, it should be noted that the variability in leaching, accessibility and pH-based enzyme activity as a function of pH of fabrication make it impossible to compare the effects of backpressure on activity between different columns, thus it is only possible to observe trends within a single type of column. It is noted that for columns fabricated at pH values of 6.9 and 7.3, the activity increases linearly with backpressure, likely due to more rapid diffusion of substrate to the enzyme as pressure increases. However, the activity in the mesoporous column prepared at pH 7.6 shows a negative deviation from linearity at high backpressure (>2000 psi), which is consistent with a transition from diffusion controlled to reaction controlled kinetics. It is noteworthy that the activity at a given flowrate (i.e. 1 ␮L/min) also scales with the amount of protein retained in the columns, with columns that retained more protein showing higher activity. The relatively similar activity for columns produced at pH 6.9 and 7.3, even with the large differences in backpressure, suggest that in each case a similar fraction of retained protein is accessible, and that changes in apparent activity may be related more to the rate of flow, which would cause faster delivery of substrate to protein and thus faster turnover. Gleason et al. have noted similar effects for surface immobilized enzyme flow reactors, wherein increased catalytic rates were observed at increased flow rates [70]. They attributed the increases in turnover rates to deple-

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minimized as a result of the higher backpressures obtained using continuous flow of substrate, which should give KM values much closer to that obtained in solution. Moreover, bioreactor studies using immobilized enzymes on rotating discs have shown that increased rotation velocity, and hence decreased diffusion limitations, also leads to decreases in KM [71]. The catalytic rate constant (kcat ) for the entrapped ␥-GT, which is defined by Eq. (2), can also be estimated by normalizing the Vmax values to account for the total amount of enzyme [E]T in a 10 cm column, taking in account the amount of protein leaching: kcat = Fig. 7. The reaction kinetics for a mesoporous ␥-GT column made with 2.5% (w/v) PEG at pH 7.6, at different flow rates: 250, 500, 1000 nL/min.

tion of the product-containing boundary layer thereby allowing increased assess of substrate to the surfaced exposed enzyme. Such diffusion boundaries are certainly applicable for materials containing large macropores where bulk flow can exist. Another potential issue that could arise is changes in the catalytic properties of the enzymes as a function of flowrate and/or backpressure. To address this issue, the catalytic constants were obtained for ␥-GT within columns prepared at pH 7.6, since these showed the highest activity and the largest range of accessible backpressures. Fig. 7 shows substrate saturation curves for the ␥-GT bioreactor column formed at pH 7.6, and Table 2 summarizes the kinetic parameters calculated using the Michaelis equation (Eq. (1)) as compared to the enzyme in solution, where Vmax is the maximal rate, [S] the substrate concentration and KM is the Michaelis constant: Vmax [S] (1) v= KM + [S] It can be seen that the apparent KM decreases with increasing flow rate, indicative of higher affinity for the substrate, and that the KM values were all lower than the value obtained in solution. This was unlike previous results obtained for ␥-GT in bulk DGS derived silica monoliths where a KM of 5.7 mM was obtained, which was roughly 2.5-fold higher than the solution value [31]. For bulk monolithic samples increased KM values are often attributed to slow mass transfer and substrate partitioning effects arising from interactions of the substrate with the silica matrix [31]. However, partitioning and mass transfer effects are Table 2 Kinetic parameters of ␥-GT bioreactor column fabricated with 2.5% PEG and pH 6.9 Flow rate (nL/min)

KM (mM)

250 500 1000 Solution

1.78 1.23 1.01 1.98

Vmax (nM s−1 ) 777 818 1030 257

kcat a (s−1 ) 0.62 0.65 0.82 5.41

Solution values are given as reference. a Based on [E] = 1350 nM in columns (6.6 pmol ␥-GT in a 10 cm column, assuming 4.91 ␮L internal volume and 84% protein retention), and [E] = 47 nM in solution (10.7 pmol ␥-GT in a 225 ␮L solution assay volume). kcat values assume that all entrapped enzyme is accessible.

Vmax [E]T

(2)

Comparison of the solution and column data shows that the entrapped ␥-GT had a kcat that was, on average, eight-fold lower than that of the enzyme in solution, indicating that the entrapped enzyme was not as efficient as the free enzyme at processing substrate, due to inaccessibility and/or a fraction of inactive protein. Liquid-to-silica mass transfer limitations can lead to reduced catalysis rates for enzymatic flow reactors [72] and such decreases are typical for most entrapped enzyme systems [63]. The increase in Vmax and kcat with flow rate is consistent the substrate either accessing more protein due to pressure-induced intrusion of the mobile phase into the smaller pores, or accessing the protein at a higher rate owing to more rapid flow conditions. Overall, the higher apparent affinity and catalytic constants obtained at higher flow rates, coupled with the reduction in analysis time that is inherent in operation at increased flow rates, indicate that operation of columns under such conditions is optimal. However, for mesoporous columns the flowrate is limited by the relatively high backpressure. Further work will be required to more carefully map out the flowrate, backpressure and enzyme activity values for columns formed between pH 7.3 and 7.6 to determine the ideal composition that allows both high protein retention and activity and a wider range of accessible flow rates. 4. Conclusions Our results demonstrate the importance of the processing conditions, and in particular processing pH, on the structural features and catalytic performance of enzyme-doped monolithic silica columns. When utilizing biocompatible processing conditions under aqueous conditions, processing at neutral pH with a relatively low PEG/silica ratio will provide a bicontinuous macroporous structure capable of high flow rates at low backpressure. However, as indicated by the ␥-GT bioreactor column results, such materials can lead to significant leaching of enzyme, and hence suboptimal column performance. Adjustment of pH toward slightly more basic conditions (pH 7.6) leads to a reduction in macropore volume, but it is still possible to obtain flow of eluent, albeit at higher backpressure. Such columns show excellent retention of protein, and the high backpressure provides increased enzyme activity owing to pressure-induced intrusion of the mobile phase and analytes into the mesopores where the protein is entrapped. Further adjustment of pH in the range of 7.3–7.6 may result in columns with

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reduced backpressure and higher flowrates that still retain significant amounts of protein. Acknowledgements The authors thank MDS-Sciex, the Natural Sciences and Engineering Research Council of Canada, the Canada Foundation for Innovation and the Ontario Innovation Trust for financial support of this work. TRB holds an NSERC Canada Graduate Scholarship. JDB holds the Canada Research Chair in Bioanalytical Chemistry. References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17]

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