Materials and Methods

4.2.1 Immobilization Protocols

The published protocol from Ahn, et al.33 _{was followed (Figure 4.1) to produce a batch of}

trypsin immobilized particles. The published protocol involved 40 mg of trypsin (TPCK treated, Sigma Aldrich P/N T1426) in 2 mL of 50 mM trisodium citrate at pH 5.0. This was added to a 5 mL round bottom flask with 1 mL of ALD coupling solution (Sterogene Bioseparations,

Carlsbad, CA) which contained 1 M sodium cyanoborohydride. The crosslinker (110 μ of triethoxysilyl butyraldehyde Tech-90, Gelest Inc.) was added and the mixture was rotated for 2 hr at room temperature. After 2 hr, 0.3 g of non-bonded bridge ethyl hybrid (BEH) silica particles from Waters Corporation was added and the mixture was rotated for 30 min.

Approximately 1 mL of 2 M sodium sulfate in 50 mM trisodium citrate (pH 5) was used to salt out the reaction as it was left to rotate overnight at room temperature. The next day the reaction was quenched with 1 mL of 1 M ethanolamine and rotated for 2 hr. Finally the particle slurry was washed with 2 aliquots of 5 mL 50 mM trisodium citrate followed by 2 aliquots of 5 mL 1 M sodium chloride. A final wash with water (at pH 4 with trifluoroacetic acid) was performed and the slurry was stored at 5°C.

For the UNC revised trypsin immobilization protocol (Figure 4.2), approximately 0.3 g of non-bonded bridged ethyl hybrid (BEH) silica particles (5 µm, 300 Å) from Waters Corporation (Milford, MA) were added to a 5 mL round bottom flask and kept under a constant flow of nitrogen. Then 110 μ of triethoxysilyl butyraldehyde (Tech-90, Gelest Inc., Morrisville, PA) in 2 mL of ethanol (Fisher Scientific, Pittsburgh, PA) was added. This was allowed to rotate for 2

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hr at room temperature. The particles were rinsed with pH 9, 50 mM ammonium bicarbonate (Sigma Aldrich, St. Louis, MO) to remove excess linker and 40 mg of trypsin (Sigma Aldrich, TPCK treated, P/N T1426) in 2 mL 50 mM ammonium bicarbonate was added. The ALD

coupling solution (1 mL of 1 M NaCNBH3, Sterogene Bioseparations, Carlsbad, CA) was added

and the solution was allowed to rotate for 2 hr at room temperature. Afterwards, 1 mL of 1 M ethanolamine was added to quench the reaction and stirred for 30 min at room temperature. The particles were washed with 50 mM ammonium bicarbonate to remove excess trypsin and ethanolamine then stored in water brought to pH 4 with trifluoroacetic acid (TFA, Sigma Aldrich) at 5°C.

4.2.2 Determining Trypsin Activity of Particles in a Slurry

To determine activity of the immobilized trypsin, standard trypsin substrate BAEE (Nα-

benzoyl-L-arginine ethyl ester hydrochloride, Sigma Aldrich, P/N B4500) was used. A 1 min in- solution digestion was performed with 1.5 m of 0.25 mM BAEE and 100 μ of particle slurry (slurry concentration was 30 mg particles per milliliter of water). This was compared to a 1 min digestion of trypsin in solution (50 μg) with 1.5 m of 0.25 mM BAEE. When the digestion times were complete the samples were centrifuged at 15,000 rpm (1 min) and the supernatant was injected onto a PLRP-S reverse phase column (300 Å, 5 µm, 250 x 4.6 mm, Agilent Technologies, Santa Clara, CA) for UV analysis of BAEE (Figure 4.7). Although the free

(unbound) trypsin sample did not contain particles it followed the same centrifugation protocol

for consistency. Trypsin hydrolyzes BAEE producing Nα-benzoyl-L-arginine (BA) and ethanol.

BAEE and BA were chromatographically separated on the PLRP-S reversed phase column at 80°C and observed at 253 nm (Figure 4.8). The mobile phase for the reverse phase separation of BAEE/BA consisted of water and acetonitrile with 0.1% trifluoroacetic acid (TFA, Fisher) and the gradient is outlined in Table 4.1.

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4.2.3 Determining the Amount of Trypsin Immobilized

In order to determine the amount of enzyme immobilized on the silica substrate, a bicinchoninic acid (BCA assay) was performed according to the Pierce Micro BCA Protein Assay

Reagent Kit (#23235).35 _{For the procedure, 1 mL of each standard (solutions of known free un-}

bound trypsin concentrations 0 to 50 μg/m ) and unknowns (slurries of known particle concentration, stored as 0.6 g particles/10 mL solution) were combined with 1 mL of working reagent from the assay kit. The samples were placed on a shaker for 2 hr at room temperature, centrifuged, and the absorbance measured at 562 nm. By plotting the calibration curve for the absorbance with known trypsin concentration, the concentration of trypsin on the particles can

be determined.35 _{An example calibration curve is shown in Figure 4.9.}

4.2.4 Packing the Trypsin IMER

Once the particles produced by the UNC immobilization procedure were determined to be the most active, the protocol was scaled up by a factor of 2 (0.6 g of particles) to pack into a chromatographic column. An Isco D-Series syringe pump module (Model 260D, Teledyne ISCO, Lincoln, NE) was used to pack the particles into an empty HPLC column assembly (2.1 mm x 30 mm x ¼” O.D., Restek, Bellefonte, PA). The syringe pump was connected to a pressure vessel which held the particles in a slurry (60 mg/mL in 50 mM ammonium

bicarbonate, pH 8). The column was placed on top of the vessel with the outlet frit in place. The entire apparatus was rinsed with 50 mM ammonium bicarbonate prior to column packing. The column was packed at 3,000 psi for 30 min and the pressure was increased to 5,000 psi for 30 min to ensure proper packing for a range of HPLC pressures. When not in use the column was rinsed with water (pH adjusted to 4 with trifluoroacetic or formic acid) and stored at 5°C. At this pH and temperature, the trypsin activity can be conserved.

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