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New Analytical Method Approaches for Enzyme Characterization

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Pancreatic enzymes are essential for hydrolytic intraluminal breakdown of macronutrients into smaller, absorbable metabolites.  Porcine pancreatic extract is used to treat pancreatic enzyme deficiency.  It is often prescribed for people with cystic fibrosis, chronic inflammation of the pancreas, or blockages of the pancreas or common bile duct caused by cancer.  It is also taken by people who have had their pancreas removed or who have had gastrointestinal bypass surgery.  Pancrease is taken to help with digestion of proteins, starches, and fats.

Several valuable enzymes are contained in a pancreas.  Their physical, chemical and biochemical properties and substrate specificity are closely similar.  The molecular weights of trypsin, chymotrypsin and elastase are 23 800, 25 000 and 25 900 respectively.  The active sites of all of these enzymes are serine and because of this no simple method of separation exists.  The characteristics and purification of trypsin and chymotrypsin have been intensively studied but this is not the case for elastase.  Several methods for the separation and purification of pancreatic enzymes have been reported but all have major disadvantages.  Until recently, there has been no single analytical method available that can sufficiently separate all the components in the extracts and be used for quality control in support of the manufacturing process.

Enzymes contained in pancreatic enzymes can be chromatographically separated from other product components using reverse phase High Performance Liquid Chromatography (HPLC) and detected using a UV detector.  HPLC allows for sample separation by typically using 5 µm particle chemistries.  However, the limits of resolution and sensitivity are always observed.

Ultra Performance Liquid Chromatography (UPLC) technology is designed for outperforming traditional or optimized HPLC.  Unlike HPLC, sub-2 µm particle chemistries are used and give higher efficiency, resolution and separation with a much wider range of linear velocities, flow rates and back pressures.  Besides, column length can be reduced with the same efficiency and increase in throughput can be generated with no loss in resolution.  Furthermore, flow rate is reduced with no loss in efficiency and UPLC with sub-2 µm particles provides up to 70% higher sensitivity.

By taking advantages of UPLC technology, this facilitates the development of a LC/UV method for separating and detecting major pancreatic enzymes.

An enzyme chromatographic profile by UPLC/UV is achieved by optimizing the choice of column, gradient of mobile phases and run time.  The first challenge is to choose a suitable column.  A UPLC BEH C4 column can be evaluated as it has 300 Å pore size which effectively allows separation of large molecules.  However, the resolution is not the best.  A UPLC BEH Phenyl column will provide better separation because it has trifunctional C₆ alkyl ether between the phenyl ring and the silyl functionality.

The gradient of the mobile phases is critical and controls the run time.  In order to avoid potential degradation of pancreatic enzymes during analysis, a shorten run time is essential.  It is a challenge as better resolution normally requires longer run time.  A shallow gradient could be chosen to increase or maintain resolution.  However, a longer run time would be required.  Therefore, a compromise has to be made on run time.  Delicate gradient has to be prepared to provide better resolution of peaks.

A preliminary run should be performed on a UPLC BEH C4 column and caution must be taken in choosing a mobile phase.  Typically, many chromatograms must be obtained by different gradients and run times to satisfy the resolution of peaks.  Moving to a UPLC BEH Phenyl column can provide better resolution of the peak.  As compared with chromatograms from HPLC, more peaks will be obtained and be well resolved.

A UPLC HILIC column can also be used to separate proteins presented in pancreatic enzyme samples.  The preliminary results demonstrate that the pH of the mobile phase plays a critical role for chromatographic profiling.  The profile obtained from usage of a HILIC column is not as good as that demonstrated by using phenyl column.

A long run time typically seen in the beginning can be shortened by fine tuning the gradient, which can be reduced by more than half the time without a significant impact on the resolution of peaks.

Different diluents with different pH's can be used to prepare the sample solution.  For samples prepared in H₂O, early eluted peaks disappear and peaks at ~ 90 minutes are merged.  In addition, peaks from 36–66 minutes detected are less intensive in comparison with the samples prepared in 0.1 % TFA in H₂O.   In 0.1 % NH₄OH in water, more early eluted peaks are observed and a duplex peak is observed instead of three resolved peaks around 90 minutes.  Like samples prepared in H₂O, peaks detected are less intensive than for the sample prepare by an HPLC method.

Various experimental conditions using UPLC BEH Phenyl Column 50 mm have been attempted in order to shorten the run time without affecting the resolution.  However, data obtained from several trials with different gradients and flow rates demonstrate that the resolution is not as good as those obtained from an 80 minute run by using 150 mm phenyl column.

As part of an FDA recommendation, Capillary Electrophoresis methods (SDS non-reduced, SDS reduced and cIEF) may be required in order to support the analysis of a protein profile in pancreatic enzyme extracts.