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Using CE, HPLC and UPLC for Peptide Drug Product Characterization

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Capillary Electrophoresis (CE) is a relatively new analytical technique for the separation and analysis of small and large molecules such as peptides and is complementary to existing approaches such as High Performance Liquid Chromatography (HPLC).  The advantages of CE over HPLC include:

• Relatively small sample volume requirements,

• Simpler experimental set-up

• Easy and rapid method development

• Reduced sample pre-treatment and running costs

• Lower solvent demand

• Rapid separation

The separation mechanisms are widely different in CE and HPLC and are based on electrophoretic mobility and partitioning differences, respectively.  These two techniques are often used in combination when assessing drug quality.  Good agreement between CE and HPLC results would support a comprehensive evaluation of the quality of therapeutic peptide dosage forms.  CE has been employed to monitor protein profiles in enzyme extract drug products that would be beneficial in the assessment of drug quality and support a Forced Degradation study.  A forced degradation study for enzyme extracts is required in order to determine the degradation pathway, identify potential impurities and make an assignment about the drug stability.

The influence of various parameters on the peaks separation in the CE electrophergrams such as buffer pH and concentration, applied voltage, injection time and capillary length must be investigated to optimize the methodology of extract proteins and their impurities separation.

The preliminary development work must be based on the utilization of a gel mixture as well as a buffer system.  The exploration of a number of gel mixtures and buffer systems must be undertaken to determine whether different gel mix-buffer system could provide better CE conditions that facilitate protein separations and can also improve peaks symmetry.

A Comparison of Separation using CE, HPLC and UPLC:

Higher efficiency and better resolution is observed with CE as compared with HPLC.  However, the data obtained from CE is inconsistent with those that are observed in UPLC.

Separation of the same analytes by HPLC, UPLC requires an organic solvent which may compromise the resolutions between peaks.  In addition, no organic solvents are used in CE, making it more representative of the dosage form, more environmentally friendly and more economical than HPLC and UPLC.  Although different, the results from the two approaches are complementary.  The complementary nature is demonstrated by comparison of an electropherograms alone with HPLC and UPLC chromatograms.  The fact that these two methods may demonstrate different elution patterns at similar pH values for the same sample suggests that CE may provide additional confirmatory information to that provided by HPLC or UPLC.

For enzyme extracts, Capillary Electrophoresis methods provide for more efficient separation of these enzymes and their equilibrium products and impurities.  The aqueous conditions used in this these types of studies mimic the matrix at which these enzyme extracts would exist after reconstitution and after administration.  These conditions cannot be duplicated with HPLC due to the presence of the needed organic solvent and the poor peak shape obtained for these analytes at high pH.  These CE conditions should more accurately reflect purity.

The influence of the buffer pH and concentration, length of capillary and sample injection time on the separation should be systematically examined.  The buffer pH can an important parameter that can be manipulated to optimize selectivity for enzyme extracts by CE.

These methods must be verified according to ICH and USP guidelines.  Linearity, accuracy, precision, limit of detection, limit of semi-quantification and selectivity should be determined to assess the suitability of the method.  Sample injection volumes for CE are lower, whereas considerably larger sample volumes are typically required for an HPLC method.

Finally, an additional advantage of CE over HPLC is the use of relatively small volumes of the background electrolyte, as compared with a mixed organic mobile phase for HPLC.  CE is therefore more economical and environmentally safe than HPLC.  The simplicity associated with CE and the use of inexpensive capillary columns, coupled with the above advent ages, makes capillary electrophoresis a valuable compliment to existing techniques for analysis of enzyme extracts.