John Frenz

Characterization of human growth hormone by capillary electrophoresis

Production of proteins by recombinant DNA technology for use as pharmaceuticals requires the use of the most powerful tools of analytical protein chemistry in order to confirm purity and identity of the product and reliability of the process. Capillary electrophoresis is an emerging technology that shows high sensitivity and selectivity and may have promise in this application. The technique combines the instrumental control and quantification features of high-performance liquid chromatography with the separating power of electrophoresis, and thereby has attracted broad interest. In this report, human growth hormone expressed in bacteria has been analyzed by both free zone electrophoresis and isoelectric focusing in a coated capillary to demonstrate the separation of the native molecule from its deamidated variant. A capillary zone electrophoretic tryptic map has also been developed and characterized. This map complements the widely employed reversed-phase high-performance liquid chromatography tryptic mapping systems that are important in protein characterization. Certain drawbacks to capillary zone electrophoresis compared to other analytical methods are noted, including relatively poor reproducibility and low sample tolerance. For applications as demonstrated here, however, the speed, separating power and sensitivity of the technique compensate for these shortcomings.

Effect of electric field on liquid chromatographic separation of peptide digests. Combining capillary separation techniques.

A system is described which allows operation of a range of capillary based liquid phase separations including capillary electrophoresis, isocratic and gradient capillary electrochromatography, isocratic and gradient capillary liquid chromatography and electrically assisted gradient capillary liquid chromatography. The system was coupled to electrospray ionization mass spectrometry in the electrically assisted capillary liquid chromatography mode to investigate the effect of applied voltage on the selectivity in peptide mapping separations. Analyses were performed on tryptic digests of recombinant human growth hormone and tissue plasminogen activator. The results show a small but useful effect on selectivity that can be used to fine tune specific separations.

High-performance displacement chromatography-mass spectrometry of tryptic peptides of recombinant human growth hormone

The combination of high-performance displacement chromatography with continuous flow fast atom bombardment (FAB)-mass spectrometry (MS) offers a means of overcoming the sample capacity limitations imposed by the low flow-rates tolerated in microbore systems employed for directly coupled liquid chromatography-MS. Displacement chromatography is performed at high concentrations with the same equipment and columns as typically used in chromatography at low concentrations. By using this mode of chromatography with a solution of cetyltrimethylammonium bromide as the displacer, the capacity of a reversed-phase column can be increased 50- to 100-fold for separation of a tryptic digest of biosynthetic human growth hormone. Despite the high load, the use of displacement chromatography allowed high-resolution separation of the complex mixture of eighteen major components. On-line analysis by continuous flow FAB-MS yielded high-quality spectra of these peptides and demonstrated that sharp, single-component bands can be obtained in this separation. Along with the major fragments, the chromatogram showed other peptides originating from protein variants in the sample, from non-specific cleavage in the enzymatic digest or from autolysis of trypsin. On-line analysis also allowed selective ion monitoring of the column effluent for individual peptides and confirmed the high efficiency and resolution obtained by preparative displacement separations on HPLC columns and equipment.

Rapid method for monitoring galactosylation levels during recombinant antibody production by electrospray mass spectrometry with selective-ion monitoring.

This report describes a simple and rapid method to determine the relative amounts of glycoforms differing in terminal galactose on a recombinant antibody produced in Chinese hamster ovary (CHO) cells. The method uses a single quadrupole mass spectrometer coupled to an HPLC system to quantify the glycoform amounts found on a recombinant antibody that binds to the human CD20 antigen. Samples from the recombinant antibody process are reduced and injected directly into the HPLC system where the heavy and light chain antibody fragments, as well as host-cell protein contaminants, are separated chromatographically. Mass-selective detection is performed in the selected-ion monitoring (SIM) mode to monitor the most abundant (38+) ions corresponding to the glycoforms found on the heavy chain of the recombinant antibody. Results obtained using the assay demonstrate good sensitivity, linearity and reproducibility. Comparison to a method using capillary electrophoresis (CE) of the labeled free oligosaccharides demonstrates similar quantitation of the glycoforms in the recombinant antibody. The LC-MS method provides a simple and rapid means for accurately quantifying antibody glycoforms directly from cell culture and other process samples.

Characterization of a tryptic digest by high-performance displacement chromatography and mass spectrometry☆

High-performance displacement chromatography (HPDC) provides a means of increasing the capacity of a chromatographic column, while maintaining the resolution afforded by high-performance liquid chromatographic (HPLC) instruments. The high capacity and high resolution of HPDC can be exploited in tryptic mapping to facilitate the characterization of a protein preparation. In this manner, minor constituents of the mixture, which may be difficult to isolate by conventional chromatographic methods, can be obtained in sufficient amounts to permit chemical characterization by established techniques. The isolation by HPDC of peptides obtained by digestion of recombinant human growth hormone (rhGH) and the subsequent characterization of the peptides are described. The identification of certain of these peptides revealed information on the specificity of trypsin for the substrate, rhGH, and for autolysis. Fractions from the HPDC tryptic map were collected and analyzed by electrospray ionization mass spectrometry (ESI-MS) either directly or following further separation by gradient elution HPLC. Fragment ions observed in the ESI mass spectra facilitated identification of peptides obtained by HPDC tryptic mapping.

High performance capillary electrophoresis

The application of recombinant-DNA methods for the production of therapeutic proteins has, over the past decade, driven the development of new technology for the analysis and characterization of biological molecules. High performance capillary electrophoresis (HPCE) has generated enormous interest among biochemists, analytical chemists and chromatographers, and is emerging as an extremely high-resolution separation technique, that may rival high performance liquid chromatography (HPLC) in its efficiency and breadth of application.