Frank Moffatt

Approaches towards the quantitative analysis of peptides and proteins by reversed-phase high-performance liquid chromatography in the absence of a pure reference sample

A reversed-phase HPLC protocol for the quantitative analysis of peptides and proteins is presented. It is applicable to purified samples and potentially to crude biological extracts. The key feature is that an analytically pure reference sample of the analyte is not required because the extinction coefficient for the UV absorbance at 280 nm can be accurately estimated from the amino acid sequence. The concentration of a protein can therefore be calculated from the peak area relative to an internal standard. Sources of error and limitations of the method are systematically considered. Tryptophan containing peptides gave closer agreement to expected values than those with only tyrosine. It was found that analogous, previously used methods could not be directly applied to lower wavelengths.

Robustness of iCIEF methodology for the analysis of monoclonal antibodies: An interlaboratory study

An international team including 12 laboratories from 11 independent biopharmaceutical companies in the United States and Switzerland was formed to evaluate the precision and robustness of imaged capillary isoelectric focusing for the charge heterogeneity analysis of monoclonal antibodies. The different laboratories determined the apparent pI and the relative distribution of the charged isoforms for a representative monoclonal antibody sample using the same capillary isoelectric focusing assay. Statistical evaluation of the data was performed to determine within and between laboratory consistencies and outlying information. The apparent pI data generated for each charged variant peak showed very good precision between laboratories with RSD values of less than 0.8%. Similarly, the RSD for the therapeutic monoclonal antibody charged variants percent peak area values are less than 11% across different laboratories using different analyst, different lots of ampholytes and multiple instruments. These results validate the appropriate use of imaged capillary isoelectric focusing in the biopharmaceutical industry in support of process development and regulatory submissions of therapeutic antibodies.

Characterisation of electroosmotic flow in capillary electrochromatography columns

Currently available capillary electrochromatography (CEC) instrumentation using UV-Vis detection dictates the use of duplex columns. Due to discontinuities (electric field strength and conductivity) that arise at the boundary between the packed and open sections in these columns, the determination of the electroosmotic flow (EOF) is complicated. Thiourea has been found to be an accurate EOF marker under the conditions employed in this study. By injecting this compound onto a fully packed column and comparing the obtained mobilities with those calculated from measured zeta potential values a value for tortuosity has been obtained. The use of laser Doppler velocimetry (LDV) for the measurement of zeta potential has been found to be the most direct and rapid method of characterising silica support materials in terms of electroosmotic mobility. The open section in duplex CEC columns has been shown to influence the actual column flow-rate. The EOF measured using duplex columns of varying packed and open section lengths have been compared with those obtained for a fully packed column.

Capillary electrochromatography for pesticide analysis: effects of environmental matrices.

An insecticide, pirimicarb, and a fungicide, azoxystrobin, were analyzed by capillary electrochromatography. Nine environmental matrices derived from soil, plant and animal extracts were used. After a series of 311 consecutive injections with no washing between injections, retention times increased by ca. 0.5 min. The use of exaggerated application rates for metabolism studies enabled the detection of xenobiotic pesticide degradates by UV absorbance. A 1.2 mm path length, high‐sensitivity flow cell gave a gain in sensitivity; however, a further increase in sensitivity of at least two orders of magnitude is required for pesticide residue analysis. Analyte stacking using large volume injections of aqueous samples led to a large increase in retention times.

Influence of the unpacked section on the chromatographic performance of duplex strong anion-exchange columns in capillary electrochromatography

This work describes initial investigations of strong anion-exchange (SAX) packing materials for capillary electrochromatography (CEC). The use of SAX phases in CEC is theoretically appealing for the analysis of negatively charged species. The reversed direction of the electroosmotic flow (EOF) generated by SAX phases (in comparison to reversed phases and strong cation-exchange phases) means that negative species can migrate with the EOF, not against it, hence the analysis times, of such species should be decreased and efficiencies improved. Duplex CEC columns (the standard for instruments using UV detection) consist of a packed and an unpacked section. Using common reversed-phase packing materials the direction of the EOF in both sections is co-linear, however when normal fused-silica capillaries are packed with SAX material the direction of the EOF in the two sections oppose one another. It has been shown, using conventional duplex CEC columns and fully packed CEC-MS columns that the opposing direction of EOF causes a massive degradation in column performance. Consequentially, it is demonstrated that if the EOF in the open section of the duplex SAX column can be controlled via pH or capillary derivatisation then good, reproducible CEC can be performed on anionic species using SAX packed CEC columns.

Capillary electrochromatography-applicability to the analysis of pesticides and compounds of environmental concern and the theory of electroosmosis

SummaryThe applicability of capillary electrochromatography to the automated analysis of pesticides and phthalate esters that are of environmental concern was assessed. Reversed phase packing materials were compared. Column to column and run to run reproducibility was established. Peak height with an internal standard gave the best reproducibility. Faster analysis than alternative HPLC methods was demonstrated for a mixture of the insecticide pirimicarb and related pyrimidines. The relationship between the concentration of an analyte in a sample and at the detector was determined by the use of radio-labelled14C-pirimicarb. The volume fraction of the liquid zone was 0.64. The possibility of electroosmosis through the pores is discussed with reference to the Rice-Whitehead model for electroosmotic flow in a capillary. A new parameter, the effective pore size is used in equations for electroosmosis through porous packings.

Determination and control of low-level amino acid misincorporation in human thioredoxin protein produced in a recombinant Escherichia coli production system.

Escherichia coli is used extensively in the production of proteins within biotechnology for a number of therapeutic applications. Here, we discuss the production and overexpression of the potential biopharmaceutical human thioredoxin protein (rhTRX) within E. coli. Overexpression of foreign molecules within the cell can put an enormous amount of stress on the translation machinery. This can lead to a misfiring in the construction of a protein resulting in populations differing slightly in amino acid composition. Whilst this may still result in a population of active molecules being expressed, it does present significant problems with molecules that are destined for clinical applications. Amino acid misincorporation of this subset could potentially result in antibodies being raised to these unnatural proteins. Cross‐reaction with a patients endogenous thioredoxin could then lead to an autoimmune phenomena and serious health implications. Generally, the issue of misincorporation appears not to be a routine regulatory concern (see ICH Q6B guidelines). Therefore, amino acid misincorporation may not have been detected, much less explored in the clinic as the occurrence or absence of these random errors is not routinely reported. Using current technologies based on proteomics, the ability to find misincorporation critically depends upon the criteria for matching theoretical and experimental mass spectrometry data. Additionally, isolation and extraction of these mistranslated proteins from the production process is both difficult and expensive. Therefore, it is advantageous to find routes for removing their production during the upstream phase. In this study, we show how modern proteomic technology can be used to identify and quantify amino acid misincorporation. Using these techniques we have shown how manipulation of gene sequence and scoping of fermentation media composition can lead to the reduction and elimination of these misincorporations in rhTRX. Biotechnol. Bioeng. 2012; 109:1987–1995.

Effect of ionic strength on perfusive flow in capillary electrochromatography columns packed with wide-pore stationary phases

The use of wide-pore stationary phases in capillary electrochromatography has shown exceptional increases in separation efficiency in conjunction with high electroosmotic flow. These effects are due to the perfusive flow mechanism which is primarily controlled by the ionic strength of the mobile phase. Good correlation between calculated values of electrochemical double-layer thickness and efficiency data have also been obtained. Reduced plate height values of <0.5 have been observed with pore sizes of 4000 A. In addition, electroosmotic flow mobility twice that of 3 microm Spherisorb ODS-1 has been obtained.