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.

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 patient's 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.

Determination of antifungal proteins in soil by liquid chromatography.

A liquid chromatography method was developed for the determination of antifungal/antimicrobial proteins Rs-AFP1 and Dm-AMP1 in sandy loam soils. The extraction of these highly basic proteins was achieved by mechanical shaking with aqueous Tris buffer pH 9 containing guanidinium thiocyanate salt (4.1 M), EDTA and nonionic polyoxyethylene 20 cetyl ether, Brij-58 detergent. The extracts were cleaned up on Oasis HLB polymer solid-phase extraction cartridges and quantified by liquid chromatography fluorescence detection based on the fluorescence properties of the tryptophan content of these proteins. The detector response was linear for 0.3-10 microg mL(-1). Procedural recoveries were tested in the range 10-100 mg kg(-1). The limit of quantification was 10 mg kg(-1 )protein in the soil sample representing the lowest validated fortification level. The antifungal proteins were found to be stable in soil extract tested up to 9 days when stored at 4 degrees C.