Dyes of several classes were investigated as candidates for use in a multiplex, four-decay fluorescence detection scheme for DNA sequencing. The dyes include nitrobenzofuran dyes, rhodamine dyes, fluorescein dyes, cyanine dyes, Nile Red, and BODIPY dyes. Based on the results of fluorescence spectral and lifetime studies, an initial set of four dyes was selected for further study: NBD-aminohexanoic acid (NBD-HA, r = 1.1 ns), tetramethyl-rhodamine, methyl ester (r = 2.2 ns), rhodamine green (r = 4.3 ns), and BODIPY 505/515 (r = 5.9 ns). Limits of lifetime detection of the four dyes were investigated, and lifetime resolution was demonstrated for mixtures of the free dyes in batch solution. Lifetime of dye-labeled DNA primers also were determined in batch solution and detected on-the-fly in capillary electrophoresis (CE). Conjugation of the dyes to DNA improved the resolution of their individual lifetimes in mixtures in batch measurements. When attached to the primer, tetramethyl-rhodamine exhibited biexponential decay with a dominant lifetime of 3.8 ns, making it unsuitable for four-decay sequencing. Contact with the CE gel lengthened the lifetime of NBD-HA-labeled primer from 1.3 to 2.1 ns but did not affect the lifetimes of the other dyes. Lifetime detectability of labeled primers at individual points along an electrophoretic peak in the attomole range.

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.

Interlaboratory comparisons are essential to bringing emerging technologies into biopharmaceutical industry practice and regulatory acceptance. As a result, an international team including 12 laboratories from 10 independent biopharmaceutical companies in the United States and Switzerland was formed to evaluate the precision and robustness of capillary isoelectric focusing (CIEF) to assess the charge heterogeneity of monoclonal antibodies. The different laboratories determined the apparent pI and the relative distribution of the charge isoforms of a representative monoclonal antibody (rMAb) sample using the same CIEF method. Statistical evaluation of the data was performed to determine within and between-laboratory consistencies and outlying information. The apparent pI data generated for each charge variant peak showed very good precision between laboratories with percentage of RSD values of B0.5%. Similarly, the percentage of RSD for the rMAb charge variants percent peak area values are B4.4% across different laboratories with different analysts using different lots of ampholytes and multiple instruments. Taken together, these results validate the appropriate use of CIEF in the biopharmaceutical industry in support of regulatory submissions.

No abstract

Mixtures of dye-labeled, M13-forward DNA primers were separated by capillary gel electrophoresis and detected on-the-fly, using fluorescence lifetime measurements, to evaluate four-decay detection for multiplex DNA sequencing. Three different four-dye systems were used, two that were excited at 488 nm and one that was excited at 514 nm. Each dye-labeled primer was identified on the basis of the lifetime of the conjugated dye using nonlinear least squares or the maximum entropy method to analyze the lifetime data. Overlapping electrophoretic peaks were generated by making multiple injections of mixtures of the dye-labeled primers. The overlapping peaks were resolved by fitting the data to two-, three- or four-component lifetime models used in nonlinear least-squares analysis in which each lifetime component was fixed to the predetermined lifetime of the corresponding dye-labeled primer. In two of the dye systems, the lifetimes of the four dye-labeled primers were sufficiently different to allow peak resolution. In the other dye system, addition of 10% DMSO to the run buffer changed the lifetime of one dye-labeled primer, allowing it to be resolved from another dye-labeled primer with similar lifetime.