Long, accurate reads are an important factor for high-throughput de novo DNA sequencing. In previous work from this laboratory, a separation matrix of high-weight-average molecular mass (HMM) linear polyacrylamide (LPA) at a concentration of 2% (w/w) was used to separate 1000 bases of DNA sequence in 80 min with an accuracy close to 97% (Carrilho, E.; et al. Anal. Chem. 1996, 68, 3305-3313). In the present work, significantly improved speed and sequencing accuracy have been achieved by further optimization of factors affecting electrophoretic separation and data processing. A replaceable matrix containing a mixture of 2.0% (w/w) HMM (9 MDa) and 0.5% (w/w) low-weight-average molecular mass (50 kDa) LPA was employed to enhance the separation of DNA sequencing fragments in CE. Experimental conditions, such as electric field strength and column temperature, as well as internal diameter of the capillary column, have been optimized for this mixed separation matrix. Under these conditions, in combination with energy-transfer (BigDye) dye-labeled primers for high signal-to-noise ratio and a newly developed expert system for base calling, the electrophoretic separation of 1000 DNA sequencing fragments of both standard (M13mp18) and cloned single-stranded templates from human chromosome 17 could be routinely achieved in less than 55 min, with a base-calling accuracy between 98 and 99%. Identical read length, accuracy, and migration time were achieved in more than 300 consecutive runs in a single column.

We present here a method for the rapid determination of the intact mass of noncovalently associated antibody heavy chains (HC) and light chains (LC) which result from the attachment of drug conjugates to interchain cysteine residues. By analyzing the antibody-drug conjugate (ADC) using native desalting conditions, we maintain the intact bivalent structure of the ADC, which ordinarily would decompose as a consequence of denaturing chromatographic conditions typically used for liquid chromatographic-mass spectrometric (LC-MS) analysis. The mass of the desalted ADC is subsequently determined using standard desolvation and ionization conditions. Methods presented previously in the literature for analyzing interchain cysteinyl-linked ADCs are either not amenable to online mass spectrometry or result in the denaturing dissociation of conjugated HC and LC during chromatographic separation and subsequent mass measurement. We have avoided this outcome with our method and have successfully and routinely obtained intact mass measurement of IgG1 mAbs conjugated with maleimidocaproyl-monomethyl Auristatin F (mcMMAF) and valine-citrulline-monomethyl Auristatin E (vcMMAE) at interchain cysteine residues. Our results thus represent the first reported direct measurement of the intact mass of an ADC conjugated at interchain cysteine residues.

Glycan characterization of therapeutic proteins is of utmost importance due to the role of carbohydrates in protein stability, half-life, efficacy and mechanism of action. The primary assay for characterization and lot release of N-linked glycans on glycoprotein products at Genentech, Inc., is a capillary electrophoresis (CE) based assay, wherein PNGase F-released, APTS-labeled glycans are separated by CE with laser induced fluorescence (LIF) detection. With the growing number of new molecular entities in the pipeline, a fast and direct characterization approach is of increasing importance. This paper describes the development of CE-MS technology with on-line LIF detection that allows identification of major and minor glycan species (1-5% of total glycans) by providing accurate mass information. Data is presented for therapeutic rMAbs which presented previously unidentified, minor peaks during routine CE-LIF analysis. CE-LIF-MS was then used to provide accurate mass on these species, identifying CE peaks corresponding to sialylated (G1 + NANA, G2 + NANA), afucosylated (G0-GlcNAc-fucose) and low-level isomers of major APTS-labeled glycans G0, G1, G1' and G2.

In previous work, a capillary electrophoresis sodium dodecyl sulfate (CE-SDS) method using precolumn labeling and laser-induced fluorescence (LIF) detection was developed at Genentech Inc. as part of the control system for the quality control release of a recombinant monoclonal antibody (rMAb) (Hunt, G.; Nashabeh, W. Anal. Chem. 1999, 71, 2390-2397.). In the current work, a generic and quantitative CE-SDS assay with LIF detection of rMAbs with improved accuracy and precision is described. The implementation of an alkylating step with iodoacetamide and optimization of the incubation temperature and time, in the presence of SDS, greatly decrease any thermally induced fragmentation of nonreduced labeled rMAb samples. In addition, a quantitative study of the effects of sample buffer pH on rMAb fragmentation is also presented. Furthermore, the performance of alternative CE-SDS polymer solutions and instrumentation for quantitative analysis of rMAbs is shown in this article. The validation of this method, under the guidelines of the International Committee on Harmonization (ICH), demonstrates that the assay quantitatively determines the consistency of rMAb manufacture as it relates to size heterogeneity and product purity.

A method for the cleanup of Sanger DNA sequencing reaction products for capillary electrophoresis analysis with replaceable polymer solutions has been developed. A poly(ether sulfone) ultrafiltration membrane pretreated with linear polyacrylamide was first used to remove template DNA from the sequencing samples. Then, gel filtration in a spin column format (two columns per sample) was employed to decrease the concentration of salts below 10 microM in the sample solution. The method was very reproducible and increased the injected amount of the sequencing fragments 10-50-fold compared to traditional cleanup protocols. Using M13mp18 as template, the resulting cleaned-up single DNA sequencing fragments could routinely be separated to more than 1000 bases with a base-calling accuracy of at least 99% for 800 bases. The method is simple and universal and can be easily automated. In the following paper, a systematic study to determine quantitatively the effects of the sample solution components such as high-mobility ions (e.g., chloride and dideoxynucleotides) and template DNA on the injected amount and separation efficiency of the sequencing fragments is presented.