Traditionally, CE with SDS (CE-SDS) places many restrictions on sample composition. Requirements include low salt content, known initial sample concentration, and a narrow window of final sample concentration. As these restrictions require buffer exchange for many sample types, sample preparation is often tedious and yields poor sample recoveries. To improve capacity and streamline sample preparation, an automated robotic platform was developed using the PhyNexus Micro-Extractor Automated Instrument (MEA) for both the reduced and nonreduced CE-SDS assays. This automated sample preparation normalizes sample concentration, removes salts and other contaminants, and adds the required CE-SDS reagents, essentially eliminating manual steps during sample preparation. Fc-fusion proteins and monoclonal antibodies were used in this work to demonstrate benefits of this approach when compared to the manual method. With optimized conditions, this application has demonstrated decreased analyst “hands on” time and reduced total assay time. Sample recovery greater than 90% can be achieved, regardless of initial composition and concentration of analyte.

Manufacturing-induced disulfide reduction has recently been reported for monoclonal human immunoglobulin gamma (IgG) antibodies, a widely used modality in the biopharmaceutical industry. This effect has been tied to components of the intracellular thioredoxin reduction system that are released upon cell breakage. Here, we describe the effect of process parameters and intrinsic molecule properties on the extent of reduction. Material taken from cell cultures at the end of production displayed large variations in the extent of antibody reduction between different products, including no reduction, when subjected to the same reduction-promoting harvest conditions. Additionally, in a reconstituted model in which process variables could be isolated from product properties, we found that antibody reduction was dependent on the cell line (clone) and cell culture process. A bench-scale model using a thioredoxin/thioredoxin reductase regeneration system revealed that reduction susceptibility depended on not only antibody class but also light chain type; the model further demonstrates that the trend in reducibility was identical to DTT reduction sensitivity following the order IgG1λ > IgG1κ > IgG2λ > IgG2κ. Thus, both product attributes and process parameters contribute to the extent of antibody reduction during production.

High levels of translational errors, both truncation and misincorporation in an Fc‐fusion protein were observed. Here, we demonstrate the impact of several commercially available codon optimization services, and compare to a targeted strategy. Using the targeted strategy, only codons known to have translational errors are modified. For an Fc‐fusion protein expressed in Escherichia coli , the targeted strategy, in combination with appropriate fermentation conditions, virtually eliminated misincorporation (proteins produced with a wrong amino acid sequence), and reduced the level of truncation. The use of full optimization using commercially available strategies reduced the initial errors, but introduced different misincorporations. However, truncation was higher using the targeted strategy than for most of the full optimization strategies. This targeted approach, along with monitoring of translation fidelity and careful attention to fermentation conditions is key to minimizing translational error and ensuring high‐quality expression. These findings should be useful for other biopharmaceutical products, as well as any other transgenic constructs where protein quality is important. Biotechnol. Bioeng. 2012; 109: 2770–2777. © 2012 Wiley Periodicals, Inc.