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The productivity of mammalian cell culture expression systems is critically important to file production of biopharmaceuticals. In rids study, a high-producing Chinese hamster ovary cell culture which was transfected with the apoptosis inhibitor Bcl-[X.sub.L] gene was compared to a low-producing control that was not transfected. Shotgun proteomics was used to compare the high and low-producing fed-batch cell cultures at different growth time points. The goals of this study were twofold; it would be of value to find a biomaker that could predict cell lines with higher growth efficiency and to gain mechanistic insights into the effects of the introduction of a foreign gene flint is known to have growth regulating properties in human cells. A total of 392 proteins were identified in lifts study, and 32 of these proteins were determined to be differentially expressed. In the high-producing cell culture, several proteins related to protein metabolism were upregulated, such as eukaryotic translation initiation factor 3 and ribosome 40S. In addition, several intermediate filament proteins such as vimentin and annexin, as well as histone H1.2 and H2A, were down-regulated in the high producer. The expression of these proteins may be indicative of cellular productivity. A growth inhibitor, galectin-1, was downregulated in the high producer, which may be linked to the expression of Bcl-[X.sub.L]. The molecular chaperone BiP was upregulated significantly in the high producer and may indicate an unfolded protein response due to endoplasmic reticulum (ER) stress. Several proteins involved in regulation of the cell cycle such as RACK1 and GTPase Ran were found to be differentially expressed, which may be due to a differentially controlled cell cycle between low- and high-producing cell cultures.

Unlike small-molecule drugs, the conformational properties of protein biopharmaceuticals in solution are influenced by a variety of factors that are not solely defined by their covalent chemical structure. Since the conformation (or higher order structure) of a protein is a major modulator of its biological activity, the ability to detect changes in both the higher order structure and conformational dynamics of a protein, induced by an array of extrinsic factors, is of central importance in producing, purifying, and formulating a commercial biopharmaceutical with consistent therapeutic properties. In this study we demonstrate that two complementary mass spectrometry-based approaches (analysis of ionic charge-state distribution and hydrogen/deuterium exchange) can be a potent tool in monitoring conformational changes in protein biopharmaceuticals. The utility of these approaches is demonstrated by detecting and characterizing conformational changes in the biopharmaceutical product interferon [beta]-1a (IFN-[beta]-1a). The protein degradation process was modeled by inducing a single chemical modification of IFN-[beta]1a (alkylation of its only free cysteine residue with N-ethylmaleimide), which causes significant reduction in its antiviral activity. Analysis of IFN-[beta]1a ionic charge-state distributions unequivocally reveals a significant decrease of conformational stability in the degraded protein, while hydrogen/deuterium exchange measurements provide a clear indication that the higher order structure is affected well beyond the covalent modification site. Importantly, neither technique required that the location or indeed the nature of the chemical modification be known prior to or elucidated in the process of the analysis. In contrast, application of the standard armamentarium of biophysical tools, which are commonly employed for quality control of protein pharmaceuticals, met with very limited success in detection and characterization of conformational changes in the modified IFN-[beta]1a. This work highlights the role mass spectrometry can and should play in the biopharmaceutical industry beyond the presently assigned task of primary structure analysis.

A competitive immunoassay for cortisol based on capillary electrophoresis (CE) and laser-induced fluorescence is described. The work involved the production of assay reagents and the development of separation conditions allowing for routine analysis of serum samples. Fluorescein-labeled cortisol was synthesized and purified. Fab fragments were produced from mouse monoclonal anti-cortisol antibody and purified using a POROS cation exchange chromatography column. After incubation of these reagents with serum, free and bound labeled antigen were separated by CE with high reproducibility. No prior sample cleanup of the serum samples was necessary. Serum calibration curves were established and used for the quantitation of cortisol in serum. The results demonstrate feasibility for a cortisol assay based on CE operating directly on serum samples.

Gradient ion-exchange chromatography (IEC) was interfaced with a low-angle laser light scattering photometer (LALLS) and ultraviolet (UV) and refractive index (RI) detectors connected in series for on-line determination of the differential refractive index (dn/dc) of proteins and eventually their molecular weights ([M.sub.w]'s). Interfacing of gradient HPLC with a RI detector was made possible by using two isorefractive buffers, which helped generate stable baselines for the LALLS and RI detectors. An optically modified, laser based RI detector was used for determination of dn/dc. On-line determinations of dn/dc required smaller amounts of sample compared to off-line determinations (3--4 mg vs 200 mg). Also, dn/dc could be measured by making one repetitive injection of the sample, if the molar absorptivity of the sample was known. dn/dc values determined on-line were in close agreement with those determined off-line. Additionally, percent recovery of the sample was calculated on-line, and this provided true concentration detected at the UV detector, which was then used for determination of [M.sub.W]., The interfacing of gradient ion-exchange chromatography with a low-angle laser light scattering photometer and ultraviolet and refractive index detectors is discussed. Two isorefractive buffers were used to enable the the interface. Change in the refractive index of the solution was determined by an optically modified laser based refractive index detector. The efficacy of the determinations was exhibited.