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To characterize combinatorial chemical libraries of small drug compounds, an automated column switching system incorporating an immunoaffinity extraction (IAE) column and two reversed-phase HPLC columns was coupled to a triple-quadrupole mass spectrometer. A Protein G column and antibodies to benzodiazepines were used to screen library components. A pH change in the mobile phase eluted the benzodiazepine - antibody complexes onto a C-18 restricted access media (RAM) column, thereby separating the selected benzodiazepines from the antibody. In a final step, backflushing the RAM column eluted the benzodiazepines onto a C-8 analytical reversed-phase column for separation before detection and preliminary structural characterization using ion spray mass spectrometry (MS) and tandem mass spectrometry (MS/MS). A known 19-component library and an unknown 20-component library were analyzed. Full-scan IAE/LC/LC/MS and IAE/LC/LC/MS/MS chromatograms suggested the feasibility of this combination of techniques, although the antibodies used were not highly specific. Inspection of MS/MS spectra of components in the unknown library compared to the MS/MS spectrum of a known standard (chlordiazepoxide) identified a subclass of benzodiazepines. Product ions of the known standard and an unknown benzodiazepine were successively captured and fragmented (M[S.sup.n] experiments) using an ion-trap mass spectrometer off-line, which confirmed that the unknown was an analogue of chlordiazepoxide.

The DNA binding protein Tus contains a proline-repeat segment from residues 223-243. Its structure has been observed to be similar to that of a 20 amino acid peptide, acetyl-PQNAKLKIKRPVKVQPIARRVY-amide (TPPI). In order to determine the structure of Tus, the structure of TPPI was determined in 50% trifluroethanol by nuclear magnetic resonance experiments. The results revealed a novel helical structure having 5.6 residues per turn. This structure is amphiphilic with one side consisting of a proline pipe helix.

Combines immunoaffinity extraction and two-dimensional, reversed-phase chromatography with ion spray MS and tandem MS to separate, detect, and characterize known 19-component and unknown 20-component benzodiazepine libraries. Binding of benzodiazepines by an antibody; Flexibility of the multicolumn system.

The photodegradation of a human IgG1 monoclonal antibody has been examined in a high concentration (100 mg/mL) liquid formulation. It was observed that a yellowish color is generated when the formulation is exposed to intense and prolonged light exposure, and this discoloration occurs along with a loss in bioactivity. Extensive analytical characterization was performed to determine light induced degradation pathways that occur during exposure to intense light of ICH photodegradation conditions. It has been shown that the monoclonal antibody undergoes a combination of physical and chemical reactions under these conditions, including covalent aggregate formation, fragmentation at the hinge region, oxidation of Trp, His, and Met residues, and deamidation of Asn residues. Oxidation of Trp 94 and deamidation of Asn 93, located in the light chain CDR region, correlates with loss of bioactivity under these conditions. A series of formulation experiments were performed to elucidate the impact of the extent of light exposure, oxygen, protein concentration, and solution pH on the photostability of the formulation. Results demonstrated that photodegradation of the IgG, after intensive light exposure, can be prevented by proper secondary packaging. In addition, it is also shown that a high concentration, liquid dosage form of a human monoclonal antibody is stable upon exposure to the ambient light conditions encountered during routine manufacturing, long-term storage, and administration with proper design of formulation conditions, the primary container as well as the secondary package. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3117–3130, 2009 [ABSTRACT FROM AUTHOR]