MAGE A3, which belongs to the family of cancer-testis antigens, is an attractive target for adoptive therapy given its reactivation in various tumors and limited expression in normal tissues. We developed an affinity-enhanced T cell receptor (TCR) directed to a human leukocyte antigen (HLA)–A*01–restricted MAGE A3 antigen (EVDPIGHLY) for use in adoptive therapy. Extensive preclinical investigations revealed no off-target antigen recognition concerns; nonetheless, administration to patients of T cells expressing the affinity-enhanced MAGE A3 TCR resulted in a serious adverse event (SAE) and fatal toxicity against cardiac tissue. We present a description of the preclinical in vitro functional analysis of the MAGE A3 TCR, which failed to reveal any evidence of off-target activity, and a full analysis of the post-SAE in vitro investigations, which reveal cross-recognition of an off-target peptide. Using an amino acid scanning approach, a peptide from the muscle protein Titin (ESDPIVAQY) was identified as an alternative target for the MAGE A3 TCR and the most likely cause of in vivo toxicity. These results demonstrate that affinity-enhanced TCRs have considerable effector functions in vivo and highlight the potential safety concerns for TCR-engineered T cells. Strategies such as peptide scanning and the use of more complex cell cultures are recommended in preclinical studies to mitigate the risk of off-target toxicity in future clinical investigations.

To detect sub-ppb levels of the antibiotic chloramphenicol in honey matrix, a convenient method of extraction and measurement using liquid chromatography with detection by tandem mass spectrometry (LC/MS/MS) was developed. Honey samples fortified with chloramphenicol and isotopically labeled chloramphenicol were extracted using diatomaceous-based supported liquid-liquid extraction cartridges to generate a standard calibration curve. Four MS/MS transitions were used for quantification and four other transitions for confirmation of chloramphenicol. The limit of detection for chloramphenicol was 0.05 ng/g and the lower limit of quantification was 0.1 ng/g. Several commercial honey samples were analyzed for chloramphenicol content using this method.

Glycoforms of glargine expressed in Pichia pastoris were isolated by high-performance liquid chromatography and analyzed by a series of chemical and mass spectrometric methods for the identification of various glycoforms, glycosylation position, nature and structure of glycans. Reduction and alkylation, peptide mapping techniques were used to decipher the amino acid site at which glycosylation had taken place. Chemical methods were coupled with mass spectrometry techniques such as electrospray ionization and matrix-assisted laser desorption/ionization for identification of the glycosylation site.

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A strategy is developed for the identification of isocephalomannine in the presence of alkali metal ion adducts and other cephalomannine isomers in a paclitaxel active pharmaceutical ingredient. Intact molecular ion analyses and a sub-structural study have been performed for the differentiation of isocephalomannine (2-debenzoylpaclitaxel-2-pentenoate) from cephalomannine and 7-epi-cephalomannine. A comparative study of the cephalomannine isomers was carried out using molecular ions (MS) and fragmentation patterns (MS/MS) for sub-structural analysis. An attempt has been made to identify isocephalomannine in Cremophor(R) EL formulations.