Identification and evaluation of cell- growth-inhibiting bDtBPP-analogue degradation products from phosphite antioxidants used in polyolefin bioprocessing materials.

The inhibiting effect of the secondary phosphite antioxidant degradation product bis(2,4-di-tert-butylphenyl)phosphate (bDtBPP) on cell growth is well-known. The present study describes structurally related compounds which are likely to be formed from similar widely used phosphite antioxidants used in materials for the manufacturing of single-use (SU) equipment. Two potential candidates of such compounds-3,3',5,5'-tetra-tert-butyl-2,2'-dihydroxybiphenylphosphate (TtBBP) and bis(p-nonylphenyl)phosphate (bNPP)-were identified by chromatography and mass spectrometry followed by synthesis and X-ray structure elucidation. Additionally, the formation of TtBBP was confirmed in an analytical degradation study and its migration from SU bioprocessing material was estimated. The cytotoxicity evaluation by means of cell culture spiking experiments and flow cytometry analysis revealed that' even if cell growth was inhibited by all the compounds to some extent, bDtBPP showed the most severe effect and stoods out from the other two degradants investigated. Graphical abstract.

Quantitative characterization of leachables sinks in biopharmaceutical downstream processing.

This article demonstrates, on a quantitative level, that leachables - potentially accumulated during a biopharmaceutical manufacturing process - will be significantly reduced/removed during four key downstream process steps: cell removal using centrifugation or depth filtration, sterile filtration and virus filtration. Eight common leachables model compounds (LMCs) were spiked into typical feed solutions containing buffer and proteins and were analyzed post-processing in the supernatant or filtrates by HPLC-UV. The clearance rates were calculated as the quotient between the scavenged and initially spiked amount of each leachable. High clearance rates were found for hydrophobic LMCs for all investigated downstream operation steps. It is shown that the removal of cells and cell debris from a culture broth reduces the amount of LMCs almost completely after centrifugation or depth filtration. Also, sterilizing-grade and virus filtration provided a high scavenger effect to most of the LMCs. In contrast, only one hydrophilic acid was not significantly scavenged by the described operations. The possibility to include leachables sinks to a process qualification and risk mitigation concept is explained.

Capillary Electrophoresis in Metabolomics.

Metabolomics is an analytical toolbox to describe (all) low-molecular-weight compounds in a biological system, as cells, tissues, urine, and feces, as well as in serum and plasma. To analyze such complex biological samples, high requirements on the analytical technique are needed due to the high variation in compound physico-chemistry (cholesterol derivatives, amino acids, fatty acids as SCFA, MCFA, or LCFA, or pathway-related metabolites belonging to each individual organism) and concentration dynamic range. All main separation techniques (LC-MS, GC-MS) are applied in routine to metabolomics hyphenated or not to mass spectrometry, and capillary electrophoresis is a powerful high-resolving technique but still underused in this field of complex samples. Metabolomics can be performed in the non-targeted way to gain an overview on metabolite profiles in biological samples. Targeted metabolomics is applied to analyze quantitatively pre-selected metabolites. This chapter reviews the use of capillary electrophoresis in the field of metabolomics and exemplifies solutions in metabolite profiling and analysis in urine and plasma.

Optimizing a ultrahigh pressure liquid chromatography-time of flight-mass spectrometry approach using a novel sub-2µm core-shell particle for in depth lipidomic profiling of Caenorhabditis elegans.

Lipid profiling or lipidomics is currently applied in many different research fields. It refers to the global analysis of a samples lipid content using different analytical chemistry methods, with mass spectrometry as the mostly employed technology. We developed a comprehensive in-depth analysis method for the lipidome of the soil-dwelling nematode Caenorhabitis elegans, a widely used model organism. Four different columns were compared with a generic gradient and a novel sub-2-µm core-shell column, Waters Cortecs C18, showed superior performance in case of chromatographic peak characteristics, e.g. plate numbers and number of detected lipid features. Retention time deviation was generally less than 1% within one column and below 5% for columns from different batches. Intensity variation was lower than 30% for most detected features. Improved chromatographic separation showed enhanced resolution for isomeric lipids and allowed collection of highly detailed MS/MS spectra for lipid identification. In total 1304 lipid features were detected in positive ionization mode and 265 in negative mode. Lipids from different classes were annotated and MS/MS spectra obtained by data dependent fragmentation were used for identification purposes.