Large-Scale Assessment of Extractables and Leachables in Single-Use Bags for Biomanufacturing.

Single-use technologies (SUTs) are widely used during biopharmaceutical manufacture as disposable bioreactors or media and buffer storage bags. Despite their advantages, the risk of release of extractable and leachable (E&Ls) substances is considered an important drawback in adopting disposables in the biomanufacturing process. E&Ls may detrimentally affect cell viability or productivity or may persist during purification and present a risk to the patient if remaining in the final drug product. In this study, 34 plastic films from single-use bags (SUBs) for cell cultivation were extracted with selected solvents that represent reasonable worst-case conditions for most typical biomanufacturing applications. SUBs were incubated at small-scale under accelerated-aging conditions that represented standard operational conditions of use. Leachables analysis was performed following dispersive liquid-liquid microextraction (DLLME) for analyte preconcentration and removal of matrix interference. Resulting extracts were characterized by GC-headspace for volatiles, high resolution GC-Orbitrap-MS/MS for semivolatiles, high resolution LC-Orbitrap-MS/MS for nonvolatiles, and ICP-MS for trace elemental analysis. Multivariate statistical analysis of the analytical data revealed significant correlations between the type and concentration of compounds and bags features including brand, manufacturing date and polymer type. The analytical data demonstrates that, over recent years, the nature of E&Ls has been altered due to the implementation of manufacturing changes and new types of polymers and may change further with the future advent of regulations that will limit or ban the use of certain raw materials and additives. The broad E&L database generated herein facilitates toxicological assessments from a biomanufacturing standpoint and provides practical guidelines for confident determination of E&Ls to enable screening and elimination of nonsatisfactory films for single use bioprocessing.

Assessing sample extraction efficiencies for the analysis of complex unresolved mixtures of organic pollutants: A comprehensive non-target approach.

The comprehensive extraction recovery assessment of organic analytes from complex samples such as oil field produced water (PW) is a challenging task. A targeted approach is usually used for recovery and determination of compounds in these types of analysis. Here we suggest a more comprehensive and less biased approach for the extraction recovery assessment of complex samples. This method combines conventional targeted analysis with a non-targeted approach to evaluate the extraction recovery of complex mixtures. Three generic extraction methods: liquid-liquid extraction (Lq), and solid phase extraction using HLB cartridges (HLB), and the combination of ENV+ and C8 (ENV) cartridges, were selected for evaluation. PW was divided into three parts: non-spiked, spiked level 1, and spiked level 2 for analysis. The spiked samples were used for targeted evaluation of extraction recoveries of 65 added target analytes comprising alkanes, phenols, and polycyclic aromatic hydrocarbons, producing absolute recoveries. The non-spiked samples were used for the non-targeted approach, which used a combination of the F-ratio method and apex detection algorithm. Targeted analysis showed that the use of ENV cartridges and the Lq method performed better than use of HLB cartridges, producing absolute recoveries of 53.1 ± 15.2 for ENV and 46.8 ± 13.2 for Lq versus 19.7 ± 6.7 for HLB. These two methods appeared to produce statistically similar results for recoveries of analytes, whereas they were both different from the produced recoveries via the HLB method. The non-targeted approach captured unique features that were specific to each extraction method. This approach generated 26 unique features (mass spectral ions), which were significantly different between samples and were relevant in differentiating each extract from each method. Using a combination of these targeted and non-targeted methods we evaluated the extraction recoveries of the three extraction methods for analysis of PW.

Problems in the application of the three-step BCR sequential extraction to low amounts of sediments: an alternative validated route.

Poor recoveries are obtained if the BCR three-step sequential extraction is applied to 100 mg specimens rather than to 1 g. It is observed that analytes are lost during each phase separation which is carried out via centrifugation and can be hardly quantitatively performed on 100 mg sediment specimens. An alternative procedure, which is carried out on a single empty SPE column and involves separation by filtration, is developed to solve this problem. The proposed method is validated on 100 mg samples of certified sediment (BCR-701), but could be potentially used for even lower sediment specimens. Problems related to pH stability during step 2 and its influence on recoveries is also reported.

Application of double-spike isotope dilution for the accurate determination of Cr(III), Cr(VI) and total Cr in yeast.

A method is presented for the simultaneous determination of Cr(III) and Cr(VI) in yeast using species-specific double-spike isotope dilution (SSDSID) with anion-exchange liquid chromatography (LC) separation and sector field inductively coupled plasma mass spectrometric (SF-ICP-MS) detection. Total Cr is quantitated using ID SF-ICP-MS. Samples were digested on a hot plate at 95+/-2 degrees C for 6 h in an alkaline solution of 0.5 M NaOH and 0.28 M Na2CO3 for the determination of Cr(III) and Cr(VI), whereas microwave-assisted decomposition with HNO3 and H2O2 was used for the determination of total Cr. Concentrations of 2,014+/-16, 1,952+/-103 and 76+/-48 mg kg-1 (one standard deviation, n=4, 3, 3), respectively were obtained for total Cr, Cr(III) and Cr(VI) in the yeast sample. Significant oxidation of Cr(III) to Cr(VI) (24.2+/-7.6% Cr(III) oxidized, n=3) and reduction of Cr(VI) to Cr(III) (37.6+/-6.5% Cr(VI) reduced, n=3) occurred during alkaline extraction and subsequent chromatographic separation at pH 7. Despite this significant bidirectional redox transformation, quantitative recoveries for both Cr(III) and Cr(VI) were achieved using the SSDSID method. In addition, mass balance between total Cr and the sum of Cr(III) and Cr(VI) concentrations was achieved. Method detection limits of 0.3, 2 and 30 mg kg-1 were obtained for total Cr, Cr(VI) and Cr(III), respectively, based on a 0.2-g sub-sample.