Within the biopharmaceutical industry, recombinant plasmid DNA is used both as a raw material (e.g. in lentiviral and AAV vector production) as well as an active ingredient (e.g. in DNA vaccines). Consequently, many analytical laboratories are routinely involved with plasmid DNA topoisoform qualitative analysis and quantification. In order to reliably determine plasmid topology, one must ensure that the methodology employed can reliably, precisely and accurately measure qualitatively and quantitatively all topological isoforms. Presented here are an anion-exchange high-performance liquid chromatography (AEC) and an agarose gel electrophoresis (AGE)-based method developed for this purpose. The strategies undertaken to overcome the respective typical problems of limited linear range of quantitation (for AGE) and isoform resolution (for AEC) are described. Also presented is a subsequent direct comparison (for assay precision/accuracy) of these two methods, as well as a package of species characterization [by chloroquine-AGE, enzymatic digestion, multi-angle laser light-scattering (MALLS) and electron microscopy] undertaken to confirm the identity of a minor supercoiled dimeric concatamer observed by both approaches.

BACKGROUNDA current challenge in bioprocessing is the ability to analyse critical quality attributes such as aggregation without prior purification. This study evaluated the use of fluorescent dyes (Bis‐ANS, SYPRO Orange, Thioflavin T and ProteoStat) to characterise mAb aggregates in Chinese hamster ovary clarified cultures.RESULTSThe null and mAb culture supernatants showed an increase in fluorescence intensity over the duration of the culture. The null cultures on day 14 saw a rapid increase in fluorescence intensity; day 10 to day 14, Bis‐ANS and Thioflavin T had average increases of 21% and 48%, respectively, whereas ProteoStat and SYPRO Orange showed an average increase of 60%. Higher fluorescence intensity on day 14 with the null cultures, also correlated with loss of viability.CONCLUSIONFluorescent dyes are not a specific indicator of mAb aggregation, but rather an indicator of overall protein aggregation or high molecular weight species. SYPRO Orange was more sensitive at detecting very large molecular weight species and ProteoStat seemed better suited to smaller aggregates. Although the assay cannot be used to measure mAb aggregates in cell culture, it could be used to aid cell line selection in maximising viabilities and minimising the amount of aggregates. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Human cytochrome P450 2B6 ( CYP2B6 ) metabolizes the prodrug cyclophosphamide ( CPA ) to produce phosphoramide mustard that cross -links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 ( designated``MetXia -P450'' ) and used it to transduce the human tumor cell lines HT29 and T47D. MetXia -P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide -treated cells transduced with MetXia -P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3 -D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed -enzyme prodrug therapy ( GDEPT ). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450 -based GDEPT strategy mediated by a direct retroviral gene transfer in vivo. Cancer Gene Therapy ( 2001 ) 8, 473 ± 482

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