4 research outputs found
Assessment of the percentage of full recombinant adeno-associated virus particles in a gene therapy drug using CryoTEM
In spite of continuous development of gene therapy vectors with thousands of drug candidates in clinical drug trials there are only a small number approved on the market today stressing the need to have characterization methods to assist in the validation of the drug development process. The level of packaging of the vector capsids appears to play a critical role in immunogenicity, hence an objective quantitative method assessing the content of particles containing a genome is an essential quality measurement. As transmission electron microscopy (TEM) allows direct visualization of the particles present in a specimen, it naturally seems as the most intuitive method of choice for characterizing recombinant adeno-associated virus (rAAV) particle packaging. Negative stain TEM (nsTEM) is an established characterization method for analysing the packaging of viral vectors. It has however shown limitations in terms of reliability. To overcome this drawback, we propose an analytical method based on CryoTEM that unambiguously and robustly determines the percentage of filled particles in an rAAV sample. In addition, we show that at a fixed number of vector particles the portion of filled particles correlates well with the potency of the drug. The method has been validated according to the ICH Q2 (R1) guidelines and the components investigated during the validation are presented in this study. The reliability of nsTEM as a method for the assessment of filled particles is also investigated along with a discussion about the origin of the observed variability of this method
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A metabolic CRISPR-Cas9 screen in Chinese hamster ovary cells identifies glutamine-sensitive genes
Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism
Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering
Hackl M, Jakobi T, Blom J, et al. Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering. Journal of Biotechnology. 2011;153(1-2):62-75.Chinese hamster ovary (CHO) cells are the predominant cell factory for the production of recombinant therapeutic proteins. Nevertheless, the lack in publicly available sequence information is severely limiting advances in CHO cell biology, including the exploration of microRNAs (miRNA) as tools for CHO cell characterization and engineering. In an effort to identify and annotate both conserved and novel CHO miRNAs in the absence of a Chinese hamster genome, we deep-sequenced small RNA fractions of 6 biotechnologically relevant cell lines and mapped the resulting reads to an artificial reference sequence consisting of all known miRNA hairpins. Read alignment patterns and read count ratios of 5' and 3' mature miRNAs were obtained and used for an independent classification into miR/miR* and 5p/3p miRNA pairs and discrimination of miRNAs from other non-coding RNAs, resulting in the annotation of 387 mature CHO miRNAs. The quantitative content of next-generation sequencing data was analyzed and confirmed using qPCR, to find that miRNAs are markers of cell status. Finally, cDNA sequencing of 26 validated targets of miR-17-92 suggests conserved functions for miRNAs in CHO cells, which together with the now publicly available sequence information sets the stage for developing novel RNAi tools for CHO cell engineering