Determination of linearized pDNA template in mRNA production process using HPLC.

The recent clinical success of messenger RNA (mRNA) technology in managing the Covid pandemic has triggered an unprecedented innovation in production and analytical technologies for this therapeutic modality. mRNA is produced by enzymatic transcription of plasmid DNA (pDNA) using polymerase in a cell-free process of in vitro transcription. After transcription, the pDNA is considered a process-related impurity and is removed from the mRNA product enzymatically, chromatographically, or by precipitation. Regulatory requirements are currently set at 10 ng of template pDNA per single human dose, which typically ranges between 30 and 100 µg. Here, we report the development of a generic procedure based on enzymatic digestion and chromatographic separation for the determination of residual pDNA in mRNA samples, with a limit of quantification of 2.3 ng and a limit of detection of less than 0.1 ng. The procedure is based on enzymatic degradation of mRNA and anion exchange HPLC separation, followed by quantification of residual pDNA with a chromatographic method that is already widely adopted for pDNA quality analytics. The procedure has been successfully applied for in-process monitoring of three model mRNAs and a self-amplifying RNA (saRNA) and can be considered a generic substitution for qPCR in mRNA in-process control analytical strategy, with added benefits that it is more cost-efficient, faster, and sequence agnostic.

Differences in N-glycosylation of recombinant human coagulation factor VII derived from BHK, CHO, and HEK293 cells.

UNLABELLED: BACKGROUND & METHODS: Recombinant factor VII (rFVII), the precursor molecule for recombinant activated FVII (rFVIIa), is, due to its need for complex post translational modifications, produced in mammalian cells. To evaluate the suitability of a human cell line in order to produce rFVII with post-translational modifications as close as possible to pdFVII, we compared the biochemical properties of rFVII synthesized in human embryonic kidney-derived (HEK)293 cells (HEK293rFVII) with those of rFVII expressed in Chinese hamster ovary (CHO, CHOrFVII) and baby hamster kidney (BHK, BHKrFVII) cells, and also with those of plasma derived FVII (pdFVII), using various analytical methods. rFVII was purified from selected production clones derived from BHK, CHO, and HEK293 cells after stable transfection, and rFVII isolates were analyzed for protein activity, impurities and post-translational modifications. RESULTS & DISCUSSION: The analytical results showed no apparent gross differences between the various FVII proteins, except in their N-linked glycosylation pattern. Most N-glycans found on rFVII produced in HEK293 cells were not detected on rFVII from CHO and BHK cells, or, somewhat unexpectedly, on pdFVII; all other protein features were similar. HEK293rFVII glycans were mainly characterized by a higher structural variety and a lower degree of terminal sialylation, and a high amount of terminal N-acetyl galactosamines (GalNAc). All HEK293rFVII oligosaccharides contained one or more fucoses (Fuc), as well as hybrid and high mannose (Man) structures. CONCLUSIONS: From all rFVII isolates investigated, CHOrFVII contained the highest degree of sialylation and no terminal GalNAc, and CHO cells were therefore assumed to be the best option for the production of rFVII.

Nuclear flow FISH: isolation of cell nuclei improves the determination of telomere lengths.

Understanding telomere biology is of utmost importance for aging and cancer research. An essential tool is the determination of telomere length, which traditionally is done by telomere restriction fragment analysis, a laborious and time consuming method. Therefore, large efforts have been made to establish alternative methods like flow FISH analysis. This method, combining fluorescence in situ hybridization with a telomere specific peptide nucleic acid probe and flow cytometry, measures single cells, is suitable for analysis of non-dividing cells, and can be performed within 24 h. However, when performing flow FISH analysis with normal human kidney epithelial cells, we observed strong variation of autofluorescence at different population doubling levels, especially at replicative senescence, which limits the suitability of this method for the analysis of normal human cells. Since molecules responsible for autofluorescence are predominantly accumulating in the cytoplasm, we decided to isolate the nuclei to perform flow FISH analysis. With this novel nuclear flow FISH (NFF) technique we were able to minimize autofluorescence and its variability, thereby improving the signal-to-noise ratio and consequently, allowing the determination of telomere length during in vitro aging with high accuracy. Moreover, NFF will find broader applications, whenever in situ hybridization signals have to be quantitated.

Establishment of a strategy for the rapid generation of a monoclonal antibody against the human protein SNEV (hNMP200) by flow-cytometric cell sorting.

The screening for antigen-specific hybridoma cells with adequate production rates is still a time-, labour- and money-consuming procedure. A reduction in cell culture testing by specifically selecting those fused cells that produce antibody could therefore make hybridoma technology more attractive, even for small research groups or for newly discovered proteins at an early stage of research. Additional problems, such as the requirement to produce sufficient amounts of the unknown protein at a purity that allows specific immunisation of mice and testing of the resulting hybridoma clones, also need to be overcome. Here we present a new strategy to isolate rapidly and efficiently monoclonal antibodies against new proteins, for which only sequence information at the DNA level is known. The strategy consists of fusion of the protein to a hexa-His-tag to allow easy purification, production in yeast and insect cells to reduce background immunisation with host cell proteins and the selection of IgG-producing hybridoma cells by flow-cytometric cell sorting using the affinity matrix secretion assay technique.

Screening for improved cell performance: selection of subclones with altered production kinetics or improved stability by cell sorting.

One of the major problems in the biotechnology industry is the selection of cell lines well suited for production of biopharmaceutical proteins. Usually, the most important selection criterion is the cell specific production rate. Nevertheless, a good producer cell line should have a number of additional advantageous properties, which allow the cell line to perform well in the type of bioreactor chosen for the process. However, the time and work required to select for high production rates as well as the lack of methods to specifically select for other cellular properties, usually prevents researchers from including such criteria into their screening program. With the Single Cell Secretion Assay it is possible to measure the specific production rates of individual cells by catching secreted product in an artificial matrix applied to the cell surface. Flow cytometric cell sorting then allows selection of rare cells with high production rates, which occur at frequencies as low as 10(-6). By combining this method with culture conditions that bring out a desired cellular property, we were able to isolate subclones with similar production rates, but improved performance from a recombinant Chinese hamster ovary cell line producing a human monoclonal antibody. The two desired cellular properties screened for were a non-growth associated production kinetic and improved stability in the absence of selective pressure.