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      Development of a high‐throughput scale‐down model in Ambr® 250 HT for plasmid DNA fermentation processes

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          Abstract

          Recent advances in messenger ribonucleic acid (mRNA) vaccines and gene therapy vectors have increased the need for rapid plasmid DNA (pDNA) screening and production within the biopharmaceutical industry. High‐throughput (HT) fermentor systems, such as the Ambr® 250 HT, can significantly accelerate process development timelines of pDNA upstream processes compared to traditional bench‐scale glass fermentors or small‐scale steam‐in‐place (SIP) fermentors. However, such scale‐down models must be qualified to ensure that they are representative of the larger scale process similar to traditional small‐scale models. In the current study, we developed a representative scale‐down model of a Biostat® D‐DCU 30 L pDNA fermentation process in Ambr® 250 HT fermentors using three cell lines producing three different constructs. The Ambr scale‐down model provided comparable process performance and pDNA quality as the 30 L SIP fermentation process. In addition, we demonstrated the predictive value of the Ambr model by two‐way qualification, first by accurately reproducing the prior trends observed in a 30 L process, followed by predicting new process trends that were then successfully reproduced in the 30 L process. The representative and predictive scale‐down Ambr model developed in this study would enable a faster and more efficient approach to strain/clone/host‐cell screening, pDNA process development and characterization studies, process scale‐up studies, and manufacturing support.

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          Most cited references35

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          Is Open Access

          Improving accuracy of cell and chromophore concentration measurements using optical density

          Background UV–vis spectrophotometric optical density (OD) is the most commonly-used technique for estimating chromophore formation and cell concentration in liquid culture. OD wavelength is often chosen with little thought given to its effect on the quality of the measurement. Analysis of the contributions of absorption and scattering to the measured optical density provides a basis for understanding variability among spectrophotometers and enables a quantitative evaluation of the applicability of the Beer-Lambert law. This provides a rational approach for improving the accuracy of OD measurements used as a proxy for direct dry weight (DW), cell count, and pigment levels. Results For pigmented organisms, the choice of OD wavelength presents a tradeoff between the robustness and the sensitivity of the measurement. The OD at a robust wavelength is primarily the result of light scattering and does not vary with culture conditions; whereas, the OD at a sensitive wavelength is additionally dependent on light absorption by the organism’s pigments. Suitably robust and sensitive wavelengths are identified for a wide range of organisms by comparing their spectra to the true absorption spectra of dyes. The relative scattering contribution can be reduced either by measurement at higher OD, or by the addition of bovine serum albumin. Reduction of scattering or correlation with off-peak light attenuation provides for more accurate assessment of chromophore levels within cells. Conversion factors between DW, OD, and colony-forming unit density are tabulated for 17 diverse organisms to illustrate the scope of variability of these correlations. Finally, an inexpensive short pathlength LED-based flow cell is demonstrated for the online monitoring of growth in a bioreactor at culture concentrations greater than 5 grams dry weight per liter which would otherwise require off-line dilutions to obtain non-saturated OD measurements. Conclusions OD is most accurate as a time-saving proxy measurement for biomass concentration when light attenuation is dominated by scattering. However, the applicability of OD-based correlations is highly dependent on the measurement specifications (spectrophotometer model and wavelength) and culture conditions (media type; growth stage; culture stress; cell/colony geometry; presence and concentration of secreted compounds). These variations highlight the importance of treating literature conversion factors as rough approximations as opposed to concrete constants. There is an opportunity to optimize measurements of cell pigment levels by considering scattering and absorption-dependent wavelengths of the OD spectrum.
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            DNA polymorphism detectable by restriction endonucleases.

            M Nei, F Tajima (1981)
            Data on DNA polymorphisms detected by restriction endonucleases are rapidly accumulating. With the aim of analyzing these data, several different measures of nucleon (DNA segment) diversity within and between populations are proposed, and statistical methods for estimating these quantities are developed. These statistical methods are applicable to both nuclear and nonnuclear DNAs. When evolutionary change of nucleons occurs mainly by mutation and genetic drift, all the measures can be expressed in terms of the product of mutation rate per nucleon and effective population size. A method for estimating nucleotide diversity from nucleon diversity is also presented under certain assumptions. It is shown that DNA divergence between two populations can be studied either by the average number of restriction site differences or by the average number of nucleotide differences. In either case, a large number of different restriction enzymes should be used for studying phylogenetic relationships among related organisms, since the effect of stochastic factors on these quantities is very large. The statistical methods developed have been applied to data of Shah and Langley on mitochondrial (mt)DNA from Drosophila melanogaster, simulans and virilis. This application has suggested that the evolutionary change of mtDNA in higher animals occurs mainly by nucleotide substitution rather than by deletion and insertion. The evolutionary distances among the three species have also been estimated.
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              Oxygen uptake rate in microbial processes: An overview

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                Author and article information

                Journal
                Biotechnology Progress
                Biotechnology Progress
                Wiley
                8756-7938
                1520-6033
                March 18 2024
                Affiliations
                [1 ] BioProcess Research & Development Pfizer Inc. Chesterfield Missouri USA
                Article
                10.1002/btpr.3458
                3ff6649e-5466-48fb-bd60-27c6207dd38b
                © 2024

                http://onlinelibrary.wiley.com/termsAndConditions#vor

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