![]() Finally, a simulation run accounting for variability expected in larger-scale production was executed to provide a prediction model. The method involved five PPs at the Escherichia coli DH5α fermentation step to investigate the impact on CQAs, which is supercoiled content, and performance attributes (PAs), which are volumetric yield and specific yield. ![]() In this study, we employed DSD method to characterize the process of porcine DNA vaccine production. Requiring a minimum number of runs as few as 2m + 1 or 2m + 3, for m variable factors when m is even and odd number, respectively.ĭSD has become widely used in diverse applications including paint manufacturing, green energy, and biotechnology. ![]() This design method contains several desirable properties ( Jones and Nachtsheim, 2011 Xiao et al., 2012 Erler et al., 2013 Nguyen and Stylianou, 2013 Tai et al., 2015) An alternative one-step design method named definitive-screening design (DSD) was introduced by Jones and Nachtsheim. This requires many experimental runs to gain sufficient data for further analysis, resulting in taking more times and resources ( Abu-Absi et al., 2010 Erler et al., 2013). ![]() Conventional DoE scheme is first via screening designs to determine significant main effects followed by response-surface models to justify the design space. By performing Design of Experiments (DoE), it is allowed to use a minimum number of experimental runs where all experimental parameters studied are varied simultaneously to obtain sufficient information ( Mandenius and Brundin, 2008 Montgomery, 2017). This process characterization applies Quality by Design principle to help establish a rational and cost-effective approach on process design and optimization ( ICH, 2009 McCurdy, 2011). Therefore, it is indispensable to demonstrate process robustness with identified critical process parameters (PPs) and critical quality attributes (CQAs) during the process development phase. The US Food and Drug Administration recommends that at least 80% supercoiled content shall be obtained as this has superior biological activity as compared to other plasmid forms ( Urthaler et al., 2005 U.S. The production process for DNA vaccine consists of several steps with aims of achieving high quantity and quality to meet product specifications. There are also a number of DNA vaccines undertaking clinical studies for human uses including GX188E and VGX-3100 for human papillomavirus ( Cheng et al., 2018), a prime/boost of DNA.Mel3 with MVA.Mel3 for advanced metastatic melanoma cancer treatment ( Dangoor et al., 2010) and INO-4800 for COVID-19 ( Smith et al., 2020). To date, there are six DNA vaccines approved for veterinary applications, which include preventive vaccines for West Nile virus infection in horses ( Davidson et al., 2005), hematopoietic necrosis virus infection in salmon ( Garver et al., 2005), therapeutic cancer vaccine for dogs ( Bergman et al., 2006), a growth hormone gene therapy to increase litter survival in breeding pig sows ( Person et al., 2008), pancreas disease infection in Atlantic salmon ( EMA, 2017), and H5N1 in chicken with conditional license ( Agrilabs, 2017). DNA vaccination is genetically engineered DNA containing a transgene that expresses a specific antigen into cells or tissues ( Ingolotti et al., 2010). Gene immunization including DNA vaccines has become an attractive approach for vaccination because of its well-documented safety unlike live attenuated viral vaccine, the absence of specific immune responses to the plasmid, and its absence of genetic integration ( Robinson, 2000 Wahren and Liu, 2014). Using the definitive-screening design, there were 16 runs, including 3 additional center points to create the predictive model, which then was used to simulate the operational ranges for capability analysis. The parameters of interest were temperature, pH, dissolved oxygen, cultivation time, and feed rate. Design of Experiments (DoE) was employed to determine process parameters that have impacts on a critical quality attribute of the product, which is the active form of plasmid DNA referred as supercoiled plasmid DNA content, as well as the performance attributes, which are volumetric yield and specific yield from fermentation. Thus, we, herein, presented a systematic approach for process characterization of fed-batch Escherichia coli DH5α fermentation producing a porcine DNA vaccine. The demand for large-quantity production of DNA vaccines also increases. Plasmid DNA is a vital biological tool for molecular cloning and transgene expression of recombinant proteins however, decades ago, it has become an exceptionally appealing as a potential biopharmaceutical product as genetic immunization for animal and human use.
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