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We sought to engineer mammalian cells to secrete nuclease activity as a step toward removing the need to purchase commercial nucleases as process additions in bioprocessing of AAV5 and AAV9 as gene therapy vectors. Engineering HeLa cells with a serratial nuclease transgene did not bring about nuclease activity in surrounding media whereas engineering serum-free, suspension-adapted HEK293F cells with a staphylococcal nuclease transgene did result in detectable nuclease activity in surrounding media of the resultant stable transfectant cell line, "NuPro-1S." When cultivated in serum-free media, NuPro-1S cells yielded 3.06 × 10 10 AAV5 viral genomes (vg)/mL via transient transfection, compared with 3.85 × 10 9 vg/mL from the parental HEK293F cell line. AAV9 production, followed by purification by ultracentrifugation, yielded 1.8 × 10 13 vg/mL from NuPro-1S cells compared with 7.35 × 10 12 vg/mL from HEK293F cells. AAV9 from both HEK293F and NuPro-1S showed almost identical ability to transduce cells embedded in a scaffold tissue mimic or cells of mouse neonate brain tissue in vivo . Comparison of agarose gel data indicated that the DNA content of AAV5 and AAV9 process streams from NuPro-1S cells was reduced by approximately 60% compared with HEK293F cells. A similar reduction in HEK293F cells was only achievable with a 50 U/mL Benzonase treatment., Competing Interests: The authors declare no competing interests., (© 2024 The Authors.)

We engineered HEK293T cells with a transgene encoding tetracycline-inducible expression of a Staphylococcus aureus nuclease incorporating a translocation signal. We adapted the unmodified and nuclease-engineered cell lines to grow in suspension in serum-free media, generating the HEK293TS and NuPro-2S cell lines, respectively. Transient transfection yielded 1.19 × 10 6 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 10 6 TU/mL from HEK293TS cells. DNA ladder disappearance revealed medium-resident nuclease activity arising from NuPro-2S cells in a tetracycline-inducible manner. DNA impurity levels in lentiviral material arising from NuPro-2S and HEK293TS cells were undetectable by SYBR Safe agarose gel staining. Direct measurement by PicoGreen reagent revealed DNA to be present at 636 ng/mL in lentiviral material from HEK293TS cells, an impurity level reduced by 89% to 70 ng/mL in lentiviral material from NuPro-2S cells. This reduction was comparable to the 23 ng/mL achieved by treating HEK293TS-derived lentiviral material with 50 units/mL Benzonase.

At present lentiviral vector production for cell and gene therapy commonly involves transient plasmid transfection of mammalian cells cultivated in serum-containing media and addition of exogenous nuclease to reduce host cell and plasmid DNA impurities. Switching from serum-containing media to chemically-defined, serum free media, and minimising the number of process additions, are both increasingly regarded as necessary steps for simplifying and potentially automating lentiviral vector bioprocessing in future. Here we adapted human embryonic kidney 293T (HEK293T) cells to grow in serum-free media and also modified these cells with transgenes designed to encode a secreted nuclease activity. Stable transfection of HEK293T cells with transgenes encoding the Staphylococcus aureus nuclease B (NucB) open reading frame with either its native secretion signal peptide, the murine Igκ chain leader sequence or a novel viral transport fusion protein, all resulted in qualitatively detectable nuclease activity in serum-free media. Serum-free transient transfection of human embryonic kidney HEK293T cells stably harbouring the transgene for NucB with its native secretion signal produced active lentivirus in the presence of medium-resident nuclease activity. This lentivirus material was able to transduce the AGF-T immortal T cell line with a green fluorescent protein reporter payload at a level of 2.05 × 10 5 TU/mL (±3.34 × 10 4 TU/mL). Sufficient nuclease activity was present in 10 μL of this unconcentrated lentivirus material to degrade 1.5 μg DNA within 2 h at 37 °C, without agitation - conditions compatible with lentivirus production. These observations demonstrate that lentiviral vector production, by transient transfection, is compatible with host cells harbouring a nuclease transgene and evidencing nuclease activity in their surrounding growth media. This work provides a solid basis for future investigations, beyond the scope of this present study, in which commercial and academic groups can apply this approach to therapeutic payloads and potentially omit exogenous nuclease bioprocess additions., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier Ltd.)

Transmission electron microscopy (TEM) is a gold standard analytical method for nanoparticle characterization and is playing a valuable role in virus-like particle (VLP) characterization extending to other biological entities such as viral vectors. A dedicated TEM facility is a challenge to both small and medium-sized enterprises (SMEs) and companies operating in low-and-middle income countries (LMICs) due to high start-up and running costs. A low-voltage TEM solution with assisted image acquisition and analysis such as the MiniTEM system, coupled with Vironova Imaging and Analysis Software (VIAS) could provide an affordable and practical alternative. The MiniTEM system has a small footprint and software that enables semi-automated data collection and image analysis workflows using built-in deep learning methods (convolutional neural networks) for automation in analysis, increasing speed of information processing and enabling scaling to larger datasets. In this perspective we outline the potential and challenges in the use of TEM as mainstream analytical tool in manufacturing settings. We highlight the rationale and preliminary findings from our proof-of-concept study aiming to develop a method to assess critical quality attributes (CQAs) of VLPs and facilitate adoption of TEM in manufacturing settings. In our study we explored all the steps, from sample preparation to data collection and analysis using synthetic VLPs as model systems. The applicability of the method in product development was verified at pilot-scale during the technology transfer of dengue VLPs development from a university setting to an LMIC- based vaccine manufacturing company, demonstrating the applicability of this analytical technique to VLP vaccine characterization., Competing Interests: SH-G and MC-D were employed by the company Vironova AB. Vironova AB provides electron microscopy-based imaging and analysis services, as well as TEM hardware and image analysis software for this purpose. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 De Sá Magalhães, De Santis, Hussein-Gore, Colomb-Delsuc and Keshavarz-Moore.)

Vaccination is of paramount importance to global health. With the advent of the more recent pandemics, the urgency to expand the range has become even more evident. However, the potential limited availability and affordability of vaccines to resource low- and middle-income countries has created a need for solutions that will ensure cost-effective vaccine production methods for these countries. Pichia pastoris ( P. pastoris ) (also known as Komagataella phaffii) is one of the most promising candidates for expression of heterologous proteins in vaccines development. It combines the speed and ease of highly efficient prokaryotic platforms with some key capabilities of mammalian systems, potentially reducing manufacturing costs. This review will examine the latest developments in P. pastoris from cell engineering and design to industrial production systems with focus on vaccine development and with reference to specific key case studies.

In this study, we have demonstrated that the type and feeding regimen of amino acids have a significant impact on the quality as well as the quantity of DNA vectors produced. Nutrient pool and factorial design experiments were carried out in order to identify the amino acids involved in increased biomass and induction of plasmid amplification. Leucine, glycine, and histidine were responsible for increased biomass and leucine starvation in the presence of histidine was implicated in plasmid amplification. Supercoiling of the plasmid was optimized using a dual feeding strategy. As a result of this, a fed-batch fermentation strategy for the production of a 6.9 kb plasmid, pSVß, in Escherichia coli DH5α was developed. In batch fermentation, a maximum plasmid yield of 39.4 mg/L equivalent to 11.3 mg/g dry cell weight (DCW) was achieved with casein hydrolysate limitation. About 90% of plasmid was in the supercoiled (SC) form after 31 hr of fermentation but only remained so for a short period, leading to a very brief window for harvesting cells at scale. Subsequently, a fed-batch fermentation using a dual feeding strategy was employed. A mean maximum plasmid yield of 44 mg/L equivalent to 9.1 mg plasmid/g DCW was achieved. After 25 hr, 90% of plasmid was in the SC form and remained at this level for the remaining 10 hr of the fermentation, allowing adequate time for the harvesting of cells without the loss of supercoiling of product. This study emphasized that optimizing fermentation strategy and identifying the essential nutrients are beneficial for bioprocessing of plasmid DNA for therapeutic applications., (© 2019 American Institute of Chemical Engineers.)

This study investigates how sorbitol/methanol mixed induction affects fermentation performance, dewatering characteristics of cells during harvesting and the profile of host cell proteins (HCP) in the process fluid when producing the target recombinant protein aprotinin. Compared to standard methanol induction, sorbitol/methanol (1:1, C-mol/C-mol) mixed induction improved cellular viability from 92.8 ± 0.3% to 97.7 ± 0.1% although resulted in a reduced product yield from 1.65 ± 0.03 g L -1 to 1.12 ± 0.07 g L -1 . On the other hand, average oxygen consumption rate (OUR) dropped from 241.4 ± 21.3 mmol L -1  h -1 to 145.5 ± 6.7 mmol L -1  h -1 . Cell diameter decreased over time in the mixed induction, resulting in a D 50 value of 3.14 μm at harvest compared to 3.85 μm with methanol. The reduction in cell size enhanced the maximum dewatering efficiency from 78.1 ± 3.9% to 84.5 ± 3.3% as evaluated by using an established ultra scale-down methodology that models pilot and industrial scale disc stack centrifugation. Seventy host cell proteins (HCPs) were identified in clarified supernatant when using sorbitol/methanol mixed induction regimen. The total number of HCPs identified with standard methanol induction was nearly one hundred. The downstream process advantage of the mixed induction lies in improved product purity by reducing both cell mortality and level of released whole cell proteins. This needs to be balanced and optimised against the observed reduction in product yield during fermentation., (Copyright © 2019. Published by Elsevier Inc.)

There is an increased interest in plasmid DNA as therapeutics. This is evident in the number of ongoing clinical trials involving the use of plasmid DNA. In order to be an effective therapeutic, high yield and high level of supercoiling are required. From the bioprocessing point of view, the supercoiling level potentially has an impact on the ease of downstream processing. We approached meeting these requirements through plasmid engineering. A 7.2 kb plasmid was developed by the insertion of a bacteriophage Mu strong gyrase-binding sequence (Mu-SGS) to a 6.8 kb pSVβ-Gal and it was used to transform four different E. coli strains, and cultured in order to investigate the Mu-SGS effect and dependence on strain. There was an increase of over 20% in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The extent of supercoiling was examined using superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of -0.022, and pSVβ-Gal -0.019, in both strains. This study has shown that plasmid modification with the Mu-phage SGS sequence has a beneficial effect on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing.

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed-batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled-up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale-up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:58-68, 2018., (© 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers.)

Objectives: To reduce unwanted Fab' leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab' fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives., Results: We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab' grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab' coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab' leakage relative to the original autonucleolytic Fab' strain with OmpA-fused staphylococcal nuclease., Conclusions: We successfully rescued Fab' leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab' fragment to the surrounding growth media.

Background: The production of recombinant proteins containing disulfide bonds in Escherichia coli is challenging. In most cases the protein of interest needs to be either targeted to the oxidizing periplasm or expressed in the cytoplasm in the form of inclusion bodies, then solubilized and re-folded in vitro. Both of these approaches have limitations. Previously we showed that soluble expression of disulfide bonded proteins in the cytoplasm of E. coli is possible at shake flask scale with a system, known as CyDisCo, which is based on co-expression of a protein of interest along with a sulfhydryl oxidase and a disulfide bond isomerase. With CyDisCo it is possible to produce disulfide bonded proteins in the presence of intact reducing pathways in the cytoplasm., Results: Here we scaled up production of four disulfide bonded proteins to stirred tank bioreactors and achieved high cell densities and protein yields in glucose fed-batch fermentations, using an E. coli strain (BW25113) with the cytoplasmic reducing pathways intact. Even without process optimization production of purified human single chain IgA 1 antibody fragment reached 139 mg/L and hen avidin 71 mg/L, while purified yields of human growth hormone 1 and interleukin 6 were around 1 g/L. Preliminary results show that human growth hormone 1 was also efficiently produced in fermentations of W3110 strain and when glucose was replaced with glycerol as the carbon source., Conclusions: Our results show for the first time that efficient production of high yields of soluble disulfide bonded proteins in the cytoplasm of E. coli with the reducing pathways intact is feasible to scale-up to bioreactor cultivations on chemically defined minimal media.