Optimisation of an alkaline lysis process for a plug- and play plasmid DNA production

Renewed interest in plasmid DNA (pDNA) production is the result of its role in the supply chain of novel biopharmaceuticals.” pDNA lies at the heart of viral and mRNA vector production because it provides the coding sequences for gene-based advanced therapy medicinal products (ATMPs). Its manufacture is therefore critical to both the supply and success of these products”1. One main bottleneck in processing of pDNA is the lysis step that still rely on techniques developed for laboratory-based DNA extraction. A robust process adaptable to a range of DNA sizes and scales remain to be fully realised. These include control of the alkaline environment, potential heterogeneity in a large vessel, change in the rheological properties of the process fluid and potentially shear impact if the molecule is large. Here, we focused on a batch lysis operation in a 1 L reactor. Three parameters studied were pH, mixing time and agitation speed. Using a central composite design for optimisation of these factors, we obtained the maximum plasmid yield (13 mg/gcells, R2=92%) and supercoiling content (91%, R2=95%) at 0.2M NaOH, mixing time of 15 minutes and tip speed of 2.3 m/s. These results will benchmark the design of plug- and- play platforms. 1.T. Hitchcock 2021, Genetic Engineering & Biotechnology News 2nd August

Enhancing the productivity of supercoiled plasmid upstream bioprocessing through plasmid engineering

This study was set out to develop an approach for producing highly supercoiled plasmid DNA. Potentially, the level of supercoiling can have an impact on ease of downstream processing. A 7.2kb plasmid was developed by cloning of Bacteriophage-Mu Strong gyrase-binding sequence (Mu-SGS) into 6.8kb pSVβ-Gal. Four E. coli strains were transformed with both the modified pSVβ-Gal398 plasmid and pSVβ-Gal. Small scale fermentations and analysis were carried out in triplicate cultures to screen for best performing strains. Two of the four strains selected amplified the plasmids efficiently. There was over 20% increase in the total plasmid yield with pSVβ-Gal398 in both strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The two strains were investigated further in shake flasks. Increases in supercoiling and plasmid yield were also observed. The extent of supercoiling was examined by superhelical density quantification, with pSVβ-Gal398 maintaining a supercoil density of -0.022 and pSVβ-Gal -0.019 in both strains. The compactness of the plasmid DNA was also quantified by hydrodynamic diameter measurement using the Nanoparticle Tracking Analysis (NTA) and it was observed that pSVβ-Gal398 was more compact with a Dh of 40-59nm compared to pSVβ-Gal with Dh of 70-90nm for both strains examined. The report of this study has shown that plasmid engineered to contain 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. References: Hassan, S., Keshavarz‐Moore, E., & Ward, J. (2016). A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy. Biotechnology and bioengineering, 113(9), 2064-2071. Yau, S. Y., Keshavarz‐Moore, E., & Ward, J. (2008). Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large‐scale processes. Biotechnology and bioengineering, 101(3), 529-544

Optimisation of an alkaline lysis process for a plug- and play plasmid DNA production

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Application of filtration blocking models to describe fouling and transmission of large plasmids DNA in sterile filtration

Sterile filtration is considered as a final step in processing pharmaceutical grade plasmid DNA. During the development of the filtration process, fundamental understanding on the mechanism of fouling is critical to improve filtration operations. The mechanism of fouling of pQR150 (20 kb) and pGEc47 (56 kb) plasmids DNA during constant pressure filtration inside 0.22 μm PVDF membrane is experimentally investigated. The decline of filtrate flux as a function of time is analysed using the framework of classical and combined blocking models. The results for both plasmids indicate a transition between fouling mechanisms. Initially, during the early part of the filtration, the intermediate blocking model provided the best fit of the experimental results suggesting that fouling of the membrane was mainly caused by deposition of particles onto its surface. Afterwards, the result trends were best captured by the standard blocking model indicating that internal fouling of the membrane was the dominant fouling mechanism. A study of the transmission of both plasmids shows a significant reduction of plasmid transmission which coincides with the transition of the fouling mechanism from intermediate to standard blocking. The study highlights how the fouling behaviour of large plasmid DNA during sterile filtration is determined by the complex interplay between the flexibility of the molecules and the internal structure of the membrane

Scale-Up and Bioprocessing of Phages

A profusion of new applications for phage technologies has been developed within the last few years, stimulating investigations into the large-scale production of different phages. Applications such as antibiotic replacement, phages as gene therapy vectors, phages as vaccines, diagnostics using filamentous phages and novel optical applications such as the phage laser may need grams to kilogrammes of phage in the future. However, many of the techniques that are used for the growth and purification of bacteriophage at small scale are not transferable to large-scale production facilities of phage in industrial processes. In this chapter, the stages of production that need to be carried out at scale are examined for the efficient large-scale fermentation of the filamentous phage M13 and the Siphoviridae phage lambda (λ). A number of parameters are discussed: the multiplicity of infection (MOI) of phage to host cells, the impact of agitation on the initial infection stages, the co-growth with phage rather than static attachment, the use of engineered host cells expressing nuclease, the optimisation of both the quantity and the physiology of the E. coli inoculum and phage precipitation methods

Microbial platform for vaccine production for low and medium income countries (LMICs): 2 case studies

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Characterisation and process verification studies in a miniature bioreactor used as a predictive tool to scale-up an industrial process

There is continuous pressure on the pharmaceutical industry to acquire a thorough understanding of their product and process, using a quality by design (QbD) approach, in order to speed up the timescales for approval of a therapy to market. One way to achieve this is to conduct detailed characterization studies using a miniature model, such as a bioreactor or a single-use micro-well plate system, in order to identify the key parameters which have an impact on the scale-up of a bioprocess. The design and development of such a representative miniature model, using integrated sensors for monitoring of process conditions on-line, is vital for scaling purposes and allows for parallelization and automation whereby multiple parameters can be tested in a high-throughput fashion. This concept has been applied to a downstream process for the recovery of periplasmic Fab’ from E.coli cells, a therapeutic recombinant protein used to treat autoimmune diseases. The aim of the research is to address the key challenges of scaling by characterizing a 20mL small scale model in terms of mixing performance and fluid dynamics and identifying which factors, such as shear stress, fluid velocity and spatial distribution of cells have the most impact on scaling. In the study, novel, advanced techniques, such as the dual pH indicator system for mixing time (DISMT) and particle image velocimetry (PIV), were used to characterise the fluid dynamics in the small scale bioreactor. The bioreactor was fitted with a miniature temperature and pH probe to observe conditions, and process verification studies were conducted in order to compare performance at different scales for a variety of operating conditions. The results show that the small scale bioreactor was able to successfully mimic an industrially relevant heat extraction process up to 10,000 fold scale with good accuracy on the basis of constant volumetric power input. Assays were used to analyse the quantity and quality of the product and impurities, and scanning electron microscopy was used to examine the morphology of cells at different stages of the process. The study was further extended to 24 deep square micro-well plates and initial data suggests that the shaken system may also be used to scale-down 10,000 fold, and therefore indicates the feasibility and applicability of these single-use plates as a predictive tool for scaling up. The poster will discuss how fluid dynamic studies may be used to understand and improve scale-up and will additionally address alternative methods which may be used to minimise the variation observed in the feed material and hence its impact on subsequent unit operations

Effect of the oxygen transfer rate on oxygen-limited production of plasmid DNA by Escherichia coli

Oxygen limitation can increase the pDNA yield in cultures of Escherichia coli. Nevertheless, such effect has not been studied systematically. Namely, only cultures at low DOT have been performed, excluding important factors like the oxygen transfer rate (OTR). Moreover, to the best of our knowledge, there is no information regarding the impact of oxygen availability on the topology of the plasmid. The supercoiling of DNA requires energy and it is hypothesized that oxygen availability will affect the produced isoforms. In the present study, we performed fully aerobic and oxygen-limited cultures of E. coli bearing a high copy number plasmid. Cultures at OTRmax values of 10, 14, 30, 45 (for oxygen-limited cultures) and 110 mmol L-1 h-1 (for aerobic cultures) were performed in microtiter plates with DOT, pH, biomass (measured as scattered light) and NADH fluorescence online monitoring. To further investigate the impact of oxygen limitation on pDNA topology, an E. coli strain constitutively expressing the Vitreoscilla hemoglobin (VHb) was used. VHb is known to improve aerobic respiration and consequently ATP generation at low oxygen availability. Our results show that the pDNA yields on biomass (YpDNA/X) were inversely proportional to the OTRmax for both strains, and increased more than two-fold in cultures at the lowest OTRmax, compared to aerobic cultures. Expression of VHb resulted in lower YpDNA/X, compared to cultures of the parent strain. The strain expressing the VHb displayed higher specific growth rates at OTRmax of 10, 14 and 30 mmol L-1 h-1, compared to the parent strain. However, at OTRmax of 45 and 110 mmol L-1 h-1, the growth rate of the parent strain was higher. In general, the specific NADH fluorescence was lower in cultures of the engineered strain, which can be associated to a more oxidized intracellular state, in agreement with the proposed effect of VHb on the cellular metabolism. The pDNA supercoiled fraction (SCF) was maximum in cultures at OTRmax of 30 mmol L-1 h-1, reaching 92.9 % for the wild type strain and 98.7 % for the strain expressing VHb, while no linearized pDNA was detected. This condition was replicated in a 1 L stirred tank bioreactor (STB) for W3110 recA-, due to the higher productivity of this strain. The performance of cultures in the STB was very similar to that of cultures in the MTP concerning accumulated fermentative by-products, cell growth and pDNA production and SCF. Altogether, these results show the existence of an optimal OTRmax for oxygen-limited production of plasmid DNA. Furthermore, we demonstrate that studies in microtiter plates are excellent to predict culture performance of STB and to scale-up plasmid DNA production cultures

Serum-free lentiviral vector production is compatible with medium-resident nuclease activity arising from adherent HEK293T host cells engineered with a nuclease-encoding transgene

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 × 105 TU/mL (±3.34 × 104 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