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

A cell engineering strategy to enhance supercoiled plasmid DNA production for gene therapy

With the recent revival of the promise of plasmid DNA vectors in gene therapy, a novel synthetic biology approach was used to enhance the quantity, (yield), and quality of the plasmid DNA. Quality was measured by percentage supercoiling and supercoiling density, as well as improving segregational stability in fermentation. We examined the hypothesis that adding a Strong Gyrase binding Site (SGS) would increase DNA gyrase-mediated plasmid supercoiling. SGS from 3 different replicons, (the Mu bacteriophage and two plasmids, pSC101 and pBR322) were inserted into the plasmid, pUC57. Different sizes of these variants were transformed into E. coli DH5α, and their supercoiling properties and segregational stability measured. A 36% increase in supercoiling density was found in pUC57-SGS, but only when SGS was derived from the Mu phage and was the larger sized version of this fragment. These results were also confirmed at fermentation scale. Total % supercoiled monomer was maintained to 85-90%. A two-fold increase in plasmid yield was also observed for pUC57-SGS in comparison to pUC57. pUC57-SGS displayed greater segregational stability than pUC57-cer and pUC57, demonstrating a further potential advantage of the SGS site. These findings should augment the potential of plasmid DNA vectors in plasmid DNA manufacture. This article is protected by copyright. All rights reserved

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

Pichia pastoris (Komagataella phaffii) as a Cost-Effective Tool for Vaccine Production for Low- and Middle-Income Countries (LMICs)

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

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

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Influence of Pichia pastoris cellular material on polymerase chain reaction performance as a synthetic biology standard for genome monitoring

Advances in synthetic genomics are now well underway in yeasts due to the low cost of synthetic DNA. These new capabilities also bring greater need for quantitating the presence, loss and rearrangement of loci within synthetic yeast genomes. Methods for achieving this will ideally; i) be robust to industrial settings, ii) adhere to a global standard and iii) be sufficiently rapid to enable at-line monitoring during cell growth. The methylotrophic yeast Pichia pastoris (P. pastoris) is increasingly used for industrial production of biotherapeutic proteins so we sought to answer the following questions for this particular yeast species. Is time-consuming DNA purification necessary to obtain accurate end-point polymerase chain reaction (e-pPCR) and quantitative PCR (qPCR) data? Can the novel linear regression of efficiency qPCR method (LRE qPCR), which has properties desirable in a synthetic biology standard, match the accuracy of conventional qPCR? Does cell cultivation scale influence PCR performance? To answer these questions we performed e-pPCR and qPCR in the presence and absence of cellular material disrupted by a mild 30s sonication procedure. The e-pPCR limit of detection (LOD) for a genomic target locus was 50 pg (4.91 × 103 copies) of purified genomic DNA (gDNA) but the presence of cellular material reduced this sensitivity sixfold to 300 pg gDNA (2.95 × 104 copies). LRE qPCR matched the accuracy of a conventional standard curve qPCR method. The presence of material from bioreactor cultivation of up to OD600 = 80 did not significantly compromise the accuracy of LRE qPCR. We conclude that a simple and rapid cell disruption step is sufficient to render P. pastoris samples of up to OD600 = 80 amenable to analysis using LRE qPCR which we propose as a synthetic biology standard

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

Application of Magnetic Field for Improvement of Microbial Productivity

Continued attempt by the industry and research sectors to improve productivity of commercially viable microbial products fall into three general approaches including microbial-based (e.g. isolation, selection, and manipulation of microbes as higher producers), environmental-based (e.g. media development), and bioreactor/bioprocessbased studies. Application of electromagnetic field to microbial cultures is a recent bioprocess-based technique. Current literature shows some effects on characteristics of microbial species (fungi and bacteria). These include enhancement of ethanol production capacity of Saccharomyces cerevisiae, citric acid and cellulase production by Aspergillus niger species and insulase production by Geotrichum candidum after the cultures were exposed to electromagnetic field. In this paper we report the application of electromagnetic field to cultures of Bacillus licheniformis to enhance productivity of bacitracin, a water-soluble branched polypeptide used as an antimicrobial agent against grampositive and some gram-negative bacteria. Electromagnetic field was applied on cultures of B. licheniformis in stirred tank reactors (STRs) with working volume of 1.5 litres circulating into an in-house designed and constructed magnetic field generator with low magnetic field intensity. The experiments were carried out both with and without pH control of the culture. Samples were assayed for bacitracin concentration to confirm the effects of electromagnetic field. The microbial growth and pH profiles were also monitored. The results showed that circulation of culture at flow rate of 10 mL.min-1 into magnetic field with 10 millitesla intensity leads to an increase in bacitracin concentration. The increase was higher when the pH of the culture was controlled compared to non-controlled culture. The highest percentage increase in bacitracin concentration was 36 % after 35 hours without pH control, while the highest bacitracin percentage increase obtained from the controlled culture under pH 7 exposed to electromagnetic field, was almost 89 % after 43 hours

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