Efficient soluble expression of disulfide bonded proteins in the cytoplasm of Escherichia coli in fed-batch fermentations on chemically defined minimal 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 IgA1 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.

Improving Fab' fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA.

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.

Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation.

Numerous therapeutic proteins are expressed in Escherichia coli and targeted to the periplasm in order to facilitate purification and enable disulfide bond formation. Export is normally achieved by the Sec pathway, which transports proteins through the plasma membrane in a reduced, unfolded state. The Tat pathway is a promising alternative means of export, because it preferentially exports correctly folded proteins; however, the reducing cytoplasm of standard strains has been predicted to preclude export by Tat of proteins that contain disulfide bonds in the native state because, in the reduced state, they are sensed as misfolded and rejected. Here, we have tested a series of disulfide-bond containing biopharmaceuticals for export by the Tat pathway in CyDisCo strains that do enable disulfide bond formation in the cytoplasm. We show that interferon α2b, human growth hormone (hGH) and two antibody fragments are exported with high efficiency; surprisingly, however, they are efficiently exported even in the absence of cytoplasmic disulfide formation. The exported proteins acquire disulfide bonds in the periplasm, indicating that the normal disulfide oxidation machinery is able to act on the proteins. Tat-dependent export of hGH proceeds even when the disulfide bonds are removed by substitution of the Cys residues involved, suggesting that these substrates adopt tertiary structures that are accepted as fully-folded by the Tat machinery.

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 three 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 percentage supercoiled monomer was maintained to 85-90%. A twofold 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. Biotechnol. Bioeng. 2016;113: 2064-2071. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

Importin-7 mediates nuclear trafficking of DNA in mammalian cells.

Eukaryotic cells have the ability to uptake and transport endogenous and exogenous DNA in their nuclei, however little is known about the specific pathways involved. Here we show that the nuclear transport receptor importin 7 (imp7) supports nuclear import of supercoiled plasmid DNA and human mitochondrial DNA in a Ran and energy-dependent way. The imp7-dependent pathway was specifically competed by excess DNA but not by excess of maltose-binding protein fused with the classical nuclear localizing signal (NLS) or the M9 peptides. Transport of DNA molecules complexed with poly-l-lysine was impaired in intact cells depleted of imp7, and DNA complexes remained localized in the cytoplasm. Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls. Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response. Our results provide evidence that imp7 is a non-redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response.

A non-chromatographic method for the removal of endotoxins from bacteriophages.

The Ff filamentous bacteriophages show potential as a new class of therapeutics, displaying utility in materials science as well as pharmaceutical applications. These phages are produced by the infection of E. coli, a Gram-negative bacterium which unavoidably sheds endotoxins into the extracellular space during growth. Since endotoxin molecules are highly immunoreactive, separation from the phage product is of critical importance, particularly those developed for human therapeutic use. The properties of M13, one of the Ff group, present a purification challenge chiefly because the standard scalable method for endotoxin removal from proteins-anion exchange chromatography-is not applicable due to pI similarity between the particles. This article examines the potential of polyethylene glycol (PEG)-NaCl precipitation as a scalable method for the separation of endotoxins from phage M13. Precipitation of M13 by 2% (w/v) PEG 6 000, 500 mM NaCl reduced endotoxin contamination of the phage product by 88%, but additional precipitation rounds did not maintain this proportional decrease. Dynamic light scattering was subsequently used to determine the effectiveness of a detergent to disassociate endotoxin molecules from M13. As a result, PEG-NaCl precipitation was supplemented with up to 2% (v/v) Triton X-100 to improve separation. A 5.7 log10 reduction in endotoxin concentration was achieved over three rounds of precipitation whilst retaining over 97% of the phage. This method compares favorably with the well-known ATPS (Triton X-114) technique for endotoxin removal from protein solutions.

Breakage of transgenic tobacco roots for monoclonal antibody release in an ultra-scale down shearing device.

Transgenic tobacco roots offer a potential alternative to leaves for monoclonal antibody (MAb) production. A possible method for extraction of MAbs from roots is by homogenization, breaking the roots into fragments to release the antibody. This process was assessed by shearing 10 mm root sections ("roots") in a 24 mL ultra-scale down shearing device, including an impeller with serrated blade edges, intended to mimic the action of a large-scale homogenizer. Size distributions of the remaining intact roots and root fragments were obtained as a function of shearing time. The data suggest that about 36% of the roots could not be broken under the prevailing conditions and, beyond these unbreakable roots, the fragmentation was approximately first order with respect to intact root number. It was postulated that root breakage in such a high shearing device was due to root-impeller collisions and the particle size data suggest that roots colliding with the impeller were completely fragmented into debris particles of the order of 0.1 mm in length. IgG release normalized to release by grinding appeared to lag behind the number of roots that had fragmented, suggesting that a process of leakage followed fragmentation in the ultra-scale down shearing device.

Engineering an Autonucleolytic Mammalian Suspension Host Cell Line to Reduce DNA Impurity Levels in Serum-Free Lentiviral Process Streams.

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 × 106 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 106 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.

An autonucleolytic suspension HEK293F host cell line for high-titer serum-free AAV5 and AAV9 production with reduced levels of DNA impurity.

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 × 1010 AAV5 viral genomes (vg)/mL via transient transfection, compared with 3.85 × 109 vg/mL from the parental HEK293F cell line. AAV9 production, followed by purification by ultracentrifugation, yielded 1.8 × 1013 vg/mL from NuPro-1S cells compared with 7.35 × 1012 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.

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.

Recombinant monoclonal antibody yield in transgenic tobacco plants is affected by the wounding response via an ethylene dependent mechanism.

Variability in recombinant IgG yield in transgenic tobacco plants has previously been observed in relation to leaf position, and is interpreted as a function of ageing and the senescence process, leading to increasing protein degradation. Here, similar findings are demonstrated in plants of different ages, expressing IgG but not IgG-HDEL, an antibody form that accumulates within the endoplasmic reticulum. Antibody yields declined following wounding in young transgenic plants expressing IgG but not in those expressing IgG-HDEL. However, in mature IgG plants, the opposite was demonstrated, with significant boosts in yield, while mature IgG-HDEL plants could not be boosted. The lack of response in IgG-HDEL plants suggests that the changes induced by wounding occur post-translationally, and the findings might be explained by wounding responses that differ in plants according to their developmental stages. Plant mechanisms involved in senescence and wounding overlap to a significant degree and compounds such as ethylene, jasmonic acid and salicylic acid are important for mediating downstream effects. Treatment of transgenic plants with ethylene also resulted in a decrease in recombinant IgG yield, which was consistent with the finding that wounded plants could induce lower IgG yields in neighbouring non-wounded plants. Treatment with 1-MCP, an ethylene antagonist, abrogated the IgG yield drop that resulted from wounding, but had no effect on the more gradual IgG yield loss associated with increasing plant age.

Growth and productivity impacts of periplasmic nuclease expression in an Escherichia coli Fab' fragment production strain.

Host cell engineering is becoming a realistic option in whole bioprocess strategies to maximize product manufacturability. High molecular weight (MW) genomic DNA currently hinders bioprocessing of Escherichia coli by causing viscosity in homogenate feedstocks. We previously showed that co-expressing Staphylococcal nuclease and human Fab' fragment in the periplasm of E. coli enables auto-hydrolysis of genomic DNA upon cell disruption, with a consequent reduction in feedstock viscosity and improvement in clarification performance. Here we report the impact of periplasmic nuclease expression on stability of DNA and Fab' fragment in homogenates, host-strain growth kinetics, cell integrity at harvest and Fab' fragment productivity. Nuclease and Fab' plasmids were shown to exert comparable levels of growth burden on the host W3110 E. coli strain. Nuclease co-expression did not compromise either the growth performance or volumetric yield of the production strain. 0.5 g/L Fab' fragment (75 L scale) and 0.7 g/L (20 L scale) was achieved for both unmodified and cell-engineered production strains. Unexpectedly, nuclease-modified cells achieved maximum Fab' levels 8-10 h earlier than the original, unmodified production strain. Scale-down studies of homogenates showed that nuclease-mediated hydrolysis of high MW DNA progressed to completion within minutes of homogenization, even when homogenates were chilled on ice, with no loss of Fab' product and no need for additional co-factors or buffering.

Investigation of the impact of Tat export pathway enhancement on E. coli culture, protein production and early stage recovery.

The twin arginine translocation (Tat) pathway occurs naturally in E. coli and has the distinct ability to translocate folded proteins across the inner membrane of the cell. It has the potential to export commercially useful proteins that cannot be exported by the ubiquitous Sec pathway. To better understand the bioprocess potential of the Tat pathway, this article addresses the fermentation and downstream processing performances of E. coli strains with a wild-type Tat system exporting the over-expressed substrate protein FhuD. These were compared to strains cell-engineered to over-express the Tat pathway, since the native export capacity of the Tat pathway is low. This low capacity makes the pathway susceptible to saturation by over-expressed substrate proteins, and can result in compromised cell integrity. However, there is concern in the literature that over-expression of membrane proteins, like those of the Tat pathway, can impact negatively upon membrane integrity itself. Under controlled fermentation conditions E. coli cells with a wild-type Tat pathway showed poor protein accumulation, reaching a periplasmic maximum of only 0.5 mg L⁻¹ of growth medium. Cells over-expressing the Tat pathway showed a 25% improvement in growth rate, avoided pathway saturation, and showed 40-fold higher periplasmic accumulation of FhuD. Moreover, this was achieved whilst conserving the integrity of cells for downstream processing: experimentation comparing the robustness of cells to increasing levels of shear showed no detrimental effect from pathway over-expression. Further experimentation on spheroplasts generated by the lysozyme/osmotic shock method--a scaleable way to release periplasmic protein--showed similar robustness between strains. A scale-down mimic of continuous disk-stack centrifugation predicted clarifications in excess of 90% for both intact cells and spheroplasts. Cells over-expressing the Tat pathway performed comparably to cells with the wild-type system. Overall, engineering E. coli cells to over-express the Tat pathway allowed for greater periplasmic yields of FhuD at the fermentation scale without compromising downstream processing performance.

High-yield export of a native heterologous protein to the periplasm by the tat translocation pathway in Escherichia coli.

Numerous high-value recombinant proteins that are produced in bacteria are exported to the periplasm as this approach offers relatively easy downstream processing and purification. Most recombinant proteins are exported by the Sec pathway, which transports them across the plasma membrane in an unfolded state. The twin-arginine translocation (Tat) system operates in parallel with the Sec pathway but transports substrate proteins in a folded state; it therefore has potential to export proteins that are difficult to produce using the Sec pathway. In this study, we have produced a heterologous protein (green fluorescent protein; GFP) in Escherichia coli and have used batch and fed-batch fermentation systems to test the ability of the newly engineered Tat system to export this protein into the periplasm under industrial-type production conditions. GFP cannot be exported by the Sec pathway in an active form. We first tested the ability of five different Tat signal peptides to export GFP, and showed that the TorA signal peptide directed most efficient export. Under batch fermentation conditions, it was found that TorA-GFP was exported efficiently in wild type cells, but a twofold increase in periplasmic GFP was obtained when the TatABC components were co-expressed. In both cases, periplasmic GFP peaked at about the 12 h point during fermentation but decreased thereafter, suggesting that proteolysis was occurring. Typical yields were 60 mg periplasmic GFP per liter culture. The cells over-expressed the tat operon throughout the fermentation process and the Tat system was shown to be highly active over a 48 h induction period. Fed-batch fermentation generated much greater yields: using glycerol feed rates of 0.4, 0.8, and 1.2 mL h(-1), the cultures reached OD(600) values of 180 and periplasmic GFP levels of 0.4, 0.85, and 1.1 g L(-1) culture, respectively. Most or all of the periplasmic GFP was shown to be active. These export values are in line with those obtained in industrial production processes using Sec-dependent export approaches.

Quality assessment of virus-like particle: A new transmission electron microscopy approach.

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.

Study of robustness of filamentous bacteriophages for industrial applications.

The development of a whole new class of industrial agents, such as biologically based nanomaterials and viral vectors, has raised many challenges for their large-scale manufacture, principally due to the lack of essential physical data and bioprocessing knowledge. A new example is the promise of filamentous bacteriophages and their derivatives. As a result, there is now an increasing need for the establishment of strong biochemical engineering foundations to serve as a guide for future manufacture. This article investigates the effect of high-energy fluid flow on filamentous bacteriophage M13 to determine its robustness for large-scale processing. By the application of well-understood ultra scale-down predictive techniques, the viability of bacteriophage M13 was studied as a measure of its robustness and as a function of energy dissipation rate and fluid conditions. These experiments suggested that despite being perceived as a relatively fragile molecule in the literature, bacteriophage M13 should tolerate processing conditions in existing large-scale equipment designs. No loss of viability was noted up to a maximum energy dissipation rate of 2.9 × 10(6) W kg(-1) . Furthermore, significant losses above this threshold only occurred over periods well in excess of the exposure times expected in a bioprocess environment. Filamentous bacteriophages may therefore be regarded as a viable process material for industrial applications.

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.

A study of D-lactate and extracellular methylglyoxal production in lactate re-utilizing CHO cultures.

In large-scale mammalian cell culture, the key toxic by-products assessed and monitored are lactate and ammonia. Often no distinction between the two isoforms of lactate is made. Here, we present profiles of both D- and L-lactate. D-Lactate is the end molecule of the methylglyoxal pathway. D-Lactate unlike L-lactate is not re-utilized, and although during normal culture time frames it represents one-tenth of total lactate, during lactate re-use it represents nearly 35%. This indicates significant carbon flow through pathways not associated with primary metabolites. We have observed that the behavior of D-lactate is radically different from that of L-lactate with the level of one isoform changing, whilst the concentration of the other remains constant. This is an example of an alternate carbon flow pathway containing metabolic intermediates that may potentially have a detrimental effect on cells. The activity of the methylglyoxal pathway when measured as a proportion of glucose consumption in this study far exceeds any previously reported. This highlights the potential importance of "non-primary" metabolisms to long lifespan mammalian fermentation practices.

Sorbitol/methanol mixed induction reduces process impurities and improves centrifugal dewatering in Pichia pastoris culture.

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 ±â€¯0.3% to 97.7 ±â€¯0.1% although resulted in a reduced product yield from 1.65 ±â€¯0.03 g L-1 to 1.12 ±â€¯0.07 g L-1. On the other hand, average oxygen consumption rate (OUR) dropped from 241.4 ±â€¯21.3 mmol L-1 h-1 to 145.5 ±â€¯6.7 mmol L-1 h-1. Cell diameter decreased over time in the mixed induction, resulting in a D50 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 ±â€¯3.9% to 84.5 ±â€¯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.

Application of Plasmid Engineering to Enhance Yield and Quality of Plasmid for Vaccine and Gene Therapy.

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 pSVß-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 pSVß-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 pSVß-Gal398 maintaining a superhelical density of -0.022, and pSVß-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.