Production of Recombinant Protein Using the HeLa S3-Vaccinia Virus Expression System: Bioreactor Perfusion and Effects of Post-Infection Temperature

Bleckwenn, Nicole A.; Bentley, William E.; Shiloach, Joseph

doi:10.1271/bbb.69.1065

Cited by 3 publications

(4 citation statements)

References 26 publications

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“…Hadas, Einav, Fishov, and Zaritsky () previously hypothesized that by preventing cell replication, at a lower temperature of infection, the host cells become larger and therefore have more available binding sites for phage. In addition, Bleckwenn et al () suggested that a temperature reduction may aid in viral protein synthesis and further exploratory work here would be beneficial. Our results show that there is a significant increase in productivity in bacteriophage bioprocessing in relation to the temperature during infection and future work intends to investigate the underpinning biological mechanisms for this increase.…”

Section: Resultsmentioning

confidence: 89%

“…Hadas, Einav, Fishov, and Zaritsky (1997) previously hypothesized that by preventing cell replication, at a lower temperature of infection, the host cells become larger and therefore have more available binding sites for phage. In addition, Bleckwenn et al (2005) suggested that a temperature reduction may aid in viral protein synthesis and further exploratory work here would be beneficial. Our results…”

Section: Infection Temperature Investigationmentioning

confidence: 94%

“…The temperature of infection has not been widely studied to date with minimal studies examining its effect on phage titer. Grieco, Wong, Dunbar, MacGillivray, and Curtis (2012) showed that a reduction in temperature can improve the phage titer achieved, whilst Bleckwenn, Bentley, and Shiloach, (2005) hypothesized that a reduction in temperature aids viral protein synthesis. Due to the phage infection mechanism, reducing the temperature may aid in the integration of phage DNA leading to an improvement in the production of phage and allow it to become more efficient thereby producing higher titers.…”

Section: Studies Have Shown That Approximately 54% Of Predominantmentioning

confidence: 99%

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A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

Ali

Rafiq

Ratcliffe

2019

Biotech & Bioengineering

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Therapeutic bacteriophages are emerging as a potential alternative to antibiotics and synergistic treatment of antimicrobial‐resistant infections. This is reflected by their use in an increasing number of recent clinical trials. Many more therapeutic bacteriophage is being investigated in preclinical research and due to the bespoke nature of these products with respect to their limited infection spectrum, translation to the clinic requires combined understanding of the biology underpinning the bioprocess and how this can be optimized and streamlined for efficient methods of scalable manufacture. Bacteriophage research is currently limited to laboratory scale studies ranging from 1–20 ml, emerging therapies include bacteriophage cocktails to increase the spectrum of infectivity and require multiple large‐scale bioreactors (up to 50 L) containing different bacteriophage–bacterial host reactions. Scaling bioprocesses from the milliliter scale to multi‐liter large‐scale bioreactors is challenging in itself, but performing this for individual phage‐host bioprocesses to facilitate reliable and robust manufacture of phage cocktails increases the complexity. This study used a full factorial design of experiments approach to explore key process input variables (temperature, time of infection, multiplicity of infection, agitation) for their influence on key process outputs (bacteriophage yield, infection kinetics) for two bacteriophage–bacterial host bioprocesses (T4 – Escherichia coli; Phage K – Staphylococcus aureus). The research aimed to determine common input variables that positively influence output yield and found that the temperature at the point of infection had the greatest influence on bacteriophage yield for both bioprocesses. The study also aimed to develop a scaled down shake‐flask model to enable rapid optimization of bacteriophage batch bioprocessing and translate the bioprocess into a scale‐up model with a 3 L working volume in stirred tank bioreactors. The optimization performed in the shake flask model achieved a 550‐fold increase in bacteriophage yield and these improvements successfully translated to the large‐scale cultures.

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Section: Resultsmentioning

confidence: 89%

Section: Infection Temperature Investigationmentioning

confidence: 94%

Section: Studies Have Shown That Approximately 54% Of Predominantmentioning

confidence: 99%

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A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

Ali

Rafiq

Ratcliffe

2019

Biotech & Bioengineering

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“…Previous work has suggested that using an infection temperature of 28ºC is more beneficial to the phage infection compared to 37ºC and higher titres of phage can be attained (Greico et al, 2012; Ali et al, 2019). Additionally, other studies show that a lower infection temperature may be more beneficial for phage and viral propagation (Bleckwenn et al, 2005;Tokman et al, 2016). Within the few studies that consider fermentation, there have been no published studies into the conditions at which the point of infection takes place.…”

Section: Introductionmentioning

confidence: 99%

Improving phage titre through examining point of infection

Ali¹,

Rafiq²,

Ratcliffe³

2020

Preprint

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Bacteriophages are viruses that cause the lysis of bacteria. They have recently been used to combat antimicrobial resistant infections as an alternative therapy to antibiotics. Their production and propagation, however, remains understudied and will be key to obtaining titres required for future clinical studies and research. Previous work suggests that temperature of infection significantly influences the production process and output yield of phage, with a reduction in temperature from 37ºC to 28ºC resulting in significant increases in productivity for multiple host-phage systems. The current study aimed to build upon this previous work by examining different temperature conditions at the point of infection to determine the effect on harvest phage titre improvements. Investigations were conducted at different culture scales ranging from 20mL shake flasks to 3L stirred tank bioreactor cultures to investigate process differences when scaling from laboratory bench-scale to initial industrial scale fermentations. Additionally, the kinetics of phage infection were investigated. In small scale cultures, the greatest phage bursts and harvest titres were generated by maintaining cultures under static 28 o C incubation during infection compared to agitation and temperature reduction from 37 o C. Investigating the dynamics around the point of infection will help to inform scalable processes for manufacture of phage for a variety of applications ranging from direct therapeutic application to self-assembling bio-templates for nanostructure synthesis.

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Vectors for recombinational cloning and gene expression in mammalian cells using modified vaccinia virus Ankara

Pradeau‐Aubreton¹,

Ruff²,

Garnier³

et al. 2010

Analytical Biochemistry

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Production of Recombinant Protein Using the HeLa S3-Vaccinia Virus Expression System: Bioreactor Perfusion and Effects of Post-Infection Temperature

Cited by 3 publications

References 26 publications

A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

A scaled‐down model for the translation of bacteriophage culture to manufacturing scale

Improving phage titre through examining point of infection

Vectors for recombinational cloning and gene expression in mammalian cells using modified vaccinia virus Ankara

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