Development of a generic reversed-phase liquid chromatography method for protein quantification using analytical quality-by-design principles

Kopp, Julian; Zauner, Florian B.; Pell, Andreas; Hausjell, Johanna; Humer, Diana; Ebner, Julian; Herwig, Christoph; Spadiut, Oliver; Slouka, Christoph; Pell, Reinhard

doi:10.1016/j.jpba.2020.113412

Cited by 29 publications

(17 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Mobile phase was composed of water (ultrapure) (eluent A) and acetonitrile (eluent B) both supplemented with 0.1% (v/v) trifluoride acetic acid. Details on the method are given in Kopp et al (2020) . For quantification of soluble protein, a size exclusion (=SEC) chromatography principle was applied, using a X-bridge column (Waters Corporation, ıMilford, MA, United States).…”

Section: Methodsmentioning

confidence: 99%

The Lazarus Escherichia coli Effect: Recovery of Productivity on Glycerol/Lactose Mixed Feed in Continuous Biomanufacturing

Kittler

Kopp

Veelenturf

et al. 2020

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

Continuous cultivation with Escherichia coli has several benefits compared to classical fed-batch cultivation. The economic benefits would be a stable process, which leads to time independent quality of the product, and hence ease the downstream process. However, continuous biomanufacturing with E. coli is known to exhibit a drop of productivity after about 4–5 days of cultivation depending on dilution rate. These cultivations are generally performed on glucose, being the favorite carbon source for E. coli and used in combination with isopropyl β- D -1 thiogalactopyranoside (IPTG) for induction. In recent works, harsh induction with IPTG was changed to softer induction using lactose for T7-based plasmids, with the result of reducing the metabolic stress and tunability of productivity. These mixed feed systems based on glucose and lactose result in high amounts of correctly folded protein. In this study we used different mixed feed systems with glucose/lactose and glycerol/lactose to investigate productivity of E. coli based chemostats. We tested different strains producing three model proteins, with the final aim of a stable long-time protein expression. While glucose fed chemostats showed the well-known drop in productivity after a certain process time, glycerol fed cultivations recovered productivity after about 150 h of induction, which corresponds to around 30 generation times. We want to further highlight that the cellular response upon galactose utilization in E. coli BL21(DE3), might be causing fluctuating productivity, as galactose is referred to be a weak inducer. This “Lazarus” phenomenon has not been described in literature before and may enable a stabilization of continuous cultivation with E. coli using different carbon sources.

show abstract

Section: Methodsmentioning

confidence: 99%

The Lazarus Escherichia coli Effect: Recovery of Productivity on Glycerol/Lactose Mixed Feed in Continuous Biomanufacturing

Kittler

Kopp

Veelenturf

et al. 2020

Front. Bioeng. Biotechnol.

Self Cite

View full text Add to dashboard Cite

show abstract

“…The flow rate and the column temperature of the final method were 0.4 mL/min and 70 • C, respectively. Details on the measurement methodology is given in [74].…”

Section: Ib Titermentioning

confidence: 99%

Production of Active Recombinant Hyaluronidase Inclusion Bodies from Apis mellifera in E. coli Bl21(DE3) and characterization by FT-IR Spectroscopy

Schwaighofer

Ablasser

Lux

et al. 2020

IJMS

Self Cite

View full text Add to dashboard Cite

The bacterium E. coli is one of the most important hosts for recombinant protein production. The benefits are high growth rates, inexpensive media, and high protein titers. However, complex proteins with high molecular weight and many disulfide bonds are expressed as inclusion bodies (IBs). In the last decade, the overall perception of these IBs being not functional proteins changed, as enzyme activity was found within IBs. Several applications for direct use of IBs are already reported in literature. While fluorescent proteins or protein tags are used for determination of IB activity to date, direct measurements of IB protein activity are scacre. The expression of recombinant hyaluronidase from Apis mellifera in E. coli BL21(DE3) was analyzed using a face centered design of experiment approach. Hyaluronidase is a hard to express protein and imposes a high metabolic burden to the host. Conditions giving a high specific IB titer were found at 25 °C at low specific substrate uptake rates and induction times of 2 to 4 h. The protein activity of hyaluronidase IBs was verified using (Fourier transform) FT-IR spectroscopy. Degradation of the substrate hyaluronan occurred at increased rates with higher IB concentrations. Active recombinant hyaluronidase IBs can be immediately used for direct degradation of hyaluronan without further down streaming steps. FT-IR spectroscopy was introduced as a method for tracking IB activity and showed differences in degradation behavior of hyaluronan dependent on the applied active IB concentration.

show abstract

“…SpA concentrations were analyzed in triplicate by reversed phase high performance liquid chromatography (RP‐HPLC) 18 . Protein purity (i.e.…”

Section: Methodsmentioning

confidence: 99%

Economic and ecological benefits of a leaky E. coli strain for downstream processing: a case study for staphylococcal protein A

Kastenhofer

Cataldo

Ebner

et al. 2021

J of Chemical Tech & Biotech

Self Cite

View full text Add to dashboard Cite

BACKGROUND Downstream processing of soluble recombinant proteins from Escherichia coli is complicated by the need to access the intracellular product by cell disruption and to separate the target protein from impurities, particularly host cell protein, DNA, endotoxins and lipids. We previously demonstrated the ability of the E. coli X‐press strain to leak high amounts of product to the culture medium without sacrificing viability. In this case study, we assessed the economic and ecological benefit of this strain for downstream processing in direct comparison to the industrial standard E. coli BL21(DE3). Staphylococcal Protein A was used as a model protein. We performed recombinant protein production, primary recovery and capture by anion exchange chromatography at laboratory scale, and used the data obtained for estimating costs and resource consumption by economic modeling. RESULTS After primary recovery, the X‐press process resulted in a 1.5‐fold higher product purity, a 150‐fold lower DNA, 3.5‐fold lower endotoxin and 3.4‐fold lower lipid load compared to BL21(DE3). Consequently, anion exchanger binding capacity was increased 2.7‐fold and purity and concentration of the eluate also was increased. Extracellular protein production with X‐press resulted in a 25% reduction of costs and a 36% reduction of both water usage and water‐related CO2 emissions compared to intracellular production with BL21(DE3). CONCLUSIONS This case study performed with stapyhlococcal Protein A demonstrated the potential of E. coli X‐press to reduce costs for downstream processing and improve the environmental footprint by simplified primary recovery, lower impurity load and consequently higher chromatographic efficiency. © 2021 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

show abstract

Development of a generic reversed-phase liquid chromatography method for protein quantification using analytical quality-by-design principles

Cited by 29 publications

References 25 publications

The Lazarus Escherichia coli Effect: Recovery of Productivity on Glycerol/Lactose Mixed Feed in Continuous Biomanufacturing

The Lazarus Escherichia coli Effect: Recovery of Productivity on Glycerol/Lactose Mixed Feed in Continuous Biomanufacturing

Production of Active Recombinant Hyaluronidase Inclusion Bodies from Apis mellifera in E. coli Bl21(DE3) and characterization by FT-IR Spectroscopy

Economic and ecological benefits of a leaky E. coli strain for downstream processing: a case study for staphylococcal protein A

Contact Info

Product

Resources

About