scholarly journals A Flow-Through Chromatographic Strategy for Hepatitis C Virus-Like Particles Purification

Processes ◽  
2020 ◽  
Vol 8 (1) ◽  
pp. 85 ◽  
Author(s):  
Ricardo J. S. Silva ◽  
Mafalda G. Moleirinho ◽  
Ana S. Moreira ◽  
Alex Xenopoulos ◽  
Paula M. Alves ◽  
...  
Keyword(s):  
Hepatitis C ◽  
Expression System ◽  
Cell Protein ◽  
Operational Parameters ◽  
Virus Like Particles ◽  
Radial Flow Chromatography ◽  
Flow Through ◽  
Cell Expression ◽  
Insect Cell Expression ◽  
The Impact

Biopharmaceuticals are currently becoming one of the fastest growing segments of the global pharmaceutical industry, being used in practically all branches of medicine from disease treatment to prevention. Virus-like particles (VLP) hold tremendous potential as a vaccine candidate due to their anticipated immunogenicity and safety profile when compared to inactivated or live attenuated viral vaccines. Nevertheless, there are several challenges yet to be solved in the development and manufacturing of these products, which ultimately can increase time to market. Suchlike virus-based products, the development of a platform approach is often hindered due to diversity and inherent variability of physicochemical properties of the product. In the present work, a flow-through chromatographic purification strategy for hepatitis C VLP expressed using the baculovirus-insect cell expression system was developed. The impact of operational parameters, such as residence time and ionic strength were studied using scaled-down models and their influence on the purification performance was described. The flow-through strategy herein reported made use of radial-flow chromatography columns packed with an anion exchanger and was compared with a bind and elute approach using the same chromatography media. Overall, by selecting the optimal operational setpoints, we were able to achieve higher VLP recoveries in the flow-through process (66% versus 37%) with higher removal of DNA, baculovirus and host-cell protein (92%, 99% and 50% respectively).

scholarly journals Virus-Like Particles Produced Using the Brome Mosaic Virus Recombinant Capsid Protein Expressed in a Bacterial System

2021 ◽  
Vol 22 (6) ◽  
pp. 3098
Author(s):  
Aleksander Strugała ◽  
Jakub Jagielski ◽  
Karol Kamel ◽  
Grzegorz Nowaczyk ◽  
Marcin Radom ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Mosaic Virus ◽  
Capsid Protein ◽  
Self Assembly ◽  
Environmental Changes ◽  
Expression System ◽  
Brome Mosaic Virus ◽  
Virus Like Particles ◽  
Assembly Principles ◽  
The Impact

Virus-like particles (VLPs), due to their nanoscale dimensions, presence of interior cavities, self-organization abilities and responsiveness to environmental changes, are of interest in the field of nanotechnology. Nevertheless, comprehensive knowledge of VLP self-assembly principles is incomplete. VLP formation is governed by two types of interactions: protein–cargo and protein–protein. These interactions can be modulated by the physicochemical properties of the surroundings. Here, we used brome mosaic virus (BMV) capsid protein produced in an E. coli expression system to study the impact of ionic strength, pH and encapsulated cargo on the assembly of VLPs and their features. We showed that empty VLP assembly strongly depends on pH whereas ionic strength of the buffer plays secondary but significant role. Comparison of VLPs containing tRNA and polystyrene sulfonic acid (PSS) revealed that the structured tRNA profoundly increases VLPs stability. We also designed and produced mutated BMV capsid proteins that formed VLPs showing altered diameters and stability compared to VLPs composed of unmodified proteins. We also observed that VLPs containing unstructured polyelectrolyte (PSS) adopt compact but not necessarily more stable structures. Thus, our methodology of VLP production allows for obtaining different VLP variants and their adjustment to the incorporated cargo.

Protein production using the baculovirus-insect cell expression system

2013 ◽  
Vol 30 (1) ◽  
pp. 1-18 ◽  
Author(s):  
A. Contreras-Gómez ◽  
A. Sánchez-Mirón ◽  
F. García-Camacho ◽  
E. Molina-Grima ◽  
Y. Chisti
Keyword(s):  
Insect Cell ◽  
Protein Production ◽  
Expression System ◽  
Insect Cell Expression System ◽  
Cell Expression ◽  
Insect Cell Expression ◽  
Cell Expression System

Activation of recombinant trp by thapsigargin in Sf9 insect cells

1994 ◽  
Vol 267 (5) ◽  
pp. C1501-C1505 ◽  
Author(s):  
L. Vaca ◽  
W. G. Sinkins ◽  
Y. Hu ◽  
D. L. Kunze ◽  
W. P. Schilling
Keyword(s):  
Expression System ◽  
Cation Selectivity ◽  
Drosophila Protein ◽  
Insect Cell Expression System ◽  
Cell Expression ◽  
Insect Cell Expression ◽  
Mammalian Protein ◽  
Entry Mechanism ◽  
Proline Rich Region ◽  
Cell Expression System

The mammalian protein responsible for Ca2+ release-activated current (Icrac) may be homologous to the Drosophila protein designated trp. Thus the activity of trp, and another Drosophila protein designated trp-like or trpl, may be linked to depletion of the internal Ca2+ store via the so-called capacitative Ca2+ entry mechanism. To test this hypothesis, the effect of thapsigargin, a selective inhibitor of the endoplasmic reticulum Ca2+ pump, on trp- and trpl-induced whole cell membrane current was determined using the baculovirus Sf9 insect cell expression system. The results demonstrate that trp and trpl form Ca(2+)-permeable cation channels. The trpl encodes a nonselective cation channel that is constitutively active under basal nonstimulated conditions and is unaffected by thapsigargin, whereas trp is more selective for Ca2+ than Na+ and is activated by depletion of the internal Ca2+ store. Although evaluation of cation selectivity suggests that trp is not identical to the channel responsible for Icrac, these channels must share some structural feature(s) since both are activated by thapsigargin. A unique proline-rich region in the COOH-terminal tail of trp, which is absent in trpl, may be necessary for capacitative Ca2+ entry.

scholarly journals Improving Chinese hamster ovary host cell protein ELISA using Ella®: an automated microfluidic platform

BioTechniques ◽  
2020 ◽  
Vol 69 (3) ◽  
pp. 186-192
Author(s):  
Kathleen Van Manen-Brush ◽  
Jacob Zeitler ◽  
John R White ◽  
Paul Younge ◽  
Samantha Willis ◽  
...  
Keyword(s):  
Host Cell ◽  
Chinese Hamster Ovary ◽  
Expression System ◽  
Chinese Hamster ◽  
Cell Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Host Cell Proteins ◽  
Common Technique ◽  
Automated Instrument ◽  
Cell Expression

Chinese hamster ovary (CHO) cells are a mammalian cell line used in the production of therapeutic proteins. Host cell proteins (HCPs) are process-related impurities that are derived from the host cell expression system. During biopharmaceutical drug development, removal of HCPs is required. Enzyme-linked immunosorbent assay (ELISA) is a common technique to quantitate HCPs, but is a labor-intensive process that takes up to 7 h. Ella® is an automated instrument that utilizes microfluidics and glass nanoreactors to quantitate HCPs in 75 min using similar ELISA reagents. The antibodies and antigens are captured on three distinct glass nanoreactors, resulting in sensitive reproducible data. Our results indicate that Ella quantitates CHO HCPs with precision, accuracy, sensitivity and trends comparable with our traditional CHO HCP ELISA.

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