scholarly journals Insect High FiveTM cell line development using site-specific flipase recombination technology

2021 ◽  
Author(s):  
Mafalda M Dias ◽  
João Vidigal ◽  
Daniela P Sequeira ◽  
Paula M Alves ◽  
Ana P Teixeira ◽  
...  
Keyword(s):  
Cell Line ◽  
Trichoplusia Ni ◽  
Scale Up ◽  
Transient Gene Expression ◽  
Suspension Cultures ◽  
Host Cells ◽  
Vector System ◽  
Small Scale ◽  
Heterologous Proteins ◽  
Protein Model

Abstract Insect Trichoplusia ni High FiveTM (Hi5) cells have been widely explored for production of heterologous proteins, traditionally mostly using the lytic baculovirus expression vector system (BEVS), and more recently using virus-free transient gene expression systems. Stable expression in such host cells would circumvent the drawbacks associated with both systems when it comes to scale-up and implementation of more efficient high-cell density process modes for the manufacturing of biologics. In this work, we combined Flipase (Flp) recombinase-mediated cassette exchange (RMCE) with fluorescence-activated cell sorting (FACS) for generating a stable master clonal Hi5 cell line with the flexibility to express single or multiple proteins of interest from a tagged genomic locus. The 3-step protocol herein implemented consisted of (i) introducing the RMCE docking cassette into the cell genome by random integration followed by selection in Hygromycin B and FACS (Hi5-tagging population), (ii) eliminating cells tagged in loci with low recombination efficiency by transfecting the tagging population with an eGFP-containing target cassette followed by selection in G418 and FACS (Hi5-RMCE population), and (iii) isolation of pure eGFP-expressing cells by FACS and expansion to suspension cultures (Hi5-RMCE master clone). Exchangeability of the locus in the master clone was demonstrated in small-scale suspension cultures by replacing the target cassette by one containing a single protein (i.e. iCherry, as an intracellular protein model) or two proteins (i.e. influenza HA and M1 for virus-like particles production, as an extracellular protein model). Overall, the stable insect Hi5 cell platform herein assembled has the potential to assist and accelerate biologics development.

scholarly journals Multi-omics profiling of CHO parental hosts reveals cell line-specific variations in bioprocessing traits

2019 ◽  
Author(s):  
Meiyappan Lakshmanan ◽  
Yee Jiun Kok ◽  
Alison P. Lee ◽  
Sarantos Kyriakopoulos ◽  
Hsueh Lee Lim ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Cho Cells ◽  
Cell Cycle Progression ◽  
Chinese Hamster ◽  
Suspension Cultures ◽  
Host Cells ◽  
Phenotypic Traits ◽  
Cell Factories

AbstractChinese hamster ovary (CHO) cells are the most prevalent mammalian cell factories for producing recombinant therapeutic proteins due to their ability to synthesize human-like post-translational modifications and ease of maintenance in suspension cultures. Currently, a wide variety of CHO host cell lines have been developed; substantial differences exist in their phenotypes even when transfected with the same target vector. However, relatively less is known about the influence of their inherited genetic heterogeneity on phenotypic traits and production potential from the bioprocessing point of view. Herein, we present a global transcriptome and proteome profiling of three commonly used parental cell lines (CHO-K1, CHO-DXB11 and CHO-DG44) in suspension cultures and further report their growth-related characteristics, and N- and O-glycosylation patterns of host cell proteins (HCPs). The comparative multi-omics analysis indicated that some physiological variations of CHO cells grown in the same media are possibly originated from the genetic deficits, particularly in the cell cycle progression. Moreover, the dihydrofolate reductase deficient DG44 and DXB11 possess relatively less active metabolism when compared to K1 cells. The protein processing abilities and the N- and O-glycosylation profiles also differ significantly across the host cell lines, suggesting the need to select host cells in a rational manner for the cell line development on the basis of recombinant protein being produced.

scholarly journals Bioreactor production of rVSV-vectored vaccines in Vero cell suspension cultures

2021 ◽  
Author(s):  
Sascha Kiesslich ◽  
Gyoung Kim ◽  
Chun Fang Shen ◽  
Chil-Yong Kang ◽  
Amine Kamen
Keyword(s):  
Vero Cell ◽  
Cell Line ◽  
Virus Disease ◽  
Fixed Bed ◽  
Suspension Cultures ◽  
Suspension System ◽  
Small Scale ◽  
Adherent Cell ◽  
Viral Particles ◽  
Viral Vaccine

The Vero cell line is the most used continuous cell line in viral vaccine manufacturing. This adherent cell culture platform requires the use of surfaces to support cell growth, typically roller bottles or microcarriers. We have recently compared the production of rVSV-ZEBOV on Vero cells between microcarrier and fixed-bed bioreactors. However, suspension cultures are considered superior with regards to process scalability. Therefore, we further explore the Vero suspension system for rVSV-vectored vaccine production. Previously, this suspension cell line was only able to be cultivated in a proprietary medium. Here, we expand the adaptation and bioreactor cultivation to a serum-free commercial medium. Following small scale optimization and screening studies, we demonstrate bioreactor productions of highly relevant vaccines and vaccine candidates against Ebola virus disease, HIV and COVID-19 in the Vero suspension system. rVSV-ZEBOV, rVSV-HIV and rVSVInd-msp-SF-Gtc can replicate to high titers in the bioreactor, reaching 3.87 × 107 TCID50/mL, 2.12 × 107 TCID50/mL and 3.59 × 109 TCID50/mL, respectively. Further, we compare cell specific productivities, and the quality of the produced viruses by determining the ratio of total viral particles to infectious viral particles

scholarly journals Ginsenoside content in suspension cultures of Panax quinquefolium L. cultivated in shake flasksand stirred-tank bioreactor

2018 ◽  
Vol 72 (1) ◽  
pp. 15 ◽  
Author(s):  
Ewa Kochan ◽  
Sylwia Caban ◽  
Grażyna Szymańska ◽  
Piotr Szymczyk ◽  
Anna Lipert ◽  
...  
Keyword(s):  
Large Scale ◽  
Raw Materials ◽  
Scale Up ◽  
Fold Increase ◽  
Suspension Cultures ◽  
Stirred Tank ◽  
Small Scale ◽  
Panax Quinquefolium ◽  
Stirred Tank Bioreactor ◽  
Shake Flasks

<p>Plant suspension cultures are described as a source for the acquisition of medicinal secondary metabolites which in the future may become an alternative to traditional raw materials. This study demonstrates that the cell cultures of one of the ginseng species – Panax quinquefolium L. synthesize ginsenosides, which are triterpene saponins having a multidirectional pharmacological effects. Tested suspension cultures were run on a small scale in the shake flasksand in scale up of the process in a 10-liter stirred tank. In the shake flasks,the highest biomass yield (2.28 gl-1 for dry and 33.99 gl-1 for fresh weight) was reached on day 30 of culture, and the highest content of saponins (2.66 mg g -1 dw) was determined on day 28 of culture. In the bioreactor, nearly 2.67 and 3-fold increase of respectively dry and fresh biomass was recorded in relation to the inoculum. Large-scale cultures synthesized protopanaxatriol derivatives such as Rg1 and Re ginsenosides, however, no saponins belonging to the protopanaxadiol derivatives were reported.</p>

scholarly journals Maize Streak Virus Coat Protein Is Karyophyllic and Facilitates Nuclear Transport of Viral DNA

1999 ◽  
Vol 12 (10) ◽  
pp. 894-900 ◽  
Author(s):  
H. Liu ◽  
M. I. Boulton ◽  
C. L. Thomas ◽  
D. A. M. Prior ◽  
K. J. Oparka ◽  
...  
Keyword(s):  
Coat Protein ◽  
Nuclear Transport ◽  
Transient Gene Expression ◽  
Maize Streak Virus ◽  
Host Cells ◽  
Immunofluorescent Staining ◽  
Vector System ◽  
Viral Gene ◽  
Streak Virus ◽  
Viral Dna

Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculovirus vector system, and also in tobacco protoplasts with a cauliflower mosaic virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1-labeled viral ss or ds DNA, was microinjected into maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.

Procaryotic Expression of Single-Chain Variable-Fragment (scFv) Antibodies: Secretion in L-Form Cells of Proteus mirabilis Leads to Active Product and Overcomes the Limitations of Periplasmic Expression in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4862-4869 ◽  
Author(s):  
Jörg F. Rippmann ◽  
Michaela Klein ◽  
Christian Hoischen ◽  
Bodo Brocks ◽  
Wolfgang J. Rettig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proteus Mirabilis ◽  
Binding Activity ◽  
Host Cells ◽  
Heterologous Proteins ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Active Product ◽  
Periplasmic Expression

ABSTRACT Recently it has been demonstrated that L-form cells ofProteus mirabilis (L VI), which lack a periplasmic compartment, can be efficiently used in the production and secretion of heterologous proteins. In search of novel expression systems for recombinant antibodies, we compared levels of single-chain variable-fragment (scFv) production in Escherichia coliJM109 and P. mirabilis L VI, which express four distinct scFvs of potential clinical interest that show differences in levels of expression and in their tendencies to form aggregates upon periplasmic expression. Production of all analyzed scFvs in E. coli was limited by the severe toxic effect of the heterologous product as indicated by inhibition of culture growth and the formation of insoluble aggregates in the periplasmic space, limiting the yield of active product. In contrast, the L-form cells exhibited nearly unlimited growth under the tested production conditions for all scFvs examined. Moreover, expression experiments with P. mirabilis L VI led to scFv concentrations in the range of 40 to 200 mg per liter of culture medium (corresponding to volume yields 33- to 160-fold higher than those with E. coli JM109), depending on the expressed antibody. In a translocation inhibition experiment the secretion of the scFv constructs was shown to be an active transport coupled to the signal cleavage. We suppose that this direct release of the newly synthesized product into a large volume of the growth medium favors folding into the native active structure. The limited aggregation of scFv observed in the P. mirabilis L VI supernatant (occurring in a first-order-kinetics manner) was found to be due to intrinsic features of the scFv and not related to the expression process of the host cells. The P. mirabilis L VI supernatant was found to be advantageous for scFv purification. A two-step chromatography procedure led to homogeneous scFv with high antigen binding activity as revealed from binding experiments with eukaryotic cells.

scholarly journals Solid Form and Phase Transformation Properties of Fexofenadine Hydrochloride during Wet Granulation Process

Pharmaceutics ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 802
Author(s):  
Suye Li ◽  
Hengqian Wu ◽  
Yanna Zhao ◽  
Ruiyan Zhang ◽  
Zhengping Wang ◽  
...  
Keyword(s):  
Phase Transition ◽  
Phase Transformation ◽  
Water Content ◽  
Scale Up ◽  
Small Scale ◽  
Dissolution Behavior ◽  
Wet Granulation ◽  
Water Addition ◽  
Granulation Process ◽  
And Performance

The quality control of drug products during manufacturing processes is important, particularly the presence of different polymorphic forms in active pharmaceutical ingredients (APIs) during production, which could affect the performance of the formulated products. The objective of this study was to investigate the phase transformation of fexofenadine hydrochloride (FXD) and its influence on the quality and performance of the drug. Water addition was key controlling factor for the polymorphic conversion from Form I to Form II (hydrate) during the wet granulation process of FXD. Water-induced phase transformation of FXD was studied and quantified with XRD and thermal analysis. When FXD was mixed with water, it rapidly converted to Form II, while the conversion is retarded when FXD is formulated with excipients. In addition, the conversion was totally inhibited when the water content was <15% w/w. The relationship between phase transformation and water content was studied at the small scale, and it was also applicable for the scale-up during wet granulation. The effect of phase transition on the FXD tablet performance was investigated by evaluating granule characterization and dissolution behavior. It was shown that, during the transition, the dissolved FXD acted as a binder to improve the properties of granules, such as density and flowability. However, if the water was over added, it can lead to the incomplete release of the FXD during dissolution. In order to balance the quality attributes and the dissolution of granules, the phase transition of FXD and the water amount added should be controlled during wet granulation.

SUBMERGED CITRIC ACID FERMENTATION OF SUGAR BEET MOLASSES: INCREASE IN SCALE

1955 ◽  
Vol 1 (5) ◽  
pp. 299-311 ◽  
Author(s):  
R. Steel ◽  
C. P. Lentz ◽  
S. M. Martin
Keyword(s):  
Citric Acid ◽  
Scale Up ◽  
Aeration Rate ◽  
Small Scale ◽  
Acid Fermentation ◽  
Inoculum Level ◽  
Cross Sectional ◽  
Factors Affecting ◽  
Beet Molasses ◽  
Initial Ph

Factors affecting the production of citric acid in the submerged fermentation of ferrocyanide-treated beet molasses by Aspergillus niger were studied in 2.5 and 36 liter fermenters. The small fermenters were used to determine the effects of changes in sterilization technique, phosphate supplement, ferrocyanide treatment, inoculum level, initial pH, fermentation temperature, and aeration rate. The relation between ferrocyanide concentration and inoculum level was also studied. Four different samples of molasses were fermented successfully. An average yield of 8.2% citric acid (64% conversion) was obtained from 51 small-scale fermentations. Comparable yields were obtained in the large fermenters under comparable conditions. Most of the information obtained with the small fermenters was applicable to the larger-scale fermenters, but in the latter the fermentation was significantly more stable. Aeration was the main problem in the scale-up and aeration rates approximately double those calculated on a fermenter cross-sectional area basis were required for comparable results in the large fermenters.

scholarly journals Sustainable Second-Generation Bioethanol Production from Enzymatically Hydrolyzed Domestic Food Waste Using Pichia anomala as Biocatalyst

2020 ◽  
Vol 13 (1) ◽  
pp. 259
Author(s):  
Ioanna Ntaikou ◽  
Georgia Antonopoulou ◽  
Gerasimos Lyberatos
Keyword(s):  
Food Waste ◽  
Second Generation ◽  
Scale Up ◽  
Pichia Anomala ◽  
Small Scale ◽  
Amylolytic Enzymes ◽  
Second Generation Bioethanol ◽  
Saccharification Efficiency ◽  
Ethanol Yields ◽  
Domestic Food

In the current study, a domestic food waste containing more than 50% of carbohydrates was assessed as feedstock to produce second-generation bioethanol. Aiming to the maximum exploitation of the carbohydrate fraction of the waste, its hydrolysis via cellulolytic and amylolytic enzymatic blends was investigated and the saccharification efficiency was assessed in each case. Fermentation experiments were performed using the non-conventional yeast Pichia anomala (Wickerhamomyces anomalus) under both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) modes to evaluate the conversion efficiencies and ethanol yields for different enzymatic loadings. It was shown that the fermentation efficiency of the yeast was not affected by the fermentation mode and was high for all handlings, reaching 83%, whereas the enzymatic blend containing the highest amount of both cellulolytic and amylolytic enzymes led to almost complete liquefaction of the waste, resulting also in ethanol yields reaching 141.06 ± 6.81 g ethanol/kg waste (0.40 ± 0.03 g ethanol/g consumed carbohydrates). In the sequel, a scale-up fermentation experiment was performed with the highest loading of enzymes in SHF mode, from which the maximum specific growth rate, μmax, and the biomass yield, Yx/s, of the yeast from the hydrolyzed waste were estimated. The ethanol yields that were achieved were similar to those of the respective small scale experiments reaching 138.67 ± 5.69 g ethanol/kg waste (0.40 ± 0.01 g ethanol/g consumed carbohydrates).

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