scholarly journals Expression of Recombinant Factor IX Using the Transient Gene Expression Technique

2019 ◽  
Author(s):  
Jafar Vatandoost ◽  
Mohammad Amin Azimifar
Keyword(s):  
Gene Expression ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Transient Gene Expression ◽  
Factor Ix ◽  
Cell Factory ◽  
Scale Production ◽  
Hek Cells ◽  
Large Scale Production ◽  
A Cell

Background: Pilot and large-scale production of recombinant proteins require the presence of stable clones, but the process of selecting stable clones is time consuming. Moreover, continuous clone culturing in large-scale production may cause loss of incoming plasmid and recombinant genes. Considering the advancements in Transient Gene Expression (TGE) technology, the large-scale expression of factor IX (FIX) was investigated in HEK cells by the TGE technique. Materials and Methods: HEK cells were seeded in a cell factory, and then transfected by pcDNA-hFIX plasmid using calcium phosphate co-precipitation method. Stable HEKhFIX cells were also seeded in a cell factory, separately. After adding vitamin K, recombinant FIX was quantified in conditioned media using an ELISA. Moreover, its functional activity was assayed using an aPTT test. Results: The results showed that the expression and activity of FIX by TGE technology was, respectively, 1.6 and 1.5 times higher than that obtained through stable HEK-FIX cells. Since calculating the specific activity revealed that for all time periods it is 0.2 mU/ng, so the increase in activity is due to the increase in the amount of FIX. Conclusions: HEK cells with higher transfectability seemed to be an appropriate alternative for transient expression for large-scale protein production. Furthermore, if rapid expression of recombinant proteins is intended, TGE can replace costly and lowyield methods.

A new Procedure for Large Scale Production and Freezing of Lymphokine Activated Killer (LAK) Cells to be used in Adoptive Immunotherapy of Cancer

1988 ◽  
Vol 74 (5) ◽  
pp. 523-530 ◽  
Author(s):  
Carlo Gambacorti-Passerini ◽  
Marina Radrizzani ◽  
Licia Rivoltini ◽  
Edoardo Marchesi ◽  
Fernando Ravagnani ◽  
...  
Keyword(s):  
Adoptive Immunotherapy ◽  
Large Scale ◽  
Interleukin 2 ◽  
Peripheral Blood Lymphocytes ◽  
Cell Factory ◽  
Scale Production ◽  
Lymphokine Activated Killer Cells ◽  
Large Scale Production ◽  
Recombinant Interleukin 2 ◽  
Immunotherapy Of Cancer

A new procedure for activation of peripheral blood lymphocytes (PBL) with recombinant interleukin 2 (rIL2) is described. PBL obtained by leukapheresis were subjected to NH4Cl (ACK) treatment to clear erythrocyte contamination; Ficoll separation was not performed. PBL were subsequently seeded in 10-floor multitrays (Cell Factory™, CF), gasified and incubated at 37 °C for 3-4 days in a humidified 5% CO2 atmosphere. This procedure achieved an activation (evaluated as cytotoxicity and proliferation) comparable with that obtained by culturing PBL in small flasks. Optimal activation of PBL was achieved in CF even in the presence of granulocyte contamination of up to 40%. It was also possible to freeze, thaw and recover most of the frozen cells and their cytotoxic activity. With this procedure therefore large quantities of lymphokine activated killer cells (LAK) can be easily produced to be used in adoptive immunotherapy trials.

Plants as factories for production of biopharmaceutical and bioindustrial proteins: lessons from cell biologyThis review is one of a selection of papers published in the Special Issue on Plant Cell Biology.

2006 ◽  
Vol 84 (4) ◽  
pp. 679-694 ◽  
Author(s):  
Allison R. Kermode
Keyword(s):  
Recombinant Proteins ◽  
Protein Function ◽  
Cell Biology ◽  
Large Scale ◽  
Production Systems ◽  
Plant Cells ◽  
Scale Production ◽  
Functional Protein ◽  
Large Scale Production ◽  
Engineer Plant

Transgenic plants, seeds, and cultured plant cells are potentially one of the most economical systems for large-scale production of recombinant proteins for industrial and pharmaceutical uses. Biochemical, technical, and economic concerns with current production systems have generated enormous interest in developing plants as alternative production systems. However, various challenges must be met before plant systems can fully emerge as suitable, viable alternatives to current animal-based systems for large-scale production of biopharmaceuticals and other products. Aside from regulatory issues and developing efficient methods for downstream processing of recombinant proteins, there are at least two areas of challenge: (1) Can we engineer plant cells to accumulate recombinant proteins to sufficient levels? (2) Can we engineer plant cells to post-translationally modify recombinant proteins so that they are structurally and functionally similar to the native proteins? Attempts to improve the accumulation of a recombinant protein in plant cells require an appreciation of the processes of gene transcription, mRNA stability, processing, and export, and translation initiation and efficiency. Likewise, many post-translational factors must be considered, including protein stability, protein function and activity, and protein targeting. Moreover, we need to understand how the various processes leading from the gene to the functional protein are interdependent and functionally linked. Manipulation of the post-translational processing machinery of plant cells, especially that for N-linked glycosylation and glycan processing, is a challenging and exciting area. The functions of N-glycan heterogeneity and microheterogeneity, especially with respect to protein function, stability, and transport, are poorly understood and this represents an important area of cell biology.

Large-Scale Production and Properties of a Solvent-Detergent-Treated Factor IX Concentrate from Human Plasma

Vox Sanguinis ◽  
1988 ◽  
Vol 55 (4) ◽  
pp. 202-210 ◽  
Author(s):  
C. Michalski ◽  
F. Bal ◽  
T. Burnouf ◽  
M. Goudemand
Keyword(s):  
Human Plasma ◽  
Large Scale ◽  
Factor Ix ◽  
Scale Production ◽  
Large Scale Production

scholarly journals Preparation of factor IX deficient human plasma by immunoaffinity chromatography using a monoclonal antibody

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 664-670 ◽  
Author(s):  
AH Goodall ◽  
G Kemble ◽  
DP O'Brien ◽  
E Rawlings ◽  
F Rotblat ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Human Plasma ◽  
Large Scale ◽  
High Capacity ◽  
Factor Ix ◽  
Affinity Column ◽  
Scale Production ◽  
Large Scale Production ◽  
Normal Human ◽  
Coagulation Assay

Abstract A murine hybridoma clone is described that grows continuously in culture and produces a monoclonal antibody we have called Royal Free Monoclonal Antibody to factor IX No. 1 (RFF-IX/1). This has high affinity for a coagulation site on factor IX. RFF-IX/1 immobilised on sepharose can be used to deplete factor IX from normal human plasma. This immunoaffinity depleted plasma is indistinguishable from severe Christmas disease plasma and can be used as the substrate in a one stage coagulation assay for factor IX. The affinity column has high capacity and can be regenerated so that large scale production from normal plasma of factor IX deficient plasma as a diagnostic reagent is now feasible.

scholarly journals Assessment of Medicago Based Systems for the Production of Human Proteins: Microscopy Analysis of the Subcellular Deposition Patterns of the Recombinant Product

2012 ◽  
Vol 18 (S5) ◽  
pp. 11-12 ◽  
Author(s):  
R.B. Santos ◽  
A.S. Pires ◽  
H.S. Silva ◽  
and R. Abranches
Keyword(s):  
Recombinant Proteins ◽  
Large Scale ◽  
Scale Production ◽  
Deposition Patterns ◽  
Large Scale Production ◽  
Recombinant Product ◽  
Therapeutic Value ◽  
Human Proteins ◽  
Microscopy Analysis ◽  
Whole Plants

The use of transgenic plants for the large scale production of recombinant proteins with commercial and therapeutic value has emerged as an alternative to conventional platforms. Plant based systems, including whole plants and plant cell cultures offer many advantages particularly regarding safety and cost effectiveness. In our laboratory we have been using the model plant Medicago truncatula as a system to express recombinant proteins with a variety of applications.

Strategies for Large-Scale Production of Recombinant Proteins in Filamentous Fungi

2005 ◽  
pp. 225-237 ◽  
Author(s):  
Heidi Sisniega ◽  
José-Luis Río ◽  
María-José Amaya ◽  
Ignacio Faus
Keyword(s):  
Filamentous Fungi ◽  
Recombinant Proteins ◽  
Large Scale ◽  
Scale Production ◽  
Large Scale Production
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