Download Bioproduction of recombinant protein

1. Bioinformatic and
Gene expression
2. Viral based vectors
3. In vitro cell assays
4. In vivo models
Bioproduction of recombinant protein
Vectalys is an R&D company with a state-of-the-art technology platform for customized viral vector production.
The company has developed a unique process enabling the production of high titer high purity lentiviral vectors for
optimal knock-down or over expression in relevant models: primary and stem cells for target gene validation and specific
tissue for animal models.
Here, we present the results of our bioproduction models.
Firstly, we determine the best conditions for CHO-S cell
transduction (Rate of transduced cells and fluorescent
intensity level) using concentrated GFP lentiviral suspension. Secondly, protein of interest is produced using
cDNA-His tagged-expressing lentiviral vectors, the supernatant is harvested and western blot analysis performed.
Material and methods
Transduction setting
CHO-S cells are mixed with GFP-expressing lentiviral
vectors using a range of multiplicity of infection (MOI)
from 0 to 100. Cells are then analyzed by cytometry 5
days post-transduction.
cDNA-His-tagged transduction
CHO-S and HEK293 Freestyle cells are transduced with
cDNA- His-tag expressing lentiviral vectors at MOI 40.
Cells are cultured at 37°C / 8% CO2/140 RPM.
> Western blot analysis: For the detection of the se-
creted His tag-protein, supernatant is harvested, then
concentrated (ultrafiltration membrane, 10kD cut-off)
or not before western blot analysis using an anti-His
antibody.
Results
CHO-S transduction setting
A
CHO-S Transduction by GFP-expressing lentiviral vector
8000
120
7000
100
6000
80
5000
4000
60
3000
40
2000
20
1000
0
0
0
Validation of transduction
Transduction is confirmed by two methods:
> RNA analysis: One week after transduction, cells are
pelleted prior to total RNA extraction.
10
20
40
60
80
100
MOI
Fluorescence intensity
% GFP positive cells
Fluorescence analysis of CHO-S Transduction setting. Transduction
efficiency according to MOI, analyzed by cytometry (A) or
microscopy (B)
% GFP Positive Cells
Vectalys has developed innovative and efficient methods
of using viral vectors for the production of recombinant
protein using Hamster (CHO-S, cells more commonly
used) or Human (HEK293 Freestyle) cells.
Reverse transcription (RT) is then performed with 1µg of
total RNA in a final volume of 20µL before PCR is carried
out using 2µL of RT. 5µL of PCR product are taken at
cycles 25, 30 and 35 for gene of interest and GAPDH (as
loading control).
Fluorescence Intensity
Introduction
Bioproduction of recombinant protein
B
3. In vitro cell assays
Our results show that :
MOI 0
MOI 10
> Lentiviral transduction is very effective from MOI 20
however the expression level can increase further to
reach the maximum with MOI 80.
MOI 40
MOI 60
> The mRNA expression level is very strong.
> We can detect « hard-to-produce » proteins by wes-
tern blot using an anti-His antibody.
MOI 80
MOI 100
> We can produce protein from Human and Hamster
cells, displaying their own glycosylations
Conclusion
Validation of transduction
Characterization of transduced cell line by analyses of
expression of mRNA (A) and protein (B).
RNA analysis (A)
Vectalys can efficiently transduce cells in suspension and
drive the expression level by modifying the MOI to construct a cell line dedicated to protein production.
The customized cell lines can be constructed both for
validation of candidate genes and recombinant protein
production.
The gene transfer using lentiviral methods has two main
benefits:
> stable cell lines are established in a very short time,
without antibiotic use,
> integrated copy number is controlled.
This technology can be used with other cell lines, such as
CHO-DG44, CHO-K1, HEK293, CHO-FS, etc...
Western blot analysis (B)
15 µL of crude or concentrated supernatant are loaded into SDSPAGE. Proteins of interest are transferred onto PVDF membrane
and detected using an anti-His Tag antibody
Vectalys provides ready-to-use stable cell lines with or
without serum. These established stable cell lines are
available for clonal selection, scale up and management
of all parameters throughout the production process.