If in the ProteoCool n°29 I extensivelly show the properties of the mammalian Expi293 expression
system, today I would like to briefly report you the further improvement in
terms of productivity that I was able to obtain thanks to the ExpiCHO cell expression system.
Example 1: Expression of a mouse igG2a-CL-k recombinant
monoclonal antibodies
(both heavy and light chain were cloned in
pcdna3.4) with 2 different leader peptides)
Protocol (in brief):
1)
15 ml of cells were transfected in a 125 cap vented flask
2)
Cultures timelines:
1)
Cell were centrifuged 20’ at 3000g and surnatant was filtered at 0,22uM
2)
Surnatant is added of 1.5ml of PBS 10X
3)
Surnantat is loaded on a Poliprep gravity column containing 300ul of
Protein G FF resin
5b) For the Expi-CHO productions, the
Flow-Through from the column was loaded in a 2nd protein column to recover all
the mab
4)
Column is washed with 50CV of PBS
5)
Mab was eluted with glycine 100mM ph2.7 on a Tris 1M pH=9 solution
6)
For Expi-CHO cells due to high expression the
7)
Mab was concentrated with amicon ultra 50Kda and buffer exchanged in PBS
8)
Mab was quantified with nanodrop measuring the 280nm absorbance and the
igG quantification mode.
Results:
!! ExpiCHO vs EXpi293 --> More than 10 fold improvement!!
Example 2: Production of recombinant human CTLA-4 (1-161 domain)
(carrying a
C-terminal 6XHis tag cloned in pcdna 3.4)
Protocol (in brief)
1)
15 ml of cells were transfected in a 125 cap vented flask
2)
Cultures timelines: Same that reported in the Example
3)
Cell were centrifuged 20’ at 3000g and surnatant was filtered at 0,22uM
4)
Surnatant is added of the same volume of IMAC binding buffer (Tris 20mM,
imidazole 10mM, NaCL300mM ph=8)
5)
Surnantat is loaded on a Poliprep gravity column containing 300ul of Ni-sepharose
FF
6)
Column is washed with 50CV of Binding buffer
7)
CTLA-4 was eluted with 1ml of IMAC elute buffer (Tris 20mM, imidazole
300mM, NaCl 300mM pH=8.
8) CTLA-4 was quantified with nanodrop measuring
the 280nm absorbance. CTLA-4 extinction coefficient was calculated by
ProtParamTool
Results:
Summary:
Pro/Cons Expi-CHO vs Expi293
Low DNA amount required for transfection (0,5ug/ml vs 1ug/ml)
Low temperature during protein expression(may help production of thermostable proteins)
Use in the preclinical studies the same cell line (eg CHO) that will be
used for the manufacturing of clinical lot and that represent the gold standard
strain for biomanufacturing and reduce risks associated to strain transition
High growth rate at
37°C and higher tendency to aggregate: Routine cells passages require more attention to
perform the cell count and cell dilution.
Longer culture times (at least 8day vs
4day)
PMT (eg
glycosylation profile) may be slightly different from the human cells (eg HEK293 or
Expi293)
2 different CO2 incubator shaker are required: shakr n°1 fixed at 37°C 120rpm 8%CO2 for the cell line propagation a shaker n°2 fixed at 32°C 120rpm 5%CO2 for cell transfection
Expensive or cheaper?
It depends from the yields. In
the reported examples, much cheaper!
To date culture media and transfection
reagents are slightly similar
1liter Expi293 mediumà 340euro vs 1liter ExpiCHO medium à 466euro
1liter transfection kit Expi293 Ã 1128
euro vs 1liter transfection kit Expi-CHO Ã 1236 euro
Those costs may seem very high (about
2000euro/liter) but it depends from yields and complexity of each target.
Expi293 and ExpiCHO allow to you to produce high
yields of proteins (eg protein rich of S-S bonds) and/or full length antibodies those are almost
impossible to be produced in similar yields (and correct folding) using less
expensive organisms (as E.coli)
