ProteoCool Pills#14: Hints for cell propagation and recombinant protein expression using Expi-CHO and Expi293 (WT and GNTI-)

For many years the application of the traditional adherent mammalian cell cultures for recombinant protein production was difficult and applicable only in those approaches requiring very low protein amount (ug’s) because cell scale up was very difficult, transfection not very efficient and the final volumetric yields (mg/litre) very low. 

This limitation forces the scientist to use other simple eukaryotes expression systems as Pichia Pastoris or Baculo virus for the production of mammalian protein in mg scale.

However, in the last 20 years some technological improvements as:

 1) The adaptation of several cell lines to suspension cultures;

 2) The development of more efficient transfection agents;

 3) The development of serum free media where the cells are able to growth at high cell density;

Improve drastically the performances of the mammalian expression systems;

As I already mention in previous presentations, theExpi293 and Expi-CHO date, represent 2 of the most performing mammalian transient expression systems. 

If it is true that those systems are quite expensive, on the other hand they are very simple to use and guarantee, high yields also in shake flask. 

For some recombinant protein, Expi293 in shake flask were able to provide me up 300mg/yields. 

Values that until some years ago were reachable only using E. coli expression in high cell density set-up (e.g. feed-batch fermenters)

Expi-CHO are able to provide up to 100-200 mg/l of full length recombinant antibodies. Full length antibodies are big (150KDa), composed from 2 chains and contains several disulfide bonds, their expression in bacterial expression system is very challenging.

However, to reach those results details, as;

-             - The selection of the optimal shake flask format and right culture volume;

-              -  The selection of an antibiotic not affecting cell growth and protein expression;

-                The selection of the optimal signal peptide for protein secretion into the cell surnatant;

can make a huge difference!!

The selection of the right shake flask and culture volume

Expi293F and Expi293 GNT- cells :

In small scale (up to 60ml culture) Expi293 are not so sensitive to the flask format and presence of baffle:

For example, in our experience Expi293 are growing well in both

-        Erlenmeyer flaskscup vented plain (cost ~ 8€/flask for 125ml format)

-        Erlenmeyer flasks cup vented baffled

-        Thomson Optimun growth flaks  (cost ~ 8€/flask for 125ml format and 30€/flask for 1,6l)

Optimal culture volumes: 

125ml flask volume for 30ml Expi293 transfection and 250ml flask volume for 60ml Expi293 transfection

For culture scale up (eg 200-500ml culture),  the Thomson Optimun growth flaks eems to perform better probably  because it guarantee better CO2 and oxygen exchange.

My preferred format is 200-300ml transfection in 1.6liter flask and generally we do not exceed the 500ml single transfection (using a 2.4 litre flask) because in high volumes we more frequent bacterial contamination was observed, therefore in case of large culture volume e.g. 1-2liter, I prefer to split the culture using more flask in parallel.

ExpiCHO:

ExpiCHO show an high tendency to aggregate and are much more susceptible to  the flask format.

Thomson Optimun growth flaks https://htslabs.com/og/ that contain small baffles seem to represent the best compromise and are the format more able to prevent the cell aggregation during the cell maintenance passages.

N.B: Expi-CHO are able to growth up to very high cell density (>10milion/ml) but to avoid cell aggregation is it better do not exceed the 6 milion/ml during the cell propagation passages

Optimal culture volumes: 

   125ml flask for 25ml transfection 

   250ml flask for 50 ml trasfection

    1,8liter flask for 150-300ml tranfection

In all cases we use the max titer protocol à 8day of trasfection at 32°C

The selection of the right antibiotics to prevent cell contamination.

The Expi293 and ExpiCHO Thermofisher manuals do not suggest the addition of any antibiotics to prevent bacterial contaminations. However after that we experienced some culture contaminations, considering the high  cost of the reagents (more than 1000euro/Liter)  we decided to perform some  transfection trials in the presence of  antibiotics to see if they affect or not the cell viability, growth rate and recombinant protein expression

Expi293 are not affected by:

-        Pen/strep (thermo cod. 15070-063) diluted 1:100 (50 unit/ml penicillin and 50ug/ml Streptomycin)  

-        Gentamicin (Sigma cod. G1272) diluted 1:200 (50ug/ml)

Generally, we use for cell propagation and transfection the Pen/Strep 

Expi293 GNT- are not affected by Gentamicin (Sigma cod. G1272) diluted 1:200 (50ug/ml) while reduction in cell growth rate and viability was observed in presence of Pen/Strep;

Expi-CHO are not affected by Pen/strep (thermo cod. 15070-063) diluted 1:100 while reduction in cell growth rate and viability was observed in presence of Gentamicin diluted 200 times.