ProteoCool Pills#15: An alternative way to fix the sticky pad to the Eppendorf S41i CO2 shaker

In the last 20 years, thanks to the adaptation in suspension of several cell lines and to the development of more efficient transfection agents (eg lipofectamines) and culture media, the scientist were able to obtain amazing progresses in the performance of mammalian expression systems, 

As I already reported in the ProteoCool n° 29 and in the tips of the week#8 , modern cell lines, as  ExpiCHO and Expi293, are able to produce high levels (up to 300-500mg/l) of recombinant antigens and/or antibodies in the culture surnatants also in shaking flask culture. 

The availability of an orbital shaker able to maintain 5-8% of CO₂ atmosphere is essential during cell propagation and maintenance.

Differently to bacteria, the cell lines do not require high shaking rates (generally 120-125rpm) but are more prone to contamination, therefore since the cleanliness is essential, the use of sticky pads, those can be easily removed and sainted with ethanol or isopropanol solutions is preferable respect than the conventional flask clamps.

To my knowledge Eppendorf S41i (previously New Brunswick) and  the Infors multitron  are the mostly used for mammalian cell cultures.

In this presentation I would like to share with you,  my experience with the Eppendorf S41i

In our lab we have 2 Eppendorf S41i shakers:

- 1st setted at 37°C- 8% CO₂ and 120rpm for propagation of both Expi293 and ExpiCHO and for transfection of Expi293 cells;

-  2nd setted at 32°C- 5%CO₂ and 120rpm for the transfection of the ExpiCHO cells; 

The first shaker was acquired in 2016-2017 while the second in the 2020. 

Our older S41i shaker carry a platforms characterized by a smooth surface and the sticky pads attach well  directly to its surface

                   while our newest  S41i shaker carry a platforms characterized by a rough surface and the                         sticky pads don’t stick well to its surface.

                   Eppendorf sell a Stickpad ADAPTOR KIT which contains 2 metal smooth plates

                                but this kit seems to be designed for a different shaker format since

               the sizes of those plates are not appropriate to cover the entire area of the shaking platform.

Since the Eppendorf S41i is smallest than other shaking incubator, e.g. the multitron, 

 giving up such a large part of the plate surface represent an important limitation that may 

induce the scientist to choose a different shaker for their laboratories. 

In this post, I would like to show how we were able to cover with the sticky pad the entire shaking platform of both our shakers

           You can use the fixing screws supplied with the conventional flask clamps or wtih the Stickpad ADAPTOR KIT  to drilling and fixing the sticky pads directly on the black  shaker platform.

Standard Washers with 6mm in diamteter

 those can acquired in the most hardware stores /e,g  Leroy Merlin, Obi, Bricoman, Brico ) to ensure the sticky pad integrity and extend the life time of this solution.

N.B I'm happy to share with you that, as responce to this post, Eppendorf specify  that all the platforms actually produced are chatacterized by a smooth surface and the sticky pads are able to attach directly to it. 

 

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.

 

 

 

 

 

ProteoCool Pills#8: Expi-CHO a powerful cell line for transient expression of biologics

 

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 à1128 euro vs 1liter transfection kit Expi-CHO à1236 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)

 

ProteoCool Pills#6: Rapid protein Expression and solubility check using a 24 tip-horn sonicator

 

The use of high cell density media (e.g Enpresso reported in the ProteoCool n°7 or YGR reported in the ProteoCool n°32 ) for protein expression using E.coli allows to scale down culture volumes and easily express many clones in parallel.

Protein expression and solubility check can become a limiting step and the set-up of a simple and reliable protocol is essential.

Automation by using robotic platform is a possible solution for a real high-throughput approach, (e.g a screening projects that require to process  thousands samples for week) but in the most cases just the implementation of a optimized and rational protocol based on the use of multi-channel pipettes and 96-deep well plates can allow to manipulate in parallel up to hundreds samples/week with low cost and high protocol flexibility.

The lysis method used in the small scale test need to be similar to the ones that will be applied for the purification of the protein from rest of biomass in case of positive results, and therefore Sonication or is preferable respect than freeze-thaw cycles (work well in small scale but is not scalable) and chemical/enzymatic lysis ( expensive in scale-up)

Protocool

1) About 8OD of bacterial cells (for Enpresso correspond to 800ul not induced or 500ul induced 8h or 300 ul induced 24h) were transferred from culture into the 96 Deepwell plate following the scheme reported below (to fit the 24 tip sonicator horm)

2) Plate were centrifuged for 20' at 3700 rpm and 4°C

3) After centrifugation the surnatants were discharged 

4)    The pellets were resuspended adding 960ul of cell Lysis/binding buffer ( eg: Tris 20mM, NaCl 300mM, imidazole 10mM pH=8 for His-tagged protein that will be purified by IMAC) and mixed 3-5 times with the multichannel to allow complete pellet re-suspension. 

2)    Cell Lysis by sonication using the 24-tip horn:

Sonication cicle: 30’’ ON/45’’ OFF , 20 cycles  50% power – Qsonica Q700 sonicator.

The plate need be placed in ICE during the sonication to avoid sample heating.

3)    From each well 60ul of sonicated solution (Total fraction) e transferred in PCR plate no skirted containing 20ul of Loading sample buffer 4X ( eg. Thermo scientific cod. NP0008 ) with reducing agent.

4)    The 96-deepwell plate were centrifuged at 20’ a 4000rpm 4°C;

5)    From each well 60ul of surnatant (Souble fraction) e transferred in PCR plate no skirted containing 20ul of Loading sample buffer 4X ( eg. Thermo scientific cod. NP0008 ) with reducing agent.

6)    The no skirted PCR plate is closed by aluminum foil and incubated 5’ a 95°C in a PCR machine.

7)   12ul of each samples were loaded in a 26well 4-12% SDS-page gel.

Example of results: