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#12: Rapid insertion/deletion/replacement of N-terminal signal peptide sequences using PIPE cloning

 In both eukaryotic and prokaryotic cells, all proteins are synthesized in cytoplasm. 

Proteins that are destined to enter into the secretory pathway are usually endowed with an N-terminal signal peptides (SPs, known also as N terminal leader sequences): the signal peptide those are short peptides and usually have a length of 16–30 amino acids.

After directing proteins to their specific locations, SPs are removed by signal peptidases

The presence or absence of the SPs allow to direct the expression of the protein in different cellular compartments:

- E.coli,  a protein with-out SPs will be directed in the cytoplasm, while the addiction of a SPs (as pelB, OmpA signal peptides) of the signal will  direct the protein into the periplasm;

-In Gram positive bacteria (as baccilus) and mammalian cells (as HEK293, CHO) the addiction of N terminal SPs direct the protein in the culture surnatant.

Since the accumulation of recombinant proteins in the cytoplasm may lead to the formation of inclusion bodies or protein degradation via proteases and the recombinant protein folding may also affected from the reducing conditions of the cell compartment (eg E.coli cytoplasm is strongly reducing and not compatible with S-S bond formation), the selection of the right Signal peptides play a decisive role in the industrial production of recombinant proteins.

It has been shown that using different homologous or heterologous signal peptides can affect the yields of recombinant proteins. Selecting a proper signal peptide to increase the secretion efficiency becomes a common methodology to optimize the production of recombinant protein

The availability of a simple cloning method to readily add, replace and modify a signal peptide sequences in an expression clone is therefore an essential tool to screen for the best protein/antibody productivity.

As already mentioned in the ProteoCool n°1: Cloning methods overview; The PIPE cloning is a nice method to manipulate expression vectors and perform mutagenesis, insertion and/or deletion or some vector regions.

With a single vector PCR is possible to insert dna fragments up to 80-100bp in any  vector region and therefore it can be applied also to the insertion of replacement of signal peptides in the vector of interest.

Using the PIPE cloning i  was able to insert pelB (MKYLLPTAAAGLLLLAAQPAMA)  and ompA (MKKTAIAIAVALAGFATVAQA) signal peptides in several pet15 clones for E.coli expression and other mammalian signal peptide in pcdna 3.4 clones for expression in Expi293 and Expi-CHO.

Of couse a similar approach could be performed to insert/delete or replace other short sequences as:

 - 6x His Tag

- Avi Tag

- Protease digestion sites (eg TEV, Eterokinase, Fatt.Xa)

or delete:

-N- or C- teminal region of your gene;

-N or C- terminal fusions (eg GFP, GST, MBP, etc)

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PIPE cloning steps:

1) Vector amplification by PCR  (V-PCR)

2) PCR digestion with dpnI (to remove template vector background)

3) Trasfrom the vector into the MACH1 E.coli cells

4) Plasmid extraction from at least 4 colonies and DNA sequencing

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                                            Tips to perform the Vector PCR:

PCR reaction:

My preferred DNA polymerases: Kapa Hifi (Roche) or Clone amp (Takara)

                       Theoretically you can use all high fidelity polimerases that do not add poly AA

DNA template --> less than 0.1ng/reaction 

(higher template amount may result in background colonies with the original template)

PCR volume --> 25ul/reaction are more than enough considering that for PIPE reaction 1-2ul are normally enough and PCR purification is not required.

PCR cycle:

Elongation time --> >1minute/kb also if the datasheet of the Taq suggest shorter extension time to exploit the 3-' --> 5' exonuclease activity that improve the formation of incomplete extension that are require for PIPE cloning.

Do not insert the final elongation time step.

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V-PCR trasfromation in E.coli cells (PIPE reaction)

PIPE cloning do not require PCR purification.

1)  Mix 2 ul of the V-PCR in 20ul of chemically competent MACH1 cells (thermo cod. )and incubate the mixture in ice 30 minutes;

N.B:  If the V-PCR band intensity is very high  (as in the reported picture)  yon can try also to dilute the plasmid 5 times and perform a second trasfromation with 2ul of the diluted plasmid in 20ul of cells because too much DNA can reduce the trasformation efficiency)

2) Incubate the cells 1' at 42°C

3) Transfer the cell in ice

4) Add 250ul of SOC or LB sterile media (with out antibiotic)

5) incubate the cells at 37°C - 180/600rpm (in a thermomixer or incubator shaker) 

6) Plate all the cells in LB-agar plates containing the proper selection antibiotic

(eg 100mg/l ampicillin for pet21 or pcdna 3.4,  50mg/l of kanamicin for pet24 clones)

7) incubate the plates O/N at 37°C

Primers design

Annealing regions à 18-26bp with an annealing temperature salt adjusted (calculated with Oligocalculator)  of 58-62°C if is possible)

Flanking regions –> length up to 60bp. Overlapping regions have to be between 15 and 20bp

Is not mandatory to add flanking regions in both forward and reverse primer to create the 16bp overlapping. It can be done also adding the flanking region in just 1 of the 2 primers. I choose one or the other solution on the basis of the length and Tm of the relative annealing regions.

----------------------------------------------------------------------------------------------------------------------------------------------------                                                                                      A great thanks to  

Roberto Petracca

an amazing supervisor who introduced me to the PIPE cloning

i was very lucky to shave with him many years in the Novartis Reseach centre in Siena 

References: 

Klock HE, Lesley SA. The Polymerase Incomplete Primer Extension (PIPE) method applied to high-throughput cloning and site-directed mutagenesis. Methods Mol Biol. 2009;498:91-103.

                    

 

ProteoCool Pills#10: Use of 2F-Peracetyl-Fucose to produce low fucosilated monoclonal antibodies with Expi293 and ExpiCHO cell lines

Therapeutic monoclonal antibodies are the fastest growing class of therapeutics for the treatment of various cancers and inflammatory disorders. The Fc region of human IgG antibodies interacts with multiple Fcγ receptor (FcγR) and complement proteins and mediates immune effector functions, which are important for many therapeutic applications, e.g. elimination of targeted cells via antibody-dependent cellular-cytotoxicity (ADCC), -phagocytosis (ADCP) or complement-dependent cytotoxicity (CDC).

To date, therapeutic IgG antibodies (either approved or in clinical development) belong to the IgG1, IgG2 or IgG4 subclasses. Each IgG isotype has a distinct binding affinity to the various FcγRs, which are expressed differently on immune cells. 

In cancer immunotherapy, some IgG1 antibodies (eg ipilimumab) rely on the Fc-mediated immune effector function, dependent cellular cytotoxicity (ADCC), as the major mode of action to deplete tumor cells.

It is well-known that this effector function is modulated by the N-linked glycosylation (N297) in the Fc region of the antibody.

In fact, if the N297A mutation  is able to reduced FcγR-binding and abolish ADCC activity on the other hand,  the absence of core fucose on the Fc N-glycan has been shown to increase IgG1 Fc binding affinity to the FcγRIIIa present on immune effector cells such as natural killer cells and lead to enhanced ADCC activity.

Various strategies have focused on producing afucosylated antibodies to improve therapeutic efficacy. While engineering of the cell lines (eg FUT8 deletion: @Potelligent or @GlymaxX Technologies) are required for scale up production of recombinant afucosilated mabs, for small lab scale for production of mgs, the supplementation of 2F-Peracetyl-Fucose is a simple and efficient solution to produce “low fucosilated mabs” to be used in functional in vivo and in vitro assay.

Media supplementation with 200 µM of  2F-Peracetyl-Fucose allow to produce low fucosilated mabs with transient transfection in Expi293 or ExpiCHO cells) just before cell transfection (as reported on Chakraborty et.al for the Expi293 cells) allow to produce a mab with low fucosilation level (as reported in Fig1) and enhanced ADCC activity.

n.b: The results of ELISA assay on mabs produced from Expi293 and ExpiCHO seem to suggest that the ExpiCHO show a decreased fucosilation level respect than Expi293 also in absence of the 2F paracetyl fucose. 

Preparation of 2F-Peracetyl-Fucose stock solution:

2F-Peracetyl-Fucose is supplied as powder (10mg). It can be directly resuspended in the glass bottle with 1mM DMSO to obtain a 34.2mM stock solution (171X) that could be stored at -20°C.

584µl of 34.2mM of 2F-Peracetyl-Fucose were added to 100 ml of cells just before the transient transfection with Expifectamines

Prons:

Very Simple!!

Concerns:

Expensive: Cost of  2F-Peracetyl-Fucose: ~ 1000euro/liter of transfection make this approach feasible only for small lab scale productions

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)