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#7:Small scale IMAC purification from Expi293 or Expi-CHO culture surnatant

 

The Expi293 (see ProteoCool n° ) and Expi-CHO Expression System (Thermo) provides high-yield recombinant protein production in mammalian cells     by combining high-density an optimized chemically defined media and transfection protocol.

Generally 'i'm performing the  small scale expression and solubility trials in 2ml scale culture (in 6 well plates)

The availability of a simple IMAC Small scale purification protocol is essential in the preliminary expression tests to detect those proteins that are poorly expressed and/or co-localize with other proteins secreted in the cell surnatant.

 

Here I report a simple and rapid protocol that can allow to you to perform small IMAC purification for up to 24 sample in parallel in short time (1-2h):

1.      The cells were Transferred in a 2ml Eppendorf or 24 deep well plates (depend from the number of sample tested);

2.      Centrifugation at 300g for 5;

        a)      Cell Pellet: resuspended in 2ml of binding buffer, diluted 1/3 and loaded in SDS page; 

       b)     Surnatant à go to step 3;

3.Centrifugation 10’ at maximum speed to completely remove cell debris (12000g in Eppendorf 2ml0

                              a)      Pellet  discharged;

b)     Surnatant were diluted 1:1 with the IMAC binding buffer (Tris 20mM, Nacl300mM, imidazole 10mM pH=8) and loaded in the purification column* (go to step5);

* Buffer exchange is not necessary to obtain good binding to the IMAC coloumn;

4.      Assembly of the purification column:

-   Biorad Bio-Spin (cod.7326008) loaded with 100ul of Ni-Sepharose FF (Ni-Sepharose FF - Cytiva cod.17531801*)

*The same protocol was also performed using Ni- Sepharose Excel resin (GE cod. E17371201) those theoretically was developer for purification of protein from the mammalian cell suratant but in our hands the standard Ni-sepharose seems to provide best results in terms of protein purity;

-   Column was washed (for gravity) with 2 ml of milliQ water to remove the ethanol.

-   Column was equilibrated with 2ml of IMAC Binding buffer;

    Using a simple 4 position tube rack (VWR cod. 525-0951) and a large volume adjustable spacer multipipette (e.g RAININ LA6-1200XLS) up to 32 protein sample can be easily loaded (step 5) and  washed in parallel (step 6)

5.      Protein sample were loaded into the equilibrated column and F.T discharged.

6.      Columns were washed with 6ml of IMAC binding buffer and buffer discharged

7.      Elution was performed with 300ul (3CV) of elution buffer (Tris 20mM, NaCL 300mM, imidazole 300mM pH=8) and the eluted fraction is collected in a 2 ml Eppendorf tube.

8.       To collect all the elute columns were centrifuged 2’ at 3000rpm.

Example of result obtained with this protocol:

where: 

C- are non transfected cells

 C+ are Expi293 transfected with a 10His-tagged protein cloned in pcdna3.4 with an N_terminal leader sequence for secretion in mammalian cells.

The culture volumes and the column size to be used in the scale up need to be carefully select on the basis of the expression level observed in the small-scale trials and of the amount of protein that we need to produce.

Eg:

<0,5mg of protein: 100 of Ni-sepharose resin in a Biorad Bio-Spin coloumn

0,5-1mg of protein: 200-300ul Ni-sepharose resin in a Biorad Poliprep (cod. #7311550)  Gravity flow ;

1-10 mg of protein:1-2ml di of Ni-Sepharose resin in a empty PD-10 gravity flow (a peristaltic pump could be used to perform sample loading, wash and elution)  

>10mg of protein   prepacked 5ml  His-trap FF Crude connected to a peristaltic pump or AKTA purification system

Differently from the expression test, when we would like to purify protein, all the fractions need to be collected and loaded in the SDS-page to follow the protein and washing steps with buffers contain intermediate imidazole concentration (e.g. 20mM for 6xHis-tagged protein or 30mM for 10xHis tagged proteins) are included  in order to improve  final protein purity.

The columns if correctly stored (must not go DRY and stored at 4°C in water or water/ethanol 20% for long time)  are reusable for several time. Metal stripping and regeneration could be performed as already shown in the ProteoCool n° 25: Simple IMAC regeneration 

 

ProteoCool n°32 High Cell density protein expression in E.coli using YGR media

 

is now available on https://proteocool.blogspot.com/