Production of Monoclonal Antibodies

Laboratory of Cellular Oncology, NCI

•In the past, our lab has had some trouble with the stability of stocks of monoclonal antibodies (mAbs) purified out of cell culture supernatant.  We therefore prefer to share the hybridoma cells themselves.  This has the advantage of providing investigators with a renewable source of the mAb. 

Unfortunately, most of our hybridomas are addicted to IL-6 supplement.  IL-6 can be provided in the form of a media supplement called Hybridoma Fusion and Cloning Supplement (HFCS, Roche# 11363735001)).  For IL-6-addicted hybridomas, attempting to thaw the culture without HFCS will result in extremely slow growth rates or, in some cases, mass cell death.  Do not attempt to culture hybridomas without HFCS.

Culturing hybridomas

1)   Prepare HFCS-supplemented medium.  To RPMI add:  

1x HFCS supplement (Roche# 11363735001)

55 µM 2-Mercaptoethanol (Invitrogen)

Antibiotic, such as Primocin (500x)

Glutamax-I (Invitrogen)

Non-essential amino acids (Invitrogen)

10% fetal calf serum (FCS)

Aside from the HFCS, which is strictly required for some hybridomas, all other media components can be omitted without any major consequences.  The complete medium can be stored at -80 ºC for at least a year without loss of IL-6 activity.

2)   Thaw cells and place into 25 ml of HFCS medium supplemented with an additional 10% FCS (i.e., a total of 20% FCS) in a T-75 flask. It may be helpful to spin the cells out of the DMSO cryoprotectant medium.  This can be done by adding the thawed cells to 10ml of RPMI with 10% FCS and spinning 200xg for 5 minutes.  However, cells seem to do fine if the thawed DMSO-containing suspension is diluted into at least 25ml of medium.

3)   Incubate the cells with the culture flask set in an upright position.  Culturing with the flask upright facilitates removal and replacement of supernatant. Upright culture also makes it easier to triturate (pipet) any loosely attached hybridoma cells off the plastic.

4)   After the initial thaw, the culture can be expanded by adding more HFCS medium.  Hybridomas are sensitive to overcrowding.  The culture should be split and fed as soon as the supernatant becomes orange verging on yellow.  If the culture medium is yellow, mass die-off may occur after feeding the culture.

5)   After recovery from thaw, freeze down at least a few vials of cells.  We use the following freeze medium:

50% conditioned supernatant (retained from the culture being frozen)

40% FCS

10% DMSO

The cells should be spun down, resuspended in reserved conditioned medium, and transferred to gasketed cryo tubes.  Chill the suspension for 15-30 minutes in the refrigerator and add an equal volume of chilled 80% FCS/20% DMSO (to achieve the final concentrations listed above).  Freeze the chilled suspension in a “Mr Frosty” (or similar cryo device) pre-chilled to 4ºC. 

6)   Expand the remaining cell culture to generate conditioned supernatant containing the mAb of interest.  See step F below.

Generation of hybridomas

A) Immunization

1)   Generate recombinant protein to be used as immunogen. Virus-like particles can be produced in 293TT cells using standard methods.  For production of proteins in bacteria, we’ve had good luck with the maltose binding protein system (NEB).  The MBP fusion partner helps improve the solubility of many recombinant antigens.  We generally engineer a TEV cleavage site between the MBP and the fusion partner. See David Waugh’s TEV page.

2)   Emulsify ~10 µg of antigen with complete Freund’s adjuvant.  Immunize each mouse with ~50 µl of antigen suspension each subcutaneously.

3)   If the immunogen is a VLP or other regularly arrayed repetitive immunogen, plan to sacrifice the mice two weeks after priming. Do not use a booster dose for VLP immunogens.  For reasons we don’t understand, hybridomas from animals boosted with regularly arrayed antigens are unstable and unclonable (see PMID: 20598728).

4)   For regular immunogens, such as recombinant polyomavirus T antigens, boost the mice with ~10µg of antigen in incomplete Freund’s adjuvant after 4-6 weeks. Sacrifice the mice three days after the boost.  Optional:  if you’re not in a hurry, wait 4-6 more weeks after the boost and perform a second boost.  Sacrifice mice three days after the second boost.  Theoretically, the longer dosing schedule may yield monoclonals with better affinity.

5)   Harvest spleens and lymph nodes.  Transfer organs to an Eppendorf tube and mince into fine pieces using sterilized scissors.  Add 1 ml of digestion medium (RPMI with 2% fetal calf serum, 0.5 mg/ml collagenase A, and 0.1 mg/ml DNase I).  DNase-I stock (Roche) is 10 mg/ml.  Collagenase stock (Roche) is 20 mg/ml.  Incubate cells in digestion medium 5 minutes at 37ºC.

6)    Resuspend the minced organ pieces.  Transfer suspension to a 70 µm mesh filter (Falcon 352350) in a sterile petri dish. Press the cells through the mesh using the rubber end of a 3ml syringe plunger.  Rinse the mesh with 10ml of RPMI containing 2% FCS.

7)   Pellet the cells at 150 x g for 10 minutes. Gently resuspend the cell pellet in 1 ml (per spleen) of ACK red blood cell lysis buffer (Lonza/Biowhittaker #10-548E).  Incubate suspension at room temperature 5-10 minutes.  Add 13 ml of RPMI/10% FCS.  Re-pellet the cells.  The pellet should be cream colored (not red).

8)   Resuspend the cells in 10ml of RPMI (no FCS).  Count cells.  Expect about 100 million splenocytes from each mouse.  Chill on ice until SP2/mIL6 cells are ready.

B) Preparation of myeloma fusion partner

7)   Obtain myeloma SP2/mIL-6 from ATCC (catalog number CRL-2016).

8)   Prepare HFCS medium.  To RPMI add:  

1x HFCS supplement (Roche# 11363735001)

55 µM 2-Mercaptoethanol (Invitrogen)

Primocin (InVIVOgen)(use Primocin stock as 500x)

Glutamax-I (Invitrogen)

10% FCS

9)   Thaw cells and place into 50ml of HFCS medium supplemented with an additional 10% FCS.  Incubate the cells in a T-225 flask set in an upright position.  Culturing with the flask upright facilitates removal and replacement of supernatant.  Upright culture also makes it easier to triturate loosely attached cells off the plastic.

10)After the initial thaw, the culture can be expanded by adding RPMI with 10% FCS and without HFCS.  SP2/mIL-6 are sensitive to overcrowding.  The culture should be split and fed as soon as the supernatant becomes orange verging on yellow.

11)Expand the culture to contain 100 million SP2/mIL6 cells in HFCS medium.  This will take a week, or a bit longer if plain RPMI/10% FCS is used.  The culture may need to be carried in two T-225 flasks. 

C) Perform fusion

1)   Roughly follow the steps in the Roche HFCS package insert.

2)   Set up a small 37ºC water bath in the hood. Warm the PEG-1500 solution (Roche# 10783641001) to 37ºC.  Warm 25 ml of plain RPMI to 37ºC.  Warm 50ml of HFCS-containing medium to 37ºC.  Warm centrifuge (or just the 50 ml tube insert) to 25ºC.

3)   Mix 100 million SP2/mIL-6 with 100 million immunized mouse splenocytes and wash into plain RPMI (no FCS) in a 50 ml conical centrifuge tube.  In our experience, the total number of cells and the ratio between the splenocytes and SP2/mIL-6 fusion partner seem not to matter very much.

4)   Remove the RPMI.  Gently tap the tube to resuspend the pellet.  Warm the suspension to 37ºC.

5)   Add 1.5ml of PEG solution dropwise over the course of 90 seconds while gently swirling the tube containing the cells in the 37º water bath.  Swirl for an additional 30 seconds.

6)   Add pre-warmed RPMI as instructed in HFCS instructions (slowly over the course of several minutes)

7)   Spin cells down in an uncooled centrifuge.

8)   Incubate the pelleted cells at 37ºC for 5 minutes, then remove supernant.

9)   Gently resuspend cells in 100 ml of pre-warmed HFCS medium containing a total of 20% FCS and 1x HAT (ATCC# 69-X).  Note: the instructions on the HAT label are somewhat confusing.  The vial should be reconstituted by adding 2 ml of sterile water. This produces a 500x drug stock. The stock drops out of solution upon storage at 4ºC, but it’s easy to resuspend the precipitate, which quickly redissolves upon dilution into medium.

10) Distribute suspension into 10x 96-well plates at 100µl/well

11) Incubate the cultures 5 days.  Feed by adding 100µl of fresh HFCS medium with 1x HAT per well.  It may be helpful to occasionally agitate the plate (gently pat, shake back and forth, swirl, or all of the above).  This will help break down large clumps of cells.  Cells in the middle of clumps become unhealthy and may die.

D) Screen

1)   Culture supernatants can be screened for the presence of antigen-reactive hybridomas 8-10 days after fusion.  

2)   Coat Immulon 2HB plates overnight with 50 ng of recombinant antigen in 100 µl of PBS per well.   

3)   Block the well by adding 150 µl of PBS with 1% nonfat dry milk (blotto).  Incubate 2 hours, then wash plate with PBS + 0.05% Tween-20.  

4)   Shake wash buffer out of plate, whack plate on paper towels, then add 75µl of blotto per well.

5)   Add 25µl of hybridoma supe to each well.  Use fresh tips for each well (this operation will use many boxes of tips).  Put the plates in a 37º CO2 incubator for 30 minutes, then move plates to a rocker or shaker at room temperature for 30 minutes.  

6)   Wash away supe, probe with goat-anti-mouse-HRP secondary (BioRad)

7)   Wash away secondary.  Develop ELISA with ABTS substrate (Roche).

Screening notes:

•The “anti-IgG” secondary we use has enough light-chain reactivity to detect IgM.  However, the OD is usually dramatically lower for IgM hybridomas than for IgG hybridomas (i.e., faintly positive wells are often IgM instead of IgG).

•MBP is very immunogenic.  If an MBP fusion protein was used for immunization, it’s very likely that a majority of antibodies will target MBP.  If the antigen cannot readily be separated from the MBP (e.g. by proteolytic cleavage and processing over Q resin (Pierce #90010)), it is possible to prevent the detection of MBP-specific antibodies by adding an excess of free MBP during primary antibody binding.  Add free MBP (NEB #E8046L) or an irrelevant MBP fusion protein to the binding buffer (step D-4) at a concentration of 10 µg/ml (1 µg/well).  A better alternative would be to screen the hybridomas using an antigen purified using a different affinity tag (e.g., His tag).

•It’s often a good idea to screen for a subset of mAbs likely to work in Western.  To screen for such mAbs using ELISA, first denature the antigen before coating plates.  

A)  Starting with purified antigen stock, add reagent stock solutions to achieve final concentrations of roughly 2% SDS, 50mM DTT, 25mM EDTA, and 50mM Tris pH 8.5.

-For example, take 100µl of recombinant antigen at 0.5 mg/ml.  Add: 33µl of 10% SDS, 10 µl of 1M DTT, 10 µl of 0.5M EDTA, 10µl of 1M Tris pH 8.5

B)  Incubate the mixture at 65ºC for 10 minutes.

C)  Dilute the mixture at least 1:500 in bicarb buffer (Sigma #C-3041).

-For example, dilute antigen mixture above into 100ml of bicarb buffer and use it to coat 10 H2B plates.  

It’s important to dilute away the SDS, which might compete against the antigen for plastic binding.

E) Clone hybridomas

1)   Choose ~24 ELISA-positive wells and expand the selected wells into a 24-well plate.  Use RPMI-HFCS supplemented with HT instead of HAT.  If the hybridomas were dense and/or growing as large balls of cells, there may be significant die-off after splitting them into the 24-well plate.

Note: hybridomas can be somewhat sticky.  The 96-well culture should be thoroughly resuspended by pipetting up and down all around the well.  Try to avoid scraping the pipet tip on the culture plastic (this can shred cells) and try to avoid foaming.  Check a few wells on the microscope to verify that your technique is effectively dislodging nearly all cells from the plastic.

Important: the selection drug aminopterin persists within the cell longer than hypoxanthine and thymidine.  This is a problem because hypoxanthine and thymidine are necessary antidotes for aminopterin.  The solution to this problem is to “wean” the cells off of aminopterin by passaging them for a week or so in medium containing HT supplement (ATCC# 71-X) instead of HAT.  Once the aminopterin has been diluted (or decayed) away, the cells can be carried in medium without drugs.

2)   When the 24-well culture is growing healthily, suspend and freeze down a tube of cells.  To freeze, mix 500 µl of suspended cells with 400 µl of FCS and add 100 µl of DMSO.  Place vials in Mr. Frosty or other cryo-cooler and put at -80, then archive in liquid nitgrogen.

3)   Dilute the cells into 10 ml HFCS medium (with HT) at a density of 20 cells per ml.  Extensively triturate the cell suspension to ensure that clumps or doublets of cells have been broken up.  Distribute the suspension into a 96-well plate at 100µl (~2 cell) per well.

4)   Screen supes from the entire plate.  Sometimes, the desired hybridoma is represented in only a few percent of cells in the starting culture.  Thus, this initial round of cloning uses higher density plating (2 cells per well) to screen a larger number of clones.

5)   Expand positive wells and freeze down as above.

6)   Repeat the cloning at a density of 0.2 cells per well (2 cells per ml).  Two rounds of cloning are generally enough to achieve clonality.

7)   Test the mAb using a quick isotyping kit.  If the kit shows more than one isotype, it is generally an indication that the culture is not clonal.  Perform another round of cloning at low density (0.2 cells per well).

F)  Produce mAbs

1)   Although some hybridomas can adapt to growth in RPMI/10% FCS (without HFCS supplement), most are addicted to the HFCS supplement and will stop growing (or maybe even die) when HFCS is removed.

2)   Conditioned supernatants of hybridoma cultures perform well in various types of assays when diluted 1:10 or 1:100.  The mAb is usually present at a concentration of 1-10 µg/ml.  To make conditioned supernatant, simply allow the hybridoma culture to grow to near-confluence in HFCS-supplemented medium.  The conditioned medium should be orange verging on yellow.  Remove the conditioned supernatant and spin out any suspended cells. Transfer the clarified supe to a fresh tube and buffer to neutral pH by adding 10 mM Hepes (starting from 1M stock pH 7.5, Invitrogen).  Conditioned supe can be stored at 4ºC for at least a couple of years with little loss of antibody activity.

3)   NIH rules now make it extremely difficult to generate traditional ascites.  Unfortunately, the alternative approach of purifying mAbs out of culture supernatant is a pain in the neck.  Cow antibodies present in fetal calf serum bind to protein A and protein G and will therefore compete against the mAb for binding to these resins.  Some mAbs have kappa chains that bind protein L.  Since protein L does not bind cow antibodies, protein L resin may provide an easy way to purify mAbs out of FCS-containing medium. Unfortunately, this approach is only possible if the mAb happens to have a protein L-binding kappa chain (this must be determined empirically). If purification is necessary (e.g. for making direct-conjugates) and the mAb does not bind protein L, use the following procedure:

A)   Grow up roughly 10⁸cells in RPMI/10% FCS/HFCS medium.  

B)   Transfer the cells into 50-100ml of Hybridoma-SFM(Invitrogen).  Since most hybridomas will divide very slowly in Hybridoma-SFM, it’s important to expand the culture to a large number of cells in HFCS+FCS medium before transferring into serum free conditions.  For some hybridomas, it may be necessary to supplement the Hybridoma-SFM medium with HFCS supplement to prevent mass die-off.  The HFCS supplement does not seem to contain significant amounts of unwanted antibodies.

C)  Use a Pierce protein G kit to purify the mAb out of the serum-free conditioned supernatant.

Important: the purified antibody must be dialyzed or gel filtered to remove the protein G elution buffer after purification. Although the Tris neutralization step called for in the protein G kit brings the purified antibody to near neutral pH, the pH will gradually drift down during storage at 4ºC.  The gradual return to acidic (pH 5) conditions can result in destruction of antibody function.  It’s therefore important to exchange the purified antibody into a more stable (amine-free) buffer system (e.g., 50 mM phosphate 7.5, PBS, or HBSS).

D)  If larger amounts of antibody are needed, it may be useful to concentrate antibodies out of larger volumes of conditioned culture supernatant using ammonium sulfate (see instructions for Pierce# 45216).  Ammonium sulfate stocks of mAbs seem to be pretty stable.