The overall goal of this method is to develop a high-yielding serum free expression system for the lab scale production of a therapeutic antibody. This method can address key considerations in the design of an antibody expression system such as producing a stable cell line, the addition of supplements, and optimizing purification strategies. The main advantage of this technique is that producing a stable cell line adapted to serum media will yield robust and reproducible quantities of therapeutic antibody.
To begin, culture HEK-293 cells according to standard protocols in serum-free medium supplemented with 0.1%nonionic surfactant, in Erlenmeyer flasks. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide at 120 rotations per minute. Maintain the culture at two times 10 to the fifth cells per milliliter and subculture every fourth day.
On the day prior to transpection, seed HEK-293 cells at three times 10 to the fifth cells per milliliter in the wells of a 12 vial plate in two milliliters of DMEM with 10%heat inactivated FBS. On the day of transpection, check that the cells have reached 80 to 90%confluency. Dilute 1.25 micrograms of vector DNA per well, in 300 microliters of transpection medium.
Then use 300 microliters of transpection medium to dilute 2.5 microliters of one milligram per milliliter of polyethylenimine, or PEI solution, per well. Incubate both solutions at room temperature for 5 minutes. Next, add the diluted DNA to the diluted PEI.
Gently mix, and incubate the solution at room temperature for fifteen minutes. Then add 50 microliters of the DNA PEI mixture drop wise to each well of the plate. Gently rock the plate to distribute the transpection mixture then incubate the plate at 37 degrees Celsius and 5%carbon dioxide for 24 hours.
The following day, add 50 micrograms per milliliter of hygromycin B per well and return the plate to the incubator for 10 days. Following transpection, successful stable transfectants will remain viable and adherent. To carry out serum-free adaptation, replace the medium in the wells with 1/4 the volume of serum-free medium and 3/4 the volume of DMEM with 10%FBS.
Incubate the cultures for four days. Then replace the medium with half the volume of serum-free medium and half the volume of DMEM with 10%FBS. And incubate for an additional four days.
Now, replace the medium with 3/4 the volume of serum-free medium and 1/4 the volume DMEM supplemented with 10%FBS. And incubate for an additional four days. Finally, replace the medium with serum-free medium supplemented with 0.1%of a nonionic surfactant and incubate for four more days.
Following the successful adaptation of stable cells, use a pipette to gently mix the suspension cultures in a 12 well plate and pool the cells in a separate tube. With the hemocytometer, enumerate the pooled cells and seed the cells in 30 milliliters of serum-free medium in a flask at two times 10 to the fifth cells per milliliter. Culture the cells at 37 degrees Celsius and 5%carbon dioxide at 120 rotations per minute.
Subculture the cells and expand every fourth day. To compare antibody yields from unsupplemented and nutrient-supplemented cultures, seed cells at two times 10 to fifth cells per milliliter in 100 milliliters of serum-free medium in two flasks. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide at 120 rotations per minute for 24 hours.
To the nutrient-supplemented culture, add tryptone to a final concentration of 0.5%then use serum-free medium to equalize the volume of the unsupplemented culture. Incubate the cells for an additional seven days. From the eighth day of culturing forward, count the cells daily using a hemocytometer to monitor cell viability.
Once the cell viability reaches less than 80%harvest the culture by centrifugation at 3000 times G for 15 minutes. Then filter sterilized a supernatant through a 0.22 micrometer filter. Store the supernatants at four degrees Celsius in the short term or freeze at negative 20 degrees Celsius long term.
After preparing buffers and initializing the FPLC system according to the text protocol, use the method editor to set up the run steps as follows. Equilibrate the system with five column volumes, or CV of binding buffer. Load the sample onto the column and collect the sample flow through.
And then use five CV of binding buffer to wash the system. Use five CV of elution buffer to elute the column via isocratic fractionation. And collect the purified antibody samples as fractions using the fraction collector.
Finally, equilibrate the system with five CV of binding buffer and collect the flow through into a waste container. Once the method set up is complete, specify the volume of conditioned medium to be a applied to the column and save the method. Next, submerge the sample pump tubing into the vessel containing the conditioned medium.
Then prepare a separate container to collect sample flow through via the specified outlet tubing. Insert collection tubes in the fraction collector and add neutralization buffer to each collection tube. In the system control module of the software, open the method to be used.
Then click start to initiate the run. When the purification is complete, in the evaluation module, check the resulting chromatogram. Combine all the protein containing fractions into a tube and then use PBS and a centrifugal filter device with a 30 kilodalton cut-off to concentrate the sample and carry out buffer exchange.
Finally, use the BCA assay to measure the antibody concentration according to the manufacturer's instructions. Stable production of trastuzumab bar transfected HEK-293 cells was confirmed using biolayer interferometry as presented in this figure. An IGG standard curve was generated by measuring the binding rate between an IGG antibody standard and a protein A biosensor.
The crude supernatant sample was similarly measured and its concentration interpolated at 25 micrograms per milliliter from the standard curve. As shown in this chromatogram, measurement of PH conductivity and system pressure are monitored during sample loading, column washing, and finally, elution of the bound protein. Three buffers were tested for optimal binding of trastuzumab to the column and washing to maximize elution yield.
Scouting the optimal elution PH and buffer revealed that the antibody eluded more efficiently at a lower PH using 0.1 molar glycine HCL or citric acid. In addition, elution peaks with 0.1 molar glycine HCL appeared less broadened when compared with 0.1 molar citric acid at a similar PH.The purification chromatograms of batch overgrow cultures are shown here. The tryptone supplemented culture yielded 3.8 milligrams and the unsupplemented culture 1.7 milligrams of trastuzumab as measured following buffer exchange and concentration.
Finally, this STS page shows that trastuzumab grown in unsupplemented and tryptone supplemented conditions results in similar antibody profiles under non-reducing and reducing conditions. Once optimized, this process of producing a stable cell line that is adapted to serum-free culture and harvesting practical quantities of antibody can be done in under six weeks if it is performed without mishap. While attempting this procedure, it's important to critically monitor the cells during the adaption process.
The four day turnover is a recommendation and if cells are struggling at a particular adaption step, then subculture at the previous adaption step before proceeding. Following this procedure, other methods such as size exclusion HPLC, ELISA, and western blotting can be performed in order to further characterize and confirm the antibody product. After watching this video, you should have a good understanding of how to produce a stable cell line and adapt it to serum-free conditions in pursuit of developing a high-yielding expression system for the lab scale production of antibody protein.
