Editing 293F Protein Production (section)

=== Methods<br><br>  ===

==== Cell Culture<br>  ====

1. HEK-293 cells are subcultivated every 3–4 days by inoculation in 100 mL Ex-cell® 293 medium (when used for cell culture, the medium contains L-glutamine and phenol red in a 250-mL square-shaped glass bottle at an initial cell density of 0.3 × 10^6 cells/mL.<br>2. Determine the cell density and viability by trypan blue staining using a Neubauer hemocytometer chamber and an inverted phase contrast microscope.<br>3. After cell counting, transfer 3–4 × 107 cells into a 50-mL centrifuge tube and centrifuge at 500 × g for 5 min in a standard tabletop centrifuge.<br> 

4. The medium is removed by aspiration or decanting. The cell pellet is resuspended in 10 mL of Ex-cell® 293 medium and transferred to a 250-mL square-shaped bottle containing 90 mL of prewarmed Ex-cell® 293 medium.<br>5. Attach the bottle to a platform mounted on an orbital shaker using double-sided adhesive transfer tape (3M) and agitate at 110 rpm at 37°C in a 5% CO2 atmosphere without humidity. Keep the cap of the bottle opened about one quarter of a turn.<br> 

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==== Cell Expansion for Transfection<br><br>  ====

1. Count cells one day before transfection.<br>2. Transfer 6 × 10^8 cells into a 250-mL centrifuge bottle. For this step, it is necessary to have two 100-mL cultures.<br>3. Centrifuge the cells for 10 min at 1,000 rpm at room temperature.<br>4. Remove the medium by aspiration or decanting and gently resuspend the cell pellet in 50 mL of prewarmed Ex-cell® 293 medium.<br>5. Transfer 25 mL into each of two 1-L square-shaped glass bottles with 275 mL of Ex-cell® 293 medium. The starting cell density of each culture is about 1 × 10^6 cells/mL.<br>6. Place the bottles on an orbital shaker and incubate at 37°C overnight with agitation at 110 rpm. Keep the bottle caps open one quarter of a turn.<br><br> 

==== Transfection<br><br>  ====

1. Count cells in the two 1-L bottles.<br>2. Transfer a total of 1 × 10^9 (1 billion) cells from the two overnight cultures into two 500-mL centrifuge bottles (Corning) and centrifuge at 1,000 rpm for 10 min at room temperature.<br>3. Remove the medium by aspiration or decanting and resuspend the cells from the two centrifuge bottles in a total volume of 50 mL by addition of prewarmed RPMI 1640 medium containing 0.1% Pluronic® F-68. The cell density after resuspension is 20 × 10^6 (20 million) cells/mL.<br>4. Transfer the cells to a 250-mL cylindrical glass bottle.<br>5. Add 1.25 mg of plasmid DNA to the culture and mix gently by swirling the bottle. For the example described here, 612 ug of pXLGHEK-RhLC, 612 ug pXLGHEK-RhHC, and 25 ug pEGFP-N1 (49:49:2 w/w/w ratio) are added to the culture.<br> 

6. Add 3.75 mL linear 25-kDa PEI solution (1 mg/mL) to the culture and gently mix by swirling the bottle.<br>'''1 billion cells:&nbsp; 1.25 mg DNA + 3.75 mg PEI ---&gt; 50 ml of 20 million cells/ml. ''''''PEI to DNA ratio is 3'''.<br> 

7. The bottle is agitated in an incubator shaker at 110 rpm at 37°C in a 5% CO2 atmosphere without humidity with the bottle cap slightly opened.<br>8. After 3 h of incubation, transfer the cells to a 5-L cylindrical glass bottle containing '''950 mL''' of prewarmed Ex-cell® 293 medium.<br> 

'''Final volumn is 1000 ml.'''<br>9. '''Add 7 mL of 0.5 M VPA to achieve a final concentration of 3.75 mM.'''<br>10. Incubate the culture as in step 7.<br>11. After 1 day of incubation, the analysis of transfection efficiency is performed on a small aliquot (20 uL) by flow cytometry on a Guava EasyCyte (Millipore). The sample is diluted 1:10 in PBS and the percentage of GFP-expressing cells is determined by counting at least 10,000 cells.<br><br> 

==== <u>Small Scale (10 mL transfection)</u><br>  ====

Resuspend 10 × 10^6 cells in 0.5 mL of RPMI 1640<br> 

Add 12.5 ug plasmid DNA and 37.5 uL PEI (1 mg/mL stock solution). <br> 

After transfection add 9.5-mL Ex-cell 293 medium.<br> 

Add 70 uL of 0.5 M VPA 24 hours later.<br> 

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==== <u>Medium Scale (100 mL transfection)</u><br><br>  ====

Resuspend 100 × 10^6 cells in 5 mL of RPMI 1640.<br> 

Add 125 ug plasmid DNA and 375 uL PEI (1 mg/mL stock solution). <br> 

After transfection add&nbsp; 95 mL Ex-cell 293 medium.<br> 

Add 700 uL of 0.5 M VPA 24 hours later.<br> 

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==== Analysis of Antibody Production<br><br>  ====

1. To measure recombinant antibody accumulation over time, 100 uL aliquots are taken daily during the production phase. After centrifugation to remove cells, the antibody concentration in each sample is measured by sandwich ELISA.<br>2. Coat a 96-well ELISA plate overnight at 4°C with 100 uL of goat anti-human kappa light chain IgG diluted in PBS (coating solution).<br>3. Remove the coating solution with a multichannel pipettor and wash each well three times with 200 uL of washing buffer. The final wash is performed just<br>before the samples are loaded into the wells. After the final wash, tap the plate on a paper towel to remove any remaining wash solution.<br>4. Samples from the culture are diluted 1:10 in blocking buffer, and then 200 uL of each is loaded in triplicate onto the plate. Two serial two-fold dilutions in blocking buffer (100 uL sample + 100 uL blocking buffer) are done directly on the plate.<br>5. Load the antibody standard as serial 1:2 dilutions in triplicate on the plate.<br>6. Incubate the plate for 1 h at 37°C and then remove the samples using a multichannel pipetter.<br>7. Wash each well three times with 200- uL washing buffer as in step 3.<br>8. Add AP-conjugated goat anti-human gamma chain IgG diluted 1,000-fold in PBS to each well. For each plate, dilute 11 uL of antibody in 11 mL of PBS and add 100 uL to each well.<br>9. Incubate the plate for 1 h at 37°C.<br>10. Remove solutions and wash each well three times in 200 uL washing buffer as in step 3.<br>11. Add 100 L substrate solution to each well and cover the plate with aluminum foil.<br>12. Incubate the plate for 15 min at room temperature with gentle agitation.<br>13. Stop the reaction by addition of 100 L of 3 M NaOH.<br>14. Measure the absorbance at 405 nm using a microplate reader.<br>15. Determine the antibody concentration in each sample after generation of the standard curve from the absorbance of the standard antibody samples.<br><br> 

==== <br>Harvest and Purification of the Recombinant Antibody<br><br>  ====

<br>1. At the end of the production phase of 7–12 days, cells are harvested by transferring the culture into two 500-mL centrifuge bottles (Corning).<br>2. Centrifuge at 2,000 × g for 15 min at 4°C. <br>3. Recover the supernatant by decanting into a 1-L bottle.<br> 

4. Remove any additional cell debris by filtration with a 500-mL filter unit with a 0.45- m membrane (Corning) and transfer the filtrate into two 500-mL centrifuge bottles (Corning).<br> 

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'''If protein has 6His tag, Ni beads can be used for purification: dialyse filtered medium against 25 mM phosphate buffer saline (PBS, pH8.0) at 4 °C o/ before loading to Ni column.''' 

<br>5. Equilibrate the Streamline-rProtein A beads. For 1 L of supernatant containing about 1 g of antibody, pipette 20 mL beads into a 250-mL centrifuge bottle. Wash the beads three times by addition of 200 mL of 20 mM sodium phosphate (pH 7.5) to the bottle. Mix gently by inverting several times. Let the<br>beads settle to the bottom of the bottle and carefully remove most of the buffer by decanting, paying attention not to lose the beads.<br>6. With a 10-mL pipette, transfer the washed beads along with the remaining buffer to the two 500-mL centrifuge bottles with the cell culture medium and incubate overnight with agitation on an STR4 rotator at 4°C.<br>7. Let the beads settle by gravity to the bottom of the conical centrifuge bottles and then transfer them to a chromatography column with a 10-mL pipette. Let the beads deposit at the bottom of the column. Remove the tip of the column to allow liquid flow.<br>8. After the medium has drained from the column, wash the beads with 10 column volumes (CVs) of 20 mM sodium phosphate (pH 7.5) and collect the flow-through in a clean beaker.<br>9. Elute the antibody from the beads with 5 CVs of 100 mM sodium citrate (pH 3.0) and collect in a 250-mL bottle.<br>10. Add one volume of 1 M Tris–HCl (pH 8.0) to the eluate. Store at 4°C until the solution is ready to be concentrated.<br>11. Regenerate the column with 5–10 CVs of 100 mM sodium citrate buffer (pH 3.0).<br>12. Reequilibrate the column with 5 CVs of 20 mM sodium phosphate (pH 7.5).<br>13. Remove the beads from the column by adding 20 mM sodium phosphate buffer (pH 7.5) with 20% ethanol and pouring them in a 50-mL centrifuge tube.<br>14. Store at 4°C.<br>15. Concentrate the partially purified antibody and change the buffer to PBS using a Centricon Plus-70 (Millipore) according to the manufacturer’s protocol.<br>16. Determine the antibody concentration by measuring absorbance at 280 nm. Convert absorbance units to concentration with the conversion factor (1 U A280 = 1 mg/mL antibody).<br><br>