Editing 293F Protein Production (section)

==== <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>