1. Expression and purification of antibodies

1. Maintenance of CHO cells
   1. Grow CHO cells in FreeStyle CHO Media supplemented with commercially available 1x glutamine supplement at 37 °C shaking at 130 rpm with 5% CO₂ using delong Erlenmeyer flasks, either glass or disposable.
      NOTE: It is highly recommended to use a baffled flask with a vented cap for increased agitation and to improve gas transfer during shaking conditions. Significantly reduced antibody yield has been obtained with regular flasks (non-baffled) due to limited agitation of the suspension culture.
   2. Maintain the cell number between 1-5 x 10⁶ cells/mL with >95% cell viability. If the cell number increases over 5 x 10⁶ cells/mL, split the cells. Never allow CHO cells to reach below 0.2 x 10⁶ cells/mL.
2. Transfection of CHO cells
   1. Grow CHO cells in 200 mL of media (2 x 10⁶ cells/mL) in delong Erlenmeyer baffled flasks.
      NOTE: Suspension cultures grown in baffled flasks have always produced higher protein yields vs when grown in non-baffled flasks.
   2. In a 15 mL tube, take 5 mL of CHO FreeStyle media and add 50 μg of VH clone DNA and 75 μg of VL clone DNA. Vortex to mix well.
   3. Incubate the DNA mixture at room temperature for 5 min.
      NOTE: Longer incubation reduces the protein yields.
   4. Add 750 μL of 1 mg/mL polyethyleneimine (PEI) stock to the DNA solution and aggressively vortex the mixture for 30s. Incubate at room temperature for an additional 5 min.
      NOTE: PEI must be made fresh. Multiple freeze thaw cycle of PEI reduces overall yield significantly.
   5. Add the entire mixture of DNA and PEI to the cells while manually shaking the flask. Immediately incubate the delong Erlenmeyer baffled flasks with cells at 37 °C shaking at 130 rpm.
3. Expression
   NOTE: The antibody has a secretory signal peptide engineered to its N-terminal end, which helps antibodies to be secreted out into the media.
   1. Grow the transfected cells at 37 °C, with shaking at 130 rpm on Day 1.
   2. On Day 2, add 2 mL of 100x anti-clumping agent and 2 mL of 100x anti-bacterial-anti-mycotic solution. Shift the flask to a lower temperature (32-34 ˚C), with shaking at 130 rpm.
   3. On every fifth day, add 10 mL of Tryptone N1 feed and 2 mL of 100x glutamine supplement.
   4. Keep counting the cells every third day using a hemocytometer after staining an aliquot of cells with a trypan blue stain. Ensure that the cell viability stays above 80%.
   5. On Day 10 or 11, harvest the medium for antibody purification. Spin the culture at 3000 x g, 4 °C for 40-60 min and then filter the clear media using 0.22 µM bottle filters.
4. Purification
   NOTE: Antibody purification is performed using a commercially available Protein-A column (see Table of Materials), using a peristaltic pump.
   1. Equilibrate the column with two-column volume of binding buffer (20 mM sodium phosphate at pH 7.4).
   2. Pass the filtered media containing antibody (obtained in step 1.3.5) through the column at the flow rate of 1 mL/min.
   3. Wash the column with a two-column volume of binding buffer.
   4. Elute the antibody into 500 μL fractions using 5 mL elution buffer (30 mM sodium acetate at pH 3.4).
   5. Neutralize the pH of the eluted antibody by adding 10 μL of neutralization buffer (3 M sodium acetate at pH 9) per fraction.
      NOTE: Fraction numbers 3-6 contain most of the antibody. It is advisable to keep all the fractions in case the antibody is eluted in a later fraction than expected.
   6. Measure the concentration of purified antibody using a spectrophotometer by selecting the default protocol for IgG. The final concentration of the antibody is obtained in mg/mL by considering the molecular weight and absorption coefficient.