Recombinant protein expression and purification for monoclonal antibody generation

1. P 3 —a Protein Production Platform for mAb Generation : a Series of Case Studies Part IV Carlo Ciatto, PhD [email_address]

2. Cases 6 and 7

3. Purified antibody probed with anti-IgG (H+L) Cell lysates probed with purified antibody and Integra supernatant (+): stimulated (-): non-stimulated 6: One antibody or two antibodies? Compared to Integra flask supernatant, 201B purified antibody reacts with extra bands when used to probe cell lysates. When analyzed by WB, the purified antibody itself shows up as two bands. Is the purified antibody contaminated with a second one, or are the two bands just different glycosylated forms? Pure Supe

4. 1: purified antibody 2: precipitated material (elutes in the void volume) 3: antibody/peptide complex 4: unbound peptide and antibody One antibody! Gel filtration analysis with analytical Superdex200 column II ary : goat anti-mouse IgG (H+L) 1 2 3 4 1 201B purified antibody a 2 3 4 Antibody/peptide mix c BSA-peptide (immunization antigen) b

6. Two antibodies! Stepwise elution from protein A with decreasing pH. Acidic elution—typical of IgG2b—reveals presence of correct antibody, despite lack of reactivity with isotyping strip