It was expected that most of the constructs would express to some degree, so as time was key, small scale expression grows were omitted in favour of medium scale so that steps weren’t repeated and time lost. Expression was quickly assessed using a Coomassie -stained SDS-PAGE gel of the impurified culture supernatant as a protein-free culture medium was used. Expression levels were sufficiently high to visualise without purification. This was critically important to enable purification to start with confidence. Two wild type Fabs were grown at 2L scale so that a purification method could be developed, whilst 1L grows of the other 22 constructs followed, ready for processing as soon as the method was ready.
The Fabs did not contain a purification tag however they had a basic Isoelectric Point (pI) which allowed capture on a Cation Exchange column in low pH buffer. Optimisation of a pH/salt gradient allowed sufficient purity for each of the wild type Fabs, and eluted protein was immediately buffer exchanged on a desalting column into the desired assay buffer.
Using a range of chromatography equipment and efficient time management, the method was applied and, when necessary, fine-tuned for the remaining constructs. A QC package (mass spectrometry, analytical SEC, SDS PAGE, A280) was completed for each antibody to confirm purity, identity and quality. More than 10mg of all 24 constructs with >95% purity was supplied to BiVictriX to meet their deadline. In many cases expression yields of 100-200mg/L were obtained.