Time critical purification of multiple Fab constructs for assay
As a leading protein purification company, BiVictriX required us to develop a method to purify 10mg each of 24 different antibody-binding fragments (Fabs) to be tested in assays. Once analysed by BiVictriX, a smaller number of chosen constructs were to be scaled up by us to generate 100 mgs for use in their conjugation experiments.
BiVictriX had a tight deadline and we had limited time in which to deliver the proteins. We needed a smooth process to carry out multiple HEK293 cell cultures, develop the purification methodology and generate sufficient quantities of all 24 proteins to >95% purity.
Our Solution
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.
The Impact
Careful planning, method streamlining and timely responses resulted in the provision of all 24 proteins with full QC to BiVictriX in line with their schedule. This enabled assay data to be generated quickly and constructs chosen for purification scale-up for the next stage in their process.
“…Peak Proteins Fabs are behaving much better than those generated elsewhere, both in terms of purity and binding affinity. We will be sure to let people know the quality of your work….”Tiffany Thorn, CEO BiVitriX
