DOI: 10.6084/m9.figshare.31438251.v1
title: 3in1: an ultra-fast phage display biopanning method overcoming the biopanning bias and yielding more diverse binders

description: Phage display combined with biopanning is a powerful method for the discovery of therapeutic antibodies. Classic biopanning is lengthy and labor-intensive, requiring at least five days of work. The biopanning process is plagued by a bias toward binders with propagation advantages, causing a loss in diversity. We present a more efficient biopanning technique named 3in1 biopanning. Using non-denaturing elution and skipping the intermediary phage amplification steps, 3in1 biopanning performs all biopanning rounds in a single day and overcomes the inherent biopanning bias. 3in1 is five times faster than conventional biopanning and yields up to six times more unique hits with a wide range of affinities. It shortens lead discovery time by a week and reduces consumables and waste. We demonstrate that this novel biopanning technique is suitable for naive-synthetic and immune libraries against a variety of targets. Phage display is a classic method to discover new therapeutic antibodies. It modifies bacteriophage to present antibody fragments on their surface. With this method, large libraries of 10<sup>9</sup> antibodies can be built. Libraries are incubated with target antigen immobilized on a solid support and phage that display antibodies specific to the target are captured on the solid support, isolated and amplified. This cycle is repeated two to three times to yield a population enriched for binders. This process typically takes five days. We present a faster method to complete this process in a single day and yield a higher number of unique antibodies than the classic method. We describe an ultra-fast method for phage display biopanning. As with classic biopanning, phage display libraries are incubated with immobilized antigen, unbound phage are washed away and phage displaying antibodies specific to the target antigen are captured on the solid support. Instead of using a proteolytic elution, such as trypsin, this method uses non-denaturing acid elution. The eluate is neutralized and the phage are used for the next round of biopanning without intermediate amplification. With this method, we obtain fully enriched and highly diverse output populations in a single day. A biopanning method for phage display called 3in1 biopanning allows to perform three rounds of biopanning in a single day instead of five days.3in1 biopanning uses nondestructive acid elution and omits amplification between biopanning rounds.3in1 biopanning yields comparable enrichment and hit rates to classic biopanning.3in1 biopanning tends to give a higher sequence diversity than classic biopanning by eliminating the amplification bias.3in1 reduces the amount of biological and plastic waste generated during biopanning.3in1 yields a broader range of affinities, while classic panning selectively retains stronger binders and purges weaker binders A biopanning method for phage display called 3in1 biopanning allows to perform three rounds of biopanning in a single day instead of five days. 3in1 biopanning uses nondestructive acid elution and omits amplification between biopanning rounds. 3in1 biopanning yields comparable enrichment and hit rates to classic biopanning. 3in1 biopanning tends to give a higher sequence diversity than classic biopanning by eliminating the amplification bias. 3in1 reduces the amount of biological and plastic waste generated during biopanning. 3in1 yields a broader range of affinities, while classic panning selectively retains stronger binders and purges weaker binders