Aggregation Mitigation Case Study

  • Re-engineering

    Case Study #1

    Following expression and Protein A purification the candidate sequence was shown to have a high and unacceptable levels of aggregation. The selected sequence was re-engineered by targeting aggregation hotspots and other motifs involved in aggregation.  Specific substitutions were made to reduce the aggregation propensity of the molecule. New variants were expressed, purified and characterized in parallel with the wild-type. Aggregation was successfully reduced in all variants by up to 80% compared to the wild type.

    Figure 1.  Antibody variants were re-engineered to reduce aggregation, measured by SE-HPLC. Wild-type (WT) on the far right shows approximately 9% aggregation (high), post-Protein A purification. Aggregation of variants is reduced by up to 80% (variant 3). The number of amino acid changes necessary to reduce aggregation (in this instance) is highlighted to indicate that re-engineering is kept to a minimum (where possible). Aggregation was reduced for all variants.

  • Binding Affinity

    Case Study #1

    Re-engineered variants were analysed for the retention of binding affinity. All variants, including those where substitutions had been made in the CDR, retained binding affinity comparable to that of wild-type.

    Figure 2.  Binding affinity of mAbs following re-engineering. All mAb variants show comparable biniding affinity to the wild-type.

  • Productivity

    Case Study #1

    High aggregation is often associated with lower productivity. Re-engineered variants were monitored for productivity compared to wild-type, and in all cases an increase in relative productivity was observed.

    Figure 3.  The relative productivity of each variant following re-engineering to reduce aggregation.