Engineer pI

The Engineer pI tool allows for the identification and mutation of surface-exposed, charged residues to shift the isoelectric point (pI) of an antibody while monitoring the impact on humanness scores.

Accessing the Tool

Select exactly one antibody in the Project View which has an associated PDB file. Go to the Edit menu and select Engineer pI. This will open the Engineer pI workspace in a new tab.

Using the Tool

Targeting Residues

Select Shift Direction: Choose whether to shift the pI "Higher" (targeting Acidic residues D, E) or "Lower" (targeting Basic residues K, R, and H).
Select Replacement Pool: Choose which residues to include in the search. The default is all but those being shifted from.
Select SASA Cutoff: Adjust the Solvent Accessible Surface Area (SASA) cutoff to target only residues whose sidechains are sufficiently exposed (default is 10.0 Å²).
Click Calculate Targets to generate a list of residues that meet the shift and exposure criteria.

At this point, the Target Mutants tab will be populated with a grid of residues that meet the shift and exposure criteria. The tool suggests the highest likelihood mutation from the Replacement Pool for each position based on AbLang probabilities.

Target Mutants

Creating Mutation Designs

Select the desired residues in the Target Mutants tab.
Set Max Combinations to limit the number of variants generated.
Set Max Humanness Loss to limit lowering of the humanness score (relative to the parent) to achieve the pI shift.
Click Build Combinations in the Target Mutants tab to generate a set of combinatorial variants based on the selected residues.

Pay attention to the number of variants in the upper right corner of the Target Mutants tab to be generated. If the number is too large, the tool will take a long time to process.

During Build Combinations all possible combinatorial variants are built from the selected residues. If the variant's humanness score is beyond the cutoff it is discarded. After humanness, all variants must have a pI shifted in the desired direction. The resulting variants are sorted by pI (rounded to the nearest 0.01) and then sorted by Humanness score (high to low), and the top Max Combinations variants are shown in the Combinations tab.

The Max Combinations and Max Humanness Loss parameters may be adjusted after evaluation of the generated variants and Build Combinations re-run.

Combinations

Reviewing and Building Variants

Select the desired variants to be built in the Combinations tab.
Click Build FASTA to generate and download a FASTA file of the targeted variants.
Click Build Selected to build and process the selected variants into the project view.