Isoelectric point, net charge and amino acid analysis of experimentally validated therapeutic antibodies

The isoelectric point (pI) of an antibody is known to affect its non-specific interactions and repulsive self-interactions. However, analytical outcomes for the pI of a large number of therapeutic antibodies remain unexplored. In this study, we explored the pI and net charge of variable heavy (VH), variable light (VL), CDR (complementarity determining regions) and whole IgG on a large number of therapeutic antibodies, additionally amino acids distribution in the CDR regions were also analyzed. A total of 708 experimentally validated antibodies from the Thera-SAbDab database were analyzed in this study. Analysis of the antibody dataset showed that the pI of the whole IgG sequence is between 5 and 9, while the majority was in the intermediate range between 7 and 9 (86.7%). The charge had a wide range from - 10 to 12, with the majority falling between the charges 2-6 (53.4%). However, the combined pI score of the CDRs of light chains (60%) as well as for the heavy chains (67%) was observed in the range of 4-6. The amino acid composition analysis of CDR regions revealed that most of the amino acids in the light chain are uncharged-polar (46.3%) followed by hydrophobic-aliphatic (28.4%), while in the heavy chain; it is hydrophobic-aliphatic (35.2%) followed by uncharged-polar (24.6%). In conclusion, the pI and net charge analysis of therapeutic antibodies are crucial for understanding pharmacokinetic properties. Moreover, amino acid composition of the light and heavy chain CDR regions has a significant impact on the pI and charge of the entire IgG antibody.

The online version contains supplementary material available at 10.1007/s40203-025-00356-y.