Method for discovery of alternative antigen specific antibody variants

Herein is reported a method for selecting a variant of a parental antibody variable domain encoding nucleic acid, wherein the parental antibody variable domain amino acid sequence encoded by said encoding nucleic acid has at least one developability hot spot, the method comprising the steps of (i) providing a multitude of DNA-containing samples (genomic material of antibody secreting B-cell) each including one or more antibody variable domain encoding nucleic acids; (ii) performing PCR amplification of said antibody variable domain encoding nucleic acids of (i) using consensus sequence-specific primers to obtain amplification products (wherein said consensus sequence-specific primers bind to consensus sequences that are common to a plurality of genes within the genetic loci set, thereby generating a pool of amplification products); (iii) sequencing a plurality of said amplification products obtained in step (ii) in order to determine the relative proportion of each nucleotide at each position in a sequencing read; (iv) performing a sequence alignment between the sequencing read results of (iii) and the parental antibody variable domain encoding nucleic acid; (v) performing a sequence-identity/homology-based ranking of the antibody variable domain encoding nucleic acids in said sequence alignment with the parental antibody variable domain encoding nucleic acid being the perfect/template/reference sequence; and (vi) selecting the variant antibody variable domain encoding nucleic acid based on the sequence ranking of step (v), whereby the variant selected in step (vi) is selected so that the developability hot-spot is removed.