To demonstrate primary structure comparability between ABP-450 and the reference product (Botox^®) to support biosimilarity.

Two independent methodologies were used:

1.  Genomic Sequence Alignment: The DNA sequence encoding the Botulinum neurotoxin type A1 (BoNT/A1) gene in the ABP-450 production strain was obtained and compared to that of the published reference product sequence.

2.  LC-MS Peptide Mapping: A highly sensitive LC-MS method was developed to overcome the challenge of low active pharmaceutical ingredient (API) levels in the presence of high excipient content in the drug product.  Using this approach, a side-by-side comparison of peptide fragments of ABP-450 drug substance, ABP-450 drug product, and Botox^® was performed, including the 150 kDa core neurotoxin (BoNT/A1) and accessory proteins (NTNH, HA70, HA33, and HA17).  

Genomic analysis confirmed identical gene-to-protein translation of BoNT/A1 between ABP-450 and Botox^®. The LC-MS peptide mapping results demonstrated high peptide coverage from 93.5% to 99.3% in BoNT/A1 and accessory proteins (NTNH, HA70, HA33, and HA17). No variant peptides were observed, and peptide mass patterns across multiple lots were indistinguishable between ABP-450 and Botox^®.

The dual-method approach confirmed that ABP-450 and Botox^® share identical primary structures at both the genomic and proteomic levels. These findings support the molecular comparability required for biosimilarity.