TABLE I

Genetic Methods for Characterizing Clonal Origin^{^a}

Method	Production Cell Line	Analytical Subclones
Southern Blots	Traditionally used to compare MCB with EOP cells. Accepted methodology for BLA filing.	A single shared hybridization band in a suitable number of analytical subclones can provide assurance of clonal origin.
FISH	Karyotype analysis of individual cells from the MCB can identify unique consistent integration sites to support clonal origin.	Not necessary, as integration site consistency can be detected in MCB.
NGS	Whole genome sequencing and TLA can provide detailed transgene and integration site DNA sequence.	Unique transgene integration sites or sequence variant markers can be used as clone-specific markers for subsequent PCR-based assays.
NGS	TLA provides greater sequence coverage of the targeted transgene and flanking genome and can detect low frequency gene of interest sequence variants.	Markers identified in a suitable number of analytical subclones can provide assurance of clonal origin. These NGS methods could be performed directly on analytical subclones but are data and cost intensive compared with PCR-based assays.
PCR	Inverse and splinkerette PCR can be used to identify transgene genomic integration fusion junctions and flanking sequences.	Genomic integration site junctions are unique identifiers that can be used for comparing a suitable number of analytical subclones to provide assurance of clonal origin.