Use of polymerase chain reaction (PCR) in the development of a recombinant organism.

Tryptic mapping of a purified recombinant antibody expressed in a mammalian cell line demonstrated low level heterogeneity in the heavy chain. Amino acid analysis and N-terminal sequencing of these fragments revealed that a conversion of tyrosine (Y) to glutamine (Q) had occurred at residue 376. DNA sequencing of 30 clones of the original expression construct indicated that the chance of the Y376Q variant being present in the original plasmid was low, < 5%. Once the gene had been cloned into a mammalian cell system, sequencing of 30 clones is considered ineffective for screening large numbers of subclones. Consequently, an alternative method was developed to directly probe total mammalian DNA for the presence of cloned variant. The method described here uses a polymerase chain reaction (PCR) based assay optimizing the ability of Taq polymerase to distinguish whether the 3' end of primers have completely annealed with template DNA during PCR cycling. Ten percent of the subclones producing high levels of the variant were identified and experiments indicated that the Y to Q conversion had developed during transfection of the antibody gene into the mammalian cell system. PCR was shown to be useful as a quick screen for verification of a cloned variant. Tryptic mapping is still considered the most sensitive and appropriate analytical tool for assessing genetic heterogeneity of recombinant cell lines.

Assessing genetic heterogeneity in production cell lines: detection by peptide mapping of a low level Tyr to Gln sequence variant in a recombinant antibody.

A recombinant antibody directed against the human epidermal growth factor receptor-2 extracellular domain was subjected to detailed structural characterization. Heterogeneity in the heavy chain was demonstrated by recovery of two forms of a tryptic peptide, with either glutamine or the expected tyrosine at residue 376. Subsequent experiments indicated that the Y376Q variant developed during transfection of the antibody heavy and light chain genes into Chinese hamster ovary cells. Levels of the Y376Q variant (range: 27% to 1%) in the purified antibody were inversely proportional to cell age. The established cell line was subcloned and found to be heterogeneous by polymerase chain reaction analysis of cell extracts and protein analysis of the purified antibody. Ten percent of subclones produced high levels of the Y376Q variant while 90% of the subclones produced antibody with only the expected heavy chain sequence. This report demonstrates the utility of peptide mapping as a sensitive tool for assessing genetic heterogeneity of recombinant cell lines.