RESUMO
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.
Assuntos
Receptores ErbB/imunologia , Variação Genética , Glutamina , Cadeias Pesadas de Imunoglobulinas/biossíntese , Tirosina , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Linhagem Celular , Cromatografia Líquida de Alta Pressão , Clonagem Molecular/métodos , Cricetinae , Técnicas de Cultura/métodos , Primers do DNA , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Mapeamento de Peptídeos , Reação em Cadeia da Polimerase/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Transfecção , TripsinaRESUMO
Vero cells were grown in microcarrier culture and subsequently infected with herpes simplex virus (type 1). Amino acid analyses were conducted on culture supernatants using a rapid reverse-phase high performance liquid chromatography method. The utilization of amino acids by the cells during cell growth and virus production phases was examined. During cell growth, glutamine and histidine were rapidly depleted (80-90%) by Vero cells while arginine and tryptophan-menthionine (peaks not resolved) were only moderately reduced (ca. 40%). In contrast, a marked increase in the concentrations of alanine and serine in the culture medium was observed. During virus production, the rapid depletion of glutamine, as well as the release of alanine and serine, was again observed, while the concentrations of isoleucine, lysine, threonine and valine increased only moderately.