RESUMO
This white paper, which is the 10th in a series intended to address issues associated with the development of therapeutic oligonucleotides, examines the subject of product-related impurities. The authors consider chemistry and safety aspects and advance arguments in favor of platform approaches to impurity identification and qualification. Reporting, identification, and qualification thresholds suitable for product-related impurities of therapeutic oligonucleotides are proposed.
Assuntos
Técnicas de Química Analítica/métodos , Química Farmacêutica/métodos , Contaminação de Medicamentos/prevenção & controle , Oligonucleotídeos/análise , Animais , Desenho de Fármacos , Indústria Farmacêutica/normas , Feminino , Humanos , Limite de Detecção , Masculino , Camundongos , Modelos Animais , Oligonucleotídeos/síntese química , Oligonucleotídeos/uso terapêutico , Segurança do Paciente/normasRESUMO
Protective antigen (PA), the receptor-binding moiety of anthrax toxin, contains two calcium atoms buried within domain 1(') (amino acid residues 168-258). We showed that these ions are stably bound and exchange with free 45Ca(2+) only slowly (t(1/2) approximately 4.0 h). Dissociation is the rate-limiting step. PA(63), the heptameric prepore form of PA, showed a slightly higher exchange rate than the monomeric intact protein. Exchange by this form was retarded by binding of the enzymatic moieties of the toxin, but was unaffected by reducing the pH to 5.0, a condition known to trigger conversion of the prepore to the pore form. These results are consistent with the hypothesis that bound Ca(2+) within PA plays primarily a structural role, maintaining domain 1(') in a conformation that allows PA(63) to oligomerize and bind the enzymatic moieties of the toxin.