RESUMO
One of the important steps in developing protein therapeutics is the determination of their preliminary PK in vivo. These data are essential to design optimal dosing in animal models prior to progressing to clinical trials in man. The quantitative detection of protein therapeutics in serum is traditionally performed by ELISA, which has the prerequisite of the availability of the appropriate monoclonal antibodies. We have developed an alternative method using polyclonal antibodies immobilized on ProteinChip Arrays and SELDI-TOF mass spectrometry. This method has an advantage over ELISA since it provides simultaneously information on the clearance rate of the protein and it's in vivo processing. We compared these two methods using a RANTES variant, [(44)AANA(47)]-RANTES as the test protein in this study. Using SELDI-TOF mass spectrometry, we were able to establish that the protein is readily oxidized in serum, and moreover is processed in vivo to produce a truncated 3-68 protein, and undergoes a further cleavage to produce the 4-68 protein. These modifications are not identified by ELISA, whilst the serum exposure profiles determined by the two methods show essentially similar protein concentration values.
Assuntos
Quimiocina CCL5/sangue , Quimiocina CCL5/metabolismo , Análise Serial de Proteínas , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Quimiocina CCL5/farmacocinética , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB CRESUMO
CST3 is the coding gene for cystatin C (CysC). CST3 B/B homozygosity is associated with an increased risk of developing Alzheimer disease. We performed CysC analysis on human primary skin fibroblasts obtained from donors carrying A/A, A/B, and B/B CST3. Pulse-chase experiments demonstrated that the release of the B variant of CysC has a different temporal pattern compared to that of the A one. Fibroblasts B/B homozygous displayed a reduced secretion of CysC due to a less efficient cleavage of the signal peptide, as suggested by high-resolution Western blot analysis and by in vitro assay. In the brain, the reduced level of CysC may represent the molecular factor responsible for the increased risk of Alzheimer disease.