RESUMO
Coagulation factor IX (FIX) is a complex post-translationally modified human serum glycoprotein and high-value biopharmaceutical. The quality of recombinant FIX (rFIX), especially complete γ-carboxylation, is critical for rFIX clinical efficacy. Bioreactor operating conditions can impact rFIX production and post-translational modifications (PTMs). With the goal of optimizing rFIX production, we developed a suite of Data Independent Acquisition Mass Spectrometry (DIA-MS) proteomics methods and used these to investigate rFIX yield, γ-carboxylation, other PTMs, and host cell proteins during bioreactor culture and after purification. We detail the dynamics of site-specific PTM occupancy and structure on rFIX during production, which correlated with the efficiency of purification and the quality of the purified product. We identified new PTMs in rFIX near the GLA domain which could impact rFIX GLA-dependent purification and function. Our workflows are applicable to other biologics and expression systems, and should aid in the optimization and quality control of upstream and downstream bioprocesses.
Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Coagulantes/isolamento & purificação , Meios de Cultura/metabolismo , Fator IX/isolamento & purificação , Células Cultivadas , Cromatografia de Fase Reversa , Humanos , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteômica , Controle de Qualidade , Proteínas Recombinantes/isolamento & purificação , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Carga de TrabalhoRESUMO
The appropriate regimen of platelet inhibitors that should be used in patients with immune thrombocytopenia purpura (ITP; formerly called idiopathic thrombocytopenic purpura) who are undergoing percutaneous coronary intervention is unclear. We report the case of a patient with ITP who underwent two separate coronary interventions. The first involved the use of aspirin and a cutting balloon to treat obstructive disease of the left circumflex. When the patient presented with restenosis, he received eptifibatide, clopidogrel, and an intracoronary stent. He is currently 16 months removed from his second procedure and remains physically active without any anginal symptoms. Percutaneous revascularization in patients with ITP remains a challenge and this therapeutic approach, while ultimately successful in the patient, requires further validation.