RESUMO
One of the most important structural features of recombinant monoclonal antibodies produced in mammalian cells is the N-linked oligosaccharide profile. These profiles impact recombinant therapeutics in a multitude of ways affecting distribution, efficacy, and immunogenicity. High mannose, alpha-gal and other oligosaccharide species are highly immunogenic and in most cases should be minimized during manufacturing. A recombinant monoclonal antibody, h5G1.1, was produced in NS0 and CHO cell lines and tested to identify changes in the N-linked oligosaccharide profiles caused from a change in cell line. Traditional peak analysis using HPLC with fluorescence detection was augmented by mass spectrometric analysis. Nano LC-MS following tryptic digestion corroborated HPLC findings of the presence of several alpha-gal oligosaccharide species in the recombinant IgG (rIgG) from NS0 cell line. Both cell lines possessed rIgGs with complex and small amounts of high mannose glycans.
Assuntos
Anticorpos Monoclonais/química , Cromatografia Líquida de Alta Pressão/métodos , Oligossacarídeos/análise , Espectrometria de Fluorescência/métodos , Animais , Células CHO , Cricetinae , Cricetulus , Espectrometria de Massas/métodos , Proteínas Recombinantes/químicaRESUMO
A number of HPLC and mass spectrometric techniques are used to characterize post-translational modification in recombinant monoclonal antibodies (MAbs) using the intact glycoprotein and free glycans. LC separation utilizing fluorescent detection technique allows tentative structural assignment of MAb oligosaccharides. Intact molecular weight analysis via electrospray allows for an accurate mass determination and observation of the native glycoform mass envelope. N-linked oligosaccharides are then analyzed by MALDI-ToF. Their structures are further confirmed by analyzing the fragmentation patterns formed by MS/MS. All these techniques provide useful information when performed in isolation. However, the combined information allows for definitive and robust characterization of the N-linked glycans from recombinant MAbs.
Assuntos
Anticorpos Monoclonais/química , Cromatografia Líquida de Alta Pressão/métodos , Oligossacarídeos/química , Espectrometria de Fluorescência/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Proteínas Recombinantes/químicaRESUMO
Abnormal production and accumulation of amyloid-beta peptide (Abeta) plays a major role in the pathogenesis of Alzheimer's disease (AD). beta-secretase (BACE1) is responsible for the cleavage at thebeta-site in amyloid beta protein precursor (AbetaPP/APP) to generate the N-terminus of Abeta. Here we report the stepwise identification and characterization of a novel APP-beta-site mutant, "NFEV" (APP_NFEV) in vitro and in cells. In vitro, the APP_NFEV exhibits 100-fold enhanced cleavage rate relative to the "wild-type" substrate (APPwt) and 10-fold increase relative to the Swedish-type mutation variant (APPsw). In cells, it was preferably cleaved among 24 APP beta-site mutations tested. More importantly, the APP_NFEV mutant failed to generate any detectable Abeta peptides in BACE1-KO mouse fibroblast cells. The production of Abeta peptides was restored by co-transfecting human BACE1, demonstrating that BACE1 is the only enzyme responsible for the processing of APP_NFEV in these cells. Analysis of APP_NFEV cleavage products secreted in the media revealed that in cells BACE1 cleaves APP_NFEV at the position between NF and EV, identical to that observed in vitro. A BACE inhibitor blocked the processing of the APP_NFEV beta-site in vitro and in cells. Our data indicates that the "NFEV" mutant is not only an enhanced substrate for BACE1 in vitro, but also a specific substrate for BACE1 in cells.
Assuntos
Peptídeos beta-Amiloides , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidases/genética , Fragmentos de Peptídeos , Mutação Puntual/genética , Doença de Alzheimer/enzimologia , Doença de Alzheimer/genética , Doença de Alzheimer/imunologia , Secretases da Proteína Precursora do Amiloide , Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/biossíntese , Peptídeos beta-Amiloides/genética , Animais , Anticorpos Monoclonais/imunologia , Ácido Aspártico Endopeptidases/metabolismo , Modelos Animais de Doenças , Endopeptidases , Ativação Enzimática/fisiologia , Fibroblastos/metabolismo , Regulação Enzimológica da Expressão Gênica , Técnicas In Vitro , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Especificidade por Substrato , TransfecçãoRESUMO
The amyloid beta peptides (Abeta) are the major components of the senile plaques characteristic of Alzheimer's disease. Abeta peptides are generated from the cleavage of amyloid precursor protein (APP) by beta- and gamma-secretases. Beta-secretase (BACE), a type-I transmembrane aspartyl protease, cleaves APP first to generate a 99-amino acid membrane-associated fragment (CT99) containing the N terminus of Abeta peptides. Gamma-secretase, a multi-protein complex, then cleaves within the transmembrane region of CT99 to generate the C termini of Abeta peptides. The production of Abeta peptides is, therefore, dependent on the activities of both BACE and gamma-secretase. The cleavage of APP by BACE is believed to be a prerequisite for gamma-secretase-mediated processing. In the present study, we provide evidence both in vitro and in cells that BACE-mediated cleavage between amino acid residues 34 and 35 (Abeta-34 site) in the Abeta region is dependent on gamma-secretase activity. In vitro, the Abeta-34 site is processed specifically by BACE1 and BACE2, but not by cathepsin D, a closely related aspartyl protease. Moreover, the cleavage of the Abeta-34 site by BACE1 or BACE2 occurred only when Abeta 1- 40 peptide, a gamma-secretase cleavage product, was used as substrate, not the non-cleaved CT99. In cells, overexpression of BACE1 or BACE2 dramatically increased the production of the Abeta 1-34 species. More importantly, the cellular production of Abeta 1-34 species induced by overexpression of BACE1 or BACE2 was blocked by a number of known gamma-secretase inhibitors in a concentration-dependent manner. These gamma-secretase inhibitors had no effect on enzymatic activity of BACE1 or BACE2 in vitro. Our data thus suggest that gamma-secretase cleavage of CT99 is a prerequisite for BACE-mediated processing at Abeta-34 site. Therefore, BACE and gamma-secretase activity can be mutually dependent.