RESUMEN
Low levels of alanine to serine sequence variants were identified in an IgG4 monoclonal antibody by ultra/high performance liquid chromatography and tandem mass spectrometry. The levels of the identified sequence variants A183S and A152S, both in the light chain, have been determined to be 7.8-9.9% and 0.5-0.6%, by extracted ion currents of the tryptic peptides L16 and L14, respectively. The A183S variant was confirmed through tryptic map spiking experiments using synthetic peptide, SDYEK, which incorporated Ser at the position of native Ala in the tryptic peptide L16. Both mutations were also observed by endoproteinase Asp-N peptide mapping. The variant level of A183S was also quantified by LC-UV with detection at 280nm and fluorescence detection of tyrosine residues on the tryptic peptides. The results from LC-MS, UV, and fluorescence detection are in close agreement with each other. The levels of the sequence variants are comparable among the antibody samples manufactured at different scales as well as locations, indicating that the variants' levels are not affected by manufacture scale or locations. DNA sequencing of the master cell bank revealed the presence of mixed bases at position 183 encoding both wild and mutated populations, whereas bases encoding the minor sequence variant at position 152 were not detected. The root cause for A152S mutation is not yet clearly understood at this moment.
Asunto(s)
Alanina/química , Anticuerpos Monoclonales/química , Inmunoglobulina G/química , Mapeo Peptídico/métodos , Serina/química , Alanina/análisis , Alanina/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/genética , Células CHO , Cromatografía Líquida de Alta Presión/métodos , Cricetinae , Cricetulus , ADN Complementario/genética , Humanos , Inmunoglobulina G/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Serina/análisis , Serina/genética , Espectrometría de Masas en Tándem/métodos , Tripsina/químicaRESUMEN
Epothilone F, 21-hydroxyl-epothilone B, is an intermediate in the synthesis of BMS-310705, an antitumor compound that has been evaluated in Phase I clinical trials. A bioconversion process utilizing the Gram-positive bacterium Amycolatopsis orientalis was used to prepare epothilone F from epothilone B. In order to improve the yield of epothilone F, a mutagenesis program was performed with the goal of engineering the epothilone-B hydroxylase (EBH) enzyme to improve the yield of epothilone F through oxidative biotransformation. The mutations in EBH increased the yield of epothilone F from 21% in the recombinant expression system to higher than 80% utilizing the best EBH mutants. The studies described here show how a homology model of EBH was used to obtain an understanding of the possible mechanism that led to improved yield of epothilone F in the mutated enzymes. A novel aspect of this study is that it provides some insight into how mutations distant from the binding site can affect enzyme activity.
Asunto(s)
Dominio Catalítico/genética , Epotilonas/metabolismo , Oxigenasas de Función Mixta/genética , Mutagénesis Sitio-Dirigida , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Simulación por Computador , Sistema Enzimático del Citocromo P-450 , Oxigenasas de Función Mixta/química , Oxigenasas de Función Mixta/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
Degenerate PCR primers were used to amplify cytochrome P450 gene fragments from the high-GC gram-negative bacteria Amycolatopsis orientalis, which catalyzes the hydroxylation of epothilone B to produce epothilone F. The amplified fragments were used as hybridization probes to identify and clone two intact cytochrome P450 genes. The expression of one of the cloned genes in a Streptomyces lividans transformant resulted in the biotransformation of epothilone B to epothilone F. The conversion of epothilone B to epothilone F by the S. lividans transformant was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy.