RESUMEN
A rapid reversed-phase HPLC separation of recombinant human immunoglobulin gamma 2 (IgG2) disulfide isomers using columns packed with superficially porous particles is reported. Under optimal conditions, a separation of monoclonal IgG2 disulfide isomers was achieved in 10 min using a Poroshell™ 300SB-C8 column via a combination of high column temperature (85°C), mobile phases with high eluotropic strength (e.g. isopropanol) and high flow rate (1.5 mL/min). Thermodynamic stability analyses of chromatographically enriched IgG2 disulfide isomers revealed differences in their individual denaturation temperatures, which correlate with the observed temperature-dependent refinement of peak profiles by reversed-phase HPLC. This reversed-phase HPLC method in conjunction with other orthogonal analytical techniques (e.g. capillary gel electrophoresis, peptide mapping, ion exchange chromatography, etc.) is being used to characterize disulfide isomers in the development of therapeutic IgG2 antibodies.
Asunto(s)
Cromatografía Líquida de Alta Presión , Disulfuros/química , Inmunoglobulina G/química , Inmunoglobulina G/aislamiento & purificación , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Humanos , Isomerismo , Modelos Moleculares , Porosidad , Conformación Proteica , Temperatura , TermodinámicaRESUMEN
A CGE method for monitoring the disulfide isomer distribution characteristic of IgG2 MAbs is presented. Disulfide heterogeneity of MAbs has been studied using various chromatographic and electrophoretic methods. Although CGE operates using a different selectivity mechanism from that of sorption chromatographic techniques, similar trends are present in the data, which allow the CGE method to be used as a complementary method for studying disulfide isomer distribution. This article focuses on the optimization of a capillary-based gel electrophoresis method that can be used to support antibody development including bioprocess optimization, antibody characterization, release, and formulation stability assessment.
Asunto(s)
Anticuerpos Monoclonales/química , Disulfuros/análisis , Electroforesis Capilar/métodos , Inmunoglobulina G/química , Biofarmacia/métodos , Disulfuros/química , IsomerismoRESUMEN
Under stressed conditions such as prolonged exposure to high pH, the C-terminal disulfide bridge in bovine somatotropin (bST) is susceptible to a base catalyzed beta-elimination reaction. This reaction converts the disulfide bond to a dehydroalanine residue with loss of a sulphur atom. Two altered species were isolated in pure form and determined to be generated from this dehydroalanine intermediate. One is a monomeric lanthionyl bST (L-bST) with a thioether linkage, and the other is an inter-molecular disulfide linked dimer containing a lysinoalanine. These two novel structures were unambiguously determined using various techniques including enzymatic digestion, amino acid sequencing and analysis, and mass spectrometry. The monomeric L-bST was demonstrated to be equipotent to normal bST in a hypox rat assay, thus showing that formation of lanthionine in place of this disulfide bond does not affect it bioactivity.
Asunto(s)
Alanina/análogos & derivados , Hormona del Crecimiento/química , Alanina/química , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía por Intercambio Iónico , Hormona del Crecimiento/aislamiento & purificación , Concentración de Iones de Hidrógeno , Lisinoalanina/química , Datos de Secuencia Molecular , Mapeo Peptídico , Conformación Proteica , Multimerización de Proteína , Análisis de Secuencia de Proteína , Sulfuros/química , Espectrometría de Masas en TándemRESUMEN
Human growth hormone (hGH) is not only a valuable recombinant therapeutic protein for hormone deficiency indications, but is also an extensively characterized molecule both from recombinant bacterial systems and as circulating in humans. We describe the characterization of hGH produced in three different plant systems: tobacco cell culture, soy seed, and maize seed. The data indicate highest production in the maize seed system, with continued productivity over multiple generations, and when bred to a new host genotype for improved productivity. Purification indicated significant material of the correct structure from both plant cell culture and maize seed, with maize seed also showing correct activity relative to that produced by Escherichia coli. However, all systems showed some proteolyzed hGH, with data from gel electrophoresis, mass spectrometry, and peptide mapping localizing to a region of the protein also prone to cleavage in some other systems. Together, the data indicate the dependence of recombinant protein accumulation on posttranslational processes in different host systems.
