RESUMO
Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.
Assuntos
Imunoglobulina G/química , Imunoglobulina G/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Estudos de Viabilidade , Humanos , Radical Hidroxila/farmacologia , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Dados de Sequência Molecular , Mutação , Estabilidade Proteica , Proteólise/efeitos dos fármacosRESUMO
We report a case study of an IgG1 with a unique basic charge variant profile caused by C-terminal proline amidation on either one or two heavy chains. The proline amidation was sensitive to copper ion concentration in the production media during cell culture: the higher the Cu ( 2+) ion concentration, the higher the level of proline amidation detected. This conclusion was supported by the analysis of samples that revealed direct correlation between the proline amidation level observed from peptide maps and the level of basic peaks measured by imaged capillary isoelectric focusing and a pH gradient ion-exchange chromatography method. The importance of these observations to therapeutic antibody production is discussed.
Assuntos
Amidas/metabolismo , Anticorpos Monoclonais/química , Cobre/farmacologia , Imunoglobulina G/química , Amidas/química , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Células CHO , Cromatografia por Troca Iônica , Cobre/análise , Cricetinae , Meios de Cultura/química , Humanos , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Focalização Isoelétrica , Mapeamento de Peptídeos , Prolina/química , Força Próton-MotrizRESUMO
We report a case study of characterization of a non-enzymatically glycated IgG1 using reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and mass spectrometry (MS). Glycation was found to occur nonspecifically at multiple sites in both the light and heavy chains. The glycated light and heavy chains result in wider peaks eluting late in the reducing CE-SDS profile; in particular, the glycated light chain behaved as a shoulder peak detected by either ultraviolet (UV) or laser-induced fluorescence (LIF) signals. The glycated species can be enriched by boronate affinity chromatography. Analyzing the enriched samples by reversed phase high-performance liquid chromatography in line with time-of-flight MS (RP-HPLC-TOF/MS) revealed adducts of +162 and +324 Da to both the light and heavy chains, suggesting the presence of multiple glycation sites. Tryptic peptide mapping and tandem mass sequencing were used to identify two glycation sites on each of the light and heavy chains.
Assuntos
Eletroforese Capilar/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Imunoglobulina G/química , Animais , Anticorpos Monoclonais/química , Células CHO , Cromatografia de Afinidade/métodos , Cromatografia Líquida de Alta Pressão/métodos , Cricetinae , Cricetulus , Glicosilação , Espectrometria de Massas/métodos , Mapeamento de Peptídeos/métodosRESUMO
Susceptibility of tryptophan (Trp) in a complementarity-determining region (CDR) to oxidation is a significant issue for recombinant monoclonal antibody (mAb) therapeutics due to the clinical efficacy and stability concerns. Here we present a case study using hydrophobic interaction chromatography (HIC) to separate an oxidized Trp containing population of an IgG1. The best separation was achieved using dual Dionex ProPac HIC-10 columns, and the oxidized Trp population was isolated as a separated pre-peak. Peptide map analysis revealed that the oxidized Trp is located in a heavy chain CDR. In addition, the HIC method was capable of monitoring the oxidation status of the CDR Trp, as the oxidation rate of the CDR Trp measured by HIC directly correlated with the results of the peptide maps. The same method conditions were also capable of separating oxidized methionine (Met) and isomerization/deamidation products, which co-elute as another pre-peak at a different retention time from the oxidized Trp species. These observations indicate that the HIC procedure can be utilized to monitor the oxidative status of the CDR Trp in the IgG1.