RESUMO
In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab')2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen.
Assuntos
Anticorpos Monoclonais , Eletroforese Capilar/métodos , Focalização Isoelétrica/métodos , Proteínas Recombinantes , Animais , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/química , Células CHO , Cromatografia Líquida de Alta Pressão , Cricetinae , Cricetulus , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/química , Proteínas Recombinantes/análise , Proteínas Recombinantes/químicaRESUMO
Process changes are inevitable in the life cycle of recombinant monoclonal antibody therapeutics. Products made using pre- and post-change processes are required to be comparable as demonstrated by comparability studies to qualify for continuous development and commercial supply. Establishment of comparability is a systematic process of gathering and evaluating data based on scientific understanding and clinical experience of the relationship between product quality attributes and their impact on safety and efficacy. This review summarizes the current understanding of various modifications of recombinant monoclonal antibodies. It further outlines the critical steps in designing and executing successful comparability studies to support process changes at different stages of a product's lifecycle.
Assuntos
Anticorpos Monoclonais/química , Química Farmacêutica/métodos , Química Farmacêutica/normas , Garantia da Qualidade dos Cuidados de Saúde/métodos , Proteínas Recombinantes/química , Descoberta de Drogas , Humanos , Processamento de Proteína Pós-TraducionalRESUMO
A streamlined method has been developed for the isolation and analysis of polycyclic aromatic hydrocarbons in avian blood cells and plasma utilizing quick, easy, cheap, effective, rugged, and safe extraction in combination with novel phospholipid cleanup technology. A variety of traditional extraction and cleanup techniques have been employed in the preparation and analysis of polycyclic aromatic hydrocarbonsin a variety of matrices; liquid-liquid partitioning, solid-phase extractions, gel permeation chromatography, and column chromatography are all effective techniques, however they are laborious and time consuming processes that require large amounts of solvent. Using quick, easy, cheap, effective, rugged, and safe extraction coupled with phospholipid cleanup, samples can be quickly screened while maintaining high throughput and sensitivity. With a liquid chromatography approach, analysis times may be kept short at 16 min while maintaining high analyte recovery. Recoveries in quality control samples ranged from 70 to 109%, with average surrogate recoveries of 80.6 ± 1.10%. The result of using a quick, easy, cheap, effective, rugged, and safe extraction approach in conjunction with phospholipid cleanup is a methodology that significantly reduces sample preparation time and solvent use while maintaining high sensitivity and reproducibility.
