RESUMO
Recombinant antibodies produced in Chinese hamster ovary (CHO) cells often exhibit a slight yellow-brown color, but the molecular basis for this color has remained elusive. We predicted that the color results from post-translational modifications on the antibody, because colored species were found to coelute with antibody products during size exclusion chromatography. Previously, modification by oxidation and advanced glycation end products (AGEs) had been shown to give rise to colored and fluorescent species whose spectral properties are in agreement with the spectral properties of CHO-derived recombinant antibodies. To test whether these modifications give rise to the color exhibited in our CHO-derived antibodies, we searched for 8 different oxidation and 28 different glycation and AGE modifications by mass spectrometry in a variety of samples exhibiting varying color intensities. Oxidation and glycation modifications correlated weakly with color in a subset of samples, but several AGEs exhibited a strong correlation with product color in all samples tested. This strong correlation with sample color was verified for a specific AGE, carboxymethyllysine, by ELISA, thus validating the mass spectrometry data. These data indicate that AGEs are at least partially responsible for the color seen in CHO-derived recombinant antibodies.
Assuntos
Anticorpos Monoclonais/química , Produtos Finais de Glicação Avançada/química , Imunoglobulina G/química , Lisina/análogos & derivados , Processamento de Proteína Pós-Traducional , Animais , Anticorpos Monoclonais/metabolismo , Células CHO , Cromatografia em Gel , Cor , Cricetulus , Ensaio de Imunoadsorção Enzimática , Produtos Finais de Glicação Avançada/metabolismo , Imunoglobulina G/metabolismo , Lisina/química , Espectrometria de Massas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
The multi-attribute method (MAM), a liquid chromatography-mass spectrometry (LC-MS)-based peptide mapping method, has gained increased interest and applications in the biopharmaceutical industry. MAM can, in one method, provide targeted quantitation of multiple site-specific product quality attributes, as well as new peak detection. In this review, we focus on the scientific and regulatory considerations of using MAM in product quality attribute monitoring and quality control (QC) of therapeutic proteins. We highlight MAM implementation challenges and solutions with several case studies, and provide our perspective on the opportunities to use MS in QC for applications other than standard peptide mapping-based MAM.