Recovery & Purification
Purification Process Development for a Light-Sensitive Monoclonal Antibody
Light exposure of biopharmaceuticals may lead to chemical and physical degradation by photooxidation of tryptophan, tyrosine, phenylalanine, and cysteine/cystine. An early photo-stability assessment demonstrated that a mAb had a rapid activity loss when the protein was exposed to white and ultra-violet light. This observation led to challenges for purification process development that included determining the mAb degradation rate in varying process intermediate conditions, determining light exposure control limits to minimize inactive mAb formation, and designing a polishing chromatography step to reduce inactive mAb, formed during processing, to acceptable levels. A photo-stability study of the mAb in process intermediates showed unacceptable degradation rates at room temperature. This result led to a decision to actively minimize light exposure to process material s during the manufacture of all development and clinical materials. Peptide analysis of the isolated inactive mAb revealed that activity loss was caused by the oxidation of two tryptophan residues present at the antigen binding region. Tryptophan oxidation could lead to a localized change is surface hydrophobicity for an impacted protein. This difference in local surface hydrophobicity between the mAb and the oxidized mAb was successfully exploited to separate the two variants using hydrophobic interaction chromatography. The optimized chromatography process delivered active mAb that met design process manufacturing goals for mAb purity and yield. In conclusion, a robust manufacturing process was successfully developed through understanding of protein degradation rates in process intermediates and by applying peptide-level characterization information to guide purification optimization strategy.