Sequence variants (SVs) are protein isoforms that contain one or more unintended amino acid substitutions. They can arise at a single amino acid site due to a genetic (RNA/DNA) mutation or at multiple amino acid locations, potentially due to translational errors, also referred to as misincorporations. The ability to detect SVs in protein biotherapeutics is critical due to their potential impact on structural/functional characteristics, safety and efficacy. Trypsin peptide mapping with liquid chromatography-ultrahigh resolution tandem mass spectrometry (LC-MS/MS) provides the ideal workflow for the detection, identification, and relative quantitation of both genetic and translational SVs. LC-MS/MS complements next-generation sequencing (NGS) of product cDNA and amino acid analysis (AAA) of cell culture medium during clone selection and process optimization in providing sensitive, comprehensive screening to strategically prevent/minimize SVs and ensure high product quality.
The occurrence of genetic SVs was evaluated using Sanger sequencing and LC/MS. In this work, mAbs with known high and low-level genetic SVs were studied at various cell culture conditions including scale, process and cell age. While scale and process had no significant impact on genetic SV levels, low-level SVs were found to decrease with cell age whereas high level SVs remained constant.
Multiple cell culture process options and the final process conditions are analyzed via LC-MS/MS prior to lock-down of the manufacturing process. Additionally, the cell culture medium (days in culture) for all small scale, pilot and clinical batches are analyzed by AAA to ascertain amino acid nutrient levels, which provides indirect monitoring of possible misincorporation situations. For mAbs with confirmed misincorporations, AAA and LC-MS/MS-peptide mapping results primarily correlated with amino acid nutrient depletion.