High-Throughput Protein Quantitation for Process Optimization

Rapid, accurate and cost-effective quantitation of monoclonal antibodies (mAbs) is essential for bioprocessing. There are currently more than 140 mAbs approved by the FDA as biotherapeutic agents, representing the most rapidly growing class of new drugs. Despite advances in downstream processing technology, affinity purification of monoclonal antibodies using Protein A chromatography is still predominantly utilized.

To use Protein A resin as productively as possible it’s important to load the resin close to its dynamic binding capacity (DBC). The DBC is normally determined with small sorbent volumes before scaling up to the process level. Scientists at Pall Life Sciences investigated the effects of residence time, and mAb concentration on Protein A dynamic binding capacity. The knowledge of DBC under these conditions allowed them to scale-up the process robustly, while maintaining high resin productivity.

To measure the DBC of Protein A for mAb, feedstock is loaded onto the column until binding sites become saturated and antibody product begins to leak through. As contaminant proteins in the CHO feedstock have a large absorbance, it is impossible to accurately determine the breakthrough of mAb using UV A280. To follow the antibody breakthrough, fractions of the column flowthrough were collected, and the samples analyzed by Protein A HPLC and with a Protein A biolayer interferometry (BLI) assay.

The latter using the Sartorius Octet® R8 BLI system. The analytical methods were compared using several metrics including process time, preparation time, cost per sample, dynamic range, precision, accuracy, limit of detection (LoD) and limit of quantitation (LoQ). The results confirmed that Octet® BLI systems provide a fast, accurate and economical means of quantifying monoclonal antibodies, and decrease process development efforts.