Abstract
In the next 10 years, the pharmaceutical industry anticipates that revenue from biotherapeutics will overtake those generated from small drug molecules. Despite effectively treating a range of chronic and life-threatening diseases, the high cost of biotherapeutics limits their use. For biotherapeutic monoclonal antibodies (mAbs), an important production cost is the affinity resin used for protein capture. Cleaning-in-place (CIP) protocols aim to optimise the lifespan of the resin by slowing binding capacity decay. Binding assays can determine resin capacity from the mobile phase, but do not reveal the underlying causes of Protein A ligand degradation. The focus needs to be on the stationary phase to examine the effect of CIP on the resin. To directly determine both the local Protein A ligand concentration and conformation on two Protein A resins, we developed a method based on attenuated total reflection (ATR) Fourier transform infrared (FTIR) spectroscopy. ATR-FTIR spectroscopic imaging revealed that applying a carefully controlled load to agarose beads produces an even and reproducible contact with the internal reflection element. This allowed detection and quantification of the binding capacity of the stationary phase. ATR-FTIR spectroscopy also showed that Protein A proteolysis does not seem to occur under typical CIP conditions (below 1 M NaOH). However, our data revealed that concentrations of NaOH above 0.1 M cause significant changes in Protein A conformation. The addition of >0.4 M trehalose during CIP significantly reduced NaOH-induced ligand unfolding observed for one of the two Protein A resins tested. Such insights could help to optimise CIP protocols in order to extend resin lifetime and reduce mAb production costs.