Impact of freeze–thaw processes on monoclonal antibody platform process development

Abstract

Freezing of cell culture supernatant (CCS) is a standard procedure in process development of monoclonal antibody (mAb) platform processes as up‐ and downstream development are usually separated. In the manufacturing process of mAb, however, freezing is avoided, which poses the question of comparability and transferability from process development to manufacturing. In this case study, mAb CCS from Chinese hamster ovary (CHO) cells is frozen and thawed in a novel active freezing device and subsequently captured by protein A chromatography. Critical quality attributes such as host cell protein (HCP) concentration and soluble mAb dimer shares have been monitored throughout the case study. Furthermore, cryo‐concentration of individual proteins was investigated. The main factors that drive cryo‐concentration are diffusion and natural convection. Natural convection in freezing processes was found to increase at warmer freezing temperatures and thus slower freezing, leading to higher concentration gradients from top to bottom of a freezing chamber. The freeze concentration was dependent on protein size and correlated to diffusivity, where smaller proteins are exposed to higher cryo‐concentration. Our results suggest that as a result of freezing processes, large particles based on mAb and specific host cell proteins (HCPs) expressing a certain affinity to mAbs are formed that have to be removed before purification. This leads to a significant improvement in HCP reduction by the protein A step, when compared with reference samples, where twice as much HCP remained in the eluate. Furthermore, HCP and mAb dimer concentrations in protein A eluate were dependent on the freezing temperature. As a conclusion, CCS should be frozen as rapidly as possible during process development to minimize issues of transferability from process development to manufacturing.

Raman spectroscopy as a process analytical technology to investigate biopharmaceutical freeze concentration processes

“…As shown in Equation (1), the amount of solute encapsulated at the freezing front is dependent on the solute concentration at the freezing front. Thus, larger proteins may experience overall less freeze concentration as they are entrapped by the freezing front to a higher degree, which agrees with the freeze concentration of different proteins in the cell culture supernatant (Weber et al, 2021). Kolhe and Badkar (2011) also reported significant differences in sugar and mAb freeze concentration for solutions frozen in bottles.…”