Thermal and pH stress dictate distinct mechanisms of monoclonal antibody aggregation.

Protein aggregation is a significant challenge in the development of monoclonal antibodies (mAbs), which can be exacerbated by stress conditions encountered along its production pipeline. In this study, we examine how thermal and pH stress conditions influence mAb aggregation mechanisms. We observe a complex interplay between these factors that significantly affects mAb stability, particularly under combined stress conditions. The mAb aggregates formed also varied distinctly in size and properties depending on the pH and thermal conditions, suggesting differences in their underlying mechanisms. Using a combination of experimental methods and kinetic modelling, we found that acidic pH conditions primarily promoted aggregation via the mAb unfolding step, while higher temperature conditions facilitated the formation of larger aggregates via monomer-independent cluster-cluster aggregation steps. These insights underscore the importance of extrinsic stress conditions in determining mAb aggregation propensity, and potentially provides a quantitative framework to holistically assess this across various accelerated stress conditions for the development of stable biologics.

Biomass specific perfusion rate as a control lever for the continuous manufacturing of biosimilar monoclonal antibodies from CHO cell cultures.

Continuous manufacturing enables high volumetric productivities of biologics such as monoclonal antibodies. However, it is challenging to maintain both high viable cell densities and productivities at the same time for long culture durations. One of the key controls in a perfusion process is the perfusion rate which determines the nutrient availability and potentially controls the cell metabolism. Cell Specific Perfusion Rate (CSPR) is a feed rate proportional to the viable cell density while Biomass Specific Perfusion Rate (BSPR) is a feed rate proportional to the biomass (cell volume multiply by cell density). In this study, perfusion cultures were run at three BSPRs in the production phase. Low BSPR favored a growth arresting state that led to gradual increase in cell volume, which in turn led to an increase in net perfusion rate proportional to the increase in cell volume. Consequently, at low BSPR, while the cell viability and cell density decreased, high specific productivity of 55 pg per cell per day was achieved. In contrast, the specific productivity was lower in bioreactors operating at a high BSPR. The ability to modulate the cell metabolism by using BSPR was confirmed when the specific productivity increased after lowering the BSPR in one of the bioreactors that was initially operating at a high BSPR. This study demonstrated that BSPR significantly influenced cell growth, metabolism, and productivity in cultures with variable cell volumes.

Leveraging an advanced simulated moving bed approach to achieve 3-component separation for enhanced impurity removal in a non-affinity cation exchange capture step.

One of the key challenges in downstream bioprocessing is to obtain products of high purity in a productive fashion through the effective removal of process and product related impurities. While a classical simulated moving bed (SMB) system operation can typically achieve a 2-component separation between the weakly bound impurities and target species, here we present an advanced SMB approach that can achieve a 3-component separation, including the removal of the strongly bound impurities from the target species. As a proof-of-concept, we demonstrate the enhanced removal of strongly bound host cell proteins (HCP) from the target monoclonal antibody (mAb) through the utilisation of the advanced SMB approach in a non-affinity cation exchange (CEX) capture step. In this way, 1 less polishing step was required to achieve the therapeutic requirements of < 100 ppm HCP and the overall process recovery was increased by ~ 6% compared to the corresponding process that utilised a batch CEX operation. The non-affinity CEX capture platform technology established through the utilisation of the advanced SMB approach presented here can potentially be further applied to address the downstream processing challenges presented by other challenging biotherapeutic modalities to yield a final target product with improved purity and recovery.