Enabling end-to-end continuous biomanufacturing by exploring integration approaches of continuous TFF

The Downstream Processing (DSP) production/process line comprises of several chromatographic process unit operations (techniques for protein isolation and purification), several liquid filtrations process unit operations and final Ultrafiltration/Diafiltration (UF/DF) process unit operation (process for product concentration to the desired final protein concentration and for exchange into final formulation buffering condition). UF/DF step is conventionally performed with the batch tangential flow filtration (TFF). However, several pump passes and a recirculation skid are required to achieve the target concentration in conventional TFF. Thus, it is impractical to implement batch TFF as continuous UF/DF step or in-line concentration step between different DSP chromatographic steps. In this work the use of single-pass tangential flow filtration (SPTFF) will be examined as continuous TFF step and as the integrational process unit operation allowing both final UF/DF step and in-line protein concentration before chromatographic step. SPTFF integration approach will be explored as continuous TFF delivering a new robust solution with a potential to overcome manufacturing bottlenecks and thus enabling the end-to-end fully continuous biomanufacturing. In this study, the process stability, volumetric concentration factor (VCF) ranges, and process economics of operating SPTFF as continuous in-line UF/DF will be explored in detail. Additionally, the placement of the SPTFF before Protein A capture chromatography unit operation will be explored. This allows coupling of these two steps and as such the potential solution to overcome low and variable protein concentration in the harvest to intensify continuous process. Limitations and requirements for a continuous TFF will be identified. Unique challenges to use technology in a fully continuous UF/DF process step, which is at an earlier phase in development, will also be presented. Please click Additional Files below to see the full abstract

Designing scalable ultrafiltration/diafiltration process of monoclonal antibodies via mathematical modeling by coupling mass balances and Poisson–Boltzmann equation

Ultrafiltration/diafiltration (UF/DF) operations are employed for achieving the desired therapeutic monoclonal antibody (mAb) formulations. Due to electrostatic interactions between the charged proteins, solute ions, and uncharged excipients, the final pH and concentration values are not always equal to those in the DF buffer. At high protein concentrations, typical for industrial formulations, this effect becomes predominant. To account for challenges occurring in industrial environments, a robust mathematical framework enabling the prediction of pH and concentration profiles throughout the UF/DF process is provided. The proposed mechanistic model combines a macroscopic mass balance approach with a molecular approach based on a Poisson–Boltzmann equation dealing with electrostatic interactions and accounting for protein exclusion volume effect. The mathematical model was validated with experimental data of two commercially relevant mAbs obtained from an industrial UF/DF process using scalable laboratory equipment. The robustness and flexibility of the model were tested by using proteins with different isoelectric points and net charges. The latter was determined via a titration curve, enabling realistic protein charge-pH evaluation. In addition, the model was tested for different DF buffer types containing both monovalent and polyvalent ions, with various types of uncharged excipients. The model generality enables its implementation for the UF/DF processes of other protein varieties