Theoretical analysis of excipient concentrations during the final ultrafiltration/diafiltration step of therapeutic antibody

Abstract

Diafiltration of a protein solution into a new buffer is a common final step in biopharmaceutical manufacturing. However, the excipient concentrations in the retentate are not always equal to their corresponding concentrations in the new buffer (diafiltration buffer). This phenomenon was observed repeatedly during diafiltration of different therapeutic monoclonal antibodies in which the concentrations of histidine and either sorbitol or sucrose (depending on which was chosen for the diafiltration buffer) in the retentate were lower than in the diafiltration buffer. Experimental studies and theoretical analyses of the ultrafiltration/diafiltration (UF/DF) step were carried out to determine the primary causes of the phenomenon and to develop a mathematical model capable of predicting retentate excipient concentrations. The analyses showed that retentate histidine concentration was low primarily because of repulsive charge interactions between positively-charged histidine molecules and positively-charged protein molecules, and that volume exclusion effects were secondary for like-charged molecules. The positively-charged protein molecules generate an electrical potential that cause an uneven distribution of charged histidine molecules. This interaction was used to construct a mathematical model based on the Poisson-Boltzmann equation. The model successfully predicted the final histidine concentration in the diafiltered product (retentate) from the UF/DF development and production runs, with good agreement across a wide range of protein and histidine concentrations for four therapeutic monoclonal antibodies. The concentrations of uncharged excipients (sorbitol or sucrose) were also successfully predicted using previously established models, with volume exclusion identified as the primary cause of differences in uncharged excipient concentrations in the retentate and diafiltration buffer.

Predicting diafiltration solution compositions for final ultrafiltration/diafiltration steps of monoclonal antibodies

“…At a protein concentration of 100 g/L, for example, only 60% of the diafiltration buffer histidine was measured in the retentate for both MAbs, and models predicted the same amount of exclusion. Model predictions at concentrations greater than 150 g/L are not presented, as EDL overlap occurs under those conditions and the model assumptions are not satisfied (Miao et al, 2009). While the model is not valid in this region, the data points at concentrations >150 g/L continue the trend of decreasing histidine levels with increasing protein concentration.…”

“…For uncharged excipients, the volume exclusion model alone was successfully applied to predict the retentate concentrations of sucrose and sorbitol. Other studies applied a model based on Donnan equilibrium to model the concentration of charged excipients (Stoner et al, 2004); however, our group took a different approach as the Donnan-based model required fit parameters and it is uncertain if equilibrium is established across the membrane during constant flow during our UF/DF operations (Miao et al, 2009).…”

“…In the original model (Miao et al, 2009), an approximation that histidine carried a net positive charge was applied (e.g., all in a conjugate acid state) hence pH could not be predicted. In the current study we follow an approach similar to that presented by Bolton et al (2008), and apply the Henderson-Hasselbalch equation in conjunction the original Poisson-Boltzmann and volume exclusion models.…”

“…However, at high protein concentrations the final pH and excipient concentrations of the diafiltered retentate are not always equal to those in the diafiltration buffer. Due to combined effects of electrostatic origin and volume exclusion, both the pH and excipient levels (both charged and uncharged) can shift significantly from that of the diafiltration buffer (Miao et al, 2009;Stoner et al, 2004).…”

“…In our group's previous work (Miao et al, 2009), models applying the Poisson-Boltzmann equation in combination with volume exclusion were applied to predict the concentration of charged excipients in retentate fractions. Here the electric potential around the protein resulted in partial exclusion of like-charged excipients within the electrical double layer (EDL) of the protein, and differences in the final retentate composition from the diafiltration buffer composition were successfully predicted.…”