Suffering High Protein Sieving Losses? It Might Be The Buffer
By Aylin Mohammadzadehmarandi, Department of Chemical Engineering, The Pennsylvania State University

Ultrafiltration and diafiltration (UFDF) are critical steps in the formulation of biopharmaceutical proteins, where achieving high protein retention is paramount. However, subtle changes in formulation buffers can have a profound impact on protein sieving losses, thereby influencing process efficiency and product quality.
Our recent research sheds light on how specific buffers, such as phosphate, acetate, citrate, and histidine, affect protein retention during ultrafiltration. This is especially relevant for bioprocess engineers aiming to optimize separation processes while maintaining protein integrity.
Understanding the interplay between buffer composition and protein biophysical properties offers practical insights for addressing challenges in protein formulation. This article explores key findings from our study on the effects of buffers on the retention of bovine serum albumin (BSA), a model protein, and provides actionable strategies to minimize sieving losses in ultrafiltration processes.
Our research found that the difference can be substantial. A UFDF process would have 95.7% yield in histidine buffer but only 87.3% in phosphate buffer. This difference of about 10% yield, retained after controlling sieving loss, can affect total mAb/Fab production costs.
Research Aimed To Help Industry Reduce Sieving Loss
Protein sieving during ultrafiltration depends on the interaction between the membrane and the protein, influenced by factors such as buffer composition, pH, and ionic strength. Our study revealed that phosphate buffer at pH 4.8 significantly reduced BSA retention compared to acetate, citrate, and histidine buffers. This reduction was linked to changes in the hydrodynamic diameter of BSA, as measured by dynamic light scattering (DLS). Further analysis using circular dichroism spectroscopy and differential scanning calorimetry confirmed that phosphate buffer induced minor conformational changes in BSA, leading to a smaller effective size.
The motivation behind this work was to provide a deeper understanding of buffer-induced protein behavior, which is often overlooked during process design. By identifying the conditions that minimize sieving losses, we aim to equip biopharmaceutical manufacturers with strategies to enhance process efficiency.
Common Buffer At The Heart Of Losses For Model Protein BSA
The ability of ultrafiltration membranes to retain proteins is a cornerstone of biopharmaceutical process development. Protein sieving losses occur when proteins pass through the membrane pores, potentially compromising yield and purity. Our study focused on quantifying the effects of various buffers on protein retention during ultrafiltration of BSA, a widely studied model protein.
We observed that phosphate buffer at pH 4.8 exhibited the highest sieving losses among the tested buffers. This finding was surprising given that phosphate is a commonly used buffer in protein formulations. To understand this behavior, we measured the hydrodynamic diameter of BSA using dynamic light scattering. Results showed a small decrease in diameter when BSA was in phosphate buffer compared to other buffers. This reduction in size suggests that phosphate buffer induced slight conformational changes in BSA, making it easier for the protein to pass through the membrane pores.
Further investigations using circular dichroism spectroscopy and differential scanning calorimetry revealed subtle changes in BSA’s secondary and tertiary structures in the presence of phosphate buffer. These structural changes were consistent with the observed decrease in hydrodynamic diameter and increased sieving losses.
In contrast, acetate, citrate, and histidine buffers maintained higher protein retention. These buffers either preserved the native conformation of BSA or promoted slight expansions in its hydrodynamic diameter, reducing the likelihood of membrane permeation. These findings highlight the critical role of buffer selection in minimizing protein sieving losses during ultrafiltration.
From a practical standpoint, our results emphasize the need for careful buffer optimization in the early stages of process development. For example, switching from phosphate to histidine buffer at similar pH conditions for BSA solution could significantly improve protein retention without requiring changes to the membrane or operating conditions. This approach not only enhances process performance but also reduces downstream recovery steps and associated costs.
More Research Required
Despite the insights gained from our study, several challenges remain. One major limitation is that our findings are based on a single model protein (BSA). While BSA is commonly used in bioprocess research, its behavior may not fully represent the complexity of other therapeutic proteins, such as monoclonal antibodies or fusion proteins. Therefore, additional studies are needed to generalize these results across a broader range of proteins and operating conditions. However, we analyzed the DLS characteristics of a challenging mAb provided by our industrial collaborator. We evaluated its size in various buffers and identified that it exhibited the smallest size in the buffer that resulted in higher sieving efficiency.
Another obstacle is the potential variability in membrane properties. Factors such as pore size distribution, surface charge, and fouling behavior can influence protein retention independently of buffer composition. Thus, membrane characterization should be integrated into any optimization strategy.
Lastly, changes in buffer composition can have downstream implications, such as altered protein stability, solubility, or compatibility with subsequent purification steps. These factors must be carefully evaluated to ensure that improvements in ultrafiltration do not negatively impact overall product quality.
Conclusion
Buffer composition plays a pivotal role in controlling protein sieving losses during ultrafiltration. Our findings underscore the importance of selecting buffers that preserve protein conformation and minimize hydrodynamic size reductions. While phosphate buffers are widely used, alternatives such as acetate, citrate, and histidine buffers may offer superior performance by enhancing protein retention for specific protein solution.
For biopharmaceutical manufacturers, these insights provide a practical framework for optimizing ultrafiltration processes. By carefully tailoring buffer conditions, it is possible to reduce product losses, improve process efficiency, and maintain protein integrity. Future research should focus on extending these findings to more complex proteins and multi-component systems to further enhance our understanding of buffer-protein interactions in ultrafiltration.
About The Author:
Aylin Mohammadzadehmarandi is a Ph.D. candidate in chemical engineering at Penn State University, specializing in high-performance countercurrent membrane purification (HPCMP) as a novel approach for continuous platform for therapeutic purification. Additionally, part of her research focuses on the effect of buffer excipients on protein sieving loss. She is the recent recipient of the Women in Chemical Engineering Award from AIChE, the Best Presentation Award from the MAST (Membrane Applications Science and Technology) Center, and the Leadership Scholarship from Penn State University. Her industrial experience includes three years of research and development in downstream and formulation development at CinnaGen Biopharma (Iran), where she developed advanced protein characterization and purification methods for monoclonal antibodies.