Why do pH levels sometimes change during diafiltration, even when the starting and diafiltration buffers have the same pH?

During diafiltration, pH shifts can occur due to the non-ideal behavior of buffer solutions. This non-ideality is influenced by ionic strength, which affects the activity coefficients of buffer components. Changes in ionic strength modify the pKa of the buffer, leading to pH variations, even when the initial feed and diafiltration buffers are set to the same pH. These shifts can impact the stability of the final biopharmaceutical product. These non-idealities arise because of electrostatic interactions and molecular forces that influence the effective equilibrium of acid-base reactions.

How does ionic strength influence the behavior of buffers during the diafiltration process?

Ionic strength significantly affects buffer behavior during diafiltration. As ionic strength changes (e.g., during buffer exchange), the activity coefficients of buffer components shift, which alters the pKa of the buffer. For example, when diafiltering from a high-salt phosphate buffer to a low-salt histidine buffer, the pKa of the phosphate buffer increases, leading to a rise in pH. The Debye-Hückel theory helps explain how ionic strength affects ion activity and, consequently, pH.

What strategies can be used to maintain stable pH levels during diafiltration?

To achieve stable pH during diafiltration, it's recommended to match the ionic strength of the feed and diafiltration buffers. This minimizes changes in activity coefficients and pKa, resulting in a more stable pH profile. However, if matching ionic strength isn't feasible due to formulation requirements, careful consideration of buffer properties and process parameters is crucial. This approach minimizes unwanted pH changes and helps maintain product quality.

What is the Debye-Hückel theory, and how does it apply to pH shifts during diafiltration?

The Debye-Hückel theory explains how ionic strength influences ion activity in solutions. It demonstrates that electrostatic interactions alter the activity coefficients of ions, affecting the pKa of buffers. Understanding the Debye-Hückel theory provides insights into predicting and controlling pH shifts during diafiltration. The Debye-Hückel theory explains non-ideal behvaior, while pKa describes the acid dissociation constant. By matching feed and diafiltration buffers, the impact of pKa and unwanted pH shifts are reduced.

Surreal illustration of pH instability in biopharmaceutical diafiltration.

Diafiltration pH Drift: Why Your Bioprocess Buffers Aren't Behaving

Q: Why are phosphate buffers especially prone to pH shifts during diafiltration, and how can this be managed?

Phosphate buffers are susceptible to ionic strength effects because they contain multivalent ions. When the ionic strength changes during diafiltration with a phosphate buffer, the pKa of the buffer shifts, leading to pH variations. For instance, when transitioning from a high-salt phosphate buffer to a low-salt histidine buffer, the pKa of the phosphate buffer increases, raising the pH. Similarly, a higher salt concentration in the diafiltration buffer can decrease the pKa, lowering the pH. These effects underscore the importance of managing ionic strength to stabilize pH when using phosphate buffers in bioprocessing.

Samir D’Costa in Tech & Innovation December 2025 • 5 min read.

"Uncover the hidden causes of pH variations during diafiltration and how to control them for stable biopharmaceutical formulations."

Diafiltration is a critical process in the production of biopharmaceuticals, serving as the final step in formulation by facilitating buffer exchange. In this process, it's common to use a constant volume to precisely control the concentrations of retained solutes. However, achieving consistent pH levels throughout diafiltration can be surprisingly challenging. Recent studies have shown that even when the initial feed and diafiltration buffers are set to the same pH, variations can arise during the process, which can lead to instability of the final formulated product.

Traditionally, these pH shifts have been attributed to interactions between buffer species and the protein product. However, there's growing evidence that the reasons can be largely independent of protein concentration, particularly in the early stages of diafiltration. The key lies in understanding the non-ideal behavior of buffer solutions, which is influenced by factors such as ionic strength. These unexpected shifts can directly influence the quality and stability of biopharmaceutical products.

This article aims to help uncover the critical insights into how buffer non-idealities affect pH during diafiltration. It will also explore strategies to control and minimize these variations, focusing on understanding the influence of ionic strength and optimizing buffer selection. By delving into the science behind diafiltration pH shifts, we aim to equip bioprocessing professionals with the knowledge to enhance process robustness and ensure the quality of their biopharmaceutical products.

The Science Behind the Shift: Non-Ideal Buffers and Ionic Strength

Surreal illustration of pH instability in biopharmaceutical diafiltration.

The driving force behind pH variations during diafiltration stems from the non-ideal behavior of buffer solutions. Ideal solutions assume that interactions between molecules are uniform, which isn't the case in real-world scenarios. Buffers, especially those containing multivalent ions such as phosphate and citrate, exhibit activity coefficients that deviate from unity, particularly at higher concentrations. These deviations are a direct result of electrostatic interactions and other molecular forces that influence the effective equilibrium of acid-base reactions.

Ionic strength plays a pivotal role in modulating buffer behavior. As ionic strength changes during diafiltration (e.g., when exchanging a high-salt buffer for a low-salt one), the activity coefficients of buffer components shift. This, in turn, affects the pKa (acid dissociation constant) of the buffer, leading to a change in pH. The Debye-Hückel theory provides a framework for understanding these relationships, demonstrating how electrostatic interactions alter the activity coefficients of ions in solution.

Debye-Hückel Theory: Explains how ionic strength affects ion activity.
Activity Coefficients: Deviation from ideal behavior due to electrostatic forces.
Impact on pKa: Changes in ionic strength modify buffer pKa, causing pH shifts.

For instance, phosphate buffers, commonly used in bioprocessing, are particularly susceptible to ionic strength effects. When diafiltering from a high-salt phosphate buffer to a low-salt histidine buffer, the pKa of the phosphate buffer increases, leading to an initial rise in pH. Conversely, if the diafiltration buffer has a much higher salt concentration, the pKa of the phosphate will decrease, leading to a reduction in pH. This highlights the need to carefully consider the ionic strength of both the feed and diafiltration buffers to minimize unwanted pH changes.

Strategies for Stable pH During Diafiltration

Controlling pH shifts during diafiltration requires a proactive approach that considers buffer properties and process parameters. One of the most effective strategies is to match the ionic strength of the feed and diafiltration buffers. This minimizes changes in activity coefficients and pKa, resulting in a more stable pH profile throughout the process. However, matching ionic strength isn't always practical, especially when specific formulation requirements dictate low-salt conditions.

Another approach is to use a staged diafiltration process, where the ionic strength of the diafiltration buffer is gradually reduced over time. This can help to mitigate abrupt pH changes and provide better control over the final formulation. Additionally, the choice of buffer system can significantly impact pH stability. Some buffers, such as histidine, exhibit less sensitivity to ionic strength compared to phosphate, making them a better option for certain applications. Always consider factors like buffering capacity, compatibility with the protein, and regulatory requirements.

Finally, modeling and simulation can be invaluable tools for predicting and optimizing pH profiles during diafiltration. By incorporating non-ideal buffer behavior and ionic strength effects into the model, it's possible to identify critical process parameters and design a robust diafiltration process that minimizes pH variations and ensures product quality. Implementing these insights from research will help to help bioprocessing professionals stabilize product formulation and sustain consistent results.