Buffer preparation

Importance of pH in cell culture

Of the variables that influence successful cell culture and fermentation, a key factor to consider is pH. Changes in acidity or alkalinity can cause undesired effects like conformational changes in proteins. pH variation may also lead to the denaturation and destruction of proteins, as well as impaired cell functioning.

Buffers

Buffers are chemicals that prevent liquids from having major fluctuations in their acidic properties. Different buffer systems have different pKa ranges to accommodate the various pH ranges needed for final buffers. A major application of buffers in cell culture is the maintenance of a stable pH as cells grow and produce both lactic acid and carbon dioxide.

Apart from regulating pH values in cell culture media, buffers also regulate pH values in solutions used in bioprocessing, including the chromatography steps. Precise control of buffer pH and ionic strength is particularly critical for ion exchange chromatography (IEX), where ionized side groups interact with the charged ligand on the chromatography resin or other stationary phase.

Buffers comprise a proton donor like acetic acid and a conjugate base, such as the acetate ion. When added to a solution, the buffer system can take up or release hydrogen ions with ease. This allows control of the free-floating hydrogen ion concentration within the pKa range of that buffer system.

Buffer preparation FAQs

Below are answers to some frequently asked questions about buffer preparation.

How is a buffer solution prepared?

Prepare buffer solutions either by mixing an acid with its conjugate base or a base with a conjugate acid. Buffers are prepared manually, by adding acids (e.g., HCl) and bases (e.g., NaOH) to reach the desired pH. They can also be prepared using an automated buffer management solution.

How do you prepare a buffer for ion exchange chromatography?

In IEX, a sample with a target molecule, such as a protein, and contaminants is adjusted to a specific pH. The pH is critical to ensure that the net surface charge of the target molecule is either above or below the isoelectric point, depending on what type of ion exchanger is used. The sample is loaded onto a column at low ionic strength. The conditions are then altered to desorb the bound substances differently. A typical elution increases the salt concentration or changes the pH in a gradient.

To ensure that the separation performs as expected time after time, it’s important to control both the pH and ionic strength of the loaded sample as well as buffers used during the chromatography run. Automated buffer preparation solutions are well-suited to this task.

What is buffer automation?

Buffer automation is the production of buffers from stocks using automated systems that minimize the need for human handling. The two types of automated buffer production techniques are inline dilution and inline conditioning.

What are the benefits of buffer automation?

Since automating the process saves time, it improves the efficiency of media preparation in upstream bioprocessing. And it eliminates downstream bottlenecks. Buffer automation also increases the accuracy of the final buffers, which minimizes variation in biologic production.

What's the difference between inline dilution and inline conditioning?

Inline dilution is a buffer preparation technique that involves the dilution of concentrated buffers with water. This capability comes with some chromatography systems that bring multiple process streams together for dilution. On the other hand, inline conditioning is the formulation of buffers from a stock solution of an acid, base, salt, and water for injection (WFI).

Inline buffer dilution requires a different multi-component buffer concentrate to produce each final buffer solution. In contrast, inline conditioning uses a limited number of stock solutions that each have only one component. Multiple final buffers can be prepared automatically from these same stocks. And it saves facility footprint, because there’s no need to stock buffer concentrates for all the final buffers required. This latter point is especially important when producing large quantities of a biologic.