pH: Are You in Control of a Moving Target?

Further, in a hollow-fiber reactor system, researchers found gradients in nutrient concentrations, which could eventually lead to gradients in pH and dissolved oxygen [12]. As this research suggests, understanding the effects of variations in culture pH on cell functions and properties is essential for each new culture.

Protein Aggregation

Careful pH control can also help reduce protein aggregation, commonly found during biopharmaceutical manufacturing. Protein aggregation can take many forms. For example, reversible protein aggregation (e.g., as occurs in a monoclonal antibody to VEGF) results from relatively weak covalent protein interactions that shifts the equilibrium between monomer and higher order forms. This equilibrium can be affected by a simple change in extracellular pH [13].

Sensor Function and Drift

One additional challenge for the pH measurement in bioprocessing is the cleaning process. The fermentor or bioreactor must be sterilized prior to the start of either process to ensure that no cross-batch or unwanted microbial contamination is present. In addition, pH sensors undergo a two-point calibration using buffer solutions. Residual buffer chemicals must be removed prior to start of the batch. The Clean in Place system (CIP) is used prior to Steam in Place (SIP). Exposure to high temperature steam and rapid thermal shock during SIP can significantly affect the pH sensor’s function.

Different culture batches will have different optimal setpoints, and the pH levels may change when the batch moves to different phases. With classical glass pH electrodes, a number of factors may result in pH values drifting, diminishing confidence in measurements. These include include fouling or coated junctions on the reference electrode.

Two-point buffer calibration cannot be performed with sensors that are mounted in a vessel that has already been sterilized, since the probe cannot be removed to recalibrate with buffer standards. Therefore, the net result is to perform more frequent off-line pH tests and then standardize the on-line measurement to the off-line values (one-point calibration). This is more pronounced in longer batches where the well-trained personnel must be on site to properly perform the tasks.

Once a therapeutic protein has been produced from a cell culture (or other expression system), it has to be further purified to reduce or remove host cell proteins, viruses and process-related impurities. To purify and concentrate the required protein, orthogonal purification techniques are used, which exploit differences in affinity, charge, size or other properties that distinguish the desired from undesired product.

The protein will experience a wide range of pH, ionic strength and concentration during this process. While these are all critical, they must also be closely controlled to prevent the loss of product from processes such as aggregation of the protein.

Where QbD Fits In

The implementation of process analytical technology (PAT) to facilitate biopharmaceutical Quality by Design (QbD) is critical to maintaining consistency in critical environmental parameters such as pH. In the past 10 years, protein expression titers have increased 1000 times to the g/L range. Downstream bioprocessing involving buffer preparation and delivery has had to increase proportionally to keep pace. With the development of more sophisticated technologies and procedures to produce and deliver industrial quantities of buffers with the requisite accuracies, feedback control of critical parameters such as pH becomes more important.

In the protein purification and separation process, the use of ion-exchange columns is a critical step. These function through the separation of proteins based on their net surface charge at a specific pH with separation based on the reversible interaction between a charged protein and an oppositely charged chromatographic medium. Key considerations are the following:

•    Protein with 0 net charge: pI = pH will not bind to a charged medium
•    Protein with pI < pH will bind to a positively charged medium or anion exchanger
•    Protein with pI > pH will bind to a negatively charged medium or cation exchanger

Elution of the proteins can occur using either a continuous or a stepwise salt or pH gradient. pH gradient separation requires 1-2 pH unit change to separate two proteins of different charge. The titration curve between acid and base, whether strong or weak, however, is not linear. This obviates the use of pH gradients during a chromatographic run to resolve proteins with similar pI.

Typically, the blending of a weak acid such as acetic acid and weak base such as ammonium hydroxide results in a sigmoidal titration curve with a steep rise in pH from pH 6.5 to pH 9 over an extremely short period of time when using mass-flow. However, it has been demonstrated that using a PAT-based system involving the careful feedback-control of the acid delivery can generate a linear pH gradient that could promote more efficient chromatographic separation [14].

In summary, pH measurement plays a significant role in all aspects of biopharmaceutical production. The accurate control of pH is necessary for maintaining high quality standards mandated under cGMP. With the introduction of single-use manufacturing technologies has come a new challenge to the manufacturers of pH sensors. Newer technologies are being introduced and must keep improving to keep pace with rapidly evolving demands in the industry.

About the Author
Jim Wilkins joined Sensorin in 2009 as Chief Technology Officer. Previously, he served as Director of Technology Assessment and Licensing at Genentech; as Professor of Chemical Engineering at Yale; and as Vice President of Process Development and Manufacturing at Alexion Pharmaceuticals. Dr. Wilkins earned his B.A. at the University of Texas at Austin; his Ph.D. at the University of Tennessee; and was a post-doctoral fellow and faculty member in biophysics at the Johns Hopkins University.

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