pH: Are You in Control of a Moving Target?

Effect on Expression Levels

One of the challenges for the industry is that each individual cell line requires optimization of its own limits of tolerance, as these can vary dramatically from one cell line to another. Although the vast majority of bioreactors utilize CHO cells, the requirement for pH optimization is not necessarily reduced by knowledge of prior cell line behavior.

Studies have also focused on the effect of pH on the expression levels of critical cell-surface proteins involved in the mediation of critical functions such as viral infectivity, cell-cell communication and recognition of cancer cells. It has been demonstrated that changes in extracellular pH can modify the expression levels of certain cell surface receptors. Also, decreasing extracellular pH resulted in an increase in the expression of CD13 in HL60 cells while having no impact on mRNA levels for CD13, suggesting that the effect of pH was on the protein synthesis or protein trafficking to the cell surface. In addition, a decrease in extracellular pH had a profound impact on cell metabolism. Cells cultured at pH 7.0 and 7.2 exhibited glucose consumption and lactate production rates that were from 20% to 40% lower than those of the pH 7.4 cultures [3].

This trend of decreasing glucose consumption and lactate production rates with decreasing culture pH was also observed in experiments done with HeLa cells [4] and hybridoma cells [5,6]. One study found that as the culture pH decreased from 7.6 to 6.8, hybridoma cells exhibited a decrease in growth rate, as well as in glucose consumption and lactate production rates [6]. This is in contrast to the observation on HL60 cells, where as the culture pH decreased from 7.4 to 7.0, the HL60 growth rate was largely unaffected, while a 20-40% decrease in glucose consumption and lactate production rates was observed.

Possible reasons for the observed overall better yields (i.e., more cells per mole of glucose) could be due to utilization of a higher percentage of other nutrients at lower pH, a decrease in metabolic energy requirements (including cell maintenance), or more likely, complex events involving several metabolic and membrane processes.

Clearly, pH is an important bioprocessing parameter in the large-scale culture of mammalian cells, as it affects cell physiology, protein expression and quality, and cell differentiation.

Glycosylated Products

Protein glycosylation is of critical importance in the efficacy of therapeutic proteins. N-linked glycosylation of the protein therapeutic with N-acetylneuraminic acid (aka sialic acid), a modification that is closely linked to circulatory half-life, is a typical marker for product quality assessment. Glycosylated therapeutics currently on the market includes blockbusters such as Avastin, Enbrel, Erbitux and Herceptin, to name a few. Obtaining a consistent glycoform profile in production is critical due to regulatory concerns. Complete characterization of a therapeutic molecule typically includes thorough characterization of its carbohydrate structures using a combination of enzymatic procedures, liquid chromatography and mass spectrometry.

Mammalian expression systems, particularly CHO cells, are the preferred method for producing glycoproteins at commercial scale, because their innate processing machinery, including protein glycosylation, closely resembles that in humans. However, the cell type, culture process and media can have critical effects on the glycosylation profile. Most of the effects are impacted via the metabolic process that occurs within the host cells, which is critically dependent on the pH. For example, ammonium ions, which can accumulate as a byproduct of glutamine and asparagine metabolism, can drive an increase in intracellular pH and a decrease in terminal sialylation which, in turn, can dramatically impact pharmacokinetics and efficacy [7].

Bioreactor pH has also been found to affect protein glycosylation. For example, in hybridoma cells, shifts in pH can affect both the galactosylation and sialylation of the resulting monoclonal antibody. Increasing the pH to 7.4 and above appears to promote the production of digalacto-complex-type glycans in HEPES buffered cultures, while lower pH promotes the formation of mono- or agalacto-type-complex glycans [8].

The proportion of acidic isoforms of Erythropoietin increase with decreasing culture pH with an optimal range of 6.8-7.2 favoring sialylation. Of note, however, is that the effect of pH is not always consistent, as a higher pH has the opposite effect with polysialic acid attached to neural cell adhesion molecules expressed on CHO cell surfaces [9].

Stem Cell Processes

With the growing importance of stem cells as potential therapeutic agents, interest in understanding and optimizing stem cell processing is increasing significantly. Almost all mammalian cell culture expansion cultures are run at fairly standard conditions (pH 7.4; 20% oxygen, 5% carbon dioxide and 37C), but different stem cell cultures proliferate and differentiate at different optima. For instance, higher pH (pH 7.6) was found to enhance the differentiation and maturation of megakaryocytic progenitors and, also, to favor erythroid differentiation [10]. In contrast, granulopoeisis is optimal at lower pH (pH 7.21), which enhances granulocyte colony-stimulating factor receptor (G-CSFR) expression and granulocyte proliferation and differentiation [11].

Unless pH is properly controlled, problems with cellular quality and/or uniformity may result. Effective pH control is not always easy to achieve, because pH gradients can result from spatial inhomogeneity in the cell culture. In addition, the extracellular pH may vary dramatically depending upon the culture system, especially as the cell density increases. Recent studies have shown that, within six hours of feeding, Chinese hamster fibroblasts in T-flasks, had a local pH of 6.5, even though the bulk pH remained at 7.6. Clearly, the use of gentle agitation in bioreactor tanks can provide greater homogeneity within the extracellular milieu.