Removal of Ammonia

Several techniques were investigated to remove ammonia during cell culture and to improve culture performance. These techniques involve the use of adsorbents, gas exchange and ion exchange membranes, and electrodialysis. Although the systems can be placed in the bioreactor, they are normally used as a loop system (Fig. 9). Cell culture fluid is recycled through a column, where ammonia is removed. In the case of adsorbents, the recycle is halted routinely to allow the regeneration of the column. The column is regenerated by stripping the ammonia from the adsorbents, using a stripping solution. The methods using gas and ion exchange membranes and electrodialysis, on the other hand, can be run continuously. In these cases, the ammonia fixing-stripping solution is continuously recycled on the other side of the membrane.

Use of Adsorbents. Several resins and natural adsorbents were investigated to selectively remove ammonia from cell culture media. Carbonell et al. (150) and Capiau-mont et al. (151) used natural adsorbents such as Clinop-tilolite to adsorb ammonia in a column where cells culture media is cycled through. The operation of the adsorbent had to be interrupted periodically to regenerate the column. Although these columns removed ammonia from the culture, the cell culture performance did not change significantly. Similar results were obtained when Zeolite (Phillipsite-Gismondine) and ion exchange resins were used as adsorbents (77). On the other hand, Nayve et al. (152,153) and Matsumura and Nayve (154) obtained good results in hybridoma cultures by combining the adsorption method and other removal systems. In fact, Matsumura and Nayve (154) obtained a viable cell concentration of 25 million cells/mL in a perfusion system with ammonia removal.

Membrane Amm°rna

Membrane Amm°rna

Figure 9. Ammonia removal from cell culture using adsorbents, gas or ion exchange membranes, or electrodialysis.

The use of adsorbents to remove ammonia requires complicated systems. The regeneration of the column prohibits continuous operation and requires complicated process control. These systems have to be proven to be reliable and effective for commercial scale operation.

Gas Exchange Membranes. Another method of ammonia removal from cell culture uses gas exchange membranes (such as polypropylene) where ammonia is stripped from the solution because of its gaseous properties (155-158). These systems were first developed for microbial systems and for chemical processes (159-162). The efficiency of ammonia removal in these systems depends on the concentration gradient for gaseous ammonia across the membrane (Fig. 10). The ammonia removal rate (R) is given by:

where k is the mass transfer coefficient, A is the surface area, and [NH3]c and [NH3]s are the concentrations of gaseous ammonia, in the cell culture and receiving side, respectively. The concentration gradient can be maximized by maintaining a very low gaseous ammonia concentration on the receiving side of the membrane ([NH3]s r 0). In most cases, the transported ammonia is irreversibly converted by either enzymatic or chemical reactions (e.g., protoni-zation). On the cell culture side, the concentration of gaseous ammonia ([NH3]c) is determined by the total ammonia concentration. Gaseous ammonia is at equilibrium with ammonium ion, and the culture pH determines the concentration of ammonia in gaseous form:

where [NH+]T is the total ammonia concentration (ionized and gas) and pKa is the ionization constant. At typical culture conditions (pH below 7.5), gaseous ammonia constitutes less than 1% of the total ammonia concentration (pKa = 9.2 at 37 °C). Thus, only a minute fraction of ammonia is available for its removal. The efficiency of ammonia re-

Cell Gas exchange Ammonia culture membrane fixation

Membrane Removal Ammonia
Figure 10. Ammonia removal from cell culture using gas exchange membranes. The rate of removal depends on the ammonia gas concentration difference.

moval can be increased by increasing the pH on the cell culture side. Higher pH shifts the equilibrium to ammonia gas and increases the concentration gradient. However, this method cannot be utilized fully because the cells require a tight pH range for growth and maintenance. Increasing pH also affects the media components, and high pH can precipitate proteins, including the product.

The removal of ammonia using gas exchange membranes was tested in a variety of hybridoma lines (7,81,151,163) in an effort to increase antibody yield. Although the final cell density was reported to have increased, the effect on production was not significant. Ammonia removal, however, did result in an alteration in cell metabolism.

Ion Exchange Membranes. Ammonia can be selectively removed by ion (cation) exchange membranes. Contrary to gas exchange membranes, ion exchange membranes remove ammonium ion instead ofgaseous ammonia. As mentioned before, ammonia in the cell culture is almost 99% in ammonium ion form, so the removal via ion exchange membranes should be more effective.

The use of autoclavable cation exchange membranes (DuPont, Wilmington, Del., Nafion 417) was described by Thommes et al. (164). Ammonium ions bind to the fixed anions in the membrane and pass into the strip-fixation solution, where they are deprotonized or stripped from the solution by pervaporation (Fig. 11). The elimination of ammonia on the strip-fixation side and a high concentration of ammonium ions on the cell culture side establishes an efficient concentration gradient so the removal ofammonia is very effective. Thommes et al. (164) reported an increase in cell density and antibody production in a murine hybridoma culture. The problem with the ion exchange membrane is the simultaneous removal of some other cations from the culture. Some cations such as calcium and magnesium can also precipitate as bicarbonates as a result of ion exchange (56).

Electrodialysis. Another technique for ammonia removal involves the use of an electrokinetic mechanism utilizing electrophoresis. Chang and Wang (82,165) applied a continuous DC electrical field to remove charged ammonia and lactate from the culture of ATCC CRL 1606 hybridoma cells. At a current density of 50 A/m2, almost all ammonia in the culture could be removed. This ammonia removal system allowed the use of 4 X concentrated media. The system allowed enhancement of cell density and antibody production significantly. The applied current did not cause any detrimental effect on the cells. Removal of ammonia increased the glutamine consumption rate. The electrodial-ysis system also removed lactate and minimized lactate related inhibition.

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