While not rigidly true for all mammalian cell processes, it is generally accepted and observed that high growth rates are usually linked to lower per cell, or specic productivity rates [15]. When cells are focusing their metabolic precursors and cel­lular energy on biomass production (division), rates of recombinant protein produc­tion are generally lower. This is part of the reason that in fed-batch cultures the majority of protein production occurs in the last several days of the batch after most division is complete, and the cell density is much higher than earlier in the culture. So, these two constraints are actually somewhat synergistic. Minimizing the cell bleed once the target steady-state cell density is reached can minimize product losses, and simultaneously maintaining a lower cellular growth rate can help keep per cell productivity high. Low cell growth rate=low cell bleed rate. By increasing the nutrient content of the perfusion media and checking the culture for residual nutrient levels occasionally, one can ensure that sufcient nutrients are always avail­able to the cells [16]. In this case then the growth rate of cells in a perfusion culture is directly controlled by the rate of removal of metabolic wastes and other inhibitors of cell division. Lower perfusion rates lead to more accumulation of inhibitors of cell growth which ultimately leads to lower cellular growth rates. A slow reduction in perfusion rate during process development can then lead to a slow reduction in growth rate, increased productivity and reduced medium usage. However, the dif­culty of continuously decreasing the perfusion rate (while increasing the available nutrients in that perfusion medium) is that the most signicant benets in per cell productivity tend to come at very low growth rates which may be difcult to main­tain [17]. A minor process deviation, even one that might not be readily detectable, might be sufcient to push cells into a no-growth state, out of the steady-state condi­tion, and result in a culture with a changing and decreasing cell density. Additionally, mammalian cells in culture are continuously undergoing slight epigenetic changes as they continuously adapt to environmental factors [18], making their long-term responses to culture conditions slightly unpredictable.

For many of the reasons stated in the previous paragraph, while a sustainable, or true steady-state perfusion process has many advantages, there are considerable challenges in the design and operation of such a process with a high cell-specic

Our laboratory has found that this dynamic perfusion/HILVOP process achieves specic productivity (per cell) rates near that of an optimized fed-batch process and signicantly higher than sustainable perfusion processes that we have explored for several cell lines [17]. The overall volumes of perfusion media used are moderate and signicantly lower than most sustainable “steady-state” perfusion processes. Because the cell density is much higher than fed-batch and the specic productivity (picograms/cell/day) is usually similar to a fed batch, the overall volumetric

When in a highly proliferative state and therefore dividing quickly, most mamma­lian cell lines in exvivo culture tend to exhibit some degree of “aerobic glycolysis,” meaning that in spite of readily available oxygen, cells shift to metabolize signi­cant amounts of glucose directly to lactic acid [21]. This metabolic state may have advantages during fast growth as large amounts of various biochemicals may be drawn off from the glycolytic pathway for the production of needed precursors of DNA, RNA, lipids or other cellular building blocks required for accelerated bio­mass production. One can imagine that in tumorigenic growth, there would be sig­nicant competitive advantages to fast growth in that the tumor cells themselves do not have to trouble with the lactic acid produced as it is merely carried away to other tissues, including liver and muscle, by the nearly endless supply of fresh blood pro­vided by the host organism.

24, 25]. While such a practice might slow cell growth, the slower accumulation of

lactate sometimes allows the growth phase to be extended longer, resulting ulti­mately in a higher IVCD, or at a minimum a healthier biomass than would have

Other groups have experimented with alternative carbon sources to glucose such as galactose, fructose, or mannose, but while those substitutions often do reduce the formation of lactate it is usually at the expense of fast growth rates and results in considerably reduced peak cell densities [35–38]. Additional researchers tried to slow the rate of six-carbon sugar membrane transport with chemical inhibitors or genetic engineering techniques such as amplication, suppression, addition, or silencing of enzymes or substrate membrane transporters [39–41]. Because in most cases the cells eventually reverted to their original wild-type metabolism, practical success with these techniques was not achieved.

After experiments in our laboratory demonstrated that the accumulation of lactate in our CHO cell fed-batch cultures was the primary factor suppressing continued cell division and productivity [23], we built on controlled glucose feeding experiments conducted earlier [25]. After realizing that the uptake of lactic acid from the bulk culture uid was sufcient to result in a signicant rise in pH detectable by conven­tional in-situ pH probes, we developed a feedback control system for lactate that would prove to be easy to manage and implement even in the largest bioreactors in common use (13,000L) in our production facilities. It was discovered that cells in culture would “signal” a low level of glucose in the bulk culture by taking up lactic acid from the surrounding culture uid. The switch to consumption of lactic acid was fast enough and of sufcient magnitude that the bulk culture increase in pH was enough to trigger a feed pump to add a small amount of glucose to the culture before the cells were overly starved for glucose. Starting with a low level of glucose in the initial medium in the production bioreactor, this control loop strategy, later given the acronym HiPDOG, for Hi-end pH delivery of glucose (shown as a hypothesized sequence of events in Fig.4.2 below) would then turn on and off the glucose feed

pump periodically throughout the growth phase of the culture. Somewhat surpris­ingly, with a sufciently tuned system, the growth rate of a fed-batch culture utiliz­ing the HiPDOG control strategy was found to be nearly identical to that of parallel cultures which never experienced glucose limitation. The net effect of the glucose/ lactic acid control scheme resulted in zero net lactic acid formation during the growth phase once the control system rst activated as the glucose concentration rst fell into a limiting range. Additionally, since little to no titrant is added in such a process, the “base titrant ion penalty” mentioned above never occurs.

Because a rapidly expanding fed-batch culture requires many other nutrients, it was found to be useful to also include these nutrients in the glucose feed being delivered by the HiPDOG control scheme. In this way, cells in a fed-batch culture would precisely control their own rate of feeding without any operator intervention. The amount of nutrients delivered would of course depend upon the glucose con­centration in the feed since only the delivery of glucose was actually being con­trolled by the culture. The higher the concentration of glucose in the feed, the less volume of feed that would be delivered, and vice-versa. Finding the right ratio of glucose to other nutrients allowed for an optimum delivery rate to the culture, main­taining all amino acids and other components above growth limiting concentrations, yet not leading to excess accumulation which could lead to toxicity, excess ammo­nium production if amino acids were used as a carbon source, or merely high osmotic strength.