culture science would then be to determine the other by-products and devise meth­odologies to limit or control their formation. Easier said than done! We will touch on such efforts later in this chapter, but for now remind the reader that “the solution to pollution is dilution,” or in the case of cell culture…perfusion. Perfusion is the brute force method that will solve nearly any waste product accumulation problem. However, if the waste product is merely lactate, perfusion is a very inefcient method of keeping lactate below a growth inhibitory concentration [29, 46]. A much more efcient way to implement perfusion is to allow cells to control their own perfusion rate and simultaneously reduce net lactate production to near zero. This can be accomplished by modifying and extending the HiPDOG control scheme to work with perfusion [9]. The technique can reduce by several fold the rate of perfu­sion required to maintain a culture in an exponential growth state and allow us to coin yet another cute acronym, HiPCOP, or Hi-end pH Control Of Perfusion. Instead of controlling the rate of nutrient addition to a fed-batch culture, now when the pH reaches a high-end set point (indicating the culture is limited for glucose) a pump is activated that delivers perfusion media containing glucose to the culture, while a second pump maintains the volume of the culture constant by removing an equal amount of liquid from the culture through a cell retention device. In a similar man­ner to the HiPDOG control, the addition of perfusion media via HiPCOP stops when the pH drops back below the high-end set point as a small amount of lactic acid is produced. However, at this point the astute cell culture scientist would realize the fundamental problem with this this approach. The control of lactic acid forma­tion would almost be too good in this case. Recall that when HiPDOG was used with fed-batch cultures the lactate concentration would slowly fall throughout the course of a culture since other acidic metabolic by-products partially interfered with the control. Even with many dozens of CHO cell lines tested with HiPDOG in fed batch, provided the initial glucose level of the culture was high enough, the level of lactate never fell too low for the HiPDOG control scheme to continue to operate effectively during the growth phase. However, in the case of perfusion, with the simultaneous reduction in lactate concentrations just due to the perfusion operation itself (the ushing of the pollution, remember?), reduction in lactate to near zero could be a real possibility and might cause the HiPCOP control scheme to fail, overly restricting the perfusion rate and slowing cell growth.

Upon realizing this complication, in our laboratory we began adding sodium-L­lactate to the incoming perfusion medium at low levels to ensure that there would always be a low level of lactate in the perfusion bioreactor culture so that the HiPCOP control scheme would not fail. While almost a blasphemous proposal to add lactate to a culture when for the last few decades we were trying to minimize lactate forma­tion in mammalian cell culture, it seemed pragmatic initially. We found, however, that pure sodium-L-lactate is not an inexpensive raw material. Far less expensive is the racemic mixture of D/L-lactic acid which is a by-product of certain industrial processes, and seems to be well tolerated when added to cell cultures. However, since the D form of lactic acid is not readily consumed by mammalian cultures, it would be an extra ion contributing to osmotic strength and the chemical mixture itself would be another raw material requiring suitable sourcing. In continued exper­imentation and understanding of the chemical and biochemical processes occurring

Many of the more potent inhibitors were found to be by-products of amino acid catabolism. It was found that simply keeping the levels of a number of amino acids below a certain upper threshold level, while simultaneously not allowing them to be completely depleted, could minimize the formation of some inhibitors and enable additional cell growth [42]. However, as with early efforts of cell culture scientists to measure and precisely feed glucose and keep it in a lower range to limit lactate formation, it was realized that other metabolic engineering approaches might be more straightforward and could be implemented across the board for CHO host cell lines. It was realized that three of the inhibitors: isovalerate, 2-methylbutyrate, and isobutyrate, which were primarily formed from leucine, isoleucine, and valine catabolism, respectively, shared the same enzyme at the beginning of their catabolic pathway. Efforts were made to knock out the enzyme—branched chain amino acid transaminase-1, or BCAT1. The resulting CHO cell lines grew to higher cell densi­ties, maintained higher cell viability longer, and when transfected with the appropri­ate genetic elements also produced higher levels of recombinant proteins in fed-batch and perfusion cultures than CHO cells without the knockout [48].

volume and efcient utilization of the production bioreactor are typically the prin­cipal limiting factor that determines the overall annual productivity of any manufac­turing facility. Therefore, there is incentive to have that vessel lled with the highest density of healthy cells at all times. Any time used during the expansion of the cell mass in the production bioreactor is potentially wasted time. With this in mind it is logical to minimize the time in the production bioreactor during which the cells are dividing. N-1 (or seed bioreactor) perfusion can help to achieve this.