Cell-culture–related in vitro recombinant protein production is currently a $70-billion/year business. In 2007, biotech drug sales grew by 12.5%, twice as fast as standard pharmaceuticals (1). Current ongoing efforts to maximize productivity in both time and volume directly affect the scale and capital investment required for a bioreactor suite. As cells reach higher concentrations more quickly while each cell pumps out more product than ever before, the number and scale of bioreactors can be reduced. To that end, not only cell engineering, but also culture media and related chemical and physical environments are used to assist cells in reaching peak performance quickly and maintaining such a high level as long as possible.

Most culture schemes currently involve some form of nutrient supplementation after inoculation. Numerous approaches have been tried. Only by considering a number of variables can an informed decision be made to yield maximum cell productivity of a specific protein in specific cells using specific techniques. This three-part review elucidates some prominent nutrient supplementation options.

PRODUCT FOCUS: PROTEINS

PROCESS FOCUS: PRODUCTION

WHO SHOULD READ: MANUFACTURING AND PROCESS DEVELOPMENT

KEYWORDS: CULTURE MEDIA, FED-BATCH, PERFUSION, MEDIA SUPPLEMENTS, AND DESIGN OF EXPERIMENTS

LEVEL: INTERMEDIATE

Photo 1:

Photo 1: (Click to enlarge)

The basic goal of nutrient supplementation is to add depleting nutrients to cell cultures that maintain the cells in a viable and productive state as long as possible. Fed-batch and other supplementation protocols use concentrates of essential nutrients. When NaCl and other unconsumed components are omitted, more needed components can be added to a culture before osmolality increases become culture limiting. However, careful consideration is required because for some cells, phosphates and lipids (for example) can be optimized for additional productivity gains. Most attempts at fed-batch supplementation can yield a twofold or higher gain in productivity, but to consider a nutrient supplement, it is necessary to consider its relation to the type of cell culture production system used.

Overview

The entire process of designing, using, and verifying a nutrient supplementation strategy is dynamically interrelated and should not be thought of as a unidirectional series of events. For this discussion of component parts, however, Figure 1 suggests an overview. After choosing the type of bioreactor and supplementation process, the supplement's chemical composition needs to be determined. A number of techniques are available, some offering significant savings in time and potential for testing a wide range of options. As provisional formulations are assessed, scale-up should be addressed. In addition to testing with larger bioreactors, scale-down models and biochemical simulations offer further help in increasing proficiency to help companies arrive at a final formulation and feeding protocol.

Figure 1: (Click to enlarge)

Matching Supplement to Bioreactor System

Most biomanufacturing platforms involve either a batch or a fed-batch protocol, but other production options (Photo 1) offer advantages to be considered. Table 1 shows the major bioreactor operational supplementation modes with the main characteristics of each. Optimal supplementation results require different formulations and feeding strategies because different bioreactor types work best with different kinds of supplements.Table 1: Nutrient delivery options for bioreactors

Table 1: Nutrient delivery options for bioreactors (Click to enlarge)

In fed-batch culture, for instance, supplements should be as concentrated as possible to reduce the volume of additions because they are cumulative (volume is not withdrawn until the end of culture). So waste product accumulation may become culture-limiting. With controlled-fed perfusion culture, less concentrated supplements are used because the supplement serves not only to feed cells, but also to remove waste products for extended cultures. If a supplement is too concentrated, the low flow rate that would be acceptable for providing sufficient nutrients to the cells and that also would provide a concentrated end product would not sufficiently “flush” waste products out of the bioreactor to allow for extended culture.

Batch culture involves no supplementation. Cultures are inoculated, followed for cell viability, and harvested at the appropriate time. This is the simplest process, but culture longevity is limited by exhaustion of nutrients and build-up of waste products. As explained below, some wastes will be used toward the end of the culture.

Fed-Batch Supplementation: A typical fed-batch protocol involves adding concentrated supplemental components to a culture after inoculation. The concentrates are designed to supply needed nutrients that are consumed by the culture. Addition at the time of inoculation would increase osmolality during the growth phase, which can sometime negatively affect cell expansion. In addition, increased waste product concentrations may result from either medium component degradation due to higher initial concentrations or less efficient metabolic pathways from excess nutrient levels. Nutrient supplements usually don't contain components that are not depleted by cells (e.g., most salts) because they would further increase osmolality, reducing the amount of supplementation possible. Although in general good results are the norm when adding supplements after inoculation, it has also been observed that comparable results may be obtained by adding a portion at inoculation.

Operational complexity is low for fed-batch processes, but the timing of specific volume additions needs to be optimized for each cell clone. Culture longevity can be increased from days for a batch up to one to two weeks with a “typical” fed-batch scheme; however, product is held for that time at incubation temperatures, so degradation must be assessed. Product concentration is higher than for batch culture, but culture waste products can affect product quality, and bioreactor homeostasis is low.

Continuous Supplementation: A culture maintained under continuous supplementation refers to continually adding 1× medium because cell concentration increases with no attempt to maintain or concentrate cells within the bioreactor. This process is more complicated than fed-batch with periodic additions, but it is operationally much less complex than perfusion. In short, 1× medium enters the bioreactor while the same volume of spent media and cells exit.

Continuous supplementation reduces the proportion of cells entering production phase at any given time because of the continual flush-out of cell populations, which results in lower titers. For labile products, continuous removal may be an advantage because product can be stored refrigerated before purification. With continuous supplementation, waste products are minimized but titers are low and purification volumes are relatively high.

Perfusion culture involves maintaining cells within a bioreactor through which 1× medium perfuses. This requires a cell concentration methodology such as spin filters, hollow fibers, acoustic membranes, or continuous centrifugation. Each of those techniques offers advantages and disadvantages. And each perfusion option will concentrate cells to different levels, which requires different supplementation strategies.

Spin filters may become plugged at some point during a process run and have to be replaced. In hollow-fiber technology, cells are maintained in an extracapillary space, with 1× medium perfusing through the capillaries. Almost tissue-dense cell masses form in the extracapillary space outside hollow fibers, yielding exceptionally high productivity per bioreactor unit volume, but nutrient and oxygen levels vary from one end of the unit to the other. Acoustic membrane technology has scale-up considerations that may preclude its use at large volumes. Nutrient delivery at very high volume exchanges has been shown to reduce cell viability presumably due to lack of self-feeding (2). Continuous centrifugation may also have scale-up issues, although multiple centrifuge units can be used.

Once a system is validated, several distinct advantages come from perfusion technologies. Cultures can be maintained for extended periods of weeks to a month or more. An equilibrium is established in which cell concentrations build and then are maintained at levels higher than other culture methods can accommodate. Viability generally holds at a relatively constant level, assuming continuous nutrient supply and waste product removal.

With hollow fibers, because cells are concentrated in the extracapillary space and depending on the molecular weight cutoff of the fibers, product may pass across the membrane and be withdrawn. It thus transfers to the perfusing 1× medium, which reduces the potential for degradation of sensitive products. One advantage for stable products is to choose a membrane cut-off limit that will keep the product in the extracapillary space. This provides for exceedingly high product concentration, which can vastly reduce the volume of supernatant to purify.

With spin filters, acoustic membranes, and continuous centrifugation operated in perfusion mode, waste products can be maintained at low levels by operating at relatively high perfusion rates. Product storage at refrigerated temperatures is possible, although purification volumes are high. Bioreactor homeostasis can be excellent in perfusion processes.

Controlled-Fed Perfusion: One additional nutrient delivery option is controlled-fed perfusion (3,4). This process combines advantages of fed-batch with those of perfusion. It involves lowering the rate of a basic 1× perfusion process by admitting smaller volumes of concentrated nutrients. The main advantage of this technique is a reduction of the large volumetric requirements of 1× media. As long as product solubility is maintained, advantages would be smaller purification volumes (higher product concentrations) with decent waste product removal and adequate nutrient levels. Success would require a relatively fine-tuned balance of reduced 1× media perfusion volumes with addition of feed supplement concentrate to maintain nutrient levels consistent with maximal cell viability and productivity. This may be the most operationally complicated form of nutrient supplementation.

Many different protein production platform options are available. Although fed-batch is the most common form of manufacture involving supplements, other options may offer specific advantages such as removal of sensitive or toxic components from cell cultures. Not only the nutrient supplement formulation, but also the feeding strategy will depend on the system used.