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It can be very complicated and time-consuming, and will require expertise and resources. In this section, we outline the common as well as novel methods used for SF medium development. Details of each strategy can be found in the referenced literature.
In the early s, efforts were initiated to eliminate serum and animal-derived components from the culture media used to produce human therapeutics. At that time, two different strategies were commonly utilized for the development of SF media:. The last decade saw the development of many new genomic and automated screening tools. These advancements, as well as an improved understanding of mammalian cell culture, allowed novel concepts and approaches to be applied to the development of SF and CD media.
Three representative strategies are briefly summarized below:. There are several advantages of utilizing a chemically defined CD media, specifically CD media has less lot-to-lot variability compared to media with undefined components, such as hydrolysates [ 48 ].
Also, CD media allows the scientist to have more insight into the metabolic state of the cells. For example, the metabolism of short peptides by mammalian cells is not well understood, thus media containing them is not desirable [ 48 ]. One of the primary drawbacks of CD media is that most CD media formulations typically do not support cell growth as well as media formulations with undefined components [ 49 ]. However, using similar methods to those detailed in the previous section, Ma et al.
Huang et al. These experiments were followed by additional rounds of CD optimization experiments using trace metals and vitamins. Ma et al. The concentrations of amino acids in this simplified formulation were further optimized through the use of spent media analysis. This final optimized formulation improved the culture longevity and decreased lactate accumulation while simultaneously doubling the final titer [ 48 ].
The top-down approach implemented by Ma et al. A cell culture scientist may not have the wherewithal to attain the optimal cell culture media and feed formulation using a top-down approach and instead will have to implement a more empirical methodology. Many therapeutic proteins are produced using genetically modified mammalian cells, as described in the preceding sections.
This section describes the basic design and function of bioreactors used for suspended mammalian cell culture. Bioreactors should provide a sterile environment, adequate mixing, ease of operation, and control of temperature, pH, and dissolved oxygen. Traditionally, these requirements were met using glass or stainless-steel stirred tank systems.
At production scale, therapeutic proteins are primarily produced in stainless-steel stirred tank bioreactors. Stirred tank bioreactors are generally glass or stainless-steel tanks with an impeller to provide mixing. Air or oxygen is usually bubbled through the media to supply oxygen to the cells.
An example of a stirred tank bioreactor is depicted in Fig. Sterility is obviously an important issue in mammalian cell culture, and therefore traditional glass or stainless-steel stirred tank systems require extensive cleaning and sterilization. A significant portion of the labor involved with operating these systems is related to cleaning and sterilizing. Mixing in Stirred Tank Bioreactors. Adequate mixing is essential to suspend the cells and to facilitate heat and mass transfer.
Historically, however, due to concerns regarding the sensitivity of mammalian cells to hydrodynamic stress, most stirred tank bioreactors were agitated just enough to keep the cells in suspension [ 51 ]. This low level of mixing can result in large concentration gradients of pH, oxygen, and other nutrients.
Ozturk [ 52 ] demonstrated this experimentally by adding base to a poorly mixed bioreactor. The base was added to the top of the bioreactor, and because of poor mixing, a high pH region was created at the top. Proper impeller selection and sizing will improve mixing.
Generally, the diameter of the impeller should be approximately one third to one half of the tank diameter. Retrofitting existing bioreactors with different impellers, however, may prove difficult because the motor driving the impellers may not produce enough torque to turn them. Adequate baffling will also improve mixing; baffles prevent solid body rotation and vortex formation [ 53 ].
Oxygen can be introduced to the culture in many different ways. Membrane aeration provides efficient oxygen transfer with minimum shear damage to the mammalian cells and minimal foaming. However, due to the design complexity and the difficulty involved in cleaning and sterilizing membrane reactors, membrane aeration has limited utility in large-scale bioreactor systems.
Sparger aeration offers high oxygen transfer rates and is widely used in both bench and production-scale bioreactors.
Since oxygen is only sparingly soluble in water, a large surface area is needed to maximize diffusion of oxygen into the cell culture media. Frit spargers with micro-pores provide a large surface area for diffusion, but this type of sparger can cause foaming problems at large scale, and frit spargers are not effective removing dissolved carbon dioxide [ 54 , 55 ]. Traditional large-hole-ring spargers tend to have fewer issues with foaming, remove dissolved carbon dioxide more efficiently, and are easier to clean.
Therefore, traditional spargers are often used in production-scale stirred tank bioreactors [ 54 , 56 ]. Sensors are required to adequately monitor bioreactor performance. Ideally, one would want online sensors to minimize the number of samples to be taken from the bioreactor and to automate the bioreactor process.
Most bioreactors have autoclavable pH and dissolved oxygen D. An online pCO 2 probe is commercially available from Mettler-Toledo. Probes are also commercially available that determine viable cell density by measuring the capacitance of a cell suspension. Data from perfusion and batch cultures indicate that these probes are reasonably accurate at cell concentrations greater than 0.
The accuracy of the capacitance biomass probes can be improved by using dielectric spectroscopy data [ 59 ]. One of the major drawbacks of the stainless-steel stirred tank bioreactors is the difficulty of cleaning and sterilizing the vessels. Disposable bioreactors are now commercially available that are based on the use of presterilized plastic bags. There are two primary designs of disposable bioreactors, rocker motion bags and stirred tanks.
For the rocker motion bioreactors, a sterile bag is partially filled with cell culture media and is then pressurized with a mixture of carbon dioxide and air. The bag is placed on a platform that rocks back and forth creating waves inside the bag. Currently, rocker bags are available in sizes up to L culture volume and have been proven for the Good Manufacturing Practice GMP production of human therapeutics.
Rocker bioreactors have been primarily used as batch culture for inoculum preparation [ 61 ] and transient production; however, internal perfusion filters can be applied, allowing the option of use for high-density perfusion culture. Stirred tank single-use bioreactors are the other major class of disposable bioreactors, and are the most popular type of disposable production bioreactor [ 62 ]. Disposable stirred tank bioreactors usually consist of a cylindrical presterilized bag, supported by a stainless-steel shell, with either mechanically or magnetically coupled drive shafts turning axial flow impellers.
The bag also contains either a drilled hole or a frit sparger, depending on the vendor, and an apparatus to insert autoclaved pH and DO probes or presterilized optical pH and DO patches. These patches contain a luminescent dye that changes color in proportion to changes in pH or dissolved oxygen. The color shifts of these dyes are detected using an external fluorometer [ 63 , 64 ].
Disposable bioreactors are available in sizes up to L and have been shown to give comparable cell culture performance, titer, and product quality as stainless-steel bioreactors [ 65 ]. Industrial mammalian cell culture can be divided into three primary modes of operation: batch, fed-batch, and perfusion.
Each mode of operation has its attributes and drawbacks, which are described in detail below. Batch culture is the simplest of the three modes to operate. Cells are inoculated into media, and with the exception of agitation, temperature, pH, and D.
Batch processes are easy to operate and require the least optimization effort of the three modes. Also, some protein products are degraded in the media during the batch process. Fed-batch processes start out as batch cultures; after a few days of growth — when a crucial nutrient is depleted — a concentrated solution of nutrients is added to the media. However, one needs to optimize the contents of the feed solution as well as the feeding strategy.
Similar to batch cultures, fragile proteins may be degraded during the course of the culture. Perfusion cultures can be considered continuous cultures, since conditioned media is continually removed from the bioreactor. The cells are separated from the medium using a cell retention device and are returned to the bioreactor.
The cell-free medium is collected for later protein purification. There are a variety of cell retention devices available, including spin filters, alternating tangential flow ATF filtration, acoustic separators, continuous centrifuges, and gravity settlers. Spin filters and, to a lesser extent, acoustic settlers are the two most commonly used cell retention devices for large-scale perfusion cultures [ 67 ], and recently many new perfusion-based large-scale processes are using ATF filtration [ 68 ].
Perfusion cultures usually last several weeks, but require a longer time for process optimization, and more effort in bioreactor operation than either batch or fed-batch cultures. However, the volumetric productivity is typically ten times greater than that of fed-batch cultures [ 70 ]. Since conditioned media is continually removed from the bioreactor, labile proteins can be separated immediately from cell proteases and other components that can cause degradation of the product.
Some companies have explored the use of perfusion bioreactors in conjunction with novel purification schemes to create a continuous process, whereby the product is purified immediately after it is produced in the bioreactor [ 71 ]. Mammalian cell culture processes must be tightly controlled to attain acceptable cell density, maximize product titer, and maintain acceptable product quality. Slight deviations in pH, temperature, nutrient, or catabolite concentrations can cause irreversible damage to the cells.
This section covers the effects of pH, shear stress, catabolite, and carbon dioxide accumulation on cell growth and product formation, and discusses the importance of controlling glucose and glutamine concentrations in fed-batch and perfusion cultures. A brief discussion of scale-up heuristics in mammalian cell culture is also included. General Parameter Values.
The optimal pH range for mammalian cell growth is 6. The oxygen demand for mammalian cells is 0. Shear Stress. Because mammalian cells lack a cell wall and are larger than bacteria, they are more susceptible to hydrodynamic forces, or shear stress. Several studies have investigated the effects of shear stress on mammalian cells [ 72 — 75 ].
Many indicate that the action of the impeller alone does not decrease the viability of suspension-adapted mammalian cells [ 72 , 75 , 76 ]. Some bioprocess engineers in industry have observed a few cell lines that appear to be less robust, and anecdotally might have been damaged by the impeller. However, bubble rupture does cause sufficient hydrodynamic force to kill all the cells attached to the bubble [ 75 ].
The effects of bubble rupture can be greatly reduced by the addition of surfactants, such as Pluronic F Pluronic F renders cell to bubble adhesion thermodynamically unfavorable, so the cells do not adhere to bubbles [ 77 ]. However, Pluronic F offers very little shear protection; cells rupture at the same level of hydrodynamic force regardless of the Pluronic F concentration [ 78 ].
Cells attached to microcarriers, however, are very susceptible to shear stress and can quite easily be removed and killed by the action of the impeller [ 74 ]. As mentioned in Sect. Cell lysis occurs at extreme pH; however, even moderate deviations from the optimal pH may be detrimental. Osman et al. Antibody titers increased when the pH setpoint was reduced from 7. The best way to eliminate transient deviations from optimal pH is to improve mixing reduce the mixing time ; this can be achieved by increasing the agitation or aeration rate, adding baffles, or optimizing the impeller design or placement.
Catabolite Accumulation. Several byproducts of cellular metabolism accumulate during the course of a bioreactor run. Many of these catabolites, such as lactate, ammonia, and carbon dioxide, are detrimental to cell growth and protein production.
Lao and Toth [ 81 ] pointed out the difficulty in completely decoupling the effects of lactate accumulation from the effects of increased osmolality osmolality increases with increasing lactate concentration.
For some cell lines, the effects of lactate accumulation can be mitigated by keeping glucose levels low, which can be achieved by optimizing media composition and feeding strategies [ 48 , 83 ], respectively. Other nutrient feeding strategies may limit lactate accumulation without limiting the glucose concentration in the culture [ 84 ]. In cell culture, ammonia is produced as a cellular metabolite and is converted from glutamine in the media through deamination.
An ammonia concentration of 20 mM, and possibly less, can inhibit cell growth, induce apoptosis, and alter glycosylation in certain cell lines [ 85 — 87 ]. The primary method of reducing ammonia concentrations in fed-batch and perfusion cultures is to optimize feeding strategies. Genzel et al. Carbon dioxide is a product of cellular respiration. In mammalian cell culture, carbon dioxide and sodium bicarbonate are normally used to control bioreactor pH.
Elevated partial pressure of carbon dioxide pCO 2 hinders cell growth and protein production [ 88 — 90 ].
As with lactate accumulation, the effect of elevated pCO 2 is difficult to completely decouple from the effect of elevated osmolality [ 88 , 90 ]. Mostafa and Gu [ 92 ] were able to reduce pCO 2 in a L culture and nearly double the titer by increasing the sparge rate and using an open pipe instead of a sparger. Effects of shear force, pH deviation, and accumulation of lactate, ammonia, and carbon dioxide on cell culture.
Temperature Shifts. Culture temperature is one of the primary control parameters in mammalian cell culture. Fox et al. The temperature shift occurred toward the end of the exponential growth phase of the cultures, approximately 3—4 days after inoculation in a batch culture. Different cell lines and culture conditions may have different optimal time points for the temperature shift. However, finding the optimal time point is important and worth investing the time required.
In general, fed-batch cultures are initially operated in batch mode until a key nutrient s is exhausted. Then, a solution containing the nutrient s is added to the media. A common approach is to use partial concentrates i. A useful rule for choosing the nutrient setpoint concentrations is that it should provide enough nutrients to support cell growth and product production while avoiding formation of toxic levels of catabolites due to excess feeding.
Another method is to determine which media components are depleted during the culture and add those particular nutrients to the bioreactor independently [ 97 , 98 ]. Wong et al. Using this strategy, they maximized cell viability and density while decreasing accumulation of lactate and other catabolites.
Recent advances in proteomics and metabolomics have given researchers additional insight into the internal machinery of CHO cells, and could potentially lead to more targeted approaches to media and feed optimization [ ]. Another common approach to nutrient feed optimization relies on the use of non-animal-derived hydrolysates, such as soy, wheat gluten, and yeast [ 49 ]. Hydrolysates are undefined, and variability between lots of the same type of hydrolysate is a concern [ 49 ].
The use of size exclusion filtration can reduce the lot-to-lot variability of hydrolysates [ ]. After a series of optimization experiments, based on statistical design of experiments, Kim and Lee were able to increase titer by approximately two- to three-fold using a mixture of soy, wheat, and yeast hydrolysates. In perfusion bioreactors, culture is removed from the bioreactor at certain times. The cells are separated from the conditioned media, the conditioned media is collected, and the cells are returned to the bioreactor.
Perfusion bioreactors can be operated in a variety of modes. The simplest mode is to consistently remove a certain amount of conditioned media each day i. This mode is relatively easy to control. However, as the cell density increases, the required nutrient level may not be met. Also, the protein product tends to become diluted in the collected supernatant [ 52 , 87 ]. Another mode of perfusion operation is to remove relatively small volumes of media at a time and replace the volume with a concentrated solution of nutrients.
This mode is similar to fed-batch bioreactors. Sophisticated analysis and control schemes have been developed for this type of perfusion bioreactor [ , ]. A third mode of perfusion bioreactor operation attempts to maintain a pseudo-steady state cell density, after an initial growth period. Dowd et al. This mode of perfusion reduces the frequency of sampling and analysis required to maintain a set nutrient concentration, but requires a well-characterized online cell density probe and a well-calibrated pump control scheme.
Warikoo et al. In other words, Warikoo et al. When scaling up a process to large scale, it is important to maintain the same physical and chemical conditions as in small scale. The chemical conditions include pH, oxygen level, concentration of medium components, and concentrations of toxic metabolites. These must be monitored and controlled to keep the cells in the proper physiological environment.
The physical conditions include the bioreactor configuration and the power provided to the bioreactor. In scale-up, it is advisable to preserve a similar geometrical configuration of the bioreactor in order to facilitate duplication of mixing patterns. Impellers are an important physical component in a stirred bioreactor; they convert mechanical energy to hydrodynamic motion and generate the turbulence required to keep the cells in suspension and achieve sufficient mass transfer.
At large scale, efficient oxygen delivery and carbon dioxide stripping become increasingly difficult due to suboptimal mixing and relatively low air sparge rates [ 54 , 55 ]. At the same time, the energy generated at the tip of the impeller blades must be limited, as certain cell lines can be damaged by the elevated shear force. Therefore, to achieve good mixing and minimize possible cell damage from high shear force, one must determine the proper impeller shape, ratio of impeller to vessel diameter, and impeller tip speed [ 54 ].
The mixing time as well as the oxygen and carbon dioxide mass transfer rates can be correlated to the power per unit volume of the reactor, also known as the average or overall energy dissipation rate [ 55 , ]. Maintaining constant power per unit volume is a commonly used scale-up strategy because of its simplicity.
Other strategies include keeping the average shear force experienced by the cells constant, or keeping the maximal shear force constant the shear force experienced by the cells when passing the impeller tip.
In addition to scale-up models, scale-down models are widely used to establish the operating ranges of critical large-scale process variables [ , ]. Conducting the many experiments required to define a validated range for each parameter is not feasible at large scale. Newer Post Older Post. Subscribe to: Post Comments Atom. Search Silhouette School. About Me. Craft Tools I Use. You can read Silhouette School's Terms of Use here.
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