Abstract:
A method for continuously preparing a medium formulation mixes a diluent with a plurality of chemically incompatible concentrate solutions in such a manner that none of the ingredients of the concentrate solutions chemically react in an adverse manner. The method utilizes a static mixing chamber to add the concentrate solutions to the diluent stream sufficiently in advance of one another so that adverse chemical reactions do not occur. The method also adjusts a pH level of the diluent prior to adding any of the concentrate solutions to the diluent.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     1. Field of the Invention  
         [0002]     The present invention relates to the field of cell culture medium formulations, and more specifically, to methods for continuously preparing cell culture medium formulations and buffered salt solutions from selected subgroups of medium concentrates.  
         [0003]     2. Background Art  
         [0004]     Cell culture medium formulation provide nutrients necessary to maintain and grow cells in a controlled, artificial and in vitro environment. Characteristics and compositions of the cell culture mediums vary depending on the particular cellular requirements. Important parameters include osmolarity, pH, and nutrient formulations.  
         [0005]     Medium formulations have been used to grow a number of cell types including animal, plant and bacterial cells. Cells grown in culture medium catabolize available nutrients and produce useful biological substances such as monoclonal antibodies, hormones, growth factors and the like. Such products have therapeutic applications and, with the advent of recombinant DNA technology, cells can be engineered to produce large quantities of these products. Thus, the ability to grow cells in vitro is not only important for the study of cell physiology, it is necessary for the production of useful substances which may not otherwise be obtained by cost-effective means.  
         [0006]     Cell culture medium formulations have been well documented in the literature and a number of medium are commercially available. Typical nutrients in cell culture medium formulations include amino acids, salts, vitamins, trace metals, sugars, lipids and nucleic acids. Often, particularly in complex medium formulations, stability problems result in toxic products and/or lower effective concentrations of required nutrients, thereby limiting the functional life-span of the culture medium. For instance, glutamine is a constituent of almost all medium formulations that are used in the culturing of mammalian cells in vitro. Glutamine decomposes spontaneously into pyrrolidone carboxylic acid and ammonia. The rate of degradation can be influenced by pH and ionic conditions but in cell culture medium, formation of these breakdown products cannot be avoided (Tritsch et al.,  Exp. Cell Research,  28:360-364(1962)).  
         [0007]     Wang et al. (In Vitro, 14:(8):715-722 (1978)) have shown that photoproducts such as hydrogen peroxide, which are lethal to cells, are produced in Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM). Riboflavin and tryptophan or tyrosine are components necessary for formation of hydrogen peroxide during light exposure. Because most mammalian culture medium formulations contain riboflavin, tyrosine and tryptophan, toxic photoproducts are likely produced in most cell culture mediums.  
         [0008]     To avoid these problems, researchers make medium formulations on an “as needed” basis, and avoid long term storage of the culture medium. Commercially available medium formulations, typically in dry powder form, serve as a convenient alternative to making the medium formulations from scratch, i.e., adding each nutrient individually, and also avoids some of the stability problems associated with liquid medium formulations. However, only a limited number of commercial culture medium formulations are available, except for those custom formulations supplied by the manufacturer.  
         [0009]     Although dry powder medium formulations may increase the shelf-life of some medium formulations, there are a number of problems associated with dry powdered medium formulations, especially in large scale application. Production of large volumes requires storage facilities for the dry powder, not to mention the specialized kitchens necessary to mix and weigh the nutrient components. Due to the corrosive nature of dry powder medium ingredients, mixing tanks must be periodically replaced.  
         [0010]     There exists a need to lower the cost of production of biological substances. Efficient and cost effective methods to stabilize liquid cell culture medium formulations as well as the development of convenient methods to produce 1× medium formulations would be an important development in the field of cell culture medium technology.  
         [0011]     One such development in the field of cell culture medium formulations is the development of liquid medium concentrates as is disclosed in U.S. Pat. No. 5,474,931 issued to DiSorbo et al. on Dec. 12, 1995 (“DiSorbo”). DiSorbo discloses a method of subgrouping medium formulations into stable, compatible components that can be solubilized at high concentrations (10× to 100×). Concentrated culture medium formulations (2-10×) or 1× cell culture medium formulations can be prepared by mixing a sufficient amount of the concentrated subgroup solutions with each other and with a sufficient amount of a diluent (water, buffer, etc.).  
         [0012]     Escalating demand for large volumes of nutrient medium and buffered salt solutions and increasing pressure to minimize batch-associated costs, such as sterile filtration and quality release testing, has driven a requirement for increased production batch sizes of liquid medium. As a result, stainless steel formulation tanks of 5000-10,000 liters for preparation of large batches of liquid medium or buffered salt solutions have become relatively common. However, scale-up manufacture of these fluids in this manner presents challenges regarding product quality and economy.  
         [0013]     What is needed is a system and method for providing continuous, online preparation of large volumes of biological fluids (e.g., liquid medium, buffered salt solutions, etc.) within a highly controlled manufacturing system.  
       BRIEF SUMMARY OF THE INVENTION  
       [0014]     The present invention is a system and method for continuous, online preparation of cell culture medium formulations from selected subgroups of medium concentrates. In particular, a computer controlled system controls the flow of a diluent and one or more concentrated solutions into a static mixing chamber wherein the diluent and the concentrated solutions are mixed to form the cell culture medium formulations.  
         [0015]     The present invention is able to formulate a cell culture medium from concentrated solution subgroups including an acid soluble concentrate solution subgroup, a group I salts solution concentrate subgroup, a group II salts solution concentrate subgroup, and a base soluble solution concentrate subgroup. Furthermore, the present invention is able to adjust the pH of the cell culture medium using either an acid solution or a base (caustic) solution.  
         [0016]     In particular, the present invention is able to mix the concentrated solution subgroups with the diluent in a manner such that the ingredients of the concentrated solution subgroups do not adversely react chemically with one another.  
         [0017]     One feature of the present invention is the preparation of large quantities of 1× cell culture medium (100,000 liters or more) while requiring only one quality control test. By increasing the size of the “batch,” the present invention reduces the per liter cost of cell culture medium.  
         [0018]     Another feature of the present invention is the increased consistency in the 1× cell culture medium. Statistical analyses have demonstrated that the present invention is able to provide 1× cell culture medium with homogeneity within batches of ±2.0%. Furthermore, the present invention provides improved precision between production runs of 1× cell culture medium manufactured from identical concentrate solutions of ±3.0%.  
         [0019]     Still another feature of the present invention is-a clean in place (CIP) and a steam in place (SIP) system which allows various components of the present invention to be sanitized and sterilized according to current good manufacturing practices (cGMP).  
         [0020]     Further features and advantages of the present invention, as well as the structure and operation of various embodiments of the present invention, are described in detail below with reference to the accompanying drawings. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES  
       [0021]     The present invention is described with reference to the accompanying drawings. In the drawings, like reference numbers indicate identical or functionally similar elements. Additionally, the left-most digit(s) of a reference number identifies the drawing in which the reference number first appears.  
         [0022]      FIG. 1  illustrates an automated liquid manufacturing system (ALMS) according to the present invention.  
         [0023]      FIG. 2  illustrates a diluent system according to a preferred embodiment of the present invention.  
         [0024]      FIG. 3  illustrates a medium mixing system according to a preferred embodiment of the present invention.  
         [0025]      FIG. 4  illustrates a medium surge vessel according to one embodiment of the present invention.  
         [0026]      FIG. 5  illustrates a pre-filtration system and a sterile filtration system according to a preferred embodiment of the present invention.  
         [0027]      FIGS. 6A and 6B , respectively, illustrate a front view and a right side view of a medium mixing chamber according to a preferred embodiment of the present invention.  
         [0028]      FIG. 7  illustrates an isometric view of a portion of the medium mixing chamber according to a preferred embodiment of the present invention.  
         [0029]      FIG. 8  illustrates an example of a computer control system useful for controlling the operation of the present invention.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0030]     In the description that follows, a number of terms conventionally used in the field of cell culture medium are utilized extensively. In order to provide a clear and consistent understanding of the specification and claims, and the scope to be given such terms, the following definitions are provided.  
         [0031]     Ingredients. The term “ingredients” refers to any compound, whether of chemical or biological origin, that can be used in cell culture medium to maintain or promote the growth or proliferation of cells. The terms “component,” “nutrient,” and “ingredient” can be used interchangeably and are all meant to refer to such compounds. Typical ingredients that are used in cell culture medium formulations include amino acids, salts, metals, sugars, lipids, nucleic acids, hormones, vitamins, fatty acids, proteins and the like. Other ingredients that promote or maintain growth of cells in vitro can be selected by those of skill in the art, in accordance with the particular need.  
         [0032]     Cell Culture. By “cell culture” is meant cells or tissues that are maintained, cultured or grown in an artificial, in vitro environment.  
         [0033]     Culture Vessel. Glass, plastic or metal containers of various sizes that can provide an aseptic environment for growing cells are termed “culture vessels.” 
         [0034]     Cell Culture Medium. The phrases “cell culture medium” or “culture medium” or “medium formulation” or “cell culture medium formulation” refer to a nutritive solution for culturing or growing cells. The ingredients that comprise such medium formulations may vary depending on the type of cell to be cultured. In addition to nutrient composition, osmolarity and pH are considered important parameters of culture medium formulations.  
         [0035]     Compatible Ingredients. Each ingredient used in cell culture medium formulations has unique physical and chemical characteristics. By “compatible ingredients” is meant those medium nutrients which can be maintained in solution and form a “stable” combination. A solution containing “compatible ingredients” is said to be “stable” when the ingredients do not degrade or decompose substantially into toxic compounds, or do not degrade or decompose substantially into compounds that can not be utilized or catabolized by the cell culture. Ingredients are also considered “stable” if degradation can not be detected or when degradation occurs at a slower rate when compared to decomposition of the same ingredient in a 1× cell culture medium formulation. Glutamine, for example, in 1× medium formulations, is known to degrade into pyrrolidone carboxylic acid and ammonia. Glutamine in combination with divalent cations are considered “compatible ingredients” since little or no decomposition can be detected over time.  
         [0036]     Compatibility of medium ingredients, in addition to stability measurements, are also determined by the “solubility” of the ingredients in solution. The term “solubility” or “soluble” refers to the ability of an ingredient to form a solution with other ingredients. Ingredients are thus compatible if they can be maintained in solution without forming a measurable or detectable precipitate. Thus, the term “compatible ingredients” as used herein refers to the combination of particular culture medium ingredients which, when mixed in solution either as concentrated or 1× medium formulations, are “stable” and “soluble.” 
         [0037]     1× Formulation. A cell culture medium is composed of a number of ingredients and these ingredients vary from medium to medium. A “1× formulation” or “1× medium formulation” is meant to refer to any aqueous solution that contains some or all ingredients found in a cell culture medium. The “1× formulation” can refer to, for example, the cell culture medium or to any subgroup of ingredients for that medium. The concentration of an ingredient in a 1× solution is about the same as the concentration of that ingredient found in the cell culture formulation used for maintaining or growing cells. Cell culture medium formulations used to grow cells are 1× formulation by definition. When a number of ingredients are present (as in a subgroup of compatible ingredients), each ingredient in a 1× formulation has a concentration about equal to the concentration of those ingredients in a cell culture medium. For example, RPMI 1640 culture medium contains, among other ingredients, 0.2 g/l L-arginine, 0.05 g/l L-asparagine, and 0.02 g/l L-aspartic acid. A “1× formulation” of these amino acids, which are compatible ingredients according to the present invention, contains about the same concentrations of these ingredients in solution. Thus, when referring to a “1× formulation,” it is intended that each ingredient in solution has the same or about the same concentration as that found in the cell culture medium being described. The concentrations of medium ingredients in a 1× formulation are well known to those of ordinary skill in the art, See  Methods For Preparation of Media, Supplements and Substrate For Serum - Free Animal Cell Culture,  Allen R. Liss, N.Y. (1984), which is incorporated by reference herein in its entirety. The osmolarity and/or pH, however, may differ in a 1× formulation compared to the culture medium, particularly when fewer ingredients are contained by the 1× formulation.  
         [0038]     10× Formulation. A “10× formulation” refers to a solution wherein each ingredient in that solution is about 10 times more concentrated than the same ingredient in the cell culture medium formulation. RPMI 1640 medium, for example, contains, among other things, 0.3 g/l L-glutamine. By definition, a “10× formulation” contains about 3.0 g/l glutamine. A “10× formulation” may contain a number of additional ingredients at a concentration about 10 times that found in the 1× culture medium. As will be apparent, “25× formulation,” “50× formulation” and “100× formulation” designate solutions that contain ingredients at about 25, 50 or 100 fold concentrations, respectively, as compared to a 1× cell culture medium. Again, the osmolarity and pH of the medium formulation and concentrated formulation may vary.  
         [0000]     Automated Liquid Manufacturing System  
         [0039]     According to the present invention, an automated liquid manufacturing system (ALMS) continuously prepares medium products (e.g., cell culture medium, buffered salt solutions, salt solutions, buffers, etc.) having various formulations (e.g., 1-10×) by mixing one or more concentrate solution subgroups together with a diluent (e.g. water, buffer, etc.). The amount of concentrated solution and amount of diluent needed may vary depending on the concentration of each subgroup, the number of subgroups, and the desired concentration of the final medium product. One of ordinary skill in the art can easily determined a sufficient volume of a diluent and a sufficient volume of the concentrated solutions to prepare the desired medium product.  
         [0040]     The pH of the desired medium product may also be adjusted by the addition of acid or base. The medium product, however, may not require any adjustment, especially if the pH of the medium product as prepared is within the desired pH range. Osmolarity of the medium product can also be adjusted after mixing the concentrated solutions with the diluent. Typically, the desired, osmolarity may be predetermined and adjustments in the salt concentration of the concentrated solutions may be made to prepare a final medium product with the desired osmolarity.  
         [0041]     The present invention also provides for on-line sanitization and sterilization in place as required by current good manufacturing practices (cGMP). The sanitization operation is commonly referred to as “clean in place,” and sterilization operation is commonly referred to as “steam in place.” These operations are discussed in further detail below.  
         [0042]     According to the present invention, sufficient amounts of each concentrate solution subgroup are continuously admixed with sufficient amounts of a diluent in a mixing chamber, while the resulting medium product is continuously removed. The following describes various aspects of the present invention and the manner in which they accomplish the continuous preparation of medium product.  
         [0043]      FIG. 1  illustrates a system level block diagram of an automated liquid manufacturing system (ALMS)  100  according to the present invention. ALMS  100  includes a concentrate system  110 , a diluent system  120 , a medium mixing system  130 , a medium surge vessel  140 , a prefiltration system  150 , a sterile filtration system  160  and a fill system  170 . Sterile filtration system  160  and fill system  170  operate in a clean area  180 . In addition to the above-mentioned system components, a preferred embodiment of the present invention includes a waste disposal system  190 . Each of these components of ALMS  100  will be discussed in further detail below.  
         [0044]     A preferred embodiment of the present invention is controlled by a computer control system  105 . For ease of illustration, connections between computer control system  105  and the various components of ALMS  100  have not been shown. Needless to say, each of the components of ALMS  100  has some subcomponent, be it a valve, a pump, a sensor, etc., that is connected to computer control system  105  and used to control the operation of ALMS  100  as would be apparent. Computer control system  105  is described in further detail below.  
         [0000]     Concentrate System  
         [0045]     Concentrate system  110  provides one or more concentrate solutions  115  to ALMS  100 . Specifically, concentrate system  110  provides concentrate solutions  115  to medium mixing system  130 . Concentrate system  110  may perform this task in a variety of ways. In one embodiment of the present invention, concentrate system may provide concentrate solutions  115  in a manner similar to that described in commonly owned U.S. Pat. No. 5,474,931 issued to DiSorbo et al. on Dec. 12, 1995, which is incorporated herein by reference as if reproduced below in its entirety. DiSorbo discloses a method for producing liquid medium concentrates in compatible subgroups. According to this embodiment of the present invention, concentrate solutions  115  are chemically stable 50× formulations of liquid medium concentrates.  
         [0046]     These subgroups include the following: an acid soluble concentrate solution subgroup, a group I salts concentrate solution subgroup, a group II concentrate solution subgroup, and a base soluble concentrate solution subgroup. In addition, sodium hydroxide may be prepared as a concentrate solution subgroup although this is not necessary. The acid soluble concentrate solution subgroup referred to herein is essentially equivalent-to the acid-soluble subgroup referred to in DiSorbo; the group I salts concentrate solution subgroup referred to herein is essentially equivalent to the glutamine-containing subgroup referred to in DiSorbo; the group II salts concentrate solution subgroup referred to herein is essentially equivalent to the weak acid-base soluble subgroup referred to in DiSorbo; and the base soluble concentrate solution subgroup referred to herein is essentially equivalent to the alkali-soluble subgroup referred to in DiSorbo. The remaining subgroups referred to in DiSorbo are treated as reserve concentrate solutions for purposes of the present invention.  
         [0047]     In this embodiment, the subgroups are formulated and “kited” according to published procedures as would be apparent. After being prepared according to these procedures the-subgroups are stored in intermediate storage vessels for use by ALMS  100 .  
         [0048]     In another embodiment of the present invention, concentrate system  110  provides preformulated and prepackaged concentrate solutions  115 . These concentrate solutions  115  are purchased from a manufacturer of such concentrate solutions such as are available from Life Technologies, Incorporated, 3175 Staley Road, Grand Island, N.Y., 716/774-6700. In addition, concentrated subgroups for buffered salts can be obtained from Life Technologies as acid soluble concentrate solution subgroups and base soluble concentrate solution subgroups. This embodiment permits a manufacturer of medium products to purchase concentrate solutions  115  without itself having the facilities to manufacture or produce such concentrate solutions  115 .  
         [0049]     In yet another embodiment of the present invention, concentrate system  110  provides an on-line concentrate solution  115  as a part of a continuous manufacturing process in which concentrate solutions  115  are produced directly from raw materials and passed directly to ALMS  100  without an intermediate storage device such as that described in DiSorbo.  
         [0050]     As would be apparent to one skilled in the art, other types of concentrate solutions  115  are available other than the subgroups described above. Furthermore, other means for providing concentrate solution  115  to ALMS  100  may be available as would also be apparent.  
         [0000]     Diluent System  
         [0051]     Diluent system  120  provides a diluent  125  to ALMS  100 . In particular, diluent system  120  provides diluent  125  to medium mixing system  130 . Diluent  125  may be any solution or liquid that may be used to dilute concentrate solutions  115 . Such diluents include water, buffers, salt solutions, etc. In a preferred embodiment of the present invention, diluent  125  is water, most preferably, water for injection. However, any diluent  125  may be used in ALMS  100  that appropriately dilutes concentrate solutions  115  according to the particular needs of the medium product manufacturer.  
         [0052]     A preferred embodiment of diluent system  120  is illustrated in  FIG. 2 . In this embodiment of the present invention, diluent system  120  includes an ambient water for injection (WFI) tank  210 , a hot WFI tank  220 , a control valve  215 , a control valve  225 , and a WFI break tank  230 . WFI break tank  230  includes a level indicator  250  and a spray ball  240 .  
         [0053]     The purpose of WFI break tank  230  is to provide an atmospheric break between the plant water system and ALMS  100  as required by current good manufacturing practices (cGMP). In addition, WFI break tank  230  assures removal of entrained air from ambient WFI tank  210  and hot WFI tank  220  prior to their introduction to ALMS  100 .  
         [0054]     In one embodiment of the present invention, ambient WFI tank  210  is not a tank. Rather, ambient WFI tank  210  is directly connected to the plant&#39;s water system. In other embodiments of the present invention, ambient WFI tank  210  may actually be a tank. This may be the case, for example, when a diluent  125  other than water is used, or when a particular type of water is required (e.g. deionized, distilled, sterile, etc.). Hot WFI tank  220  provides hot water to ALMS  100  during a clean-in-place (CIP) operation which is discussed in further detail below.  
         [0055]     Valve  215  and valve  225  control the flow of ambient water from ambient WFI tank  210  and hot water from hot WFI tank  220 , respectively, to WFI break tank  230 . In a preferred embodiment of the present invention, WFI break tank  230  provides ambient water as diluent  125  to ALMS  100 .  
         [0056]     Level indicator  250  monitors a level of diluent  125  in WFI break tank  230 . Level indicator  250  is monitored by computer control system  105  to maintain an appropriate level of diluent  125  in WFI break tank  230 .  
         [0057]     Spray ball  240  is a part of the CIP operation which is discussed in further detail below. Spray ball  240  provides a mechanism for cleaning the inside of WFI break tank  230  during the CIP operation.  
         [0000]     Medium Mixing System  
         [0058]     Medium mixing system  130  is shown in further detail in  FIG. 3 . Medium mixing system  130  includes a static mixing chamber  310 , a diluent input pump  320 , a diluent flow indicator  325 , a CIP divert valve  330 , a series of concentrate solution pumps  340  (shown as concentrate solution pumps  340 A-H), a first pH sensor  361 , a second pH sensor  362 , a conductivity sensor  363 , a UV absorbance sensor  364 , an output flow indicator  365 , a diverter valve  370 , and a back flow preventer valve  375 . Each of these elements of medium mixing system  130  is described in further detail below.  
         [0059]     Medium mixing system  130  receives diluent  125  and one or more concentrate solutions  115  and mixes them in mixing chamber  310 . Medium mixing system  130  accomplishes this in a manner such that none of the ingredients of concentrate solutions  115  adversely chemically react with one another or with diluent  125 . By “adversely chemically react” it is meant that the ingredients react 1) to form an irreversible precipitate; 2) to cause degradation in one or more components of the concentrate solutions; 3) to cause certain components to become inactivated; or 4) to cause any other condition that would result in an unacceptable medium product  135 .  
         [0060]     Diluent input pump  320  controls the flow of diluent  125  into static mixing chamber  310 . This flow is measured by diluent flow indicator  325 . Diluent flow indicator  325  permits computer control system  105  to monitor the flow of diluent  125  and thereby, control diluent input pump  320 . Back flow preventer valve  375  prevents diluent  125  from flowing backwards from static mixing chamber  
         [0061]     Based on the flow of diluent  125  into static mixing chamber  310 , computer control system  105  controls the flows of concentrate solutions  115  (shown as concentrate solutions  115 A-H) into static mixing chamber  310  via concentrate solution pumps  340  (shown as concentrate solution pumps  340 A-H). The flow of each of concentrate solutions  115 A-H is controlled to be proportional to the flow of diluent  125  into static mixing chamber  310  according to a formulation of a desired medium product.  
         [0062]     Sensors  361 ,  362 ,  363 ,  364  and  365  monitor a medium product  135  output from static mixing chamber  310  to ensure that particular parameters associated with medium product  135  are within acceptable levels associated with the desired medium product. These sensors are coupled to computer control system  105  which monitors these parameters of medium product  135  to ensure that proper mixing of concentrate solutions  115 A-H and diluent  125  is being accomplished. If the medium product is within the acceptance levels, medium product  135  passes to medium surge vessel  140 . If not, computer control system  105  diverts medium product  135  to waste disposal system  190  via diverter valve  370 . This allows medium mixing system  130  to guarantee an acceptable medium product  135 . For example, when ALMS  100  starts up preparation of a particular medium product  135 , the initial output of static mixing chamber  310  may not be within the acceptance levels for the particular medium product. Thus, this portion of the output is diverted to waste disposal system  190 . When the output of static mixing chamber  310  enters into the acceptable levels (i.e., the operation reaches a “steady state”), the output from static mixing chamber  310  is passed to medium surge vessel  140 .  
         [0063]     In a preferred embodiment of the present invention, first pH sensor  361  and second pH sensor  362  are placed in close proximity to each other and as close to static mixing chamber  310  as possible, and prior to sensors  363 ,  364  to ensure that the proper pH levels of medium product  135  is being achieved.  
         [0064]     Conductivity sensor  363  measures the ionic character of medium product  135 . In particular, conductivity sensor  363  measures the resistivity of the flow of medium product  135 . Conductivity sensor  363  is useful for determining the quality of medium product  135 , especially for salt solutions.  
         [0065]     UV absorbance sensor  364  measures an amount of ultraviolet light that passes through the flow of medium product  135 . UV absorbance sensor  364  is useful for detecting the presence of precipitates within medium product  135 . UV absorbance sensor  364  can also be used to measure a concentration of a particular component as an on-line measurement of concentrate addition and mixing quality.  
         [0066]     As would be apparent to one skilled in the art, other types of sensors may be implemented in medium mixing system  130  to measure other levels of other parameters associated with medium product  135 .  
         [0067]     In a preferred embodiment of the present invention, concentrate solution pumps  340 A-H are extremely precise variable speed pumps. In particular, concentrate solution pumps  340 A-F are capable of delivering 0 to 3 liters of fluid per minute with ±1.0% or better accuracy. Concentrate solution pumps  340 G-H are capable of delivering 0 to 3.5 liters of fluid per minute with ±1.0% accuracy. A preferred embodiment of the present invention uses pumps which are manufactured by IVEK, North Springfield, Vt.  
         [0068]     In a preferred embodiment of the present invention, concentrate solution  115 A and concentrate solution  115 B are reserved for providing an acid solution and a base solution, respectively, to static mixing chamber  310 . Hence, referring to these as “concentrate solutions” may be considered a misnomer. However, as would be apparent, solutions, liquids, etc., other that “concentrate solutions” may be introduced in this manner to static mixing chamber  310  as would be apparent.  
         [0069]     In this preferred embodiment of the present invention, acid solution  115 A and caustic solution  115 B adjust a pH level of diluent  125  according to specifications required by the production of medium product  135 . The addition of either acid solution  115 A or caustic solution  115 B to diluent  125  is done first so that the proper pH level of diluent  125  can be achieved prior to the addition of other concentrate solutions  115 C-H.  
         [0070]     As shown in  FIG. 3 , diluent  125  enters static mixing chamber  310  and begins “mixing” sufficiently prior to the addition of any concentrate solutions  115 A-H. This ensures that static mixing chamber  310  can provide a “turbulent diluent stream” from diluent  125  to enhance the overall mixing process between diluent steam  125  and concentrate solution  115 A-H. The turbulent diluent stream is produced from diluent  125  by being forced past a series of baffles within static mixing chamber  310  as is well understood by those in the art. Also, the introduction of a last concentrate solution  115 H occurs sufficiently prior to the end of static mixing chamber  310  so that last concentrate solution  115 H can be sufficiently mixed in turbulent diluent stream. As discussed above, the output of static mixing chamber  310  is medium product  135 .  
         [0071]     As shown in  FIG. 3 , static mixing chamber  310  includes a series of injection ports  315  (shown as injection ports  315 A- 315 H). Injection ports  315  introduce concentrate solutions  115  into static mixing chamber  310 . In particular, injection ports  315  introduce concentrate solutions  115  into turbulent diluent stream  125 .  FIG. 6  shows a mechanical drawing of static mixing chamber  310  in further detail.  
         [0072]      FIG. 6A ,  FIG. 6B , and  FIG. 7  illustrate static mixing chamber  310  in greater detail. In particular,  FIGS. 6A and 6B  are mechanical drawings showing a front view and a right side view, respectively, of static mixing chamber  310 .  FIG. 7  is an isometric drawing of static mixing chamber  310 . As shown in  FIGS. 6A, 6B , and  7 , static mixing chamber  310  includes a series of injection ports  315 . In particular, static mixing chamber  310  includes two groupings of radially disposed injection ports shown as injection ports  315 C,  315 D, and  315 E and injection ports  315 F,  315 G, and  315 H. In addition, as shown in  FIGS. 6A and 6B , static mixing chamber  310  also includes two additional injection ports  315 A and  315 B.  
         [0073]     Injection ports  315 C,  315 D, and  315 E are described as being radially disposed around static mixing chamber  310 . By “radially disposed” it is meant that injection ports  315 C,  315 D, and  315 E are located on a common circumference around static mixing chamber  310 . That is, injection ports  315 C,  315 D, and  315 E are located at an approximately equal distance from the upstream end of static mixing chamber  310 . Preferably, injection ports  315 C,  315 D, and  315 E are spaced equally about the common circumference of static mixing chamber  310 . Thus, for the case of three injection ports, the injection ports  315 C,  315 D, and  315 E are space at 120 degree increments. Other embodiments may provide for non-equal spacings about the common circumference.  
         [0074]     In one embodiment of the present invention, the injection ports are essentially disposed both “linearly” and “radially” from one another. Such would be the case, for example, where the injection ports were disposed in spiral fashion about static mixing chamber  310 . Depending on the length of the spiral, the injection ports could be considered linearly disposed, radially disposed, or both. Injection ports  315 F,  315 G, and  315 H are also radially disposed around static mixing chamber  310 . In addition, this group of injection ports, both individually and collectively, is “linearly disposed” along the fluid flow path of static mixing chamber  310  from injection ports  315 C,  315 D, and  315 E as shown in  FIG.6 . In other words, injection ports  315 F,  315 G, and  315 H are located at an approximately equal distance from the upstream end of static mixing chamber  310 , where this distance is sufficiently different from the distance from the upstream end of static mixing chamber  310  to injection ports  315 C,  315 D, and  315 E.  
         [0075]     In the particular embodiment shown in  FIG. 6  and  FIG. 7 , three injection ports are radially disposed from one another in each of the two groups of injection ports. As would be apparent to one skilled in the art, additional injection ports may be included within each group, limited by two parameters. The first parameter is the number of injection ports that can physically, or mechanically, fit around static mixing chamber  310 . The second parameter is the number of injection ports that can be used to introduce concentrate solutions  115  to diluent  125  without the ingredients of concentrate solutions  115  adversely chemically reacting with one another. As also would be apparent, fewer injection ports may be included within each group.  
         [0076]     In addition to changing the number of injection ports within each radially disposed group, the number of radially disposed groups may also be changed. The number of radially disposed groups of injection ports is also limited by the same parameters as described above as would be apparent.  
         [0077]     As shown in  FIG. 6  and  FIG. 7 , diluent  125  flows from the upstream end of static mixing chamber  310  toward the downstream end of static mixing chamber  310 . Thus, as diluent  125  flows through static mixing chamber  310 , diluent  125  encounters injection ports  315 A and  315 B first, followed by injection ports  315 C,  315 D and  315 E, and finally, injection ports  315 F,  315 G and  315 H.  
         [0078]     As thus described, static mixing chamber  310  provides two manners in which different concentrate solutions  115  can be added to diluent  125 . The first manner is to add the different concentrate solutions  115  by using injection ports that are radially disposed from one another such as injection ports  315 F,  315 G,  315 H or injection ports  315 C,  315 D and  315 E. The second manner in which different concentrate solutions  115  can be added to diluent  125  is by using injection ports  315  that are linearly disposed from one another such as injection ports  315 C and  315 F. In either case, an injection port  315  adds a concentrate solution  115  to diluent  125  in a manner such that the concentrate solution  115  becomes sufficiently diluted by diluent  125  prior to encountering any other concentrate solution  115  added from a different injection port  315 . This prevents any adverse chemical reaction between the ingredients of the two concentrate solutions.  
         [0079]     While this is true in general, the order of introduction of certain concentrate solutions  115  to diluent  125  from a particular injection port configuration are preferred, while other orders of introduction are discouraged. For example, medium product  135  that includes a base soluble concentrate solution and a group II salts concentrate solution are preferably prepared by introducing these two concentrate solutions into diluent  125  by radially disposed injection ports. Doing so improves the microenvironment chemistry of the resulting medium product  135 .  
         [0080]     Also, medium product  135  that includes a group II salts concentrate solution and an acid soluble concentrate solution are preferably prepared by introducing these two concentrate solutions into diluent  125  from linearly disposed injection ports  315 . Introducing these two concentrate solutions from injection ports that are radially disposed from one another is detrimental to product quality and may create an irreversible precipitation of critical cell culture medium components rendering the resulting medium product inactive.  
         [0081]     In a preferred embodiment of the present invention, the following injection ports  315  concentrate solution  115  pairings are used: acid soluble concentrate solutions are introduced by injection port  315 D; group I salts concentrate solutions are introduced by injection port  315 E; group II salts concentrate solutions are introduced by injection port  315 G; base soluble concentrate solutions are introduced by injection port  315 H; acid solutions for adjusting pH are introduced by injection port  315 A; and base (caustic) solutions for adjusting pH are introduced by injection port  315 B. If sodium hydroxide concentrate solutions are used, they are preferably introduced by injection port  315 F. Otherwise, injection port  315 F is reserved for other concentrate solutions not included above. Injection port  315 C is also reserved for other concentrate solutions not included above.  
         [0000]     Medium Surge Vessel  
         [0082]      FIG. 4  illustrates medium surge vessel  140  in greater detail. Medium surge vessel  140  includes a medium surge tank  410 , an agitation system  420 , a level indicator  430 , a temperature control system  450 , and a pH sensor  470 . Medium product  135  from medium mixing system  130  enters medium surge tank  410  which provides a buffering mechanism for ALMS  100 . In other words, medium surge vessel  140  provides a “buffer” between the continuous operation of medium mixing system  130  and the discontinuous operation of downstream components of ALMS  100  such as fill system  170 . Thus, medium product  135  from medium mixing system is permitted to accumulate in medium surge vessel  140  when, for example, fill system  170  is temporarily shutdown to change fill containers.  
         [0083]     An amount of medium product  135  in medium surge tank  410  is monitored by computer control system  105  via fill indicator  430 . Depending on the level of medium product  135  in medium surge tank  410 , computer control system  105  adjusts the output rate of medium product  135  from medium mixing system  130 .  
         [0084]     A pH level of medium product  135  is measured by pH sensor  470  as medium product  135  leaves medium surge tank  410 . This permits computer control system  105  to monitor and ensure the quality of medium product  135 .  
         [0085]     In one embodiment of the present invention, agitation system  420  is used to provide agitation (i.e., mixing) to medium product  135  within medium surge tank  410 . In one embodiment, agitation system  420  provides continuous mixing of medium product  135  in medium surge tank  410 . In another embodiment, agitation system  420  provides mixing of medium product in medium surge tank  410  after a particular level is reached or some other parameter. Agitation system  420  may or may not be required in order to maintain medium product  135  in a homogeneous state. In a preferred embodiment of the present invention, agitation system  420  is not used.  
         [0086]     In one embodiment of the present invention, temperature control system  450  controls the temperature of medium product  135  within medium surge tank  410 . Temperature control system  450  operates so as to maintain a particular temperature of medium product  135  in medium surge tank  410 . Various means of controlling the temperature of the contents of medium surge tank  410  are available as would be apparent. In one embodiment of the present invention, glycol is circulated through an outer tank (not shown) around medium surge tank  410  thereby maintaining a particular temperature of the contents within medium surge tank  410 . In a preferred embodiment of the present invention, temperature control system  450  is not used.  
         [0087]     In one embodiment of the present invention, compressed air  460  is provided to medium surge tank  410  to maintain a given head pressure within medium surge tank  410 . Compressed air  460  is used to provide sufficient pressure to move medium product  135  through medium surge tank into prefiltration system  150 . In a preferred embodiment of the present invention, the head pressure is maintained between 6 and 10 p.s.i.g. Other embodiments may utilize gases other than air, such as nitrogen, to provide the head pressure as well as to prevent the outgasing from medium product  135  as would be apparent.  
         [0088]     Diverter valve  445  is controlled by computer control system  105  to implement the CIP operation as will be discussed below. Diverter valve  445  diverts fluid to spray ball  440  in order to clean the inside of medium surge tank  410  during the CIP operation.  
         [0000]     Filtration System  
         [0089]      FIG. 5  illustrates prefiltration system  150  and sterile filtration system  160  in further detail. Prefiltration system  150  includes a prefiltration pump  510  and a prefiltration filter  520 . Prefiltration system  150  receives medium product  145  from medium surge tank  140 . Prefiltration pump  510  pumps medium product  145  through a non-sterile prefilter filter  520 . Prefilter filter  520  is a filter membrane that provides variable filtration of medium product  145 . Depending upon the particular medium product  145  being prepared, the filter membrane is selected to filter particles that may range between 0.1 and 2 microns.  
         [0090]     Medium product that has been filtered by prefiltration system  150  enters sterile filtration system  160 . As shown in  FIG. 5 , sterile filtration system  160  operates in a clean area  180 . Sterile filtration system  160  includes two sterilizing filters  530 A and  530 B in a parallel configuration followed by a final sterilizing filter  540 . This particular configuration of sterilizing filters provides redundant 0.1 or 0.2 micron filtration for medium product  145 . Filtered medium product  165  is output from sterile filtration system  160  and enters fill system  170 .  
         [0091]     Sterilizing filters  530  and final sterilizing filter  540  are steam sterilized via a steam in place operation which is discussed in further detail below. In a preferred embodiment, the sterilizing filters are steam sterilized prior to manufacturing a new batch of cell culture medium formulation.  
         [0000]     Fill System  
         [0092]     As shown, fill system  170  is also contained within clean area  180 . Fill system  170  provides aseptic connections in clean area  180  so that multiple medium product containers can be filled outside of clean area  180 .  
         [0093]     In one embodiment of present invention, fill system  170  provides a mechanism whereby multiple containers (i.e., sterile bags, carboys, glass bottles, drums, etc.) can be filled. In another embodiment of the present invention, fill system  170  may not be required or may be modified. For example, an embodiment of ALMS  100  may be implemented to provide medium product  145  directly to a bioreactor as would be apparent.  
         [0094]     Diverter valves  505 ,  525  and  545  are controlled by computer control system  105  and used during the CIP operation as will be discussed below. The diverter valves provide a mechanism to flush unwanted medium product through to waste disposal system  190  as well as to provide mechanisms to clean and product purge prefilter  520  and sterilizing filters  530 A,  530 B and  540 .  
         [0000]     ALMS Process Capability  
         [0095]     In a preferred embodiment of the present invention, ALMS  100  is designed to operate with flow rates between 1,000 and 3,000 liters or medium product per hour. Other embodiments of the present invention may have different flow rates depending upon the sizing and accuracy of, for example, concentrate solution pumps  340 , diluent input pump  320 , and static mixing chamber  310 .  
         [0096]     In a preferred embodiment of the present invention, medium product  165  has an intra-run homogeneity with a precision tolerance of ±2.0%. Precision between production runs of medium product  165  from identical concentrated materials is ±3.0%. Furthermore, a pH fluctuation of medium product  165  is within ±0.1 units.  
         [0000]     Clean In Process (CIP) and Steam In Place (SIP) Process Operations  
         [0097]     ALMS  100  is designed for on-line sanitization and sterilization in place as required. The sanitization operation is commonly referred to as “clean in place.” The sterilization operation using steam under pressure is commonly referred to as “steam in place.” A typical operation will require sanitization of the entire system including WFI brake tank  230  and steam sterilization of sterile filtration system  160  as well as fill system  170 .  
         [0098]     Sanitization of ALMS  100  includes the flushing of the entire ALMS  100  with hot water from hot WFI  220 . Hot water from hot WFI  220  is routed through ALMS  100  via diverter valves (e.g., diverter valve  145 , diverter valve  505 , diverter valve  525 , diverter valve  545 , etc.) to and through spray balls (e.g., spray ball  240  and spray ball  440 ), and recirculated from fill system  170  to media mixing system  130  via an appropriate conduit (shown as line  175  in  FIG. 1 ) to flush ALMS  100 . In one embodiment of the present invention, caustic solution is added to hot water from hot WFI  220  via static mixing chamber  310  to provide a hot caustic sanitization of ALMS  100 . The hot caustic is recirculated, neutralized with acid and sent to waste disposal system  190 .  
         [0099]     For sterilizing ALMS  100 , steam is introduced at the sterile filtration system  160  via a steam input port  550  located inside clean area  180 . Steam flows through sterile filtration system  160 , including sterilizing filters  530  and final sterilizing filter  540 , and fill system  170 , and heats these components to sterilization temperatures. The temperature is monitored at appropriate points and sterilization is confirmed using well known time/temperature parameters as would be apparent.  
         [0100]     The by-products of the sanitization process are routed to waste disposal system  190  as shown in various figures. In one embodiment of the present invention, waste disposal system treats any by-products of ALMS  100  by appropriate measures so as not to introduce any harmful products into the plant&#39;s waste disposal system as would be apparent.  
         [0000]     Computer Control System  
         [0101]     In various embodiments of the present invention, computer control system  105  is implemented using hardware, software or a combination thereof and may be implemented in a computer system or other processing system. In fact, in one embodiment, the invention is directed toward a computer system capable of carrying out the functionality described herein. An example computer system  802  is shown in  FIG. 8 . Computer system  802  includes one or more processors, such as processor  804 . Processor  804  is connected to a communication bus  806 . Various software embodiments are described in terms of this example computer system. After reading this description, it will become apparent to a person skilled in the relevant art how to implement the invention using other computer systems and/or computer architectures.  
         [0102]     Computer system  802  also includes a main memory  808 , preferably random access memory (RAM), and may also include a secondary memory  810 . Secondary memory  810  may include, for example, a hard disk drive  812  and/or a removable storage drive  814 , representing a floppy disk drive, a magnetic tape drive, an optical disk drive, etc. Removable storage drive  814  reads from and/or writes to a removable storage unit  818  in a well known manner. Removable storage unit  818 , represents a floppy disk, magnetic tape, optical disk, etc. which is read by and written to by removable storage drive  814 . As will be appreciated, removable storage unit  818  includes a computer usable storage medium having stored therein computer software and/or data.  
         [0103]     In alternative embodiments, secondary memory  810  may include other similar means for allowing computer programs or other instructions to be loaded into computer system  802 . Such means can include, for example, a removable storage unit  822  and an interface  820 . Examples of such can include a program cartridge and cartridge interface (such as that found in video game devices), a removable memory chip (such as an EPROM, or PROM) and associated socket, and other removable storage units  822  and interfaces  820  which allow software and data to be transferred from the removable storage unit  818  to computer system  802 .  
         [0104]     Computer system  802  can also include a communications interface  824 . Communications interface  824  allows software and data to be transferred between computer system  802  and external devices. Examples of communications interface  824  can include a modem, a network interface (such as an Ethernet card), a communications port, a PCMCIA slot and card, etc. Software and data transferred via communications interface  824  are in the form of signals which can be electronic, electromagnetic, optical or other signals capable of being received by communications interface  824 . Signals  826  are provided to communications interface via a channel  828 . Channel  828  carries signals  826  and can be implemented using wire or cable, fiber optics, a phone line, a cellular phone link, an RF link and other communications channels.  
         [0105]     In this document, the terms “computer program medium” and “computer usable medium” are used to generally refer to media such as removable storage device  818 , a hard disk installed in hard disk drive  812 , and signals  826 . These computer program products are means for providing software to computer system  802 .  
         [0106]     Computer programs (also called computer control logic) are stored in main memory and/or secondary memory  810 . Computer programs can also be received via communications interface  824 . Such computer programs, when executed, enable the computer system  802  to perform the features of the present invention as discussed herein. In particular, the computer programs, when executed, enable processor  804  to perform the features of the present invention. Accordingly, such computer programs represent controllers of the computer system  802 .  
         [0107]     In an embodiment where the invention is implement using software, the software may be stored in a computer program product and loaded into computer system  802  using removable storage drive  814 , hard drive  812  or communications interface  824 . The control logic (software), when executed by processor  804 , causes processor  804  to perform the functions of the invention as described herein.  
         [0108]     In another embodiment, the invention is implemented primarily in hardware using, for example, hardware components such as application specific integrated circuits (ASICs). Implementation of the hardware state machine so as to perform the functions described herein will be apparent to persons skilled in the relevant art(s).  
         [0109]     In yet another embodiment, the invention is implemented using a combination of both hardware and software.  
       Conclusion  
       [0110]     While the invention has been particularly shown and described with reference to several preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined in the appended claims.