Method for culturing adherent cells in multi-parallel bioreactors

Abstract

The present invention relates to a method for culturing adherent cells in multi-parallel bioreactors using PET carrier strips for the optimization of adherent cell growth and production parameters.The present invention also relates to a method for propagating viruses and vectors from adherent cells in multi-parallel bioreactors for the optimization of production processes.The present invention relates to a method for culturing adherent cells in multi-parallel bioreactors for the optimization of adherent cell growth and production parameters.

Description

[Technical Field]

[0001] [ka] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 869,050, filed July 1, 2019, which is incorporated by reference herein in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to a method for culturing adherent cells in multi-parallel bioreactors for the optimization of adherent cell growth and production parameters. The present invention also relates to a method for propagating viruses and vectors from adherent cells in multi-parallel bioreactors for the optimization of production processes. [Background technology]

[0003] Background of the Invention Multi-parallel bioreactors (e.g., AMBR systems (Sartorius)) are fully automated, single-use bioreactors that can be utilized for process development and optimization of suspension cell growth parameters and conditions in a rapid timeframe. Multi-parallel bioreactors have historically been used to perform multiple simultaneous design of experiments (DOE) because they utilize small amounts of resources and reagents, have the ability to perform experiments at a fraction of the cost, and at a higher throughput than can be performed in traditional reactors. However, these systems are currently only available for suspension cell platforms. As various biological agents (e.g., viruses, viral vectors) are better adapted to be grown in adherent cells, procedures to make these multi-parallel bioreactors compatible with adherent cells for DOE and production procedures and parameter optimization associated with adherent bioreactors are essential. Summary of the Invention [Means for solving the problem]

[0004] Brief summary of the invention The disclosed procedure provides a novel method for using a solid attachment platform for adherent cells in multi-parallel bioreactors to optimize growth and production parameters of adherent cells.

[0005] In a first aspect, the present invention provides a process for growing adherent cells in a containment box of a multi-parallel bioreactor, the process comprising the steps of seeding the adherent cells onto a PET strip held in a culture dish, transferring the adherent cells on the PET carrier strip to the containment box of the multi-parallel bioreactor, and growing the adherent cells in the containment box at an impeller speed of between 200 rpm and 1200 rpm.

[0006] In another aspect, the process of the present invention further comprises the step of transferring the adherent cells on the PET carrier strip to a storage box of the multi-parallel bioreactor and harvesting the adherent cells 3 to 10 days later.

[0007] In another aspect of the present invention, the process of the present invention further comprises the steps of infecting the adherent cells on the PET carrier strip with at least one virus or virus particle, incubating the adherent cells on the PET carrier strip with the virus, and harvesting the virus.

[0008] In yet another aspect of the present invention, the process of the present invention further comprises treating the cells with at least one vector that produces a biological factor, incubating the adherent cells on the PET carrier strip with the vector, and harvesting the biological factor.

[0009] Other features, advantages, and aspects of the process of the present invention will become apparent to those skilled in the art from the following detailed description, examples, and claims. However, the detailed description and examples, while indicating preferred embodiments of the invention, are given for illustrative purposes only. Various changes and modifications within the spirit and scope of the disclosed invention will become apparent to those skilled in the art upon reading and understanding the description provided herein. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1 illustrates the PET carrier strips in a 6-well plate for cell seeding. Three PET strips were placed in one well of a 6-well plate and covered with 1 mL of medium. Cells were then added to the medium well and incubated overnight at 37°C.

[0011] [Figure 2] FIG. 2 shows the insertion of the PET carrier strip with adherent cells into the containment box of the multi-parallel bioreactor.

[0012] [Figure 3] Figure 3 illustrates a PET carrier strip with adherent cells in medium within the containment box of the multi-parallel bioreactor. The containment box contains an impeller. The impeller setting is set to the lowest possible impeller speed. The red circle indicates the impeller in the containment box.

[0013] [Figure 4] Figure 4 illustrates the PET carrier strips with adherent cells in the AMBR containment box while in the bioreactor. Impeller speeds of approximately 300 rpm and 1000 rpm were optimal for growth of cells attached to the PET strips.

[0014] [Figure 5]FIG. 5 is a graph illustrating the attachment and growth of different cell densities on PET carrier strips within 24 hours.

[0015] [Figure 6] FIG. 6 is a graph illustrating the growth of Vero cells adhered to PET carrier strips and incubated in the containment box described above at 37° C. for 72 hours with an impeller speed of 300 rpm.

[0016] [Figure 7] FIG. 7 is a graph illustrating the growth of Vero cells adhered to PET carrier strips and incubated in a containment box at 37° C. for 6 days at agitation levels of 300 rpm, 650 rpm, and 1000 rpm.

[0017] [Figure 8] Figure 8 is an illustration of the response shape of a VCD proof-of-concept experiment using the AMBR system. The data show optimal cell growth when cells are seeded at between 10,000 and 12,500 cells / cm², harvested on day 3, and agitated at 37°C between 300 and 350 rpm in the AMBR containment box.

[0018] [Figure 9] Figure 9 illustrates the design space failure probability model used to show the maximum percent change in cell growth success. The model shows the maximum failure probability and success probability of cell growth when accounting for seeding density and impeller speed. Green indicates the lowest % failure probability and red indicates the highest % failure probability.

[0019] [Figure 10A]10A-C illustrate the evaluation of various metabolite parameters from different cell seeding densities. Glutamine, NH4, O2, CO2, glucose, and lactate were evaluated when cells were seeded at various cell densities. These metabolite parameters indicate the overall health of the cells, and no differences in these parameters were observed based on seeding density. [Figure 10B] 10A-C illustrate the evaluation of various metabolite parameters from different cell seeding densities. Glutamine, NH4, O2, CO2, glucose, and lactate were evaluated when cells were seeded at various cell densities. These metabolite parameters indicate the overall health of the cells, and no differences in these parameters were observed based on seeding density. [Figure 10C] 10A-C illustrate the evaluation of various metabolite parameters from different cell seeding densities. Glutamine, NH4, O2, CO2, glucose, and lactate were evaluated when cells were seeded at various cell densities. These metabolite parameters indicate the overall health of the cells, and no differences in these parameters were observed based on seeding density.

[0020] [Figure 11] Figure 11 illustrates virus production from adherent cells seeded on PET strips in a multi-parallel vessel. Virus production is shown at dissolved oxygen concentrations of 85%, 40%, 20%, and 10%, with virus production increasing with decreasing dissolved oxygen.

[0021] [Figure 12A] FIG. 12A illustrates a graph of metabolite changes after the adherent cells on the PET carrier strip in the AMBR system were infected with recombinant vesicular stomatitis virus (rVSV).

[0022] [Figure 12B] FIG. 12B depicts a graph showing glutamine upregulation after rVSV infection. DETAILED DESCRIPTION OF THE INVENTION

[0023] Detailed Description of the Invention The present invention provides methods for using adherent cells in multi-parallel bioreactors.

[0024] The following corresponds to the detailed description section of this application.

[0025] Where an indefinite or definite article is used when referring to a singular noun (e.g., "a" and "an," or "the," "this," "the"), this includes the plural of that noun unless something else is specifically stated. In the context of the present invention, the term "about" or "approximate" indicates an interval of accuracy that a person skilled in the art would understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation of ±10%, and preferably ±5%, from the indicated numerical value.

[0026] The present invention includes a process for growing adherent cells in a containment box of a multi-parallel bioreactor, comprising the steps of seeding the adherent cells onto a PET carrier strip held in a culture dish; transferring the adherent cells on the PET carrier strip to the containment box of the multi-parallel bioreactor; and growing the adherent cells in the containment box at an impeller speed of between 200 rpm and 1200 rpm.

[0027] As used herein, the term "bioreactor" refers to a device that supports a biologically active environment in which biological processes, such as the propagation of viruses and vectors, can occur under controlled conditions. Bioreactors can be designed for small-scale culture (e.g., those used in laboratories) and large-scale bioreactors containing vessels or vats for producing and harvesting biological macromolecules (e.g., vaccine viruses, antigens, and vectors) on a pilot plant or commercial scale. Bioreactors can be used to grow both suspension and adherent cells. The bioreactor is a controlled environment in which oxygen, nitrogen, carbon dioxide, and pH levels can be regulated. Parameters such as oxygen, pH, temperature, and biomass are measured at periodic intervals.

[0028] As used herein, the term "multiparallel bioreactor" refers to a device in which at least two bioreactor vessels are run in parallel. As used herein, a "containment box" refers to the vessel of a multiparallel bioreactor. A multiparallel bioreactor may have 6 to 100 containment boxes. Preferably, the multiparallel bioreactor has 12 to 24 containment boxes. A multiparallel bioreactor may be fully automated so that each containment box can be controlled for media filling, inoculation, sampling, and feeding. The containment boxes may be single-use disposable vessels. Each containment box can be individually controlled for temperature, pH, and impeller speed. Each containment box may include components, including, but not limited to, sensor ports for continuous monitoring of pH and dissolved oxygen (DO), an impeller for stirring / agitation, feed tubes for media / reagent addition, gas delivery tubes for N2, O2, and air delivery, and a sample port for sample removal.

[0029] Examples of commercially available multi-parallel bioreactors that can be used in the processes of the present invention include, but are not limited to, the AMBR 15, AMBR 250, Solida Biotech parallel bioreactor, and xCubio bioreactor.

[0030] The bioreactor capacity is the volume of medium that can be held in the bioreactor. The multi-parallel bioreactor capacity is the capacity of each containment box. The multi-parallel bioreactor capacity or "capacity," as used herein, can range from 5 mL to about 5 L. The capacity can be about 2 mL to about 10 mL, about 5 mL to about 50 mL, about 25 mL to about 100 mL, about 75 mL to about 500 mL, about 250 mL to about 750 mL, or about 600 mL to about 1000 mL. Preferably, the capacity can be 15 mL or 250 mL. More preferably, the containment box volume is 15 mL.

[0031] The multi-parallel bioreactor may be a closed-loop. As used herein, "closed-loop" refers to a process system with equipment designed and operated so that products are not exposed to the room environment and materials can be introduced or removed from the closed-loop system but in a manner that avoids exposing the products to the room environment. "Multi-parallel bioreactor metabolite" or "metabolite" refers to a metabolite produced by the adherent cells during the cell growth phase and virus or vector propagation phase, which can be monitored in the multi-parallel bioreactor and includes, but is not limited to, NH4, carbon dioxide, glutamine, glucose, and lactate. "Multi-parallel bioreactor condition" or "condition" refers to a condition of the multi-parallel bioreactor that can be monitored or adjusted during the growth of the adherent cells or the propagation of the virus or vector. Examples of conditions include, but are not limited to, pH, temperature, DO, and cell density.

[0032] As used herein, a "carrier" refers to any solid support matrix to which the adherent cells can attach. The carrier can be of any shape, including, but not limited to, a strip, a sheet, a fiber, a filament, a sphere, or any combination thereof. Preferably, the carrier is in the shape of a strip. The carrier can be made of any material, including, but not limited to, polystyrene, polyethylene, polyethylene terephthalate (PET), polypropylene, polyester, polycarbonate, polyamide, polyurethane, glass, ceramic, metal, acrylamide, silica, silicone, cellulose, dextran, collagen, and glycosaminoglycans. The present invention also contemplates materials for the support matrix that are not yet known or that may become known to those skilled in the art in the future. The materials can be used by themselves or with other materials. Preferably, the carrier is made of PET.

[0033] The above carrier is 1cm 2 ~about 50cm 2 The carriers may provide different average growth areas ranging from about 1 cm 2 ~about 10cm 2 , about 5cm 2 ~about 50cm 2 , about 25cm 2 ~Approx. 100cm 2 , about 50cm 2 ~about 500cm 2 , about 250cm 2 ~Approx. 750cm 2 , about 600cm 2 ~About 1000cm 2 Preferably, the carrier provides an average growth area of about 5 cm 2 ~20cm 2 More preferably, the carrier provides an area of about 13.9 cm 2 Most preferably, the carrier is a PET strip containing a high surface area, which is approximately 13.9 cm 2This provides a growth area of 3D area, which is increased due to the woven PET fibers within the strip, creating an environment that promotes high density growth of adherent cell growth.

[0034] Agitation, which involves stirring or moving the culture medium inside the containment box, can be performed to supply nutrients to the cells within the containment box and increase the DO concentration in the culture medium within the containment box. The agitation can be performed by a device such as a propeller or impeller. Agitation is preferably performed by using an impeller within the containment box. An "impeller" is a rotor for increasing the pressure of a fluid flow. Each vessel of the multi-parallel vessel can have at least one impeller. The impeller speed or agitation speed can be controlled. The impeller speed or agitation speed can range from 200 rpm to 2000 rpm. Preferably, the impeller speed or agitation speed used during the cell growth and proliferation phases can be from 200 rpm to 1000 rpm. More specifically, the impeller speed or agitation speed is 300 rpm.

[0035] As used herein, "culture media" or "media" refers to the liquid used to culture adherent cells in the containment box. The media used in the disclosed procedures can contain various components that support the growth of adherent cells, including, but not limited to, amino acids, vitamins, organic and inorganic salts, and carbohydrates. The media can be serum-free media, which are media formulated without any animal serum. When used, serum-free media are selected from, but are not limited to, DMEM, DMEM / F12, Medium 199, MEM, RPMI, OptiPRO SFM, VP-SFM, VP-SFM AGT, HyQ PF-Vero, and MP-Vero. The culture media can also be animal-free media, i.e., they do not contain any products of animal origin. The culture media can also be protein-free media, i.e., the media are not formulated with proteins.

[0036] Adherent cells are cells that adhere to a surface in culture conditions, and anchorage may be required for their growth; they may also be referred to as anchorage-dependent cells. Adherent cells suitable for the procedures of the present disclosure include, but are not limited to, Madin-Darby canine kidney epithelial cells (MDCK), Madin-Darby bovine kidney epithelial cells (MDBK), chicken or quail cells, PerC6 cells, 3T3 cells, NTCT cells, CHO cells, PK15 cells, MDBK cells, LLC-MK2 cells, MRC-5 cells, 293 cells, Hela cells, HEK293 cells, or combinations or modifications thereof. Preferred adherent cells are anchorage-dependent cells that can be grown on carriers such as PET strips, although suspension cells that can be adapted to grow as adherent cells can also be used. More preferably, the anchorage-dependent cells of the present disclosure are Vero cells. Selecting adherent host cells to use the processes of the present disclosure is within the knowledge of one of ordinary skill in the art.

[0037] The adherent cells can be pre-seeded or seeded on a carrier (e.g., a PET strip) before being transferred to the storage box of the multi-parallel bioreactor. The adherent cells can be seeded on a carrier held in a culture dish (e.g., but not limited to, a Petri dish, a 6-well culture plate, or a 12-well plate). Preferably, the adherent cells can be seeded on a carrier held in a 6-well culture plate containing 1 mL of culture. The adherent cells can be incubated with the strip at 37°C for 8 to 48 hours. Preferably, the adherent cells can be incubated with the strip at 37°C for 8 hours.

[0038] The number of adherent cells seeded onto the carrier to carry out the procedure of the present disclosure is approximately 10,000 viable cells / cm. 2 ~Approx. 50,000 live cells / cm 2 The PET carrier strips act as a medium to allow cell attachment. The strips contain a high surface area, creating an environment that promotes high-density growth of adherent cell growth. The use of these strips in the containment box of the multi-parallel bioreactor provides a novel platform for utilizing this microsystem for both cell and virus growth or process optimization of adherent bioreactors.

[0039] The virus of the process of the present disclosure can be a virus, a virus antigen, or a virus vector, or a combination or modification thereof.The virus can be a whole virus or a virus antigen, but is not limited to, selected from the group consisting of vesicular stomatitis virus (VSV), adenovirus, influenza virus, chikungunya virus, Ross River virus, hepatitis A virus, vaccinia virus and recombinant vaccinia virus, Japanese encephalitis virus, herpes simplex virus, cytomegalovirus (CMV), rabies virus, West Nile virus, yellow fever virus, and chimeras thereof, and rhinovirus and reovirus.Preferably, the virus can be VSV.

[0040] The adherent cells can be inoculated with a vector to produce a biological factor. As used herein, "vector" refers to any agent capable of delivering and expressing a nucleic acid molecule in a host cell (e.g., the adherent cells). A vector can be any suitable nucleic acid molecule that can be introduced into the cell or integrated into the cellular genome of the adherent cells. Types of vectors include, but are not limited to, plasmids, viruses, cosmids, or episomes, including naked DNA. The vectors of the present invention can be viral vectors, such as modified vaccinia virus Ankara (MVA), rVSV, adeno-associated virus (AAV), lentivirus, retrovirus, or adenovirus. The recombinant protein expressed by the viral vector can be a viral protein, a bacterial protein, or any therapeutic recombinant protein. More preferably, the recombinant protein produced by the viral vector is a viral protein. The vectors of the present invention can be expression vectors, which can be nucleic acid molecules containing a promoter and other sequences necessary to drive the expression of a desired gene or DNA sequence.

[0041] Specific metabolites can be evaluated in the cell-containing PET carrier strip in the AMBR storage box. For example, metabolites (such as, but not limited to, glutamine, NH4, O2, CO2, glucose, and lactate) can be evaluated in each storage box containing the adherent cells. The specific patterns of metabolite consumption and production can be used to evaluate the cell-containing PET carrier strip in the AMBR system.

[0042] The procedures of the present invention can be used in DOE studies or small-scale bioreactor operations for production optimization. Non-limiting examples of applications of the procedures of the present invention include media development, process optimization to make the process scalable, strain selection, and vector screening. [Example]

[0043] Example Example 1 PET Strip Seeding in 6-well Plates Vero cells were grown on PET carrier strips (each measuring approximately 2.5 cm x 0.7 cm, but with an increased three-dimensional area due to woven PET fibers within the strips, resulting in approximately 13.9 cm per strip). 2 Three PET strips were placed in each well of a 6-well plate and covered with 1 mL of medium (Figure 1). Vero cells were placed in 1 cm of the PET strips. 2 1 x 10 per 4 , 1.5×10 4 , and 2 × 10 4 Different seeding densities of live cells were added. 1 mL of medium was added to the wells and the cells were incubated with the strips overnight at 37°C.

[0044] Example 2: Cell growth on PET carrier strips in a containment box Each of the three strips prepared according to Experiment 1 was placed into an AMBR containment box (one PET carrier strip with Vero cells attached per containment box), and the amber impeller setting was set to the lowest impeller speed. The impeller in the AMBR containment box is shown in Figure 3, and the containment box in the AMBR bioreactor is shown in Figure 4. 1 x 10 cells were placed in a 6-well plate as described in Experiment 1 for the PET strips. 4 , 1.5×10 4 , and 2 × 10 4 live cells / cm 2 Seeding of cells in the AMBR box resulted in cell attachment and growth within the AMBR containment box (Figure 5).

[0045] After programming the AMBR system, cell growth was found to be promoted on the PET carrier strips with gentle agitation (between 300 rpm and 1,000 rpm) (Figures 8 and 9). As a proof-of-concept for utilizing this novel system for DOE testing, the PET strips were seeded at various cell densities and harvested between 3 and 10 days post-seeding at various agitation speeds. Data from 4D response geometries showed that cells were grown at 10,000 cells / cm. 2 The results show that optimal cell growth occurs when cells are inoculated at 10,000 to 12,500 cells / cm and harvested 3 days after inoculation, and agitated between 300 and 350 rpm at 37°C inside an AMBR containment box (Figure 8). As confirmation, the design space failure probability model predicts optimal cell growth between 10,000 and 12,500 cells / cm. 2 showed that seeding cells between 100 and 150 rpm and agitating the cells between 300 and 487 rpm promoted the highest probability of successful cell growth (Figure 9).

[0046] Experiment 3: Measurement of metabolites during cell growth As a proof of concept, specific metabolites were evaluated in cell-containing PET carrier strips in an AMBR containment box. Glutamine, NH4, O2, CO2, glucose, and lactate were evaluated (Figure 10). The data show specific patterns of metabolite consumption and production in this system, further demonstrating that the system can be used to evaluate cell-containing PET carrier strips in an AMBR system. Similar data was observed when evaluating agitation speed (data not shown).

[0047] Taken together, these data suggest a novel mechanism for process optimization of adherent cells using PET carrier strips and the AMBR system (which is designed for suspension cell culture optimization). The system allows for measurement of cell growth and various metabolites. This is important for evaluating various conditions for process optimization of adherent cell culture bioreactor systems (Figure 10). While not previously described, this system is useful for determining the optimization parameters required for cell growth, virus production, antibody production, etc.

[0048] Experiment 4: Virus production from adherent cells on PET strips in a multi-parallel bioreactor The adherent cells were infected with virus, and viral growth from the adherent cells growing on the strips was measured. The adherent cells on the strips were infected with VSV virus as described in Experiments 1-3. The virus was propagated under different conditions, including growth after growing the adherent cells at impeller speeds of 300 rpm and 480 rpm (data not shown), as well as at different DO levels. VSV was successfully propagated in the adherent cells on the PET carrier strips in the multi-parallel bioreactor under all conditions tested. An increase in viral growth was observed when DO was reduced from 85% to 10%, as observed by assay (FIG. 11).

[0049] Experiment 5: Measurement of metabolites and conditions during virus growth Vero cells were grown and infected with VSV as described in Experiments 1-4. Metabolites and conditions (e.g., glutamine, NH4, O2, CO2, glucose, lactate, and pH) were measured after infection and during VSV growth in Vero cells (Figure 12A). Glutamine was upregulated after infection without the addition of any medium. Glutamine upregulation can be used as a metabolic parameter to indicate positive viral infection in Vero cells (Figure 12B).

[0050] Conclusions from Experiments 1-5: Overall, these data demonstrate that cells attached to PET strips can be used in a multi-parallel bioreactor (e.g., AMBR suspension platform) to conduct DOE studies or small-scale bioreactor runs for production process optimization. This represents a novel use of a system not previously described and demonstrates a range of applications, from optimizing cell growth conditions to optimizing virus, protein, and / or antibody production parameters from adherent cells growing on PET strips, through virus / vector infection or transfection procedures. The resulting process optimization data can be utilized to establish parameters to be used in adherent bioreactor systems (i.e., iCELLis or Univercells reactor systems). The PET-AMBR adherent strategy described above provides a means to acquire extensive amounts of data in a high-throughput and cost-effective manner using 24 or 48 small-scale bioreactors in parallel, which can be used to replace numerous parallel adherent bioreactor runs, which utilize significant resources and time. This system allows for increased flexibility and an enhanced decision-making process, which is helpful when dealing with complex biological therapeutic, vaccine, and prophylactic development and production. Furthermore, multiple adherent bioreactor runs conducted in parallel for process optimization result in higher production costs; therefore, incorporating the use of the novel PET-AMBR adherent optimization strategy results in faster production timelines and lower overall production costs, two factors of critical importance in the pharmaceutical market. In certain embodiments, for example, the following items are provided: (Item 1) 1. A process for growing adherent cells in a containment box of a multi-parallel bioreactor, the process comprising: seeding the adherent cells onto a carrier held in a culture dish; transferring the adherent cells on the carriers to a containment box of the multi-parallel bioreactor; growing the adherent cells in a containment box while stirring the medium at an impeller speed between 200 rpm and 1200 rpm; A process that encompasses. (Item 2) 2. The process of claim 1, wherein the carrier is a PET strip. (Item 3) 3. The process according to item 1 or 2, wherein the culture dish is a 6-well plate. (Item 4) 3. A process for optimizing cell growth of adherent cells in the containment box of the multi-parallel bioreactor of item 1 or 2, said process comprising: a step of transferring the adherent cells on the carriers to a storage box of the multi-parallel bioreactor and harvesting the adherent cells 3 to 10 days later; A process that further encompasses (Item 5) 3. A process for growing adherent cells in the containment box of the multi-parallel bioreactor of item 1 or 2, said process comprising: infecting the adherent cells on the carrier with at least one virus or virus particle; incubating the adherent cells on the carrier with the virus; and harvesting the virus; A process that further encompasses (Item 6) 3. A process for growing adherent cells in the containment box of the multi-parallel bioreactor of item 1 or 2, said process comprising: treating the cells with at least one vector that produces a biological factor; incubating the adherent cells on the carrier with the vector; harvesting the biological factor; A process that further encompasses (Item 8) 7. The process of item 6, wherein the vector is a viral vector selected from the group consisting of modified vaccinia virus Ankara (MVA), vesicular stomatitis virus (VSV), adeno-associated virus (AAV), lentivirus, retrovirus, and adenovirus. (Item 9) 6. The process of items 1, 2, 3, 4, and 5, wherein the adherent cells are selected from the group consisting of Madin-Darby canine kidney epithelial (MDCK) cells, Madin-Darby bovine kidney epithelial (MDBK) cells, chicken or quail cells, PerC6 cells, 3T3 cells, NTCT cells, CHO cells, PK15 cells, MDBK cells, LLC-MK2 cells, MRC-5 cells, HEK293 cells, Hela cells, or combinations or modifications thereof. (Item 10) 10. The process according to items 1 to 9, wherein the adherent cells are Vero cells. (Item 11) 10. The process according to items 1 to 9, wherein the adherent cells are HEK293 cells. (Item 12) Item 1. The process of item 1, wherein the impeller speed ranges from 300 rpm to 1000 rpm. (Item 13) Item 13. The process of item 1 or item 12, wherein the impeller speed is 300 rpm. (Item 14) The growth area of the PET strip is 10 cm 2 ~15cm 2 The process according to item 2, ranging from (Item 15) The growth area of the PET strip is 13.9 cm 2 Item 3. The process according to item 2, wherein (Item 16) 3. The process according to item 2, wherein the PET strip is made from an interwoven fiber. (Item 17) 18. The process of items 1 to 17, wherein the adherent cells are grown in a closed-loop manufacturing system. (Item 18) 18. The process according to items 1 to 17, wherein the multi-parallel bioreactor has at least two containment boxes. (Item 19) 19. The process of items 1 to 18, wherein the multi-parallel bioreactor has 24 containment boxes. (Item 20) 17. The process according to items 1 to 16, wherein the multi-parallel bioreactor is selected from the group comprising AMBR 15, AMBR 250, Solida Biotech parallel bioreactor, and xCubio bioreactor. (Item 21) 17. The process of items 1 to 16, wherein the process is used to perform DOE studies or small-scale bioreactor operations for production process optimization. (Item 22) 22. The process according to item 21, wherein the production process to be optimized is cell growth, virus production, or antibody production.

Claims

1. 1. A process for growing adherent cells in a multi-parallel bioreactor, the process comprising: seeding the adherent cells in a medium in a first container onto a carrier, which is a polyethylene terephthalate (PET) carrier; transferring the adherent cells on the PET carriers to a second vessel within a multi-parallel bioreactor; and growing the adherent cells in the second vessel while agitating the medium in the second vessel at an impeller speed of between 300 rpm and 487 rpm; A process that encompasses.

2. The process of claim 1 , wherein the PET carrier is a PET strip.

3. a. transferring the adherent cells on the carriers to the second container within the multi-parallel bioreactor and harvesting the adherent cells 3 to 10 days later; 3. The process of claim 1 or claim 2, further comprising:

4. a. infecting the adherent cells on the carrier with at least one virus or virus particle; b. Incubating the adherent cells on the carrier with the virus or virus particles to result in virus production from the adherent cells; and c. harvesting the virus; The process of any one of claims 1 to 3, further comprising:

5. 5. The process of claim 4, wherein the cells are incubated at reduced dissolved oxygen levels of 85%, 40%, 20%, or 10% dissolved oxygen, wherein incubating the cells at the reduced dissolved oxygen levels increases the virus production.

6. a. treating the adherent cells on the carrier with at least one vector that produces a biological factor; b. Incubating the adherent cells on the carrier with the at least one vector; c. harvesting the biological factor; The process of any one of claims 1 to 5, further comprising:

7. The process of claim 6 , wherein the vector is naked DNA, a plasmid, a virus, a cosmid, or an episome.

8. 8. The process of any one of claims 1 to 7, wherein the adherent cells are selected from the group consisting of Madin-Darby canine kidney epithelial (MDCK) cells, Madin-Darby bovine kidney epithelial (MDBK) cells, chicken or quail cells, PerC6 cells, 3T3 cells, NTCT cells, CHO cells, PK15 cells, LLC-MK2 cells, MRC-5 cells, HEK293 cells, Hela cells, or combinations thereof.

9. The process according to any one of claims 1 to 7, wherein the adherent cells are Vero cells.

10. The process of any one of claims 1 to 9, wherein the adherent cells are grown in a closed-loop manufacturing system.

11. The process of any one of claims 1 to 10, wherein the second vessel comprises a storage box within the multi-parallel bioreactor, and the multi-parallel bioreactor has at least two storage boxes, or the multi-parallel bioreactor has 24 storage boxes.

12. The process described in any one of claims 1 to 11, wherein the process is used to perform design of experiments (DOE) studies or small-scale bioreactor operations for an optimized production process, the optimized production process including cell growth, virus production, and / or antibody production.

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