The authors would like to thank Scott Lute for the analytical support they provided. Partial internal funding and support for this work was provided by the CDER Critical Path Program (CA #1-13). This project was supported in part by an appointment to the Internship/Research Participation Program at the Office of Biotechnology Products, U.S. Food and Drug Administration, administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and FDA.

<ol><li>Köhler, G., Milstein, C. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/1172191">Continuous cultures of fused cells secreting antibody of predefined specificity.</a> Nature. 256, 495-497 (1975).</li>
<li>Buss, N. A., Henderson, S. J., McFarlane, M., Shenton, J. M., de Haan, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Monoclonal+antibody+therapeutics%3A+history+and+future%5BTitle%5D)+AND+%22Current+Opinion+in+Pharmacology%22%5BJournal%5D)">Monoclonal antibody therapeutics: history and future.</a> Current Opinion in Pharmacology. 12 (5), 615-622 (2012).</li>
<li>Jakobovits, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+fully+human+antibodies+by+transgenic+mice%5BTitle%5D)+AND+%22Current+Opinion+in+Biotechnology%22%5BJournal%5D)">Production of fully human antibodies by transgenic mice.</a> Current Opinion in Biotechnology. 6 (5), 561-566 (1995).</li>
<li>Maksimenko, O. G., Deykin, A. V., Khodarovich, Y. M., Georgiev, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Use+of+Transgenic+Animals+in+Biotechnology%3A+Prospects+and+Problems%5BTitle%5D)+AND+%22Acta+Naturae%22%5BJournal%5D)">Use of Transgenic Animals in Biotechnology: Prospects and Problems.</a> Acta Naturae. 5 (1), 33-46 (2013).</li>
<li>Jayapal, K. P., Wlaschin, K. F., Hu, W., Yap, M. G. <a target="_blank" href="https://www.researchgate.net/publication/279541139_Recombinant_Protein_Therapeutics_from_CHO_Cells_-_20_Years_and_Counting">Recombinant protein therapeutics from CHO cells-20 years and counting.</a> Chemical Engineering Progress. 103 (10), 40(2007).</li>
<li>Velugula-Yellela, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Impact+of+media+and+antifoam+selection+on+monoclonal+antibody+production+and+quality+using+a+high+throughput+micro-bioreactor+system%5BTitle%5D)+AND+%22Biotechnology+Progress%22%5BJournal%5D)">Impact of media and antifoam selection on monoclonal antibody production and quality using a high throughput micro-bioreactor system.</a> Biotechnology Progress. , (2017).</li>
<li>Foltz, I. N., Karow, M., Wasserman, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evolution+and+Emergence+of+Therapeutic+Monoclonal+Antibodies%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Evolution and Emergence of Therapeutic Monoclonal Antibodies.</a> Circulation. 127 (22), 2222-2230 (2013).</li>
<li>Kondragunta, B., Drew, J. L., Brorson, K. A., Moreira, A. R., Rao, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+clone+selection+using+high-throughput+bioreactors%5BTitle%5D)+AND+%22Biotechnology+Progress%22%5BJournal%5D)">Advances in clone selection using high-throughput bioreactors.</a> Biotechnology Progress. 26 (4), 1095-1103 (2010).</li>
<li>Altamirano, C., Paredes, C., Cairo, J., Godia, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Improvement+of+CHO+cell+culture+medium+formulation%3A+simultaneous+substitution+of+glucose+and+glutamine%5BTitle%5D)+AND+%22Biotechnology+Progress%22%5BJournal%5D)">Improvement of CHO cell culture medium formulation: simultaneous substitution of glucose and glutamine.</a> Biotechnology Progress. 16 (1), 69-75 (2000).</li>
<li>Selvarasu, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Combined+in+silico+modeling+and+metabolomics+analysis+to+characterize+fed-batch+CHO+cell+culture%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">Combined in silico modeling and metabolomics analysis to characterize fed-batch CHO cell culture.</a> Biotechnology and Bioengineering. 109 (6), 1415-1429 (2012).</li>
<li>Seth, G., Ozturk, S., Zhang, C. <a target="_blank" href="https://www.amazon.com/Cell-Culture-Bioprocess-Engineering-Wei-Shou/dp/0985662603">Cell Culture Bioprocess Engineering</a>. Hu, W. -S. , Wei-Shou Hu. Ch. 4 97-126 (2012).</li>
<li>McKeehan, W. M. K., Ham, R. <a target="_blank" href="http://journals.sagepub.com/doi/pdf/10.1177/000456328201900613">The Growth Requirements of Vertebrate Cells In vitro</a>. Ham, R., Waymouth, C., Chapple, P. , Ch. 223 223-243 (1981).</li>
<li>Jordan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+culture+medium+improvement+by+rigorous+shuffling+of+components+using+media+blending%5BTitle%5D)+AND+%22Cytotechnology%22%5BJournal%5D)">Cell culture medium improvement by rigorous shuffling of components using media blending.</a> Cytotechnology. 65 (1), 31-40 (2013).</li>
<li>Castro, P. M. L., Hayter, P. M., Ison, A. P., Bull, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+a+statistical+design+to+the+optimization+of+culture+medium+for+recombinant+interferon-gamma+production+by+Chinese+hamster+ovary+cells%5BTitle%5D)+AND+%22Applied+Microbiology+and+Biotechnology%22%5BJournal%5D)">Application of a statistical design to the optimization of culture medium for recombinant interferon-gamma production by Chinese hamster ovary cells.</a> Applied Microbiology and Biotechnology. 38 (1), 84-90 (1992).</li>
<li>Liu, C. -H., Chu, I. M., Hwang, S. -M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factorial+designs+combined+with+the+steepest+ascent+method+to+optimize+serum-free+media+for+CHO+cells%5BTitle%5D)+AND+%22Enzyme+and+Microbial+Technology%22%5BJournal%5D)">Factorial designs combined with the steepest ascent method to optimize serum-free media for CHO cells.</a> Enzyme and Microbial Technology. 28 (4-5), 314-321 (2001).</li>
<li>Parampalli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Developement+of+serum-free+media+in+CHO-DG44+cells+using+a+central+composite+statistical+design%5BTitle%5D)+AND+%22Cytotechnology%22%5BJournal%5D)">Developement of serum-free media in CHO-DG44 cells using a central composite statistical design.</a> Cytotechnology. 54 (1), 57-68 (2007).</li>
<li>Hsu, W. -T., Aulakh, R. P., Traul, D. L., Yuk, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+microscale+bioreactor+system%3A+a+representative+scale-down+model+for+bench-top+bioreactors%5BTitle%5D)+AND+%22Cytotechnology%22%5BJournal%5D)">Advanced microscale bioreactor system: a representative scale-down model for bench-top bioreactors.</a> Cytotechnology. 64 (6), 667-678 (2012).</li>
<li>Janakiraman, V., Kwiatkowski, C., Kshirsagar, R., Ryll, T., Huang, Y. -M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+high-throughput+mini-bioreactor+system+for+systematic+scale-down+modeling%2C+process+characterization%2C+and+control+strategy+development%5BTitle%5D)+AND+%22Biotechnology+Progress%22%5BJournal%5D)">Application of high-throughput mini-bioreactor system for systematic scale-down modeling, process characterization, and control strategy development.</a> Biotechnology Progress. 31 (6), 1623-1632 (2015).</li>
<li>Kim, B. J., Diao, J., Shuler, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mini-scale+bioprocessing+systems+for+highly+parallel+animal+cell+cultures%5BTitle%5D)+AND+%22Biotechnology+Progress%22%5BJournal%5D)">Mini-scale bioprocessing systems for highly parallel animal cell cultures.</a> Biotechnology Progress. 28 (3), 595-607 (2012).</li>
<li>Legmann, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+predictive+high-throughput+scale-down+model+of+monoclonal+antibody+production+in+CHO+cells%5BTitle%5D)+AND+%22Biotechnology+and+Bioengineering%22%5BJournal%5D)">A predictive high-throughput scale-down model of monoclonal antibody production in CHO cells.</a> Biotechnology and Bioengineering. 104 (6), 1107-1120 (2009).</li>
<li>Schäpper, D., Alam, M. N. H. Z., Szita, N., Lantz, A. E., Gernaey, K. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Application+of+microbioreactors+in+fermentation+process+development%3A+a+review%5BTitle%5D)+AND+%22Analytical+and+Bioanalytical+Chemistry%22%5BJournal%5D)">Application of microbioreactors in fermentation process development: a review.</a> Analytical and Bioanalytical Chemistry. 395 (3), 679-695 (2009).</li>
<li>Hegab, H. M., ElMekawy, A., Stakenborg, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Review+of+microfluidic+microbioreactor+technology+for+high-throughput+submerged+microbiological+cultivation%5BTitle%5D)+AND+%22Biomicrofluidics%22%5BJournal%5D)">Review of microfluidic microbioreactor technology for high-throughput submerged microbiological cultivation.</a> Biomicrofluidics. 7 (2), 021502(2013).</li>
<li>Rameez, S., Mostafa, S. S., Miller, C., Shukla, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+miniaturized+bioreactors+for+cell+culture+process+development%3A+Reproducibility%2C+scalability%2C+and+control%5BTitle%5D)+AND+%22Biotechnology+Progress%22%5BJournal%5D)">High-throughput miniaturized bioreactors for cell culture process development: Reproducibility, scalability, and control.</a> Biotechnology Progress. 30 (3), 718-727 (2014).</li>
<li>Xu, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Characterization+of+TAP+Ambr+250+disposable+bioreactors%2C+as+a+reliable+scale-down+model+for+biologics+process+development%5BTitle%5D)+AND+%22Biotechnology+Progress%22%5BJournal%5D)">Characterization of TAP Ambr 250 disposable bioreactors, as a reliable scale-down model for biologics process development.</a> Biotechnology Progress. 33 (2), 478-489 (2017).</li>
<li>Delouvroy, F., et al. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3981575/">Evaluation of the advanced micro-scale bioreactor (ambr™) as a highthroughput tool for cell culture process development.</a> BMC Proceedings. 7 (6), 1-3 (2013).</li>
<li>Kelly, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimizing+performance+of+semi-continuous+cell+culture+in+an+ambr15+microbioreactor+using+dynamic+flux+balance+modeling%5BTitle%5D)+AND+%22Biotechnology+Progress%22%5BJournal%5D)">Optimizing performance of semi-continuous cell culture in an ambr15 microbioreactor using dynamic flux balance modeling.</a> Biotechnology Progress. , (2017).</li>
</ol>

Disclaimer: This publication reflects the views of the author and should not be construed to represent FDA's views or policies.

Running the automated micro bioreactor system properly and efficiently involves the timely execution of multiple automated steps. One of the most important parts of running the system is programming the software. If there is any error while writing the program, there will be serious errors in the experiment that may result in unexpected changes in the process, feeding strategy, sampling strategy, or final product quality which may invalidate the findings of the study. Another important aspect of running the system is to place and tighten the clamp plate correctly to ensure proper DO control. The most common indication that the clamp plate has been tightened unevenly is unexpected variations in DO measurements for vessels 1, 6, 7 and 12 (corner reactor vessels). Overall DO instability indicates a loosening of gaskets at the gas inlet lines in the clamp plates. This scenario may impede reaching the DO set point. Another common pitfall to avoid when starting an experiment is letting the cells sit too long during the inoculation step, causing them to settle. The less time the cells spend sitting, the less chance there is that progressively lower inoculum cell counts are added chronologically to the reactor vessels which can induce significant bias that unwittingly harms study results. It is better to inoculate in multiple stages, i.e.&#160;inoculate each culture station one after another with pause steps in between so the cells are not sitting in the inoculation plate for longer than 15 minutes.
Concerning day-to-day use, maintaining sterility is vital. Although the system is in a biological safety cabinet, sterility is not guaranteed due to frequent movement in and out of the hood. Consequently, everything that goes in the hood must be sprayed with 70% IPA. Secondly, it is essential to ensure that minimal foaming occurs during the culture; media can clog gassing and exhaust lines, leading to damage of the clamp plate and even core components below. Preventive anti-foam addition steps are critical in any micro bioreactor program design. In the event of a &#34;foam out&#34; it would be beneficial to follow the manufacturers cleaning protocol and can prevent permanent damage of clamp plates. Alternatively, use of non-sparged vessels may be beneficial for lower cell densities or when running in batch mode as higher surface to volume ratio enables efficient oxygen even with lack of a sparger. However, non-sparged vessels might not be useful for high cell density or perfusion cultures as the head space is insufficient to keep up with the cultures growing consumption of oxygen.
There are numerous advantages provided by the microbioreactor system, as it enables multiple controlled cultures to be run in parallel at a small scale with greater control than shake flasks.17&#160;Therefore, the system facilitates the execution of screening studies, DoEs, high throughput clone studies and transfection studies. Automated liquid handling also reduces analyst-to-analyst variability while simultaneously minimizing tedious and time-intensive labor for trained personnel. While there are several advantages to the system, there are several key disadvantages that should be considered. First, a culture volume of 15 mL significantly limits in-process sampling and final harvest material, and multiple alternative small scale bioreactors (up to 500 mL) have recently become available. One recent advancement to the system is the integration of the automated microscale bioreactor with the BioProfile FLEX 2 analyzer from Nova Biomedical, which mitigates the in process-sampling issue by reducing sample volume for cell density and nutrient analysis. Benefits can include quick setup and virtually no cleaning leading to operational savings, however the cost of disposable units should be considered for long term projects as it may be costlier to purchase the units than the reusable conventional systems.
The method discussed in this paper is primarily suitable for batch mode cell culture, but can be modified depending on the needs of the user. Each culture station has independent control of temperature, while DO and pH can be varied at the level of individual reactor vessels. Sartorius also offers DoE planning software designed specifically to allow experiments to be tailored for the micro bioreactor system. Large scale DoE studies using the new DoE software provided by the manufacturer can help in media and supplement optimization. Although not used here, the microbioreactor system also enables fed-batch studies. The system has not yet been optimized for perfusion cell cultures. However, there have been limited studies and trials to mimic perfusion cell culture operation in the current micro bioreactor system.26&#160;This method can be modified to mimic high density perfusion cultures by cell settling. By varying the height to which the pipet is inserted into the reactor and by optimizing settling time, the media can be removed and replenished to mirror profusion mode of culture. There are new products in this developing area that may work better than the system presented here if perfusion mode of culture is desired.
In summary, this study demonstrates the use of automated micro-bioreactors and associated analytical for CHO cell culture operations to produce and characterize a model IgG1 Monoclonal antibody. It emphasizes the role small scale micro-bioreactors play in bioprocess manufacturing and their impact on cell culture development and media screening. While there are many advantages to using an automated small scale system, to fully realize their benefits process understanding and analytical characterization is imperative. This study provides the user with a guideline for using an automated microscale reactor system, that can be developed and improved per individual research needs.

Monoclonal antibodies (mAbs) were first produced in mouse hybridoma cells in 19751. Since then, an increase in the development of recombinant protein production has taken place to humanize mAbs to increase in vivo safety and efficacy2,3,4. Most of the recombinant protein production processes employ Chinese Hamster Ovary (CHO) cells for the ease with which they can be adapted to serum free media, their ability to produce proteins with similar post-translational modifications to that of an innate human protein&#160;and their dependability as host cells5,6.
Demand is growing to deliver product faster, and for larger patient populations with consistent quality. In addition to the economic benefits, the repertoire of diseases treated by mAbs is increasing, which now includes autoimmune diseases, post-transplantation complications, arthritis, and cancers7.&#160;Average yields for modern commercial mAb production lines are typically in the range of 5-6 g/L and continue to rise5. In part, this has been accomplished through CHO cell engineering and improved production line screening using high-throughput bioreactors8. However, most increases in protein production have been attributed to process improvements, including advancements in media optimization, cell culture conditions, and enhanced feeding strategies7,9,10. Nutrient supplementation is essential not only for proper cell growth but also for efficient production of high quality protein. Furthermore, cells require the stoichiometric addition of specific nutrients, requiring additional understanding for feeding strategy optimization6,11. Traditional optimization methods include individual media component titration and media blending with mixture designs. However, these methods are time consuming, labor intensive, and involve risks associated with human error12,13.
Media optimization studies previously relied on shake flasks and 1-2 L bioreactors&#160;which may be prohibitively expensive in terms of raw materials and human capital. Microplates have also been used&#160;but these methods provide limited scalability. Furthermore, this may still require multiple time-consuming runs that introduce batch-to-batch variability&#160;which obscures the CQA variability caused by media composition and feeding strategy14,15,16. Thus, the need for high-throughput and highly consistent parallel bioreactor systems emerged17,18,19,20.
With the significant expense associated with the operation of traditional bench-scale bioreactors (0.5-5 L), microbioreactors offer a cost-reducing alternative for assessing the production of biologically derived drugs.21&#160;Bench-scale stirred-tank bioreactors are dependable and provide dense data via sensory arrays. Feedback control systems allow for easy oversight of operation. However, the assembly, calibration, cleaning, labor costs, substrate costs, and sterilization requirements make bench-scale stirred-tank bioreactors expensive and labor intensive to operate. Shake flasks and microtiter plates remove some of the cost and labor problems associated with larger scale bioreactors, but these alternatives provide weak control over processing conditions and produce low density data, often only end-point measurements.22
Alternatively, microbioreactors utilize a small working volume to provide a scale-down approach to cell line and upstream process development. The scale of microbioreactor experiments can significantly decrease running cost through lower utilization of power, substrate, labor, space, and utilities.23&#160;Microbioreactors are like shake flasks in that they are easy to handle due to their size, but they retain the advantages of traditional bench-scale bioreactors through their online feedback control of pH, temperature, dissolved oxygen, and acid/base consumption&#160;as well as their real-time data output of quality parameters including off gas composition. Microbioreactor scale allows for high-throughput screening capability, which can be useful for clone selection and process development.24
The Advanced Microscale Bioreactor has been shown to be an effective tool for CHO cell culture process characterization and development18. Herein, an automated ambr15 system, consisting of 48 microbioreactors in parallel, that&#160;have been shown to be comparable to classical stirred tank reactors in scale up studies,25 was used in a manner analogous to prior work that optimized the media composition for a CHO-DG44 cell line producing a model chimeric IgG16. The effects of varying media conditions on growth and titer were compared, and analyzed. In this paper a general guideline to run the microbioreactor system and analysis of crude media samples has been presented.

<table><tbody><tr><td>CHO DG44 Cell Line</td><td>Invitrogen</td><td>A1100001</td><td></td></tr><tr><td>ambr 15 automated microbioreactor system</td><td>Sartorius</td><td>001-2804</td><td>automated micro bioreactor</td></tr><tr><td>ambr 15 Cell Culture 24 Disposable Bioreactors - Sparged</td><td>Sartorius</td><td>001-2B80</td><td></td></tr><tr><td>1 mL disposable pipette tips, sterilized</td><td>Sartorius</td><td>A-0040</td><td></td></tr><tr><td>5 mL disposable pipette tips, sterilized</td><td>Sartorius</td><td>A-0039</td><td></td></tr><tr><td>24 Well deep well plates</td><td>Sartorius</td><td>A-0038</td><td></td></tr><tr><td>1 Well plates</td><td>Sartorius</td><td>A-0068</td><td></td></tr><tr><td>Vi-Cell XR cell counter</td><td>Beckman Coulter</td><td>731050</td><td>automated cell counter</td></tr><tr><td>EX-CELL&amp;nbsp;Antifoam (gamma irradiated)</td><td>Sigma-Aldrich</td><td>59920C-1B</td><td></td></tr><tr><td>CD OptiCHO AGT Medium</td><td>Thermo Fisher Scientific</td><td>A1122205</td><td></td></tr><tr><td>200 mM L-glutamine</td><td>Corning</td><td>25-005-CV</td><td></td></tr><tr><td>100X Penicillin/Streptomycin</td><td>Corning</td><td>30-001-CI</td><td></td></tr><tr><td>125 mL F-Bottom Shake Flasks (Sterile, Vented)</td><td>Fisher Scientific</td><td>PBV12-5</td><td></td></tr><tr><td>125 mL glass Spinner Flasks</td><td>Corning Life Sciences Glass</td><td>4500-125</td><td></td></tr><tr><td>250 mL PP Conical Centrifuge Tubes (Sterile)</td><td>Nalgene (Thermo Scientific)</td><td>376814</td><td></td></tr><tr><td>TC20 Automated Cell Counter</td><td>BioRad Laboratories, Inc.</td><td>1450103</td><td></td></tr><tr><td>Trypan Blue</td><td>Sigma-Aldrich</td><td>T8154</td><td></td></tr><tr><td>10x PBS</td><td>Corning</td><td>46-013-CM</td><td></td></tr><tr><td>BioProfile FLEX Analyzer</td><td>Nova Biomedical</td><td>49418</td><td>Nutrient Analyzer</td></tr><tr><td>Octet Red 96</td><td>Pall Fort&amp;eacute;Bio&amp;nbsp;</td><td>99-0042</td><td>Protein A Biosensor</td></tr><tr><td>Protein A Dip and Read Biosensors</td><td>Pall Fort&amp;eacute;Bio&amp;nbsp;</td><td>18-5010</td><td></td></tr><tr><td>Polypropylene 96-well Microplate, F-bottom, Chimney-style, Black</td><td>Greiner Bio-One</td><td>655209</td><td></td></tr></tbody></table>

use of high-throughput automated microbioreactor system for production of model igg1 in cho cells

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of experimental conditions while minimizing potential process variability. Applications of this approach include: clone screening, temperature and pH shifts,&#160;media and supplement optimization. Furthermore, the small reactor volumes are conducive to large Design of Experiments that investigate a wide range of conditions. This allows upstream processes to be significantly optimized before scale-up where experimentation is more limited in scope due to time and economic constraints. Automated microscale bioreactor systems offer various advantages over traditional small scale cell culture units, such as shake flasks or spinner flasks. However, during pilot scale process development&#160;significant care must be taken to ensure that these advantages are realized. When run with care, the system can enable high level automation, can be programmed to run DOE&#39;s with a higher number of variables&#160;and can reduce sampling time when integrated with a nutrient analyzer or cell counter. Integration of the expert-derived heuristics presented here, with current automated microscale bioreactor experiments can minimize common pitfalls that hinder meaningful results. In the extreme, failure to adhere to the principles laid out here can lead to equipment damage that requires expensive repairs. Furthermore, the microbioreactor systems have small culture volumes&#160;making characterization of cell culture conditions difficult. The number and amount of samples taken in-process in batch mode culture is limited as operating volumes cannot fall below 10 mL. This method will discuss the benefits and drawbacks of microscale bioreactor systems.

Monitoring critical process parameters and other cell culture parameters throughout the cell cultures&#39; operation is an important aspect in bioprocessing. The cell counter and nutrient analyzer were used to quantify five attributes that characterize cell growth, nutrient consumption and byproduct formation. The cell counts were obtained daily for all culture conditions. The average viable cell densities and viabilities are as seen in Figure 3 along with their &#177;1 SD interval. The nutrient and byproduct profiles are also shown with the &#177;1 SD interval through the stationary phase of the cultures. The slope of this profile represents the average glucose and glutamine consumption, and lactate production, rates. Overall, these results demonstrate the feasibility of monitoring these attributes in the microbioreactor system; as well as the capability of the microbioreactor system to maintain these parameters within a tight range.
The total productivity of the cell cultures was quantified using a proteinA biosensor system after the harvested cell culture media was passed through a 0.22 micron PVDF filter. The specific productivity per cell ranged from 0.87 pg/cell-d to 1.15 pg/cell-d as seen in Figure 4. Based on these results a wide array of conditions can be investigated to select media composition and feeding strategies that maximize the amount of protein produced for a minimal investment in experimental procedures.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58231/58231fig1.jpg" /> 
Figure 1:&#160;Layout of the microbioreactor system with 4 culture stations (CS) having 12 reactor vessels each. <a href="https://www.jove.com/files/ftp_upload/58231/58231fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58231/58231fig2.jpg" /> 
Figure 2:&#160;Example sample plate set-up for a basic quantitation with regeneration experiment on the proteinA biosensor system.&#160;row 1 (red &#34;R&#34;) is reserved for reference sample (i.e.&#160;matrix absent analyte); row 2 (aquamarine) is a set of standards (concentrations are in &#181;g/mL); rows 3 and 4 (orange) are sets of low and high positive controls (&#34;PL&#34; and &#34;PH,&#34; respectively); rows 5 through 10 (purple) contain unknown samples; rows 11 and 12 (grey) are program-default positions for regeneration (&#34;R&#34;) and neutralization (&#34;N&#34;) buffers. <a href="https://www.jove.com/files/ftp_upload/58231/58231fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58231/58231fig3.jpg" /> 
Figure 3:&#160;a) Average viable cell density and viability profiles over the age of a batch. &#177; 1 SD is also shown to indicate the tight control of cell growth in the microbioreactor systems. b) Average nutrient profile for glucose and glutamine as well as the average byproduct profile for lactate. &#177; 1 SD is also shown to indicate tight control over media composition in the microbioreactor systems. (N=3, all conditions were run in triplicate). <a href="https://www.jove.com/files/ftp_upload/58231/58231fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58231/58231fig4.jpg" /> 
Figure 4:&#160;Representative box-plot of average specific productivity of the various media conditions. (N=3, all conditions were run in triplicate) <a href="https://www.jove.com/files/ftp_upload/58231/58231fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells at JoVE.com

Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Automated microscale bioreactors (15 mL) can be a useful tool for cell culture engineers. They facilitate the simultaneous execution of a wide variety of ...

use-high-throughput-automated-microbioreactor-system-for-production

Research

JoVE Journal

Bioengineering

11.0K Views.  U.S. Food and Drug Administration. A detailed protocol for the concurrent operation of 48 parallel cell cultures under varied conditions in a microbioreactor system is presented. Cell culture process, harvest and subsequent antibody titer analysis are described.

Video: Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells

Laboratory Scale Production and Purification of a Therapeutic Antibody

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the antibody genes followed by stable transfection into a suitable cell line. The stable clones are subjected to screening in order to select those clones with desired production and growth characteristics. This is a critical albeit time-consuming step in the process. This protocol considers vector selection and sourcing of antibody sequences for the expression of a therapeutic antibody. The methods describe preparation of vector DNA for stable transfection of a suspension variant of human embryonic kidney 293 (HEK-293) cell line, using polyethylenimine (PEI). The cells are transfected as adherent cells in serum-containing media to maximize transfection efficiency, and afterwards adapted to serum-free conditions. Large scale production, setup as batch overgrow cultures is used to yield antibody protein that is purified by affinity chromatography using an automated fast protein liquid chromatography (FPLC) instrument. The antibody yields produced by this method can provide sufficient protein to begin initial characterization of the antibody. This may include in vitro assay development or physicochemical characterization to aid in the time-consuming task of clonal screening for lead candidates. This method can be transferable to the development of an expression system for the production of biosimilar antibodies.

This protocol describes the production of a therapeutic antibody in a mammalian expression system. The methods described include preparation of vector DNA, stable transfection and serum-free adaptation of a human embryonic kidney 293 cell line, set up of large scale cultures and purification using affinity chromatography.

Ensuring the successful production of a therapeutic antibody begins early on in the development process. The first stage is vector expression of the ...

Biochemistry

A High-throughput Automated Platform for the Development of Manufacturing Cell Lines for Protein Therapeutics

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing requirement for drug supplies. The generation of production cell lines has traditionally involved manual operations that are labor-intensive, low-throughput and vulnerable to human errors. We report here an integrated high-throughput and automated platform for development of manufacturing cell lines for the production of protein therapeutics.
The combination of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has enabled a high-throughput and more efficient cell line development process. In this operation, production host cells are first transfected with an expression vector carrying the gene of interest 1, followed by the treatment with a selection agent. The stably-transfected cells are then stained with fluorescence-labeled anti-human IgG antibody, and are subsequently subject to flow cytometry analysis 2-4. Highly productive cells are selected based on fluorescence intensity and are isolated by single-cell sorting on a BD FACSAria. Colony formation from single-cell stage was detected microscopically and a series of time-laps digital images are taken by CloneSelect Imager for the documentation of cell line history. After single clones have formed, these clones were screened for productivity by ELISA performed on a TECAN Freedom EVO liquid handling system. Approximately 2,000 - 10,000 clones can be screened per operation cycle with the current system setup.
This integrated approach has been used to generate high producing Chinese hamster ovary (CHO) cell lines for the production of therapeutic monoclonal antibody (mAb) as well as their fusion proteins. With the aid of different types of detecting probes, the method can be used for developing other protein therapeutics or be applied to other production host systems. Comparing to the traditional manual procedure, this automated platform demonstrated advantages of significantly increased capacity, ensured clonality, traceability in cell line history with electronic documentation and much reduced opportunity in operator error.

A high-throughput, automated platform of manufacturing cell line development for producing protein therapeutics is described. Implementation of BD FACS Aria Cell Sorter, CloneSelect Imager and TECAN Freedom EVO liquid handling system has demonstrated significantly increased processing capacity in cell line development with improved cell line quality and high reproducibility.

The fast-growing biopharmaceutical industry demands speedy development of highly efficient and reliable production systems to meet the increasing ...

Medicine

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Immunology and Infection

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Here, we present a detailed procedure to run a Design of Experiment in an automated micro-bioreactor followed by cell harvest and protein quantification using a Protein A column.

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain ...

Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response without any interference such as low protein expression, enzyme inactivity, or structural changes. This makes APN an attractive candidate in the development of vaccines that selectively target the mucosal epithelium. Previous studies have shown that APN is a receptor protein for both enterotoxigenic Escherichia coli (E. coli) F4 and transmissible gastroenteritis virus. Thus, APN shows promise in the development of antibody-drug conjugates or novel vaccines based on APN-specific antibodies. In this study, we compared production of APN-specific monoclonal antibodies (mAbs) using traditional hybridoma technology and recombinant antibody expression method. We also established a stably transfected Chinese hamster ovary (CHO) cell line using pIRES2-ZSGreen1-rAbs-APN and an E. coli expression BL21(DE3) strain harboring the pET28a (+)-rAbs-APN vector. The results show that antibodies expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and mAbs produced using hybridomas could recognize and bind to the APN protein. This provides the basis for further elucidation of the APN receptor function for the development of therapeutics targeting different APN-specific epitopes.

The recombinant antibody protein expressed in pIRES2-ZSGreen1-rAbs-APN-CHO cells and monoclonal antibodies produced using traditional hybridoma technology can recognize and bind to the porcine aminopeptidase N (APN) protein.

Porcine aminopeptidase N (APN), a membrane-bound metallopeptidase abundantly present in small intestinal mucosa, can initiate a mucosal immune response ...

Automated and High-throughput Microbial Monoclonal Cultivation and Picking Using the Single-cell Microliter-droplet Culture Omics System

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors are hindered by cumbersome procedures and low throughput, impeding the rapid progress of microbial culturomics research. To address these challenges, we had successfully developed the Single-cell Microliter-droplet Culture Omics System (MISS cell), an automated high-throughput platform that utilizes droplet microfluidic technology for microbial monoclonal isolation, cultivation, and screening. This system can generate a large number of single-cell droplets and cultivate, screen, and collect monoclonal colonies in a short time, facilitating an integrated process from microbial isolation to picking. In this protocol, we demonstrated its application using the isolation and cultivation of human gut microbiota as an example and compared the microbial isolation efficiency, monoclonal culture performance, and screening throughput using the solid-plate culture method. The experimental workflow was simple, and reagent consumption was very low. Compared to solid-plate culture methods, the MISS cell could cultivate a greater diversity of gut microbiota species, offering significant potential and value for microbial culturomics research.

This protocol describes how to use the Single-cell Microliter-droplet Culture Omics System (MISS cell) to perform microbial monoclonal isolation, cultivation, and picking. The MISS cell achieves an integrated workflow based on droplet microfluidic technology, which offers excellent droplet monodispersity, high parallel cultivation, and high-throughput biomass detection.

Pure bacterial cultures are essential for the study of microbial culturomics. Traditional methods based on solid plates, well plates, and micro-reactors ...

Production and Purification of Recombinant Antibodies from Mammalian Cells

Source: Shivange, Gururaj, et al. <a href="https://app.jove.com/v/60727">Analyzing Tumor and Tissue Distribution of Target Antigen-Specific Therapeutic Antibody.</a> J. Vis. Exp.(2020).
The video explains the process of producing antibodies in mammalian host cells, from gene expression to secretion. It involves transcription, translation, signal recognition, correct folding, and eventual release into the extracellular medium via secretory vesicles.

1. Expression and purification of antibodies

	Maintenance of CHO cells
	
		Grow CHO cells in FreeStyle CHO Media supplemented with commercially available ...

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Production and Purification

A Method to Produce Recombinant Antibodies Against Metallopeptidase

Source: Xia, P. et al., <a href="https://app.jove.com/v/62437">Production of Monoclonal Antibodies Targeting Aminopeptidase N in the Porcine Intestinal Mucosal Epithelium.</a>&#160;J. Vis. Exp.&#160;(2021)
This video demonstrates a procedure for the production of recombinant antibodies against metallopeptidase. Recombinant plasmids carrying genes for the antibody, green fluorescent protein, and antibiotic resistance are transfected into epithelial cells with lipofectamine. The transfected cells are selected and cultivated to generate recombinant antibodies.

1. Preparation of porcine aminopeptidase N (APN) protein antigen
NOTE: The pET28a (+)-APN-BL21 (DE3) strain and the APN stably expressed cells ...

Antibody Production from a Batch Overgrowth Culture of Transfected Mammalian Cells

Source: Elgundi, Z. et al., <a href="https://app.jove.com/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a>&#160;J. Vis. Exp.&#160;(2017)
The video demonstrates a method for producing therapeutic antibodies using stably transfected mammalian cells. Tryptone supplementation stimulates cell division, resulting in overgrowth and supporting a higher antibody yield. The antibody-containing medium is harvested, filtered, and stored for further use.