Name: use of high-throughput automated microbioreactor system for production of model igg1 in cho cells
Uploaded: 2018-09-28T13:00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about Use of High-Throughput Automated Microbioreactor System for Production of Model IgG1 in CHO Cells at JoVE.com

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.

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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 border="0" cellpadding="0" cellspacing="0" style="width:899px;" width="898">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:373px;">CHO DG44 Cell Line</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:157px;">A1100001</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ambr 15 automated microbioreactor system</td>
			<td>Sartorius</td>
			<td>001-2804</td>
			<td>automated micro bioreactor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ambr 15 Cell Culture 24 Disposable Bioreactors - Sparged</td>
			<td>Sartorius</td>
			<td>001-2B80</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 mL disposable pipette tips, sterilized</td>
			<td>Sartorius</td>
			<td>A-0040</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5 mL disposable pipette tips, sterilized</td>
			<td>Sartorius</td>
			<td>A-0039</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">24 Well deep well plates</td>
			<td>Sartorius</td>
			<td>A-0038</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Well plates</td>
			<td>Sartorius</td>
			<td>A-0068</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Vi-Cell XR cell counter</td>
			<td>Beckman Coulter</td>
			<td>731050</td>
			<td>automated cell counter</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EX-CELL&#160;Antifoam (gamma irradiated)</td>
			<td>Sigma-Aldrich</td>
			<td>59920C-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CD OptiCHO AGT Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1122205</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">200 mM L-glutamine</td>
			<td>Corning</td>
			<td>25-005-CV</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100X Penicillin/Streptomycin</td>
			<td>Corning</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">125 mL F-Bottom Shake Flasks (Sterile, Vented)</td>
			<td>Fisher Scientific</td>
			<td>PBV12-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">125 mL glass Spinner Flasks</td>
			<td>Corning Life Sciences Glass</td>
			<td>4500-125</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">250 mL PP Conical Centrifuge Tubes (Sterile)</td>
			<td>Nalgene (Thermo Scientific)</td>
			<td>376814</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TC20 Automated Cell Counter</td>
			<td>BioRad Laboratories, Inc.</td>
			<td>1450103</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypan Blue</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10x PBS</td>
			<td>Corning</td>
			<td>46-013-CM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">BioProfile FLEX Analyzer</td>
			<td>Nova Biomedical</td>
			<td>49418</td>
			<td>Nutrient Analyzer</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Octet Red 96</td>
			<td>Pall Fort&#233;Bio&#160;</td>
			<td>99-0042</td>
			<td>Protein A Biosensor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Protein A Dip and Read Biosensors</td>
			<td>Pall Fort&#233;Bio&#160;</td>
			<td>18-5010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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 ...

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