The authors would like to thank Scott Lute for the analytical support he provided. Partial internal funding and support for this work is provided by the CDER Critical Path Program (CA #1-13). This project is 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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This publication reflects the views of the author and should not be construed to represent FDA's views or policies.

HCCF contains debris and large particles that can clog and destroy costly instrumentation, thus culture clarification is needed before further downstream processing. Centrifugation is generally the first approach to separate cells and other insoluble particles from proteins followed by filtration. This filtered HCCF is then subjected to Fast Protein Liquid Chromatography (FPLC) for purification. Purification of HCCF from automated microbioreactors to obtain the product is an important step in downstream processing. Here, a benchtop FPLC system with a protein A column is used to obtain monoclonal antibodies from the HCCF. Analytics for upstream processes can provide useful insight into cell behavior and guide bioprocess design, helping to obtain a consistent and reliable quality product. Analytics also allows us to link Critical Quality Attributes (CQAs) to upstream and downstream processes. Presented here are four assays that are commonly used in the characterization of monoclonal antibodies. These techniques are robust, reliable, and readily deployable for process and product analysis from a variety of upstream sources which are only partially purified and may still contain residual levels of DNA and HCP.
When cleaning up samples for analytics, an important balance must be struck between creating a sample that is sufficiently clean for analysis while preserving the variability present in the bioreactor. The two most common contaminants impacting product are DNA and HCP, which can be checked by measuring the ratio absorbance at 260/280 nm and through SDS-PAGE or &#181;CE-SDS. The assays presented here are not sensitive to low levels of DNA content. The purity of the product is &#62;95% pure, as determined by &#181;CE-SDS.
Charge variant analysis with a microcapillary electrophoresis system provides a high-throughput method to identify charge variants, with chips and reagents that are relatively easy to implement. The nature of the technique and the chemistry of the labeling reagent are both sensitive to excipients and other primary amines, thus requiring a desalting step for most sample matrixes. From experience, low levels of DNA co-migrate with the free-dye from the labeling reaction and do not impact the quality of results. While variability of basic, main, and acidic peak quantification is typically &#60;1%, higher levels of DNA and other contaminants can increase the variability of the assay. It is extremely important to be consistent with the protein labeling and ensure the prompt use of DMF after removal from the bottle and being mixed with the dye. Lysine and/or histidine standards are recommended as labeling controls. Over time and depending on sample quality, chips can foul or lose the coating on the microfluidics channels, leading to greater noise, the presence of ghost peaks, and greater sample-to-sample variation. To identify this occurrence, blanks and a system suitability standard (i.e. NISTmAb) were concurrently analyzed with the samples at regular intervals. When chip issues arise, the chips can be washed with the storage solution or replaced.
The methods used for glycan analysis of therapeutic glycoproteins primarily involves liquid chromatography (LC) and/or mass spectrometry (MS), with lectin microarray analysis gaining popularity as a third option25. The method described in this paper uses both LC and MS, which has benefits and disadvantages. Mass spectrometric methods have the advantage of mass verification of the analyzed glycans, which is not possible with LC-based methods using a fluorescent detection output or lectin microarrays. This method uses LC and fluorescence detection to assign glycan identities using retention time comparison to a dextran ladder standard. Fluorescence monitoring allows for increased sensitivity and quantification due to the ease of its detection, where MS alone might not be able to quantify low abundance species due to the poor ionization efficiency of oligosaccharides. The mass information from MS is used to confirm glycan identities, but the processing software does not use mass information as the primary assignment criteria. Hence, without reproducible chromatography and easily resolvable peaks, this method can suffer regarding glycan assignments. Fortunately, the mass information can help with glycan assignments even in situations when the chromatography is subpar, such as shifts in retention time that hinder reproducible glycan assignments. If this method is used without MS, the chromatography must be at the highest level since mass information cannot be used to correct for residence time drift.
The amino acid analysis method described here utilizes LC-MS for rapid quantitation of underivatized amino acids in crude cell culture media. Alternative amino acid analysis methods require amino acid derivatization agents to enable UV detection26. The LC-MS method offers important advantages over the LC-UV method: it allows for identification based on both retention time and ion mass as opposed to the LC-UV method, which is limited by a lack of mass characterization. Furthermore, the LC-MS method offers time and reproducibility advantages, as the LC-UV method requires a time-consuming derivatization reaction, which may impart sample variability27. However, the injection of crude cell culture media in the LC-MS method can cause detrimental effects on MS signal due to ion skimmer fouling. A calibration ladder is injected frequently as a system suitability check, and sample order is randomized to prevent bias in the data.
The cell culture process for antibody production using microbioreactors is previously described9. In this study, detailed protocols for monoclonal antibody characterization methods that maximize data acquired from limited sample volumes are well-defined. Limited amounts of harvested cell culture fluid can sometimes restrict the product information acquired and selection of the right analytical procedures to obtain product quality data is essential. Analytics are important to link together upstream process parameters to the changes in product quality. Here, a guideline is provided for users to characterize mAbs when working with microbioreactors.

Protein therapeutics are being used to treat a growing variety of medical conditions including tissue transplant complications, autoimmune disorders, and cancers1. Since 2004, the United States Food and Drug Administration (USFDA) has documented an increasing proportion of biologic license applications (BLAs) of all approvals regulated by the Center for Drug Evaluation and Research (CDER), with BLAs accounting for over 25% in 2014 and 20152.
Considering this expanding market, biopharmaceutical manufacturers are challenged with quickly delivering more product with consistent quality. Efforts to augment product yield have focused on CHO cell engineering and production line screening, though the most significant improvements are due to advances in media/feed strategy optimization and cell culture environmental controls1,3,4,5 during the manufacturing process.
Since mAbs are produced in a biological system, there can be inherent protein variability. Antibody composition can be altered post-translationally, such as glycosylation or impacted by degradation or enzymatic reactions. These structural variations may provoke dangerous immune reactions or alter antibody binding, which in turn can reduce or eliminate the intended therapeutic function5. Thus, critical quality attributes (CQAs) of monoclonal antibodies - N-glycan profile, charge variant distribution, and the percentage of antibody in monomeric form - are regularly monitored and controlled as part of a Quality by Design (QbD) approach during manufacturing processes1,6. In a regulated production environment, therapeutic proteins must meet acceptance criteria to be licensed as an approved commercial drug product7. The methods presented herein would typically be part of the quality characterization process for an antibody7,8, and any protein scientist will be familiar with their usage.
In prior work9, the application and operation of microbioreactors for high throughput screening of cell culture conditions in upstream bioprocessing has been described. The purified product obtained from the varying media conditions is subjected to N-glycan analysis using LC-MS. Glycosylation patterns of therapeutic proteins can be detected and characterized using LC-MS techniques10,11, and the presence of various glycan species has been linked to bioprocess parameters such as feed strategy, pH, and temperature12. The effect of the varying media conditions on product quality, indicated by the percentage of the resulting IgG in monomeric form, is also evaluated with Size Exclusion Chromatography- Multi-Angle Light Scattering (SEC-MALS)13,14,15. The charge variant profile represents a number of modifications16 that could impact the function of a product. Microcapillary zone electrophoresis (mCZE) is a technique that offers a considerably faster analysis time compared to traditional cation exchange (CEX) chromatography and capillary isoelectric focusing (cIEF) methods used for charge variant analysis17,18. Spent bioreactor media was analyzed to track amino acid consumption during protein production as it relates to changes in the antibody&#39;s identifying attributes19,20,21,22,23.
Protein analytics allow us to identify critical process parameters (CPPs) based on the relationships between process inputs and changes in CQAs. During bioprocess development, identifying and measuring CPPs fundamentally demonstrates process control and ensures that the product has not changed, which is essential in highly regulated manufacturing environments. In this paper, analytical techniques to measure some of the biochemical characteristics of the protein most pertinent to product CQAs (N-glycan profile, charge variants, and size homogeneity) are presented.

<table><tbody><tr><td>CHO DG44 Cell Line</td><td>Invitrogen</td><td>A1100001</td><td></td></tr><tr><td>Akta Avant 25</td><td>General Electric Life Sciences</td><td>28930842</td><td></td></tr><tr><td>Pro Sep vA Ultra Chromatography Resin</td><td>Millipore Sigma</td><td>115115830</td><td>Purification Stationary Phase</td></tr><tr><td>Omnifit 10cm Column</td><td>Diba Fluid Intelligence</td><td>006EZ-06-10-AA</td><td>Housing for Stationary Phase</td></tr><tr><td>Tris Base</td><td>Fisher Scientific</td><td>BP154-1</td><td></td></tr><tr><td>Superloop 10 mL</td><td>GE Healthcare</td><td>18-1113-81</td><td></td></tr><tr><td>&amp;micro;Dawn Multi Angle Light Scattering Detector</td><td>Wyatt</td><td>WUDAWN-01</td><td></td></tr><tr><td>0.22 &amp;micro;m Millex GV Filter Unit PVDF Membrane</td><td>Merck Millipore</td><td>SLGV033RB</td><td></td></tr><tr><td>10X Phosphate Buffered Saline</td><td>Corning</td><td>46-013-CM</td><td></td></tr><tr><td>12 mL Syringe</td><td>Covidien</td><td>8881512878</td><td></td></tr><tr><td>1290 Infinity Binary Pump</td><td>Agilent Technologies</td><td>G4220A</td><td></td></tr><tr><td>1290 Infinity DAD&amp;nbsp;</td><td>Agilent Technologies</td><td>G4212A</td><td></td></tr><tr><td>1290 Infinity Sampler</td><td>Agilent Technologies</td><td>G4226A</td><td></td></tr><tr><td>1290 Infinity Thermostat</td><td>Agilent Technologies</td><td>G1330B</td><td></td></tr><tr><td>1290 Infinity Thermostatted Column Compartment</td><td>Agilent Technologies</td><td>G1316C</td><td></td></tr><tr><td>15 mL Falcon tube</td><td>Corning Inc.</td><td>352097</td><td></td></tr><tr><td>150 uL Glass Inserts with Polymer Feet&amp;nbsp;</td><td>Agilent Technologies</td><td>5183-2088</td><td></td></tr><tr><td>50 mL Falcon tube</td><td>Corning Inc.</td><td>352070</td><td></td></tr><tr><td>96-Well Plate</td><td>Bio-Rad</td><td>127737</td><td></td></tr><tr><td>Acetic Acid</td><td>Sigma-Aldrich</td><td>695072</td><td></td></tr><tr><td>Acetonitrile</td><td>Fisher Chemical</td><td>BPA996-4</td><td></td></tr><tr><td>ACQUITY I-Class UPLC BSM</td><td>Waters Corporation</td><td>18601504612</td><td></td></tr><tr><td>ACQUITY I-Class UPLC Sample Manager</td><td>Waters Corporation</td><td>186015000</td><td></td></tr><tr><td>ACQUITY UPLC FLR Detector</td><td>Waters Corporation</td><td>176015029</td><td></td></tr><tr><td>Amicon Ultra-4 100 kDa centrifugal filters</td><td>Merck Millipore</td><td>UFC810096</td><td></td></tr><tr><td>Amino Acid Standard, 1 nmol/&amp;micro;L&amp;nbsp;</td><td>Agilent Technologies</td><td>5061-3330</td><td></td></tr><tr><td>Amino Acid Supplement</td><td>Agilent Technologies</td><td>5062-2478</td><td></td></tr><tr><td>Ammonium Formate Solution - Glycan Analysis</td><td>Waters Corporation</td><td>186007081</td><td></td></tr><tr><td>Blue Screw Caps with Septa</td><td>Agilent Technologies</td><td>5182-0717</td><td></td></tr><tr><td>CD OptiCHO AGT Medium</td><td>Thermo Fisher Scientific</td><td>A1122205</td><td></td></tr><tr><td>Centrifuge Tubes</td><td>Eppendorf</td><td>22363352</td><td></td></tr><tr><td>Charge Variant Chip</td><td>Perkin Elmer</td><td>760435</td><td></td></tr><tr><td>Charge Variant Reagent Kit</td><td>Perkin Elmer</td><td>CLS760670</td><td></td></tr><tr><td>Chromatography Water (MS Grade)</td><td>Fisher Chemical</td><td>W6-4</td><td></td></tr><tr><td>Dimethylformamide</td><td>Thermo Scientific</td><td>20673</td><td></td></tr><tr><td>Extraction Plate Manifold for Oasis 96-Well Plates</td><td>Waters Corporation</td><td>186001831</td><td></td></tr><tr><td>Formic Acid</td><td>Fisher Chemical</td><td>A117-50</td><td></td></tr><tr><td>GlycoWorks RapiFlour-MS N-Glycan Starter Kit - 24 Sample</td><td>Waters Corporation</td><td>176003712</td><td></td></tr><tr><td>GXII Buffer Tubes</td><td>E&amp;amp;K Scientific</td><td>697075- NC</td><td></td></tr><tr><td>GXII Detection Window Cleaning Cloth</td><td>VWR</td><td>21912-046</td><td></td></tr><tr><td>GXII HT Touch</td><td>Perkin Elmer</td><td>CLS138160</td><td></td></tr><tr><td>GXII Ladder Tubes</td><td>Genemate</td><td>C-3258-1</td><td></td></tr><tr><td>GXII Lint-Free Swab</td><td>ITW Texwipe</td><td>TX758B</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher Scientific</td><td>A144-500</td><td></td></tr><tr><td>Intact mAb Mass Check Standard</td><td>Waters Corporation</td><td>186006552</td><td></td></tr><tr><td>Intrada Amino Acid Column 150 x 2 mm</td><td>Imtakt</td><td>WAA25</td><td></td></tr><tr><td>NanoDrop One Microvolume UV-Vis Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>840274100</td><td></td></tr><tr><td>Optilab UT-rEX Differential Refractive Index Detector</td><td>Wyatt</td><td>WTREX-11</td><td></td></tr><tr><td>Perchloric acid</td><td>Aldrich Chemistry</td><td>311421</td><td></td></tr><tr><td>Pipet Tips with Microcapillary for Loading Gels</td><td>Labcon</td><td>1034-960-008</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><tr><td>RapiFlour-MS Dextran Calibration Ladder</td><td>Waters Corporation</td><td>186007982</td><td></td></tr><tr><td>Screw Top Clear Vial 2mL</td><td>Agilent Technologies</td><td>5182-0715</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>S271-1</td><td></td></tr><tr><td>Sodium Iodide</td><td>Sigma Aldrich</td><td>383112</td><td></td></tr><tr><td>TSKgel UP-SW3000 4.6mm ID x 30 cm L</td><td>Tosoh Biosciences</td><td>003449</td><td></td></tr><tr><td>UNIFI Scientific Information System</td><td>Waters Corporation</td><td>667005138</td><td></td></tr><tr><td>Vacuum Manifold Shims</td><td>Waters Corporation</td><td>186007986</td><td></td></tr><tr><td>Vacuum Pump</td><td>Waters Corporation</td><td>725000604</td><td></td></tr><tr><td>Xevo G2 Q-ToF</td><td>Waters Corporation</td><td>186005597</td><td></td></tr><tr><td>Zeba Spin Desalting Column, 0.5 mL</td><td>Thermo Scientific</td><td>89883</td><td></td></tr></tbody></table>

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.

The harvested cell culture fluid from the automated microscale bioreactor is purified using fast protein liquid chromatography (FPLC), as seen in Figure 1 and the purified proteins&#39; critical quality attributes (CQAs) were characterized by various downstream analytical methods. This is a key benefit of the automated microbioreactor system; differences in CQAs can be rapidly assessed across a wide range of conditions. N-glycan data from CHO-produced mAbs that are processed by mass spectrometry should appear like the chromatograms shown in Figure 2. The figure depicts a comparison between two chromatograms showing that the mannose 5 peak (M5) from one sample is considerably lower. If only a noisy baseline is observed instead of peaks, this may mean that the chromatography setup is faulty or that the procedure is not successful. Using controls, troubleshooting can be simplified. First, assess the FLR peaks from the dextran ladder; these peaks indicate that the chromatographic system is working correctly. Next, compare the experimentally obtained peaks with those obtained from a processed intact mAb standard. If peaks from the standard are visible, but no sample peaks are identified, then the mAb samples were not processed correctly. This may be due to SDS or nucleophile presence in the buffer interfering with N-glycan labeling and purification.
SEC-MALS can be used to assess two more CQAs: the aggregation profile and the molecular weight of the antibody. A representative SEC-MALS chromatogram is comparable to the one shown in Figure 3. The molecular mass distribution and the absolute molecular weight were determined using the required software with an extinction coefficient of 1.37 mL*(mg*cm)-1 and a dn/dc of 0.185 mL/g. As peak calling and setting the baseline in the software is performed manually, results may vary slightly from user to user. The absolute molecular weight of monomeric IgG1 from Figure 3 is 1.504 x 105 Da &#177; 0.38% (blue) and the higher order complex is 7.799 x 105 Da &#177; 3.0% (red). The polydispersity of the aggregates is much greater than that of the monomer, as indicated by the red molar mass distribution of Peak 1 (Figure 3). The small quantity of sample and importance of aggregation as a CQA make this technique a highly valuable complementary analytical tool to the automated microbioreactor system.
The result of mCZE is an electropherogram, such as in Figure 4, which shows the charge variant profile for a monoclonal antibody. The profile is a unique signature for the protein being investigated and is highly sensitive to the operating pH. Also visible is a free-dye peak to the left of the charge variant profile. When establishing an operating pH, there is some discretion to the operator to balance resolution and signal; in addition, the operator must ensure good separation from the free-dye peak which migrates at ~30 s. The sample can be desalted after labelling to remove this peak, though this leads to a significant loss in signal. Once an operating pH is established, the sample profiles can be compared. While generally consistent, changes in labeling efficiency or differences in excipients can lead to minor differences in the migration of a sample and the charge variant profile making electropherograms hard to directly compare. Instead, the method of comparison is usually based on the percentages of basic, main, and acidic species. In this case, relative differences as small as 1-2% can be identified using mCZE.
Amino acid consumption can be monitored to determine if depletion is causing changes in CQAs. Chromatogram readouts from the mass spectrometer can be used to evaluate the successful creation of a calibration curve for the absolute quantification of amino acids in crude bioreactor media samples. Figure 5 depicts two total ion chromatograms (TIC) and one extracted ion chromatogram (XIC) as representative results during this process. In Figure 5A, the TIC shown depicts the background signal from the buffer system as only a water blank was injected. Figure 5B depicts a representative TIC of the amino acid standard where, when compared to the water blank, small peaks that correspond to the individual amino acid species can be observed (such as lysine at 7.96 minutes). To integrate the peak and facilitate the quantification of peak area (and therefore the concentration), the XIC is used where only the signal from a defined &#34;chromatogram mass window&#34; is displayed. Depending on the sensitivity of the instrument and the quality of the chromatographic separation, the optimal mass window will have to be determined by the user. In this example (Figure 5C), the XIC of lysine (m/z = 147.1144) with a mass window of 10 ppm is shown where lysine in the amino acid standard elutes off the column at 8.03 minutes.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58947/58947fig1.jpg" /> 
Figure 1. Representative chromatogram of the purification scheme using the Fast Protein Liquid Chromatography (FPLC) technique. Purification method phases corresponding to volume (mL) are labeled along the x-axis. UV absorbance at 280 nm (mAU y-axis, solid line) is monitored throughout the purification cycle. Non-specifically bound impurities are displaced by increasing conductivity (mS/cm y-axis, dashed line) during the High Salt Wash. Antibody is eluted from Protein A column with the introduction of elution buffer (Conc B, dotted line) when the pH decreases to 4 (not shown). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58947/58947fig1large.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/58947/58947fig2.jpg" /> 
Figure 2.&#160;A representative fluorescence chromatogram obtained from tagged glycans that are mass verified. The x-axis is retention time (minutes) while the y-axis is signal intensity. The peak at 14.94 min represents the Mannose 5 (M5) glycan, where a large difference between the M5 signal strength can be observed between the two samples that are overlaid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58947/58947fig2large.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/58947/58947fig3.jpg" /> 
Figure 3.&#160;Molecular weight distribution of IgG1 monoclonal antibody. Chromatogram of an intact IgG1 monoclonal antibody separated by size exclusion chromatography in 1x PBS (pH7.4). Absorbance is monitored at 280 nm (black; left axis) and light scattering and refractive index detectors were used to calculate the absolute molecular weight of each peak (red and blue; right axis). High Molecular Weight species are indicated with the peak labeled &#34;HMW&#34;. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58947/58947fig3large.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/58947/58947fig4.jpg" /> 
Figure 4. Charge variant profile of a IgG1 monoclonal antibody. This electropherogram is generated on a mCZE platform. A free-dye peak migrates at ~30 s and is well separated from the IgG1. For quantification, peaks were split into basic, main, and acidic species using instrument data analysis software. The red line outlines the integrated peak areas. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58947/58947fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58947/58947fig5.jpg" /> 
Figure 5.&#160;Representative results of the ion chromatograms for mass spectrometry-based amino acid analysis of crude bioreactor media. The x-axis is time (minutes) while the y-axis is signal intensity (A) A water blank serves as the negative control and reveals the background signal observed over the course of the liquid chromatography gradient (B) The 225 pmol/&#181;L amino acid standard is used here as a positive control, as the individual peaks observed in this total ion chromatogram represent the different amino acids of the standard mix being resolved chromatographically (C) A representative extracted ion chromatogram for m/z 147.1144, which is lysine. The 7.96 min peak in B corresponds to the 8.03 peak in C of lysine. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58947/58947fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System at JoVE.com

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

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 ...

purification-analytics-monoclonal-antibody-from-chinese-hamster-ovary

Nanyang Technological University

Research

JoVE Journal

Bioengineering

14.6K Views.  U.S. Food and Drug Administration. Dit protocol richt zich op de analyse van beperkte monsters verkregen uit microbioreactoren om essentiële informatie over de kritische kwaliteitskenmerken van de producten te extraheren. Een ander voordeel van deze technieken is dat ze minimale tijden van analyse gebruiken om de belangrijkste kenmerken te bepalen die de kwaliteitsparameters van het vervaardigde product dicteren. Demonstrerende procedure zijn David Powers, Talia Faison en Phillip Angart, onderzoekers van mijn laboratorium.

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

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 Murine Monoclonal Antibodies by Hybridoma Technology

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding molecules is time-consuming and laborious due to the complicated handling and lack of alternatives. The aim of this protocol is to provide one standard method for the generation of monoclonal antibodies using hybridoma technology. This technology combines two steps. Step 1 is an appropriate immunization of the animal and step 2 is the fusion of B lymphocytes with immortal myeloma cells in order to generate hybrids possessing both parental functions, such as the production of antibody molecules and immortality. The generated hybridoma cells were then recloned and diluted to obtain stable monoclonal cell cultures secreting the desired monoclonal antibody in the culture supernatant. The supernatants were tested in enzyme-linked immunosorbent assays (ELISA) for antigen specificity. After the selection of appropriate cell clones, the cells were transferred to mass cultivation in order to produce the desired antibody molecule in large amounts. The purification of the antibodies is routinely performed by affinity chromatography. After purification, the antibody molecule can be characterized and validated for the final test application. The whole process takes 8 to 12 months of development, and there is a high risk that the antibody will not work in the desired test system.

An optimized protocol is presented for the generation of monoclonal antibodies based on the hybridoma technology. Mice were immunized with an immunoconjugate. Spleen cells were fused by PEG and an electric impulse with immortal myeloma cells. Antibody-producing hybridoma cells were selected by HAT and antigen-specific ELISA screening.

Monoclonal antibodies are universal binding molecules and are widely used in biomedicine and research. Nevertheless, the generation of these binding ...

Immunology and Infection

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.

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 ...

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 ...

Rapid Antibody Glycoengineering in Chinese Hamster Ovary Cells

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. However, monoclonal antibody functionality and half-life may be reduced by the type and distribution of host-specific glycosylation. Attempts to produce superior antibodies have inspired the development of genetically modified producer cells that synthesize glyco-optimized antibodies. Glycoengineering typically requires the generation of a stable knockout or knockin cell line using methods such as clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9. Monoclonal antibodies produced by engineered cells are then characterized using mass spectrometric methods to determine if the desired glycoprofile has been obtained. This strategy is time-consuming, technically challenging, and requires specialists. Therefore, an alternative strategy that utilizes streamlined protocols for genetic glycoengineering and glycan detection may assist endeavors toward optimal antibodies. In this proof-of-concept study, an IgG-producing Chinese hamster ovary cell served as an ideal host to optimize glycoengineering. Short interfering RNA targeting the Fut8 gene was delivered to Chinese hamster ovary cells, and the resulting changes in FUT8 protein expression were quantified. The results indicate that knockdown by this method was efficient, leading to a ~60% reduction in FUT8. Complementary analysis of the antibody glycoprofile was performed using a rapid yet highly sensitive technique: capillary gel electrophoresis and laser-induced fluorescence detection. All knockdown experiments showed an increase in afucosylated glycans; however, the greatest shift achieved in this study was ~20%. This protocol simplifies glycoengineering efforts by harnessing in silico design tools, commercially synthesized gene targeting reagents, and rapid quantification assays that do not require extensive prior experience. As such, the time efficiencies offered by this protocol may assist investigations into new gene targets.

The glycosylation pattern of an antibody determines its clinical performance, thus industrial and academic efforts to control glycosylation persist. Since typical glycoengineering campaigns are time- and labor-intensive, the generation of a rapid protocol to characterize the impact of glycosylation genes using transient silencing would prove useful.

Recombinant monoclonal antibodies bind specific molecular targets and, subsequently, induce an immune response or inhibit the binding of other ligands. ...

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 ...

Antibody Generation: Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4 
1 Microbiology, Immunology, and Cancer Biology Graduate Program, University of Minnesota, Minneapolis, MN 55455 
2 Center for Immunology, University of Minnesota, Minneapolis, MN 55455 
3 Department of Urology, University of Minnesota, Minneapolis, MN 55455 
4 Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455
Polyclonal antibodies are defined as a collection of antibodies directed against different antigenic determinants of an antigen or several antigens (1). While polyclonal antibodies are powerful tools for identifying biological molecules, there is one important limitation - they are unable to distinguish between antigens that share antigenic determinants. For example, when bovine serum albumin is used to immunize an animal, B cells with different surface Ig will respond to different antigenic determinants on bovine serum albumin. The result is a mixture of antibodies in the antiserum. Because bovine serum albumin shares some epitopes with human serum albumin in evolutionarily conserved regions of the protein, this anti-bovine serum albumin antiserum will also react with human serum albumin. Therefore, this antiserum will not be useful for distinguishing between bovine and human serum albumins.
Several approaches have been taken to overcome the specificity issue of polyclonal antisera. One is by absorbing the unwanted antibodies by passing the antiserum through a chromatography column of immobilized antigens (2). This method is tedious and frequently unable to completely remove the unwanted antibodies. Another approach is to isolate individual antibody-producing B cells and expand them in culture. However, like most normal untransformed cells, B cells do not survive in long-term culture.
To overcome the inability of B cells to survive in culture, one approach is to prepare a myeloma-B cell hybridoma. In 1847, Henry Bence-Jones discovered that patients with multiple myeloma, a lymphoid tumor, produced a large quantity of antibodies (3). B cells in these patients have become malignant and grow uncontrollably. Since the malignant B cells are derived from a single clone, they are identical and produce only a single type of antibody (i.e., a monoclonal antibody, or mAb). However, most of these myeloma cells produce antibodies of unknown specificities. In 1975, by fusing a myeloma cell to a B cell, Cesar Milstein and Georges Kohler succeeded in producing a hybridoma that can be cultured indefinitely in vitro and produces an unlimited number of monoclonal antibodies of known antigenic specificity (4). The rationale behind their approach is to combine the immortal properties of the myeloma cell and the antibody producing properties of the B cell. Their technique revolutionized antibody production and provides a powerful means for identification and purification of biological molecules using monoclonal antibodies.
Generally, preparing a monoclonal antibody requires several months. The general procedure includes the following steps:
<ol>
	<li>Immunization and screening of antibody titer</li>
	<li>Fusion of antibody-producing B cells and myeloma cells</li>
	<li>Selective growth of the hybridoma</li>
	<li>Screening the hybridomas for producing the desired monoclonal antibody</li>
	<li>Cloning by limiting dilution - a process whereby cells are diluted to a concentration to statistically allow for less than 1 cell to be added to the wells of a 96-well plate. Some wells will end up with 0 cells and some will have 1 cell. The wells with seeded with 1 cell will eventually grow into a monoclonal population of cells.</li>
	<li>Growth of the hybridoma and preparation of monoclonal antibody</li>
</ol>
This protocol focuses on the last step - growth of the hybridoma and preparation of the monoclonal antibody. The antibody is purified from the culture supernatant by ammonium sulfate precipitation (often referred to as salting out) - a commonly used method of removing proteins from a solution. Proteins in solution form hydrogen bonds, along with other hydrophilic interactions, with water through their exposed polar and ionic groups. When concentrations of small, highly charged ions (such as ammonium or sulfate) are added, these groups compete with the proteins for binding to water. This removes the water molecules from the protein and decreases its solubility, resulting in precipitation of the protein.

Producing Monoclonal Antibodies Using Hybridomas

Source: Frances V. Sjaastad1,2, Whitney Swanson2,3, and Thomas S. Griffith1,2,3,4
1 Microbiology, Immunology, and Cancer Biology Graduate Program, ...

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A Technique to Purify Monoclonal Antibodies Produced by Chinese Hamster Ovary Cells

Source: Velugula-Yellela, S. R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58947">Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System.</a> J. Vis. Exp. (2019).
This video demonstrates an assay for purifying monoclonal antibodies from cell culture fluid harvested from Chinese Hamster Ovarian cells. It employs a porous glass resin with immobilized protein A, which binds to the Fc region of the antibodies in the sample, effectively separating them from contaminants. The use of a low-pH elution buffer weakens the interaction, facilitating the retrieval of purified antibodies.

1. Purification of antibody
NOTE: The equilibration buffer for the in-house antibody is 25 mM Tris, 100 mM NaCl, pH 7.5. The elution buffer used is 0.1 M ...

JoVE Encyclopedia of Experiments

Antibody-Based Technologies

Production and Purification

A Technique to Assess the In Vivo Localization of Tumor-Specific Antibodies

Source: Shivange, G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/60727">Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody.</a> J. Vis. Exp. (2020).
This video demonstrates a technique to study the in vivo localization of tumor-specific antibodies in a mouse tumor xenograft model. Upon subcutaneously injecting cancer cells into mice for tumor formation, fluorescently-labeled antibodies are injected that bind to tumor-specific receptors, enabling in vivo assessment of antibody localization via imaging.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Immunodiagnostics

Imaging and Visualization Techniques

An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Source: Elgundi, Z., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55153">Laboratory Scale Production and Purification of a Therapeutic Antibody.</a> J. Vis. Exp. (2017).
This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System
NOTE: The following procedure can ...

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

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